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Malaria-transmitting mosquitoes are continuously exposed to microbes, including their midgut microbiota. This naturally acquired microbial flora can modulate the mosquito's vectorial capacity by inhibiting the development of Plasmodium and other human pathogens through an unknown mechanism. We have undertaken a comprehensive functional genomic approach to elucidate the molecular interplay between the bacterial co-infection and the development of the human malaria parasite Plasmodium falciparum in its natural vector Anopheles gambiae. Global transcription profiling of septic and aseptic mosquitoes identified a significant subset of immune genes that were mostly up-regulated by the mosquito's microbial flora, including several anti-Plasmodium factors. Microbe-free aseptic mosquitoes displayed an increased susceptibility to Plasmodium infection while co-feeding mosquitoes with bacteria and P. falciparum gametocytes resulted in lower than normal infection levels. Infection analyses suggest the bacteria-mediated anti-Plasmodium effect is mediated by the mosquitoes' antimicrobial immune responses, plausibly through activation of basal immunity. We show that the microbiota can modulate the anti-Plasmodium effects of some immune genes. In sum, the microbiota plays an essential role in modulating the mosquito's capacity to sustain Plasmodium infection.

Control of infection with Mycobacterium tuberculosis (Mtb) requires Th1-type immunity, of which CD8+ T cells play a unique role. High frequency Mtb-reactive CD8+ T cells are present in both Mtb-infected and uninfected humans. We show by limiting dilution analysis that nonclassically restricted CD8+ T cells are universally present, but predominate in Mtb-uninfected individuals. Interestingly, these Mtb-reactive cells expressed the Valpha7.2 T-cell receptor (TCR), were restricted by the nonclassical MHC (HLA-Ib) molecule MR1, and were activated in a transporter associated with antigen processing and presentation (TAP) independent manner. These properties are all characteristics of mucosal associated invariant T cells (MAIT), an \"innate\" T-cell population of previously unknown function. These MAIT cells also detect cells infected with other bacteria. Direct ex vivo analysis demonstrates that Mtb-reactive MAIT cells are decreased in peripheral blood mononuclear cells (PBMCs) from individuals with active tuberculosis, are enriched in human lung, and respond to Mtb-infected MR1-expressing lung epithelial cells. Overall, these findings suggest a generalized role for MAIT cells in the detection of bacterially infected cells, and potentially in the control of bacterial infection.

  [...]a mounting body of evidence indicates the presence of additional or complementary mechanisms such as intracellular targeting of cytoplasmic components crucial to proper cellular physiology (Figure 2) [7], [8]. [...]the initial interaction between the peptides and the microbial cell membrane would allow them to penetrate into the cell to bind intracellular molecules, resulting in the inhibition of cell wall biosynthesis and DNA, RNA, and protein synthesis. [...]insights into the mechanisms employed by AMPs will facilitate new approaches to discover and develop pharmacologic agents.

Aedes aegypti, the mosquito vector of dengue viruses, utilizes its innate immune system to ward off a variety of pathogens, some of which can cause disease in humans. To date, the features of insects' innate immune defenses against viruses have mainly been studied in the fruit fly Drosophila melanogaster, which appears to utilize different immune pathways against different types of viruses, in addition to an RNA interference-based defense system. We have used the recently released whole-genome sequence of the Ae. aegypti mosquito, in combination with high-throughput gene expression and RNA interference (RNAi)-based reverse genetic analyses, to characterize its response to dengue virus infection in different body compartments. We have further addressed the impact of the mosquito's endogenous microbial flora on virus infection. Our findings indicate a significant role for the Toll pathway in regulating resistance to dengue virus, as indicated by an infection-responsive regulation and functional assessment of several Toll pathway-associated genes. We have also shown that the mosquito's natural microbiota play a role in modulating the dengue virus infection, possibly through basal-level stimulation of the Toll immune pathway.

Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immunocompromised patients. Macrophages and neutrophils are known to kill conidia, whereas hyphae are killed mainly by neutrophils. Since hyphae are too large to be engulfed, neutrophils possess an array of extracellular killing mechanisms including the formation of neutrophil extracellular traps (NETs) consisting of nuclear DNA decorated with fungicidal proteins. However, until now NET formation in response to A. fumigatus has only been demonstrated in vitro, the importance of neutrophils for their production in vivo is unclear and the molecular mechanisms of the fungus to defend against NET formation are unknown. Here, we show that human neutrophils produce NETs in vitro when encountering A. fumigatus. In time-lapse movies NET production was a highly dynamic process which, however, was only exhibited by a sub-population of cells. NETosis was maximal against hyphae, but reduced against resting and swollen conidia. In a newly developed mouse model we could then demonstrate the existence and measure the kinetics of NET formation in vivo by 2-photon microscopy of Aspergillus-infected lungs. We also observed the enormous dynamics of neutrophils within the lung and their ability to interact with and phagocytose fungal elements in situ. Furthermore, systemic neutrophil depletion in mice almost completely inhibited NET formation in lungs, thus directly linking the immigration of neutrophils with NET formation in vivo. By using fungal mutants and purified proteins we demonstrate that hydrophobin RodA, a surface protein making conidia immunologically inert, led to reduced NET formation of neutrophils encountering Aspergillus fungal elements. NET-dependent killing of Aspergillus-hyphae could be demonstrated at later time-points, but was only moderate. Thus, these data establish that NET formation occurs in vivo during host defence against A. fumigatus, but suggest that it does not play a major role in killing this fungus. Instead, NETs may have a fungistatic effect and may prevent further spreading.

  Summary Points * In most countries, tuberculosis (TB) notification is twice as high in men as in women. * Although there is clear evidence that socioeconomic and cultural factors leading to barriers in accessing health care may cause undernotification in women, particularly in developing countries, biological mechanisms may actually account for a significant part of this difference between male and female susceptibility to TB. * The role of biological gender has been determined in a number of infectious and noninfectious diseases. Key Research Actions on Sex Bias in TB * Parallel and homogeneous epidemiological surveys in human populations from different geographic and ethnic backgrounds to dissect simultaneously the various factors possibly contributing to the sex bias in TB in the most exhaustive manner, including: * Sociocultural components: income, stigmatization, awareness, etc. * Behavioural components: smoking, alcohol and drug abuse, exposure to toxic dusts at the work place, dietary differences, etc. * Biological components: sex hormones, genetic background * Detailed follow-ups of sex hormone profiles in men and women presenting TB, as well as in the corresponding healthy contacts exposed to the same environmental pressures * Development of an appropriate animal model that mimics the sex bias observed in TB in humans for subsequent in vivo dissection of the influence of sex hormones in castrated and hormone-reconstituted animals on immune response to M. tuberculosis and disease outcome * Development of suitable in vitro cell models to investigate the influence of sex hormones and immune modulators (cytokines and nutrients such as iron, vitamin D, etc.) on the immune response to M. tuberculosis (see Figure 3) * Genome-wide association studies in populations from diverse geographic areas, involving large cohorts of clinically well-defined TB cases and appropriate controls, stratified by sex * Genome-wide gene expression profiling in different in vitro and ex vivo biological settings (e.g., monocyte-derived phagocytes, blood samples, lung biopsies, broncho-alveolar lavages) from male and female TB patients and relevant controls [Figure omitted, see PDF] Figure 3.

Host defence against infection requires a range of innate and adaptive immune responses that may lead to tissue damage. Such immune-mediated pathologies can be controlled with appropriate T regulatory (Treg) activity. The aim of the present study was to determine the influence of gut microbiota composition on Treg cellular activity and NF- Kappa B activation associated with infection. Mice consumed the commensal microbe Bifidobacterium infantis 35624 followed by infection with Salmonella typhimurium or injection with LPS. In vivo NF- Kappa B activation was quantified using biophotonic imaging. CD4 super(+)CD25 super(+)Foxp3 super(+) T cell phenotypes and cytokine levels were assessed using flow cytometry while CD4 super(+) T cells were isolated using magnetic beads for adoptive transfer to naive animals. In vivo imaging revealed profound inhibition of infection and LPS induced NF- Kappa B activity that preceded a reduction in S. typhimurium numbers and murine sickness behaviour scores in B. infantis-fed mice. In addition, pro-inflammatory cytokine secretion, T cell proliferation, and dendritic cell co-stimulatory molecule expression were significantly reduced. In contrast, CD4 super(+)CD25 super(+)Foxp3 super(+) T cell numbers were significantly increased in the mucosa and spleen of mice fed B. infantis. Adoptive transfer of CD4 super(+)CD25 super(+) T cells transferred the NF- Kappa B inhibitory activity. Consumption of a single commensal micro-organism drives the generation and function of Treg cells which control excessive NF- Kappa B activation in vivo. These cellular interactions provide the basis for a more complete understanding of the commensal-host-pathogen trilogue that contribute to host homeostatic mechanisms underpinning protection against aberrant activation of the innate immune system in response to a translocating pathogen or systemic LPS. Author Summary The normal response to infection is rapid and effective clearance of pathogenic microbes. However, this immune response may occasionally cause collateral inflammatory damage to host tissue and in severe cases, such as systemic sepsis, results in organ failure. Various cellular mechanisms, including regulatory T cells, protect against aggressive immune responses. However, environmental agents which promote regulatory T cells are not well understood. We and others have previously shown that non-pathogenic or commensal micro-organisms can protect the host from aberrant pro-inflammatory activity within the gut, but the influence of these microbes on regulatory T cells in the context of systemic infection has not been examined. In this study, we demonstrate that consumption of a single commensal bacterium induces regulatory T cells in vivo which protect the host from pathogen-induced inflammatory responses by limiting activation of the pro-inflammatory transcription factor NF- Kappa B via the toll-like receptor 4 (TLR-4) pathway. This report conclusively demonstrates a cellular and molecular basis for the commensal-host-pathogen trilogue resulting in enhanced protection from systemic infection whilst limiting pro-inflammatory damage mediated by activation of the innate immune system.

The bacterial pathogen Staphylococcus aureus seeds abscesses in host tissues to replicate at the center of these lesions, protected from host immune cells via a pseudocapsule. Using histochemical staining, we identified prothrombin and fibrin within abscesses and pseudocapsules. S. aureus secretes two clotting factors, coagulase (Coa) and von Willebrand factor binding protein (vWbp). We report here that Coa and vWbp together are required for the formation of abscesses. Coa and vWbp promote the non-proteolytic activation of prothrombin and cleavage of fibrinogen, reactions that are inhibited with specific antibody against each of these molecules. Coa and vWbp specific antibodies confer protection against abscess formation and S. aureus lethal bacteremia, suggesting that coagulases function as protective antigens for a staphylococcal vaccine.

Many enteropathogenic bacteria target the mammalian gut. The mechanisms protecting the host from infection are poorly understood. We have studied the protective functions of secretory antibodies (sIgA) and the microbiota, using a mouse model for S. typhimurium diarrhea. This pathogen is a common cause of diarrhea in humans world-wide. S. typhimurium (S. tm(att), sseD) causes a self-limiting gut infection in streptomycin-treated mice. After 40 days, all animals had overcome the disease, developed a sIgA response, and most had cleared the pathogen from the gut lumen. sIgA limited pathogen access to the mucosal surface and protected from gut inflammation in challenge infections. This protection was O-antigen specific, as demonstrated with pathogens lacking the S. typhimurium O-antigen (wbaP, S. enteritidis) and sIgA-deficient mice (TCRβ(-/-)δ(-/-), J(H) (-/-), IgA(-/-), pIgR(-/-)). Surprisingly, sIgA-deficiency did not affect the kinetics of pathogen clearance from the gut lumen. Instead, this was mediated by the microbiota. This was confirmed using 'L-mice' which harbor a low complexity gut flora, lack colonization resistance and develop a normal sIgA response, but fail to clear S. tm(att) from the gut lumen. In these mice, pathogen clearance was achieved by transferring a normal complex microbiota. Thus, besides colonization resistance ( = pathogen blockage by an intact microbiota), the microbiota mediates a second, novel protective function, i.e. pathogen clearance. Here, the normal microbiota re-grows from a state of depletion and disturbed composition and gradually clears even very high pathogen loads from the gut lumen, a site inaccessible to most \"classical\" immune effector mechanisms. In conclusion, sIgA and microbiota serve complementary protective functions. The microbiota confers colonization resistance and mediates pathogen clearance in primary infections, while sIgA protects from disease if the host re-encounters the same pathogen. This has implications for curing S. typhimurium diarrhea and for preventing transmission.

The intestinal ecosystem is formed by a complex, yet highly characteristic microbial community. The parameters defining whether this community permits invasion of a new bacterial species are unclear. In particular, inhibition of enteropathogen infection by the gut microbiota ( = colonization resistance) is poorly understood. To analyze the mechanisms of microbiota-mediated protection from Salmonella enterica induced enterocolitis, we used a mouse infection model and large scale high-throughput pyrosequencing. In contrast to conventional mice (CON), mice with a gut microbiota of low complexity (LCM) were highly susceptible to S. enterica induced colonization and enterocolitis. Colonization resistance was partially restored in LCM-animals by co-housing with conventional mice for 21 days (LCM(con21)). 16S rRNA sequence analysis comparing LCM, LCM(con21) and CON gut microbiota revealed that gut microbiota complexity increased upon conventionalization and correlated with increased resistance to S. enterica infection. Comparative microbiota analysis of mice with varying degrees of colonization resistance allowed us to identify intestinal ecosystem characteristics associated with susceptibility to S. enterica infection. Moreover, this system enabled us to gain further insights into the general principles of gut ecosystem invasion by non-pathogenic, commensal bacteria. Mice harboring high commensal E. coli densities were more susceptible to S. enterica induced gut inflammation. Similarly, mice with high titers of Lactobacilli were more efficiently colonized by a commensal Lactobacillus reuteri(RR) strain after oral inoculation. Upon examination of 16S rRNA sequence data from 9 CON mice we found that closely related phylotypes generally display significantly correlated abundances (co-occurrence), more so than distantly related phylotypes. Thus, in essence, the presence of closely related species can increase the chance of invasion of newly incoming species into the gut ecosystem. We provide evidence that this principle might be of general validity for invasion of bacteria in preformed gut ecosystems. This might be of relevance for human enteropathogen infections as well as therapeutic use of probiotic commensal bacteria.