Manipulation of small Rho GTPases is a pathogen-induced process detected by NOD1.

Our innate immune system distinguishes microbes from self by detecting conserved pathogen-associated molecular patterns. However, these are produced by all microbes, regardless of their pathogenic potential. To distinguish virulent microbes from those with lower disease-causing potential the innate immune system detects conserved pathogen-induced processes, such as the presence of microbial products in the host cytosol, by mechanisms that are not fully resolved. Here we show that NOD1 senses cytosolic microbial products by monitoring the activation state of small Rho GTPases. Activation of RAC1 and CDC42 by bacterial delivery or ectopic expression of SopE, a virulence factor of the enteric pathogen Salmonella, triggered the NOD1 signalling pathway, with consequent RIP2 (also known as RIPK2)-mediated induction of NF-κB-dependent inflammatory responses. Similarly, activation of the NOD1 signalling pathway by peptidoglycan required RAC1 activity. Furthermore, constitutively active forms of RAC1, CDC42 and RHOA activated the NOD1 signalling pathway. Our data identify the activation of small Rho GTPases as a pathogen-induced process sensed through the NOD1 signalling pathway.

Malaria parasite infection compromises control of concurrent systemic non-typhoidal Salmonella infection via IL-10-mediated alteration of myeloid cell function.

Non-typhoidal Salmonella serotypes (NTS) cause a self-limited gastroenteritis in immunocompetent individuals, while children with severe Plasmodium falciparum malaria can develop a life-threatening disseminated infection. This co-infection is a major source of child mortality in sub-Saharan Africa. However, the mechanisms by which malaria contributes to increased risk of NTS bacteremia are incompletely understood. Here, we report that in a mouse co-infection model, malaria parasite infection blunts inflammatory responses to NTS, leading to decreased inflammatory pathology and increased systemic bacterial colonization. Blunting of NTS-induced inflammatory responses required induction of IL-10 by the parasites. In the absence of malaria parasite infection, administration of recombinant IL-10 together with induction of anemia had an additive effect on systemic bacterial colonization. Mice that were conditionally deficient for either myeloid cell IL-10 production or myeloid cell expression of IL-10 receptor were better able to control systemic Salmonella infection, suggesting that phagocytic cells are both producers and targets of malaria parasite-induced IL-10. Thus, IL-10 produced during the immune response to malaria increases susceptibility to disseminated NTS infection by suppressing the ability of myeloid cells, most likely macrophages, to control bacterial infection.

Interactions of the human pathogenic Brucella species with their hosts.

Brucellosis is a zoonotic infection caused primarily by the bacterial pathogens Brucella melitensis and B. abortus. It is acquired by consumption of unpasteurized dairy products or by contact with infected animals. Globally, it is one of the most widespread zoonoses, with 500,000 new cases reported each year. In endemic areas, Brucella infections represent a serious public health problem that results in significant morbidity and economic losses. An important feature of the disease is persistent bacterial colonization of the reticuloendothelial system. In this review we discuss recent insights into mechanisms of intracellular survival and immune evasion that contribute to systemic persistence by the pathogenic Brucella species.

CD4+ T cell-derived IL-10 promotes Brucella abortus persistence via modulation of macrophage function.

Evasion of host immune responses is a prerequisite for chronic bacterial diseases; however, the underlying mechanisms are not fully understood. Here, we show that the persistent intracellular pathogen Brucella abortus prevents immune activation of macrophages by inducing CD4(+)CD25(+) T cells to produce the anti-inflammatory cytokine interleukin-10 (IL-10) early during infection. IL-10 receptor (IL-10R) blockage in macrophages resulted in significantly higher NF-kB activation as well as decreased bacterial intracellular survival associated with an inability of B. abortus to escape the late endosome compartment in vitro. Moreover, either a lack of IL-10 production by T cells or a lack of macrophage responsiveness to this cytokine resulted in an increased ability of mice to control B. abortus infection, while inducing elevated production of pro-inflammatory cytokines, which led to severe pathology in liver and spleen of infected mice. Collectively, our results suggest that early IL-10 production by CD25(+)CD4(+) T cells modulates macrophage function and contributes to an initial balance between pro-inflammatory and anti-inflammatory cytokines that is beneficial to the pathogen, thereby promoting enhanced bacterial survival and persistent infection.

Salmonella uses energy taxis to benefit from intestinal inflammation.

Chemotaxis enhances the fitness of Salmonella enterica serotype Typhimurium (S. Typhimurium) during colitis. However, the chemotaxis receptors conferring this fitness advantage and their cognate signals generated during inflammation remain unknown. Here we identify respiratory electron acceptors that are generated in the intestinal lumen as by-products of the host inflammatory response as in vivo signals for methyl-accepting chemotaxis proteins (MCPs). Three MCPs, including Trg, Tsr and Aer, enhanced the fitness of S. Typhimurium in a mouse colitis model. Aer mediated chemotaxis towards electron acceptors (energy taxis) in vitro and required tetrathionate respiration to confer a fitness advantage in vivo. Tsr mediated energy taxis towards nitrate but not towards tetrathionate in vitro and required nitrate respiration to confer a fitness advantage in vivo. These data suggest that the energy taxis receptors Tsr and Aer respond to distinct in vivo signals to confer a fitness advantage upon S. Typhimurium during inflammation by enabling this facultative anaerobic pathogen to seek out favorable spatial niches containing host-derived electron acceptors that boost its luminal growth.

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.

Very long O-antigen chains enhance fitness during Salmonella-induced colitis by increasing bile resistance.

Intestinal inflammation changes the luminal habitat for microbes through mechanisms that have not been fully resolved. We noticed that the FepE regulator of very long O-antigen chain assembly in the enteric pathogen Salmonella enterica serotype Typhimurium (S. Typhimurium) conferred a luminal fitness advantage in the mouse colitis model. However, a fepE mutant was not defective for survival in tissue, resistance to complement or resistance to polymyxin B. We performed metabolite profiling to identify changes in the luminal habitat that accompany S. Typhimurium-induced colitis. This analysis suggested that S. Typhimurium-induced colitis increased the luminal concentrations of total bile acids. A mutation in fepE significantly reduced the minimal inhibitory concentration (MIC) of S. Typhimurium for bile acids in vitro. Oral administration of the bile acid sequestrant cholestyramine resin lowered the concentrations of total bile acids in colon contents during S. Typhimurium infection and significantly reduced the luminal fitness advantage conferred by the fepE gene in the mouse colitis model. Collectively, these data suggested that very long O-antigen chains function in bile acid resistance of S. Typhimurium, a property conferring a fitness advantage during luminal growth in the inflamed intestine.

Innate immune recognition of flagellin limits systemic persistence of Brucella.

Brucella are facultative intracellular bacteria that cause chronic infections by limiting innate immune recognition. It is currently unknown whether Brucella FliC flagellin, the monomeric subunit of flagellar filament, is sensed by the host during infection. Here, we used two mutants of Brucella melitensis, either lacking or overexpressing flagellin, to show that FliC hinders bacterial replication in vivo. The use of cells and mice genetically deficient for different components of inflammasomes suggested that FliC was a target of the cytosolic innate immune receptor NLRC4 in vivo but not in macrophages in vitro where the response to FliC was nevertheless dependent on the cytosolic adaptor ASC, therefore suggesting a new pathway of cytosolic flagellin sensing. However, our work also suggested that the lack of TLR5 activity of Brucella flagellin and the regulation of its synthesis and/or delivery into host cells are both part of the stealthy strategy of Brucella towards the innate immune system. Nevertheless, as a flagellin-deficient mutant of B. melitensis wasfound to cause histologically demonstrable injuries in the spleen of infected mice, we suggested that recognition of FliC plays a role in the immunological stand-off between Brucella and its host, which is characterized by a persistent infection with limited inflammatory pathology.

Intestinal inflammation allows Salmonella to use ethanolamine to compete with the microbiota.

Conventional wisdom holds that microbes support their growth in vertebrate hosts by exploiting a large variety of nutrients. We show here that use of a specific nutrient (ethanolamine) confers a marked growth advantage on Salmonella enterica serovar Typhimurium (S. Typhimurium) in the lumen of the inflamed intestine. In the anaerobic environment of the gut, ethanolamine supports little or no growth by fermentation. However, S. Typhimurium is able to use this carbon source by inducing the gut to produce a respiratory electron acceptor (tetrathionate), which supports anaerobic growth on ethanolamine. The gut normally converts ambient hydrogen sulfide to thiosulfate, which it then oxidizes further to tetrathionate during inflammation. Evidence is provided that S. Typhimurium's growth advantage in an inflamed gut is because of its ability to respire ethanolamine, which is released from host tissue, but is not utilizable by competing bacteria. By inducing intestinal inflammation, S. Typhimurium sidesteps nutritional competition and gains the ability to use an abundant simple substrate, ethanolamine, which is provided by the host.

Species-specific multiplex PCR for the diagnosis of Brucella ovis, Actinobacillus seminis, and Histophilus somni infection in rams.

BACKGROUND: Infectious ovine epididymitis results in substantial economic losses worldwide due to reproductive failure and culling of breeders. The most common causative agents of these infections are Brucella ovis, Actinobacillus seminis, and Histophilus somni. The aim of this study was to develop a multiplex PCR assay for simultaneous detection of Brucella ovis, Actinobacillus seminis, and Histophilus somni with species-specific primers applied to biological samples for molecular diagnosis of these infections. RESULTS: The multiplex assay was capable of detecting B. ovis, A. seminis, and H. somni DNA simultaneously from genomic bacterial DNA samples and pool of semen samples from experimentally infected rams. The method was highly specific since it did not amplify DNA from other bacterial species that can potentially cause epididymitis in rams as well as species phylogenetically related to B. ovis. All negative control samples were negative in PCR multiplex assay. Urine can be used as an alternative to semen samples. CONCLUSIONS: The species-specific multiplex PCR assay developed in this study can be successfully used for the detection of three of the most common bacterial causes of ovine epididymitis.

How to become a top model: impact of animal experimentation on human Salmonella disease research.

Salmonella serotypes are a major cause of human morbidity and mortality worldwide. Over the past decades, a series of animal models have been developed to advance vaccine development, provide insights into immunity to infection, and study the pathogenesis of human Salmonella disease. The successive introduction of new animal models, each suited to interrogate previously neglected aspects of Salmonella disease, has ushered in important conceptual advances that continue to have a strong and sustained influence on the ideas driving research on Salmonella serotypes. This article reviews important milestones in the use of animal models to study human Salmonella disease and identify research needs to guide future work.

Early MyD88-dependent induction of interleukin-17A expression during Salmonella colitis.

The development of T helper 17 (T(H)17) cells is a well-established adaptive mechanism for the production of interleukin-17A (IL-17A), a cytokine involved in neutrophil recruitment. However, pathways contributing to mucosal expression of IL-17A during the initial phase of a bacterial infection have received less attention. Here we used the mouse colitis model of Salmonella enterica serotype Typhimurium infection to investigate the contribution of myeloid differentiation primary response protein 88 (MyD88) to inflammation and mucosal IL-17A expression. Expression of IL-23 in the cecal mucosa during S. Typhimurium colitis was dependent on the presence of MyD88. Furthermore, initial expression of IL-17A at 24 h after S. Typhimurium infection was dependent on MyD88 and the receptor for IL-1ß. IL-23 and IL-1ß synergized in inducing expression of IL-17A in splenic T cells in vitro. In the intestinal mucosa, IL-17A was produced by three distinct T cell populations, including δγ T cells, T(H)17 cells, and CD4(-)CD8(-) T cells. The absence of IL-1ß signaling or IL-17 signaling reduced CXC chemokine expression but did not alter the overall severity of pathological lesions in the cecal mucosa. In contrast, cecal pathology and neutrophil recruitment were markedly reduced in Myd88-deficient mice during the initial phases of S. Typhimurium infection. Collectively, these data demonstrate that MyD88-dependent mechanisms, including an initial expression of IL-17A, are important for orchestrating early inflammatory responses during S. Typhimurium colitis.

Putative ATP-binding cassette transporter is essential for Brucella ovis pathogenesis in mice.

Brucella ovis is a major cause of reproductive failure in sheep, which is associated with epididymitis and infertility in rams. Importantly, B. ovis is one of the few Brucella species that is not zoonotic. Due to the scarcity of studies on B. ovis infection, a murine model of infection was developed. The roles of B. ovis genes encoding a putative hemagglutinin and an ABC transporter were investigated in the mouse model. The kinetics of B. ovis infection were similar in BALB/c and C57BL/6 mice, and both strains of mice developed multifocal microgranulomas in the liver and spleen, but only minimal colonization and histopathological changes were observed in the genital tract. Therefore, the mouse was considered a suitable infection model for B. ovis but not for B. ovis-induced genital disease. Two mutant strains were generated in this study (the ΔabcAB and Δhmg strains). The B. ovis ΔabcAB strain was attenuated in the spleens and livers of BALB/c mice compared to the wild-type (WT) strain (P < 0.001). Conversely, the Δhmg strain infected mice at the same level as WT B. ovis, suggesting that a putative hemagglutinin is not required for B. ovis pathogenesis. Additionally, the ΔabcAB strain did not survive in peritoneal macrophages, extracellularly in the peritoneal cavity, or in RAW 264.7 macrophages. Moreover, infection with the ΔabcAB strain was not lethal for male regulatory factor 1-knockout mice, whereas infection with the B. ovis WT strain was 100% lethal within 14 days postinfection. These results confirm that the predicted ABC transporter is required for the full virulence and survival of B. ovis in vivo.

Natural antibody contributes to host defense against an attenuated Brucella abortus virB mutant.

Brucella abortus is an intracellular pathogen that persists within phagocytic cells of the reticuloendothelial system. To identify in vivo interactions between B. abortus and the host that lead to persistent infection, we studied the persistence of B. abortus and an isogenic virB mutant deficient in the VirB type IV secretion system (T4SS) in knockout mice. In contrast to control mice, mice lacking B cells (Igh6(-/-)) were permissive for infection with the attenuated virB mutant. To determine the basis for this phenotype, we characterized immune functions of Igh6(-/-) mice in the context of B. abortus infection. Igh6(-/-) mice had greater numbers of extracellular bacteria in the spleen and increased early expression of proinflammatory cytokines during B. abortus infection. Further, a virB mutant, despite its wild-type level of survival, failed to elicit microgranuloma formation in the spleens of Igh6(-/-) mice, suggesting a requirement for the T4SS to elicit this pathological change. Passive transfer of immunoglobulin G from naïve mice restored the ability of Igh6(-/-) mice to control the persistence of the virB mutant by a complement-independent mechanism. Further, adoptive transfer of CD11b(+) cells from C57BL/6 mice to Igh6(-/-) mice restored the ability of the knockout mice to limit the replication of the virB mutant in the spleen, suggesting that the Igh6(-)(/)(-) mutation affects phagocyte function and that phagocyte function can be restored by natural antibody.

cDNA sequencing and expression of Nramp1 (Slc11a1) in dogs phenotypically resistant or susceptible to visceral leishmaniasis.

Nramp1 (Slc11a1) is linked to resistance to Leishmania in mice, but its role in canine leishmaniasis is not clear. In this study we sequenced the Nramp1 cDNA from dogs whose macrophages allowed or restricted intracellular growth of Leishmania chagasi. Peripheral blood monocyte-derived macrophages were isolated from 29 dogs, cultured and inoculated with L. chagasi. This approach resulted in the identification of dogs whose macrophages were resistant or susceptible to L. chagasi. Nramp1 cDNA sequences of these dogs were identical. mRNA levels of Nramp1, IFNgamma, IL-4 and the subunit p35 of IL-12 were assessed in the spleen of naturally infected symptomatic and asymptomatic dogs in comparison to uninfected controls. Although not statistically significant, asymptomatic dogs had a tendency for higher levels of Nramp1 mRNA (p = 0.11). Expression of Nramp1 was then compared between phenotypically resistant and susceptible dogs, without any significant difference between these groups.

Venereal transmission of canine visceral leishmaniasis.

Leishmania chagasi, the agent of visceral leishmaniasis in dogs in the Americas has a tropism to the male genital system, particularly the epididymis, prepuce, and glans penis, resulting in shedding of Leishmania in the semen. The goal of this study was to verify the possibility of venereal transmission of L. chagasi. Twelve Leishmania-free bitches, housed in the absence of the insect vector, copulated with multiple naturally infected dogs that were shedding Leishmania in the semen. PCR analysis of serially collected ejaculates indicated that shedding of Leishmania in the semen is intermittent. Three bitches seroconverted, and six were PCR positive by the end of the experimental period (165 days after the last copulation). These data support the notion that L. chagasi may be sexually transmitted from naturally infected dogs to susceptible bitches in the absence of the biological insect vector.

Genital lesions and distribution of amastigotes in bitches naturally infected with Leishmania chagasi.

Recent reports indicate that Leishmania chagasi has tropism to the male canine genital system, which is associated with shedding of the organism in the semen, supporting the hypothesis of venereal transmission. The aim of this study was to describe the lesions and assess parasite load in the genital system of bitches with canine visceral leishmaniasis (CanL). Symptomatic (n=5) and asymptomatic (n=5) bitches seropositive for CanL were randomly selected at the Center for Zoonosis Control (Belo Horizonte, State of Minas Gerais, Brazil). Five serologically negative, healthy, adult bitches also from the CZC were used as controls. Samples from genital organs (vulva, vagina, cervix, uterine body, uterine horns, uterine tubes, and ovaries), liver, and spleen were histologically evaluated and processed for immunodetection of Leishmania sp., and PCR. The most significant histological change was a mild to moderate vulvar dermatitis, characterized by a histio-plasma-lymphocytic infiltrate. This change was detected in all asymptomatic, four symptomatic, and three uninfected control bitches. In one symptomatic and one asymptomatic bitch intracytoplasmic amastigotes were observed within macrophages in the inflammatory infiltrate. Samples from all the segments of the genital tract were positive in at least one infected animal, in the absence of detectable amastigotes in the tissue. These findings support the notion that L. chagasi does not have genital tropism in the bitch, which is in contrast to our previous findings in naturally infected male intact dogs.

The predicted ABC transporter AbcEDCBA is required for type IV secretion system expression and lysosomal evasion by Brucella ovis.

Brucella ovis is a major cause of reproductive failure in rams and it is one of the few well-described Brucella species that is not zoonotic. Previous work showed that a B. ovis mutant lacking a species-specific ABC transporter (ΔabcBA) was attenuated in mice and was unable to survive in macrophages. The aim of this study was to evaluate the role of this ABC transporter during intracellular survival of B. ovis. In HeLa cells, B. ovis WT was able to survive and replicate at later time point (48 hpi), whereas an ΔabcBA mutant was attenuated at 24 hpi. The reduced survival of the ΔabcBA mutant was associated with a decreased ability to exclude the lysosomal marker LAMP1 from its vacuolar membrane, suggesting a failure to establish a replicative niche. The ΔabcBA mutant showed a reduced abundance of the Type IV secretion system (T4SS) proteins VirB8 and VirB11 in both rich and acid media, when compared to WT B. ovis. However, mRNA levels of virB1, virB8, hutC, and vjbR were similar in both strains. These results support the notion that the ABC transporter encoded by abcEDCBA or its transported substrate acts at a post-transcriptional level to promote the optimal expression of the B. ovis T4SS within infected host cells.

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.