RESULTS:

During conditions of nutrient limitation bacteria undergo a series of global gene expression changes to survive conditions of amino acid and fatty acid starvation. Rapid reallocation of cellular resources is brought about by gene expression changes coordinated by the signalling nucleotides' guanosine tetraphosphate or pentaphosphate collectively termed (p)ppGpp and is known as the stringent response. The stringent response has been implicated in bacterial virulence with elevated (p)ppGpp levels being associated with increased virulence gene expression. This has been observed in the highly pathogenic Francisella tularensis sub spp. tularensis SCHU S4 the causative agent of tularaemia. Here we aimed to artificially induce the stringent response by culturing F. tularensis in the presence of the amino acid analogue l-serine hydroxamate. Serine hydroxamate competitively inhibits tRNAser aminoacylation causing an accumulation of uncharged tRNA. The uncharged tRNA enters the A site on the translating bacterial ribosome and causes ribosome stalling in turn stimulating the production of (p)ppGpp and activation of the stringent response. Using the essential virulence gene iglC which is encoded on the Francisella pathogenicity island (FPI) as a marker of active stringent response we optimized the culture conditions required for the investigation of virulence gene expression under conditions of nutrient limitation. We subsequently used whole genome RNA-seq to show how F. tularensis alters gene expression on a global scale during active stringent response. Key findings included up-regulation of genes involved in virulence stress responses and metabolism and down-regulation of genes involved in metabolite transport and cell division. F. tularensis is a highly virulent intracellular pathogen capable of causing debilitating or fatal disease at extremely low infectious doses. However virulence mechanisms are still poorly understood. The stringent response is widely recognized as a diverse and complex bacterial stress response implicated in virulence. This work describes the global gene expression profile of F. tularensis SCHU S4 under active stringent response for the first time. Herein we provide evidence for an association of active stringent response with FPI virulence gene expression. Our results further the understanding of the molecular basis of virulence and regulation thereof in F. tularensis. These results also support research into genes involved in (p)ppGpp production and polyphosphate biosynthesis and their applicability as targets for novel antimicrobials.

Mg2+ has been shown to be an important signal controlling gene regulation via the PhoPQ two-component regulatory system for a range of Gram-negative bacteria including Yersinia pestis and Yersinia pseudotuberculosis. The magnesium ion transporter MgtB is part of the complex PhoPQ regulon being upregulated in response to low Mg2+. Despite the presence of other Mg2+ transport systems in Yersinia inactivation of mgtB had a significant effect on the ability of the bacteria to scavenge this crucial ion. Whereas inactivation of PhoPQ is reported to adversely affect intracellular survival we show that Y. pestis and Y. pseudotuberculosis ΔmgtB mutants survived equally as well as the respective parent strain within macrophages although they were more sensitive to killing in the Galleria model of infection. Surprisingly despite MgtB being only one member of the Mg2+ stimulon and PhoPQ controlling the expression levels of a range of genes including mgtB the Yersinia ΔmgtB mutants were more highly attenuated than the equivalent Yersinia ΔphoP mutants in mouse models of infection. MgtB may be a suitable target for development of novel antimicrobials and investigation of its role may help elucidate the contribution of this component of the PhoPQ regulon to pathogenesis.

Francisella tularensis is an intracellular pathogen which causes tularaemia. There is no licensed vaccine currently available for prophylaxis. The γ-glutamyl transpeptidase (GGT) encoded by the ggt gene has been shown to be important for the intracellular survival of F. tularensis. In this study we have constructed a ggt deletion mutant in the highly virulent F. tularensis strain SCHU S4. Characterization of the mutant strain confirmed the function of ggt and confirmed the role of GGT in cysteine acquisition. The mutant strain was highly attenuated both in vitro and in vivo using murine models of infection. Moreover we have demonstrated that the attenuated mutant is able to induce protective immunity against an F. tularensis SCHU S4 challenge and thus may be a candidate for the development of an attenuated vaccine.

As there is currently no licensed vaccine against Francisella tularensis the causative agent of tularaemia the bacterium is an agent of concern as a potential bioweapon. Although F. tularensis has a low infectious dose and high associated mortality it possesses few classical virulence factors. An analysis of the F. tularensis subspecies tularensis genome sequence has revealed the presence of a region containing genes with low sequence homology to part of the capBCADE operon of Bacillus anthracis. We have generated an isogenic capB mutant of F. tularensis subspecies tularensis SchuS4 and shown it to be attenuated. Furthermore using BALB/c mice we have demonstrated that this capB strain affords protection against significant homologous challenge with the wild-type strain. These data have important implications for the development of a defined and efficacious tularaemia vaccine.

The LysR family of transcriptional regulators represents the most abundant type of transcriptional regulator in the prokaryotic kingdom. Members of this family have a conserved structure with an N-terminal DNA-binding helix–turn–helix motif and a C-terminal co-inducer-binding domain. Despite considerable conservation both structurally and functionally LysR-type transcriptional regulators (LTTRs) regulate a diverse set of genes including those involved in virulence metabolism quorum sensing and motility. Numerous structural and transcriptional studies of members of the LTTR family are helping to unravel a compelling paradigm that has evolved from the original observations and conclusions that were made about this family of transcriptional regulators.

Francisella tularensis has been recognized as the causative agent of tularaemia for almost a century. Since its discovery in 1911 it has been shown to infect a wide range of hosts including humans. As early as the 1920s it was suggested to be an intracellular pathogen but it has proven to be an enigmatic organism whose interaction with the host has been difficult to elucidate and we still have a very limited understanding of the molecular mechanisms of virulence. However the recent availability of genome sequence data and molecular tools has allowed us to start to understand the molecular basis of F. tularensis pathogenicity and will facilitate the development of a vaccine to protect against infection.

Recently a number of attenuated mutants of Yersinia pseudotuberculosis have been identified using a bioinformatics approach. One of the target genes identified in that study was vagH which the authors now characterized further. VagH shows homology to HemK of Escherichia coli possessing methyltransferase activity similar to that of HemK and targeting release factors 1 and 2. Microarray studies comparing the wild-type and the vagH mutant revealed that the mRNA levels of only a few genes were altered in the mutant. By proteome analysis expression of the virulence determinant YopD was found to be increased indicating a possible connection between VagH and the virulence plasmid-encoded type III secretion system (T3SS). Further analysis showed that Yop expression and secretion were repressed in a vagH mutant. This phenotype could be suppressed by trans-complementation with the wild-type vagH gene or by deletion of the negative regulator yopD. Also in a similar manner to a T3SS-negative mutant the avirulent vagH mutant was rapidly cleared from Peyer's patches and could not reach the spleen after oral infection of mice. In a manner analogous to that of T3SS mutants the vagH mutant could not block phagocytosis by macrophages. However a vagH mutant showed no defects in the T3SS-independent ability to proliferate intracellularly and replicated to levels similar to those of the wild-type in macrophages. In conclusion the vagH mutant exhibits a virulence phenotype similar to that of a T3SS-negative mutant indicating a tight link between VagH and type III secretion in Y. pseudotuberculosis.

A Burkholderia pseudomallei mutant which was attenuated in a mouse model of melioidosis was identified by a signature tagged mutagenesis approach. The transposon was shown to be inserted into a gene within the capsular biosynthetic operon. Compared with the wild-type bacteria this mutant demonstrated a 105-fold increase in the median lethal dose in a mouse model and it did not react with a monoclonal antibody against high mol. wt polysaccharide of B. pseudomallei. To determine the kinetics of infection mice were dosed intraperitoneally (i.p.) and intravenously (i.v.) with mutant and wild-type bacteria. After i.p challenge the number of mutant bacteria in the peritoneal cavity declined whereas wild-type bacteria proliferated. When administered by the i.v. route the mutant was able to cause disease but the time to death was increased compared with the wild type. Mice were dosed with the mutant and subsequently challenged with wild-type B. pseudomallei but the mutant failed to induce a protective immune response.

This study describes a PCR-based approach for the production of a rationally attenuated mutant of Yersinia pestis. Degenerate primers were used to amplify a fragment encoding 91.45% of the aroA gene of Y. pestis MP6 which was cloned into pUC18. The remainder of the gene was isolated by inverse PCR. The gene was sequenced and a restriction map was generated. The Y. pestis aroA gene had 75.9% identity with the aroA gene of Yersinia enterocolitica. The cloned gene was inactivated in vitro and reintroduced into Y. pestis strain GB using the suicide vector pGP704. A stable aro-defective mutant Y. pestis GBΔaroA was isolated and its virulence was examined in vivo. The mutant was attenuated in guinea-pigs and capable of inducing a protective immune response against challenge with the virulent Y. pestis strain GB. Unusually for an aro-defective mutant the Y. pestis aroA mutant was virulent in mice with a median dose which induced morbidity or death similar to that of the wild-type although time to death was significantly prolonged.

Nineteen strains of Bacteroides fragilis were examined by negative staining for surface structures. One strain (ATCC 23745) possessed peritrichous fibrils 16 strains carried peritrichous fimbriae and two strains carried no surface structures. The fimbriae had a diameter of 2·1±0·25 nm and appeared to be ‘curly’. Only a small proportion (4 to 41% depending on the strain) of cells in a population carried fimbriae or fibrils. Strain A312 showed phase variation of fimbriae as expression of fimbriae was repressed at 20 °C and in early exponential phase at 37 °C. The fibrils on strain ATCC 23745 did not exhibit phase variation in response to changes in incubation temperature growth phase or growth in two different media. Capsules were demonstrated by the Indian ink method on 18 of the 19 strains varying in size from strain to strain and within the same population. Cultures often contained both capsulate and noncapsulate cells. All strains possessed an electron dense ruthenium red staining layer between 7·9 and 23·9 nm in width attached to the outer membrane. Cell surface hydrophobicity quantified by the hexadecane partition assay gave low values ranging from 6·6 to 52·1%. Only a few strains were able to haemagglutinate and these were only weakly active. There was no correlation between cell surface hydrophobicity haemagglutinating activity and surface structures.