Identification of Novel Virulence-Associated Genes via Genome Analysis of Hypothetical Genes

Abstract

The sequencing of bacterial genomes has opened new perspectives for identification of targets for treatment of infectious diseases. We have identified a set of novel virulence-associated genes (vag genes) by comparing the genome sequences of six human pathogens that are known to cause persistent or chronic infections in humans: Yersinia pestis, Neisseria gonorrhoeae, Helicobacter pylori, Borrelia burgdorferi, Streptococcus pneumoniae, and Treponema pallidum. This comparison was limited to genes annotated as hypothetical in the T. pallidum genome project. Seventeen genes with unknown functions were found to be conserved among these pathogens. Insertional inactivation of 14 of these genes generated nine mutants that were attenuated for virulence in a mouse infection model. Out of these nine genes, five were found to be specifically associated with virulence in mice as demonstrated by infection with Yersinia pseudotuberculosis in-frame deletion mutants. In addition, these five vag genes were essential only in vivo, since all the mutants were able to grow in vitro. These genes are broadly conserved among bacteria. Therefore, we propose that the corresponding vag gene products may constitute novel targets for antimicrobial therapy and that some vag mutants could serve as carrier strains for live vaccines.Antibacterial therapy is becoming less effective in the treatment of infections due to the spread of multiple-antibioticresistant bacterial strains (59). This has lead to a concerted effort to develop mechanistically novel antimicrobial agents. This search is being facilitated by the wealth of information accumulating in databases via different large-scale genome projects. To date, about 100 bacterial genomes, including several important pathogens have been sequenced, revealing around 25% of the open reading frames (ORFs) to be conserved hypothetical genes without known function (17). Presumably, among this group of ORFs, protein classes that constitute suitable molecular targets for novel therapeutics will be identified. In fact, a number of genome-based studies have already identified homologous gene classes from different bacteria that encode products of unknown or hypothetical function (4,7,20,55). By combining the bioinformatic approaches with mutagenesis, genes essential for in vitro viability have been identified (4,20,55). Indeed, these proteins constitute potential targets for the development of novel antibacterial agents that would serve to inhibit bacterial growth.An alternative approach would be to target genes required only for in vivo growth and not for growth outside the animal host. Finding these nontraditional targets by using a bioinformatic approach is a hitherto-unproven strategy (29). The approach presented here makes use of the fact that several obligatory pathogenic bacteria have genome sizes that are considerably smaller than those of free-living eubacteria. During evolution, these pathogenic bacteria have condensed their genomes by eliminating genes not required for growth in the animal host ...

The Glutamine Residue of the Conserved GGQ Motif in Saccharomyces cerevisiae Release Factor eRF1 Is Methylated by the Product of the YDR140w Gene

“…The effect of the two factors is cumulative, and we interpret these observations to mean that the termination machinery can tolerate the effect either of the presence of Thr rather than Ala/Ser or the lack of Gln methylation but that the accumulated effect of both factors reduces termination efficiency at some stop codons to a level incompatible with cell growth. For reasons that remain to be determined, PrmC in Yersinia pseudotuberculosis is required for virulence, although not for growth in rich liquid media (32).…”

Methylation and in vivo expression of the surface-exposed Leptospira interrogans outer-membrane protein OmpL32

“…A requirement for methylation has been demonstrated in Y. pseudotuberculosis, where an insertion mutant deficient in the VagH protein is avirulent in mice (Garbom et al, 2004). In Y. pseudotuberculosis, the VagH protein is homologous to the E. coli HemK protein (Garbom et al, 2007), an N 5 -methyltransferase (Heurgué-Hamard et al, 2002;Nakahigashi et al, 2002).…”

“…Methylation of glycolipids has been shown to be essential for Mycobacterium avium virulence in mice (Krzywinska et al, 2005), while methylation of proteins has been shown to alter the antigenicity of the outer-membrane protein OmpB from Rickettsia typhi (Chao et al, 2008), and to alter both the antigenicity of and the host T cell-mediated immune response against the heparin-binding haemagglutinin from Mycobacterium tuberculosis Temmerman et al, 2004). In addition, methylation has been demonstrated to be essential for functional type III secretion, and thus virulence, in Yersinia pseudotuberculosis (Garbom et al, 2004(Garbom et al, , 2007, and methylation of the surface protein OmpB in Rickettsia prowazaki has been suggested to be central to the pathogenesis of that bacterium (Chao et al, 2004(Chao et al, , 2007. Collectively, these investigations highlight the importance of methylation of outer-membrane surface components in bacterial virulence.…”

Screening of genes expressed in vivo after infection by Vibrio anguillarum M3

“…Va-iv-15 encodes MesJ that is involved in cell division. With a bioinformatic approach, MesJ homologue was identified and designated as virulence-associated genes in E. coli (Garbom et al 2004). It needs further study to determine whether V. anguillarum YaeT and MesJ are virulence factors.…”