Vibrio cholerae VexH encodes a multiple drug efflux pump that contributes to the production of cholera toxin and the toxin co-regulated pilus.

The resistance-nodulation-division (RND) efflux systems are ubiquitous transporters that function in antimicrobial resistance. Recent studies showed that RND systems were required for virulence factor production in Vibrio cholerae. The V. cholerae genome encodes six RND efflux systems. Three of the RND systems (VexB, VexD, and VexK) were previously shown to be redundant for in vitro resistance to bile acids and detergents. A mutant lacking the VexB, VexD, and VexK RND pumps produced wild-type levels of cholera toxin (CT) and the toxin co-regulated pilus (TCP) and was moderately attenuated for intestinal colonization. In contrast, a RND negative mutant produced significantly reduced amounts of CT and TCP and displayed a severe colonization defect. This suggested that one or more of the three uncharacterized RND efflux systems (i.e. VexF, VexH, and VexM) were required for pathogenesis. In this study, a genetic approach was used to generate a panel of V. cholerae RND efflux pump mutants in order to determine the function of VexH in antimicrobial resistance, virulence factor production, and intestinal colonization. VexH contributed to in vitro antimicrobial resistance and exhibited a broad substrate specificity that was redundant with the VexB, VexD, and VexK RND efflux pumps. These four efflux pumps were responsible for in vitro antimicrobial resistance and were required for virulence factor production and intestinal colonization. Mutation of the VexF and/or VexM efflux pumps did not affect in vitro antimicrobial resistance, but did negatively affect CT and TCP production. Collectively, our results demonstrate that the V. cholerae RND efflux pumps have redundant functions in antimicrobial resistance and virulence factor production. This suggests that the RND efflux systems contribute to V. cholerae pathogenesis by providing the bacterium with protection against antimicrobial compounds that are present in the host and by contributing to the regulated expression of virulence factors.

Visualization of murine intranasal dosing efficiency using luminescent Francisella tularensis: effect of instillation volume and form of anesthesia.

Intranasal instillation is a widely used procedure for pneumonic delivery of drugs, vaccine candidates, or infectious agents into the respiratory tract of research mice. However, there is a paucity of published literature describing the efficiency of this delivery technique. In this report we have used the murine model of tularemia, with Francisella tularensis live vaccine strain (FTLVS) infection, to evaluate the efficiency of pneumonic delivery via intranasal dosing performed either with differing instillation volumes or different types of anesthesia. FTLVS was rendered luminescent via transformation with a reporter plasmid that constitutively expressed the Photorhabdus luminescens lux operon from a Francisella promoter. We then used an IVIS Spectrum whole animal imaging system to visualize FT dissemination at various time points following intranasal instillation. We found that instillation of FT in a dose volume of 10 µl routinely resulted in infection of the upper airways but failed to initiate infection of the pulmonary compartment. Efficient delivery of FT into the lungs via intranasal instillation required a dose volume of 50 µl or more. These studies also demonstrated that intranasal instillation was significantly more efficient for pneumonic delivery of FTLVS in mice that had been anesthetized with inhaled (isoflurane) vs. parenteral (ketamine/xylazine) anesthesia. The collective results underscore the need for researchers to consider both the dose volume and the anesthesia type when either performing pneumonic delivery via intranasal instillation, or when comparing studies that employed this technique.

A galU mutant of Francisella tularensis is attenuated for virulence in a murine pulmonary model of tularemia.

BACKGROUND: A number of studies have revealed that Francisella tularensis (FT) suppresses innate immune responses such as chemokine/cytokine production and neutrophil recruitment in the lungs following pulmonary infection via an unidentified mechanism. The ability of FT to evade early innate immune responses could be a very important virulence mechanism for this highly infectious bacterial pathogen. RESULTS: Here we describe the characterization of a galU mutant strain of FT live vaccine strain (LVS). We show that the galU mutant was highly attenuated in a murine model of tularemia and elicited more robust innate immune responses than the wild-type (WT) strain. These studies document that the kinetics of chemokine expression and neutrophil recruitment into the lungs of mice challenged with the galU mutant strain are significantly more rapid than observed with WT FT, despite the fact that there were no observed differences in TLR2 or TLR4 signaling or replication/dissemination kinetics during the early stages of infection. We also show that the galU mutant had a hypercytotoxic phenotype and more rapidly induced the production of IL-1ß following infection either in vitro or in vivo, indicating that attenuation of the galU mutant strain may be due (in part) to more rapid activation of the inflammasome and/or earlier death of FT infected cells. Furthermore, we show that infection of mice with the galU mutant strain elicits protective immunity to subsequent challenge with WT FT. CONCLUSIONS: Disruption of the galU gene of FTLVS has little (if any) effect on in vivo infectivity, replication, or dissemination characteristics, but is highly attenuating for virulence. The attenuated phenotype of this mutant strain of FT appears to be related to its increased ability to induce innate inflammatory responsiveness, resulting in more rapid recruitment of neutrophils to the lungs following pneumonic infection, and/or to its ability to kill infected cells in an accelerated fashion. These results have identified two potentially important virulence mechanisms used by FT. These findings could also have implications for design of a live attenuated vaccine strain of FT because sublethal infection of mice with the galU mutant strain of FTLVS promoted development of protective immunity to WT FTLVS.

Construction of a bioluminescence reporter plasmid for Francisella tularensis.

A Francisella tularensis shuttle vector that constitutively expresses the Photorhabdus luminescens lux operon in type A and type B strains of F. tularensis was constructed. The bioluminescence reporter plasmid was introduced into the live vaccine strain of F. tularensis and used to follow F. tularensis growth in a murine intranasal challenge model in real-time by bioluminescence imaging. The results show that the new bioluminescence reporter plasmid represents a useful tool for tularemia research that is suitable for following F. tularensis growth in both in vitro and in vivo model systems.

The cyclic dipeptide cyclo(Phe-Pro) inhibits cholera toxin and toxin-coregulated pilus production in O1 El Tor Vibrio cholerae.

Cyclo(Phe-Pro) is a cyclic dipeptide produced by multiple Vibrio species. In this work, we present evidence that cyclo(Phe-Pro) inhibits the production of the virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) in O1 El Tor Vibrio cholerae strain N16961 during growth under virulence gene-inducing conditions. The cyclo(Phe-Pro) inhibition of CT and TCP production correlated with reduced transcription of the virulence regulator tcpPH and was alleviated by overexpression of tcpPH.

Effect of the efflux inhibitors 1-(1-naphthylmethyl)-piperazine and phenyl-arginine-beta-naphthylamide on antimicrobial susceptibility and virulence factor production in Vibrio cholerae.

OBJECTIVES: The aim of the study was to test the hypothesis that the efflux pump inhibitors (EPIs) 1-(1-naphthylmethyl)-piperazine (NMP) and phenyl-arginine-beta-naphthylamide (PAbetaN) can inhibit the Vibrio cholerae resistance-nodulation-division (RND) family efflux systems, and thereby render V. cholerae susceptible to antimicrobial agents and inhibit the production of the virulence factors cholera toxin (CT) and the toxin coregulated pilus (TCP). METHODS: The susceptibility of V. cholerae to antimicrobial compounds was determined in the presence or absence of NMP and PAbetaN. Transcriptional reporters were used to assess the effects of NMP and PAbetaN on the expression of the genes encoding the virulence factor regulators TcpP and ToxT, whereas CT and TCP production were determined by ELISA using GM1 ganglioside-coated microtitre plates and TcpA Western immunoblotting, respectively. RESULTS: NMP and PAbetaN potentiated antimicrobial compounds that were substrates for the V. cholerae RND efflux systems. PAbetaN exhibited complete inhibition of the RND efflux systems for Triton X-100 and deoxycholate, but partial inhibition of the efflux systems for cholate and erythromycin. NMP exhibited partial inhibition for all compounds tested except for SDS. The presence of NMP reduced the MIC of SDS to a level that was lower than that observed in an RND efflux-deficient strain, whereas the SDS MIC was unaffected by the presence of PAbetaN. Neither EPI potentiated polymyxin B, penicillin, ampicillin or chloramphenicol. Both NMP and PAbetaN inhibited the production of CT and the TCP and appeared to have additional virulence gene repressing activity independent of RND efflux inhibition. CONCLUSIONS: RND efflux inhibitors represent potential novel therapeutics for the treatment of cholera.

Vibrio cholerae RND family efflux systems are required for antimicrobial resistance, optimal virulence factor production, and colonization of the infant mouse small intestine.

Vibrio cholerae is a gram-negative human intestinal pathogen that causes the diarrheal disease cholera. Humans acquire cholera by ingesting V. cholerae-contaminated food or water. Upon ingestion, V. cholerae encounters several barriers to colonization, including bile acid toxicity and antimicrobial products of the innate immune system. In many gram-negative bacteria, resistance to the antimicrobial effects of these products is mediated by RND (resistance-nodulation-division) family efflux systems. In this study we tested the hypothesis that the V. cholerae RND efflux systems are required for antimicrobial resistance and virulence. The six V. cholerae genes encoding RND efflux pumps were deleted from the genome of the O1 El Tor strain N16961, resulting in the generation of 14 independent RND deletion mutants, including one RND-null strain. Determination of the antimicrobial susceptibilities of the mutants revealed that the RND efflux systems were responsible for resistance to multiple antimicrobial compounds, including bile acids, antimicrobial peptides, and antibiotics. VexB (VC0164) was found to be the RND efflux pump primarily responsible for the resistance of V. cholerae to multiple antimicrobial compounds in vitro. In contrast, VexD (VC1757) and VexK (VC1673) encoded efflux pumps with detergent-specific substrate specificities that were redundant with VexB. A strain lacking VexB, VexD, and VexK was attenuated in the infant mouse model, and its virulence factor production was unaffected. In contrast, a V. cholerae RND-null strain produced significantly less cholera toxin and fewer toxin-coregulated pili than the wild type and was unable to colonize the infant mouse. The decreased virulence factor production in the RND-null strain was linked to reduced transcription of tcpP and toxT. Our findings show that the V. cholerae RND efflux systems are required for antimicrobial resistance, optimal virulence factor production, and colonization of the infant mouse.

The AcrAB RND efflux system from the live vaccine strain of Francisella tularensis is a multiple drug efflux system that is required for virulence in mice.

The ability of bacterial pathogens to infect and cause disease is dependent upon their ability to resist antimicrobial components produced by their host, such as bile acids, fatty acids and other detergent-like molecules, and products of the innate immune system (e.g. cationic antimicrobial peptides). Bacterial resistance to the antimicrobial effects of such compounds is often mediated by active efflux systems belonging to the resistance-nodulation-division (RND) family of transporters. RND efflux systems have been implicated in antibiotic resistance and virulence extending their clinical relevance. In this report the hypothesis that the Francisella tularensis AcrAB RND efflux system contributes to antimicrobial resistance and pathogenesis has been tested. A null mutation was generated in the gene encoding the AcrB RND efflux pump protein of the live vaccine strain of F. tularensis. The resulting mutant exhibited increased sensitivity to multiple antibiotics and antimicrobial compounds. Murine challenge experiments revealed that the acrB mutant was attenuated. Collectively these results suggest that the F. tularensis AcrAB RND efflux system encodes a multiple drug efflux system that is important for virulence.

Identification of Francisella tularensis lipoproteins that stimulate the toll-like receptor (TLR) 2/TLR1 heterodimer.

The innate immune response to Francisella tularensis is primarily mediated by TLR2, though the bacterial products that stimulate this receptor remain unknown. Here we report the identification of two Francisella lipoproteins, TUL4 and FTT1103, which activate TLR2. We demonstrate that TUL4 and FTT1103 stimulate chemokine production in human and mouse cells in a TLR2-dependent way. Using an assay that relies on chimeric TLR proteins, we show that TUL4 and FTT1103 stimulate exclusively the TLR2/TLR1 heterodimer. Our results also show that yet unidentified Francisella proteins, possibly unlipi-dated, have the ability to stimulate the TLR2/TLR6 heterodimer. Through domain-exchange analysis, we determined that an extended region that comprises LRR 9-17 in the extra-cellular portion of TLR1 mediates response to Francisella lipoproteins and triacylated lipopeptide. Substitution of the corresponding LRR of TLR6 with the LRR derived from TLR1 enables TLR6 to recognize TUL4, FTT1103, and triacylated lipopeptide. This study identifies for the first time specific Fran-cisella products capable of stimulating a proinflammatory response and the cellular receptors they trigger.

Immunization with heat-killed Francisella tularensis LVS elicits protective antibody-mediated immunity.

Francisella tularensis (FT) has been classified by the CDC as a category A pathogen because of its high virulence and the high mortality rate associated with infection via the aerosol route. Because there is no licensed vaccine available for FT, development of prophylactic and therapeutic regimens for the prevention/treatment of infection is a high priority. In this report, heat-killed FT live vaccine strain (HKLVS) was employed as a vaccine immunogen, either alone or in combination with an adjuvant, and was found to elicit protective immunity against high-dose FT live vaccine strain (FTLVS) challenge. FT-specific antibodies produced in response to immunization with HKLVS alone were subsequently found to completely protect naive mice against high-dose FT challenge in both infection-interference and passive immunization experiments. Additional passive immunization trials employing serum collected from mice immunized with a heat-killed preparation of an O-antigen-deficient transposon mutant of FTLVS (HKLVS-OAg(neg)) yielded similar results. These findings demonstrated that FT-specific antibodies alone can confer immunity against high-dose FTLVS challenge, and they reveal that antibody-mediated protection is not dependent upon production of LPS-specific antibodies.

Transketolase A, an enzyme in central metabolism, derepresses the marRAB multiple antibiotic resistance operon of Escherichia coli by interaction with MarR.

The Escherichia coli marRAB operon specifies two regulatory proteins, MarR (which represses) and MarA (which activates expression of the operon). The latter controls expression of multiple other chromosomal genes implicated in cell physiology, multiple drug resistance and virulence. Using randomly cloned E. coli DNA fragments in the bacterial adenylate cyclase two-hybrid system, we found that transketolase A (TktA) interacts with MarR. Purified (6H)-TktA immobilized on NiNTA resin-bound MarR. Overexpression or deletion of tktA showed that TktA interfered with MarR repression of the marRAB operon. Deletion of tktA increased antibiotic and oxidative stress susceptibilities, while its overexpression decreased them. Hydrogen peroxide induced tktA at 1 h treatment, while an increase in marRAB expression occurred only after 3 h exposure. This increase was dependent on the presence of tktA. Two MarR mutations which eliminated MarR binding to the marRAB operator and one which decreased dimerization of MarR had no effect on MarR interaction with TktA in the two-hybrid system. However, the interaction was disrupted by one of the three tested superrepressor mutant MarR proteins known to increase MarR binding to DNA. TktA inhibition of repression by MarR demonstrates a previously unrecognized level of control of the expression of marRAB operon.

Innate immune response to Francisella tularensis is mediated by TLR2 and caspase-1 activation.

Francisella tularensis, a gram-negative, facultative, intracellular bacterium, is the etiologic agent of tularemia and a category A bioterrorism agent. Little is known about the mechanism of pathogenesis of tularemia. In this paper, we describe the interaction of the live vaccine strain of F. tularensis with the innate immune system. We have found that in human and mouse dendritic cells, F. tularensis elicited a powerful inflammatory response, characterized by production of a number of cytokines and chemokines. Using cells derived from TLR2-deficient mice and in vitro transfection assays, we demonstrated that this response was mediated by TLR2 and did not require the LPS-binding protein. F. tularensis appeared to activate TLR2/TLR1 and TLR2/TLR6 heterodimers. IL-1beta secretion, a reflection of caspase-1 activation, was induced by live but not heat-killed F. tularensis, despite the fact that both forms of the bacterium equally induced the IL-1beta transcript. Our results identified activation of TLR2 and caspase-1 as the two main cellular pathways responsible for the inflammatory response to F. tularensis.

The Bla2 beta-lactamase from the live-vaccine strain of Francisella tularensis encodes a functional protein that is only active against penicillin-class beta-lactam antibiotics.

Francisella tularensis ssp. tularensis is a category A select agent and the causal organism for the zoonotic disease tularemia. The vast majority of F. tularensis isolates are beta-lactamase-positive. beta-lactamase production is widely believed to be responsible for the inefficacy of beta-lactams in the treatment of tularemia. In this study, we report the cloning and characterization of the two chromosomally encoded F. tularensis ssp. holarctica live-vaccine strain (LVS) beta-lactamases. The two LVS beta-lactamases were homologous to F. tularensis Schu S4 open reading frames FTT0681c and FTT0611c and have been named bla1 (LVS) and bla2 (LVS), respectively. Recombinant expression in Escherichia coli suggested that bla1 (LVS) did not encode a functional beta-lactamase, whereas bla2 (LVS) encoded a functional beta-lactamase that hydrolyzed penicillins but was inactive against third-generation cephalosporins, including cefprozil. As both LVS and Schu S4 were susceptible to cefprozil, we developed three new shuttle vectors based on selection for the production of the Bla(shv-2) extended-spectrum beta-lactamase with cefprozil. The resulting shuttle vectors were suitable for recombinant gene expression and complementation studies in LVS and Schu S4.

Characterization of the Vibrio cholerae vexAB and vexCD efflux systems.

Vibrio cholerae is an important human pathogen that causes the diarrheal disease cholera. Colonization of the human host is dependent upon coordinated expression of several virulence factors in response to as yet unknown environmental cues. Bile acids have been implicated in the in vitro regulation of several V. cholerae genes, including those involved in motility, chemotaxis, outer membrane protein production, and virulence factor production. Bile is toxic to bacteria and colonization of the intestinal tract is dependent upon bacterial resistance to bile acids. We have identified and characterized two bile-regulated RND-family efflux systems, named here vexAB and vexCD, that are involved in V. cholerae bile resistance. Mutational analysis revealed that the vexAB system is responsible for in vitro intrinsic resistance of V. cholerae to multiple antimicrobial compounds, including bile acids. In contrast, the vexCD efflux system was specific for certain bile acids and detergents and functioned in conjunction with the vexAB system to provide V. cholerae with high-level bile resistance. Mutants containing deletion of vexB, vexD, and vexB-vexD were able to efficiently colonize the infant mouse suggesting that these efflux systems were dispensable for V. cholerae growth in the small intestines of infant mice.