Intracellular Gene Expression Profile of Listeria monocytogenes

Abstract

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 wholegenome microarray and mutant analyses. We found that ϳ17% 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.Listeriosis is a food-borne disease with high mortality rates. Listeria monocytogenes, the causative agent, can survive in diverse habitats, including extracellular abiotic and intracellular environments, and infect a large number of vertebrate and invertebrate hosts. All of the identified virulence genes in L. monocytogenes are known to be under the direct or partial control of positive regulatory factor A (PrfA) (9, 34). Entry into nonprofessional phagocytes is mediated by surface-associated gene products internalin A and B (12, 31). Early after internalization, the bacteria disrupt the phagosomal membrane of the host by expressing a pore-forming toxin, listeriolysin (Hly), and a phospholipase (PlcA) to access the cytoplasm of the host cell. Intracellular movement of the bacteria inside the host cell is mediated by ActA, which polymerizes the host actin molecules and propels itself inside the cytosol of the host cell. Spreading from one cell to another is dependent on hemolysin (Hly) and another phospholipase (PlcB) (39). Mutations have been introduced into virulence genes of L. monocytogenes that lead to debilitating phenotypes in the mouse model of infection. In addition, a hexose phosphate transporter, UhpT, that is required for efficacious intracellular growth has recently been described (11). Rece...

Listeria monocytogenesl‐forms respond to cell wall deficiency by modifying gene expression and the mode of division

“…An example is the lmo2484-87 operon, homologous to the stress response regulator psp operon of B. subtilis, which is induced under osmotic stress conditions (Elderkin et al, 2002;Studholme and Dixon, 2003). In contrast, the downregulated qox operon results in decreased synthesis of the oxygeninduced cytochrome d complex (Chatterjee et al, 2006). These findings can be interpreted as a response of the L-forms to their markedly reduced surface-to-volume ratio, and agrees with the rule that enlargement of cells generally results in metabolic downregulation (Koch and Wang, 1982;Hess and Mikhailov, 1995).…”

The arsenal of virulence factors deployed byListeria monocytogenesto promote its cell infection cycle

“…Interestingly, the secA2 gene, as well as genes encoding SecA2-secreted proteins, are coordinately regulated in infected cells and host organs. 112,153,187,208 In particular, it was observed an upregulation of most genes encoding SecA2 substrates, including all the SecA2 components and SecA2-secreted virulence factors, in bacteria infecting mouse spleens, 112 which strongly suggests their involvement in pathogenesis.…”

“…153 Proteomic analyses unveiled that PrsA2 appears to have been adapted to facilitate L. monocytogenes survival under conditions of increased protein secretion, such as occurs during PrfA activation within the host cell cytosol. 154 The oligopeptide-binding protein OppA was shown to mediate the transport of oligopeptides, and to be involved in intracellular survival in macrophages and in bacterial growth in organs of infected mice.…”

“…This putative PrfA cofactor is yet to be identified, but links between carbon metabolism and PrfA-dependent transcription suggest that host nutrient availability may work as an intracellular localization signal for L. monocytogenes. 22,186 Interestingly, a large proportion of the genes controlled by PrfA, and in particular the major virulence factors, were shown to be highly upregulated during infection of macrophages 153 and mouse organs. 112,187 A recent temporal transcriptome analysis revealed that σ B regulates a large and diverse set of genes (nearly 200) that are predicted to function in stress tolerance, carbohydrate metabolism, transport and cell envelope processes, and virulence.…”

“…Indeed, most VirR-regulated genes (13 of 17), including virR, the dlt operon and mprF, were shown upregulated when Listeria infect mouse spleen, 112 with 10 of these genes being also upregulated in infected macrophages. 153 Reversely, 12 VirR-regulated genes are downregulated in bacteria present in the mouse intestine lumen. 187 MogR, DegU and GmaR are three regulators involved in the control of flagella production.…”