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Burkholderia pseudomallei, the etiological agent of melioidosis in humans and animals, often occupies environmental niches and infection sites characterized by limited concentrations of oxygen. Versatile genomic features enable this pathogen to maintain its physiology and virulence under hypoxia, but the crucial regulatory networks employed to switch from oxygen dependent respiration to alternative terminal electron acceptors (TEA) like nitrate, remains poorly understood. Here, we combined a Tn5 transposon mutagenesis screen and an anaerobic growth screen to identify a two-component signal transduction system with homology to RegAB. We show that RegAB is not only essential for anaerobic growth, but also for full virulence in cell lines and a mouse infection model. Further investigations of the RegAB regulon, using a global transcriptomic approach, identified 20 additional regulators under transcriptional control of RegAB, indicating a superordinate role of RegAB in the B. pseudomallei anaerobiosis regulatory network. Of the 20 identified regulators, NarX/L and a FNR homolog were selected for further analyses and a role in adaptation to anaerobic conditions was demonstrated. Growth experiments identified nitrate and intermediates of the denitrification process as the likely signal activateing RegAB, NarX/L, and probably of the downstream regulators Dnr or NsrR homologs. While deletions of individual genes involved in the denitrification process demonstrated their important role in anaerobic fitness, they showed no effect on virulence. This further highlights the central role of RegAB as the master regulator of anaerobic metabolism in B. pseudomallei and that the complete RegAB-mediated response is required to achieve full virulence. In summary, our analysis of the RegAB-dependent modulon and its interconnected regulons revealed a key role for RegAB of B. pseudomallei in the coordination of the response to hypoxic conditions and virulence, in the environment and the host. Author summary: Oxygen often becomes a highly limiting factor within tissues, in particular during inflammation and abscesses formation, leading to hypoxic or even strictly anaerobic conditions. Consequently, B. pseudomallei, a highly neglected and difficult to treat human pathogen has to adjust its metabolism accordingly in order to survive in oxygen-restricted environments. Here, we identify key regulators of anaerobic B. pseudomallei metabolism and demonstrate the fundamental role of the RegAB redox sensing two-component signal transduction system as the master regulator of anaerobic physiology and its extensive effect on virulence. Further investigations of two RegAB-dependend redox regulators, NarL and FNR, allowed us to provide a first comprehensive insight into the complex regulatory network of anaerobic gene expression in B. pseudomallei. Integrating these results, we propose a regulatory model of gene expression under oxygen-limiting conditions in B. pseudomallei. The high level of conservation of the RegAB two-component system in the proteobacteria indicates that our findings are relevant for a broad range of bacteria including may important human and animal pathogens. [ABSTRACT FROM AUTHOR]

Most of the current knowledge on Burkholderia pseudomallei-induced inflammasome activation and cell death in macrophages is derived from murine systems. Little is known about the involved bacterial structures and mechanisms in primary human macrophages. This is of particular relevance since murine and human macrophages as well as primary cells and cell lines differ in many aspects of inflammasome activation, including the proteins involved in the recognition of bacterial patterns. In this study, we therefore aimed (i) to establish an in vitro B. pseudomallei infection model with human monocyte-derived primary macrophages from single donors as these cells more closely resemble macrophages in the human host and (ii) to analyze B. pseudomallei-triggered cell death and bacterial elimination in those cells. Our results show that B. pseudomallei-infected primary human macrophages not only release the inflammasome-independent pro-inflammatory cytokines IL-8 and TNF-α, but are also engaged in canonical inflammasome activation as evidenced by caspase-1 and gasdermin D processing. Absence of the B. pseudomallei T3SS-3 needle protein BsaL, a potent activator of the canonical inflammasome, abolished lytic cell death, reduced IL-1β release, and caspase-1 and gasdermin D processing. IFN-γ, known to promote non-canonical inflammasome activation, did not influence pyroptosis induction or IL-1β release from infected primary human macrophages. Nevertheless, it reduced intracellular B. pseudomallei loads, an effect which was partially antagonist by the inhibition of NADPH oxidase. Overall, our data implicate T3SS-3 dependent inflammasome activation and IFN-γ induced immune mechanisms as critical defense mechanisms of human macrophages against B. pseudomallei. In addition, our infection model provides a versatile tool to study human host-pathogen interactions and has the potential to elucidate the role of human individual genetic variations in B. pseudomallei infections. Author summary: Considering the constantly emerging antibiotic resistance of pathogens, comprehensive analyses of immune response mechanisms against infections are urgently needed to provide the basis for novel therapeutic strategies. Studies based on primary murine cells and cell lines of murine and human origin led to advances in the understanding of immune defense mechanisms against bacterial infections including B. pseudomallei. Nevertheless, results relying on these cell types are not always transferrable to primary human cells due to e.g. pathway alterations. We established and validated a macrophage-based model system derived from human peripheral blood monocytes, which yields high amounts of genetically identical cells more closely resembling cells found in the human host. This model system provides the basis for studying the pathogenesis of B. pseudomallei in primary human macrophages and for developing new human host directed therapies avoiding pitfalls from cell lines. Using our newly established model we demonstrate, that restriction of B. pseudomallei by primary human macrophages is mediated by T3SS-3 dependent canonical inflammasome activation and IFN-γ induced intracellular bacterial restriction. [ABSTRACT FROM AUTHOR]

Background: Melioidosis, caused by Burkholderia pseudomallei, is a severe infectious disease with high mortality rates, but is under-recognized worldwide. In endemic areas, there is a great need for simple, low-cost and rapid diagnostic tools. In a previous study we showed, that a protein multiplex array with 20 B. pseudomallei-specific antigens detects antibodies in melioidosis patients with high sensitivity and specificity. In a subsequent study the high potential of anti-B. pseudomallei antibody detection was confirmed using a rapid Hcp1 single protein-based assay. Our protein array also showed that the antibody profile varies between patients, possibly due to a combination of host factors but also antigen variations in the infecting B. pseudomallei strains. The aim of this study was to develop a rapid test, combining Hcp1 and the best performing antigens BPSL2096, BPSL2697 and BPSS0477 from our previous study, to take advantage of simultaneous antibody detection. Methods and principal findings: The 4-plex dipstick was validated with sera from 75 patients on admission plus control groups, achieving 92% sensitivity and 97–100% specificity. We then re-evaluated melioidosis sera with the 4-plex assay that were previously misclassified by the monoplex Hcp1 rapid test. 12 out of 55 (21.8%) false-negative samples were positive in our new dipstick assay. Among those, 4 sera (7.3%) were Hcp1 positive, whereas 8 (14.5%) sera remained Hcp1 negative but gave a positive reaction with our additional antigens. Conclusions: Our dipstick rapid test represents an inexpensive, standardized and simple diagnostic tool with an improved serodiagnostic performance due to multiplex detection. Each additional band on the test strip makes a false-positive result more unlikely, contributing to its reliability. Future prospective studies will seek to validate the gain in sensitivity and specificity of our multiplex rapid test approach in different melioidosis patient cohorts. Author summary: The Gram-negative environmental pathogen Burkholderia pseudomallei, causes the severe disease melioidosis. It is highly endemic in southeast Asia and northern Australia, but recent studies suggest that it is also present in many other parts of the world where it is severely underreported. The latter results from the extremely variable and non-specific clinical manifestations of the disease, lack of clinical recognition, and the global scarcity of good quality laboratories to allow diagnosis from microbiological culture. This is even more unfortunate, as early diagnosis of the disease is indispensable for an effective therapy, since B. pseudomallei is intrinsically resistant to many antibiotics used for empirical treatment in endemic areas. Therefore, the development of new, standardized and sensitive tools is of high importance for both diagnostics and epidemiology. We focused on the development of a dipstick assay, which is based on the detection of serum antibodies against four B. pseudomallei specific protein antigens. Here we present a cost effective, simple and rapid melioidosis assay with improved sensitivity that does not depend on sophisticated laboratory equipment and therefore addresses most of the before mentioned obstacles and is easy to manufacture in large scales. [ABSTRACT FROM AUTHOR]

Isolation of the soil bacterium Burkholderia pseudomallei from tropical environments is important to generate a global risk map for man and animals to acquire the infectious disease melioidosis. There is increasing evidence, that the currently recommended soil culture protocol using threonine-basal salt solution with colistin (TBSS-C50) for enrichment of B. pseudomallei and Ashdown agar for subsequent subculture lacks sensitivity. We therefore investigated, if the otherwise rarely encountered erythritol catabolism of B. pseudomallei might be exploited to improve isolation of this bacterium from soil. Based on TBSS-C50, we designed a new colistin-containing medium with erythritol as the single carbon source (EM). This medium was validated in various culture protocols by analyzing 80 soil samples from 16 different rice fields in Vietnam. B. pseudomallei enrichment was determined in all culture supernatants by a specific quantitative PCR (qPCR) targeting the type three secretion system 1. 51 out of 80 (63.8%) soil samples gave a positive qPCR signal in at least one of the culture conditions. We observed a significantly higher enrichment shown by lower median cycle threshold values for B. pseudomallei in a two-step culture with TBSS-C50 for 48 h followed by EM for 96h compared to single cultures in TBSS-C50 for either 48h or 144h (p<0.0001, respectively). Accordingly, B. pseudomallei could be isolated on Ashdown agar in 58.8% (30/51) of samples after subcultures from our novel two-step enrichment culture compared to only 9.8% (5/51) after standard enrichment with TBSS-C50 for 48h (p<0.0001) or 25.5% (13/51; p<0.01) after TBSS-C50 for 144h. In the present study, we show that specific exploitation of B. pseudomallei metabolic capabilities in enrichment protocols leads to a significantly improved isolation rate of this pathogen from soil compared to established standard procedures. Our new culture method might help to facilitate the creation of environmental risk maps for melioidosis in the future. Melioidosis is a vastly underdiagnosed infectious disease caused by the bacterial soil pathogen B. pseudomallei. Global environmental suitability for B. pseudomallei has recently been predicted to cover huge areas with three billion people potentially at risk to acquire infection. Although sensitive as well as specific molecular methods are available, cultural isolation of B. pseudomallei strains from the environment will be indispensable to verify this prediction in potential endemic, low-income countries and is a prerequisite to assess the virulence potential and genetic diversity of strains. The currently applied cultural detection lacks sensitivity, which is at least partly due to a lack in selectivity allowing other highly abundant soil microorganisms to outcompete B. pseudomallei. We therefore aimed to develop a more selective medium by taking advantage of B. pseudomallei erythritol catabolism, a metabolic pathway, which is not commonly encountered in other related bacteria. Validation of our new medium with erythritol as the single carbon source was performed by molecular and cultural detection using soil samples from rice paddies in Vietnam. We observed an enhanced enrichment and selectivity resulting in the isolation of B. pseudomallei strains from soil samples, which were otherwise culture-negative when tested by the standard method. [ABSTRACT FROM AUTHOR]

The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs) upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation. [ABSTRACT FROM AUTHOR]

The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs) upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation.