Study of <i>Salmonella</i> pathogenicity mechanisms in <i>vitro</i> and in <i>vivo</i>

Salmonella enterica subsp. enterica includes many pathogenic serovars. Models of the study are S. typhimurium and S. abortusovis, a restricted serovar to sheep. Bacterial pathogen’s transmission strategies are usually connected to pathogenesis and limited information is available about the immune response of sheep to S. abortusovis and about flagellin interaction with S. abortusovis. Knowledge about those factors are mainly based on studies of S. typhimurium. Here we try to understand the role of flagella in S. abortusovis. The idea is that S. abortusovis highly regulates expression of flagella within the host, and we performed studies to clarify how S. abortusovis evade activation of the immune system. Mutations in S. abortusovis flagella genes were generated and flagella from wild type and mutant’s strains of the two different serovars were extracted using diverse media to observe differences in flagellation and host interaction. Samples were analyzed by Western Blot to determine expression of major flagellar protein. Surface expression of flagella was verified by Flow Cytometry and by Scanning Electron Microscopy. To verify the capacity of S. typhimurium and S. abortusovis to stimulate TLR5, a colorectal carcinoma cell line (T84 cells) was treated with purified flagellin and the transcriptional induction of a CXC chemokine IL-8 was measured. We showed that S. abortusovis flagella are expressed in specific media, and they induced the transcription of IL-8 in T84 cells

Transition metals and host-microbe interactions in the inflamed intestine

Host-associated microbial communities provide critical functions for their hosts. Transition metals are essential for both the mammalian host and the majority of commensal bacteria. As such, access to transition metals is an important component of host-microbe interactions in the gastrointestinal tract. In mammals, transition metal ions are often sequestered by metal binding proteins to limit microbial access under homeostatic conditions. In response to invading pathogens, the mammalian host further decreases availability of these micronutrients by regulating their trafficking or releasing high-affinity metal chelating proteins, a process termed nutritional immunity. Bacterial pathogens have evolved several mechanisms to subvert nutritional immunity. Here, we provide an overview on how metal ion availability shapes host-microbe interactions in the gut with a particular focus on intestinal inflammatory diseases

How microbiological tests reflect bacterial pathogenesis and host adaptation

Historically, clinical microbiological laboratories have often relied on isolation of pure cultures and phenotypic testing to identify microorganisms. These clinical tests are often based on specific biochemical reactions, growth characteristics, colony morphology, and other physiological aspects. The features used for identification in clinical laboratories are highly conserved and specific for a given group of microbes. We speculate that these features might be the result of evolutionary selection and thus may reflect aspects of the life cycle of the organism and pathogenesis. Indeed, several of the metabolic pathways targeted by diagnostic tests in some cases may represent mechanisms for host colonization or pathogenesis. Examples include, but are not restricted to, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella enterica, Shigella spp., and enteroinvasive Escherichia coli (EIEC). Here, we provide an overview of how some common tests reflect molecular mechanisms of bacterial pathogenesis

Staphylococcus aureus and Staphylococcus epidermidis Virulence Strains as Causative Agents of Persistent Infections in Breast Implants.

Staphylococcus epidermidis and Staphylococcus aureus are currently considered two of the most important pathogens in nosocomial infections associated with catheters and other medical implants and are also the main contaminants of medical instruments. However because these species of Staphylococcus are part of the normal bacterial flora of human skin and mucosal surfaces, it is difficult to discern when a microbial isolate is the cause of infection or is detected on samples as a consequence of contamination. Rapid identification of invasive strains of Staphylococcus infections is crucial for correctly diagnosing and treating infections. The aim of the present study was to identify specific genes to distinguish between invasive and contaminating S. epidermidis and S. aureus strains isolated on medical devices; the majority of our samples were collected from breast prostheses. As a first step, we compared the adhesion ability of these samples with their efficacy in forming biofilms; second, we explored whether it is possible to determine if isolated pathogens were more virulent compared with international controls. In addition, this work may provide additional information on these pathogens, which are traditionally considered harmful bacteria in humans, and may increase our knowledge of virulence factors for these types of infections

Host-Derived Metabolites Modulate Transcription of Salmonella Genes Involved in l-Lactate Utilization during Gut Colonization.

During Salmonella enterica serovar Typhimurium infection, host inflammation alters the metabolic environment of the gut lumen to favor the outgrowth of the pathogen at the expense of the microbiota. Inflammation-driven changes in host cell metabolism lead to the release of l-lactate and molecular oxygen from the tissue into the gut lumen. Salmonella utilizes lactate as an electron donor in conjunction with oxygen as the terminal electron acceptor to support gut colonization. Here, we investigated transcriptional regulation of the respiratory l-lactate dehydrogenase LldD in vitro and in mouse models of Salmonella infection. The two-component system ArcAB repressed transcription of l-lactate utilization genes under anaerobic conditions in vitro The ArcAB-mediated repression of lldD transcription was relieved under microaerobic conditions. Transcription of lldD was induced by l-lactate but not d-lactate. A mutant lacking the regulatory protein LldR failed to induce lldD transcription in response to l-lactate. Furthermore, the lldR mutant exhibited reduced transcription of l-lactate utilization genes and impaired fitness in murine models of infection. These data provide evidence that the host-derived metabolites oxygen and l-lactate serve as cues for Salmonella to regulate lactate oxidation metabolism on a transcriptional level

Formate oxidation in the intestinal mucus layer enhances fitness of Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium induces intestinal inflammation to create a niche that fosters the outgrowth of the pathogen over the gut microbiota. Under inflammatory conditions, Salmonella utilizes terminal electron acceptors generated as byproducts of intestinal inflammation to generate cellular energy through respiration. However, the electron donating reactions in these electron transport chains are poorly understood. Here, we investigated how formate utilization through the respiratory formate dehydrogenase-N (FdnGHI) and formate dehydrogenase-O (FdoGHI) contribute to gut colonization of Salmonella. Both enzymes fulfilled redundant roles in enhancing fitness in a mouse model of Salmonella-induced colitis, and coupled to tetrathionate, nitrate, and oxygen respiration. The formic acid utilized by Salmonella during infection was generated by its own pyruvate-formate lyase as well as the gut microbiota. Transcription of formate dehydrogenases and pyruvate-formate lyase was significantly higher in bacteria residing in the mucus layer compared to the lumen. Furthermore, formate utilization conferred a more pronounced fitness advantage in the mucus, indicating that formate production and degradation occurred predominantly in the mucus layer. Our results provide new insights into how Salmonella adapts its energy metabolism to the local microenvironment in the gut. IMPORTANCE Bacterial pathogens must not only evade immune responses but also adapt their metabolism to successfully colonize their host. The microenvironments encountered by enteric pathogens differ based on anatomical location, such as small versus large intestine, spatial stratification by host factors, such as mucus layer and antimicrobial peptides, and distinct commensal microbial communities that inhabit these microenvironments. Our understanding of how Salmonella populations adapt its metabolism to different environments in the gut is incomplete. In the current study, we discovered that Salmonella utilizes formate as an electron donor to support respiration, and that formate oxidation predominantly occurs in the mucus layer. Our experiments suggest that spatially distinct Salmonella populations in the mucus layer and the lumen differ in their energy metabolism. Our findings enhance our understanding of the spatial nature of microbial metabolism and may have implications for other enteric pathogens as well as commensal host-associated microbial communities

Infection-associated gene regulation of L-tartrate metabolism in Salmonella enterica serovar Typhimurium.

Enteric pathogens such as Salmonella enterica serovar Typhimurium experience spatial and temporal changes to the metabolic landscape throughout infection. Host reactive oxygen and nitrogen species non-enzymatically convert monosaccharides to alpha hydroxy acids, including L-tartrate. Salmonella utilizes L-tartrate early during infection to support fumarate respiration, while L-tartrate utilization ceases at later time points due to the increased availability of exogenous electron acceptors such as tetrathionate, nitrate, and oxygen. It remains unknown how Salmonella regulates its gene expression to metabolically adapt to changing nutritional environments. Here, we investigated how the transcriptional regulation for L-tartrate metabolism in Salmonella is influenced by infection-relevant cues. L-tartrate induces the transcription of ttdBAU, genes involved in L-tartrate utilization. L-tartrate metabolism is negatively regulated by two previously uncharacterized transcriptional regulators TtdV (STM3357) and TtdW (STM3358), and both TtdV and TtdW are required for the sensing of L-tartrate. The electron acceptors nitrate, tetrathionate, and oxygen repress ttdBAU transcription via the two-component system ArcAB. Furthermore, the regulation of L-tartrate metabolism is required for optimal fitness in a mouse model of Salmonella-induced colitis. TtdV, TtdW, and ArcAB allow for the integration of two cues, i.e., substrate availability and availability of exogenous electron acceptors, to control L-tartrate metabolism. Our findings provide novel insights into how Salmonella prioritizes the utilization of different electron acceptors for respiration as it experiences transitional nutrient availability throughout infection.ImportanceBacterial pathogens must adapt their gene expression profiles to cope with diverse environments encountered during infection. This coordinated process is carried out by the integration of cues that the pathogen senses to fine-tune gene expression in a spatiotemporal manner. Many studies have elucidated the regulatory mechanisms of how Salmonella sense metabolites in the gut to activate or repress its virulence program; however, our understanding of how Salmonella coordinates its gene expression to maximize the utilization of carbon and energy sources found in transitional nutrient niches is not well understood. In this study, we discovered how Salmonella integrates two infection-relevant cues, substrate availability and exogenous electron acceptors, to control L-tartrate metabolism. From our experiments, we propose a model for how L-tartrate metabolism is regulated in response to different metabolic cues in addition to characterizing two previously unknown transcriptional regulators. This study expands our understanding of how microbes combine metabolic cues to enhance fitness during infection