46 results on '"type 3 secretion"'
Search Results
2. Topology and Contribution to the Pore Channel Lining of Plasma Membrane-Embedded Shigella flexneri Type 3 Secretion Translocase IpaB
- Author
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Poyin Chen, Brian C. Russo, Jeffrey K. Duncan-Lowey, Natasha Bitar, Keith T. Egger, and Marcia B. Goldberg
- Subjects
Shigella flexneri ,type 3 secretion ,translocon ,IpaB ,topology ,Microbiology ,QR1-502 - Abstract
ABSTRACT Shigella spp. are human bacterial pathogens that cause bacillary dysentery. Virulence depends on a type 3 secretion system (T3SS), a highly conserved structure present in multiple important human and plant pathogens. Upon host cell contact, the T3SS translocon is delivered to the host membrane, facilitates bacterial docking to the membrane, and enables delivery of effector proteins into the host cytosol. The Shigella translocon is composed of two proteins, IpaB and IpaC, which together form this multimeric structure within host plasma membranes. Upon interaction of IpaC with host intermediate filaments, the translocon undergoes a conformational change that allows for bacterial docking onto the translocon and, together with host actin polymerization, enables subsequent effector translocation through the translocon pore. To generate additional insights into the translocon, we mapped the topology of IpaB in plasma membrane-embedded pores using cysteine substitution mutagenesis coupled with site-directed labeling and proximity-enabled cross-linking by membrane-permeant sulfhydryl reactants. We demonstrate that IpaB function is dependent on posttranslational modification by a plasmid-encoded acyl carrier protein. We show that the first transmembrane domain of IpaB lines the interior of the translocon pore channel such that the IpaB portion of the channel forms a funnel-like shape leading into the host cytosol. In addition, we identify regions of IpaB within its cytosolic domain that protrude into and are closely associated with the pore channel. Taken together, these results provide a framework for how IpaB is arranged within translocons natively delivered by Shigella during infection. IMPORTANCE Type 3 secretion systems are nanomachines employed by many bacteria, including Shigella, which deliver into human cells bacterial virulence proteins that alter cellular function in ways that promote infection. Delivery of Shigella virulence proteins occurs through a pore formed in human cell membranes by the IpaB and IpaC proteins. Here, we define how IpaB contributes to the formation of pores natively delivered into human cell membranes by Shigella flexneri. We show that a specific domain of IpaB (transmembrane domain 1) lines much of the pore channel and that portions of IpaB that lie in the inside of the human cell loop back into and/or are closely associated with the pore channel. These findings provide new insights into the organization and function of the pore in serving as the conduit for delivery of virulence proteins into human cells during Shigella infection.
- Published
- 2021
- Full Text
- View/download PDF
3. Translocation of Toxins by Gram-Negative Pathogens Using the Type III Secretion System
- Author
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Vermeulen, Arjan J., Tang, Yuzhou, Heuck, Alejandro P., Gopalakrishnakone, P., Editor-in-chief, Stiles, Brad, editor, Alape-Girón, Alberto, editor, Dubreuil, J. Daniel, editor, and Mandal, Manas, editor
- Published
- 2018
- Full Text
- View/download PDF
4. The Complete Genome Sequence of Xanthomonas theicola, the Causal Agent of Canker on Tea Plants, Reveals Novel Secretion Systems in Clade-1 Xanthomonads.
- Author
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Koebnik, Ralf, Burokiene, Daiva, Bragard, Claude, Chang, Christine, Saux, Marion Fischer-Le, Kölliker, Roland, Lang, Jillian M., Leach, Jan E., Luna, Emily K., Portier, Perrine, Sagia, Angeliki, Ziegle, Janet, Cohen, Stephen P., and Jacobs, Jonathan M.
- Subjects
- *
WHOLE genome sequencing , *XANTHOMONAS , *SECRETION , *BIOLOGICAL evolution , *PLASMIDS , *GENOMES , *PEPTIDES - Abstract
Xanthomonas theicola is the causal agent of bacterial canker on tea plants. There is no complete genome sequence available for X. theicola, a close relative of the species X. translucens and X. hyacinthi, thus limiting basic research for this group of pathogens. Here, we release a high-quality complete genome sequence for the X. theicola type strain, CFBP 4691T. Single-molecule real-time sequencing with a mean coverage of 264× revealed two contigs of 4,744,641 bp (chromosome) and 40,955 bp (plasmid) in size. Genome mining revealed the presence of nonribosomal peptide synthases, two CRISPR systems, the Xps type 2 secretion system, and the Hrp type 3 secretion system. Surprisingly, this strain encodes an additional type 2 secretion system and a novel type 3 secretion system with enigmatic function, hitherto undescribed for xanthomonads. Four type 3 effector genes were found on complete or partial transposons, suggesting a role of transposons in effector gene evolution and spread. This genome sequence fills an important gap to better understand the biology and evolution of the early-branching xanthomonads, also known as clade-1 xanthomonads. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
5. Topological Analysis of the Type 3 Secretion System Translocon Pore Protein IpaC following Its Native Delivery to the Plasma Membrane during Infection
- Author
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Brian C. Russo, Jeffrey K. Duncan, and Marcia B. Goldberg
- Subjects
IpaC ,Salmonella ,Shigella flexneri ,SipC ,topology ,type 3 secretion ,Microbiology ,QR1-502 - Abstract
ABSTRACT Many Gram-negative bacterial pathogens require a type 3 secretion system (T3SS) to deliver effector proteins into eukaryotic cells. Contact of the tip complex of the T3SS with a target eukaryotic cell initiates secretion of the two bacterial proteins that assemble into the translocon pore in the plasma membrane. The translocon pore functions to regulate effector protein secretion and is the conduit for effector protein translocation across the plasma membrane. To generate insights into how the translocon pore regulates effector protein secretion, we defined the topology of the Shigella translocon pore protein IpaC in the plasma membrane following its native delivery by the T3SS. Using single cysteine substitution mutagenesis and site-directed labeling with a membrane-impermeant chemical probe, we mapped residues accessible from the extracellular surface of the cell. Our data support a model in which the N terminus of IpaC is extracellular and the C terminus of IpaC is intracellular. These findings resolve previously conflicting data on IpaC topology that were based on nonnative delivery of IpaC to membranes. Salmonella enterica serovar Typhimurium also requires the T3SS for effector protein delivery into eukaryotic cells. Although the sequence of IpaC is closely related to the Salmonella translocon pore protein SipC, the two proteins have unique functional attributes during infection. We showed a similar overall topology for SipC and IpaC and identified subtle topological differences between their transmembrane α-helixes and C-terminal regions. Together, our data suggest that topological differences among distinct translocon pore proteins may dictate organism-specific functional differences of the T3SSs during infection. IMPORTANCE The type 3 secretion system (T3SS) is a nanomachine required for virulence of many bacterial pathogens that infect humans. The system delivers bacterial virulence proteins into the cytosol of human cells, where the virulence proteins promote bacterial infection. The T3SS forms a translocon pore in the membranes of target cells. This pore is the portal through which bacterial virulence proteins are delivered by the T3SS into the eukaryotic cytosol. The pore also regulates secretion of these virulence proteins. Our work defines the topology of translocon pore proteins in their native context during infection, resolves previously conflicting reports about the topology of the Shigella translocon pore protein IpaC, and provides new insights into how interactions of the pore with the T3SS likely produce signals that activate secretion of virulence proteins.
- Published
- 2019
- Full Text
- View/download PDF
6. Distinct intraspecies virulence mechanisms regulated by a conserved transcription factor.
- Author
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Connolly, James P. R., O'Boyle, Nicky, Turner, Natasha C. A., Browning, Douglas F., and Roe, Andrew J.
- Subjects
- *
TRANSCRIPTION factors , *BACTERIAL evolution , *GENETIC regulation , *ESCHERICHIA coli , *SECRETION - Abstract
Tailoring transcriptional regulation to coordinate the expression of virulence factors in tandem with the core genome is a hallmark of bacterial pathogen evolution. Bacteria encode hundreds of transcription factors forming the base-level control of gene regulation. Moreover, highly homologous regulators are assumed to control conserved genes between members within a species that harbor the same genetic targets. We have explored this concept in 2 Escherichia coli pathotypes that employ distinct virulence mechanisms that facilitate specification of a different niche within the host. Strikingly, we found that the transcription factor YhaJ actively regulated unique gene sets between intestinal enterohemorrhagic E. coli (EHEC) and extraintestinal uropathogenic E. coli (UPEC), despite being very highly conserved. In EHEC, YhaJ directly activates expression of type 3 secretion system components and effectors. Alternatively, YhaJ enhances UPEC virulence regulation by binding directly to the phase-variable type 1 fimbria promoter, driving its expression. Additionally, YhaJ was found to override the universal GAD acid tolerance system but exclusively in EHEC, thereby indirectly enhancing type 3 secretion pleiotropically. These results have revealed that within a species, conserved regulators are actively repurposed in a "personalized" manner to benefit particular lifestyles and drive virulence via multiple distinct mechanisms. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
7. A Reporter System for Fast Quantitative Monitoring of Type 3 Protein Secretion in Enteropathogenic E. coli
- Author
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Luit Barkalita, Athina G. Portaliou, Maria S. Loos, Biao Yuan, Spyridoula Karamanou, and Anastassios Economou
- Subjects
EPEC ,Type 3 secretion ,quantitative in vivo secretion assay ,alkaline phosphatase activity ,SctA-PhoA ,Biology (General) ,QH301-705.5 - Abstract
The type 3 secretion system is essential for pathogenesis of several human and animal Gram-negative bacterial pathogens. The T3SS comprises a transmembrane injectisome, providing a conduit from the bacterial cytoplasm to the host cell cytoplasm for the direct delivery of effectors (including toxins). Functional studies of T3SS commonly monitor the extracellular secretion of proteins by SDS-PAGE and western blot analysis, which are slow and semi-quantitative in nature. Here, we describe an enzymatic reporter-based quantitative and rapid in vivo assay for T3SS secretion studies in enteropathogenic E. coli (EPEC). The assay monitors the secretion of the fusion protein SctA-PhoA through the injectisome based on a colorimetric assay that quantifies the activity of alkaline phosphatase. We validated the usage of this reporter system by following the secretion in the absence of various injectisome components, including domains of the gatekeeper essential for T3SS function. This platform can now be used for the isolation of mutations, functional analysis and anti-virulence compound screening.
- Published
- 2020
- Full Text
- View/download PDF
8. Arabidopsis bZIP11 Is a Susceptibility Factor During Pseudomonas syringae Infection
- Author
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Jung-Gun Kim, Sjef Smeekens, Monika Tomar, Wolf B. Frommer, Martin C. Jonikas, Johannes Hanson, Matthew J. Prior, Mary Beth Mudgett, Jebasingh Selvanayagam, Sub Molecular Plant Physiology, and Molecular Plant Physiology
- Subjects
0106 biological sciences ,0301 basic medicine ,Physiology ,Biology ,01 natural sciences ,Microbiology ,03 medical and health sciences ,Type 3 secretion ,Xanthomonas ,Transcription (biology) ,Arabidopsis ,Plant responses to pathogens ,Pseudomonas syringae ,Arabidopsis thaliana ,BZIP transcription factor ,Transcription factor ,Secretion and cell wall changes ,Susceptibility factor ,Pseudomonas ,Biochemistry and Molecular Biology ,Botany ,General Medicine ,Botanik ,Bacterial pathogenesis ,biology.organism_classification ,QR1-502 ,030104 developmental biology ,QK1-989 ,Plant nutrient secretion systems ,Efflux ,Agronomy and Crop Science ,Biokemi och molekylärbiologi ,010606 plant biology & botany - Abstract
The induction of plant nutrient secretion systems is critical for successful pathogen infection. Some bacterial pathogens (e.g., Xanthomonas spp.) use transcription activator-like (TAL) effectors to induce transcription of SWEET sucrose efflux transporters. Pseudomonas syringae pv. tomato strain DC3000 lacks TAL effectors yet is able to induce multiple SWEETs in Arabidopsis thaliana by unknown mechanisms. Because bacteria require other nutrients in addition to sugars for efficient reproduction, we hypothesized that Pseudomonas spp. may depend on host transcription factors involved in secretory programs to increase access to essential nutrients. Bioinformatic analyses identified the Arabidopsis basic-leucine zipper transcription factor bZIP11 as a potential regulator of nutrient transporters, including SWEETs and UmamiT amino acid transporters. Inducible downregulation of bZIP11 expression in Arabidopsis resulted in reduced growth of P. syringae pv. tomato strain DC3000, whereas inducible overexpression of bZIP11 resulted in increased bacterial growth, supporting the hypothesis that bZIP11-regulated transcription programs are essential for maximal pathogen titer in leaves. Our data are consistent with a model in which a pathogen alters host transcription factor expression upstream of secretory transcription networks to promote nutrient efflux from host cells. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
- Published
- 2021
9. Salmonella Invasion of the Gut Mucosa and Subsequent Host Cell Defenses
- Author
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Fattinger, Stefan A.
- Subjects
Fluorescence microscopy ,NLRC4 ,Virulence factors ,Gasdermin ,Life sciences ,Type 3 secretion ,Gastrointestinal tract ,Salmonella ,inflammasome ,In vivo ,Electron microscopy ,Mucosal immunology ,Enterocyte ,epithelial barrier - Published
- 2022
- Full Text
- View/download PDF
10. Comprehensive identification of virulence factors required for respiratory melioidosis using Tn-seq mutagenesis
- Author
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Maria G Gutierrez, Deborah R Yoder-Himes, and Jonathan Mark Warawa
- Subjects
capsular polysaccharide ,type 6 secretion ,Mouse Infection Models ,type 3 secretion ,Tn-seq ,intubation-mediated intratracheal (IMIT) inoculation ,Microbiology ,QR1-502 - Abstract
Respiratory melioidosis is a disease presentation of the biodefense pathogen, Burkholderia pseudomallei, which is frequently associated with a lethal septicemic spread of the bacteria. We have recently developed an improved respiratory melioidosis model to study the pathogenesis of Burkholderia pseudomallei in the lung (intubation-mediated intratracheal [IMIT] inoculation), which more closely models descriptions of human melioidosis, including prominent septicemic spread from the lung and reduced involvement of the upper respiratory tract. We previously demonstrated that the Type 3 Secretion System cluster 3 (T3SS3) is a critical virulence determinant for B. pseudomallei when delivered directly into the lung. We decided to comprehensively identify all virulence determinants required for respiratory melioidosis using the Tn-seq phenotypic screen, as well as to investigate which virulence determinants are required for dissemination to the liver and spleen. While previous studies have used Tn-seq to identify essential genes for in vitro cultured B. pseudomallei, this represents the first study to use Tn-seq to identify genes required for in vivo fitness. Consistent with our previous findings, we identified T3SS3 as the largest genetic cluster required for fitness in the lung. Furthermore, we identified capsular polysaccharide and Type 6 Secretion System cluster 5 (T6SS5) as the two additional major genetic clusters facilitating respiratory melioidosis. Importantly, Tn-seq did not identify additional, novel large genetic systems supporting respiratory melioidosis, although these studies identified additional small gene clusters that may also play crucial roles in lung fitness. Interestingly, other previously identified virulence determinants do not appear to be required for lung fitness, such as lipopolysaccharide. The role of T3SS3, capsule, and T6SS5 in lung fitness was validated by competition studies, but only T3SS3 was found to be important for respiratory melioidosis when delivered as a single strain challenge, suggesting that competition studies may provide a higher resolution analysis of fitness factors in the lung. The use of Tn-seq phenotypic screening also provided key insights into the selective pressure encountered in the liver.
- Published
- 2015
- Full Text
- View/download PDF
11. Genome structural variation in
- Author
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Stephen F, Fitzgerald, Nadejda, Lupolova, Sharif, Shaaban, Timothy J, Dallman, David, Greig, Lesley, Allison, Sue C, Tongue, Judith, Evans, Madeleine K, Henry, Tom N, McNeilly, James L, Bono, and David L, Gally
- Subjects
prophage ,Prophages ,E. coli ,Pathogens and Epidemiology ,PFGE ,Escherichia coli O157 ,Shiga toxin ,Shiga Toxin 2 ,genome structure ,inversion ,optical mapping ,duplication ,cattle ,type 3 secretion ,Genomic Structural Variation ,Animals ,O157:H7 ,Research Articles - Abstract
The human zoonotic pathogen Escherichia coli O157:H7 is defined by its extensive prophage repertoire including those that encode Shiga toxin, the factor responsible for inducing life-threatening pathology in humans. As well as introducing genes that can contribute to the virulence of a strain, prophage can enable the generation of large-chromosomal rearrangements (LCRs) by homologous recombination. This work examines the types and frequencies of LCRs across the major lineages of the O157:H7 serotype. We demonstrate that LCRs are a major source of genomic variation across all lineages of E. coli O157:H7 and by using both optical mapping and Oxford Nanopore long-read sequencing prove that LCRs are generated in laboratory cultures started from a single colony and that these variants can be recovered from colonized cattle. LCRs are biased towards the terminus region of the genome and are bounded by specific prophages that share large regions of sequence homology associated with the recombinational activity. RNA transcriptional profiling and phenotyping of specific structural variants indicated that important virulence phenotypes such as Shiga-toxin production, type-3 secretion and motility can be affected by LCRs. In summary, E. coli O157:H7 has acquired multiple prophage regions over time that act to continually produce structural variants of the genome. These findings raise important questions about the significance of this prophage-mediated genome contingency to enhance adaptability between environments.
- Published
- 2021
12. Genome structural variation in Escherichia coli O157:H7
- Author
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Fitzgerald, Stephen F, Lupolova, Nadejda, Shaaban, Sharif, Dallman, Timothy J, Greig, David, Allison, Lesley, Tongue, Sue C, Evans, Judith, Henry, Madeleine K, McNeilly, Tom N, Bono, James L, Gally, David L, IRAS OH Epidemiology Microbial Agents, and IRAS OH Epidemiology Microbial Agents
- Subjects
Optical mapping ,Duplication ,Epidemiology ,Virulence ,Biology ,medicine.disease_cause ,Genome ,Microbiology ,Structural variation ,Type 3 secretion ,Genome structure ,Gene duplication ,medicine ,Genetics ,O157:H7 ,Gene ,Escherichia coli ,Molecular Biology ,Prophage ,E. coli ,Inversion ,Shiga toxin ,General Medicine ,PFGE ,H7 [O157] ,biology.protein ,Cattle - Abstract
The human zoonotic pathogen Escherichia coli O157:H7 is defined by its extensive prophage repertoire including those that encode Shiga toxin, the factor responsible for inducing life-threatening pathology in humans. As well as introducing genes that can contribute to the virulence of a strain, prophage can enable the generation of large-chromosomal rearrangements (LCRs) by homologous recombination. This work examines the types and frequencies of LCRs across the major lineages of the O157:H7 serotype. We demonstrate that LCRs are a major source of genomic variation across all lineages of E. coli O157:H7 and by using both optical mapping and Oxford Nanopore long-read sequencing prove that LCRs are generated in laboratory cultures started from a single colony and that these variants can be recovered from colonized cattle. LCRs are biased towards the terminus region of the genome and are bounded by specific prophages that share large regions of sequence homology associated with the recombinational activity. RNA transcriptional profiling and phenotyping of specific structural variants indicated that important virulence phenotypes such as Shiga-toxin production, type-3 secretion and motility can be affected by LCRs. In summary, E. coli O157:H7 has acquired multiple prophage regions over time that act to continually produce structural variants of the genome. These findings raise important questions about the significance of this prophage-mediated genome contingency to enhance adaptability between environments.
- Published
- 2021
13. Topology and Contribution to the Pore Channel Lining of Plasma Membrane-Embedded Shigella flexneri Type 3 Secretion Translocase IpaB
- Author
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Marcia B. Goldberg, Poyin Chen, Jeffrey K. Duncan-Lowey, Natasha Bitar, Brian C. Russo, and Keith T Egger
- Subjects
topology ,Virulence ,Topology ,Microbiology ,environment and public health ,Type three secretion system ,Shigella flexneri ,Bacterial Proteins ,Protein Domains ,Transferases ,Virology ,Type III Secretion Systems ,Translocase ,Humans ,Secretion ,translocon ,Dysentery, Bacillary ,biology ,Chemistry ,Effector ,IpaB ,Cell Membrane ,biology.organism_classification ,Translocon ,QR1-502 ,Transmembrane domain ,type 3 secretion ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Research Article - Abstract
Shigella spp. are human bacterial pathogens that cause bacillary dysentery. Virulence depends on a type 3 secretion system (T3SS), a highly conserved structure present in multiple important human and plant pathogens. Upon host cell contact, the T3SS translocon is delivered to the host membrane, facilitates bacterial docking to the membrane, and enables delivery of effector proteins into the host cytosol. The Shigella translocon is composed of two proteins, IpaB and IpaC, which together form this multimeric structure within host plasma membranes. Upon interaction of IpaC with host intermediate filaments, the translocon undergoes a conformational change that allows for bacterial docking onto the translocon and, together with host actin polymerization, enables subsequent effector translocation through the translocon pore. To generate additional insights into the translocon, we mapped the topology of IpaB in plasma membrane-embedded pores using cysteine substitution mutagenesis coupled with site-directed labeling and proximity-enabled cross-linking by membrane-permeant sulfhydryl reactants. We demonstrate that IpaB function is dependent on posttranslational modification by a plasmid-encoded acyl carrier protein. We show that the first transmembrane domain of IpaB lines the interior of the translocon pore channel such that the IpaB portion of the channel forms a funnel-like shape leading into the host cytosol. In addition, we identify regions of IpaB within its cytosolic domain that protrude into and are closely associated with the pore channel. Taken together, these results provide a framework for how IpaB is arranged within translocons natively delivered by Shigella during infection. IMPORTANCE Type 3 secretion systems are nanomachines employed by many bacteria, including Shigella, which deliver into human cells bacterial virulence proteins that alter cellular function in ways that promote infection. Delivery of Shigella virulence proteins occurs through a pore formed in human cell membranes by the IpaB and IpaC proteins. Here, we define how IpaB contributes to the formation of pores natively delivered into human cell membranes by Shigella flexneri. We show that a specific domain of IpaB (transmembrane domain 1) lines much of the pore channel and that portions of IpaB that lie in the inside of the human cell loop back into and/or are closely associated with the pore channel. These findings provide new insights into the organization and function of the pore in serving as the conduit for delivery of virulence proteins into human cells during Shigella infection.
- Published
- 2021
14. Genetic Analysis of the Salmonella FliE Protein That Forms the Base of the Flagellar Axial Structure
- Author
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Fabienne F. V. Chevance, Alexander J Bradshaw, Tohru Minamino, Keiichi Namba, Hee Jung Lee, Jordan J. Hendriksen, and Kelly T. Hughes
- Subjects
Salmonella typhimurium ,Protein Conformation ,Mutant ,Flagellum ,Microbiology ,Type three secretion system ,flagellum ,Bacterial Proteins ,Salmonella ,Virology ,Type III Secretion Systems ,FliE ,Inner membrane ,genetics ,structure ,Amino Acid Sequence ,Coiled coil ,Signal transducing adaptor protein ,Periplasmic space ,QR1-502 ,Cell biology ,Flagella ,type 3 secretion ,Bacterial outer membrane ,Sequence Alignment ,Research Article ,Protein Binding - Abstract
The FliE component of the bacterial flagellum is the first protein secreted through the flagellar type III secretion system (fT3SS) that is capable of self-assembly into the growing bacterial organelle. The FliE protein plays dual roles in the assembly of the Salmonella flagellum as the final component of the flagellar type III secretion system (fT3SS) and as an adaptor protein that anchors the rod (drive shaft) of the flagellar motor to the membrane-imbedded MS-ring structure. This work has identified the interactions between FliE and other proteins at the inner membrane base of the flagellar machine. The fliE sequence coding for the 104-amino-acid protein was subject to saturating mutagenesis. Single-amino-acid substitutions were generated in fliE, resulting in motility phenotypes. From these mutants, intergenic suppressor mutations were generated, isolated, and characterized. Single-amino-acid mutations defective in FliE function were localized to the N- and C-terminal helices of the protein. Motile suppressors of amino acid mutations in fliE were found in rod protein genes flgB and flgC, the MS ring gene, fliF, and one of the core T3SS genes, fliR. These results support the hypothesis that FliE acts as a linker protein consisting of an N-terminal α-helix that is involved in the interaction with the MS ring with a rotational symmetry and a C-terminal coiled coil that interacts with FliF, FliR, FlgB, and FlgC, and these interactions open the exit gate of the protein export channel of the fT3SS. IMPORTANCE The bacterial flagellum represents one of biology's most complex molecular machines. Its rotary motor spins at speeds of more than 2,000 cycles per second, and its type 3 secretion (T3S) system secretes proteins at rates of tens of thousands of amino acids per second. Within the complex flagellar motility machine resides a unique protein, FliE, which serves as an adaptor to connect a planar, inner membrane-embedded ring structure, the MS-ring, the core T3S secretion complex at the cytoplasmic base, and a rigid, axial structure that spans the periplasmic space, penetrates the outer membrane, and extends 10 to 20 microns from the cell surface. This work combines genetic mutant suppressor analysis with the structural data for the core T3S system, the MS-ring, and the axial drive shaft (rod) that transverses the periplasm to provide insight into the essential adaptor role of FliE in flagellum assembly and function.
- Published
- 2021
15. Pseudomonas aeruginosa Bacteremic Patients Exhibit Nonprotective Antibody Titers Against Therapeutic Antibody Targets PcrV and Psl Exopolysaccharide.
- Author
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Thaden, Joshua T., Keller, Ashley E., Shire, Norah J., Margarita Camara, M., Otterson, Linda, Huband, Mike, Guenther, Caitlin M., Wei Zhao, Warrener, Paul, Kendall Stover, C., Fowler Jr, Vance G., DiGiandomenico, Antonio, Camara, M Margarita, Zhao, Wei, Stover, C Kendall, and Fowler, Vance G Jr
- Subjects
- *
PSEUDOMONAS aeruginosa infections , *BISPECIFIC antibodies , *IMMUNOGLOBULIN G , *BACTEREMIA , *MONOCLONAL antibodies , *MICROBIAL exopolysaccharides , *ANTIBIOTICS , *BACTERIAL antigens , *IMMUNITY , *IMMUNOGLOBULINS , *LONGITUDINAL method , *MICROBIAL sensitivity tests , *PHAGOCYTOSIS , *PSEUDOMONAS , *PSEUDOMONAS diseases , *BACTERIAL antibodies , *PHARMACODYNAMICS - Abstract
Background: The type 3 secretion protein PcrV and Psl exopolysaccharide are promising therapeutic antibody targets against Pseudomonas aeruginosa. We examined P. aeruginosa bloodstream infection (BSI) isolates for the ability to express PcrV and Psl and evaluated corresponding patient serum for active titers to these targets.Methods: We identified 114 patients with acute P. aeruginosa BSI; 56 cases were accompanied by acute sera. Serum was evaluated for PcrV- and Psl-specific immunoglobulin G (IgG) and for cytotoxicity and opsonophagocytosis. Isolates were evaluated for susceptibility to antibiotics, expression of PcrV and Psl, and susceptibility to the anti-PcrV/Psl bispecific antibody and clinical candidate MEDI3902.Results: In-hospital mortality for patients with P. aeruginosa BSI was 39%. A total of 26% of isolates were resistant to ≥3 antibiotic classes. Although PcrV and/or Psl were detected in 99% of isolates, a majority of patients lacked active titers to PcrV (100%) and Psl (98%). In addition, MEDI3902 was active against all tested isolates.Conclusions: A vast majority of P. aeruginosa BSI isolates express PcrV and Psl; however, patient sera most often lacked IgG and functionally active responses to these targets. These results suggest that therapies directed at PcrV and Psl could be a promising approach for combating P. aeruginosa bloodstream infections. [ABSTRACT FROM AUTHOR]- Published
- 2016
- Full Text
- View/download PDF
16. Comprehensive identification of virulence factors required for respiratory melioidosis using Tn-seq mutagenesis.
- Author
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Gutierrez, Maria G., Yoder-Himes, Deborah R., and Warawa, Jonathan M.
- Subjects
MELIOIDOSIS ,BURKHOLDERIA pseudomallei ,MUTAGENESIS ,VIRULENCE of bacteria ,LIPOPOLYSACCHARIDES - Abstract
Respiratory melioidosis is a disease presentation of the biodefense pathogen, Burkholderia pseudomallei, which is frequently associated with a lethal septicemic spread of the bacteria. We have recently developed an improved respiratory melioidosis model to study the pathogenesis of Burkholderia pseudomallei in the lung (intubation-mediated intratracheal [IMIT] inoculation), which more closely models descriptions of human melioidosis, including prominent septicemic spread from the lung and reduced involvement of the upper respiratory tract. We previously demonstrated that the Type 3 Secretion System cluster 3 (T3SS3) is a critical virulence determinant for B. pseudomallei when delivered directly into the lung. We decided to comprehensively identify all virulence determinants required for respiratory melioidosis using the Tn-seq phenotypic screen, as well as to investigate which virulence determinants are required for dissemination to the liver and spleen. While previous studies have used Tn-seq to identify essential genes for in vitro cultured B. pseudomallei, this represents the first study to use Tn-seq to identify genes required for in vivo fitness. Consistent with our previous findings, we identified T3SS3 as the largest genetic cluster required for fitness in the lung. Furthermore, we identified capsular polysaccharide and Type 6 Secretion System cluster 5 (T6SS5) as the two additional major genetic clusters facilitating respiratory melioidosis. Importantly, Tn-seq did not identify additional, novel large genetic systems supporting respiratory melioidosis, although these studies identified additional small gene clusters that may also play crucial roles in lung fitness. Interestingly, other previously identified virulence determinants do not appear to be required for lung fitness, such as lipopolysaccharide. The role of T3SS3, capsule, and T6SS5 in lung fitness was validated by competition studies, but only T3SS3 was found to be important for respiratory melioidosis when delivered as a single strain challenge, suggesting that competition studies may provide a higher resolution analysis of fitness factors in the lung. The use of Tn-seq phenotypic screening also provided key insights into the selective pressure encountered in the liver. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
17. The Complete Genome Sequence of Xanthomonas theicola, the Causal Agent of Canker on Tea Plants, Reveals Novel Secretion Systems in Clade-1 Xanthomonads
- Author
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UCL - SST/ELI/ELIM - Applied Microbiology, Koebnik, Ralf, Burokiene, Daiva, Bragard, Claude, Chang, Christine, Saux, Marion Fischer-Le, Kölliker, Roland, Lang, Jillian M., Leach, Jan E., Luna, Emily K., Portier, Perrine, Sagia, Angeliki, Ziegle, Janet, Cohen, Stephen P., Jacobs, Jonathan M., UCL - SST/ELI/ELIM - Applied Microbiology, Koebnik, Ralf, Burokiene, Daiva, Bragard, Claude, Chang, Christine, Saux, Marion Fischer-Le, Kölliker, Roland, Lang, Jillian M., Leach, Jan E., Luna, Emily K., Portier, Perrine, Sagia, Angeliki, Ziegle, Janet, Cohen, Stephen P., and Jacobs, Jonathan M.
- Abstract
Xanthomonas theicola is the causal agent of bacterial canker on tea plants. There is no complete genome sequence available for X. theicola, a close relative of the species X. translucens and X. hyacinthi, thus limiting basic research for this group of pathogens. Here, we release a high-quality complete genome sequence for the X. theicola type strain, CFBP 4691T. Single-molecule real-time sequencing with a mean coverage of 264× revealed two contigs of 4,744,641 bp (chromosome) and 40,955 bp (plasmid) in size. Genome mining revealed the presence of nonribosomal peptide synthases, two CRISPR systems, the Xps type 2 secretion system, and the Hrp type 3 secretion system. Surprisingly, this strain encodes an additional type 2 secretion system and a novel type 3 secretion system with enigmatic function, hitherto undescribed for xanthomonads. Four type 3 effector genes were found on complete or partial transposons, suggesting a role of transposons in effector gene evolution and spread. This genome sequence fills an important gap to better understand the biology and evolution of the early-branching xanthomonads, also known as clade-1 xanthomonads.
- Published
- 2021
18. Arabidopsis bZIP11 Is a Susceptibility Factor during Pseudomonas syringae Infection
- Author
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Prior, Matthew J., Selvanayagam, Jebasingh, Kim, Jung-Gun, Tomar, Monika, Jonikas, Martin, Mudgett, Mary Beth, Smeekens, Sjef, Hanson, Johannes, Frommer, Wolf B., Prior, Matthew J., Selvanayagam, Jebasingh, Kim, Jung-Gun, Tomar, Monika, Jonikas, Martin, Mudgett, Mary Beth, Smeekens, Sjef, Hanson, Johannes, and Frommer, Wolf B.
- Abstract
The induction of plant nutrient secretion systems is critical for successful pathogen infection. Some bacterial pathogens (e.g., Xanthomonas spp.) use transcription activator-like (TAL) effectors to induce transcription of SWEET sucrose efflux transporters. Pseudomonas syringae pv. tomato strain DC3000 lacks TAL effectors yet is able to induce multiple SWEETs in Arabidopsis thaliana by unknown mechanisms. Because bacteria require other nutrients in addition to sugars for efficient reproduction, we hypothesized that Pseudomonas spp. may depend on host transcription factors involved in secretory programs to increase access to essential nutrients. Bioinformatic analyses identified the Arabidopsis basic-leucine zipper transcription factor bZIP11 as a potential regulator of nutrient transporters, including SWEETs and UmamiT amino acid transporters. Inducible downregulation of bZIP11 expression in Arabidopsis resulted in reduced growth of P. syringae pv. tomato strain DC3000, whereas inducible overexpression of bZIP11 resulted in increased bacterial growth, supporting the hypothesis that bZIP11-regulated transcription programs are essential for maximal pathogen titer in leaves. Our data are consistent with a model in which a pathogen alters host transcription factor expression upstream of secretory transcription networks to promote nutrient efflux from host cells.
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- 2021
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19. A Reporter System for Fast Quantitative Monitoring of Type 3 Protein Secretion in Enteropathogenic E. coli
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Spyridoula Karamanou, Maria S Loos, Anastassios Economou, Luit Moni Barkalita, Athina G. Portaliou, and Biao Yuan
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Microbiology (medical) ,Microbiology ,Article ,Type three secretion system ,03 medical and health sciences ,Type 3 secretion ,Virology ,Secretion ,alkaline phosphatase activity ,lcsh:QH301-705.5 ,030304 developmental biology ,EPEC ,0303 health sciences ,030306 microbiology ,Chemistry ,Effector ,SctA-PhoA ,Fusion protein ,Transmembrane protein ,Cell biology ,quantitative in vivo secretion assay ,Secretory protein ,lcsh:Biology (General) ,Cytoplasm ,Host cell cytoplasm ,bacteria - Abstract
The type 3 secretion system is essential for pathogenesis of several human and animal Gram-negative bacterial pathogens. The T3SS comprises a transmembrane injectisome, providing a conduit from the bacterial cytoplasm to the host cell cytoplasm for the direct delivery of effectors (including toxins). Functional studies of T3SS commonly monitor the extracellular secretion of proteins by SDS-PAGE and western blot analysis, which are slow and semi-quantitative in nature. Here, we describe an enzymatic reporter-based quantitative and rapid in vivo assay for T3SS secretion studies in enteropathogenic E. coli (EPEC). The assay monitors the secretion of the fusion protein SctA-PhoA through the injectisome based on a colorimetric assay that quantifies the activity of alkaline phosphatase. We validated the usage of this reporter system by following the secretion in the absence of various injectisome components, including domains of the gatekeeper essential for T3SS function. This platform can now be used for the isolation of mutations, functional analysis and anti-virulence compound screening. ispartof: MICROORGANISMS vol:8 issue:11 ispartof: location:Switzerland status: published
- Published
- 2020
20. Assembly and structure of the T3SS.
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Burkinshaw, Brianne J. and Strynadka, Natalie C.J.
- Subjects
- *
PROTEIN structure , *PHYTOPATHOGENIC microorganisms , *BACTERIAL cell walls , *SALMONELLA , *HOST-bacteria relationships , *BACTERIAL secretions - Abstract
Abstract: The Type III Secretion System (T3SS) is a multi-mega Dalton apparatus assembled from more than twenty components and is found in many species of animal and plant bacterial pathogens. The T3SS creates a contiguous channel through the bacterial and host membranes, allowing injection of specialized bacterial effector proteins directly to the host cell. In this review, we discuss our current understanding of T3SS assembly and structure, as well as highlight structurally characterized Salmonella effectors. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey. [Copyright &y& Elsevier]
- Published
- 2014
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21. Horizontally Transferred Genetic Elements and Their Role in Pathogenesis of Bacterial Disease.
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Gyles, C. and Boerlin, P.
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BACTERIAL diseases in animals ,ANIMAL diseases ,VETERINARY pathology ,ENTEROBACTERIACEAE ,MOBILE genetic elements - Abstract
This article reviews the roles that laterally transferred genes (LTG) play in the virulence of bacterial pathogens. The features of LTG that allow them to be recognized in bacterial genomes are described, and the mechanisms by which LTG are transferred between and within bacteria are reviewed. Genes on plasmids, integrative and conjugative elements, prophages, and pathogenicity islands are highlighted. Virulence genes that are frequently laterally transferred include genes for bacterial adherence to host cells, type 3 secretion systems, toxins, iron acquisition, and antimicrobial resistance. The specific roles of LTG in pathogenesis are illustrated by specific reference to Escherichia coli, Salmonella, pyogenic streptococci, and Clostridium perfringens. [ABSTRACT FROM PUBLISHER]
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- 2014
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22. The complete genome sequence of Xanthomonas theicola, the causal agent of canker on tea plants, reveals novel secretion systems in clade-1 xanthomonads
- Author
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Koebnik, Ralf, Burokiene, Daiva, Bragard, Claude, Chang, Christine, Fischer-Le Saux, Marion, Kölliker, Roland, Lang, Jillian, Leach, Jan, Luna, Emily, Portier, Perrine, Sagia, Angeliki, Ziegle, Janet, Cohen, Stephen Philip, Jacobs, Jonathan, UMR - Interactions Plantes Microorganismes Environnement (UMR IPME), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Université de Montpellier (UM)-Institut de Recherche pour le Développement (IRD [France-Sud]), NATURE RESEARCH CENTER VILNIUS LTU, Partenaires IRSTEA, Institut national de recherche en sciences et technologies pour l'environnement et l'agriculture (IRSTEA)-Institut national de recherche en sciences et technologies pour l'environnement et l'agriculture (IRSTEA), INSTITUTE OF LIFE SCIENCES UNIVERSITE CATHOLIQUE DE LOUVAIN LOUVAIN-LA-NEUVE BEL, Pacific Biosciences [Menlo Park], Pacific Biosciences of California, Institut de Recherche en Horticulture et Semences (IRHS), Université d'Angers (UA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-INSTITUT AGRO Agrocampus Ouest, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), DEPARTMENT OF BIOLOGY COLORADO STATE UNIVERSITY FORT COLLINS COLORADO USA, Colorado State University [Fort Collins] (CSU), University of Crete [Heraklion] (UOC), Ohio State University [Columbus] (OSU), United States Department of Agriculture National Institute of Food and Agriculture Postdoctoral Fellowship (2018-08122), for SPC, European Project, UCL - SST/ELI/ELIM - Applied Microbiology, Institut de Recherche pour le Développement (IRD [France-Sud])-Université de Montpellier (UM)-Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad), Université d'Angers (UA)-AGROCAMPUS OUEST, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Ohio State University, and Partenaires INRAE
- Subjects
Tea plant ,Evolution ,pathogen effectors ,Plant Science ,microbe-genome sequencing ,host parasite interactions ,Bacterial canker ,[SDV.BV.PEP]Life Sciences [q-bio]/Vegetal Biology/Phytopathology and phytopharmacy ,Type 3 secretion ,bacterial pathogens ,Type 2 secretion ,genomics ,Xanthomonas theicola ,Effector-encoding transposon ,Agronomy and Crop Science - Abstract
International audience; Xanthomonas theicola is the causal agent of bacterial canker on tea plants. There is no complete genome sequence available for X. theicola, a close relative of the species X. translucens and X. hyacinthi, thus limiting basic research for this group of pathogens. Here we release a highquality complete genome sequence for the X. theicola type strain, CFBP 4691T. Single-molecule real-time sequencing with a mean coverage of 264X revealed two contigs of 4,744,641 bp (chromosome) and 40,955 bp (plasmid) in size. Genome mining revealed the presence of nonribosomal peptide synthases, two CRISPR systems, the Xps type 2 secretion system, and the Hrp type 3 secretion system. Surprisingly, this strain encodes an additional type 2 secretion system and a novel type 3 secretion system with enigmatic function, hitherto undescribed for xanthomonads. Four type 3 effector genes were found on complete or partial transposons, suggesting a role of transposons in effector gene evolution and spread. This genome sequence fills an important gap to better understand the biology and evolution of the early-branching xanthomonads, also known as clade-1 xanthomonads.
- Published
- 2020
23. Context-dependent protein folding of a virulence peptide in the bacterial and host environments: structure of an SycH-YopH chaperone-effector complex.
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Vujanac, Milos and Stebbins, C. Erec
- Subjects
- *
MICROBIAL virulence , *PEPTIDES , *MOLECULAR chaperones , *YERSINIA pestis , *BACTERIAL proteins , *PROTEIN-tyrosine phosphatase , *POLYPEPTIDES - Abstract
Yersinia pestis injects numerous bacterial proteins into host cells through an organic nanomachine called the type 3 secretion system. One such substrate is the tyrosine phosphatase YopH, which requires an interaction with a cognate chaperone in order to be effectively injected. Here, the first crystal structure of a SycH-YopH complex is reported, determined to 1.9 Å resolution. The structure reveals the presence of (i) a nonglobular polypeptide in YopH, (ii) a so-called β-motif in YopH and (iii) a conserved hydrophobic patch in SycH that recognizes the β-motif. Biochemical studies establish that the β-motif is critical to the stability of this complex. Finally, since previous work has shown that the N-terminal portion of YopH adopts a globular fold that is functional in the host cell, aspects of how this polypeptide adopts radically different folds in the host and in the bacterial environments are analysed. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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24. The function of the bacterial cytoskeleton in Salmonella pathogenesis.
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Doble, Anne C., Bulmer, David M., Kharraz, Lubna, Karavolos, Michail H., and Khan, C.M. Anjam
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- *
CYTOSKELETON , *SALMONELLA enterica serovar typhimurium , *MICROBIAL virulence , *SECRETION , *PROTEINS , *BACTERIAL flagella - Abstract
The article focuses on a study which examined the effects of disrupting the bacterial cytoskeleton on the pathogenicity of Salmonella enterica serovar Typhimurium. Two pathogenicity island-encoded type 3 secretion systems (T3SSs) are involved in the virulence of salmonella enterica. Information on cell shape-determinant proteins is provided. Results of the study showed that the bacterial cytoskeleton plays a role in the expression of the SPI-1 T3SS and flagella systems.
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- 2012
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25. Induction of the Yersinia Type 3 Secretion System as an All-or-None Phenomenon
- Author
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Wiley, David J., Rosqvist, Roland, and Schesser, Kurt
- Subjects
- *
BIOLOGICAL transport , *ENTEROBACTERIACEAE , *GENE expression , *GENETIC regulation - Abstract
Abstract: Pathogenic Yersinia spp. possess a protein secretion system, designated as type 3, that plays a clear role in promoting their survival vis-à-vis the macrophage. Inductive expression of the Yersinia type 3 secretion system (T3SS), triggered either by host cell contact, or, in the absence of host cells, by a reduction in extracellular calcium ion levels, is accompanied by a withdrawal from the bacterial division cycle. Here, we analyzed Ca2+-dependent induction of the T3SS at the single-cell level to understand how Yersinia coordinates pro-survival and growth-related activities. We utilized a novel high-throughput quantitative microscopy approach as well as flow cytometry to determine how Ca2+ levels, T3SS expression, and cellular division are interrelated. Our analysis showed that there is a high degree of homogeneity in terms of T3SS expression levels among a population of Y. pseudotuberculosis cells following the removal of Ca2+, and that T3SS expression appears to be independent of the cellular division cycle. Unexpectedly, our analysis showed that Ca2+ levels are inversely related to the initiation of inductive T3SS expression, and not to the intensity of activation once initiated, thus providing a basis for the seemingly graded response of T3SS activation observed in bulk-level analyses. The properties of the system described here display both similarities to and differences from that of the lac operon first described 50 years ago by Novick and Weiner. [Copyright &y& Elsevier]
- Published
- 2007
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26. How the structural gene products of Yersinia pestis relate to virulence.
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Brubaker, Robert R.
- Subjects
YERSINIA pestis ,YERSINIA ,COMMUNICABLE diseases ,DISEASES ,PLAGUE - Abstract
Bubonic plague is the most devastating acute infectious disease known to man. The causative agent, Yersinia pestis, is now more firmly entrenched in sylvatic reservoirs throughout the world than at any time in the past. Consequently, the organism increasingly causes casual human disease and is readily available for use as a bioweapon. Recent attempts to understand the severe nature of plague have focused upon its very recent divergence from Yersinia pseudotuberculosis, an etiological instrument of chronic enteropathogenic infection. This review emphasizes that the invasive nature of plague and its dissemination by fleabite is mediated by plasmids not shared by enteropathogenic yersiniae. The basis for high lethality is considered within the context of chromosomal degeneration causing loss of normal metabolic functions and modification of virulence factors, permitting a terminal anti-inflammatory phase associated with pronounced septicemia. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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27. Salmonella typhimurium disseminates within its host by manipulating the motility of infected cells.
- Author
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Worley, Micah J., Nieman, George S., Geddes, Kaoru, and Heffron, Fred
- Subjects
- *
SALMONELLA typhimurium , *CELL motility , *PHAGOCYTES , *EPITHELIUM , *PROTEINS , *ZYXIN - Abstract
The mammalian host has a number of innate immune mechanisms designed to limit the spread of infection, yet many bacteria, including Salmonella, can cause systemic disease. Salmonella typhimurium-infected phagocytes traverse the gastrointestinal (GI) epithelium and enter the bloodstream within minutes after ingestion, thereby spreading throughout its host. Here, we provide a cellular and molecular basis for this phenomenon. We demonstrate that S. typhimurium manipulates the migratory properties of infected GI phagocytes with a type Ill secretion system. We show that one secreted effector. SrfH. interacts with the host protein TRIP6, a member of the zyxin family of adaptor proteins that regulate motility. Srf H promotes phagocyte motility in vitro and accelerates the systemic spread of infection away from the lumen of the intestine in the mouse. This is a previously uncharacterized mechanism by which an intracellular pathogen overcomes host defenses designed to immobilize infected cells. [ABSTRACT FROM AUTHOR]
- Published
- 2006
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- View/download PDF
28. Distinct intraspecies virulence mechanisms regulated by a conserved transcription factor
- Author
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Andrew J. Roe, Natasha C. A. Turner, Nicky O’Boyle, Douglas F. Browning, and James P. R. Connolly
- Subjects
Virulence Factors ,Virulence ,Biology ,medicine.disease_cause ,Microbiology ,Type three secretion system ,03 medical and health sciences ,Escherichia coli ,Type III Secretion Systems ,medicine ,Transcriptional regulation ,Uropathogenic Escherichia coli ,Gene ,Transcription factor ,Escherichia coli Infections ,030304 developmental biology ,Genetics ,Regulation of gene expression ,0303 health sciences ,Multidisciplinary ,Bacteria ,030306 microbiology ,Effector ,Escherichia coli Proteins ,regulation ,Gene Expression Regulation, Bacterial ,Biological Sciences ,niche ,type 1 fimbriae ,PNAS Plus ,type 3 secretion ,Enterohemorrhagic Escherichia coli ,Fimbriae, Bacterial ,gene expression ,Transcription Factors - Abstract
Significance Bacterial pathogens emerge by adapting mechanisms of virulence, differentiating them from their nonpathogenic progenitor. Virulence factors are often encoded on accessory genomic elements not part of the core genome and therefore must be integrated into the regulatory architecture of the cell. Here, we show that a highly conserved transcription factor in Escherichia coli has been relieved of a common purpose and adapted to regulate virulence pleiotropically in 2 distinct genetic backgrounds. This leads to enhanced virulence of both intestinal enterohemorrhagic E. coli and extraintestinal uropathogenic E. coli by exclusive mechanisms. These findings challenge the assumption that conserved transcription factors regulate common pathways maintained within a species and suggest that transcriptional repurposing creates new primary roles on an individual basis., Tailoring transcriptional regulation to coordinate the expression of virulence factors in tandem with the core genome is a hallmark of bacterial pathogen evolution. Bacteria encode hundreds of transcription factors forming the base-level control of gene regulation. Moreover, highly homologous regulators are assumed to control conserved genes between members within a species that harbor the same genetic targets. We have explored this concept in 2 Escherichia coli pathotypes that employ distinct virulence mechanisms that facilitate specification of a different niche within the host. Strikingly, we found that the transcription factor YhaJ actively regulated unique gene sets between intestinal enterohemorrhagic E. coli (EHEC) and extraintestinal uropathogenic E. coli (UPEC), despite being very highly conserved. In EHEC, YhaJ directly activates expression of type 3 secretion system components and effectors. Alternatively, YhaJ enhances UPEC virulence regulation by binding directly to the phase-variable type 1 fimbria promoter, driving its expression. Additionally, YhaJ was found to override the universal GAD acid tolerance system but exclusively in EHEC, thereby indirectly enhancing type 3 secretion pleiotropically. These results have revealed that within a species, conserved regulators are actively repurposed in a “personalized” manner to benefit particular lifestyles and drive virulence via multiple distinct mechanisms.
- Published
- 2019
29. Modeling Pneumonic Plague in Human Precision-Cut Lung Slices Highlights a Role for the Plasminogen Activator Protease in Facilitating Type 3 Secretion
- Author
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Shalynn D. Mills, Richard C. Kurten, Samantha D. Huckuntod, Roger D. Pechous, and Srijon Kaushik Banerjee
- Subjects
Pneumonic plague ,human precision-cut lung slices ,Yersinia pestis ,plasminogen activator protease ,Immunology ,pulmonary infection ,Yersinia ,Microbiology ,Bubonic plague ,Bacterial Adhesion ,Cell Line ,Type three secretion system ,Proinflammatory cytokine ,Mice ,Plasminogen Activators ,03 medical and health sciences ,stomatognathic system ,Macrophages, Alveolar ,medicine ,Animals ,Humans ,Lung ,Pathogen ,030304 developmental biology ,Plague ,0303 health sciences ,biology ,030306 microbiology ,respiratory system ,biology.organism_classification ,medicine.disease ,Molecular Pathogenesis ,3. Good health ,Mice, Inbred C57BL ,Infectious Diseases ,hPCLS ,type 3 secretion ,Host-Pathogen Interactions ,Cytokines ,Pla ,Female ,lipids (amino acids, peptides, and proteins) ,Parasitology ,pneumonic plague ,Plasminogen activator - Abstract
Pneumonic plague is the deadliest form of disease caused by Yersinia pestis. Key to the progression of infection is the activity of the plasminogen activator protease Pla. Deletion of Pla results in a decreased Y. pestis bacterial burden in the lung and failure to progress into the lethal proinflammatory phase of disease. While a number of putative functions have been attributed to Pla, its precise role in the pathogenesis of pneumonic plague is yet to be defined., Pneumonic plague is the deadliest form of disease caused by Yersinia pestis. Key to the progression of infection is the activity of the plasminogen activator protease Pla. Deletion of Pla results in a decreased Y. pestis bacterial burden in the lung and failure to progress into the lethal proinflammatory phase of disease. While a number of putative functions have been attributed to Pla, its precise role in the pathogenesis of pneumonic plague is yet to be defined. Here, we show that Pla facilitates type 3 secretion into primary alveolar macrophages but not into the commonly used THP-1 cell line. We also establish human precision-cut lung slices as a platform for modeling early host/pathogen interactions during pneumonic plague and solidify the role of Pla in promoting optimal type 3 secretion using primary human tissue with relevant host cell heterogeneity. These results position Pla as a key player in the early host/pathogen interactions that define pneumonic plague and showcase the utility of human precision-cut lung slices as a platform to evaluate pulmonary infection by bacterial pathogens.
- Published
- 2019
30. Topology and Contribution to the Pore Channel Lining of Plasma Membrane-Embedded Shigella flexneri Type 3 Secretion Translocase IpaB.
- Author
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Chen P, Russo BC, Duncan-Lowey JK, Bitar N, Egger KT, and Goldberg MB
- Subjects
- Bacterial Proteins chemistry, Bacterial Proteins genetics, Cell Membrane chemistry, Humans, Protein Domains, Shigella flexneri chemistry, Shigella flexneri genetics, Transferases genetics, Type III Secretion Systems genetics, Bacterial Proteins metabolism, Cell Membrane microbiology, Dysentery, Bacillary microbiology, Shigella flexneri metabolism, Transferases metabolism, Type III Secretion Systems chemistry, Type III Secretion Systems metabolism
- Abstract
Shigella spp. are human bacterial pathogens that cause bacillary dysentery. Virulence depends on a type 3 secretion system (T3SS), a highly conserved structure present in multiple important human and plant pathogens. Upon host cell contact, the T3SS translocon is delivered to the host membrane, facilitates bacterial docking to the membrane, and enables delivery of effector proteins into the host cytosol. The Shigella translocon is composed of two proteins, IpaB and IpaC, which together form this multimeric structure within host plasma membranes. Upon interaction of IpaC with host intermediate filaments, the translocon undergoes a conformational change that allows for bacterial docking onto the translocon and, together with host actin polymerization, enables subsequent effector translocation through the translocon pore. To generate additional insights into the translocon, we mapped the topology of IpaB in plasma membrane-embedded pores using cysteine substitution mutagenesis coupled with site-directed labeling and proximity-enabled cross-linking by membrane-permeant sulfhydryl reactants. We demonstrate that IpaB function is dependent on posttranslational modification by a plasmid-encoded acyl carrier protein. We show that the first transmembrane domain of IpaB lines the interior of the translocon pore channel such that the IpaB portion of the channel forms a funnel-like shape leading into the host cytosol. In addition, we identify regions of IpaB within its cytosolic domain that protrude into and are closely associated with the pore channel. Taken together, these results provide a framework for how IpaB is arranged within translocons natively delivered by Shigella during infection. IMPORTANCE Type 3 secretion systems are nanomachines employed by many bacteria, including Shigella , which deliver into human cells bacterial virulence proteins that alter cellular function in ways that promote infection. Delivery of Shigella virulence proteins occurs through a pore formed in human cell membranes by the IpaB and IpaC proteins. Here, we define how IpaB contributes to the formation of pores natively delivered into human cell membranes by Shigella flexneri. We show that a specific domain of IpaB (transmembrane domain 1) lines much of the pore channel and that portions of IpaB that lie in the inside of the human cell loop back into and/or are closely associated with the pore channel. These findings provide new insights into the organization and function of the pore in serving as the conduit for delivery of virulence proteins into human cells during Shigella infection.
- Published
- 2021
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- View/download PDF
31. Genome structural variation in Escherichia coli O157:H7.
- Author
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Fitzgerald SF, Lupolova N, Shaaban S, Dallman TJ, Greig D, Allison L, Tongue SC, Evans J, Henry MK, McNeilly TN, Bono JL, and Gally DL
- Subjects
- Animals, Cattle, Genomic Structural Variation, Prophages genetics, Shiga Toxin genetics, Shiga Toxin 2 genetics, Escherichia coli O157 genetics
- Abstract
The human zoonotic pathogen Escherichia coli O157:H7 is defined by its extensive prophage repertoire including those that encode Shiga toxin, the factor responsible for inducing life-threatening pathology in humans. As well as introducing genes that can contribute to the virulence of a strain, prophage can enable the generation of large-chromosomal rearrangements (LCRs) by homologous recombination. This work examines the types and frequencies of LCRs across the major lineages of the O157:H7 serotype. We demonstrate that LCRs are a major source of genomic variation across all lineages of E. coli O157:H7 and by using both optical mapping and Oxford Nanopore long-read sequencing prove that LCRs are generated in laboratory cultures started from a single colony and that these variants can be recovered from colonized cattle. LCRs are biased towards the terminus region of the genome and are bounded by specific prophages that share large regions of sequence homology associated with the recombinational activity. RNA transcriptional profiling and phenotyping of specific structural variants indicated that important virulence phenotypes such as Shiga-toxin production, type-3 secretion and motility can be affected by LCRs. In summary, E. coli O157:H7 has acquired multiple prophage regions over time that act to continually produce structural variants of the genome. These findings raise important questions about the significance of this prophage-mediated genome contingency to enhance adaptability between environments.
- Published
- 2021
- Full Text
- View/download PDF
32. Genetic Analysis of the Salmonella FliE Protein That Forms the Base of the Flagellar Axial Structure.
- Author
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Hendriksen JJ, Lee HJ, Bradshaw AJ, Namba K, Chevance FFV, Minamino T, and Hughes KT
- Subjects
- Amino Acid Sequence, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Flagella genetics, Protein Binding, Protein Conformation, Salmonella typhimurium chemistry, Salmonella typhimurium genetics, Sequence Alignment, Type III Secretion Systems chemistry, Type III Secretion Systems genetics, Type III Secretion Systems metabolism, Bacterial Proteins genetics, Flagella chemistry, Flagella metabolism, Salmonella typhimurium metabolism
- Abstract
The FliE component of the bacterial flagellum is the first protein secreted through the flagellar type III secretion system (fT3SS) that is capable of self-assembly into the growing bacterial organelle. The FliE protein plays dual roles in the assembly of the Salmonella flagellum as the final component of the flagellar type III secretion system (fT3SS) and as an adaptor protein that anchors the rod (drive shaft) of the flagellar motor to the membrane-imbedded MS-ring structure. This work has identified the interactions between FliE and other proteins at the inner membrane base of the flagellar machine. The fliE sequence coding for the 104-amino-acid protein was subject to saturating mutagenesis. Single-amino-acid substitutions were generated in fliE , resulting in motility phenotypes. From these mutants, intergenic suppressor mutations were generated, isolated, and characterized. Single-amino-acid mutations defective in FliE function were localized to the N- and C-terminal helices of the protein. Motile suppressors of amino acid mutations in fliE were found in rod protein genes flgB and flgC , the MS ring gene, fliF , and one of the core T3SS genes, fliR . These results support the hypothesis that FliE acts as a linker protein consisting of an N-terminal α-helix that is involved in the interaction with the MS ring with a rotational symmetry and a C-terminal coiled coil that interacts with FliF, FliR, FlgB, and FlgC, and these interactions open the exit gate of the protein export channel of the fT3SS. IMPORTANCE The bacterial flagellum represents one of biology's most complex molecular machines. Its rotary motor spins at speeds of more than 2,000 cycles per second, and its type 3 secretion (T3S) system secretes proteins at rates of tens of thousands of amino acids per second. Within the complex flagellar motility machine resides a unique protein, FliE, which serves as an adaptor to connect a planar, inner membrane-embedded ring structure, the MS-ring, the core T3S secretion complex at the cytoplasmic base, and a rigid, axial structure that spans the periplasmic space, penetrates the outer membrane, and extends 10 to 20 microns from the cell surface. This work combines genetic mutant suppressor analysis with the structural data for the core T3S system, the MS-ring, and the axial drive shaft (rod) that transverses the periplasm to provide insight into the essential adaptor role of FliE in flagellum assembly and function.
- Published
- 2021
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33. A Reporter System for Fast Quantitative Monitoring of Type 3 Protein Secretion in Enteropathogenic E. coli.
- Author
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Barkalita, Luit, Portaliou, Athina G., Loos, Maria S., Yuan, Biao, Karamanou, Spyridoula, and Economou, Anastassios
- Subjects
WESTERN immunoblotting ,ALKALINE phosphatase ,GRAM-negative bacteria ,CHIMERIC proteins - Abstract
The type 3 secretion system is essential for pathogenesis of several human and animal Gram-negative bacterial pathogens. The T3SS comprises a transmembrane injectisome, providing a conduit from the bacterial cytoplasm to the host cell cytoplasm for the direct delivery of effectors (including toxins). Functional studies of T3SS commonly monitor the extracellular secretion of proteins by SDS-PAGE and western blot analysis, which are slow and semi-quantitative in nature. Here, we describe an enzymatic reporter-based quantitative and rapid in vivo assay for T3SS secretion studies in enteropathogenic E. coli (EPEC). The assay monitors the secretion of the fusion protein SctA-PhoA through the injectisome based on a colorimetric assay that quantifies the activity of alkaline phosphatase. We validated the usage of this reporter system by following the secretion in the absence of various injectisome components, including domains of the gatekeeper essential for T3SS function. This platform can now be used for the isolation of mutations, functional analysis and anti-virulence compound screening. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
34. Arabidopsis bZIP11 Is a Susceptibility Factor During Pseudomonas syringae Infection.
- Author
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Prior MJ, Selvanayagam J, Kim JG, Tomar M, Jonikas M, Mudgett MB, Smeekens S, Hanson J, and Frommer WB
- Subjects
- Bacterial Proteins genetics, Plant Diseases, Pseudomonas syringae, Transcription Factors genetics, Arabidopsis genetics, Arabidopsis Proteins genetics, Solanum lycopersicum
- Abstract
The induction of plant nutrient secretion systems is critical for successful pathogen infection. Some bacterial pathogens (e.g., Xanthomonas spp.) use transcription activator-like (TAL) effectors to induce transcription of SWEET sucrose efflux transporters. Pseudomonas syringae pv. tomato strain DC3000 lacks TAL effectors yet is able to induce multiple SWEETs in Arabidopsis thaliana by unknown mechanisms. Because bacteria require other nutrients in addition to sugars for efficient reproduction, we hypothesized that Pseudomonas spp. may depend on host transcription factors involved in secretory programs to increase access to essential nutrients. Bioinformatic analyses identified the Arabidopsis basic-leucine zipper transcription factor bZIP11 as a potential regulator of nutrient transporters, including SWEETs and UmamiT amino acid transporters. Inducible downregulation of bZIP11 expression in Arabidopsis resulted in reduced growth of P. syringae pv. tomato strain DC3000, whereas inducible overexpression of bZIP11 resulted in increased bacterial growth, supporting the hypothesis that bZIP11-regulated transcription programs are essential for maximal pathogen titer in leaves. Our data are consistent with a model in which a pathogen alters host transcription factor expression upstream of secretory transcription networks to promote nutrient efflux from host cells.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
- Published
- 2021
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35. Proteomic Analysis of Potato Responding to the Invasion of Ralstonia solanacearum UW551 and Its Type III Secretion System Mutant.
- Author
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Wang B, He T, Zheng X, Song B, and Chen H
- Subjects
- Bacterial Proteins genetics, Plant Diseases, Proteomics, Type III Secretion Systems genetics, Ralstonia solanacearum, Solanum tuberosum
- Abstract
The infection of potato with Ralstonia solanacearum UW551 gives rise to bacterial wilt disease via colonization of roots. The type III secretion system (T3SS) is a determinant factor for the pathogenicity of R. solanacearum . To fully understand perturbations in potato by R. solanacearum type III effectors(T3Es), we used proteomics to measure differences in potato root protein abundance after inoculation with R. solanacearum UW551 and the T3SS mutant (UW551△ Hrc V). We identified 21 differentially accumulated proteins. Compared with inoculation with UW551△ Hrc V, 10 proteins showed significantly lower abundance in potato roots after inoculation with UW551, indicating that those proteins were significantly downregulated by T3Es during the invasion. To identify their functions in immunity, we silenced those genes in Nicotiana benthamiana and tested the resistance of the silenced plants to the pathogen. Results showed that miraculin, HBP2, and TOM20 contribute to immunity to R. solanacearum . In contrast, PP1 contributes to susceptibility. Notably, none of four downregulated proteins (HBP2, PP1, HSP22, and TOM20) were downregulated at the transcriptional level, suggesting that they were significantly downregulated at the posttranscriptional level. We further coexpressed those four proteins with 33 core T3Es. To our surprise, multiple effectors were able to significantly decrease the studied protein abundances. In conclusion, our data showed that T3Es of R. solanacearum could subvert potato root immune-related proteins in a redundant manner.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
- Published
- 2021
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36. The function of the bacterial cytoskeleton in Salmonella pathogenesis
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Anne C. Doble, Kharraz L, David M. Bulmer, Michail H. Karavolos, and C. M. A. Khan
- Subjects
Microbiology (medical) ,Immunology ,Virulence ,Flagellum ,Biology ,Models, Biological ,Microbiology ,MreB ,Type three secretion system ,Prokaryotic cytoskeleton ,03 medical and health sciences ,Bacterial Proteins ,Salmonella ,Cytoskeleton ,News and Views ,Actin ,030304 developmental biology ,0303 health sciences ,030306 microbiology ,cytoskeleton ,Pathogenicity island ,Cell biology ,virulence ,Infectious Diseases ,type 3 secretion ,Parasitology ,flagella - Abstract
Recent studies have identified cytoskeletal elements in bacteria which play important roles in cellular morphology, cell division, DNA segregation and the establishment of cell polarity. However, our understanding of the contribution the bacterial cytoskeleton makes toward virulence is lacking. The MreB protein is a bacterial homolog of eukaryotic actin and interacts intimately with MreC and MreD. We provide evidence that the Mre-based cytoskeleton directly influences pathogenicity in Salmonella. Disruption of MreC and MreD caused the downregulation of the Salmonella pathogenicity island 1 (SPI-1) type 3 secretion system (T3SS) essential for host cell-invasion, and also flagella-mediated motility. These phenotypic effects were mediated by RcsC, the sensor kinase of the Rcs phosphorelay two-component system and a major envelope stress regulator. Curiously, the SPI-2 T3SS remained viable. Our results demonstrate the importance of the integrity of the bacterial cytoskeleton for virulence, highlighting the impact of associated global regulatory mechanisms on pathogenicity.
- Published
- 2012
37. Redundant and Cooperative Roles for Yersinia pestis Yop Effectors in the Inhibition of Human Neutrophil Exocytic Responses Revealed by Gain-of-Function Approach.
- Author
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Pulsifer AR, Vashishta A, Reeves SA, Wolfe JK, Palace SG, Proulx MK, Goguen J, Bodduluri SR, Haribabu B, Uriarte SM, and Lawrenz MB
- Subjects
- Bacterial Proteins genetics, Cell Degranulation, Gain of Function Mutation, Humans, Leukotriene B4 metabolism, Neutrophils metabolism, Plague immunology, Secretory Vesicles metabolism, Type III Secretion Systems genetics, Type III Secretion Systems metabolism, Virulence Factors genetics, Yersinia pestis genetics, Yersinia pestis metabolism, Bacterial Proteins metabolism, Host-Pathogen Interactions physiology, Neutrophils physiology, Virulence Factors metabolism, Yersinia pestis pathogenicity
- Abstract
Yersinia pestis causes a rapid, lethal disease referred to as plague. Y. pestis actively inhibits the innate immune system to generate a noninflammatory environment during early stages of infection to promote colonization. The ability of Y. pestis to create this early noninflammatory environment is in part due to the action of seven Yop effector proteins that are directly injected into host cells via a type 3 secretion system (T3SS). While each Yop effector interacts with specific host proteins to inhibit their function, several Yop effectors either target the same host protein or inhibit converging signaling pathways, leading to functional redundancy. Previous work established that Y. pestis uses the T3SS to inhibit neutrophil respiratory burst, phagocytosis, and release of inflammatory cytokines. Here, we show that Y. pestis also inhibits release of granules in a T3SS-dependent manner. Moreover, using a gain-of-function approach, we discovered previously hidden contributions of YpkA and YopJ to inhibition and that cooperative actions by multiple Yop effectors are required to effectively inhibit degranulation. Independent from degranulation, we also show that multiple Yop effectors can inhibit synthesis of leukotriene B
4 (LTB4 ), a potent lipid mediator released by neutrophils early during infection to promote inflammation. Together, inhibition of these two arms of the neutrophil response likely contributes to the noninflammatory environment needed for Y. pestis colonization and proliferation., (Copyright © 2020 American Society for Microbiology.)- Published
- 2020
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38. Diarrhea, bacteremia and multiorgan dysfunction due to an extraintestinal pathogenic Escherichia coli strain with enteropathogenic E. coli genes
- Author
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Amy J. Horneman, Robert A. Kessler, Shahista Nisa, Michael S. Donnenberg, David A. Rasko, Tracy H. Hazen, and Anthony Amoroso
- Subjects
Microbiology (medical) ,DNA, Bacterial ,Diarrhea ,Male ,Short Communication ,Multiple Organ Failure ,Molecular Sequence Data ,virulence factors ,Bacteremia ,HIV Infections ,comparative genomics ,Biology ,medicine.disease_cause ,Microbiology ,Type three secretion system ,Enteropathogenic Escherichia coli ,medicine ,Immunology and Allergy ,case report ,Humans ,Escherichia coli ,Escherichia coli Infections ,Extraintestinal Pathogenic Escherichia coli ,General Immunology and Microbiology ,Hemolysin ,General Medicine ,Sequence Analysis, DNA ,Middle Aged ,medicine.disease ,Virology ,3. Good health ,hemolysin ,Infectious Diseases ,Genes, Bacterial ,type 3 secretion ,medicine.symptom ,Multiple organ dysfunction syndrome ,Genome, Bacterial - Abstract
A 55-year-old man with well-controlled HIV had severe diarrhea for 3 weeks and developed multiorgan dysfunction and bacteremia due to Escherichia coli. The genome of the patient's isolate had features characteristic of extraintestinal pathogenic E. coli and genes distantly related to those defining enteropathogenic E. coli., A novel isolate of Escherichia coli with features of extraintestinal and enteropathogenic strains was isolated from the blood of a man with multiorgan dysfunction following 3 weeks of diarrhea., Graphical Abstract Figure. A novel isolate of Escherichia coli with features of extraintestinal and enteropathogenic strains was isolated from the blood of a man with multiorgan dysfunction following 3 weeks of diarrhea.
- Published
- 2015
39. Modeling Pneumonic Plague in Human Precision-Cut Lung Slices Highlights a Role for the Plasminogen Activator Protease in Facilitating Type 3 Secretion.
- Author
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Banerjee SK, Huckuntod SD, Mills SD, Kurten RC, and Pechous RD
- Subjects
- Animals, Bacterial Adhesion, Cell Line, Cytokines metabolism, Female, Humans, Macrophages, Alveolar microbiology, Mice, Mice, Inbred C57BL, Host-Pathogen Interactions, Lung microbiology, Plague etiology, Plasminogen Activators physiology, Yersinia pestis metabolism
- Abstract
Pneumonic plague is the deadliest form of disease caused by Yersinia pestis Key to the progression of infection is the activity of the plasminogen activator protease Pla. Deletion of Pla results in a decreased Y. pestis bacterial burden in the lung and failure to progress into the lethal proinflammatory phase of disease. While a number of putative functions have been attributed to Pla, its precise role in the pathogenesis of pneumonic plague is yet to be defined. Here, we show that Pla facilitates type 3 secretion into primary alveolar macrophages but not into the commonly used THP-1 cell line. We also establish human precision-cut lung slices as a platform for modeling early host/pathogen interactions during pneumonic plague and solidify the role of Pla in promoting optimal type 3 secretion using primary human tissue with relevant host cell heterogeneity. These results position Pla as a key player in the early host/pathogen interactions that define pneumonic plague and showcase the utility of human precision-cut lung slices as a platform to evaluate pulmonary infection by bacterial pathogens., (Copyright © 2019 Banerjee et al.)
- Published
- 2019
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40. Topological Analysis of the Type 3 Secretion System Translocon Pore Protein IpaC following Its Native Delivery to the Plasma Membrane during Infection.
- Author
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Russo BC, Duncan JK, and Goldberg MB
- Subjects
- Animals, Bacterial Proteins metabolism, Cell Membrane metabolism, Cells, Cultured, HeLa Cells, Humans, Mice, Protein Binding, Protein Transport, Sheep, Shigella flexneri pathogenicity, Virulence, Antigens, Bacterial metabolism, Porins metabolism, Shigella flexneri metabolism, Type III Secretion Systems metabolism
- Abstract
Many Gram-negative bacterial pathogens require a type 3 secretion system (T3SS) to deliver effector proteins into eukaryotic cells. Contact of the tip complex of the T3SS with a target eukaryotic cell initiates secretion of the two bacterial proteins that assemble into the translocon pore in the plasma membrane. The translocon pore functions to regulate effector protein secretion and is the conduit for effector protein translocation across the plasma membrane. To generate insights into how the translocon pore regulates effector protein secretion, we defined the topology of the Shigella translocon pore protein IpaC in the plasma membrane following its native delivery by the T3SS. Using single cysteine substitution mutagenesis and site-directed labeling with a membrane-impermeant chemical probe, we mapped residues accessible from the extracellular surface of the cell. Our data support a model in which the N terminus of IpaC is extracellular and the C terminus of IpaC is intracellular. These findings resolve previously conflicting data on IpaC topology that were based on nonnative delivery of IpaC to membranes. Salmonella enterica serovar Typhimurium also requires the T3SS for effector protein delivery into eukaryotic cells. Although the sequence of IpaC is closely related to the Salmonella translocon pore protein SipC, the two proteins have unique functional attributes during infection. We showed a similar overall topology for SipC and IpaC and identified subtle topological differences between their transmembrane α-helixes and C-terminal regions. Together, our data suggest that topological differences among distinct translocon pore proteins may dictate organism-specific functional differences of the T3SSs during infection. IMPORTANCE The type 3 secretion system (T3SS) is a nanomachine required for virulence of many bacterial pathogens that infect humans. The system delivers bacterial virulence proteins into the cytosol of human cells, where the virulence proteins promote bacterial infection. The T3SS forms a translocon pore in the membranes of target cells. This pore is the portal through which bacterial virulence proteins are delivered by the T3SS into the eukaryotic cytosol. The pore also regulates secretion of these virulence proteins. Our work defines the topology of translocon pore proteins in their native context during infection, resolves previously conflicting reports about the topology of the Shigella translocon pore protein IpaC, and provides new insights into how interactions of the pore with the T3SS likely produce signals that activate secretion of virulence proteins., (Copyright © 2019 Russo et al.)
- Published
- 2019
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41. Optimization of type 3 protein secretion in enteropathogenic Escherichia coli.
- Author
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Yuan, Biao, Economou, Anastassios, and Karamanou, Spyridoula
- Subjects
- *
ESCHERICHIA coli , *GRAM-negative bacteria , *PATHOGENIC microorganisms - Abstract
The type 3 secretion system (T3SS) is a protein export pathway common to Gram-negative pathogens. It comprises a trans-envelope syringe, the injectisome, with a cytoplasm-facing translocase channel. In enteropathogenic Escherichia coli , exported substrates are chaperone-delivered to the major translocase component, EscV, and cross the membrane in strict hierarchical manner, e.g. first ‘translocators’, then ‘effectors’. The in vitro dissection of the T3SS and the determination of its structure are hampered by the low numbers of the injectisomes per cell. We have now defined an optimal M9 minimal medium and established that the per transcriptional regulator enhances the number of filamented cells, the number of injectisomes per cell and the secretion of T3S substrates. Our findings provide a valuable tool for further biochemical and biophysical analysis of the T3SS and suggest that additional improvement to maximize injectisome production is possible in future efforts. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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42. Ribosome maturation by the endoribonuclease YbeY stabilizes a type 3 secretion system transcript required for virulence of enterohemorrhagic Escherichia coli .
- Author
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McAteer SP, Sy BM, Wong JL, Tollervey D, Gally DL, and Tree JJ
- Subjects
- Enterohemorrhagic Escherichia coli genetics, Enterohemorrhagic Escherichia coli pathogenicity, Escherichia coli Proteins genetics, Gene Deletion, Gene Expression Regulation, Bacterial, Humans, Metalloproteins genetics, Models, Molecular, RNA, Bacterial genetics, RNA, Bacterial metabolism, RNA, Ribosomal, 16S genetics, RNA, Ribosomal, 16S metabolism, Ribosomes genetics, Ribosomes metabolism, Transcriptome, Type III Secretion Systems genetics, Enterohemorrhagic Escherichia coli metabolism, Escherichia coli Infections microbiology, Escherichia coli Proteins metabolism, Metalloproteins metabolism, Type III Secretion Systems metabolism
- Abstract
Enterohemorrhagic Escherichia coli (EHEC) is a significant human pathogen that colonizes humans and its reservoir host, cattle. Colonization requires the expression of a type 3 secretion (T3S) system that injects a mixture of effector proteins into host cells to promote bacterial attachment and disease progression. The T3S system is tightly regulated by a complex network of transcriptional and post-transcriptional regulators. Using transposon mutagenesis, here we identified the ybeZYX-Int operon as being required for normal T3S levels. Deletion analyses localized the regulation to the endoribonuclease YbeY, previously linked to 16S rRNA maturation and small RNA (sRNA) function. Loss of ybeY in EHEC had pleiotropic effects on EHEC cells, including reduced motility and growth and cold sensitivity. Using UV cross-linking and RNA-Seq (CRAC) analysis, we identified YbeY-binding sites throughout the transcriptome and discovered specific binding of YbeY to the "neck" and "beak" regions of 16S rRNA but identified no significant association of YbeY with sRNA, suggesting that YbeY modulates T3S by depleting mature ribosomes. In E. coli , translation is strongly linked to mRNA stabilization, and subinhibitory concentrations of the translation-initiation inhibitor kasugamycin provoked rapid degradation of a polycistronic mRNA encoding needle filament and needle tip proteins of the T3S system. We conclude that T3S is particularly sensitive to depletion of initiating ribosomes, explaining the inhibition of T3S in the Δ ybeY strain. Accessory virulence transcripts may be preferentially degraded in cells with reduced translational capacity, potentially reflecting prioritization in protein production., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2018
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- View/download PDF
43. Induction of the Yersinia type 3 secretion system as an all-or-none phenomenon
- Author
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Wiley, David J, Rosqvist, Roland, Schesser, Kurt, Wiley, David J, Rosqvist, Roland, and Schesser, Kurt
- Abstract
Pathogenic Yersinia spp. possess a protein secretion system, designated as type 3, that plays a clear role in promoting their survival vis-à-vis the macrophage. Inductive expression of the Yersinia type 3 secretion system (T3SS), triggered either by host cell contact, or, in the absence of host cells, by a reduction in extracellular calcium ion levels, is accompanied by a withdrawal from the bacterial division cycle. Here, we analyzed Ca2+-dependent induction of the T3SS at the single-cell level to understand how Yersinia coordinates pro-survival and growth-related activities. We utilized a novel high-throughput quantitative microscopy approach as well as flow cytometry to determine how Ca2+ levels, T3SS expression, and cellular division are interrelated. Our analysis showed that there is a high degree of homogeneity in terms of T3SS expression levels among a population of Y. pseudotuberculosis cells following the removal of Ca2+, and that T3SS expression appears to be independent of the cellular division cycle. Unexpectedly, our analysis showed that Ca2+ levels are inversely related to the initiation of inductive T3SS expression, and not to the intensity of activation once initiated, thus providing a basis for the seemingly graded response of T3SS activation observed in bulk-level analyses. The properties of the system described here display both similarities to and differences from that of the lac operon first described 50 years ago by Novick and Weiner.
- Published
- 2007
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44. Type III Secretion a la Chlamydia
- Author
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Peters, Jan, Wilson, David, Myers, Garry, Timms, Peter, Bavoil, Patrik, Peters, Jan, Wilson, David, Myers, Garry, Timms, Peter, and Bavoil, Patrik
- Abstract
Type III secretion (T3S) is a mechanism that is central to the biology of the Chlamydiaceae and many other pathogens whose virulence depends on the translocation of toxic effector proteins to cytosolic targets within infected eukaryotic cells. Biomathematical simulations, using a previously described model of contact-dependent, T3S-mediated chlamydial growth and late differentiation, suggest that chlamydiae contained in small non-fusogenic inclusions will persist. Here, we further discuss the model in the context of in vitro-persistent, stress-induced aberrantly enlarged forms and of recent studies using small molecule inhibitors of T3S. A general mechanism is emerging whereby both early- and mid-cycle T3S-mediated activities and late T3S inactivation upon detachment of chlamydiae from the inclusion membrane are crucial for chlamydial intracellular development.
- Published
- 2007
45. Diarrhea, bacteremia and multiorgan dysfunction due to an extraintestinal pathogenic Escherichia coli strain with enteropathogenic E. coli genes.
- Author
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Kessler R, Nisa S, Hazen TH, Horneman A, Amoroso A, Rasko DA, and Donnenberg MS
- Subjects
- Bacteremia complications, Bacteremia pathology, DNA, Bacterial chemistry, DNA, Bacterial genetics, Diarrhea complications, Diarrhea pathology, Enteropathogenic Escherichia coli classification, Enteropathogenic Escherichia coli genetics, Escherichia coli Infections pathology, Genes, Bacterial, Genome, Bacterial, HIV Infections complications, Humans, Male, Middle Aged, Molecular Sequence Data, Multiple Organ Failure pathology, Sequence Analysis, DNA, Bacteremia microbiology, Diarrhea microbiology, Enteropathogenic Escherichia coli isolation & purification, Escherichia coli Infections microbiology, Multiple Organ Failure microbiology
- Abstract
A 55-year-old man with well-controlled HIV had severe diarrhea for 3 weeks and developed multiorgan dysfunction and bacteremia due to Escherichia coli. The genome of the patient's isolate had features characteristic of extraintestinal pathogenic E. coli and genes distantly related to those defining enteropathogenic E. coli., (© FEMS 2015.)
- Published
- 2015
- Full Text
- View/download PDF
46. Assembly and structure of the T3SS
- Author
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Brianne J. Burkinshaw and Natalie C. J. Strynadka
- Subjects
Adenosine Triphosphatases ,Gram-negative bacteria ,biology ,Effector ,Membrane Proteins ,Cell Biology ,Bacterial pathogenesis ,biology.organism_classification ,Protein Structure, Tertiary ,Cell biology ,Transport protein ,Type three secretion system ,Protein Transport ,Type 3 secretion ,Bacterial secretion ,Structural biology ,Membrane protein ,Multiprotein Complexes ,Gram-Negative Bacteria ,Secretion ,Bacterial Secretion Systems ,Molecular Biology ,Bacteria ,Molecular Chaperones - Abstract
The Type III Secretion System (T3SS) is a multi-mega Dalton apparatus assembled from more than twenty components and is found in many species of animal and plant bacterial pathogens. The T3SS creates a contiguous channel through the bacterial and host membranes, allowing injection of specialized bacterial effector proteins directly to the host cell. In this review, we discuss our current understanding of T3SS assembly and structure, as well as highlight structurally characterized Salmonella effectors. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.
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- View/download PDF
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