Complementation of Listeria seeligeri with the plcA- prfA genes from L. monocytogenes activates transcription of seeligerolysin and leads to bacterial escape from the phagosome of infected mammalian cells (original) (raw)
Related papers
Applied and Environmental Microbiology, 2010
While Listeria seeligeri and L. monocytogenes contain the main Listeria virulence gene cluster, only L. monocytogenes is considered an intracellular pathogen. Initial evolutionary analyses showed that the virulence genes prfA, hly, and plcA are conserved in L. seeligeri, with specific Hly and PrfA amino acid residues showing evidence for positive selection in L. seeligeri. Our data also show that temperature-dependent transcript patterns for prfA, which encodes a transcriptional regulator of virulence genes, differed between L. monocytogenes and L. seeligeri. To further investigate the divergence of virulence gene function and regulation, L. seeligeri prfA (prfA LS ), hly (hly LS ), and plcA (plcA LS ), as well as prfA LS constructs with different prfA promoter regions, were introduced into appropriate L. monocytogenes null mutants. Only when prfA LS was under the control of the L. monocytogenes prfA promoters (P1-and P2prfA) (P1P2 LM prfA LS ) was prfA LS able to fully complement the ⌬prfA LM deletion. hly LS introduced into an L. monocytogenes background under its native promoter showed transcript levels similar to those of hly LM and was able to partially restore L. monocytogenes wild-type-level hemolysis and intracellular growth, even though Hly LM and Hly LS showed distinct patterns of cell-and supernatant-associated hemolytic activities. Our data indicate that (i) regulation of prfA expression differs between L. monocytogenes and L. seeligeri, although hly transcription is temperature dependent in both species, and (ii) PrfA and Hly functions are largely, but not fully, conserved between L. seeligeri and L. monocytogenes. Virulence gene homologues and their expression thus appear to have adapted to distinct but possibly related functions in these two species.
Molecular Microbiology, 1994
Listeria monocytogenes is a bacterial pathogen that multiplies within the cytosol of eukaryotic cells. To identify Listeria genes with preferentially intracellular expression {pic genes), a library of In917-lac insertion mutants was screened for transcriptional fusions to IacZ with higher expression inside a macrophagelike cell line than In a rich broth medium. Five pic genes with up to 100-fold induction inside cells were identified. Three of them {purH, purD and pyrE) were involved in nucleotide biosynthesis. One was part of an operon encoding an ABC (ATP-binding cassette) transporter for arginine. The corresponding mutants were not affected in intracellular growth, cell-to-cell spread or virulence, except for the transporter mutant, whose LDgo after intravenous infection of mice was twofold higher than the wild-type. The fifth gene was picA, a previously identified virulence gene that encodes a phosphatidylinositol-phospholipase C, and is cotranscribed with prfA, a gene encoding a pleiotropic transcriptional activator of known virulence genes. Although picA expression is known to depend on PrfA, a prfA promoter-/acZ fusion was highly expressed both Inside and outside cells. Furthermore, in the presence of cellobiose, a disaccharide recently shown to repress pIcA and hiy expression, picA and hiy mRNA levels were dramatically reduced without any decrease in the monocistronic prfA mRNA levels. These results demonstrate that virulence gene activation does not depend only on prfA transcript accumulation.
Molecular Microbiology, 2003
The facultative intracellular bacterial pathogen Listeria monocytogenes dramatically increases the expression of several key virulence factors upon entry into the host cell cytosol. actA , the protein product of which is required for cell-to-cell spread of the bacterium, is expressed at low to undetectable levels in vitro and increases in expression more than 200fold after L. monocytogenes escape from the phagosome. To identify bacterial factors that participate in the intracellular induction of actA expression, L. monocytogenes mutants expressing high levels of actA during in vitro growth were selected after chemical mutagenesis. The resulting mutant isolates displayed a wide range of actA expression levels, and many were less sensitive to environmental signals that normally mediate repression of virulence gene expression. Several isolates contained mutations affecting actA gene expression that mapped at least 40 kb outside the PrfA regulon, supporting the existence of additional regulatory factors that contribute to virulence gene expression. Two actA in vitro expression mutants contained novel mutations within PrfA, a key regulator of L. monocytogenes virulence gene expression. PrfA E77K and PrfA G155S mutations resulted in high-level expression of PrfAdependent genes, increased bacterial invasion of epithelial cells and increased virulence in mice. Both prfA mutant strains were significantly less motile than wild-type L. monocytogenes. These results suggest that, although constitutive activation of PrfA and PrfA-dependent gene expression may enhance L. monocytogenes virulence, it may conversely hamper the bacterium's ability to compete in environments outside host cells. 1538 L. M. Shetron-Rama et al.
Microbiology, 2003
Listeriolysin O (LLO, hly-encoded), a major virulence factor secreted by the bacterial pathogen Listeria monocytogenes, is synthesized as a precursor of 529 residues. To impair LLO secretion, the four residues of the predicted signal sequence cleavage site (EA-KD) were deleted and the mutant LLO protein was expressed in a hly-negative derivative of L. monocytogenes. Unexpectedly, the mutant protein was secreted in normal amounts in the culture supernatant and was fully haemolytic. N-terminal sequencing of the secreted LLO molecule revealed that N-terminal processing of the preprotein occurred three residues downstream of the natural cleavage site. L. monocytogenes expressing this truncated LLO showed a reduced capacity to disrupt the phagosomal membranes of bone marrow macrophages and of hepatocytes; and the mutant strain showed a 100-fold decrease in virulence in the mouse model. These results suggest that the first N-terminal residues of mature LLO participate directly in phagosom...
Molecular determinants of Listeria monocytogenes pathogenesis
Infection and immunity, 1992
Listeria monocytogenes is a rapidly growing, gram-positive, food-borne human and animal pathogen responsible for serious infections in immunocompromised individuals and pregnant women (17). The murine model of listeriosis has received enormous attention over the years because of the utility of L. monocytogenes as a model pathogen to study cell-mediated immunity. In fact, much of our current understanding of cell-mediated immunity, such as the concept of the activated macrophage, has its roots in the study of murine listeriosis . The beauty of the murine model is that it provides a highly reproducible system for the quantitation of L. monocytogenes virulence. However, until relatively recently almost nothing was known about the cell biology of intracellular growth or bacterial determinants of pathogenicity.
Molecular Microbiology, 2004
The hly-encoded listeriolysin O (LLO) is a major virulence factor secreted by the intracellular pathogen Listeria monocytogenes, which plays a crucial role in the escape of bacteria from the phagosomal compartment. Here, we identify a putative PEST sequence close to the N-terminus of LLO and focus on the role of this motif in the biological activities of LLO. Two LLO variants were constructed: a deletion mutant protein, lacking the 19 residues comprising this sequence (residues 32±50), and a recombinant protein of wild-type size, in which all the P, E, S or T residues within this motif have been substituted. The two mutant proteins were fully haemolytic and were secreted in culture supernatants of L. monocytogenes in quantities comparable with that of the wildtype protein. Strikingly, both mutants failed to restore virulence to a hly-negative strain in vivo. In vitro assays showed that L. monocytogenes expressing the LLO deletion mutant was strongly impaired in its ability to escape from the phagosomal vacuole and, subsequently, to divide in the cytosol of infected cells. This work reveals for the first time that the N-terminal portion of LLO plays an important role in the development of the infectious process of L. monocytogenes.
Infection and Immunity, 1990
We identified nonhemolytic mutants of Listeria monocytogenes that were severely deficient in their ability to invade mammalian nonprofessional phagocytes. These mutants were generated spontaneously or by means of transposon Tn916 mutagenesis. In terms of their extracellular proteins, the noninvasive mutants were deficient not only in the sulfhydryl-activated hemolysin (listeriolysin) but also in an antigenically unrelated extracellular protein with an apparent molecular weight of 32,000 which could induce opacity in egg yolk and is considered to be a phospholipase. Our results suggest the existence of a common genetic control between the expression of listeriolysin and that of other determinants, including a phospholipase and determinants involved in the ability of L. monocytogenes to enter mammalian cells.
Intracellular Gene Expression Profile of Listeria monocytogenes
Infection and Immunity, 2006
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.