Vibrio cholerae has evolved to adeptly transition between the human small intestine and aquatic environments, leading to water-borne spread and transmission of the lethal diarrheal disease cholera. Using a host model that mimics the pathology of human cholera, we applied high density transposon mutagenesis combined with massively parallel sequencing (Tn-seq) to determine the fitness contribution of >90% of all non-essential genes of V. cholerae both during host infection and dissemination. Targeted mutagenesis and validation of 35 genes confirmed our results for the selective conditions with a total false positive rate of 4%. We identified 165 genes never before implicated for roles in dissemination that reside within pathways controlling many metabolic, catabolic and protective processes, from which a central role for glycogen metabolism was revealed. We additionally identified 76 new pathogenicity factors and 414 putatively essential genes for V. cholerae growth. Our results provide a comprehensive framework for understanding the biology of V. cholerae as it colonizes the small intestine, elicits profuse secretory diarrhea, and disseminates into the aquatic environment.

Flagellar motility is an essential mechanism by which bacteria adapt to and survive in diverse environments. Although flagella confer an advantage to many bacterial pathogens for colonization during infection, bacterial flagellins also stimulate host innate immune responses. Consequently, many bacterial pathogens down-regulate flagella production following initial infection. Listeria monocytogenes is a facultative intracellular pathogen that represses transcription of flagellar motility genes at physiological temperatures (37°C and above). Temperature-dependent expression of flagellar motility genes is mediated by the opposing activities of MogR, a DNA-binding transcriptional repressor, and DegU, a response regulator that functions as an indirect antagonist of MogR. In this study, we identify an additional component of the molecular circuitry governing temperature-dependent flagellar gene expression. At low temperatures (30°C and below), MogR repression activity is specifically inhibited by an anti-repressor, GmaR. We demonstrate that GmaR forms a stable complex with MogR, preventing MogR from binding its DNA target sites. GmaR anti-repression activity is temperature dependent due to DegU-dependent transcriptional activation of gmaR at low temperatures. Thus, GmaR production represents the first committed step for flagella production in L. monocytogenes. Interestingly, GmaR also functions as a glycosyltransferase exhibiting O-linked N-acetylglucosamine transferase (OGT) activity for flagellin (FlaA). GmaR is the first OGT to be identified and characterized in prokaryotes that specifically ␤-O-GlcNAcylates a prokaryotic protein. Unlike the well-characterized, highly conserved OGT regulatory protein in eukaryotes, the catalytic activity of GmaR is functionally separable from its anti-repression function. These results establish GmaR as the first known example of a bifunctional protein that transcriptionally regulates expression of its enzymatic substrate. Flagellar motility is a fundamental mechanism by which bacteria acquire nutrients, colonize surfaces, and establish infections. Although flagellar motility confers a growth advantage in many environments, production of flagella is a complex, energy-demanding developmental process and thus is exquisitely regulated in response to many environmental cues (Aldridge and Hughes 2002;Macnab 2003). For example, flagella can enhance adherence and invasion in the early stages of host infection, yet continuous production of flagella during infection can stimulate innate immune responses (Hayashi et al. 2001;Molofsky et al. 2006;Ren et al. 2006) or impede subsequent colonization events (for review, see Ramos et al. 2004). Thus, many facultative bacterial pathogens down-regulate production of flagella shortly after infection (Akerley et al. 1995;Hughes and Galan 2002). A primary environmental cue that initiates repression of flagellar gene transcription during infection is physiological temperature (37°C) (Ott et al. 1991;Akerley and Miller 1993;Kapatral et al. 1996).Listeria...

Facultative bacterial pathogens must adapt to multiple stimuli to persist in the environment or establish infection within a host. Temperature is often utilized as a signal to control expression of virulence genes necessary for infection or genes required for persistence in the environment. However, very little is known about the molecular mechanisms that allow bacteria to adapt and respond to temperature fluctuations. Listeria monocytogenes (Lm) is a food-borne, facultative intracellular pathogen that uses flagellar motility to survive in the extracellular environment and to enhance initial invasion of host cells during infection. Upon entering the host, Lm represses transcription of flagellar motility genes in response to mammalian physiological temperature (37°C) with a concomitant temperature-dependent up-regulation of virulence genes. We previously determined that down-regulation of flagellar motility is required for virulence and is governed by the reciprocal activities of the MogR transcriptional repressor and the bifunctional flagellar anti-repressor/glycosyltransferase, GmaR. In this study, we determined that GmaR is also a protein thermometer that controls temperature-dependent transcription of flagellar motility genes. Two-hybrid and gel mobility shift analyses indicated that the interaction between MogR and GmaR is temperature sensitive. Using circular dichroism and limited proteolysis, we determined that GmaR undergoes a temperature-dependent conformational change as temperature is elevated. Quantitative analysis of GmaR in Lm revealed that GmaR is degraded in the absence of MogR and at 37°C (when the MogR:GmaR complex is less stable). Since MogR represses transcription of all flagellar motility genes, including transcription of gmaR, changes in the stability of the MogR:GmaR anti-repression complex, due to conformational changes in GmaR, mediates repression or de-repression of flagellar motility genes in Lm. Thus, GmaR functions as a thermo-sensing anti-repressor that incorporates temperature signals into transcriptional control of flagellar motility. To our knowledge, this is the first example of a protein thermometer that functions as an anti-repressor to control a developmental process in bacteria.

SummaryFlagellar motility in Listeria monocytogenes (Lm) is restricted to temperatures below 37°C due to the opposing activities of the MogR transcriptional repressor and the GmaR antirepressor. Previous studies have suggested that both the DegU response regulator and MogR regulate expression of GmaR. In this report, we further define the role of DegU for GmaR production and flagellar motility. We demonstrate that deletion of the receiver domain of DegU has no effect on flagellar motility in Lm. Using transcriptional reporter fusions, we determined that gmaR is cotranscribed within an operon initiating with fliN. Furthermore, the fliN-gmaR promoter (p fliN-gmaR) is transcriptionally activated by DegU and is also MogR-repressed. DNA affinity purification, gel mobility shift and footprinting analyses revealed that both DegU and MogR directly bind fliN-gmaR promoter region DNA and that the binding sites do not overlap. Quantitative analysis of gmaR transcripts in DmogR bacteria indicated that transcriptional activation of pfliN-gmaR by DegU is not inherently temperature-dependent. However, GmaR protein was not detectable at 37°C in DmogR bacteria, indicating that a temperature-dependent, posttranscriptional mechanism limits GmaR production to temperatures below 37°C. Our findings reveal that flagellar motility in Lm is governed by both temperature-dependent transcriptional and posttranscriptional regulation of the GmaR antirepressor.

A growing global health concern, Lyme disease has become the most common tick-borne disease in the United States and Europe. Caused by the bacterial spirochete Borrelia burgdorferi sensu lato (sl), this disease can be debilitating if not treated promptly. Because diagnosis is challenging, prevention remains a priority; however, a previously licensed vaccine is no longer available to the public. Here, we designed a six component vaccine that elicits antibody (Ab) responses against all Borrelia strains that commonly cause Lyme disease in humans. The outer surface protein A (OspA) of Borrelia was fused to a bacterial ferritin to generate selfassembling nanoparticles. OspA-ferritin nanoparticles elicited durable high titer Ab responses to the seven major serotypes in mice and non-human primates at titers higher than a previously licensed vaccine. This response was durable in rhesus macaques for more than 6 months. Vaccination with adjuvanted OspA-ferritin nanoparticles stimulated protective immunity from both B. burgdorferi and B. afzelii infection in a tick-fed murine challenge model. This multivalent Lyme vaccine offers the potential to limit the spread of Lyme disease.

Pyrazolopyrimidinediones are a novel series of compounds that inhibit growth of Helicobacter pylori specifically. Using a variety of methods, advanced analogues were shown to suppress the growth of H. pylori through the inhibition of glutamate racemase, an essential enzyme in peptidoglycan biosynthesis. The high degree of selectivity of the series for H. pylori makes these compounds attractive candidates for novel H. pylori-selective therapy.Helicobacter pylori is a gram-negative pathogen whose colonization of the human gastric mucosa can cause gastritis that can lead to peptic ulceration (23). The organism is also implicated as a causative agent for certain types of gastric cancer (20,23,24,27), and therefore, eradication of the organism is advised for people with ulcer disease (5). Recommended therapies consist of a proton pump inhibitor in combination with broad-spectrum antibacterials, but emerging resistance and poor patient compliance compromise the effectiveness of these treatments (23). Thus, alternative therapies without these issues are needed for continued successful eradication of H. pylori in patients.The non-life-threatening nature and unique disease manifestation of H. pylori infections allow highly selective therapy directed only against the specific organism. The advantage of such a selective therapy would be to limit adverse effects caused by disturbances in the microbial gut flora, thereby improving patient compliance and reducing selection for resistance in other species.A target-based research program that integrated genetic, biochemical, biophysical, and structural characterization of targets was undertaken to identify selective targets for therapy directed against H. pylori (17). Glutamate racemase (MurI), an essential enzyme in peptidoglycan biosynthesis (11,12,25) (Fig. 1), was identified through these efforts as a potentially selective target for therapy directed against H. pylori. A highthroughput screen was carried out with the enzyme, and pyrazolopyrimidinediones were identified as a class of selective H. pylori MurI inhibitors that also showed whole-cell activity (17). This study describes the microbiological characterization of this class of inhibitors.(These studies were presented in part at the 2005 Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC [10a].) MATERIALS AND METHODSBacterial strains and susceptibility testing. The bacterial strains, plasmids, and primers used in this study are listed in Table 1. Susceptibilities for H. pylori, Campylobacter coli, and Campylobacter jejuni were determined as described previously (16). MICs for anaerobic species were determined according to CLSI broth microdilution guidelines for Bacteroides fragilis (9). MICs for all other species were determined according to CLSI guidelines (10). Compounds were dissolved in dimethyl sulfoxide, and the final concentration of this solvent in all MIC assays was 2%, a concentration that was used as control.Frequency of spontaneous resistance development. Spontaneous frequenci...

SummaryPhosphate is essential for life, being used in many core processes such as signal transduction and synthesis of nucleic acids. The waterborne agent of cholera, V ibrio cholerae, encounters phosphate limitation in both the aquatic environment and human intestinal tract. This bacterium can utilize extracellular DNA (eDNA) as a phosphate source, a phenotype dependent on secreted endo‐ and exonucleases. However, no transporter of nucleotides has been identified in V . cholerae, suggesting that in order for the organism to utilize the DNA as a phosphate source, it must first separate the phosphate and nucleoside groups before transporting phosphate into the cell. In this study, we investigated the factors required for assimilation of phosphate from eDNA. We identified PhoX, and the previously unknown proteins UshA and CpdB as the major phosphatases that allow phosphate acquisition from eDNA and nucleotides. We demonstrated separable but partially overlapping roles for the three phosphatases and showed that the activity of PhoX and CpdB is induced by phosphate limitation. Thus, this study provides mechanistic insight into how V . cholerae can acquire phosphate from extracellular DNA, which is likely to be an important phosphate source in the environment and during infection.

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