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Background: Toxoplasmosis is a zoonotic disease caused by the parasite Toxoplasma gondii, a cosmopolitan intracellular parasite. It can be a risk factor for autoimmune diseases, including rheumatoid arthritis (RA). This study was designed to investigate the possible association between serological history of T. gondii infection and defined clinical manifestation of RA in Northeast of Iran. Methods: Overall, serum samples were collected from 50 RA patients and 40 healthy controls, from Qaem Hospital in Mashhad City, northeastern Iran in 2018. Seroprevalence of T. gondii infection was determined by ELISA. Results: The prevalence of anti -T. gondii IgG in RA patients 48% (24.50) was significantly higher than the control group 10% (4.40) (P

A more complete and holistic view on host-microbe interactions is needed to understand the physiological and cellular barriers that affect the efficacy of drug treatments and allow the discovery and development of new therapeutics. Here, we developed a multimodal imaging approach combining histopathology with mass spectrometry imaging (MSI) and same section imaging mass cytometry (IMC) to study the effects of

A more complete and holistic view on host-microbe interactions is needed to understand the physiological and cellular barriers that affect the efficacy of drug treatments and allow the discovery and development of new therapeutics. Here, we developed a multimodal imaging approach combining histopathology with mass spectrometry imaging (MSI) and same section imaging mass cytometry (IMC) to study the effects of Salmonella Typhimurium infection in the liver of a mouse model using the S. Typhimurium strains SL3261 and SL1344. This approach enables correlation of tissue morphology and specific cell phenotypes with molecular images of tissue metabolism. IMC revealed a marked increase in immune cell markers and localization in immune aggregates in infected tissues. A correlative computational method (network analysis) was deployed to find metabolic features associated with infection and revealed metabolic clusters of acetyl carnitines, as well as phosphatidylcholine and phosphatidylethanolamine plasmalogen species, which could be associated with pro-inflammatory immune cell types. By developing an IMC marker for the detection of Salmonella LPS, we were further able to identify and characterize those cell types which contained S. Typhimurium.

Pattern recognition receptors (PRRs) expressed in antigen-presenting cells are thought to shape pathogen-specific immunity by inducing secretion of co-stimulatory cytokines during T-cell activation, yet data to support this notion in vivo is surprisingly scarce. Here we show that the cytosolic PRR Nod-like Receptor CARD 4 (NLRC4) suppresses, rather than facilitates, effector and memory CD4(+) T-cell responses against Salmonella in mice. NLRC4 negatively regulates immunological memory by preventing delayed activation of the cytosolic PRR NLR pyrin domain 3 (NLRP3) that would otherwise amplify the production of cytokines important for the generation of Th1 immunity such as intereukin-18. Consistent with a role for NLRC4 in memory immunity, primary challenge with Salmonella expressing flagellin modified to largely evade NLRC4 recognition significantly increases protection against lethal re-challenge. This finding suggests flagellin modification to reduce NLRC4 activation enhances protective immunity which could have important implications for vaccine development against flagellated microbial pathogens.

Salmonella causes grave systemic infections in humans and other animals and provides a paradigm for other diseases in which the bacteria have both intracellular and extracellular lifestyles. New generations of vaccines rely on the essential contribution of the antibody responses for their protection. The quality, antigen specificity, and functions associated with antibody responses to this pathogen have been elusive for a long time. Recent approaches that combine studies in humans and genetically manipulated experimental models and that exploit awareness of the location and within-host life cycle of the pathogen are shedding light on how humoral immunity to Salmonella operates. However, this area of research remains full of controversy and discrepancies. The overall scenario indicates that antibodies are essential for resistance against systemic Salmonella infections and can express the highest protective function when operating in conjunction with cell-mediated immunity. Antigen specificity, isotype profile, Fc-gamma receptor usage, and complement activation are all intertwined factors that still arcanely influence antibody-mediated protection to Salmonella.

Antibiotic therapy has drastically reduced the mortality and sequelae of bacterial infections. From naturally occurring to chemically synthesized, different classes of antibiotics have been successfully used without detailed knowledge of how they affect bacterial dynamics in vivo. However, a proportion of patients receiving antimicrobial therapy develop recrudescent infections post-treatment. Relapsing infections are attributable to incomplete clearance of bacterial populations following antibiotic administration; the metabolic profile of this antibiotic-recalcitrant bacterial subpopulation, the spatio-temporal context of its emergence and the variance of antibiotic–bacterial interactions in vivo remain unclear. Here, we develop and apply a mechanistic mathematical model to data from a study comparing the effects of ciprofloxacin and ampicillin on the within-host dynamics of Salmonella enterica serovar Typhimurium in murine infections. Using the inferential capacity of our model, we show that the antibiotic-recalcitrant bacteria following ampicillin, but not ciprofloxacin, treatment belong to a non-replicating phenotype. Aligning with previous studies, we independently estimate that the lymphoid tissues and spleen are important reservoirs of non-replicating bacteria. Finally, we predict that post-treatment, the progenitors of the non-growing and growing bacterial populations replicate and die at different rates. Ultimately, the liver, spleen and mesenteric lymph nodes are all repopulated by progenitors of the previously non-growing phenotype in ampicillin-treated mice.

During the last two decades our understanding of the complex in vivo host-pathogen interactions has increased due to technical improvements and new research tools. The rapid advancement of molecular biology, flow cytometry and microscopy techniques, combined with mathematical modelling, have empowered in-depth studies of systemic bacterial infections across scales from single molecules, to cells, to organs and systems to reach the whole organism level. By tracking subpopulations of bacteria in vivo using molecular or fluorescent tags, it has been possible to reconstruct the spread of infection within and between organs, allowing unprecedented quantification of the effects of antimicrobial treatment and vaccination. This review illustrates recent advances in the study of heterogeneous traits of the infection process and illustrate approaches to investigate the reciprocal interactions between antimicrobial treatments, bacterial growth/death as well as inter- and intra-organ spread. We also discuss how vaccines impact the in vivo behaviour of bacteria and how these findings can guide vaccine design and rational antimicrobial treatment.

Background: Visceral leishmaniasis (VL) is the most severe form of leishmaniasis in Iran with high mortality rates in the case of inaccurate diagnosis and treatment. This study aimed to prepare and evaluate a new rk39 recombinant antigen from an Iranian strain of Leishmania infantum for diagnosis of VL in humans and dogs. Methods: rK39-based enzyme-linked immunosorbent assay (ELISA) was compared with the direct agglutination test (DAT) for the detection of anti L. infantum antibodies. We screened 84 human sera and 87 dog sera from clinical cases in the endemic area of Meshkin-Shahr, Iran along with 176 sera from healthy controls (collected from 86 humans and 90 dogs) during 2013 -2016. Results: Using the rK39 ELISA, a sensitivity of 85.7% (95% CI, 95-99%) and a specificity of 86.0% (95% CI, 95%-99%) were detected in human sera at a 1:800 (cut-off) titer when DAT-confirmed cases were compared with healthy controls; a sensitivity of 96.6% (95% CI, 95%-99%) and specificity of 94.4% (95% CI, 95%-99%) were found at a 1:80 (cut-off) titer compared with DAT. Kappa analysis indicated agreement between the rK39 ELISA and DAT (0.718) when using human sera at a 1:800 (cut-off) titer as well as (0.910) at a 1:80 (cut-off) titer when using dog sera (P

Background Plant lectins have long been used in biomedical research as immunomodulators against tumor cells and microbial infections. Purpose To test the ability of plant lectins ConBr (Canavalia brasiliensis) and CFL (Cratylia argentea) to activate antimicrobial and immunomodulatory activities of murine peritoneal macrophages (pMO) infected with a virulent strain of Salmonella enterica serovar Typhimurium (STm). Methods We incubated pMO with non-toxic amounts of ConBr and CFL either before (preventive schedule) or after (curative schedule) exposure to STm. Results In uninfected pMO, ConBr and CFL greatly increased levels of mRNA transcripts for IL-1β, TNF-α and IL-6 and the inducible nitric oxide synthase (iNOs), but not IL-10 and IL-12. Exposure to naive splenocytes of culture supernatants of pMO previously stimulated with CFL resulted in expression of IL-12 and IFN-γ. Both preventive and curative treatment schedules significantly reduced the intracellular load of Salmonella. Experiments in infected macrophages exposed to lectins in the preventive schedule showed that mRNA transcripts for IL-6 and TNF-α were increased by CFL, whereas ConBr enhanced IL-12 (subunit p40). In the curative schedule, CFL induced significant expression of IL-12 (p40) whereas ConBr enhanced expression IL-1β and TNF-α genes. The lectin treatments did not influence on iNOs expression in pMO infected with STm C5 regardless of the treatment schedule. Curative treatments with CFL increased approximately 130-fold expression of TLR-4 whist expression of TLR-9 was increased by treatments with ConBr. Conclusion We conclude that lectins ConBr and CFL have immunomodulatory properties that are beneficial on control of cells infected by Salmonella.

Many infectious agents enter the body using the oral route and are able to establish infections in or through the gut. For protection against most pathogens we rely on immunity to prevent or limit infection. The expression of protective immunity in the gut is normally dependent both on local (mucosal) and systemic mechanisms. In order to obtain full protection against some pathogens, particularly non-invasive micro-organisms such as Vibrio cholerae, mucosal immunity may be particularly important. There is a need to take these factors into account when designing vaccines targeting gut pathogens. Conventional parenteral vaccines (injected vaccines) can induce a degree of systemic immunity but are generally poor stimulators of mucosal responses. Thus, a basic prerequisite for designing novel vaccines against gut associated pathogens may be the requirement to induce mucosal and potentially systemic immunity.1 The most effective way to induce local immunity against infectious agents has so far proved to be direct application of vaccine antigens to mucosal surfaces (oral or intranasal delivery).2 The fact that we have so few effective oral vaccines shows that the induction of protective immunity through oral immunisation is not an easy goal to achieve as many antigens are poor oral immunogens. Here we will focus, using bacterial pathogens as examples, on some recent approaches being used to generate novel oral vaccines. Oral vaccines can be based on either live or non-living antigens. The generation of modern live oral vaccines involves the construction of genetically defined attenuated micro-organisms capable of inducing immunity in a non-harmful way. The recent improved understanding of bacterial virulence associated gene function offers the possibility of introducing multiple, defined, attenuating and stable mutations into the genome of bacterial pathogens. Furthermore, the use of precisely attenuated bacterial vectors as carriers for recombinant heterologous antigens can lead to the generation of …

Objective Mycobacterium marinum causes a rare cutaneous disease known as fish tank granuloma (FTG). The disease manifestations resemble those associated with Cutaneous Leishmaniasis (CL). The aim of this study was to determine whether FTG was the cause of cutaneous lesions in patients who were referred to the Parasitology laboratory of Imam Reza Hospital in Mashhad to be investigated for CL. Materials/Methods One hundered patients, clinically diagnosed with CL between April 2014 and March 2015, were included in this study. Ziehl-Neelsen staining was performed to identify acid-fast Mycobacterium in addition to bacterial cultures using Lowenstein-Jensen medium. Skin lesion samples were also collected and kept on DNA banking cards for PCR testing. Results Twenty-nine of the 100 individuals with skin lesions, and therefore suspected of suffering from CL, tested positive for Mycobacterium marinum by PCR. Of these, 21 (72.4) were male and 8(27.6) were female. In 97 of these cases the lesions were located on hands and fingers. These patients had a history of manipulating fish and had been in contact with aquarium water. A sporotrichoid appearance was observed in 58.6 of the patients with mycobacterial lesions; 67 of patients had multiple head appearance. Conclusion Patients suspected to have CL and who test negative for CL could be affected by FTG. Therefore, after obtaining an accurate case history, molecular diagnosis is recommended for cases that give a negative result by conventional methods. © 2019 Fata et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Although there have been great advances in our understanding of the bacterial cytoskeleton, major gaps remain in our knowledge of its importance to virulence. In this study we have explored the contribution of the bacterial cytoskeleton to the ability of Salmonella to express and assemble virulence factors and cause disease. The bacterial actin-like protein MreB polymerises into helical filaments and interacts with other cytoskeletal elements including MreC to control cell-shape. As mreB appears to be an essential gene, we have constructed a viable ΔmreC depletion mutant in Salmonella. Using a broad range of independent biochemical, fluorescence and phenotypic screens we provide evidence that the Salmonella pathogenicity island-1 type three secretion system (SPI1-T3SS) and flagella systems are down-regulated in the absence of MreC. In contrast the SPI-2 T3SS appears to remain functional. The phenotypes have been further validated using a chemical genetic approach to disrupt the functionality of MreB. Although the fitness of ΔmreC is reduced in vivo, we observed that this defect does not completely abrogate the ability of Salmonella to cause disease systemically. By forcing on expression of flagella and SPI-1 T3SS in trans with the master regulators FlhDC and HilA, it is clear that the cytoskeleton is dispensable for the assembly of these structures but essential for their expression. As two-component systems are involved in sensing and adapting to environmental and cell surface signals, we have constructed and screened a panel of such mutants and identified the sensor kinase RcsC as a key phenotypic regulator in ΔmreC. Further genetic analysis revealed the importance of the Rcs two-component system in modulating the expression of these virulence factors. Collectively, these results suggest that expression of virulence genes might be directly coordinated with cytoskeletal integrity, and this regulation is mediated by the two-component system sensor kinase RcsC., Author Summary Salmonella are major global pathogens responsible for causing food-borne disease. In recent years the existence of a cytoskeleton in prokaryotes has received much attention. In this study the Salmonella cytoskeleton has been genetically disrupted, causing changes in morphology, motility and expression of key virulence factors. We provide evidence that the sensory protein RcsC detects changes at the cell surface caused by the disintegration of the bacterial cytoskeleton and modulates expression of key virulence factors. This study provides insights into the importance of the integrity of the bacterial cytoskeleton in the ability of Salmonella to cause disease, and thus may provide a novel target for antimicrobial drugs or vaccines.

Significance Bacteria, such as nontyphoidal Salmonella, are responsible for a large global burden of disease. Due to limited need in developed countries and consequent lack of commercial incentive, vaccines are unavailable against many bacteria. Glycoconjugates constitute the standard bacterial vaccine approach, but can be costly, particularly where multivalent preparations are required. This report compares a low-cost vesicle-based technology, known as Generalized Modules for Membrane Antigens (GMMA), with glycoconjugate in bivalent vaccines against nontyphoidal Salmonella. In head-to-head immunogenicity and infection studies in mice, GMMA performed at least as well as equivalent glycoconjugate vaccine, indicating good potential of this approach. Given that many bacteria are amenable to genetic engineering for GMMA production, the GMMA strategy could provide a breakthrough for a range of needed bacterial vaccines., Nontyphoidal Salmonellae cause a devastating burden of invasive disease in sub-Saharan Africa with high levels of antimicrobial resistance. Vaccination has potential for a major global health impact, but no licensed vaccine is available. The lack of commercial incentive makes simple, affordable technologies the preferred route for vaccine development. Here we compare equivalent Generalized Modules for Membrane Antigens (GMMA) outer membrane vesicles and O-antigen-CRM197 glycoconjugates to deliver lipopolysaccharide O-antigen in bivalent Salmonella Typhimurium and Enteritidis vaccines. Salmonella strains were chosen and tolR deleted to induce GMMA production. O-antigens were extracted from wild-type bacteria and conjugated to CRM197. Purified GMMA and glycoconjugates were characterized and tested in mice for immunogenicity and ability to reduce Salmonella infection. GMMA and glycoconjugate O-antigen had similar structural characteristics, O-acetylation, and glucosylation levels. Immunization with GMMA induced higher anti–O-antigen IgG than glycoconjugate administered without Alhydrogel adjuvant. With Alhydrogel, antibody levels were similar. GMMA induced a diverse antibody isotype profile with greater serum bactericidal activity than glycoconjugate, which induced almost exclusively IgG1. Immunization reduced bacterial colonization of mice subsequently infected with Salmonella. S. Typhimurium numbers were lower in tissues of mice vaccinated with GMMA compared with glycoconjugate. S. Enteritidis burden in the tissues was similar in mice immunized with either vaccine. With favorable immunogenicity, low cost, and ability to induce functional antibodies and reduce bacterial burden, GMMA offer a promising strategy for the development of a nontyphoidal Salmonella vaccine compared with established glycoconjugates. GMMA technology is potentially attractive for development of vaccines against other bacteria of global health significance.

Summary Vaccines can serve as essential tools to prevent bacterial diseases via the induction of long‐lasting IgG responses. The efficacy of such vaccines depends on the effector mechanisms triggered by IgG. The complement system and Fc‐gamma receptors (FcγRs) can potentially play a crucial role in IgG‐mediated immunity against bacterial diseases. However, their relative importance in vivo is unclear, and has been the object of controversy and debate. In this brief study, we have used gene‐targeted mice lacking either Fcγ RI, II, II and IV or the C3 complement component as well as a novel mouse strain lacking both C3 and FcγRs to conclusively show the essential role of complement in antibody‐mediated host resistance to Salmonella enterica systemic infection. By comparing the effect of IgG2a antibodies against Salmonella O‐antigen in gene‐targeted mice, we demonstrate that the complement system is essential for the IgG‐mediated reduction of bacterial numbers in the tissues.

Introduction: Invasive non-typhoidal Salmonella (iNTS) infections cause a high burden of lethal sepsis in young children and HIV patients, often associated with malaria, anaemia, malnutrition and sickle-cell disease. Vaccines against iNTS are urgently needed but none are licensed yet.Areas covered: This review illustrates how immunology, epidemiology and within-host pathogen behaviour affect invasive Salmonella infections and highlights how this knowledge can assist the improvement and choice of vaccines.Expert Commentary: Control of iNTS disease requires approaches that reduce transmission and improve diagnosis and treatment. These are often difficult to implement due to the fragile ecology and economies in endemic countries. Vaccines will be key tools in the fight against iNTS disease. To optimise vaccine design, we need to better define protective antigens and mechanisms of resistance to disease in susceptible populations even in those individuals where innate immunity may be impaired by widesp...

Antibiotic therapy has drastically reduced the mortality and sequelae of bacterial infections. From naturally occurring to chemically synthesized, different classes of antibiotics have been successfully used without detailed knowledge of how they affect bacterial dynamics in vivo . However, a proportion of patients receiving antimicrobial therapy develop recrudescent infections post-treatment. Relapsing infections are attributable to incomplete clearance of bacterial populations following antibiotic administration; the metabolic profile of this antibiotic-recalcitrant bacterial subpopulation, the spatio-temporal context of its emergence and the variance of antibiotic–bacterial interactions in vivo remain unclear. Here, we develop and apply a mechanistic mathematical model to data from a study comparing the effects of ciprofloxacin and ampicillin on the within-host dynamics of Salmonella enterica serovar Typhimurium in murine infections. Using the inferential capacity of our model, we show that the antibiotic-recalcitrant bacteria following ampicillin, but not ciprofloxacin, treatment belong to a non-replicating phenotype. Aligning with previous studies, we independently estimate that the lymphoid tissues and spleen are important reservoirs of non-replicating bacteria. Finally, we predict that post-treatment, the progenitors of the non-growing and growing bacterial populations replicate and die at different rates. Ultimately, the liver, spleen and mesenteric lymph nodes are all repopulated by progenitors of the previously non-growing phenotype in ampicillin-treated mice.

Nontyphoidal Salmonella (NTS), particularly Salmonella enterica serovars Typhimurium and Enteritidis, is responsible for a major global burden of invasive disease with high associated case-fatality rates. We recently reported the development of a candidate O-antigen–CRM 197 glycoconjugate vaccine against S. Typhimurium. Here, using a panel of mouse monoclonal antibodies generated by the vaccine, we examined the relative efficiency of different antibody isotypes specific for the O:4 antigen of S . Typhimurium to effect in vitro and in vivo killing of the invasive African S . Typhimurium strain D23580. All O:4-specific antibody isotypes could mediate cell-free killing and phagocytosis of S . Typhimurium by mouse blood cells. Opsonization of Salmonella with O:4-specific IgA, IgG1, IgG2a, and IgG2b, but not IgM, resulted in cell-dependent bacterial killing. At high concentrations, O:4-specific antibodies inhibited both cell-free complement-mediated and cell-dependent opsonophagocytic killing of S . Typhimurium in vitro . Using passive immunization in mice, the O:4-specific antibodies provided in vivo functional activity by decreasing the bacterial load in the blood and tissues, with IgG2a and IgG2b being the most effective isotypes. In conclusion, an O-antigen–CRM 197 glycoconjugate vaccine can induce O-antigen-specific antibodies of different isotypes that exert in vitro and in vivo killing of S . Typhimurium.

Over the last ten years, isogenic tagging (IT) has revolutionised the study of bacterial infection dynamics in laboratory animal models. However, quantitative analysis of IT data has been hindered by the piecemeal development of relevant statistical models. The most promising approach relies on stochastic Markovian models of bacterial population dynamics within and among organs. Here we present an efficient numerical method to fit such stochastic dynamic models to in vivo experimental IT data. A common approach to statistical inference with stochastic dynamic models relies on producing large numbers of simulations, but this remains a slow and inefficient method for all but simple problems, especially when tracking bacteria in multiple locations simultaneously. Instead, we derive and solve the systems of ordinary differential equations for the two lower-order moments of the stochastic variables (mean, variance and covariance). For any given model structure, and assuming linear dynamic rates, we demonstrate how the model parameters can be efficiently and accurately estimated by divergence minimisation. We then apply our method to an experimental dataset and compare the estimates and goodness-of-fit to those obtained by maximum likelihood estimation. While both sets of parameter estimates had overlapping confidence regions, the new method produced lower values for the division and death rates of bacteria: these improved the goodness-of-fit at the second time point at the expense of that of the first time point. This flexible framework can easily be applied to a range of experimental systems. Its computational efficiency paves the way for model comparison and optimal experimental design., Author summary Recent advancements in technology have meant that microbiologists are producing vast amounts of experimental data. However, statistical methods by which we can analyse that data, draw informative inference, and test relevant hypotheses, are much needed. Here, we present a new, efficient inference tool for estimating parameters of stochastic models, with a particular focus on models of within-host bacterial dynamics. The method relies on matching the two lower-order moments of the experimental data (i.e., mean, variance and covariance), to the moments from the mathematical model. The method is verified, and particular choices justified, through a number of simulation studies. We then apply this moment-based inference method to experimental data, and compare the results with previously published maximum likelihood estimates.

Malaria and anaemia are key underlying factors for iNTS disease in African children. Knowledge of clinical and epidemiological risk-factors for iNTS disease has not been paralleled by an in-depth knowledge of the immunobiology of the disease. Herein, we review human and animal studies on mechanisms of increased susceptibility to iNTS in children.

The role of chitinases from the latex of medicinal shrub Calotropis procera on viability of tumor cell lines and inflammation was investigated. Soluble latex proteins were fractionated in a CM Sepharose Fast-Flow Column and the major peak (LPp1) subjected to ion exchange chromatography using a Mono-Q column coupled to an FPLC system. In a first series of experiments, immortalized macrophages were cultured with LPp1 for 24 h. Then, cytotoxicity of chitinase isoforms (LPp1-P1 to P6) was evaluated against HCT-116 (colon carcinoma), OVCAR-8 (ovarian carcinoma), and SF-295 (glioblastoma) tumor cell lines in 96-well plates. Cytotoxic chitinases had its anti-inflammatory potential assessed through the mouse peritonitis model. We have shown that LPp1 was not toxic to macrophages at dosages lower than 125 μg/mL but induced high messenger RNA expression of IL-6, IL1-β, TNF-α, and iNOs. On the other hand, chitinase isoform LPp1-P4 retained all LPp1 cytotoxic activities against the tumor cell lines with IC50 ranging from 1.2 to 2.9 μg/mL. The intravenous administration of LPp1-P4 to mouse impaired neutrophil infiltration into the peritoneal cavity induced by carrageenan. Although the contents of pro-inflammatory cytokines IL-6, TNF-α, and IL1-β were high in the bloodstreams, such effect was reverted by administration of iNOs inhibitors NG-nitro-L-arginine methyl ester and aminoguanidine. We conclude that chitinase isoform LPp1-P4 was highly cytotoxic to tumor cell lines and capable to reduce inflammation by an iNOs-derived NO mechanism.

MicroRNAs (miRNAs) are small noncoding molecules that control gene expression posttranscriptionally, with microRNA-155 (miR-155) one of the first to be implicated in immune regulation. Here, we show that miR-155-deficient mice are less able to eradicate a mucosal Citrobacter rodentium infection than wild-type C57BL/6 mice. miR-155-deficient mice exhibited prolonged colonization associated with a higher C. rodentium burden in gastrointestinal tissue and spread into systemic tissues. Germinal center formation and humoral immune responses against C. rodentium were severely impaired in infected miR-155-deficient mice. A similarly susceptible phenotype was observed in μMT mice reconstituted with miR-155-deficient B cells, indicating that miR-155 is required intrinsically for mediating protection against this predominantly luminal bacterial pathogen.

Campylobacter jejuni, the most common cause of bacterial diarrhoeal disease, is normally helical. However, it can also adopt straight rod, elongated helical and coccoid forms. Studying how helical morphology is generated, and how it switches between its different forms, is an important objective for understanding this pathogen. Here, we aimed to determine the genetic factors involved in generating the helical shape of Campylobacter. A C. jejuni transposon (Tn) mutant library was screened for non-helical mutants with inconsistent results. Whole genome sequence variation and morphological trends within this Tn library, and in various C. jejuni wild type strains, were compared and correlated to detect genomic elements associated with helical and rod morphologies. All rod-shaped C. jejuni Tn mutants and all rod-shaped laboratory, clinical and environmental C. jejuni and Campylobacter coli contained genetic changes within the pgp1 or pgp2 genes, which encode peptidoglycan modifying enzymes. We therefore confirm the importance of Pgp1 and Pgp2 in the maintenance of helical shape and extended this to a wide range of C. jejuni and C. coli isolates. Genome sequence analysis revealed variation in the sequence and length of homopolymeric tracts found within these genes, providing a potential mechanism of phase variation of cell shape.

Salmonella infections are a serious medical and veterinary problem world-wide and cause concern in the food industry. Vaccination is an effective tool for the prevention of Salmonella infections. Host resistance to Salmonella relies initially on the production of inflammatory cytokines leading to the infiltration of activated inflammatory cells in the tissues. Thereafter T- and B-cell dependent specific immunity develops allowing the clearance of Salmonella microorganisms from the tissues and the establishment of long-lasting acquired immunity to re-infection. The increased resistance that develops after primary infection/ vaccination requires T-cells cytokines such as IFNgamma TNFalpha and IL12 in addition to opsonising antibody. However for reasons that are not fully understood seroconversion and/or the presence of detectable T-cell memory do not always correlate with the development of acquired resistance to infection.Whole-cell killed vaccines and subunit vaccines are used in the prevention of Salmonella infection in animals and in humans with variable results. A number of early live Salmonella vaccines derived empirically by chemical or u.v. mutagenesis proved to be immunogenic and protective and are still in use despite the need for repeated parenteral administration. Recent progress in the knowledge of the genetics of Salmonella virulence and modern recombinant DNA technology offers the possibility to introduce multiple defined attenuating and irreversible mutations into the bacterial genome. This has recently allowed the development of Salmonella strains devoid of significant side effects but still capable of inducing solid immunity after single oral administration. Live attenuated Salmonella vaccines have been used for the expression of heterologous antigens/proteins that can be successfully delivered to the immune system. Furthermore Salmonella can transfer plasmids encoding foreign antigens under the control of eukaryotic promoters (DNA vaccines) to antigen-presenting cells resulting in targeted delivery of DNA vaccines to these cells. Despite the great recent advances in the development of Salmonella vaccines a large proportion of the work has been conducted in laboratory rodents and more research in other animal species is required.

In the present study we evaluated the role of B cells in acquired immunity toSalmonellainfection by using gene-targeted B-cell-deficient innately susceptible mice on a C57BL/6 background (Igh-6−/−).Igh-6−/−mice immunized with a live, attenuatedaroA Salmonella entericaserovar Typhimurium vaccine strain showed impaired long-term acquired resistance against the virulent serovar Typhimurium strain C5.Igh-6−/−mice were able to control a primary infection and to clear the inoculum from the reticuloendothelial system. However,Igh-6−/−mice, unlikeIgh-6+/+C57BL/6 controls, did not survive an oral challenge with strain C5 at 4 months after vaccination. Transfer of immune serum did not restore resistance inIgh-6−/−mice. Total splenocytes and purified CD4+T cells obtained fromIgh-6−/−mice 4 months after vaccination showed reduced ability to release Th1-type cytokines (interleukin 2 and gamma interferon) upon in vitro restimulation with serovar Typhimurium soluble cell extracts compared to cells obtained fromIgh-6+/+C57BL/6 control mice. Therefore, the impaired resistance to oral challenge with virulent serovar Typhimurium observed in B-cell-deficient mice, which cannot be restored by passive transfer ofSalmonella-immune serum, may be in part due to a reduced serovar Typhimurium-specific T-cell response following primary immunization.

Invasive non-typhoidal Salmonella are a common cause of invasive disease in immuno-compromised individuals and in children. Multi-drug resistance poses challenges to disease control, with a critical need for effective vaccines. Flagellin is an attractive vaccine candidate due to surface exposure and high epitope copy number, but its potential as a target for opsonophacytic antibodies is unclear.

Salmonella enterica causes a range of diseases. Salmonellae are intracellular parasites of macrophages, and the control of bacteria within these cells is critical to surviving an infection. The dynamics of the bacteria invading, surviving, proliferating in and killing macrophages are central to disease pathogenesis. Fundamentally important parameters, however, such as the cellular infection rate, have not previously been calculated. We used two independent approaches to calculate the macrophage infection rate: mathematical modelling of Salmonella infection experiments, and analysis of real-time video microscopy of infection events. Cells repeatedly encounter salmonellae, with the bacteria often remain associated with the macrophage for more than ten seconds. Once Salmonella encounters a macrophage, the probability of that bacterium infecting the cell is remarkably low: less than 5%. The macrophage population is heterogeneous in terms of its susceptibility to the first infection event. Once infected, a macrophage can undergo further infection events, but these reinfection events occur at a lower rate than that of the primary infection.

Increasingly, evidence suggests that there is a strong environmental component to the development of the autoimmune disease type 1 diabetes. Our previous data showed that NOD mice are protected from developing diabetes after infection with Salmonella typhimurium and there is some evidence that changes within the DC compartment play a crucial role in this protective effect. This paper further characterises this Salmonella-modulated protective phenotype. We find that, contrary to other infection-mediated models of type 1 diabetes protection, there was no expansion of Foxp3(+) Tregs. Furthermore, transcriptome analysis of DCs identified a distinct Salmonella-induced signature in which the inhibitory receptor PD-L1 was up-regulated. This was confirmed by flow cytometry. In vivo blockade of the PD1/PD-L1 interaction was found to ablate the protective function of Salmonella infection. These data provide evidence for a novel regulatory DC phenotype proficient at controlling autoreactive T cells for an extended duration in the NOD mouse model of diabetes.

Salmonella enterica serovar Typhi, the agent of typhoid fever in humans, expresses the surface Vi polysaccharide antigen that contributes to virulence. However, Vi expression can also be detrimental to some key steps of S. Typhi infectivity, for example, invasion, and Vi is the target of protective immune responses. We used a strain of S. Typhimurium carrying the whole Salmonella pathogenicity island 7 (SPI-7) to monitor in vivo Vi expression within phagocytic cells of mice at different times after systemic infection. We also tested whether it is possible to modulate Vi expression via the use of in vivo -inducible promoters and whether this would trigger anti-Vi antibodies through the use of Vi-expressing live bacteria. Our results show that Vi expression in the liver and spleen is downregulated with the progression of infection and that the Vi-negative population of bacteria becomes prevalent by day 4 postinfection. Furthermore, we showed that replacing the natural tviA promoter with the promoter of the SPI-2 gene ssaG resulted in sustained Vi expression in the tissues. Intravenous or oral infection of mice with a strain of S. Typhimurium expressing Vi under the control of the ssaG promoter triggered detectable levels of all IgG subclasses specific for Vi. Our work highlights that Vi is downregulated in vivo and provides proof of principle that it is possible to generate a live attenuated vaccine that induces Vi-specific antibodies after single oral administration.

Salmonella enterica is a genetically broad species harboring isolates that display considerable antigenic heterogeneity and significant differences in virulence potential. Salmonella generally exhibit an invasive potential and they can survive for extended periods within cells of the immune system. They cause acute or chronic infections that can be local (e.g. gastroenteritis) or systemic (e.g. typhoid). In vivo Salmonella infections are complex with multiple arms of the immune system being engaged. Both humoral and cellular responses can be detected and characterized, but full protective immunity is not always induced, even following natural infection. The murine model has proven to be a fertile ground for exploring immune mechanisms and observations in the mouse have often, although not always, correlated with those in other infectable species, including humans. Host genetic studies have identified a number of mammalian genes that are central to controlling infection, operating both in innate and acquired immune pathways. Vaccines, both oral and parenteral, are available or under development, and these have been used with some success to explore immunity in both model systems and clinically in humans.

Several classes and multiple subclasses of immunoglobulins are produced towards protein and polysaccharide antigens in response to Salmonella infection and play a key role in protection against systemic disease. The targeting of Salmonella to Fc receptors (FcR) on phagocytes is a key step in the antibody-mediated antibacterial functions of host cells. We wished to compare the relative efficiency of different human IgG subclasses, which targeted the Salmonella enterica OmpA surface protein in modulating the interaction of bacteria with human phagocytes. To this end, we developed a novel system by tagging OmpA with a foreign CD52 mimotope (TSSPSAD) and opsonizing the bacteria with a panel of humanized CD52 antibodies that share the same antigen-binding V-region, but have constant regions of different subclasses. Our data revealed that opsonization with all the IgG subclasses increases Salmonella uptake by human phagocytes. IgG3 resulted in the highest level of bacterial uptake and the highest average bacterial load per infected cell, which was closely followed by IgG1, then IgG4 and lastly IgG2. Phagocytosis mediated by IgG1, IgG3 and IgG4 had a higher dependency on FcγRI than FcγRIIA, whereas IgG2-mediated phagocytosis required FcγRIIA more than FcγRI. The results show that IgG binding to OmpA increases the uptake of Salmonella by human phagocytic cells and that the efficiency of this process depends both on the subclass of the IgG and the type of FcR that is available for antibody binding.

The interaction between Salmonella enterica and the host immune system is complex. The outcome of an infection is the result of a balance between the in vivo environment where the bacteria survive and grow and the regulation of fitness genes at a level sufficient for the bacteria to retain their characteristic rate of growth in a given host. Using bacteriological counts from tissue homogenates and fluorescence microscopy to determine the spread, localization, and distribution of S. enterica in the tissues, we show that, during a systemic infection, S. enterica adapts to the in vivo environment. The adaptation becomes a measurable phenotype when bacteria that have resided in a donor animal are introduced into a recipient naïve animal. This adaptation does not confer increased resistance to early host killing mechanisms but can be detected as an enhancement in the bacterial net growth rate later in the infection. The enhanced growth rate is lost upon a single passage in vitro , and it is therefore transient and not due to selection of mutants. The adapted bacteria on average reach higher intracellular numbers in individual infected cells and therefore have patterns of organ spread different from those of nonadapted bacteria. These experiments help in developing an understanding of the influence of passage in a host on the fitness and virulence of S. enterica .

Protective Th1 responses to Salmonella enterica do not develop in the absence of B cells. Using chimeric mice, we dissect the early (innate) and late (cognate) contributions of B cells to Th programming. B cell-intrinsic MyD88 signaling is required for primary effector Th1 development, whereas Ag-specific BCR-mediated Ag presentation is necessary for the development of memory Th1 populations. Programming of the primary T cell response is BCR/B cell MHC II independent, but requires MyD88-dependent secretion of cytokines by B cells. Chimeras in which B cells lack IFN-γ or IL-6 genes make impaired Th1 or Th17 responses to Salmonella.

Salmonella enterica sv. typhimurium (S. enterica sv. Typhimurium) has two metal-transporting P(1)-type ATPases whose actions largely overlap with respect to growth in elevated copper. Mutants lacking both ATPases over-accumulate copper relative to wild-type or either single mutant. Such duplication of ATPases is unusual in bacterial copper tolerance. Both ATPases are under the control of MerR family metal-responsive transcriptional activators. Analyses of periplasmic copper complexes identified copper-CueP as one of the predominant metal pools. Expression of cueP was recently shown to be controlled by the same metal-responsive activator as one of the P(1)-type ATPase genes (copA), and copper-CueP is a further atypical feature of copper homeostasis in S. enterica sv. Typhimurium. Elevated copper is detected by a reporter construct driven by the promoter of copA in wild-type S. enterica sv. Typhimurium during infection of macrophages. Double mutants missing both ATPases also show reduced survival inside cultured macrophages. It is hypothesized that elevated copper within macrophages may have selected for specialized copper-resistance systems in pathogenic microorganism such as S. enterica sv. Typhimurium.