A transcription factor contributes to pathogenesis and virulence in streptococcus pneumoniae

To date, the role of transcription factors (TFs) in the progression of disease for many pathogens is yet to be studied in detail. This is probably due to transient, and generally low expression levels of TFs, which are the central components controlling the expression of many genes during the course of infection. However, a small change in the expression or specificity of a TF can radically alter gene expression. In this study, we combined a number of quality-based selection strategies including structural prediction of modulated genes, gene ontology and network analysis, to predict the regulatory mechanisms underlying pathogenesis of Streptococcus pneumoniae (the pneumococcus). We have identified two TFs (SP_0676 and SP_0927 [SmrC]) that might control tissue-specific gene expression during pneumococcal translocation from the nasopharynx to lungs, to blood and then to brain of mice. Targeted mutagenesis and mouse models of infection confirmed the role of SP_0927 in pathogenesis and virulence, and suggests that SP_0676 might be essential to pneumococcal viability. These findings provide fundamental new insights into virulence gene expression and regulation during pathogenesis.Layla K. Mahdi, Esmaeil Ebrahimie, David L. Adelson, James C. Paton, Abiodun D. Ogunniy

Molecular cloning and transcriptional regulation of ompT , a ToxR-repressed gene in Vibrio cholerae

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72189/1/j.1365-2958.2000.01699.x.pd

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

A proteome-wide protein interaction map for Campylobacter jejuni

'Systematic identification of protein interactions for the bacterium Campylobacter jejuni using high-throughput yeast two-hybrid screens detected interactions for 80% of the organism's proteins

Genetic analysis of the interaction between Vibrio cholerae transcription activator ToxR and toxT promoter DNA.

Expression of many virulence genes in Vibrio cholerae is under the control of the ToxT protein. These include genes whose products are required for the biogenesis of the toxin-coregulated pilus, accessory colonization factor, and cholera toxin. ToxT is a member of the AraC family of transcriptional activators and is part of the ToxR regulatory cascade. ToxR is a transmembrane DNA-binding protein that is required for transcription of toxT and also can directly activate transcription of the cholera toxin operon (ctxAB). The sequences upstream of ctxAB and toxT to which ToxR binds show no obvious similarity, which implies that ToxR may be recognizing a degenerate sequence or, alternatively, a common structural motif within both binding sites. Data presented in this report demonstrate that nucleotides within the upstream half-site of an inverted repeat element in the toxT promoter are critical for ToxR-regulated activation of transcription in V. cholerae. In addition, gene fusion and DNA-binding studies with mutant ToxR proteins indicate that residues of ToxR required for binding to the ctx promoter are also required for binding to the toxT promoter. These data suggest that ToxR is not recognizing an inverted repeat sequence per se in the activation of toxT but, rather, some motif composed in part of sequences within the upstream half-site of the inverted repeat and that ToxR recognizes similar motifs within the ctxAB and toxT promoters

ToxR proteins with substitutions in residues conserved with OmpR fail to activate transcription from the cholera toxin promoter.

The ToxR protein of Vibrio cholerae is an integral membrane protein that coordinately regulates the expression of virulence genes required for successful infection. ToxR has been shown to bind directly to and activate transcription of the cholera toxin (ctx) promoter. Within the amino-terminal cytoplasmic region of ToxR, several amino acids are strictly conserved among ToxR, OmpR, and the other members of a family of bacterial regulatory proteins. To better understand the function of this region, two approaches were taken: conserved residues were changed by site-directed mutagenesis, and random mutations that eliminated ToxR-mediated transcriptional activation were isolated. Several classes of mutations were identified: those that abolish promoter DNA binding and transcriptional activation (toxR R96K, toxR R68K, and toxR R68L), those that abolish transcriptional activation but retain the ability to bind promoter DNA (toxR R96L), and those that have an intermediate phenotype (toxR R77L, toxR E51K, and toxR E51D). The toxR E51K allele had reduced activity in both Escherichia coli and V. cholerae but also exerted a dominant-negative effect over wild-type ToxR when assayed in V. cholerae. This result provides additional evidence that ToxR acts as an oligomer in the transcriptional activation process. From this mutational analysis of conserved amino acid residues within the OmpR-homologous region of ToxR, we conclude that this region is essential for transcriptional activation at the level of DNA binding and other steps that lead to activation of the ctx promoter

The virulence gene activator ToxT from Vibrio cholerae is a member of the AraC family of transcriptional activators.

Virulence gene expression in Vibrio cholerae is postulated to involve ToxR-dependent activation of the toxT gene followed by ToxT activation of virulence genes, including several of those involved in biogenesis of the toxin-coregulated pilus. ToxR is a transmembrane, DNA-binding protein which is a member of the OmpR subclass of two-component activator systems in bacteria. Data presented in this report demonstrate that ToxT is similar to the AraC family of transcriptional activators identified in a variety of gram-negative bacteria. The toxT open reading frame begins approximately 200 nucleotides from the end of the tcpF gene, which is part of a cluster of genes responsible for production of the toxin-coregulated pilus. Accumulation of toxT specific mRNA is ToxR dependent and is modulated by environmental conditions that modulate expression of the regulon. Within the intergenic region between tcpF and toxT is a potential stem-loop structure of an unusual nature which may play a role in regulating expression of toxT mRNA. Experiments with tcpF and toxT cloned behind a strong, constitutive promoter suggest that the two genes can be cotranscribed, but Northern (RNA) blot analysis of V. cholerae suggests that if they are, steady-state levels of their messages may be controlled by a posttranscriptional mechanism. Possible mechanisms for ToxR-dependent expression of toxT are discussed