The role of two-component regulatory systems in environmental sensing and virulence in Salmonella.

Adaptation to environments with constant fluctuations imposes challenges that are only overcome with sophisticated strategies that allow bacteria to perceive environmental conditions and develop an appropriate response. The gastrointestinal environment is a complex ecosystem that is home to trillions of microorganisms. Termed microbiota, this microbial ensemble plays important roles in host health and provides colonization resistance against pathogens, although pathogens have evolved strategies to circumvent this barrier. Among the strategies used by bacteria to monitor their environment, one of the most important are the sensing and signalling machineries of two-component systems (TCSs), which play relevant roles in the behaviour of all bacteria. Salmonella enterica is no exception, and here we present our current understanding of how this important human pathogen uses TCSs as an integral part of its lifestyle. We describe important aspects of these systems, such as the stimuli and responses involved, the processes regulated, and their roles in virulence. We also dissect the genomic organization of histidine kinases and response regulators, as well as the input and output domains for each TCS. Lastly, we explore how these systems may be promising targets for the development of antivirulence therapeutics to combat antibiotic-resistant infections.

Gut microbiota in health and disease.

Gut microbiota is an assortment of microorganisms inhabiting the length and width of the mammalian gastrointestinal tract. The composition of this microbial community is host specific, evolving throughout an individual's lifetime and susceptible to both exogenous and endogenous modifications. Recent renewed interest in the structure and function of this "organ" has illuminated its central position in health and disease. The microbiota is intimately involved in numerous aspects of normal host physiology, from nutritional status to behavior and stress response. Additionally, they can be a central or a contributing cause of many diseases, affecting both near and far organ systems. The overall balance in the composition of the gut microbial community, as well as the presence or absence of key species capable of effecting specific responses, is important in ensuring homeostasis or lack thereof at the intestinal mucosa and beyond. The mechanisms through which microbiota exerts its beneficial or detrimental influences remain largely undefined, but include elaboration of signaling molecules and recognition of bacterial epitopes by both intestinal epithelial and mucosal immune cells. The advances in modeling and analysis of gut microbiota will further our knowledge of their role in health and disease, allowing customization of existing and future therapeutic and prophylactic modalities.

Repression of Salmonella Host Cell Invasion by Aromatic Small Molecules from the Human Fecal Metabolome.

The human microbiome is a collection of microorganisms that inhabit every surface of the body that is exposed to the environment, generally coexisting peacefully with their host. These microbes have important functions, such as producing vitamins, aiding in maturation of the immune system, and protecting against pathogens. We have previously shown that a small-molecule extract from the human fecal microbiome has a strong repressive effect on Salmonella enterica serovar Typhimurium host cell invasion by modulating the expression of genes involved in this process. Here, we describe the characterization of this biological activity. Using a series of purification methods, we obtained fractions with biological activity and characterized them by mass spectrometry. These experiments revealed an abundance of aromatic compounds in the bioactive fraction. Selected compounds were obtained from commercial sources and tested with respect to their ability to repress the expression of hilA, the gene encoding the master regulator of invasion genes in Salmonella We found that the aromatic compound 3,4-dimethylbenzoic acid acts as a strong inhibitor of hilA expression and of invasion of cultured host cells by Salmonella Future studies should reveal the molecular details of this phenomenon, such as the signaling cascades involved in sensing this bioactive molecule.IMPORTANCE Microbes constantly sense and adapt to their environment. Often, this is achieved through the production and sensing of small extracellular molecules. The human body is colonized by complex communities of microbes, and, given their biological and chemical diversity, these ecosystems represent a platform where the production and sensing of molecules occur. In previous work, we showed that small molecules produced by microbes from the human gut can significantly impair the virulence of the enteric pathogen Salmonella enterica Here, we describe a specific compound from the human gut that produces this same effect. The results from this work not only shed light on an important biological phenomenon occurring in our bodies but also may represent an opportunity to develop drugs that can target these small-molecule interactions to protect us from enteric infections and other diseases.

Enterohepatic bacterial infections dysregulate the FGF15-FGFR4 endocrine axis.

BACKGROUND: Enterohepatic bacterial infections have the potential to affect multiple physiological processes of the body. Fibroblast growth factor 15/19 (FGF15 in mice, FGF19 in humans) is a hormone that functions as a central regulator of glucose, lipid and bile acid metabolism. FGF15/19 is produced in the intestine and exert its actions on the liver by signaling through the FGFR4-ßKlotho receptor complex. Here, we examined the in vivo effects of enterohepatic bacterial infection over the FGF15 endocrine axis. RESULTS: Infection triggered significant reductions in the intestinal expression of Fgf15 and its hepatic receptor components (Fgfr4 and Klb (ßKlotho)). Infection also resulted in alterations of the expression pattern of genes involved in hepatobiliary function, marked reduction in gallbladder bile volumes and accumulation of hepatic cholesterol and triglycerides. The decrease in ileal Fgf15 expression was associated with liver bacterial colonization and hepatobiliary pathophysiology rather than with direct intestinal bacterial pathogenesis. CONCLUSIONS: Bacterial pathogens of the enterohepatic system can disturb the homeostasis of the FGF15/19-FGFR4 endocrine axis. These results open up a possible link between FGF15/19-FGFR4 disruptions and the metabolic and nutritional disorders observed in infectious diseases.

Unlocking the microbiome.

Individual species of bacteria and yeast present in the food of wild fruit flies work together to provide the nutrients needed for larval growth.

Repression of Salmonella enterica phoP expression by small molecules from physiological bile.

Infection with Salmonella enterica serovar Typhi in humans causes the life-threatening disease typhoid fever. In the laboratory, typhoid fever can be modeled through the inoculation of susceptible mice with Salmonella enterica serovar Typhimurium. Using this murine model, we previously characterized the interactions between Salmonella Typhimurium and host cells in the gallbladder and showed that this pathogen can successfully invade gallbladder epithelial cells and proliferate. Additionally, we showed that Salmonella Typhimurium can use bile phospholipids to grow at high rates. These abilities are likely important for quick colonization of the gallbladder during typhoid fever and further pathogen dissemination through fecal shedding. To further characterize the interactions between Salmonella and the gallbladder environment, we compared the transcriptomes of Salmonella cultures grown in LB broth or physiological murine bile. Our data showed that many genes involved in bacterial central metabolism are affected by bile, with the citric acid cycle being repressed and alternative respiratory systems being activated. Additionally, our study revealed a new aspect of Salmonella interactions with bile through the identification of the global regulator phoP as a bile-responsive gene. Repression of phoP expression could also be achieved using physiological, but not commercial, bovine bile. The biological activity does not involve PhoPQ sensing of a bile component and is not caused by bile acids, the most abundant organic components of bile. Bioactivity-guided purification allowed the identification of a subset of small molecules from bile that can elicit full activity; however, a single compound with phoP inhibitory activity could not be isolated, suggesting that multiple molecules may act in synergy to achieve this effect. Due to the critical role of phoP in Salmonella virulence, further studies in this area will likely reveal aspects of the interaction between Salmonella and bile that are relevant to disease.

Polymyxin Resistance in Clinical Isolates of K. pneumoniae in Brazil: Update on Molecular Mechanisms, Clonal Dissemination and Relationship With KPC-Producing Strains.

In Brazil, the production of KPC-type carbapenemases in Enterobacteriales is endemic, leading to widespread use of polymyxins. In the present study, 502 Klebsiella pneumoniae isolates were evaluated for resistance to polymyxins, their genetic determinants and clonality, in addition to the presence of carbapenem resistance genes and evaluation of antimicrobial resistance. Resistance to colistin (polymyxin E) was evaluated through initial selection on EMB agar containing 4% colistin sulfate, followed by Minimal Inhibitory Concentration (MIC) determination by broth microdilution. The susceptibility to 17 antimicrobials was assessed by disk diffusion. The presence of blaKPC, blaNDM and blaOXA-48-like carbapenemases was investigated by phenotypic methods and conventional PCR. Molecular typing was performed by PFGE and MLST. Allelic variants of the mcr gene were screened by PCR and chromosomal mutations in the pmrA, pmrB, phoP, phoQ and mgrB genes were investigated by sequencing. Our work showed a colistin resistance frequency of 29.5% (n = 148/502) in K. pneumoniae isolates. Colistin MICs from 4 to >128 µg/mL were identified (MIC50 = 64 µg/mL; MIC90 >128 µg/mL). All isolates were considered MDR, with the lowest resistance rates observed for amikacin (34.4%), and 19.6% of the isolates were resistant to all tested antimicrobials. The blaKPC gene was identified in 77% of the isolates, in consonance with the high rate of resistance to polymyxins related to its use as a therapeutic alternative. Through XbaI-PFGE, 51 pulsotypes were identified. MLST showed 21 STs, with ST437, ST258 and ST11 (CC11) being the most prevalent, and two new STs were determined: ST4868 and ST4869. The mcr-1 gene was identified in 3 K. pneumoniae isolates. Missense mutations in chromosomal genes were identified, as well as insertion sequences in mgrB. Furthermore, the identification of chromosomal mutations in K. pneumoniae isolates belonging from CC11 ensures its success as a high-risk epidemic clone in Brazil and worldwide.

Metabolomics reveals phospholipids as important nutrient sources during Salmonella growth in bile in vitro and in vivo.

During the colonization of hosts, bacterial pathogens are presented with many challenges that must be overcome for colonization to occur successfully. This requires the bacterial sensing of the surroundings and adaptation to the conditions encountered. One of the major impediments to the pathogen colonization of the mammalian gastrointestinal tract is the antibacterial action of bile. Salmonella enterica serovar Typhimurium has specific mechanisms involved in resistance to bile. Additionally, Salmonella can successfully multiply in bile, using it as a source of nutrients. This accomplishment is highly relevant to pathogenesis, as Salmonella colonizes the gallbladder of hosts, where it can be carried asymptomatically and promote further host spread and transmission. To gain insights into the mechanisms used by Salmonella to grow in bile, we studied the changes elicited by Salmonella in the chemical composition of bile during growth in vitro and in vivo through a metabolomics approach. Our data suggest that phospholipids are an important source of carbon and energy for Salmonella during growth in the laboratory as well as during gallbladder infections of mice. Further studies in this area will generate a better understanding of how Salmonella exploits this generally hostile environment for its own benefit.

Impact of salmonella infection on host hormone metabolism revealed by metabolomics.

The interplay between pathogens and their hosts has been studied for decades using targeted approaches, such as the analysis of mutants and host immunological responses. Although much has been learned from such studies, they focus on individual pathways and fail to reveal the global effects of infection on the host. To alleviate this issue, high-throughput methods, such as transcriptomics and proteomics, have been used to study host-pathogen interactions. Recently, metabolomics was established as a new method to study changes in the biochemical composition of host tissues. We report a metabolomic study of Salmonella enterica serovar Typhimurium infection. Our results revealed that dozens of host metabolic pathways are affected by Salmonella in a murine infection model. In particular, multiple host hormone pathways are disrupted. Our results identify unappreciated effects of infection on host metabolism and shed light on mechanisms used by Salmonella to cause disease and by the host to counter infection.

The deubiquitinase activity of the Salmonella pathogenicity island 2 effector, SseL, prevents accumulation of cellular lipid droplets.

To cause disease, Salmonella enterica serovar Typhimurium requires two type III secretion systems that are encoded by Salmonella pathogenicity islands 1 and 2 (SPI-1 and -2). These secretion systems serve to deliver specialized proteins (effectors) into the host cell cytosol. While the importance of these effectors to promote colonization and replication within the host has been established, the specific roles of individual secreted effectors in the disease process are not well understood. In this study, we used an in vivo gallbladder epithelial cell infection model to study the function of the SPI-2-encoded type III effector, SseL. The deletion of the sseL gene resulted in bacterial filamentation and elongation and the unusual localization of Salmonella within infected epithelial cells. Infection with the ΔsseL strain also caused dramatic changes in host cell lipid metabolism and led to the massive accumulation of lipid droplets in infected cells. This phenotype was directly attributable to the deubiquitinase activity of SseL, as a Salmonella strain carrying a single point mutation in the catalytic cysteine also resulted in extensive lipid droplet accumulation. The excessive buildup of lipids due to the absence of a functional sseL gene also was observed in murine livers during S. Typhimurium infection. These results suggest that SseL alters host lipid metabolism in infected epithelial cells by modifying the ubiquitination patterns of cellular targets.

Effect of antibiotic treatment on the intestinal metabolome.

The importance of the mammalian intestinal microbiota to human health has been intensely studied over the past few years. It is now clear that the interactions between human hosts and their associated microbial communities need to be characterized in molecular detail if we are to truly understand human physiology. Additionally, the study of such host-microbe interactions is likely to provide us with new strategies to manipulate these complex systems to maintain or restore homeostasis in order to prevent or cure pathological states. Here, we describe the use of high-throughput metabolomics to shed light on the interactions between the intestinal microbiota and the host. We show that antibiotic treatment disrupts intestinal homeostasis and has a profound impact on the intestinal metabolome, affecting the levels of over 87% of all metabolites detected. Many metabolic pathways that are critical for host physiology were affected, including bile acid, eicosanoid, and steroid hormone synthesis. Dissecting the molecular mechanisms involved in the impact of beneficial microbes on some of these pathways will be instrumental in understanding the interplay between the host and its complex resident microbiota and may aid in the design of new therapeutic strategies that target these interactions.

Metabolic profiles of multidrug resistant and extensively drug resistant Mycobacterium tuberculosis unveiled by metabolomics.

Although treatable with antibiotics, tuberculosis is a leading cause of death. Mycobacterium tuberculosis antibiotic resistance is becoming increasingly common and disease control is challenging. Conventional drug susceptibility testing takes weeks to produce results, and treatment is often initiated empirically. Therefore, new methods to determine drug susceptibility profiles are urgent. Here, we used mass-spectrometry-based metabolomics to characterize the metabolic landscape of drug-susceptible (DS), multidrug-resistant (MDR) and extensively drug-resistant (XDR) M. tuberculosis. Direct infusion mass spectrometry data showed that DS, MDR, and XDR strains have distinct metabolic profiles, which can be used to predict drug susceptibility and resistance. This was later confirmed by Ultra-High-Performance Liquid Chromatography and High-Resolution Mass Spectrometry, where we found that levels of ions presumptively identified as isoleucine, proline, hercynine, betaine, and pantothenic acid varied significantly between strains with different drug susceptibility profiles. We then confirmed the identification of proline and isoleucine and determined their absolute concentrations in bacterial extracts, and found significantly higher levels of these amino acids in DS strains, as compared to drug-resistant strains (combined MDR and XDR strains). Our results advance the current understanding of the effect of drug resistance on bacterial metabolism and open avenues for the detection of drug resistance biomarkers.

Quorum sensing in bacterial virulence.

Bacteria communicate through the production of diffusible signal molecules termed autoinducers. The molecules are produced at basal levels and accumulate during growth. Once a critical concentration has been reached, autoinducers can activate or repress a number of target genes. Because the control of gene expression by autoinducers is cell-density-dependent, this phenomenon has been called quorum sensing. Quorum sensing controls virulence gene expression in numerous micro-organisms. In some cases, this phenomenon has proven relevant for bacterial virulence in vivo. In this article, we provide a few examples to illustrate how quorum sensing can act to control bacterial virulence in a multitude of ways. Several classes of autoinducers have been described to date and we present examples of how each of the major types of autoinducer can be involved in bacterial virulence. As quorum sensing controls virulence, it has been considered an attractive target for the development of new therapeutic strategies. We discuss some of the new strategies to combat bacterial virulence based on the inhibition of bacterial quorum sensing systems.

Inhibition of Salmonella host cell invasion by dimethyl sulfide.

We show that dimethyl sulfoxide (DMSO) inhibits Salmonella hilA expression and that this inhibition is stronger under anaerobiosis. Because DMSO can be reduced to dimethyl sulfide (DMS) during anaerobic growth, we hypothesized that DMS was responsible for hilA inhibition. Indeed, DMS strongly inhibited the expression of hilA and multiple Salmonella pathogenicity island 1 (SPI-1)-associated genes as well as the invasion of cultured epithelial cells. Because DMSO and DMS are widespread in nature, we hypothesize that this phenomenon may contribute to environmental sensing by Salmonella.

Intercellular communication in bacteria.

Bacteria have been long considered primitive organisms, with a lifestyle focused on the survival and propagation of single cells. However, in the past few decades it became obvious that bacteria can display sophisticated group behaviors. For instance, bacteria can communicate amongst themselves and with their hosts, by producing, sensing, and responding to chemical signals. By doing so, they can sense their surroundings and adapt as to increase their chances of survival and propagation. Here, we review the discovery of bacterial intercellular communication, some of the signaling molecules identified to date, the role of intercellular signaling in symbiotic and pathogenic relationships between bacteria and their hosts and its implications for the development of new therapeutic strategies against human disease.

Extraction of Small Molecules from Fecal Samples and Testingof Their Activity on Microbial Physiology.

The human body is colonized by vast communities of microbes, collectively known as microbiota, or microbiome. Although microbes colonize every surface of our bodies that is exposed to the external environment, the biggest collection of microbes colonizing humans and other mammals can be found in the gastrointestinal tract. Given the fact that the human gut is colonized by several hundred microbial species, our group hypothesized that the chemical diversity of this environment should be significant, and that many of the molecules present in that environment would have important signaling roles. Therefore, we devised a protocol to extract these molecules from human feces and test their signaling properties. Potentially bioactive extracts can be tested through addition to culture medium and analyses of bacterial growth and gene expression, among other properties. The protocol described herein provides an easy and rapid method for the extraction and testing of metabolites from fecal samples using Salmonella enterica as a model organism. This protocol can also be adapted to the extraction of small molecules from other matrices, such as cultured mammalian cells, tissues, body fluids, and axenic microbial cultures, and the resulting extracts can be tested against various microbial species.

Integrated analysis of ethionamide resistance loci in Mycobacterium tuberculosis clinical isolates.

Tuberculosis patients taking second line drugs such as ethionamide (ETH) have often experienced previous treatment failure and usually have a complex history of disease and treatment that can span decades. Mutations in the ETH activating enzyme, EthA, confer resistance through undescribed mechanisms. To explore the impact of EthA mutations on ETH resistance, data from a total of 160 ETHR isolates was analysed. The most frequently mutated positions are within regions that display sequence conservation with the active site of OTEMO, another FAD-containing NADH-binding Baeyer-Villiger monooxygenase (BVMO), or with the sugar binding site of galectin-4N. Additionally, to look at a possible role of EthR on ETH resistance we purified an EthR mutant identified in a clinical isolate, F110L, and found it to bind the ethA-ethR intergenic region with higher affinity than the wild type regulator in gel shift assays. The ability of cyclic di-GMP to enhance DNA binding is maintained in the EthR mutant. To our knowledge, this is the first ETH resistance study that combines sequence and resistance data of clinical isolates with functional and structural information.

Detection of mycobacterial infection in non-human primates using the Xpert MTB/RIF molecular assay.

Tuberculosis is a major public health concern, and diagnostic strategies applied to animal populations are scarce. As part of ongoing efforts to control tuberculosis dissemination at our animal facility, two non-human primates (NHP, Saimiri sciureus) presenting cutaneous lesions were examined for mycobacterial infection. Both animals tested positive for acid-fast bacilli and Mycobacterium tuberculosis using a molecular assay (IS6110 PCR). Animals were euthanized and several samples were tested for M. tuberculosis using the Xpert MTB/RIF assay. Many samples were positive for M. tuberculosis and rifampicin resistance, and some produced mycobacterial growth. Oral swabs from cage mates were then tested with Xpert MTB/RIF, and the majority tested positive for M. tuberculosis and rifampicin resistance, and produced growth in culture. To our knowledge, this is the first report of multidrug-resistant mycobacterial infection in NHP. Additionally, our data shows that the Xpert MTB/RIF assay can be useful as a screening tool for tuberculosis infection in NHP.