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.

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.

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.

In Vibrio parahaemolyticus, scrC participates in controlling the decision to be a highly mobile swarmer cell or a more adhesive, biofilm-proficient cell type. scrC mutants display decreased swarming motility over surfaces and enhanced capsular polysaccharide production. ScrC is a cytoplasmic membrane protein that contains both GGDEF and EAL conserved protein domains. These domains have been shown in many organisms to respectively control the formation and degradation of the small signaling nucleotide cyclic dimeric GMP (c-di-GMP). The scrC gene is part of the three-gene scrABC operon. Here we report that this operon influences the cellular nucleotide pool and that c-di-GMP levels inversely modulate lateral flagellar and capsular polysaccharide gene expression. High concentrations of this nucleotide prevent swarming and promote adhesiveness. Further, we demonstrate that ScrC has intrinsic diguanylate cyclase and phosphodiesterase activities, and these activities are controlled by ScrAB. Specifically, ScrC acts to form c-di-GMP in the absence of ScrA and ScrB; whereas ScrC acts to degrade c-di-GMP in the presence of ScrA and ScrB. The scrABC operon is specifically induced by growth on a surface, and the analysis of mutant phenotypes supports a model in which the phosphodiesterase activity of ScrC plays a dominant role during surface translocation and in biofilms.Vibrio parahaemolyticus possesses a mode of motility, called swarming, that is driven by a lateral flagellar system (laf) and is suited to rapid colonization of surfaces; the organism can also elaborate a capsular polysaccharide (cps) that enables robust biofilm formation (14,29). How a cell varies its surface molecules to adapt and proliferate in specific environments is one key survival strategy. In V. parahaemolyticus, the scrABC locus modulates the lifestyle adaptation between swarming and sticking by participating in swarming and capsular polysaccharide gene regulation (7).The scrABC operon encodes three proteins: ScrA, a potential pyridoxal-phosphate-dependent enzyme; ScrB, a potential extracellular solute-binding protein; and ScrC, a potential sensory protein (7). Fractionation experiments localize ScrA in the cytoplasm, ScrB in the periplasm, and ScrC with the cell membrane. The ScrC N terminus has two predicted transmembrane domains flanking a potential periplasmic region (ϳ300 amino acids [aa] in length). The C terminus of ScrC contains both GGDEF and EAL domains and is cytoplasmically located. These two conserved domains, named after signature amino acid motifs, are found in diguanylate cyclases and phosphodiesterases and are responsible for the formation (GGDEF) and degradation (EAL) of the nucleotide bis-3Ј,5Ј cyclic dimeric GMP (c-di-GMP) (reviewed in reference 23). The role of c-di-GMP as a signaling molecule is being described in an expanding list of organisms (reviewed in references 13, 16, 38, and 39). Enzymes controlling the level of c-di-GMP, including proteins with GGDEF and EAL domains, as well as the phosphohydrolase-associated HD-GY...

The intestinal microbiota is composed of hundreds of species of bacteria, fungi and protozoa and is critical for numerous biological processes, such as nutrient acquisition, vitamin production, and colonization resistance against bacterial pathogens. We studied the role of the intestinal microbiota on host resistance to Salmonella enterica serovar Typhimurium-induced colitis. Using multiple antibiotic treatments in 129S1/SvImJ mice, we showed that disruption of the intestinal microbiota alters host susceptibility to infection. Although all antibiotic treatments caused similar increases in pathogen colonization, the development of enterocolitis was seen only when streptomycin or vancomycin was used; no significant pathology was observed with the use of metronidazole. Interestingly, metronidazole-treated and infected C57BL/6 mice developed severe pathology. We hypothesized that the intestinal microbiota confers resistance to infectious colitis without affecting the ability of S. Typhimurium to colonize the intestine. Indeed, different antibiotic treatments caused distinct shifts in the intestinal microbiota prior to infection. Through fluorescence in situ hybridization, terminal restriction fragment length polymorphism, and real-time PCR, we showed that there is a strong correlation between the intestinal microbiota composition before infection and susceptibility to Salmonella-induced colitis. Members of the Bacteroidetes phylum were present at significantly higher levels in mice resistant to colitis. Further analysis revealed that Porphyromonadaceae levels were also increased in these mice. Conversely, there was a positive correlation between the abundance of Lactobacillus sp. and predisposition to colitis. Our data suggests that different members of the microbiota might be associated with S. Typhimurium colonization and colitis. Dissecting the mechanisms involved in resistance to infection and inflammation will be critical for the development of therapeutic and preventative measures against enteric pathogens.

We recently showed that Mycobacterium leprae (ML) is able to induce lipid droplet formation in infected macrophages. We herein confirm that cholesterol (Cho) is one of the host lipid molecules that accumulate in ML-infected macrophages and investigate the effects of ML on cellular Cho metabolism responsible for its accumulation. The expression levels of LDL receptors (LDL-R, CD36, SRA-1, SR-B1, and LRP-1) and enzymes involved in Cho biosynthesis were investigated by qRT-PCR and/or Western blot and shown to be higher in lepromatous leprosy (LL) tissues when compared to borderline tuberculoid (BT) lesions. Moreover, higher levels of the active form of the sterol regulatory element-binding protein (SREBP) transcriptional factors, key regulators of the biosynthesis and uptake of cellular Cho, were found in LL skin biopsies. Functional in vitro assays confirmed the higher capacity of ML-infected macrophages to synthesize Cho and sequester exogenous LDL-Cho. Notably, Cho colocalized to ML-containing phagosomes, and Cho metabolism impairment, through either de novo synthesis inhibition by statins or depletion of exogenous Cho, decreased intracellular bacterial survival. These findings highlight the importance of metabolic integration between the host and bacteria to leprosy pathophysiology, opening new avenues for novel therapeutic strategies to leprosy.

The Vibrio fischeri quorum-sensing signal N-3-oxohexanoyl-L-homoserine lactone (3OC6-HSL) activates expression of the seven-gene luminescence operon. We used microarrays to unveil 18 additional 3OC6-HSLcontrolled genes, 3 of which had been identified by other means previously. We show most of these genes are regulated by the 3OC6-HSL-responsive transcriptional regulator LuxR directly. This demonstrates that V. fischeri quorum sensing regulates a substantial number of genes other than those involved in light production.Quorum sensing allows a species to measure its population density and control gene expression in a population densitydependent manner. Bacterial quorum sensing involves cell-cell communication mediated by extracellular signal compounds. Various species of proteobacteria use acyl-homoserine lactones (acyl-HSLs) as quorum-sensing signals (2, 12-15, 36, 42). Acyl-HSL quorum sensing was first described in the marine luminescent bacterium Vibrio fischeri, where it controls transcription of the luminescence (lux) operon (7, 10, 11). The LuxI acyl-HSL synthase and the LuxR transcriptional activator constitute the quorum-sensing system, which controls the lux operon directly. The LuxI-generated signal is N-3-oxohexanoyl-L-HSL (3OC6-HSL), and LuxR activates the lux operon in response to this signal. The LuxR-3OC6-HSL complex binds to a 20-bp inverted repeat centered at Ϫ42.5 from the transcriptional start site of the lux operon (for reviews, see references 14, 36, and 41).Vibrio fischeri occurs at low population densities in seawater and at high densities in specific light organ symbioses with certain fish and squid (3,21,29). Quorum sensing allows V. fischeri to discern its existence in the symbiosis and activate transcription of the lux operon (4). Many proteobacteria have acyl-HSL signaling systems, which serve as global regulators of extracellular factor production and are often important for successful interactions with plant or animal hosts (26,36). Existing evidence shows that besides the lux operon, LuxR and 3OC6-HSL activate five other genes (5). However, the search for quorum-controlled genes in V. fischeri has been limited to proteomic analyses. We recently reported the complete genome sequence of a strain of V. fischeri isolated from a squid light organ (30). Thus, it is now possible to use DNA microarrays to identify LuxR-regulated V. fischeri genes on a global scale. We have designed and constructed a microarray and used it to perform an analysis of 3OC6-HSL-regulated genes in V. fischeri. We furthered this analysis by examining the activity of relevant promoters in response to 3OC6-HSL and LuxR in recombinant Escherichia coli and by analyzing the binding of LuxR to promoter DNA fragments in vitro.We used E. coli DH12S (Invitrogen, Carlsbad, CA) and V. fischeri ES114 (3). The genome sequence of V. fischeri ES114 is publicly available at http://www.ergo-light.com (30). We used Luria-Bertani broth (31) without added sodium chloride to grow E. coli, at 30°C, and seawater-tryptone broth (3) with s...

The Vibrio parahaemolyticus Scr system modulates decisions pertinent to surface colonization by affecting the cellular level of cyclic dimeric GMP (c-di-GMP). In this work, we explore the scope and mechanism of this regulation. Transcriptome comparison of ⌬scrABC and wild-type strains revealed expression differences with respect to ϳ100 genes. Elevated c-di-GMP repressed genes in the surface-sensing regulon, including those encoding the lateral flagellar and type III secretion systems and N-acetylglucosamine-binding protein GpbA while inducing genes encoding other cell surface molecules and capsular polysaccharide. The transcription of a few regulatory genes was also affected, and the role of one was characterized. Mutations in cpsQ suppressed the sticky phenotype of scr mutants. cpsQ encodes one of four V. parahaemolyticus homologs in the CsgD/VpsT family, members of which have been implicated in c-di-GMP signaling. Here, we demonstrate that CpsQ is a c-di-GMP-binding protein. By using a combination of mutant and reporter analyses, CpsQ was found to be the direct, positive regulator of cpsA transcription. This c-di-GMP-responsive regulatory circuit could be reconstituted in Escherichia coli, where a low level of this nucleotide diminished the stability of CpsQ. The molecular interplay of additional known cps regulators was defined by establishing that CpsS, another CsgD family member, repressed cpsR, and the transcription factor CpsR activated cpsQ. Thus, we are developing a connectivity map of the Scr decision-making network with respect to its wiring and output strategies for colonizing surfaces and interaction with hosts; in doing so, we have isolated and reproduced a c-di-GMP-sensitive regulatory module in the circuit. Cyclic dimeric GMP (c-di-GMP) plays a determining role in diverse adaptations of many bacteria, often by moderating switches between biofilm and planktonic lifestyles (reviewed in references 16 and 32). In Vibrio parahaemolyticus, this second messenger also participates in modulating choices; however, for this organism, we know that c-di-GMP plays a key role in influencing lifestyle decisions during growth on surfaces. In particular, it participates in the decision-making processes determining biofilm development or the profound differentiation events leading to swarming that occur during growth on surfaces. A high concentration of c-di-GMP impairs surface motility and promotes biofilm formation, whereas a smaller amount of this second messenger favors swarming. In part, the concentration of c-di-GMP that reciprocally influences swarming and capsule production is established by the membrane-bound ScrC enzyme, which contains functional GGDEF and EAL domains. Proteins with the GGDEF domain are responsible for the formation of c-di-GMP, whereas EAL or HD-GYP domain-containing proteins participate in the degradation of the second messenger (reviewed in references 12 and 18). During swarming, expression of the scrABC operon is upregulated. The enzyme ScrA produces the S signal, an autoinducer sign...