The Bicarbonate Transporter Is Essential for Bacillus anthracis Lethality

In the pathogenic bacterium Bacillus anthracis, virulence requires induced expression of the anthrax toxin and capsule genes. Elevated CO2/bicarbonate levels, an indicator of the host environment, provide a signal ex vivo to increase expression of virulence factors, but the mechanism underlying induction and its relevance in vivo are unknown. We identified a previously uncharacterized ABC transporter (BAS2714-12) similar to bicarbonate transporters in photosynthetic cyanobacteria, which is essential to the bicarbonate induction of virulence gene expression. Deletion of the genes for the transporter abolished induction of toxin gene expression and strongly decreased the rate of bicarbonate uptake ex vivo, demonstrating that the BAS2714-12 locus encodes a bicarbonate ABC transporter. The bicarbonate transporter deletion strain was avirulent in the A/J mouse model of infection. Carbonic anhydrase inhibitors, which prevent the interconversion of CO2 and bicarbonate, significantly affected toxin expression only in the absence of bicarbonate or the bicarbonate transporter, suggesting that carbonic anhydrase activity is not essential to virulence factor induction and that bicarbonate, and not CO2, is the signal essential for virulence induction. The identification of this novel bicarbonate transporter essential to virulence of B. anthracis may be of relevance to other pathogens, such as Streptococcus pyogenes, Escherichia coli, Borrelia burgdorferi, and Vibrio cholera that regulate virulence factor expression in response to CO2/bicarbonate, and suggests it may be a target for antibacterial intervention

Metabolic Adaptation of Ralstonia solanacearum during Plant Infection: A Methionine Biosynthesis Case Study

MetE and MetH are two distinct enzymes that catalyze a similar biochemical reaction during the last step of methionine biosynthesis, MetH being a cobalamin-dependent enzyme whereas MetE activity is cobalamin-independent. In this work, we show that the last step of methionine synthesis in the plant pathogen Ralstonia solanacearum is under the transcriptional control of the master pathogenicity regulator HrpG. This control is exerted essentially on metE expression through the intermediate regulator MetR. Expression of metE is strongly and specifically induced in the presence of plant cells in a hrpG- and metR-dependent manner. metE and metR mutants are not auxotrophic for methionine and not affected for growth inside the plant but produce significantly reduced disease symptoms on tomato whereas disruption of metH has no impact on pathogenicity. The finding that the pathogen preferentially induces metE expression rather than metH in the presence of plant cells is indicative of a probable metabolic adaptation to physiological host conditions since this induction of metE occurs in an environment in which cobalamin, the required co-factor for MetH, is absent. It also shows that MetE and MetH are not functionally redundant and are deployed during specific stages of the bacteria lifecycle, the expression of metE and metH being controlled by multiple and distinct signals

Phasevarions Mediate Random Switching of Gene Expression in Pathogenic Neisseria

Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes) that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression). In Haemophilus influenzae, the random switching of the modA gene controls expression of a phase-variable regulon of genes (a “phasevarion”), via differential methylation of the genome in the modA ON and OFF states. Phase-variable mod genes are also present in Neisseria meningitidis and Neisseria gonorrhoeae, suggesting that phasevarions may occur in these important human pathogens. Phylogenetic studies on phase-variable mod genes associated with type III restriction modification (R-M) systems revealed that these organisms have two distinct mod genes—modA and modB. There are also distinct alleles of modA (abundant: modA11, 12, 13; minor: modA4, 15, 18) and modB (modB1, 2). These alleles differ only in their DNA recognition domain. ModA11 was only found in N. meningitidis and modA13 only in N. gonorrhoeae. The recognition site for the modA13 methyltransferase in N. gonorrhoeae strain FA1090 was identified as 5′-AGAAA-3′. Mutant strains lacking the modA11, 12 or 13 genes were made in N. meningitidis and N. gonorrhoeae and their phenotype analyzed in comparison to a corresponding mod ON wild-type strain. Microarray analysis revealed that in all three modA alleles multiple genes were either upregulated or downregulated, some of which were virulence-associated. For example, in N. meningitidis MC58 (modA11), differentially expressed genes included those encoding the candidate vaccine antigens lactoferrin binding proteins A and B. Functional studies using N. gonorrhoeae FA1090 and the clinical isolate O1G1370 confirmed that modA13 ON and OFF strains have distinct phenotypes in antimicrobial resistance, in a primary human cervical epithelial cell model of infection, and in biofilm formation. This study, in conjunction with our previous work in H. influenzae, indicates that phasevarions may be a common strategy used by host-adapted bacterial pathogens to randomly switch between “differentiated” cell types

A Combination of Independent Transcriptional Regulators Shapes Bacterial Virulence Gene Expression during Infection

Transcriptional regulatory networks are fundamental to how microbes alter gene expression in response to environmental stimuli, thereby playing a critical role in bacterial pathogenesis. However, understanding how bacterial transcriptional regulatory networks function during host-pathogen interaction is limited. Recent studies in group A Streptococcus (GAS) suggested that the transcriptional regulator catabolite control protein A (CcpA) influences many of the same genes as the control of virulence (CovRS) two-component gene regulatory system. To provide new information about the CcpA and CovRS networks, we compared the CcpA and CovR transcriptomes in a serotype M1 GAS strain. The transcript levels of several of the same genes encoding virulence factors and proteins involved in basic metabolic processes were affected in both ΔccpA and ΔcovR isogenic mutant strains. Recombinant CcpA and CovR bound with high-affinity to the promoter regions of several co-regulated genes, including those encoding proteins involved in carbohydrate and amino acid metabolism. Compared to the wild-type parental strain, ΔccpA and ΔcovRΔccpA isogenic mutant strains were significantly less virulent in a mouse myositis model. Inactivation of CcpA and CovR alone and in combination led to significant alterations in the transcript levels of several key GAS virulence factor encoding genes during infection. Importantly, the transcript level alterations in the ΔccpA and ΔcovRΔccpA isogenic mutant strains observed during infection were distinct from those occurring during growth in laboratory medium. These data provide new knowledge regarding the molecular mechanisms by which pathogenic bacteria respond to environmental signals to regulate virulence factor production and basic metabolic processes during infection

Streptococcus iniae M-Like Protein Contributes to Virulence in Fish and Is a Target for Live Attenuated Vaccine Development

Streptococcus iniae is a significant pathogen in finfish aquaculture, though knowledge of virulence determinants is lacking. Through pyrosequencing of the S. iniae genome we have identified two gene homologues to classical surface-anchored streptococcal virulence factors: M-like protein (simA) and C5a peptidase (scpI).S. iniae possesses a Mga-like locus containing simA and a divergently transcribed putative mga-like regulatory gene, mgx. In contrast to the Mga locus of group A Streptococcus (GAS, S. pyogenes), scpI is located distally in the chromosome. Comparative sequence analysis of the Mgx locus revealed only one significant variant, a strain with an insertion frameshift mutation in simA and a deletion mutation in a region downstream of mgx, generating an ORF which may encode a second putative mga-like gene, mgx2. Allelic exchange mutagenesis of simA and scpI was employed to investigate the potential role of these genes in S. iniae virulence. Our hybrid striped bass (HSB) and zebrafish models of infection revealed that M-like protein contributes significantly to S. iniae pathogenesis whereas C5a peptidase-like protein does not. Further, in vitro cell-based analyses indicate that SiMA, like other M family proteins, contributes to cellular adherence and invasion and provides resistance to phagocytic killing. Attenuation in our virulence models was also observed in the S. iniae isolate possessing a natural simA mutation. Vaccination of HSB with the Delta simA mutant provided 100% protection against subsequent challenge with a lethal dose of wild-type (WT) S. iniae after 1,400 degree days, and shows promise as a target for live attenuated vaccine development.Analysis of M-like protein and C5a peptidase through allelic replacement revealed that M-like protein plays a significant role in S. iniae virulence, and the Mga-like locus, which may regulate expression of this gene, has an unusual arrangement. The M-like protein mutant created in this research holds promise as live-attenuated vaccine

Mathematically combined half-cell reduction potentials of low-molecular-weight thiols as markers of seed ageing

The half-cell reduction potential of the glutathione disulphide (GSSG) / glutathione (GSH) redox couple appears to correlate with cell viability and has been proposed to be a marker of seed viability and ageing. We investigated the relationship between seed viability and the individual half-cell reduction potentials (Eis) of four low-molecular11 weight (LMW) thiols in Lathyrus pratensis seeds subjected to artificial ageing: GSH, cysteine (Cys), cysteinyl-glycine (Cys-Gly) and -glutamyl-cysteine (-Glu-Cys). The standard redox potential of -Glu-Cys was previously unknown and was experimentally determined. The Eis were mathematically combined to define a LMW thiol-disulphide based redox environment (Ethiol-disulphide). Loss of seed viability correlated with a shift in Ethiol-disulphide towards more positive values, with a LD50 value of -0.90 0.093 mV M (mean SD). The mathematical definition of Ethiol-disulphide is envisaged as a step towards the definition of the overall cellular redox environment, which will need to include all known redox-couple