SvpA, a novel surface virulence-associated protein required for intracellular survival of Listeria monocytogenes

Abstract

A previously unknown protein, designated SvpA (surface virulence-associated protein) and implicated in the virulence of the intracellular pathogen Listeria monocytogenes, was identified. This 64 kDa protein, encoded by svpA, is both secreted in culture supernatants and surface-exposed, as shown by immunogold labelling of whole bacteria with an anti-SvpA antibody. Analysis of the peptide sequence revealed that SvpA contains a leader peptide, a predicted C-terminal transmembrane region and a positively charged tail resembling that of the surface protein ActA, suggesting that SvpA might partially reassociate with the bacterial surface by its C-terminal membrane anchor. An allelic mutant was constructed by disrupting svpA in the wild-type strain LO28. The virulence of this mutant was strongly attenuated in the mouse, with a 2 log decrease in the LD 50 and restricted bacterial growth in organs as compared to the wild-type strain. This reduced virulence was not related either to a loss of adherence or to a lower expression of known virulence factors, which remained unaffected in the svpA mutant. It was caused by a restriction of intracellular growth of mutant bacteria. By following the intracellular behaviour of bacteria within bone-marrow-derived macrophages by confocal and electron microscopy studies, it was found that most svpA mutant bacteria remained confined within phagosomes, in contrast to wild-type bacteria which rapidly escaped to the cytoplasm. The regulation of svpA was independent of PrfA, the transcriptional activator of virulence genes in L. monocytogenes. In fact, SvpA was down-regulated by MecA, ClpC and ClpP, which are highly homologous to proteins of Bacillus subtilis forming a regulatory complex controlling the competence state of this saprophyte. The results indicate that : (i) SvpA is a novel factor involved in the virulence of L. monocytogenes, promoting bacterial escape from phagosomes of macrophages ; (ii) SvpA is, at least partially, associated with the surface of bacteria ; and (iii) SvpA is PrfA-independent and controlled by a MecAdependent regulatory network.

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

“…The virulence of a svpA mutant was shown to be strongly attenuated in infected mice. 140 This reduced virulence was the result of an impairment in the intracellular multiplication of the mutant bacteria, as most of these remained trapped inside phagosomes, in contrast to wild-type bacteria that quickly escaped to the cytoplasm. SvpA appears thus as a virulence factor of L. monocytogenes that promotes bacterial escape from macrophage phagosomes.…”

Sortase B, a New Class of Sortase inListeria monocytogenes

“…Here we studied the role of SrtB in L. monocytogenes by constructing ⌬srtB and ⌬srtA ⌬srtB knockout mutants. We show that SrtB has a "sorting" activity distinct from that of SrtA, and we identify SvpA, a virulence factor of L. monocytogenes (8), as a target of SrtB. The respective roles of SrtA and SrtB in surface protein anchoring and in the virulence of L. monocytogenes are discussed.…”

“…Bacteria were washed twice in phosphatebuffered saline (PBS) and fixed in 3% paraformaldhehyde in PBS for 10 min. They were then labeled with an anti-SvpA polyclonal antibody (8) and an Alexa 488-conjugated secondary antibody (Molecular Probes). To study the distribution of surface proteins strongly bound to PG by using the anti-PG antibody, PBS-washed bacteria were boiled in 4% sodium dodecyl sulfate (SDS) for 30 min, further washed three times with PBS to remove traces of SDS, and processed for immunofluorescence analysis as described above for SvpA.…”

“…The mutation in svpA, initially constructed in LO28, corresponds to the deletion of a 79-bp internal fragment of svpA and insertion of a promoterless aph3 gene conferring resistence to kanamycin. It was introduced into the chromosome of EGD-e by allelic replacement, using the recombinant integrative plasmid pAUL-A-svpA⍀aphA3 described previously (8).…”

Article

“…Opp est impliqué dans les processus de sporulation et compétence chez Bacillus subtilis [17,24], de conjugaison chez Enterococcus faecalis [9,18], de virulence chez plusieurs bactéries pathogè-nes (Streptococcus pyogenes [21] ; Streptococcus gordonii [19] ; Staphylococcus aureus [5] ; Bacillus cereus [23] ; Bacillus thuringiensis [11]), de survie à basses températures et à l'intérieur de macrophages chez Listeria monocytogenes [2].…”