The GTPase CpgA is implicated in the deposition of the peptidoglycan sacculus in Bacillus subtilis.

Depletion of the Bacillus subtilis GTPase CpgA produces abnormal cell shapes, nonuniform deposition of cell wall, and five- to sixfold accumulation of peptidoglycan precursors. Nevertheless, the inherent structure of the cell wall appeared mostly unchanged. The results are consistent with CpgA being involved in coordinating normal peptidoglycan deposition.

Inactivation of a subpopulation of human neutrophils by exposure to ultrahigh-molecular-weight polyethylene wear debris.

Polymorphonuclear neutrophils, a first line of defence against invading microbial pathogens, may be attracted by inflammatory mediators triggered by ultrahigh-molecular-weight polyethylene (UHMWPE) wear particles released from orthopaedic prostheses. Phagocytosis of UHMWPE particles by neutrophils may indirectly compromise their phagocytic-bactericidal mechanisms, thus enhancing host susceptibility to microbial infections. In an in vitro assay, pre-exposure of purified human neutrophils to UHMWPE micrometre- and submicrometre-sized wear particles interfered with subsequent Staphylococcos aureus uptake in a heterogeneous way, as assessed by a dual label fluorescence microscopic assay that discriminated intracellular rhodamine-labelled UHMWPE particles from fluorescein isothiocyanate-labelled S. aureus. Indeed, a higher percentage (44%) of neutrophils having engulfed UHMWPE particles lost the ability to phagocytize S. aureus, compared with UHMWPE-free neutrophils (<3%). Pre-exposure of neutrophils to UHMWPE wear particles did not impair but rather stimulated their oxidative burst response in a chemoluminescence assay. The presence of UHMWPE wear particles did not lead to significant overall consumption of complement-mediated opsonic factors nor decreased surface membrane display of neutrophil complement receptors. In conclusion, engulfment of UHMWPE wear particles led to inactivation of S. aureus uptake in nearly half of the neutrophil population, which may potentially impair host clearance mechanisms against pyogenic infections.

Exploring the role of the CTL epitope region of listeriolysin O in the pathogenesis of Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular bacterial pathogen responsible for severe opportunistic infections in humans and animals. The secreted cholesterol-dependent cytolysin, listeriolysin O (LLO), mediates phagosomal escape and allows bacterial growth in the cytosol of infected cells. In order to identify new LLO determinants participating in bacterial pathogenesis, this study focused on a major target of LLO proteolytic cleavage in vitro, the CTL epitope region (residues 91-99). Mutations were generated by site-directed mutagenesis in the epitope or in the two clusters of positive charges flanking the epitope. Two LLO mutants (a single mutation K103A and a double mutation R89G, K90G) were normally and stably secreted by L. monocytogenes. In contrast, a mutant carrying four amino acid substitutions in the epitope itself (Y92K, D94A, E97K, Y98F) was highly susceptible to proteolytic degradation. While these three LLO mutant proteins showed a reduced haemolytic activity, they all promoted efficient phagosomal escape and intracellular multiplication in different cell types, and were non-cytotoxic. The deletion of the epitope (Delta91-99), as well as the substitution of two, three or four of the four lysine residues (K103 to K106) by alanine residues did not lead to the production of a detectable protein. These results confirm the lack of correlation between haemolytic activity and phagosomal membrane disruption. They reveal the importance of the 91-99 region in the production of a stable and functional LLO. LD(50) determinations in the mouse model suggest a possible link between LLO stability and virulence.

Gap, a mycobacterial specific integral membrane protein, is required for glycolipid transport to the cell surface.

The cell envelope of mycobacteria is a complex multilaminar structure that protects the cell from stresses encountered in the environment, and plays an important role against the bactericidal activity of immune system cells. The outermost layer of the mycobacterial envelope typically contains species-specific glycolipids. Depending on the mycobacterial species, the major glycolipid localized at the surface can be either a phenolglycolipid or a peptidoglycolipid (GPL). Currently, the mechanism of how these glycolipids are addressed to the cell surface is not understood. In this study, by using a transposon library of Mycobacterium smegmatis and a simple dye assay, six genes involved in GPLs synthesis have been characterized. All of these genes are clustered in a single genomic region of approximately 60 kb. We show by biochemical analyses that two non-ribosomal peptide synthetases, a polyketide synthase, a methyltransferase and a member of the MmpL family are required for the biosynthesis of the GPLs backbone. Furthermore, we demonstrate that a small integral membrane protein of 272 amino acids named Gap (gap: GPL addressing protein) is specifically required for the transport of the GPLs to the cell surface. This protein is predicted to contain six transmembrane segments and possesses homologues across the mycobacterial genus, thus delineating a new protein family. This Gap family represents a new paradigm for the transport of small molecules across the mycobacterial envelope, a critical determinant of mycobacterial virulence.

Role of FliF and FliI of Listeria monocytogenes in flagellar assembly and pathogenicity.

Flagellar structures have been shown to participate in virulence in a variety of intestinal pathogens. Here, we have identified two potential flagellar genes of Listeria monocytogenes: lmo0713, encoding a protein similar to the flagellar basal body component FliF, and lmo0716, encoding a protein similar to FliI, the cognate ATPase energizing the flagellar export apparatus. Expression of fliF and fliI appears to be downregulated at 37 degrees C, like that of flaA, encoding flagellin. By constructing two chromosomal deletion mutants, we show that inactivation of either fliF or fliI (i) abolishes bacterial motility and flagella production, (ii) impairs adhesion and entry into nonphagocytic epithelial cells, and (iii) also reduces uptake by bone marrow-derived macrophages. However, the DeltafliF and DeltafliI mutations have only a minor impact on bacterial virulence in the mouse model, indicating that the flagellar secretion apparatus itself is not essential for survival in this animal model. Finally, among 100 human clinical isolates of L. monocytogenes tested, we found 20 strains still motile at 37 degrees C. Notably, all these strains adhered less efficiently than strain EGD-e to Caco-2 cells at 37 degrees C but showed no defect of intracellular multiplication. These data suggest that expression of the flagella at 37 degrees C might hinder optimal adhesion to epithelial cells but has no impact on intracytosolic survival of L. monocytogenes.

The svpA-srtB locus of Listeria monocytogenes: fur-mediated iron regulation and effect on virulence.

In Listeria monocytogenes the promoter region of the svpA-srtB locus contains a well-conserved Fur box. We characterized the iron-regulation of this locus: real-time polymerase chain reaction analyses and anti-SvpA immunoblots showed that, in response to iron deprivation svpA transcription and SvpA production markedly increased (80-fold and 10-fold respectively), when initiated by either the addition of the iron chelator 2,2'-bipyridyl to BHI media, or by growth in iron-restricted minimal media. Green fluorescent protein (GFP) reporter constructs also showed increased activity of the svpA-srtB promoter in Escherichia coli (37-fold) and in L. monocytogenes (two- to threefold) when the bacteria were grown in iron-deficient conditions. A Deltafur mutant of L. monocytogenes constitutively synthesized SvpA, as well as GFP fused to the svpA-srtB promoter. Cellular fractionation data revealed that in iron-rich media wild-type SvpA was exclusively secreted to the culture supernatant. However, both the Deltafur derivative and wild-type L. monocytogenes grown in iron-deficient media anchored a fraction of the SvpA proteins (approximately 5%) to peptidoglycan, and produced a lower-molecular weight, wholly secreted form of SvpA. Together these data establish that iron availability controls transcription of the svpA-srtB locus (through Fur-mediated regulation), and attachment of SvpA to the cell wall (through SrtB-mediated covalent linkage). SvpA bears homology to IsdC, a haemin-binding protein of Staphylococcus aureus, and haemin bound to SvpA in solution. However, site-directed deletions of four structural genes and the promoter of the svpA-srtB locus did not impair haemin, haemoglobin or ferrichrome utilization in nutrition tests. We did not find strong evidence to support the notion that the svpA-srtB locus participates in haemin acquisition, as was reported for the homologous isd operon of S. aureus. Furthermore, the svpA-srtB mutant strains showed no significant attenuation of virulence in an intravenous mouse model system, but we found that the mutations reduced the persistence of L. monocytogenes in murine liver, spleen and intestines after oral administration.

Differential roles of multiple signal peptidases in the virulence of Listeria monocytogenes.

Most bacteria contain one type I signal peptidase (Spase I) for cleavage of signal peptides from exported and secreted proteins. Here, we identified a locus encoding three contiguous Spase I genes in the genome of Listeria monocytogenes. The deduced Sip proteins (denoted SipX, SipY and SipZ) are significantly similar to SipS and SipT, the major SPase I proteins of Bacillus subtilis (38% to 44% peptidic identity). We studied the role of these multiple signal peptidases in bacterial pathogenicity by constructing a series of single- and double-chromosomal knock-out mutants. Inactivation of sipX did not affect intracellular multiplication of L. monocytogenes but significantly reduced bacterial virulence (approximately 100-fold). Inactivation of sipZ impaired the secretion of phospholipase C (PC-PLC) and listeriolysin O (LLO), restricted intracellular multiplication and almost abolished virulence (LD(50) of 10(8.3)), inactivation of sipY had no detectable effects. Most importantly, a mutant expressing only SipX was impaired in intracellular survival and strongly attenuated in the mouse (LD(50) of 10(7.2)), whereas, a mutant expressing only SipZ behaved like wild-type EGD in all the assays performed. The data establish that SipX and SipZ perform distinct functions in bacterial pathogenicity and that SipZ is the major Spase I of L. monocytogenes. This work constitutes the first report on the differential role of multiple Spases I in a pathogenic bacterium and suggests a possible post-translational control mechanism of virulence factors expression.

Maturation of lipoproteins by type II signal peptidase is required for phagosomal escape of Listeria monocytogenes.

Lipoproteins of Gram-positive bacteria are involved in a broad range of functions such as substrate binding and transport, antibiotic resistance, cell signaling, or protein export and folding. Lipoproteins are also known to initiate both innate and adaptative immune responses. However, their role in the pathogenicity of intracellular microorganisms is yet poorly understood. In Listeria monocytogenes, a Gram-positive facultative intracellular human pathogen, surface proteins have important roles in the interactions of the microorganism with the host cells. Among the putative surface proteins of L. monocytogenes, lipoproteins constitute the largest family. Here, we addressed the role of the signal peptidase (SPase II), responsible for the maturation of lipoproteins in listerial pathogenesis. We identified a gene, lsp, encoding a SPase II in the genome of L. monocytogenes and constructed a deltalsp chromosomal deletion mutant. The mutant strain fails to process several lipoproteins demonstrating that lsp encodes a genuine SPase II. This defect is accompanied by a reduced efficiency of phagosomal escape during infection of eucaryotic cells, and leads to an attenuated virulence. We show that lsp gene expression is strongly induced when bacteria are still entrapped inside phagosomes of infected macrophages. The data presented establish, thus, that maturation of lipoproteins is critical for efficient phagosomal escape of L. monocytogenes, a process temporally controlled by the regulation of Lsp production in infected cells.

Modification of the signal sequence cleavage site of listeriolysin O does not affect protein secretion but impairs the virulence of Listeria monocytogenes.

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 phagosomal escape and bacterial infection.

Capacity of ivanolysin O to replace listeriolysin O in phagosomal escape and in vivo survival of Listeria monocytogenes.

Listeriolysin O (LLO, hly-encoded) is a major virulence factor secreted by the pathogen Listeria monocytogenes. The amino acid sequence of LLO shows a high degree of similarity with that of ivanolysin O (ILO), the cytolysin secreted by the ruminant pathogen Listeria ivanovii. Here, it was tested whether ILO could functionally replace LLO by expressing the gene encoding ILO under the control of the hly promoter, in an hly-deleted strain of L. monocytogenes. It is shown that ILO allows efficient phagosomal escape of L. monocytogenes in both macrophages and hepatocytes. Moreover, expression of ILO is not cytotoxic and promotes normal intracellular multiplication. In vivo, the ILO-expressing strain can multiply and persist for several days in the liver of infected mice but is unable to survive in the spleen. This work underscores the key role played by the cytolysin in the virulence of pathogenic Listeria.

Critical role of the N-terminal residues of listeriolysin O in phagosomal escape and virulence of Listeria monocytogenes.

A putative PEST sequence was recently identified close to the N-terminus of listeriolysin O (LLO), a major virulence factor secreted by the pathogenic Listeria monocytogenes. The deletion of this motif did not affect the secretion and haemolytic activity of LLO, but abolished bacterial virulence. Here, we first tested whether the replacement of the PEST motif of LLO by two different sequences, with either a very high or no PEST score, would affect phagosomal escape, protein stability and, ultimately, the virulence of L. monocytogenes. Then, we constructed LLO mutants with an intact PEST sequence but carrying mutations on either side, or on both sides, of the PEST motif. The properties of these mutants prompted us to construct three LLO mutants carrying single amino acid substitutions in the distal portion of the PEST region (P49A, K50A and P52A; preprotein numbering). Our data demonstrate that the susceptibility of LLO to intracellular proteolytic degradation is not related to the presence of a high PEST score sequence and that the insertion of two residues immediately downstream of the intact PEST sequence is sufficient to impair phagosomal escape and abolish bacterial virulence. Furthermore, we show that single amino acid substitutions in the distal portion of the PEST motif are sufficient to attenuate bacterial -virulence significantly, unravelling the critical role of this region of LLO in the pathogenesis of L. -monocytogenes.

Effect of Nramp1 on bacterial replication and on maturation of Mycobacterium avium-containing phagosomes in bone marrow-derived mouse macrophages.

Pathogenic mycobacteria prevent maturation of the phagosomes in which they reside inside macrophages and this is thought to be a major strategy allowing them to survive and multiply within macrophages. The molecular basis for this inhibition is only now beginning to emerge with the molecular characterization of the phagosome membrane enclosing these pathogens. We have used here several electron microscopy approaches in combination with counts of bacterial viability to analyse how expression of Nramp1 at the phagosomal membrane may influence survival of Mycobacterium avium and affect its ability to modulate the fusogenic properties of the phagosome in which it resides. The experiments were carried out in bone marrow-derived macrophages from wild-type 129sv (Nramp1(G169)) mice and from isogenic 129sv carrying a null mutation at Nramp1 (Nramp(1-/-)) following infection with a virulent strain of M. avium. We show here that Nramp1 expression has a bacteriostatic effect and that abrogation of Nramp1 restores the bacteria's capacity to replicate within macrophages. The combined analyses of the acquisition of endocytic contents markers delivered to early endosomes and/or lysosomes either prior to or after phagocytic uptake showed that in Nramp1-positive macrophages, M. avium was unable to prevent phagosome maturation and fusion with lysosomes but that in Nramp1-negative macrophages this capacity was restored. Several hypotheses are proposed to explain how Nramp1 could affect survival of M. avium. We also propose how the present observations could relate to the model according to which mycobacteria can prevent phagosome maturation by establishing a tight interaction with constituents of the phagosome membrane. Furthermore, these results show the importance of the choice of macrophages used as a model to study intracellular survival strategies of pathogens.

Balance between two transpeptidation mechanisms determines the expression of beta-lactam resistance in Enterococcus faecium.

The d,d-transpeptidase activity of high molecular weight penicillin-binding proteins (PBPs) is essential to maintain cell wall integrity as it catalyzes the final cross-linking step of bacterial peptidoglycan synthesis. We investigated a novel beta-lactam resistance mechanism involving by-pass of the essential PBPs by l,d-transpeptidation in Enterococcus faecium. Determination of the peptidoglycan structure by reverse phase high performance liquid chromatography coupled to mass spectrometry revealed that stepwise selection for ampicillin resistance led to the gradual replacement of the usual cross-links generated by the PBPs (d-Ala(4) --> d-Asx-Lys(3)) by cross-links resulting from l,d-transpeptidation (l-Lys(3) --> d-Asx-Lys(3)). This was associated with no modification of the level of production of the PBPs or of their affinity for beta-lactams, indicating that altered PBP activity was not required for ampicillin resistance. A beta-lactam-insensitive l,d-transpeptidase was detected in membrane preparations of the parental susceptible strain. Acquisition of resistance was not because of variation of this activity. Instead, selection led to production of a beta-lactam-insensitive d,d-carboxypeptidase that cleaved the C-terminal d-Ala residue of pentapeptide stems in vitro and caused massive accumulation of cytoplasmic precursors containing a tetrapeptide stem in vivo. The parallel dramatic increase in the proportion of l-Lys(3) --> d-Asx-Lys(3) cross-links showed that the enzyme was activating the resistance pathway by generating the substrate for the l,d-transpeptidase.

Biogenesis of Leishmania-harbouring parasitophorous vacuoles following phagocytosis of the metacyclic promastigote or amastigote stages of the parasites.

Protozoan parasites Leishmania alternate between a flagellated promastigote form and an amastigote form. In their mammalian hosts, Leishmania survive and multiply in macrophages. Both forms can be internalized by these host cells at different stages of the infectious process and eventually establish themselves within parasitophorous vacuoles exhibiting phagolysosomal properties. To determine whether the biogenesis of these organelles differs according to the parasitic stage used to initiate infection, we compared their formation kinetics after phagocytosis of either metacyclic promastigotes or amastigotes of L. amazonensis or of L. major by mouse bone-marrow-derived macrophages pre-exposed or not to IFN-gamma. After 10 minutes of contact, an accumulation of F-actin was observed around the promastigotes and amatigotes undergoing phagocytosis or those that had already been internalized. This accumulation was transient and rapidly disappeared at later times. At 30 minutes, most of the promastigotes were located in long, narrow organelles that were exactly the same shape as the parasites. The latter were elongated with their cell bodies near to the macrophage nucleus and their flagella towards the periphery. This suggests that promastigote phagocytosis mainly occurs in a polarized manner, with the cell body entering the macrophages first. Most, if not all, of the phagocytosed promastigotes were located in organelles that rapidly acquired phagolysosomal properties. At 30 minutes, lamp-1, macrosialin, cathepsins B and D were detected in 70-98% of these compartments and about 70% of them were surrounded by rab7p. These late endosome/lysosome 'markers' were recruited through fusion with late endocytic compartments. Indeed, when late endosomes/lysosomes were loaded with fluorescein dextran, 81-98% of the promastigote-harbouring compartments contained the endocytic tracer 30 minutes after infection. Electron microscopy of infected macrophages previously loaded with peroxidase confirmed that the phagosomes rapidly fused with late endocytic compartments. When the amastigote stage of L. amazonensis was used to initiate infection, the kinetics of acquisition of the different late endosome/lysosome 'markers' by the phagosomes were similar to those measured after infection with metacyclics. However, more rab7p(+)-phagosomes were observed at early time points (e.g. 90% were rab7p(+) at 30 minutes). The early endosome 'markers', EEA1 and the transferrin receptor, were hardly detected in parasite-containing compartments regardless of the parasitic stage used to infect macrophages and the time after infection. In conclusion, both metacyclic- and amastigote-containing phagosomes fuse with late endosomes/lysosomes within 30 minutes. However, with L. amazonensis, the time required for the formation of the huge parasitophorous vacuoles, which are characteristic of this species, was much shorter after infection with amastigotes than after infection with metacyclic promastigotes. This indicates that the initial fusions with late endosomes/lysosomes are followed by a stage-specific sequence of events.