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51. Genome sequence of adherent-invasive Escherichia coli and comparative genomic analysis with other E. coli pathotypes

Author

Torres Alfredo G, Ziebell Kim, Mascarenhas Mariola, Konczy Paulina, Aguilar-Valenzuela Renan, Kropinski Andrew M, Villegas Andre, Nash John HE, Karmali Mohamed A, and Coombes Brian K

Subjects
Biotechnology, TP248.13-248.65, Genetics, QH426-470
Abstract

Abstract Background Adherent and invasive Escherichia coli (AIEC) are commonly found in ileal lesions of Crohn's Disease (CD) patients, where they adhere to intestinal epithelial cells and invade into and survive in epithelial cells and macrophages, thereby gaining access to a typically restricted host niche. Colonization leads to strong inflammatory responses in the gut suggesting that AIEC could play a role in CD immunopathology. Despite extensive investigation, the genetic determinants accounting for the AIEC phenotype remain poorly defined. To address this, we present the complete genome sequence of an AIEC, revealing the genetic blueprint for this disease-associated E. coli pathotype. Results We sequenced the complete genome of E. coli NRG857c (O83:H1), a clinical isolate of AIEC from the ileum of a Crohn's Disease patient. Our sequence data confirmed a phylogenetic linkage between AIEC and extraintestinal pathogenic E. coli causing urinary tract infections and neonatal meningitis. The comparison of the NRG857c AIEC genome with other pathogenic and commensal E. coli allowed for the identification of unique genetic features of the AIEC pathotype, including 41 genomic islands, and unique genes that are found only in strains exhibiting the adherent and invasive phenotype. Conclusions Up to now, the virulence-like features associated with AIEC are detectable only phenotypically. AIEC genome sequence data will facilitate the identification of genetic determinants implicated in invasion and intracellular growth, as well as enable functional genomic studies of AIEC gene expression during health and disease.

Published
2010
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52. A novel inhibitor of Chlamydophila pneumoniae protein kinase D (PknD) inhibits phosphorylation of CdsD and suppresses bacterial replication

Author

Bulir David C, Stone Chris B, Johnson Dustin L, Coombes Brian K, and Mahony James B

Subjects
Microbiology, QR1-502
Abstract

Abstract Background We have shown previously that Chlamydophila pneumoniae contains a dual-specific Ser/Thr protein kinase that phosphorylates CdsD, a structural component of the type III secretion apparatus. To further study the role of PknD in growth and development we sought to identify a PknD inhibitor to determine whether PknD activity is required for replication. Results Using an in vitro kinase assay we screened 80 known eukaryotic protein kinase inhibitors for activity against PknD and identified a 3'-pyridyl oxindole compound that inhibited PknD autophosphorylation and phosphorylation of CdsD. The PknD inhibitor significantly retarded the growth rate of C. pneumoniae as evidenced by the presence of very small inclusions with a reduced number of bacteria as seen by electron microscopy. These inclusions contained the normal replicative forms including elementary bodies (EB), intermediate bodies (IB) and reticulate bodies (RB), but lacked persistent bodies (PB), indicating that induction of persistence was not the cause of reduced chlamydial growth. Blind passage of C. pneumoniae grown in the presence of this PknD inhibitor for 72 or 84 hr failed to produce inclusions, suggesting this compound blocks an essential step in the production of infectious chlamydial EB. The compound was not toxic to HeLa cells, did not block activation of the MEK/ERK pathway required for chlamydial invasion and did not block intracellular replication of either Chlamydia trachomatis serovar D or Salmonella enterica sv. Typhimurium suggesting that the inhibitory effect of the compound is specific for C. pneumoniae. Conclusion We have identified a 3'-pyridyl oxindole compound that inhibits the in vitro kinase activity of C. pneumoniae PknD and inhibits the growth and production of infectious C. pneumoniae progeny in HeLa cells. Together, these results suggest that PknD may play a key role in the developmental cycle of C. pneumoniae.

Published
2009
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53. RpoE fine tunes expression of a subset of SsrB-regulated virulence factors in Salmonella enterica serovar Typhimurium

Author

Coombes Brian K and Osborne Suzanne E

Subjects
Microbiology, QR1-502
Abstract

Abstract Background The survival of Salmonella enterica within the intracellular host niche requires highly co-ordinated expression of virulence effectors predominantly regulated by the SsrAB two-component regulatory system. S. enterica serovar Typhimurium mutants lacking the ssrAB genes are avirulent in mice, highlighting the importance of this regulatory system in vivo. Mutants lacking the gene encoding the alternative sigma factor σE (rpoE) are also highly attenuated for intracellular survival, pointing to a potential connection with the SsrAB regulatory system. Results In this study we demonstrate that RpoE is involved in fine-tuning the expression of a subset of SsrB-regulated genes found in the Salmonella pathogenicity island-2 (SPI-2) genetic locus that encodes a horizontally acquired type III secretion system, and unlinked genes integrated into this regulon that are required for virulence in host animals. Conclusion These data point to a potential connection between the virulence phenotype of strains lacking ssrB and rpoE, and highlight new transcriptional regulation that might be essential for appropriate temporal and spatial control of the virulence-associated type III secretion system during host infection.

Published
2009
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54. Oral infection of mice with Salmonella enterica serovar Typhimurium causes meningitis and infection of the brain

Author

Provias John, Brown Nat F, Wickham Mark E, Finlay B Brett, and Coombes Brian K

Subjects
Infectious and parasitic diseases, RC109-216
Abstract

Abstract Background Salmonella meningitis is a rare and serious infection of the central nervous system following acute Salmonella enterica sepsis. For this pathogen, no appropriate model has been reported in which to examine infection kinetics and natural dissemination to the brain. Methods Five mouse lines including C57BL/6, Balb/c, 129S6-Slc11a1tm1Mcg, 129S1/SvImJ, B6.129-Inpp5dtm1Rkh were used in the murine typhoid model to examine the dissemination of systemic Salmonella enterica serovar Typhimurium following oral infection. Results We report data on spontaneous meningitis and brain infection following oral infection of mice with Salmonella enterica serovar Typhimurium. Conclusion This model may provide a system in which dissemination of bacteria through the central nervous system and the influence of host and bacterial genetics can be queried.

Published
2007
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58. A macrophage-based screen identifies antibacterial compounds selective for intracellularSalmonellaTyphimurium

Author

Ellis, Michael J., primary, Tsai, Caressa N., additional, Johnson, Jarrod W., additional, French, Shawn, additional, Elhenawy, Wael, additional, Porwollik, Steffen, additional, Andrews-Polymenis, Helene, additional, McClelland, Michael, additional, Magolan, Jakob, additional, Coombes, Brian K., additional, and Brown, Eric D., additional

Published
2018
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61. Molecular analysis as an aid to assess the public health risk of non-O157 shiga toxin-producing Escherichia coli strains

Author

Coombes, Brian K., Wickham, Mark E., Mascarenhas, Mariola, Gruenheid, Samantha, and Karmali, Mohamed A.

Subjects
Escherichia coli -- Physiological aspects, Escherichia coli -- Genetic aspects, Polymerase chain reaction -- Analysis, Disease transmission -- Research, Bacterial toxins -- Health aspects, Biological sciences
Abstract

The identification of three genomic islands encoding non-LEE effector (nle) genes and 14 individual nle genes in non-O157 shiga toxin-producing Escherichia coli (STEC) strains is reported. A molecular risk assessment method to rapidly recognize and respond to non-O157 STEC strains is presented.

Published
2008

66. Cheats never prosper: fast-growing 'defector mutants' can threaten the success of a bacterial infection. But one bacterial species prevails over these cheats by forming a subpopulation that has shut down expression of virulence genes

Author

Mulder, David T. and Coombes, Brian K.

Subjects
Bacteria, Pathogenic -- Research, Bacteria -- Health aspects, Bacterial infections -- Health aspects -- Research, Pathogenic microorganisms -- Research, Environmental issues, Science and technology, Zoology and wildlife conservation
Abstract

Bacterial pathogens promote their survival by deploying virulence factors that modify the host environment. But these virulence proteins are energetically costly to produce, and so avirulent defector mutants that grow [...]

Published
2013

71. A Highly Effective Component Vaccine against Nontyphoidal Salmonella enterica Infections

Author

Ferreira, Rosana B. R., primary, Valdez, Yanet, additional, Coombes, Brian K., additional, Sad, Subash, additional, Gouw, Joost W., additional, Brown, Eric M., additional, Li, Yuling, additional, Grassl, Guntram A., additional, Antunes, L. Caetano M., additional, Gill, Navkiran, additional, Truong, Mimi, additional, Scholz, Roland, additional, Reynolds, Lisa A., additional, Krishnan, Laskshmi, additional, Zafer, Ahmed A., additional, Sal-Man, Neta, additional, Lowden, Michael J., additional, Auweter, Sigrid D., additional, Foster, Leonard J., additional, and Finlay, B. Brett, additional

Published
2015
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74. EXPLORATION OF STRUCTURE-SWITCHING IN THE DESIGN OF RNA APTAMER SENSORS

Author

Lau, Pui Sai, Li, Yingfu, Coombes, Brian K., Brennan, John D., Biochemistry and Biomedical Sciences, Lau, Pui Sai, Li, Yingfu, Coombes, Brian K., Brennan, John D., and Biochemistry and Biomedical Sciences

Abstract

The process of ‘‘structure-switching’’ enables biomolecular switches to function as effective biosensing tools. Biomolecular switches can be activated or inactivated by binding to a specific target that triggers a precise conformational change in the biomolecules involved. Examples of aptamer-based biomolecular switches can be found in nature. Furthermore, efforts have been made in the last decade to engineer structure-switching sensors using DNA aptamers whereby, the aptamer is coupled to a signal transduction method to generate a readout signal upon target binding to the aptamer domain. Conversely, RNA aptamers have been relatively underexplored for sensor development, largely due to its susceptibility to nuclease degradation and chemical instability. Despite these shortcomings, many RNA aptamers possess superior sensing capabilities, and the abundance of RNA aptamers provides new opportunities to further advance the field. In effect, this thesis uses a structure-switching design to demonstrate the power of RNA aptamers for fluorescence-based sensor development. Herein, we demonstrate generalizable structure-switching strategies to make use of the abundance of RNA aptamers, monitor the quality control of detection and correct detection error, as well as enhance RNA aptamer sensing capability by using regulated graphene adsorption. Furthermore, our findings have expanded for secondary applications involving collaborations with other research labs. In one application, we demonstrate that entrapment of structure-switching RNA aptamers in sol-gel material confers protection against nuclease degradation and chemical instability. In another application, we further validate the use of riboswitches, or natural structure-switching RNA aptamers, as potential targets for drug discovery. Overall, these results demonstrate the capability of RNA aptamers for sensor development. We conclude with a discussion of possible areas for further inquiry, as well as future application, Doctor of Philosophy (PhD)

Published
2014

78. CELLULAR AND MOLECULAR HOST-PATHOGEN INTERACTIONS DURING CHLAMYDIA PNEUMONIAE INFECTION

Author

Coombes, Brian K., Mahony, James B., and Medical Sciences

Subjects
Medical Sciences
Abstract

Chlamydia pneumoniae is a Gram-negative bacterial pathogen that has evolved to survive completely within the intracellular environment of a host cell. The obligate intracellular lifestyle of this bacterium necessitates an efficient invasion strategy, exemplified by a broad host cell tropism with little propensity for any single cell type and the ability to replicate within both professional phagocytic cells and cells with low phagocytic capacity. After cellular invasion, C. pneumoniae actively evades host immune defenses, establishes a parasitic relationship with the host cell and forms a microcolony by dividing within a non-fusogenic cytoplasmic vacuole called an inclusion. The varied spectrum of host cell pathways modified by C. pneumoniae suggests a complex interaction between the bacteria and the host cell. Identifying the requisite host-pathogen interactions contributing to C.pneumoniae virulence is a major research goal. While the morphological features of the chlamydial development cycle have been described in detail, our understanding of the cellular and molecular events underlying C.pneumoniae invasion and potential mechanisms of disease pathogenesis are preliminary. The work presented in this thesis examines cellular and molecular interactions between C.pneumoniae and various types of host cells during invasion and intracellular growth of chlamydiae. Some of the research questions addressed herein are framed within the context of atherosclerosis and coronary artery disease, with the a posteriori reasoning that a potential microbiologic contribution to human atherosclerosis, specifically due to C.pneumoniae infection, is suggested by several converging lines of investigation. We found that c.pneumoniae invasion of human epithelial cells requires bacterial-induced remodeling of the host acting cytoskeleton and the activation of at least two host cell signal transduction pathways. These host modifications are required for C.pneumoniae uptake but not cellular attachment, suggesting that the C.pneumoniae invasion sequence is biphastic, whereby initial attachment is rapidly followed by activation of host cell signaling and actin polymerization to facilitate uptake. Secondly, we used cDNA array technology to study the endothelial cell transcriptional response to C.pneumoniae infection and found a prominent transactivation of several cytokines, chemokines and smooth muscle cell growth factor genes during early times after infection. Furthermore, using cell culture-based experiments and an established rabbit model of C. pneumoniae-induced artherosclerosis, we showed that C. pneomoniae infection is associated with paracrine activation of smooth muscle cell proliferation and aortic intimal thickening, potentially mediated through platelet-derived growth factor. Doctor of Philosophy (PhD)

Published
2002

79. The Type 1 Fimbrial Adhesin Mediates the Interaction of Adherent-Invasive Escherichia coli with the Host

Author

Wallar, Lauren E., Coombes, Brian K., Dawn Bowdish, Karen Mossman, Biochemistry, Wallar, Lauren E., Coombes, Brian K., Dawn Bowdish, Karen Mossman, and Biochemistry

Abstract

Crohn’s Disease is a chronic inflammatory bowel disease characterized by an overzealous immune response to a microbial trigger in genetically susceptible individuals. Although this microbial trigger is unknown, Escherichia coli with adherent and invasive properties (Adherent-Invasive Escherichia coli, AIEC) is preferentially enriched in a proportion of Crohn’s Disease patients. AIEC can adhere to and invade intestinal epithelial cells and replicate intracellularly within epithelial cells and macrophages in vitro. One important colonization factor expressed by AIEC is the type 1 fimbrial adhesin protein FimH. FimH mediates colonization of CEABAC10 transgenic mice and can bind several host cell receptors including the macrophage receptor CD48 in vitro indicating a potential role for FimH in macrophage interaction. However, it was not known whether FimH contributed to phagocytosis of AIEC or colonization of wild-type mice. Here we show that FimH enhances early intracellular AIEC levels in vitro and colonization in vivo. We found that deletion of fimH may reduce intracellular AIEC burden at 2 hours post-infection and that this effect was modulated by bacteria opsonisation. Using a competitive index assay, we show that a ΔfimH mutant is unable to chronically colonize CD-1 mice at the same levels as the parental strain. Our results demonstrate that FimH is an important AIEC colonization factor and may increase interaction with macrophages. Identifying factors such as FimH which contribute to colonization and persistence will further our understanding of AIEC survival strategies within the host. Development of therapeutics targeting FimH may provide a means to reduce harmful bacteria overgrowth particularly after surgical intervention., Master of Science (MSc)

Published
2012

91. Erratum: A draft genome of Yersinia pestis from victims of the Black Death

Author

Bos, Kirsten I., primary, Schuenemann, Verena J., additional, Golding, G. Brian, additional, Burbano, Hernán A., additional, Waglechner, Nicholas, additional, Coombes, Brian K., additional, McPhee, Joseph B., additional, N.DeWitte, Sharon, additional, Meyer, Matthias, additional, Schmedes, Sarah, additional, Wood, James, additional, Earn, David J. D., additional, Herring, D. Ann, additional, Bauer, Peter, additional, Poinar, Hendrik N., additional, and Krause, Johannes, additional

Published
2011
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93. Quantitative Mass Spectrometry Catalogues Salmonella Pathogenicity Island-2 Effectors and Identifies Their Cognate Host Binding Partners

Author

Auweter, Sigrid D., primary, Bhavsar, Amit P., additional, de Hoog, Carmen L., additional, Li, Yuling, additional, Chan, Y. Alina, additional, van der Heijden, Joris, additional, Lowden, Michael J., additional, Coombes, Brian K., additional, Rogers, Lindsay D., additional, Stoynov, Nikolay, additional, Foster, Leonard J., additional, and Finlay, B. Brett, additional

Published
2011
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