RESUMO
BACKGROUND: The opportunistic food-borne gram-positive pathogen Listeria monocytogenes can exist as a free-living microorganism in the environment and grow in the cytoplasm of vertebrate and invertebrate cells following infection. The general stress response, controlled by the alternative sigma factor, sigmaB, has an important role for bacterial survival both in the environment and during infection. We used quantitative real-time PCR analysis and immuno-blot analysis to examine sigmaB expression during growth of L. monocytogenes EGD-e. Whole genome-based transcriptional profiling was used to identify sigmaB-dependent genes at different growth phases. RESULTS: We detected 105 sigmaB-positively regulated genes and 111 genes which appeared to be under negative control of sigmaB and validated 36 sigmaB-positively regulated genes in vivo using a reporter gene fusion system. CONCLUSION: Genes comprising the sigmaB regulon encode solute transporters, novel cell-wall proteins, universal stress proteins, transcriptional regulators and include those involved in osmoregulation, carbon metabolism, ribosome- and envelope-function, as well as virulence and niche-specific survival genes such as those involved in bile resistance and exclusion. Ten of the sigmaB-positively regulated genes of L. monocytogenes are absent in L. innocua. A total of 75 sigmaB-positively regulated listerial genes had homologs in B. subtilis, but only 33 have been previously described as being sigmaB-regulated in B. subtilis even though both species share a highly conserved sigmaB-dependent consensus sequence. A low overlap of genes may reflects adaptation of these bacteria to their respective environmental conditions.
Assuntos
Perfilação da Expressão Gênica/métodos , Listeria monocytogenes/genética , Regulon/genética , Fator sigma/genética , Ácidos e Sais Biliares/farmacologia , Sítios de Ligação , Transporte Biológico/genética , Cromossomos Bacterianos/genética , Metabolismo Energético/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Genômica/métodos , Immunoblotting , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Concentração Osmolar , Fator sigma/metabolismo , Fator sigma/fisiologia , Transcrição Gênica/efeitos dos fármacosRESUMO
Bacterial artificial chromosome (BAC) vectors are important tools for microbial genome research. We constructed a novel BAC vector, pUvBBAC, for replication in both gram-negative and gram-positive bacterial hosts. The pUvBBAC vector was used to generate a BAC library for the facultative intracellular pathogen Listeria monocytogenes EGD-e. The library had insert sizes ranging from 68 to 178 kb. We identified two recombinant BACs from the L. monocytogenes pUvBBAC library that each contained the entire virulence gene cluster (vgc) of L. monocytogenes and transferred them to a nonpathogenic Listeria innocua strain. Recombinant L. innocua strains harboring pUvBBAC+vgc1 and pUvBBAC+vgc2 produced the vgc-specific listeriolysin (LLO) and actin assembly protein ActA and represent the first reported cloning of the vgc locus in its entirety. The use of the novel broad-host-range BAC vector pUvBBAC extends the versatility of this technology and provides a powerful platform for detailed functional genomics of gram-positive bacteria as well as its use in explorative functional metagenomics.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Vetores Genéticos/genética , Genômica/métodos , Listeria/genética , Animais , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Genoma Bacteriano , Immunoblotting , Listeria/crescimento & desenvolvimento , Macrófagos/microbiologia , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Plasmídeos/genética , Transformação GenéticaRESUMO
The ability of Listeria monocytogenes to breach mucosal and endothelial barriers of the host during infection is a hallmark property mediated by the internalins (Inl) A and B. We examined the invasive property of several L. monocytogenes strains representing 13 serotypes. We found that invasiveness is a heterogeneous phenotype amongst L. monocytogenes serotype strains. Despite this, many of the poorly invasive and non-invasive strains of L. monocytogenes express internalins at levels comparable to those of invasive isolates. Introduction of the inlAB locus from EGD-e into several poorly invasive strains had no effect on their invasive properties. A strain from serotype 4b that exhibits highly invasive properties was further examined. Deletion of the inlAB locus abrogated invasion of this strain while reintroduction of the inlAB locus into this strain restored invasiveness. An analysis of regions flanking the inlAB locus revealed considerable differences in the strains studied. Our results suggest that efficacious entry of L. monocytogenes into eukaryotic cells is complex and requires additional factors apart from internalins. Data presented here also suggest that the inlAB locus was introduced into L. monocytogenes by horizontal gene transfer with subsequent deletion and rearrangements occurring during evolution of this species.
Assuntos
Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Fatores de Virulência/fisiologia , Virulência/genética , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Western Blotting , Cromossomos Bacterianos/genética , DNA Bacteriano/genética , Deleção de Genes , Ordem dos Genes , Teste de Complementação Genética , Células HeLa , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Sorotipagem , Sintenia , Fatores de Virulência/genéticaRESUMO
Listeria monocytogenes is a gram-positive, food-borne microorganism responsible for invasive infections with a high overall mortality. L. monocytogenes is among the very few microorganisms that can induce uptake into the host cell and subsequently enter the host cell cytosol by breaching the vacuolar membrane. We infected the murine macrophage cell line P388D1 with L. monocytogenes strain EGD-e and examined the gene expression profile of L. monocytogenes inside the vacuolar and cytosolic environments of the host cell by using whole-genome microarray and mutant analyses. We found that approximately 17% of the total genome was mobilized to enable adaptation for intracellular growth. Intracellularly expressed genes showed responses typical of glucose limitation within bacteria, with a decrease in the amount of mRNA encoding enzymes in the central metabolism and a temporal induction of genes involved in alternative-carbon-source utilization pathways and their regulation. Adaptive intracellular gene expression involved genes that are associated with virulence, the general stress response, cell division, and changes in cell wall structure and included many genes with unknown functions. A total of 41 genes were species specific, being absent from the genome of the nonpathogenic Listeria innocua CLIP 11262 strain. We also detected 25 genes that were strain specific, i.e., absent from the genome of the previously sequenced L. monocytogenes F2365 serotype 4b strain, suggesting heterogeneity in the gene pool required for intracellular survival of L. monocytogenes in host cells. Overall, our study provides crucial insights into the strategy of intracellular survival and measures taken by L. monocytogenes to escape the host cell responses.
