The gene cluster inlC2DE of Listeria monocytogenes contains additional new internalin genes and is important for virulence in mice

Raffelsbauer, Diana; Bubert, Andreas; Engelbrecht, Fredi; Scheinpflug, J.; Simm, Andreas; Hess, Jürgen; Kaufmann, Stefan H. E.; Goebel, Werner

doi:10.1007/s004380050880

Cited by 96 publications

(111 citation statements)

References 39 publications

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“…Functional implications. Several members of the LPXTG protein family have been shown to be involved in the virulence of L. monocytogenes (1,22,40). Of particular interest, internalin, which is required for entry into human Caco-2 cells in vitro, had as good as no effect on bacterial virulence in the mouse model, even upon administration by the oral route (14,22).…”

Section: Discussionmentioning

confidence: 99%

The Sortase SrtA of Listeria monocytogenes Is Involved in Processing of Internalin and in Virulence

et al. 2002

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Listeria monocytogenes is an intracellular gram-positive human pathogen that invades eucaryotic cells. Among the surface-exposed proteins playing a role in this invasive process, internalin belongs to the family of LPXTG proteins, which are known to be covalently linked to the bacterial cell wall in gram-positive bacteria. Recently, it has been shown in Staphylococcus aureus that the covalent anchoring of protein A, a typical LPXTG protein, is due to a cysteine protease, named sortase, required for bacterial virulence. Here, we identified in silico from the genome of L. monocytogenes a gene, designated srtA, encoding a sortase homologue. The role of this previously unknown sortase was studied by constructing a sortase knockout mutant. Internalin was used as a reporter protein to study the effects of the srtA mutation on cell wall anchoring of this LPXTG protein in L. monocytogenes. We show that the srtA mutant (i) is affected in the display of internalin at the bacterial surface, (ii) is significantly less invasive in vitro, and (iii) is attenuated in its virulence in the mouse. These results demonstrate that srtA of L. monocytogenes acts as a sortase and plays a role in the pathogenicity.Gram-positive bacteria are surrounded by a cell wall envelope containing attached polypeptides and polysaccharides that may interact with host cells and play a role in the virulence of pathogenic species (34). In gram-positive bacteria, several distinct mechanisms of cell wall attachment and display of surface proteins have been recently described (reviewed in reference 5). The only surface proteins known to be covalently linked to the cell wall are the LPXTG proteins, exemplified by protein A of Staphylococcus aureus (34). In a pioneer work, Schneewind et al. described a cysteine protease of S. aureus, designated sortase, which is responsible for the covalent attachment of protein A, and identified the biochemical processes allowing the anchoring of protein A to the cell wall (27). Very recently, the three-dimensional structure of the sortase of S. aureus was determined by nuclear magnetic resonance spectroscopy, thus identifying a catalytic domain responsible for the transpeptidation reaction (17). After synthesis in the bacterial cytoplasm, surface protein precursors are translocated across the membrane and the NH 2 -proximal leader peptide is removed by leader peptidase. The COOH-terminal sorting signal is first cleaved by sortase between the threonine and glycine residues of the LPXTG motif. Then, the enzyme covalently links the carboxyl of the threonine to the cell wall peptidoglycan by amide linkage (34,35,43,44,45). The sorting signal consists of a conserved LPXTG motif followed by a membrane-spanning hydrophobic domain and a tail mostly composed of positively charged residues (18, 28, 42). Notably, it was recently shown that in the gram-positive human pathogen Listeria monocytogenes, the surface proteins were also cleaved between the threonine and the glycine residue of the LPXTG motif and amide-linked to the peptidogl...

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Section: Discussionmentioning

confidence: 99%

The Sortase SrtA of Listeria monocytogenes Is Involved in Processing of Internalin and in Virulence

et al. 2002

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“…In addition to these confirmed virulence genes, three other internalins (inlC2, inlD, and inlE) also showed significant expression ratios of Ͼ2.0 under osmotic and/or stationary-phase stress (Table 2). PCR mapping showed that the L. monocytogenes strain used in our study (10403S) bears an inlC2DE operon rather than an inlGHE operon, which is present in the L. monocytogenes EGD-e strain (57). Each ORF of the inlC2DE gene cluster is potentially transcribed independently of the others (13).…”

Section: Hmmmentioning

confidence: 96%

Listeria monocytogenesσ^BRegulates Stress Response and Virulence Functions

et al. 2003

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While the stress-responsive alternative sigma factor B has been identified in different species of Bacillus, Listeria, and Staphylococcus, the B regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify B -dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidate B -dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted B -dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and ⌬sigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significant B -dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-foldhigher expression in the wild type). Of the 55 genes exhibiting B -dependent expression, 54 were preceded by a sequence resembling the B promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the B -dependent nature of a subset of eight selected promoter regions. Notably, the B -dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, B also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest that B contributes to L. monocytogenes gene expression during infection.

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“…Both of these internalins belong to a large listerial protein family which in L. monocytogenes includes at least seven more internalins (12,13,43), all of which are typical members of the leucine-rich repeat superfamily that includes many other eukaryotic and prokaryotic proteins (26,27). With the exception of InlC and InlB, the internalins have in their C-terminal portions a conserved region with an LPXTG motif, which anchors these proteins to the peptidoglycan of the bacterial cell surface (36).…”

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confidence: 99%

“…Besides InlA and InlB, only InlC, InlC2, InlD, InlE, InlF, InlG, and InlH have been studied so far to some extent (12,13,43). In contrast to InlA and InlB, none of these internalins seem to be able to induce phagocytosis by nonphagocytic mammalian cells (3).…”

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confidence: 99%

“…However, InlC may support InlA-mediated internalization into epithelial cells (3). As deletion of inlC leads to a significant reduction in L. monocytogenes virulence in the mouse model and, in particular, to significantly decreased bacterial loads in the livers of orally infected mice (13) and as an L. monocytogenes mutant with a deletion in the inlGHE gene cluster exhibits an increase in the 50% lethal dose after intravenous infection of mice and reduced replication in the livers of orally infected mice (43), it has been suggested that these internalins are involved in virulence, which has been clearly shown for InlA and InlB. The functions of all other internalins are basically unknown.…”

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confidence: 99%

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Enhanced Synthesis of Internalin A inaroMutants ofListeria monocytogenesIndicates Posttranscriptional Control of theinlABmRNA

2005

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Listeria monocytogenes mutants with deletions in aroA, aroB, or aroE exhibited strong posttranscriptional upregulation of internalin A (InlA) and InlB synthesis, which resulted in a more-than-10-fold increase in InlA-mediated internalization by epithelial Caco-2 cells and a 4-fold increase in InlB-mediated internalization by microvascular endothelial cells (human brain microvascular endothelial cells) compared to the wild-type strain. The increase in InlA and InlB production was not due to enhanced PrfA-and/or sigma factor B (SigB)-dependent inlAB transcription but was caused by enhanced translation of the inlAB transcripts in the aro mutants. All inlA(B) transcripts had a 396-nucleotide upstream 5 untranslated region (UTR). Different deletions introduced into this UTR led to significant reductions in InlA and InlB synthesis; enhanced translation of all of the truncated transcripts in the aro mutants was, however, still observed. Thus, translation of the inlAB transcripts was subject to two modes of posttranscriptional control, one mediated by the UTR structure and the other mediated by the aro mutation. The latter mode of control seemed to be related to the predominantly anaerobic metabolism of the aro mutants.

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The gene cluster inlC2DE of Listeria monocytogenes contains additional new internalin genes and is important for virulence in mice

Cited by 96 publications

References 39 publications

The Sortase SrtA of Listeria monocytogenes Is Involved in Processing of Internalin and in Virulence

The Sortase SrtA of Listeria monocytogenes Is Involved in Processing of Internalin and in Virulence

Listeria monocytogenesσ^BRegulates Stress Response and Virulence Functions

Enhanced Synthesis of Internalin A inaroMutants ofListeria monocytogenesIndicates Posttranscriptional Control of theinlABmRNA

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The gene cluster inlC2DE of Listeria monocytogenes contains additional new internalin genes and is important for virulence in mice

Cited by 96 publications

References 39 publications

The Sortase SrtA of Listeria monocytogenes Is Involved in Processing of Internalin and in Virulence

The Sortase SrtA of Listeria monocytogenes Is Involved in Processing of Internalin and in Virulence

Listeria monocytogenesσBRegulates Stress Response and Virulence Functions

Enhanced Synthesis of Internalin A inaroMutants ofListeria monocytogenesIndicates Posttranscriptional Control of theinlABmRNA

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Listeria monocytogenesσ^BRegulates Stress Response and Virulence Functions