Constitutive Activation of PrfA Tilts the Balance of Listeria monocytogenes Fitness Towards Life within the Host versus Environmental Survival

PrfA is a key regulator of Listeria monocytogenes pathogenesis and induces the expression of multiple virulence factors within the infected host. PrfA is post-translationally regulated such that the protein becomes activated upon bacterial entry into the cell cytosol. The signal that triggers PrfA activation remains unknown, however mutations have been identified (prfA* mutations) that lock the protein into a high activity state. In this report we examine the consequences of constitutive PrfA activation on L. monocytogenes fitness both in vitro and in vivo. Whereas prfA* mutants were hyper-virulent during animal infection, the mutants were compromised for fitness in broth culture and under conditions of stress. Broth culture prfA*-associated fitness defects were alleviated when glycerol was provided as the principal carbon source; under these conditions prfA* mutants exhibited a competitive advantage over wild type strains. Glycerol and other three carbon sugars have been reported to serve as primary carbon sources for L. monocytogenes during cytosolic growth, thus prfA* mutants are metabolically-primed for replication within eukaryotic cells. These results indicate the critical need for environment-appropriate regulation of PrfA activity to enable L. monocytogenes to optimize bacterial fitness inside and outside of host cells

Genomic organization of <it>Tropomodulins 2</it> and <it>4</it> and unusual intergenic and intraexonic splicing of <it>YL-1</it> and <it>Tropomodulin 4</it>

<p>Abstract</p> <p>Background</p> <p>The tropomodulins (TMODs) are a family of proteins that cap the pointed ends of actin filaments. Four TMODs have been identified in humans, with orthologs in mice. Mutations in actin or actin-binding proteins have been found to cause several human diseases, ranging from hypertrophic cardiomyopathy to immunodefiencies such as Wiskott-Aldrich syndrome. We had previously mapped <it>Tropomodulin 2 (TMOD2)</it> to the genomic region containing the gene for amyotrophic lateral sclerosis 5 (ALS5). We determined the genomic structure of Tmod2 in order to better analyze patient DNA for mutations; we also determined the genomic structure of <it>Tropomodulin 4 (TMOD4).</it></p> <p>Results</p> <p>In this study, we determined the genomic structure of <it>TMOD2</it> and <it>TMOD4</it> and found the organization of both genes to be similar. Sequence analysis of <it>TMOD2</it> revealed no mutations or polymorphisms in ALS5 patients or controls. Interestingly, we discovered that another gene, <it>YL-1,</it> intergenically splices into <it>TMOD4. YL-1</it> encodes six exons, the last of which is 291 bp from a 5' untranslated exon of <it>TMOD4.</it> We used 5' RACE and RT-PCR from <it>TMOD4</it> to identify several intergenic RACE products. <it>YL-1</it> was also found to undergo unconventional splicing using non-canonical splice sites within exons (intraexonic splicing) to produce several alternative transcripts.</p> <p>Conclusions</p> <p>The genomic structure of <it>TMOD2</it> and <it>TMOD4</it> have been delineated. This should facilitate future mutational analysis of these genes. In addition, intergenic splicing at <it>TMOD4/YL-1</it> was discovered, demonstrating yet another level of complexity of gene organization and regulation.</p