Enterotoxigenic Escherichia coli Degrades the Host MUC2 Mucin Barrier To Facilitate Critical Pathogen-Enterocyte Interactions in Human Small Intestine.

Enterotoxigenic Escherichia coli (ETEC) isolates are genetically diverse pathological variants of E. coli defined by the production of heat-labile (LT) and/or heat-stable (ST) toxins. ETEC strains are estimated to cause hundreds of millions of cases of diarrheal illness annually. However, it is not clear that all strains are equally equipped to cause disease, and asymptomatic colonization with ETEC is common in low- to middle-income regions lacking basic sanitation and clean water where ETEC are ubiquitous. Recent molecular epidemiology studies have revealed a significant association between strains that produce EatA, a secreted autotransporter protein, and the development of symptomatic infection. Here, we demonstrate that LT stimulates production of MUC2 mucin by goblet cells in human small intestine, enhancing the protective barrier between pathogens and enterocytes. In contrast, using explants of human small intestine as well as small intestinal enteroids, we show that EatA counters this host defense by engaging and degrading the MUC2 mucin barrier to promote bacterial access to target enterocytes and ultimately toxin delivery, suggesting that EatA plays a crucial role in the molecular pathogenesis of ETEC. These findings may inform novel approaches to prevention of acute diarrheal illness as well as the sequelae associated with ETEC and other pathogens that rely on EatA and similar proteases for efficient interaction with their human hosts.

Host-mediated sugar oxidation promotes post-antibiotic pathogen expansion.

Changes in the gut microbiota may underpin many human diseases, but the mechanisms that are responsible for altering microbial communities remain poorly understood. Antibiotic usage elevates the risk of contracting gastroenteritis caused by Salmonella enterica serovars, increases the duration for which patients shed the pathogen in their faeces, and may on occasion produce a bacteriologic and symptomatic relapse. These antibiotic-induced changes in the gut microbiota can be studied in mice, in which the disruption of a balanced microbial community by treatment with the antibiotic streptomycin leads to an expansion of S. enterica serovars in the large bowel. However, the mechanisms by which streptomycin treatment drives an expansion of S. enterica serovars are not fully resolved. Here we show that host-mediated oxidation of galactose and glucose promotes post-antibiotic expansion of S. enterica serovar Typhimurium (S. Typhimurium). By elevating expression of the gene encoding inducible nitric oxide synthase (iNOS) in the caecal mucosa, streptomycin treatment increased post-antibiotic availability of the oxidation products galactarate and glucarate in the murine caecum. S. Typhimurium used galactarate and glucarate within the gut lumen of streptomycin pre-treated mice, and genetic ablation of the respective catabolic pathways reduced S. Typhimurium competitiveness. Our results identify host-mediated oxidation of carbohydrates in the gut as a mechanism for post-antibiotic pathogen expansion.

The Vi capsular polysaccharide enables Salmonella enterica serovar typhi to evade microbe-guided neutrophil chemotaxis.

Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a disseminated infection, while the closely related pathogen S. enterica serovar Typhimurium (S. Typhimurium) is associated with a localized gastroenteritis in humans. Here we investigated whether both pathogens differ in the chemotactic response they induce in neutrophils using a single-cell experimental approach. Surprisingly, neutrophils extended chemotactic pseudopodia toward Escherichia coli and S. Typhimurium, but not toward S. Typhi. Bacterial-guided chemotaxis was dependent on the presence of complement component 5a (C5a) and C5a receptor (C5aR). Deletion of S. Typhi capsule biosynthesis genes markedly enhanced the chemotactic response of neutrophils in vitro. Furthermore, deletion of capsule biosynthesis genes heightened the association of S. Typhi with neutrophils in vivo through a C5aR-dependent mechanism. Collectively, these data suggest that expression of the virulence-associated (Vi) capsular polysaccharide of S. Typhi obstructs bacterial-guided neutrophil chemotaxis.

Neutrophils are a source of gamma interferon during acute Salmonella enterica serovar Typhimurium colitis.

Gamma interferon (IFN-γ) is an important driver of intestinal inflammation during colitis caused by Salmonella enterica serovar Typhimurium. Here we used the mouse colitis model to investigate the cellular sources of IFN-γ in the cecal mucosa during the acute phase of an S. Typhimurium infection. While IFN-γ staining was detected in T cells, NK cells, and inflammatory monocytes at 2 days after infection, the majority of IFN-γ-positive cells in the cecal mucosa were neutrophils. Furthermore, neutrophil depletion blunted mucosal Ifng expression and reduced the severity of intestinal lesions during S. Typhimurium infection. We conclude that neutrophils are a prominent cellular source of IFN-γ during the innate phase of S. Typhimurium-induced colitis.

Airway epithelial MyD88 restores control of Pseudomonas aeruginosa murine infection via an IL-1-dependent pathway.

The opportunistic human pathogen Pseudomonas aeruginosa causes rapidly progressive and tissue-destructive infections, such as hospital-acquired and ventilator-associated pneumonias. Innate immune responses are critical in controlling P. aeruginosa in the mammalian lung, as demonstrated by the increased susceptibility of MyD88(-/-) mice to this pathogen. Experiments conducted using bone marrow chimeric mice demonstrated that radio-resistant cells participated in initiating MyD88-dependent innate immune responses to P. aeruginosa. In this study we used a novel transgenic mouse model to demonstrate that MyD88 expression by epithelial cells is sufficient to generate a rapid and protective innate immune response following intranasal infection with P. aeruginosa. MyD88 functions as an adaptor for many TLRs. However, mice in which multiple TLR pathways (e.g., TLR2/TLR4/TLR5) are blocked are not as compromised in their response to P. aeruginosa as mice lacking MyD88. We demonstrate that IL-1R signaling is an essential element of MyD88-dependent epithelial cell responses to P. aeruginosa infection.

Identification of collaborative cross mouse strains permissive to Salmonella enterica serovar Typhi infection.

Salmonella enterica serovar Typhi is the causative agent of typhoid fever restricted to humans and does not replicate in commonly used inbred mice. Genetic variation in humans is far greater and more complex than that in a single inbred strain of mice. The Collaborative Cross (CC) is a large panel of recombinant inbred strains which has a wider range of genetic diversity than laboratory inbred mouse strains. We found that the CC003/Unc and CC053/Unc strains are permissive to intraperitoneal but not oral route of S. Typhi infection and show histopathological changes characteristic of human typhoid. These CC strains are immunocompetent, and immunization induces antigen-specific responses that can kill S. Typhi in vitro and control S. Typhi in vivo. Our results indicate that CC003/Unc and CC053/Unc strains can help identify the genetic basis for typhoid susceptibility, S. Typhi virulence mechanism(s) in vivo, and serve as a preclinical mammalian model system to identify effective vaccines and therapeutics strategies.

NTRC and chloroplast-generated reactive oxygen species regulate Pseudomonas syringae pv. tomato disease development in tomato and Arabidopsis.

Coronatine (COR)-producing pathovars of Pseudomonas syringae, including pvs. tomato, maculicola, and glycinea, cause important diseases on tomato, crucifers, and soybean, respectively, and produce symptoms with necrotic lesions surrounded by chlorosis. The chlorosis is mainly attributed to COR. However, the significance of COR-induced chlorosis in localized lesion development and the molecular basis of disease-associated cell death is largely unknown. To identify host (chloroplast) genes that play a role in COR-mediated chlorosis, we used a forward genetics approach using Nicotiana benthamiana and virus-induced gene silencing and identified a gene which encodes 2-Cys peroxiredoxin (Prxs) that, when silenced, produced a spreading hypersensitive or necrosis-like phenotype instead of chlorosis after COR application in a COI1-dependent manner. Loss-of-function analysis of Prx and NADPH-dependent thioredoxin reductase C (NTRC), the central players of a chloroplast redox detoxification system, resulted in spreading accelerated P. syringae pv. tomato DC3000 disease-associated cell death with enhanced reactive oxygen species (ROS) accumulation in a COR-dependent manner in tomato and Arabidopsis. Consistent with these results, virulent strain DC3000 suppressed the expression of Prx and NTRC in Arabidopsis and tomato during pathogenesis. However, interestingly, authentic COR suppressed the expression of Prx and NTRC in tomato but not in Arabidopsis, suggesting that COR in conjunction with other effectors may modulate ROS and cell death in different host species. Taken together, these results indicated that NTRC or Prx function as a negative regulator of pathogen-induced cell death in the healthy tissues that surround the lesions, and COR-induced chloroplast-localized ROS play a role in enhancing the disease-associated cell death.

Virulence factors perforate the pathogen-containing vacuole to signal efferocytosis.

Intracellular pathogens commonly reside within macrophages to find shelter from humoral defenses, but host cell death can expose them to the extracellular milieu. We find intracellular pathogens solve this dilemma by using virulence factors to generate a complement-dependent find-me signal that initiates uptake by a new phagocyte through efferocytosis. During macrophage death, Salmonella uses a type III secretion system to perforate the membrane of the pathogen-containing vacuole (PCV), thereby triggering complement deposition on bacteria entrapped in pore-induced intracellular traps (PITs). In turn, complement activation signals neutrophil efferocytosis, a process that shelters intracellular bacteria from the respiratory burst. Similarly, Brucella employs its type IV secretion system to perforate the PCV membrane, which induces complement deposition on bacteria entrapped in PITs. Collectively, this work identifies virulence factor-induced perforation of the PCV as a strategy of intracellular pathogens to generate a find-me signal for efferocytosis.

A virus-induced gene silencing screen identifies a role for Thylakoid Formation1 in Pseudomonas syringae pv tomato symptom development in tomato and Arabidopsis.

Pseudomonas syringae pv tomato DC3000 (Pst DC3000), which causes disease in tomato (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana), produces coronatine (COR), a non-host-specific phytotoxin. COR, which functions as a jasmonate mimic, is required for full virulence of Pst DC3000 and for the induction of chlorosis in host plants. Previous genetic screens based on insensitivity to COR and/or methyl jasmonate identified several potential targets for COR and methyl jasmonate. In this study, we utilized Nicotiana benthamiana and virus-induced gene silencing to individually reduce the expression of over 4,000 genes. The silenced lines of N. benthamiana were then screened for altered responses to purified COR. Using this forward genetics approach, several genes were identified with altered responses to COR. These were designated as ALC (for altered COR response) genes. When silenced, one of the identified genes, ALC1, produced a hypersensitive/necrosis-like phenotype upon COR application in a Coronatine-Insensitive1 (COI1)-dependent manner. To understand the involvement of ALC1 during the Pst DC3000-host interaction, we used the nucleotide sequence of ALC1 and identified its ortholog in Arabidopsis (Thylakoid Formation1 [THF1]) and tomato (SlALC1). In pathogenicity assays performed on Arabidopsis thf1 mutant and SlALC1-silenced tomato plants, Pst DC3000 induced accelerated coalescing necrotic lesions. Furthermore, we showed that COR affects ALC1 localization in chloroplasts in a COI1-dependent manner. In conclusion, our results show that the virus-induced gene silencing-based forward genetic screen has the potential to identify new players in COR signaling and disease-associated necrotic cell death.

In vivo discrimination of type 3 secretion system-positive and -negative Pseudomonas aeruginosa via a caspase-1-dependent pathway.

Microbe-associated molecular patterns are recognized by Toll-like receptors of the innate immune system. This recognition enables a rapid response to potential pathogens but does not clearly provide a way for the innate immune system to discriminate between virulent and avirulent microbes. We find that pulmonary infection of mice with type 3 translocation-competent Pseudomonas aeruginosa triggers a rapid inflammatory response, while infection with isogenic translocation-deficient mutants does not. Discrimination between translocon-positive and -negative bacteria requires caspase-1 activity in bone marrow-derived cells and interleukin-1 receptor signaling. Thus, the activation of caspase-1 by bacteria expressing type 3 secretion systems allows for rapid recognition of bacteria expressing conserved functions associated with virulence.

Pathogenicity of Pseudomonas syringae pv. tomato on tomato seedlings: phenotypic and gene expression analyses of the virulence function of coronatine.

Bacterial speck disease, which is caused by Pseudomonas syringae pv. tomato, is an economically important disease on tomato. In the present study, we show that P. syringae pv. tomato DC3000 is a pathogen of tomato seedlings, an aspect of pathogen biology that has not been previously investigated. This resulted in the development of a virulence assay on tomato seedlings that has several advantages over labor-intensive foliar assays, including a shorter growth and incubation period, ease of inoculation and handling, and rapid generation of larger sample sizes per experiment. The utility of this assay was investigated by exploring the virulence function of coronatine (COR) on tomato seedlings. Using the COR- mutant DB29 and a MAPMAN display of transcript data from TOM1 microarrays, COR-dependent expression of genes involved in secondary metabolism, polyamine biosynthesis, reactive oxygen species homeostasis, and the novel transcription factor SlNAC2 were identified. Furthermore, during pathogenesis, genes involved in photosynthetic light reactions and the Calvin-Benson cycle were strongly repressed by COR. In conclusion, we show that P. syringae pv. tomato infects tomato seedlings and that COR is required for virulence in seedlings. The seedling assay can be used in high-throughput screens for the identification of molecular targets for COR and for the identification of genes involved in pathogenesis.

Mechanisms to Evade the Phagocyte Respiratory Burst Arose by Convergent Evolution in Typhoidal Salmonella Serovars.

Typhoid fever caused by Salmonella enterica serovar (S.) Typhi differs in its clinical presentation from gastroenteritis caused by S. Typhimurium and other non-typhoidal Salmonella serovars. The different clinical presentations are attributed in part to the virulence-associated capsular polysaccharide (Vi antigen) of S. Typhi, which prevents phagocytes from triggering a respiratory burst by preventing antibody-mediated complement activation. Paradoxically, the Vi antigen is absent from S. Paratyphi A, which causes a disease that is indistinguishable from typhoid fever. Here, we show that evasion of the phagocyte respiratory burst by S. Paratyphi A required very long O antigen chains containing the O2 antigen to inhibit antibody binding. We conclude that the ability to avoid the phagocyte respiratory burst is a property distinguishing typhoidal from non-typhoidal Salmonella serovars that was acquired by S. Typhi and S. Paratyphi A independently through convergent evolution.

The phytotoxin coronatine contributes to pathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringae pv. tomato DC3000.

The roles of the phytotoxin coronatine (COR) and salicylic acid (SA)-mediated defenses in the interaction of Pseudomonas syringae pv. tomato DC3000 and tomato (Solanum lycopersicum) were investigated. Unlike findings reported for Arabidopsis thaliana, DC3000 mutants impaired for production of COR or one of its components, coronafacic acid (CFA) or coronamic acid (CMA), induced distinctly different disease lesion phenotypes in tomato. Tomato plants inoculated with the CFA- CMA- mutant DB29 showed elevated transcript levels of SlICS, which encodes isochorismate synthase, an enzyme involved in SA biosynthesis in S. lycopersicum. Furthermore, expression of genes encoding SA-mediated defense proteins were elevated in DB29-inoculated plants compared with plants inoculated with DC3000, suggesting that COR suppresses SlICS-mediated SA responses. Sequence analysis of SlICS revealed that it encodes a protein that is 55 and 59.6% identical to the A. thaliana ICS-encoded proteins AtICS1 and AtICS2, respectively. Tomato plants silenced for SlICS were hypersusceptible to DC3000 and accumulated lower levels of SA after infection with DC3000 compared with inoculated wild-type tomato plants. Unlike what has been shown for A. thaliana, the COR- mutant DB29 was impaired for persistence in SlICS-silenced tomato plants; thus, COR has additional roles in virulence that are SA independent and important in the latter stages of disease development. In summary, the infection assays, metabolic profiling, and gene expression results described in this study indicate that the intact COR molecule is required for both suppression of SA-mediated defense responses and full disease symptom development in tomato.

Loss of very-long O-antigen chains optimizes capsule-mediated immune evasion by Salmonella enterica serovar Typhi.

UNLABELLED: Expression of capsular polysaccharides is a variable trait often associated with more-virulent forms of a bacterial species. For example, typhoid fever is caused by the capsulated Salmonella enterica serovar Typhi, while nontyphoidal Salmonella serovars associated with gastroenteritis are noncapsulated. Here we show that optimization of the immune evasive properties conferred by the virulence-associated (Vi) capsular polysaccharide involved an additional alteration to the cell envelope of S. Typhi, namely inactivation of the fepE gene, encoding the regulator of very-long O-antigen chains. Introduction of the capsule-encoding viaB locus into the nontyphoidal S. enterica serovar Typhimurium reduced complement deposition in vitro and intestinal inflammation in a mouse colitis model. However, both phenotypes were markedly enhanced when the viaB locus was introduced into an S. Typhimurium fepE mutant, which lacks very-long O-antigen chains. Collectively, these data suggest that during the evolution of the S. Typhi lineage, loss of very-long O-antigen chains by pseudogene formation was an adaptation to maximize the anti-inflammatory properties of the Vi capsular polysaccharide. IMPORTANCE: Genomic comparison illustrates that acquisition of virulence factors by horizontal gene transfer is an important contributor to the evolution of enteric pathogens. Acquisition of complex virulence traits commonly involves horizontal transfer of a large gene cluster, and integration of the gene cluster into the host genome results in the formation of a pathogenicity island. Acquisition of the virulence-associated (Vi) capsular polysaccharide encoded by SPI7 (Salmonella pathogenicity island 7) was accompanied in the human-adapted Salmonella enterica serovar Typhi by inactivation of the fepE gene, encoding the regulator of very-long O-antigen chains. We show that the resulting loss of very-long O-antigen chains was an important mechanism for maximizing immune evasion mediated by the Vi capsular polysaccharide. These data suggest that successful incorporation of a capsular polysaccharide requires changes in the cell envelope of the hosting pathogen.

Streptomycin-induced inflammation enhances Escherichia coli gut colonization through nitrate respiration.

UNLABELLED: Treatment with streptomycin enhances the growth of human commensal Escherichia coli isolates in the mouse intestine, suggesting that the resident microbial community (microbiota) can inhibit the growth of invading microbes, a phenomenon known as "colonization resistance." However, the precise mechanisms by which streptomycin treatment lowers colonization resistance remain obscure. Here we show that streptomycin treatment rendered mice more susceptible to the development of chemically induced colitis, raising the possibility that the antibiotic might lower colonization resistance by changing mucosal immune responses rather than by preventing microbe-microbe interactions. Investigation of the underlying mechanism revealed a mild inflammatory infiltrate in the cecal mucosa of streptomycin-treated mice, which was accompanied by elevated expression of Nos2, the gene that encodes inducible nitric oxide synthase. In turn, this inflammatory response enhanced the luminal growth of E. coli by nitrate respiration in a Nos2-dependent fashion. These data identify low-level intestinal inflammation as one of the factors responsible for the loss of resistance to E. coli colonization after streptomycin treatment. IMPORTANCE: Our intestine is host to a complex microbial community that confers benefits by educating the immune system and providing niche protection. Perturbation of intestinal communities by streptomycin treatment lowers "colonization resistance" through unknown mechanisms. Here we show that streptomycin increases the inflammatory tone of the intestinal mucosa, thereby making the bowel more susceptible to dextran sulfate sodium treatment and boosting the Nos2-dependent growth of commensal Escherichia coli by nitrate respiration. These data point to the generation of alternative electron acceptors as a by-product of the inflammatory host response as an important factor responsible for lowering resistance to colonization by facultative anaerobic bacteria such as E. coli.

Typhoid fever: "you can't hit what you can't see".

The host restricts dissemination of invasive enteric pathogens, such as non-typhoidal Salmonella serovars, by mounting acute inflammatory responses characterized by the recruitment of neutrophils. However, some enteric pathogens, such as Salmonella enterica serovar Typhi (S. typhi), can bypass these defenses and cause an invasive bloodstream infection known as typhoid fever. Recent studies on virulence mechanisms of S. typhi suggest that tight regulation of virulence gene expression during the transition from the intestinal lumen into the intestinal mucosa enables this pathogen to evade detection by the innate immune system, thereby penetrating defenses that prevent bacterial dissemination. This example illustrates how the outcome of host pathogen interaction at the intestinal mucosal interface can alter the clinical presentation and dictate the disease outcome.

Innate immune responses to Pseudomonas aeruginosa infection.

Innate immune responses play a critical role in controlling acute infections due to Pseudomonas aeruginosa in both mice and in humans. In this review we focus on innate immune recognition and clearance mechanisms that are important for controlling P. aeruginosa in the mammalian lung, with particular attention to those that influence the outcome of in vivo infection in murine models.

A role for chloroplast-localized Thylakoid Formation 1 (THF1) in bacterial speck disease development.

Coronatine (COR), a jasmonate mimic produced by Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is required for full virulence of Pst DC3000 in tomato and Arabidopsis. COR is shown to induce a range of physiological processes including chlorosis, root growth inhibition and anthocyanin accumulation in tomato. To elucidate the host/signaling genes involved in COR-responses, we utilized a forward genetics approach using Nicotiana benthamiana and virus-induced gene silencing (VIGS) and identified genes that play a role in COR-mediated chlorosis. We designated these genes as altered COR response (ALC). When silenced, one gene designated ALC1 produced a hypersensitive/necrosis-like phenotype after COR application in a coronatine insensitive 1 (COI1)-dependent manner. In pathogenicity assays performed on Arabidopsis thylakoid formation 1 (thf1) knockout lines and SlALC1-silenced tomato plants, Pst DC3000 induced coalescing necrotic lesions in an accelerated manner. Furthermore, we showed that COR affects ALC1 localization in chloroplast in a COI1-dependent manner. In conclusion, our results show the potential of VIGS-based, forward genetic screens to identify new players in COR-mediated signal transduction.