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The enteric nervous system (ENS) comprises a complex network of neurons whereby a subset appears to be dopaminergic although the characteristics, roles, and implications in disease are less understood. Most investigations relating to enteric dopamine (DA) neurons rely on immunoreactivity to tyrosine hydroxylase (TH)-the rate-limiting enzyme in the production of DA. However, TH immunoreactivity is likely to provide an incomplete picture. This study herein provides a comprehensive characterization of DA neurons in the gut using a reporter mouse line, expressing a fluorescent protein (tdTomato) under control of the DA transporter (DAT) promoter. Our findings confirm a unique localization of DA neurons in the gut and unveil the discrete subtypes of DA neurons in this organ, which we characterized using both immunofluorescence and single-cell transcriptomics, as well as validated using in situ hybridization. We observed distinct subtypes of DAT-tdTomato neurons expressing co-transmitters and modulators across both plexuses; some of them likely co-releasing acetylcholine, while others were positive for a slew of canonical DAergic markers (TH, VMAT2 and GIRK2). Interestingly, we uncovered a seemingly novel population of DA neurons unique to the ENS which was ChAT/DAT-tdTomato-immunoreactive and expressed Grp, Calcb, and Sst. Given the clear heterogeneity of DAergic gut neurons, further investigation is warranted to define their functional signatures and decipher their implication in disease., (© 2024 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Non-LEE-encoded Effector A (NleA) is a type III secreted effector protein of enterohaemorrhagic and enteropathogenic Escherichia coli as well as the related mouse pathogen Citrobacter rodentium . NleA translocation into host cells is essential for virulence. We previously published several lines of evidence indicating that NleA is modified by host-mediated mucin-type O-linked glycosylation, the first example of a bacterial effector protein modified in this way. In this study, we use lectins to provide direct evidence for the modification of NleA by O-linked glycosylation and determine that the interaction of NleA with the COPII complex is necessary for this modification to occur.

Background: Vitamin B12 supplements typically contain doses that far exceed the recommended daily amount, and high exposures are generally considered safe. Competitive and syntrophic interactions for B12 exist between microbes in the gut. Yet, to what extent excessive levels contribute to the activities of the gut microbiota remains unclear. The objective of this study was to evaluate the effect of B12 on microbial ecology using a B12 supplemented mouse model with Citrobacter rodentium, a mouse-specific pathogen. Mice were fed a standard chow diet and received either water or water supplemented with B12 (cyanocobalamin: ~120 μg/day), which equates to approximately 25 mg in humans. Infection severity was determined by body weight, pathogen load, and histopathologic scoring. Host biomarkers of inflammation were assessed in the colon before and after the pathogen challenge., Results: Cyanocobalamin supplementation enhanced pathogen colonization at day 1 (P < 0.05) and day 3 (P < 0.01) postinfection. The impact of B12 on gut microbial communities, although minor, was distinct and attributed to the changes in the Lachnospiraceae populations and reduced alpha diversity. Cyanocobalamin treatment disrupted the activity of the low-abundance community members of the gut microbiota. It enhanced the amount of interleukin-12 p40 subunit protein (IL12/23p40; P < 0.001) and interleukin-17a (IL-17A; P < 0.05) in the colon of naïve mice. This immune phenotype was microbe dependent, and the response varied based on the baseline microbiota. The cecal metatranscriptome revealed that excessive cyanocobalamin decreased the expression of glucose utilizing genes by C. rodentium, a metabolic attribute previously associated with pathogen virulence., Conclusions: Oral vitamin B12 supplementation promoted C. rodentium colonization in mice by altering the activities of the Lachnospiraceae populations in the gut. A lower abundance of select Lachnospiraceae species correlated to higher p40 subunit levels, while the detection of Parasutterella exacerbated inflammatory markers in the colon of naïve mice. The B12-induced change in gut ecology enhanced the ability of C. rodentium colonization by impacting key microbe-host interactions that help with pathogen exclusion. This research provides insight into how B12 impacts the gut microbiota and highlights potential consequences of disrupting microbial B12 competition/sharing through over-supplementation. Video Abstract., (© 2023. The Author(s).)

Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) are gastrointestinal pathogens responsible for severe diarrheal illness. EHEC and EPEC form "attaching and effacing" lesions during colonization and, upon adherence, inject proteins directly into host intestinal cells via the type III secretion system (T3SS). Injected bacterial proteins have a variety of functions but generally alter host cell biology to favor survival and/or replication of the pathogen. Non-LEE-encoded effector A (NleA) is a T3SS-injected effector of EHEC, EPEC, and the related mouse pathogen Citrobacter rodentium. Studies in mouse models indicate that NleA has an important role in bacterial virulence. However, the mechanism by which NleA contributes to disease remains unknown. We have determined that the following translocation into host cells, a serine and threonine-rich region of NleA is modified by host-mediated mucin-type O-linked glycosylation. Surprisingly, this region was not present in several clinical EHEC isolates. When expressed in C. rodentium, a non-modifiable variant of NleA was indistinguishable from wildtype NleA in an acute mortality model but conferred a modest increase in persistence over the course of infection in mixed infections in C57BL/6J mice. This is the first known example of a bacterial effector being modified by host-mediated O-linked glycosylation. Our data also suggests that this modification may confer a selective disadvantage to the bacteria during in vivo infection., (© 2022 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Citrobacter rodentium is an attaching and effacing (A/E) pathogen used to model enteropathogenic and enterohemorrhagic Escherichia coli infections in mice. During colonization, C. rodentium must adapt to stresses in the gastrointestinal tract, such as antimicrobial peptides, pH changes, and bile salts. The Cpx envelope stress response (ESR) is a two-component system used by some bacteria to remediate stress by modulating gene expression, and it is necessary for C. rodentium pathogenesis in mice. Here, we utilized simulated colonic fluid (SCF) to mimic the gastrointestinal environment, which we show strongly induces the Cpx ESR and highlights a fitness defect specific to the Δ cpxRA mutant. While investigating genes in the Cpx regulon that may contribute to C. rodentium pathogenesis, we found that the absence of the Cpx ESR resulted in higher expression of the locus of enterocyte effacement (LEE) master regulator, ler , and that the genes yebE , ygiB , bssR , and htpX relied on CpxRA for proper expression. We then determined that CpxRA and select gene mutants were essential for proper growth in SCF when in the presence of extraneous stressors and in competition. Although none of the Cpx-regulated gene mutants exhibited marked virulence phenotypes in vivo , the Δ cpxRA mutant had reduced colonization and attenuated virulence, as previously determined, which replicated the in vitro growth phenotypes specific to SCF. Overall, these results indicate that the Δ cpxRA virulence defect is not due to any single Cpx regulon gene examined. Instead, attenuation may be the result of defective growth in the colonic environment resulting from the collective impact of multiple Cpx-regulated genes.

Parkinson's disease (PD) is a neurodegenerative disorder influenced by both genetic and environmental factors. The mechanisms leading to neurodegeneration in PD are still under investigation, with several mechanistic models currently proposed. A number of microorganisms have been associated with increased risk of PD in humans, and recent research using newly developed models has begun to elucidate how these microbes may factor into disease development. Newly identified roles for PD-associated genes in host-microbe interactions and response to infections have also recently been uncovered, providing further evidence for microbial contributions to PD. Here we summarize these recent advances in the field and discuss them in the context of both historical and emerging hypotheses for PD development, with a particular focus on the application of rodent models as systems allowing for mechanistic hypothesis testing., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Stapled peptides have the ability to mimic α-helices involved in protein binding and have proved to be effective pharmacological agents for disrupting protein-protein interactions. DNA-binding proteins such as transcription factors bind their cognate DNA sequences via an α-helix interacting with the major groove of DNA. We previously developed a stapled peptide based on the bacterial alternative sigma factor RpoN capable of binding the RpoN DNA promoter sequence and inhibiting RpoN-mediated expression in Escherichia coli . We have elucidated a structure-activity relationship for DNA binding by this stapled peptide, improving DNA binding affinity constants in the high nM range. Lead peptides were shown to have low toxicity as determined by their low hemolytic activity at 100 μM and were shown to have anti-virulence activity in a Galleria mellonella model of Pseudomonas aeruginosa infection. These findings support further preclinical development of stapled peptides as antivirulence agents targeting P. aeruginosa ., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Surgical site infections (SSIs) account for a massive economic, physiological, and psychological burden on patients and health care providers. Sutures provide a surface to which bacteria can adhere, proliferate, and promote SSIs. Current methods for fighting SSIs involve the use of sutures coated with common antibiotics (triclosan). Unfortunately, these antibiotics have been rendered ineffective due to the increasing rate of antibiotic resistance. A promising new avenue involves the use of metallic nanoparticles (MNPs). MNPs exhibit low cytotoxicity and a strong propensity for killing bacteria while evading the typical antibiotic resistance mechanisms. In this work, we developed a novel MNPs dip-coating method for PDS-II sutures and explored the capabilities of a variety of MNPs in killing bacteria while retaining the cytocompatibility. Our findings indicated that our technique provided a homogeneous coating for PDS-II sutures, maintaining the strength, structural integrity, and degradability. The MNP coatings possess strong in vitro antibacterial properties against P aeruginosa and S. aureus-varying the %of dead bacteria from ~ 40% (for MgO NPs) to ~ 90% (for Fe 2 O 3 ) compared to ~ 15% for uncoated PDS-II suture, after 7 days. All sutures demonstrated minimal cytotoxicity (cell viability > 70%) reinforcing the movement towards the use MNPs as a viable antibacterial technology., (© 2022. The Author(s).)

There is a growing appreciation that the interaction between diet, the gut microbiota and the immune system contribute to the development and progression of inflammatory bowel disease (IBD). A mounting body of scientific evidence suggests that high-fat diets exacerbate IBD; however, there is a lack of information on how specific types of fat impact colitis. The Mediterranean diet (MD) is considered a health-promoting diet containing approximately 40% total fat. It is not known if the blend of fats found in the MD contributes to its beneficial protective effects., Mice deficient in the mucin 2 gene (Muc 2 -/- ) were weaned to 40% fat, isocaloric, isonitrogenous diets. We compared the MD fat blend (high monounsaturated, 2:1 n-6:n-3 polyunsaturated and moderate saturated fat) to diets composed of corn oil (CO, n-6 polyunsaturated-rich), olive oil (monounsaturated-rich) or milk fat (MF, saturated-rich) on spontaneous colitis development in Muc2 -/- mice. The MD resulted in lower clinical and histopathological scores and induced tolerogenic CD103+ CD11b+ dendritic, Th22 and IL-17+ IL-22+ cells necessary for intestinal barrier repair. The MD was associated with beneficial microbes and associated with higher cecal acetic acid levels negatively correlated with colitogenic microbes like Akkermansia muciniphila . In contrast, CO showed a higher prevalence of mucin-degraders including A. muciniphila and Enterobacteriaceae, which have been associated with colitis., A dietary blend of fats mimicking the MD, reduces disease activity, inflammation-related biomarkers and improves metabolic parameters in the Muc2 -/- mouse model. Our findings suggest that the MD fat blend could be incorporated into a maintenance diet for colitis.

Parkinson's disease (PD) is a neurodegenerative disorder that has been shown to be influenced by the intestinal milieu. The gut microbiota is altered in PD patients, and murine studies have begun suggesting a causative role for the gut microbiota in progression of PD. We have previously shown that repeated infection with the intestinal murine pathogen Citrobacter rodentium resulted in the development of PD-like pathology in Pink1 -/- mice compared to wild-type littermates. This addendum aims to expand this work by characterizing the gut microbiota during C. rodentium infection in our Pink1 -/- PD model. We observed little disturbance to the fecal microbiota diversity both between infection timepoints and between Pink1 -/- and wild-type control littermates. However, the level of short-chain fatty acids appeared to be altered over the course of infection with butyric acid significantly increasing in Pink1 -/- mice and isobutyric acid increasing in wild-type mice.

The emergence of multidrug-resistant bacterial strains worldwide has become a serious problem for public health over recent decades. The increase in antimicrobial resistance has been expanding via plasmids as mobile genetic elements encoding antimicrobial resistance (AMR) genes that are transferred vertically and horizontally. This study focuses on Salmonella enterica , one of the leading foodborne pathogens in industrialized countries. S. enterica is known to carry several plasmids involved not only in virulence but also in AMR. In the current paper, we present an integrated strategy to detect plasmid scaffolds in whole genome sequencing (WGS) assemblies. We developed a two-step procedure to predict plasmids based on i) the presence of essential elements for plasmid replication and mobility, as well as ii) sequence similarity to a reference plasmid. Next, to confirm the accuracy of the prediction in 1750 S. enterica short-read sequencing data, we combined Oxford Nanopore MinION long-read sequencing with Illumina MiSeq short-read sequencing in hybrid assemblies for 84 isolates to evaluate the proportion of plasmid that has been detected. At least one scaffold with an origin of replication (ORI) was predicted in 61.3% of the Salmonella isolates tested. The results indicated that IncFII and IncI1 ORIs were distributed in many S. enterica serotypes and were the most prevalent AMR genes carrier, whereas IncHI2A/IncHI2 and IncA/C2 were more serotype restricted but bore several AMR genes. Comparison between hybrid and short-read assemblies revealed that 81.1% of plasmids were found in the short-read sequencing using our pipeline. Through this process, we established that plasmids are prevalent in S. enterica and we also substantially expand the AMR genes in the resistome of this species.

Salmonella comprises more than 2,600 serovars. Very few environmental and uncommon serovars have been characterized for their potential role in virulence and human infections. A complementary in vitro and in vivo systematic high-throughput analysis of virulence was used to elucidate the association between genetic and phenotypic variations across Salmonella isolates. The goal was to develop a strategy for the classification of isolates as a benchmark and predict virulence levels of isolates. Thirty-five phylogenetically distant strains of unknown virulence were selected from the Salmonella Foodborne Syst-OMICS (SalFoS) collection, representing 34 different serovars isolated from various sources. Isolates were evaluated for virulence in 4 complementary models of infection to compare virulence traits with the genomics data, including interactions with human intestinal epithelial cells, human macrophages, and amoeba. In vivo testing was conducted using the mouse model of Salmonella systemic infection. Significant correlations were identified between the different models. We identified a collection of novel hypothetical and conserved proteins associated with isolates that generate a high burden. We also showed that blind prediction of virulence of 33 additional strains based on the pan-genome was high in the mouse model of systemic infection (82% agreement) and in the human epithelial cell model (74% agreement). These complementary approaches enabled us to define virulence potential in different isolates and present a novel strategy for risk assessment of specific strains and for better monitoring and source tracking during outbreaks. IMPORTANCE Salmonella species are bacteria that are a major source of foodborne disease through contamination of a diversity of foods, including meat, eggs, fruits, nuts, and vegetables. More than 2,600 different Salmonella enterica serovars have been identified, and only a few of them are associated with illness in humans. Despite the fact that they are genetically closely related, there is enormous variation in the virulence of different isolates of Salmonella enterica Identification of foodborne pathogens is a lengthy process based on microbiological, biochemical, and immunological methods. Here, we worked toward new ways of integrating whole-genome sequencing (WGS) approaches into food safety practices. We used WGS to build associations between virulence and genetic diversity within 83 Salmonella isolates representing 77 different Salmonella serovars. Our work demonstrates the potential of combining a genomics approach and virulence tests to improve the diagnostics and assess risk of human illness associated with specific Salmonella isolates., (© Crown copyright 2020.)

Dendritic cells, cells of the innate immune system, are found in a steady state poised to respond to activating stimuli. Once stimulated, they rapidly undergo dynamic changes in gene expression to adopt an activated phenotype capable of stimulating immune responses. We find that the microRNA miR-9 is upregulated in both bone marrow-derived DCs and conventional DC1s but not in conventional DC2s following stimulation. miR-9 expression in BMDCs and conventional DC1s promotes enhanced DC activation and function, including the ability to stimulate T cell activation and control tumor growth. We find that miR-9 regulated the expression of several negative regulators of transcription, including the transcriptional repressor Polycomb group factor 6 (Pcgf6). These findings demonstrate that miR-9 facilitates the transition of DCs from steady state to mature state by regulating the expression of several negative regulators of DC function in a cell-type-specific manner., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

With more than 50% of bacteria resistant to standard antibiotics, new strategies to treat bacterial infection and colonization are needed. Based on the concept of targeting the bacteria synergistically on various fronts, it is hypothesized that an electrical insult associated with antibacterial materials may be a highly effective means of killing bacteria. In this work, an injectable conductive gel based on silk fibroin (SF) and silver nanoparticles (Ag-NPs) is synthesized, capable of coating a zone of injury, allowing the application of a low electrical current to decrease bacterial contamination. With a high conductivity of 1.5 S cm -1 , SF/Ag-NPs gels killed 80% of Escherichia coli in 1 min, no toxicity toward Chinese hamster ovary cells is observed. The mechanism of an electrical composite gel combined with electrical wound therapy is associated with silver ion (Ag + ) release, and reactive oxygen species (ROS) production. The findings in the present study show a similar Ag + release for treatment with gels and the combined effect, whereas ROS generation is 50% higher when a small electrical current is applied leading to a broad bactericidal effect., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bacterial colonization of the urogenital tract is limited by innate defenses, including the production of antimicrobial peptides (AMPs). Uropathogenic Escherichia coli (UPEC) resist AMP-killing to cause a range of urinary tract infections (UTIs) including asymptomatic bacteriuria, cystitis, pyelonephritis, and sepsis. UPEC strains have high genomic diversity and encode numerous virulence factors that differentiate them from non-UTI-causing strains, including ompT. As OmpT homologs cleave and inactivate AMPs, we hypothesized that UPEC strains from patients with symptomatic UTIs have high OmpT protease activity. Therefore, we measured OmpT activity in 58 clinical E. coli isolates. While heterogeneous OmpT activities were observed, OmpT activity was significantly greater in UPEC strains isolated from patients with symptomatic infections. Unexpectedly, UPEC strains exhibiting the greatest protease activities harbored an additional ompT-like gene called arlC (ompTp). The presence of two OmpT-like proteases in some UPEC isolates led us to compare the substrate specificities of OmpT-like proteases found in E. coli. While all three cleaved AMPs, cleavage efficiency varied on the basis of AMP size and secondary structure. Our findings suggest the presence of ArlC and OmpT in the same UPEC isolate may confer a fitness advantage by expanding the range of target substrates., (© 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

The goal of this study was to isolate, screen, and characterize Arctic microbial isolates from Expedition Fjord, Axel Heiberg Island, Nunavut, Canada capable of inhibiting the growth of foodborne and clinically relevant pathogens. Arctic bacteria were isolated from twelve different high Arctic habitats pertaining to active layer permafrost soil, saline spring sediments, lake sediments, and endoliths. This was achieved using (1) the cryo-iPlate, an innovative in situ cultivation device within active layer permafrost soil and (2) bulk plating of Arctic samples by undergraduate students that applied standard culturing methods. To mitigate the possibility of identifying isolates with already-known antibacterial activities, a cell-based dereplication platform was used. Ten out of the twelve Arctic habitats tested were found to yield cold-adapted isolates with antibacterial activity. Eight cold-adapted Arctic isolates were identified with the ability to inhibit the entire dereplication platform, suggesting the possibility of new mechanisms of action. Two promising isolates, initially cultured from perennial saline spring sediments and from active layer permafrost soil ( Paenibacillus sp. GHS.8.NWYW.5 and Pseudomonas sp. AALPS.10.MNAAK.13, respectively), displayed antibacterial activity against foodborne and clinically relevant pathogens. Paenibacillus sp. GHS.8.NWYW.5 was capable of inhibiting methicillin resistant and susceptible Staphylococcus aureus (MRSA and MSSA), Listeria monocytogenes , Salmonella enterica and Escherichia coli O157:H7. Pseudomonas sp. AALPS.10.MNAAK.13 was observed to have antagonistic activity against MRSA, MSSA, Acinetobacter baumanii , Enterococcus faecium , and Enterococcus faecalis . After whole genome sequencing and mining, the genome of Paenibacillus sp. GHS.8.NWYW.5 was found to contain seven putative secondary metabolite biosynthetic gene clusters that displayed low homology (<50% coverage, <30% identity, and e-values > 0) to clusters identified within the genome of the type strain pertaining to the same species. These findings suggest that cold-adapted Arctic microbes may be a promising source of novel secondary metabolites for potential use in both industrial and medical settings.

Parkinson's disease is a neurodegenerative disorder with motor symptoms linked to the loss of dopaminergic neurons in the substantia nigra compacta. Although the mechanisms that trigger the loss of dopaminergic neurons are unclear, mitochondrial dysfunction and inflammation are thought to have key roles 1,2 . An early-onset form of Parkinson's disease is associated with mutations in the PINK1 kinase and PRKN ubiquitin ligase genes 3 . PINK1 and Parkin (encoded by PRKN) are involved in the clearance of damaged mitochondria in cultured cells 4 , but recent evidence obtained using knockout and knockin mouse models have led to contradictory results regarding the contributions of PINK1 and Parkin to mitophagy in vivo 5-8 . It has previously been shown that PINK1 and Parkin have a key role in adaptive immunity by repressing presentation of mitochondrial antigens 9 , which suggests that autoimmune mechanisms participate in the aetiology of Parkinson's disease. Here we show that intestinal infection with Gram-negative bacteria in Pink1 -/- mice engages mitochondrial antigen presentation and autoimmune mechanisms that elicit the establishment of cytotoxic mitochondria-specific CD8 + T cells in the periphery and in the brain. Notably, these mice show a sharp decrease in the density of dopaminergic axonal varicosities in the striatum and are affected by motor impairment that is reversed after treatment with L-DOPA. These data support the idea that PINK1 is a repressor of the immune system, and provide a pathophysiological model in which intestinal infection acts as a triggering event in Parkinson's disease, which highlights the relevance of the gut-brain axis in the disease 10 .

Outer membrane vesicles (OMVs) are naturally produced by Gram-negative bacteria by a bulging of the outer membrane (OM) and subsequent release into the environment. By serving as vehicles for various cargos, including proteins, nucleic acids and small metabolites, OMVs are central to interbacterial interactions and both symbiotic and pathogenic host bacterial interactions. However, despite their importance, the mechanism of OMV formation remains unclear. Recent evidence indicates that covalent modifications of lipopolysaccharides (LPS) influence OMV biogenesis. Several enteric bacteria modify LPS with phosphoethanolamine (pEtN) using the iron-regulated PmrC (EptA) and CptA pEtN transferases. In wild-type Citrobacter rodentium , the presence of increasing subtoxic concentrations of iron was found to stimulate OMV production 4- to 9-fold above baseline. C. rodentium uses the two-component system PmrAB to sense and adapt to environmental iron. Compared to the wild type, the C. rodentium Δ pmrAB strain exhibited heightened OMV production at similar iron concentrations. PmrAB regulates transcription of pmrC (also known as eptA ) and cptA OMV production in strains lacking either pmrC ( eptA ) or cptA was similarly increased in comparison to that of the wild type. Importantly, plasmid complementation of C. rodentium strains with either pmrC ( eptA ) or cptA resulted in a drastic inhibition of OMV production. Finally, we showed that β-lactamase and CroP, two enzymes found in the C. rodentium periplasm and outer membrane (OM), respectively, are associated with OMVs. These data suggest a novel mechanism by which C. rodentium and possibly other Gram-negative bacteria can negatively affect OMV production through the PmrAB-regulated genes pmrC ( eptA ) and cptA IMPORTANCE Although OMVs secreted by Gram-negative bacteria fulfill multiple functions, the molecular mechanism of OMV biogenesis remains ill defined. Our group has previously shown that PmrC (also known as EptA) and CptA maintain OM integrity and provide resistance to iron toxicity and antibiotics in the murine pathogen Citrobacter rodentium In several enteric bacteria, these proteins modify the lipid A and core regions of lipopolysaccharide with phosphoethanolamine moieties. Here, we show that these proteins also repress OMV production in response to environmental iron in C. rodentium These data support the emerging understanding that lipopolysaccharide modifications are important regulators of OMV biogenesis in Gram-negative bacteria., (Copyright © 2019 Sinha et al.)

Citrobacter rodentium is a murine pathogen used to model the intestinal infection caused by Enteropathogenic and Enterohemorrhagic Escherichia coli (EPEC and EHEC), two diarrheal pathogens responsible for morbidity and mortality in developing and developed countries, respectively. During infection, these bacteria must sense and adapt to the gut environment of the host. In order to adapt to changing environmental cues and modulate expression of specific genes, bacteria can use two-component signal transduction systems (TCS). We have shown that the deletion of the Cpx TCS in C. rodentium leads to a marked attenuation in virulence in C3H/HeJ mice. In E. coli , the Cpx TCS is reportedly activated in response to signals from the outer-membrane lipoprotein NlpE. We therefore investigated the role of NlpE in C. rodentium virulence. We also assessed the role of the reported negative regulator of CpxRA, CpxP. We found that as opposed to the Δ cpxRA strain, neither the Δ nlpE , Δ cpxP nor the Δ nlpE Δ cpxP strains were significantly attenuated, and had similar in vivo localization to wild-type C. rodentium . The in vitro adherence of the Cpx auxiliary protein mutants, Δ nlpE , Δ cpxP , Δ nlpE Δ cpxP , was comparable to wild-type C. rodentium , whereas the Δ cpxRA strain showed significantly decreased adherence. To further elucidate the mechanisms behind the contrasting virulence phenotypes, we performed microarrays in order to define the regulon of the Cpx TCS. We detected 393 genes differentially regulated in the Δ cpxRA strain. The gene expression profile of the Δ nlpE strain is strikingly different than the profile of Δ cpxRA with regards to the genes activated by CpxRA. Further, there is no clear inverse correlation in the expression pattern of the Δ cpxP strain in comparison to Δ cpxRA . Taken together, these data suggest that in these conditions, CpxRA activates gene expression in a largely NlpE- and CpxP-independent manner. Compared to wildtype, 161 genes were downregulated in the Δ cpxRA strain, while being upregulated or unchanged in the Cpx auxiliary protein deletion strains. This group of genes, which we hypothesize may contribute to the loss of virulence of Δ cpxRA , includes T6SS components, ompF , the regulator for colanic acid synthesis, and several genes involved in maltose metabolism.

Enterobacteriaceae are a large family of Gram-negative, non-spore-forming bacteria. Although many species exist as part of the natural flora of animals including humans, some members are associated with both intestinal and extraintestinal diseases. In this review, we focus on members of this family that have important roles in human disease: Salmonella, Escherichia, Shigella, and Yersinia, providing a brief overview of the disease caused by these bacteria, highlighting the contribution of animal models to our understanding of their pathogenesis and of host genetic determinants involved in susceptibility or resistance to infection.

Non-typhoidal Salmonella is a leading cause of foodborne illness worldwide. Prompt and accurate identification of the sources of Salmonella responsible for disease outbreaks is crucial to minimize infections and eliminate ongoing sources of contamination. Current subtyping tools including single nucleotide polymorphism (SNP) typing may be inadequate, in some instances, to provide the required discrimination among epidemiologically unrelated Salmonella strains. Prophage genes represent the majority of the accessory genes in bacteria genomes and have potential to be used as high discrimination markers in Salmonella . In this study, the prophage sequence diversity in different Salmonella serovars and genetically related strains was investigated. Using whole genome sequences of 1,760 isolates of S. enterica representing 151 Salmonella serovars and 66 closely related bacteria, prophage sequences were identified from assembled contigs using PHASTER. We detected 154 different prophages in S. enterica genomes. Prophage sequences were highly variable among S. enterica serovars with a median ± interquartile range (IQR) of 5 ± 3 prophage regions per genome. While some prophage sequences were highly conserved among the strains of specific serovars, few regions were lineage specific. Therefore, strains belonging to each serovar could be clustered separately based on their prophage content. Analysis of S . Enteritidis isolates from seven outbreaks generated distinct prophage profiles for each outbreak. Taken altogether, the diversity of the prophage sequences correlates with genome diversity. Prophage repertoires provide an additional marker for differentiating S. enterica subtypes during foodborne outbreaks.

Citrobacter rodentium is an intestinal mouse pathogen widely used as a model to study the mucosal response to infection. Inbred mouse strains suffer one of two fates following infection: self-limiting colitis or fatal diarrheal disease. We previously reported that Rspo2 is a major genetic determinant of the outcome of C. rodentium infection; Rspo2 induction during infection of susceptible mice leads to loss of intestinal function and mortality. Rspo2 induction does not impact bacterial colonization, but rather, impedes the ability of the host to tolerate C. rodentium infection. Here, we performed deep RNA sequencing and systematically analyzed the global gene expression profiles of C. rodentium-infected colon tissues from susceptible and resistant congenic mice strains to determine the common responses to infection and the Rspo2-mediated dysfunction pathway signatures associated with loss of disease tolerance. Our results highlight changes in metabolism, tissue remodeling, and host defence as common responses to infection. Conversely, increased Wnt and stem cell signatures, loss of epithelial differentiation, and exaggerated CD4 + T cell activation through increased antigen processing and presentation were specifically associated with the response to infection in susceptible mice. These data provide insights into the molecular mechanisms underlying intestinal dysfunction and disease tolerance during C. rodentium infection.

The Salmonella Syst-OMICS consortium is sequencing 4,500 Salmonella genomes and building an analysis pipeline for the study of Salmonella genome evolution, antibiotic resistance and virulence genes. Metadata, including phenotypic as well as genomic data, for isolates of the collection are provided through the Salmonella Foodborne Syst-OMICS database (SalFoS), at https://salfos.ibis.ulaval.ca/. Here, we present our strategy and the analysis of the first 3,377 genomes. Our data will be used to draw potential links between strains found in fresh produce, humans, animals and the environment. The ultimate goals are to understand how Salmonella evolves over time, improve the accuracy of diagnostic methods, develop control methods in the field, and identify prognostic markers for evidence-based decisions in epidemiology and surveillance.

Citrobacter rodentium is a murine pathogen used to model intestinal infections caused by the human diarrheal pathogens enterohemorrhagic and enteropathogenic Escherichia coli During infection, bacteria use two-component systems (TCSs) to detect changing environmental cues within the host, allowing for rapid adaptation by altering the expression of specific genes. In this study, 26 TCSs were identified in C. rodentium, and quantitative PCR (qPCR) analysis showed that they are all expressed during murine infection. These TCSs were individually deleted, and the in vitro and in vivo effects were analyzed to determine the functional consequences. In vitro analyses only revealed minor differences, and surprisingly, type III secretion (T3S) was only affected in the ΔarcA strain. Murine infections identified 7 mutants with either attenuated or increased virulence. In agreement with the in vitro T3S assay, the ΔarcA strain was attenuated and defective in colonization and cell adherence. The ΔrcsB strain was among the most highly attenuated strains. The decrease in virulence of this strain may be associated with changes to the cell surface, as Congo red binding was altered, and qPCR revealed that expression of the wcaA gene, which has been implicated in colanic acid production in other bacteria, was drastically downregulated. The ΔuvrY strain exhibited increased virulence compared to the wild type, which was associated with a significant increase in bacterial burden within the mesenteric lymph nodes. The systematic analysis of virulence-associated TCSs and investigation of their functions during infection may open new avenues for drug development., (Copyright © 2017 American Society for Microbiology.)

The R-spondin family of proteins has recently been described as secreted enhancers of β-catenin activation through the canonical Wnt signaling pathway. We previously reported that Rspo2 is a major determinant of susceptibility to Citrobacter rodentium-mediated colitis in mice and recent genome-wide association studies have revealed RSPO3 as a candidate Crohn's disease-specific inflammatory bowel disease susceptibility gene in humans. However, there is little information on the endogenous expression and cellular source of R-spondins in the colon at steady state and during intestinal inflammation. RNA sequencing and qRT-PCR were used to assess the expression of R-spondins at steady state and in two mouse models of colonic inflammation. The cellular source of R-spondins was assessed in specific colonic cell populations isolated by cell sorting. Data mining from publicly available datasets was used to assess the expression of R-spondins in the human colon. At steady state, colonic expression of R-spondins was found to be exclusive to non-epithelial CD45- lamina propria cells, and Rspo3/RSPO3 was the most highly expressed R-spondin in both mouse and human colon. R-spondin expression was found to be highly dynamic and differentially regulated during C. rodentium infection and dextran sodium sulfate (DSS) colitis, with notably high levels of Rspo3 expression during DSS colitis, and high levels of Rspo2 expression during C. rodentium infection, specifically in susceptible mice. Our data are consistent with the hypothesis that in the colon, R-spondins are expressed by subepithelial stromal cells, and that Rspo3/RSPO3 is the family member most implicated in colonic homeostasis. The differential regulation of the R-spondins in different models of intestinal inflammation indicate they respond to specific pathogenic and inflammatory signals that differ in the two models and provides further evidence that this family of proteins plays a key role in linking intestinal inflammation and homeostasis.

Unlabelled: Bacterial proteases contribute to virulence by cleaving host or bacterial proteins to promote survival and dissemination. Omptins are a family of proteases embedded in the outer membrane of Gram-negative bacteria that cleave various substrates, including host antimicrobial peptides, with a preference for cleaving at dibasic motifs. OmpT, the enterohemorrhagic Escherichia coli (EHEC) omptin, cleaves and inactivates the human cathelicidin LL-37. Similarly, the omptin CroP, found in the murine pathogen Citrobacter rodentium, which is used as a surrogate model to study human-restricted EHEC, cleaves the murine cathelicidin-related antimicrobial peptide (CRAMP). Here, we compared the abilities of OmpT and CroP to cleave LL-37 and CRAMP. EHEC OmpT degraded LL-37 and CRAMP at similar rates. In contrast, C. rodentium CroP cleaved CRAMP more rapidly than LL-37. The different cleavage rates of LL-37 and CRAMP were independent of the bacterial background and substrate sequence specificity, as OmpT and CroP have the same preference for cleaving at dibasic sites. Importantly, LL-37 was α-helical and CRAMP was unstructured under our experimental conditions. By altering the α-helicity of LL-37 and CRAMP, we found that decreasing LL-37 α-helicity increased its rate of cleavage by CroP. Conversely, increasing CRAMP α-helicity decreased its cleavage rate. This structural basis for CroP substrate specificity highlights differences between the closely related omptins of C. rodentium and E. coli. In agreement with previous studies, this difference in CroP and OmpT substrate specificity suggests that omptins evolved in response to the substrates present in their host microenvironments., Importance: Omptins are recognized as key virulence factors for various Gram-negative pathogens. Their localization to the outer membrane, their active site facing the extracellular environment, and their unique catalytic mechanism make them attractive targets for novel therapeutic strategies. Gaining insights into similarities and variations between the different omptin active sites and subsequent substrate specificities will be critical to develop inhibitors that can target multiple omptins. Here, we describe subtle differences between the substrate specificities of two closely related omptins, CroP and OmpT. This is the first reported example of substrate conformation acting as a structural determinant for omptin activity between OmpT-like proteases., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Bacterial proteases are important virulence factors that inactivate host defense proteins and contribute to tissue destruction and bacterial dissemination. Outer membrane proteases of the omptin family, exemplified by Escherichia coli OmpT, are found in some Gram-negative bacteria. Omptins cleave a variety of substrates at the host-pathogen interface, including plasminogen and antimicrobial peptides. Multiple omptin substrates relevant to infection have been identified; nonetheless, an effective omptin inhibitor remains to be found. Here, we purified native CroP, the OmpT ortholog in the murine pathogen Citrobacter rodentium. Purified CroP was found to readily cleave both a synthetic fluorescence resonance energy transfer substrate and the murine cathelicidin-related antimicrobial peptide. In contrast, CroP was found to poorly activate plasminogen into active plasmin. Although classical protease inhibitors were ineffective against CroP activity, we found that the serine protease inhibitor aprotinin displays inhibitory potency in the micromolar range. Aprotinin was shown to act as a competitive inhibitor of CroP activity and to interfere with the cleavage of the murine cathelicidin-related antimicrobial peptide. Importantly, aprotinin was able to inhibit not only CroP but also Yersinia pestis Pla and, to a lesser extent, E. coli OmpT. We propose a structural model of the aprotinin-omptin complex in which Lys15 of aprotinin forms salt bridges with conserved negatively charged residues of the omptin active site., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Citrobacter rodentium is a murine intestinal pathogen used as a model for the foodborne human pathogens enterohemorrhagic Escherichia coli and enteropathogenic E. coli. During infection, these pathogens use two-component signal transduction systems to detect and adapt to changing environmental conditions. In E. coli, the CpxRA two-component signal transduction system responds to envelope stress by modulating the expression of a myriad of genes. Quantitative real-time PCR showed that cpxRA was expressed in the colon of C57BL/6J mice infected with C. rodentium. To determine whether CpxRA plays a role during C. rodentium infection, a cpxRA deletion strain was generated and found to have a colonization defect during infection. This defect was independent of an altered growth rate or a defective type III secretion system, and single-copy chromosomal complementation of cpxRA restored virulence. The C. rodentium strains were then tested in C3H/HeJ mice, a lethal intestinal infection model. Mice infected with the ΔcpxRA strain survived infection, whereas mice infected with the wild-type or complemented strains succumbed to infection. Furthermore, we found that the cpxRA expression level was higher during early infection than at a later time point. Taken together, these data demonstrate that the CpxRA two-component signal transduction system is essential for the in vivo virulence of C. rodentium. In addition, these data suggest that fine-tuned cpxRA expression is important for infection. This is the first study that identifies a C. rodentium two-component transduction system required for pathogenesis. This study further indicates that CpxRA is an interesting target for therapeutics against enteric pathogens., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Four invariant chain (Ii) isoforms assist the folding and trafficking of human MHC class II (MHCIIs). The main isoforms, Iip33 and Iip35, assemble in the ER into homo- and/or hetero-trimers. The sequential binding of up to three MHCII αβ heterodimers to Ii trimers results in the formation of pentamers, heptamers and nonamers. MHCIIs are required to overcome the p35-encoded di-arginine (RxR) ER retention motif and to allow anterograde trafficking of the complex. Here, we show that inactivation of the RxR motif requires a direct cis interaction between p35 and the MHCII, precluding ER egress of some unsaturated Ii trimers. Interestingly, as opposed to MHCII/p33 complexes, those including p35 remained in the ER when co-expressed with the NleA protein of enterohaemorrhagic Escherichia coli. Taken together, our results demonstrate that p35 influences distinctively MHCII/Ii assembly and trafficking., (Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Enterohemorrhagic Escherichia coli (EHEC) infection from food or water often results in severe diarrheal disease and is a leading cause of death globally. Outer membrane vesicles (OMVs) secreted from E. coli induce lethality in mice. The omptin outer membrane protease OmpT from E. coli inactivates antimicrobial peptides and may enhance colonization of the uroepithelium, but its precise function remains unclear. Given OmpT is an outer membrane protease, we hypothesized it may have a role in OMV biogenesis. To further characterize the effect of OmpT on OMV production, a genetic approach using wild type, an ompT deletion mutant and an ompT overexpressing construct in EHEC were employed. ompT gene deletion markedly decreased OMV production and stainable lipid but increased vesicle diameter. Conversely, ompT overexpression profoundly increased OMV biogenesis but decreased stainable lipid, protein content, and vesicle diameter. Alterations in EHEC ompT gene expression have an impact on the biogenesis, composition, and size of OMVs. Changes in ompT gene expression may dynamically alter OMV formation, composition, and diameter in response to different host environments and contribute to cell-free intercellular communication to enhance bacterial growth and survival., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are food-borne pathogens that colonize the small intestine and colon, respectively. To cause disease, these pathogens must overcome the action of different host antimicrobial peptides (AMPs) secreted into these distinct niches. We have shown previously that EHEC expresses high levels of the OmpT protease to inactivate the human cathelicidin LL-37, an AMP present in the colon. In this study, we investigate the mechanisms used by EPEC to resist human α-defensin 5 (HD-5), the most abundant AMP in the small intestine. Quantitative PCR was used to measure transcript levels of various EPEC surface structures. High transcript levels of gfcA, a gene required for group 4 capsule (G4C) production, were observed in EPEC, but not in EHEC. The unencapsulated EPEC ∆gfcA and EHEC wild-type strains were more susceptible to HD-5 than EPEC wild-type. Since the G4C is composed of the same sugar repeats as the lipopolysaccharide O-antigen, an -antigen ligase (waaL) deletion mutant was generated in EPEC to assess its role in HD-5 resistance. The ∆waaL EPEC strain was more susceptible to HD-5 than both the wild-type and ∆gfcA strains. The ∆gfcA∆waaL EPEC strain was not significantly more susceptible to HD-5 than the ∆waaL strain, suggesting that the absence of -antigen influences G4C formation. To determine whether the G4C and -antigen interact with HD-5, total polysaccharide was purified from wild-type EPEC and added to the ∆gfcA∆waaL strain in the presence of HD-5. The addition of exogenous polysaccharide protected the susceptible strain against HD-5 killing in a dose-dependent manner, suggesting that HD-5 binds to the polysaccharides present on the surface of EPEC. Altogether, these findings indicate that EPEC relies on both the G4C and the -antigen to resist the bactericidal activity of HD-5.

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are bacterial pathogens that cause severe illnesses in humans. Citrobacter rodentium is a related mouse pathogen that serves as a small animal model for EPEC and EHEC infections. EPEC, EHEC and C. rodentium translocate bacterial virulence proteins directly into host intestinal cells via a type III secretion system (T3SS). Non-LEE-encoded effector A (NleA) is a T3SS effector that is common to EPEC, EHEC and C. rodentium. NleA interacts with and inhibits the mammalian COPII complex, impairing cellular secretion; this interaction is required for bacterial virulence. Although diarrhea is a hallmark of EPEC, EHEC and C. rodentium infections, the underlying mechanisms are not well characterized. One of the essential functions of the intestine is to maintain a barrier between the lumen and submucosa. Tight junctions seal the space between adjacent epithelial cells creating this barrier. Consequently, it is thought that the disruption of intestinal epithelial tight junctions by EPEC, EHEC, and C. rodentium could result in a loss of barrier function. In this study, we demonstrate that NleA mediated COPII inhibition is required for EPEC- and C. rodentium-mediated disruption of tight junction proteins and increases in fecal water content., (Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Uropathogenic Escherichia coli (UPEC) strains are among the most prevalent causative agents of urinary tract infections. To establish infection, UPEC must overcome the bactericidal action of host antimicrobial peptides. Previously, the enterohaemorrhagic E. coli outer membrane protease, OmpT, was shown to degrade and inactivate the human antimicrobial peptide LL-37. This study aims to investigate the involvement of UPEC OmpT in LL-37 degradation. An ompT deletion mutant was generated in the prototypical UPEC strain CFT073. Western blot analysis showed that the OmpT protein level is moderate in CFT073. In agreement, OmpT was shown to partially cleave LL-37. However, no difference in the minimum inhibitory concentration of LL-37 was observed between CFT073 and the ompT mutant. Plasmid complementation of ompT, which led to increased OmpT levels, resulted in complete cleavage of LL-37 and a fourfold increase in the minimum inhibitory concentration. The analysis of other UPEC isolates showed similar OmpT activity levels as CFT073. Although UPEC OmpT can cleave LL-37, we conclude that the low level of OmpT limits its contribution to LL-37 resistance. Collectively, these data suggest that UPEC OmpT is likely accompanied by other LL-37 resistance mechanisms., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

This study identified and characterized enteropathogenic Escherichia coli (EPEC) in the Canadian food supply. Eighteen of 450 E. coli isolates from food animal sources were identified as atypical EPEC (aEPEC). Several of the aEPEC isolates identified in this study possessed multiple virulence genes, exhibited adherence and attaching and effacing (A/E) lesion formation, disrupted tight junctions, and were coclassified with the extraintestinal pathogenic E. coli (ExPEC) and enterotoxigenic E. coli (ETEC) pathotypes.

Citrobacter rodentium is a natural mouse pathogen widely used as a model for enteropathogenic and enterohemorrhagic Escherichia coli infections in humans. While C. rodentium causes self-limiting colitis in most inbred mouse strains, it induces fatal diarrhoea in susceptible strains. The physiological pathways as well as the genetic determinants leading to susceptibility have remained largely uncharacterized. Here we use a forward genetic approach to identify the R-spondin2 gene as a major determinant of susceptibility to C. rodentium infection. Robust induction of R-spondin2 expression during infection in susceptible mouse strains causes a potent Wnt-mediated proliferative response of colonic crypt cells, leading to the generation of an immature and poorly differentiated colonic epithelium with deficiencies in ion-transport components. Our data demonstrate a previously unknown role of R-spondins and Wnt signalling in susceptibility to infectious diarrhoea and identify R-spondin2 as a key molecular link between infection and intestinal homoeostasis.

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) are enteric human pathogens that colonize the large and small intestines, respectively. To establish infection EHEC and EPEC must overcome innate host defenses, such as antimicrobial peptides (AMPs) produced by the intestinal epithelium. Gram-negative pathogens have evolved different mechanisms to resist AMPs, including outer-membrane proteases that degrade AMPs. We showed that the protease OmpT degrades the human AMP LL-37 more rapidly in EHEC than in EPEC. Promoter-swap experiments showed that this is due to differences in the promoters of the two genes, leading to greater ompT expression and subsequently greater levels of OmpT in EHEC. Here, we propose that the different ompT expression in EHEC and EPEC reflects the varying levels of LL-37 throughout the human intestinal tract. These data suggest that EHEC and EPEC adapted to their specific niches by developing distinct AMP-specific resistance mechanisms.

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) are food-borne pathogens that cause severe diarrhoeal disease in humans. Citrobacter rodentium is a related mouse pathogen that serves as a small animal model for EPEC and EHEC infections. EPEC, EHEC and C. rodentium translocate bacterial virulence proteins directly into host cells via a type III secretion system (T3SS). Non-LEE-encoded effector A (NleA) is a T3SS effector that is common to EPEC, EHEC and C. rodentium and is required for bacterial virulence. NleA localizes to the host cell secretory pathway and inhibits vesicle trafficking by interacting with the Sec24 subunit of mammalian coatamer protein II complex (COPII). Mammalian cells express four paralogues of Sec24 (Sec24A-D), which mediate selection of cargo proteins for transport and possess distinct, but overlapping cargo specificities. Here, we show that NleA binds Sec24A-D with two distinct mechanisms. An NleA protein variant with greatly diminished interaction with all Sec24 paralogues does not properly localize, does not inhibit COPII-mediated vesicle budding, and does not confer virulence in the mouse infection model. Together, this work provides strong evidence that the interaction and inhibition of COPII by NleA is an important aspect of EPEC- and EHEC-mediated disease., (© 2012 Blackwell Publishing Ltd.)

Antimicrobial peptides (AMPs) are present in virtually all organisms and are an ancient and critical component of innate immunity. In mammals, AMPs are present in phagocytic cells, on body surfaces such as skin and mucosa, and in secretions and body fluids such as sweat, saliva, urine, and breast milk, consistent with their role as part of the first line of defense against a wide range of pathogenic microorganisms including bacteria, viruses, and fungi. AMPs are microbicidal and have also been shown to act as immunomodulators with chemoattractant and signaling activities. During the co-evolution of hosts and bacterial pathogens, bacteria have developed the ability to sense and initiate an adaptive response to AMPs to resist their bactericidal activity. Here, we review the various mechanisms used by Gram-negative bacteria to sense and resist AMP-mediated killing. These mechanisms play an important role in bacterial resistance to host-derived AMPs that are encountered during the course of infection. Bacterial resistance to AMPs should also be taken into consideration in the development and use of AMPs as anti-infective agents, for which there is currently a great deal of academic and commercial interest., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are food-borne pathogens that cause serious diarrheal diseases. To colonize the human intestine, these pathogens must overcome innate immune defenses such as antimicrobial peptides (AMPs). Bacterial pathogens have evolved various mechanisms to resist killing by AMPs, including proteolytic degradation of AMPs. To examine the ability of the EHEC and EPEC OmpT outer membrane (OM) proteases to degrade α-helical AMPs, ompT deletion mutants were generated. Determination of MICs of various AMPs revealed that both mutant strains are more susceptible than their wild-type counterparts to α-helical AMPs, although to different extents. Time course assays monitoring the degradation of LL-37 and C18G showed that EHEC cells degraded both AMPs faster than EPEC cells in an OmpT-dependent manner. Mass spectrometry analyses of proteolytic fragments showed that EHEC OmpT cleaves LL-37 at dibasic sites. The superior protection provided by EHEC OmpT compared to EPEC OmpT against α-helical AMPs was due to higher expression of the ompT gene and, in turn, higher levels of the OmpT protein in EHEC. Fusion of the EPEC ompT promoter to the EHEC ompT open reading frame resulted in decreased OmpT expression, indicating that transcriptional regulation of ompT is different in EHEC and EPEC. We hypothesize that the different contributions of EHEC and EPEC OmpT to the degradation and inactivation of LL-37 may be due to their adaptation to their respective niches within the host, the colon and small intestine, respectively, where the environmental cues and abundance of AMPs are different.

Cerebral malaria (CM) is a lethal neurological complication of malaria. We implemented a genome-wide screen in mutagenized mice to identify host proteins involved in CM pathogenesis and whose inhibition may be of therapeutic value. One pedigree (P48) segregated a resistance trait whose CM-protective effect was fully penetrant, mapped to chromosome 8, and identified by genome sequencing as homozygosity for a mis-sense mutation (W81R) in the FERM domain of Janus-associated kinase 3 (Jak3). The causative effect of Jak3(W81R) was verified by complementation testing in Jak3(W81R/-) double heterozygotes that were fully protected against CM. Jak3(W81R) homozygotes showed defects in thymic development with depletion of CD8(+) T cell, B cell, and NK cell compartments, and defective T cell-dependent production of IFN-γ. Adoptive transfer of normal splenocytes abrogates CM resistance in Jak3(W81R) homozygotes, an effect attributed to the CD8(+) T cells. Jak3(W81R) behaves as a dominant negative variant, with significant CM resistance of Jak3(W81R/+) heterozygotes, compared to CM-susceptible Jak3(+/+) and Jak3(+/-) controls. CM resistance in Jak3(W81R/+) heterozygotes occurs in presence of normal T, B and NK cell numbers. These findings highlight the pathological role of CD8(+) T cells and Jak3-dependent IFN-γ-mediated Th1 responses in CM pathogenesis.

Citrobacter rodentium is a natural pathogen of mice that causes intestinal hyperplasia and colitis. Resistant strains such as C57BL/6J (B6) experience a self-limiting disease that peaks between one and two weeks post infection, followed by a clearing of the infection and complete recovery. However, the inbred mouse strains C3H/HeJ (C3), C3H/HeOuJ (C3Ou) and FVB/N (FVB) are highly susceptible to C. rodentium infection and develop more severe symptoms of disease leading to high rates of mortality during infection. We have recently demonstrated through a systematic genetics approach that a single locus on proximal chromosome 15 is responsible for the susceptibility of both C3 and C3Ou mice to C. rodentium infection. We have named the locus Citrobacter rodentium infection 1 (Cri1). Here we show that Cri1 also controls susceptibility to C. rodentium in FVB mice, using a targeted method of genotyping to stratify (B6 x FVB)F2 mice according to their genotype at Cri1. Mice that inherit two copies of the resistant B6 allele have 97% cumulative survival at day 30 post-infection, whereas those that inherit one or two copies of Cri1 from the FVB parent have significantly lower rates of survival (35% and 42%, respectively). These results provide evidence for a common genetic cause of fatal infectious colitis in C3, C3Ou and FVB mice following infection with Citrobacter rodentium.

Autophagy mediates the degradation of cytoplasmic components in eukaryotic cells and plays a key role in immunity. The mechanism of autophagosome formation is not clear. Here we examined two potential membrane sources for antibacterial autophagy: the ER and mitochondria. DFCP1, a marker of specialized ER domains known as 'omegasomes,' associated with Salmonella-containing autophagosomes via its PtdIns(3)P and ER-binding domains, while a mitochondrial marker (cytochrome b5-GFP) did not. Rab1 also localized to autophagosomes, and its activity was required for autophagosome formation, clearance of protein aggregates and peroxisomes, and autophagy of Salmonella. Overexpression of Rab1 enhanced antibacterial autophagy. The role of Rab1 in antibacterial autophagy was independent of its role in ER-to-Golgi transport. Our data suggest that antibacterial autophagy occurs at omegasomes and reveal that the Rab1 GTPase plays a crucial role in mammalian autophagy.

Cranberry-derived compounds, including a fraction known as proanthocyanidins (PACs) exhibit anti-microbial, anti-infective, and anti-adhesive properties against a number of disease-causing organisms. In this study, the effect of cranberry proanthocyanidins (CPACs) on the infection of epithelial cells by two enteric bacterial pathogens, enteropathogenic Escherichia coli (EPEC) and Salmonella Typhimurium was investigated. Immunofluorescence data showed that actin pedestal formation, required for infection by enteropathogenic Escherichia coli (EPEC), was disrupted in the presence of CPACs. In addition, invasion of HeLa cells by Salmonella Typhimurium was significantly reduced, as verified by gentamicin protection assay and immunofluorescence. CPACs had no effect on bacterial growth, nor any detectable effect on the production of bacterial effector proteins of the type III secretion system. Furthermore, CPACs did not affect the viability of host cells. Interestingly, we found that CPACs had a potent and dose-dependent effect on the host cell cytoskeleton that was evident even in uninfected cells. CPACs inhibited the phagocytosis of inert particles by a macrophage cell line, providing further evidence that actin-mediated host cell functions are disrupted in the presence of cranberry CPACs. Thus, although CPAC treatment inhibited Salmonella invasion and EPEC pedestal formation, our results suggest that this is likely primarily because of the perturbation of the host cell cytoskeleton by CPACs rather than an effect on bacterial virulence itself. These findings have significant implications for the interpretation of experiments on the effects of CPACs on bacteria-host cell interactions.

Enteropathogenic Escherichia coli (EPEC) is an enteric pathogen able to cause severe diarrhea. Once adhered to the small intestine, EPEC disrupts tight junctions that are important for intestinal barrier function. This disruption is dependent on the bacterial type III secretion system, as well as the translocated effectors EspF and Map. Recently we have shown that a third type III translocated bacterial effector protein, NleA, is also involved in tight junction disruption during EPEC infection. NleA has a predicted PDZ-binding domain at its C-terminus which is proposed to be involved in protein interactions with PDZ domain containing proteins. Since several PDZ-domain-containing proteins localize to tight junctions, we hypothesized that the PDZ-binding domain of NleA might be important for its role in tight junction disruption. However, here we show that a molecular variant of NleA lacking the PDZ-binding domain behaves indistinguishably from the wild-type protein with respect to disruption of tight junctions.

Enteropathogenic Escherichia coli (EPEC) is a diarrhoeal pathogen that adheres to epithelial cells of the small intestine and uses a type III secretion system to inject effector proteins into host cells. EPEC infection leads to disruption of host intestinal tight junctions that are important for maintaining intestinal barrier function. This disruption is dependent on the bacterial type III secretion system, as well as the translocated effectors EspF and Map. Here we show that a third type III translocated bacterial effector protein, NleA, is also involved in tight junction disruption during EPEC infection. Using the drug Brefeldin A, we demonstrate that the effect of NleA on tight junction integrity is related to its inhibition of host cell protein trafficking through COPII-dependent pathways. These results suggest that NleA's striking effect on virulence is mediated, at least in part, via its role in disruption of intestinal barrier function.

The PhoPQ two-component system of the intracellular pathogen Salmonella enterica senses and controls resistance to alpha-helical antimicrobial peptides (AMPs) by regulating covalent modifications of lipid A. A homologue of the phoPQ operon was found in the genome of the murine enteric extracellular pathogen, Citrobacter rodentium. Here we report that C. rodentium PhoPQ was apparently unable to mediate activation of target genes in the presence of alpha-helical AMPs. However, these AMPs activated C. rodentium PhoPQ expressed in a S. entericaDeltaphoPQ mutant. Analysis of the outer membrane (OM) fractions of the C. rodentium wild-type and DeltaphoPQ strains led to the identification of an omptin family protease (CroP) that was absent in DeltaphoPQ. Deletion of croP in C. rodentium resulted in higher susceptibility to alpha-helical AMPs, indicating a direct role of CroP in AMP resistance. CroP greatly contributed to the protection of the OM from AMP damage by actively degrading alpha-helical AMPs before they reach the periplasmic space. Accordingly, transcriptional activation of PhoP-regulated genes by alpha-helical AMPs was restored in the DeltacroP mutant. This study shows that resistance to alpha-helical AMPs by the extracellular pathogen C. rodentium relies primarily on the CroP OM protease.

Objective: We investigated mechanisms involved in iron (Fe) transport by DMT1 (endosomal Fe(II) exporter, encoded by the Nramp2 gene) using wild-type Chinese hamster ovary (CHO) cells and Nramp2-transfected CHO cells, as well as reticulocytes from normal and mk/mk mice that have a defect in DMT1., Materials and Methods: CHO cells and reticulocytes were incubated with 59Fe bound to various ligands. The radioiron was present in its Fe(II) or Fe(III) forms or bound to transferrin (Tf), and the internalized 59Fe measured under varying experimental conditions. Additionally, 125I-Tf interaction with reticulocytes was investigated and 59Fe incorporation into their heme was determined., Results: Hyperexpression of DMT1 in CHO cells greatly increases their capacity to acquire ferrous iron. Although CHO-Nramp2 cells showed an increase in Fe(III) uptake as compared to CHO cells, they transported Fe(III) with much lower efficacy than Fe(II). In addition to their defect in Fe uptake, mk/mk reticulocytes also showed a decrease in Tf receptor levels., Conclusions: Given that CHO cells acquire iron from Fe(II)-ascorbate with much higher rates than from Fe(III)-Tf, Tf-receptor levels represent the rate-limiting step in their iron uptake. As Fe(III) transport by CHO-Nramp2 cells can be inhibited by the impermeable oxidant K3Fe(CN)6, a membrane ferric reductase is probably needed for reduction of Fe(III) to Fe(II), which is then transported by DMT1. DMT1 is not a limiting factor in Fe acquisition by normal reticulocytes and their heme synthesis.

Shiga toxin-producing Escherichia coli (STEC) strains are commensal bacteria in cattle with high potential for environmental and zoonotic transmission to humans. Although O157:H7 is the most common STEC serotype, there is growing concern over the emergence of more than 200 highly virulent non-O157 STEC serotypes that are globally distributed, several of which are associated with outbreaks and/or severe human illness such as hemolytic-uremic syndrome (HUS) and hemorrhagic colitis. At present, the underlying genetic basis of virulence potential in non-O157 STEC is unknown, although horizontal gene transfer and the acquisition of new pathogenicity islands are an expected origin. We used seropathotype classification as a framework to identify genetic elements that distinguish non-O157 STEC strains posing a serious risk to humans from STEC strains that are not associated with severe and epidemic disease. We report the identification of three genomic islands encoding non-LEE effector (nle) genes and 14 individual nle genes in non-O157 STEC strains that correlate independently with outbreak and HUS potential in humans. The implications for transmissible zoonotic spread and public health are discussed. These results and methods offer a molecular risk assessment strategy to rapidly recognize and respond to non-O157 STEC strains from environmental and animal sources that might pose serious public health risks to humans.

Enterohemorrhagic Escherichia coli (EHEC) is a water- and food-borne pathogen that causes hemorrhagic colitis. EHEC uses a type III secretion system (T3SS) to translocate effector proteins that subvert host cell function. T3SS-substrates encoded outside of the locus of enterocyte effacement are important to E. coli pathogenesis. We discovered an EHEC secreted protein, NleF, encoded by z6020 in O-island 71 of E. coli EDL933 that we hypothesized to be a T3SS substrate. Experiments are presented that probe the function of NleF and its role in virulence. Immunoblotting of secreted and translocated proteins suggest that NleF is secreted by the T3SS and is translocated into host cells in vitro where it localizes to the host cytoplasm. Infection of HeLa cells with E. coli possessing or lacking nleF and transient expression of NleF-GFP via transfection did not reveal a significant role for NleF in several assays of bacterial adherence, host cytoskeletal remodeling, or host protein secretion. However, competitive coinfection of mice with Citrobacter rodentium strains possessing or lacking nleF suggested a contribution of NleF to bacterial colonization. Challenge of gnotobiotic piglets also revealed a role for NleF in colonization of the piglet colon and rectoanal junction.