Catherine Raynaud

Analysis of the Phthiocerol Dimycocerosate Locus ofMycobacterium tuberculosis EVIDENCE THAT THIS LIPID IS INVOLVED IN THE CELL WALL PERMEABILITY BARRIER

Among the few characterized genes that have products involved in the pathogenicity of Mycobacterium tuberculosis, the etiological agent of tuberculosis, are those of the phthiocerol dimycocerosate (DIM) locus. Genes involved in biosynthesis of these compounds are grouped on a 50-kilobase fragment of the chromosome containing 13 genes. Analysis of mRNA produced from this 50-kilobase fragment in the wild type strain showed that this region is subdivided into three transcriptional units. Biochemical characterization of five mutants with transposon insertions in this region demonstrated that (i) the complete DIM molecules are synthesized in the cytoplasm of M. tuberculosis before being translocated into the cell wall; (ii) the genesfadD26 and fadD28 are directly involved in their biosynthesis; and (iii) both the drrC andmmpL7 genes are necessary for the proper localization of DIMs. Insertional mutants unable to synthesize or translocate DIMs exhibit higher cell wall permeability and are more sensitive to detergent than the wild type strain, indicating for the first time that, in addition to being important virulence factors, extractable lipids of M. tuberculosis play a role in the cell envelope architecture and permeability. This function may represent one of the molecular mechanisms by which DIMs are involved in the virulence ofM. tuberculosis.

Phospholipases C are involved in the virulence of Mycobacterium tuberculosis

Phospholipases C play a role in the pathogenesis of several bacteria. Mycobacterium tuberculosis, the causative agent of tuberculosis, possesses four genes encoding putative phospholipases C, plcA, plcB, plcC and plcD. However, the contribution of these genes to virulence is unknown. We constructed four single mutants of M. tuberculosis each inactivated in one of the plc genes, a triple plcABC mutant and a quadruple plcABCD mutant. The mutants all exhibited a lower phospholipase C activity than the wild‐type parent strain, demonstrating that the four plc genes encode a functional phospholipase C in M. tuberculosis. Functional complementation of the ΔplcABC triple mutant with the individual plcA, plcB and plcC genes restored in each case about 20% of the total Plc activity detected in the parental strain, suggesting that the three enzymes contribute equally to the overall Plc activity of M. tuberculosis. RT‐PCR analysis of the plc genes transcripts showed that the expression of these genes is strongly upregulated during the first 24 h of macrophage infection. Moreover, the growth kinetics of the triple and quadruple mutants in a mouse model of infection revealed that both mutants are attenuated in the late phase of the infection emphasizing the importance of phospholipases C in the virulence of the tubercle bacillus.

Extracellular enzyme activities potentially involved in the pathogenicity of Mycobacterium tuberculosis

To evaluate the potential contribution of extracellular enzymes to the pathogenicity of mycobacteria, the presence of selected enzyme activities was investigated in the culture filtrates of the obligate human pathogen Mycobacterium tuberculosis, M. bovis BCG, the opportunistic pathogens M. kansasii and M. fortuitum, and the non-pathogenic species M. phlei and M. smegmatis. For M. tuberculosis and M. bovis, 22 enzyme activities were detected in the culture filtrates and/or cell surfaces, of which eight were absent from the culture fluids of non-pathogens: alanine dehydrogenase, glutamine synthetase, nicotinamidase, isonicotinamidase, superoxide dismutase, catalase, peroxidase and alcohol dehydrogenase. These activities, which correspond to secreted enzymes, formed a significant part (up to 92%) of the total enzyme activities of the bacteria and were absent from the culture fluids and the cell surfaces of the non-pathogenic species M. smegmatis and M. phlei. The extracellular location of superoxide dismutase and glutamine synthetase seemed to be restricted to the obligate pathogens examined. The difference in the enzyme profiles was not attributable to the growth rates of the two groups of bacteria. The presence of the eight enzyme activities in the outermost compartments of obligate pathogens and their absence in those of non-pathogens provides further evidence that these enzymes may be involved in the pathogenicity of mycobacteria. In addition, the eight enzyme activities were demonstrated in the cell extract of M. smegmatis. Stepwise erosion of the cell surface of M. smegmatis to expose internal capsular constituents showed that the various enzyme activities, with the possible exception of superoxide dismutase, were located more deeply in the cell envelope of this bacterium. This suggests that the molecular architecture of the mycobacterial envelopes may play an important role in the pathogenicity of these organisms.

Identification of the agr Locus of Listeria monocytogenes: Role in Bacterial Virulence

ABSTRACT Listeria monocytogenes is a gram-positive facultative intracellular food-borne pathogen that can cause severe infections in humans and animals. We have recently adapted signature-tagged transposon mutagenesis (STM) to identify genes involved in the virulence of L. monocytogenes. A new round of STM allowed us to identify a new locus encoding a protein homologous to AgrA, the well-studied response regulator of Staphylococcus aureus and part of a two-component system involved in bacterial virulence. The production of several secreted proteins was modified in the agrA mutant of L. monocytogenes grown in broth, indicating that the agr locus influenced protein secretion. Inactivation of agrA did not affect the ability of the pathogen to invade and multiply in cells in vitro. However, the virulence of the agrA mutant was attenuated in the mouse (a 10-fold increase in the 50% lethal dose by the intravenous route), demonstrating for the first time a role for the agr locus in the virulence of L. monocytogenes.

Mechanisms of pyrazinamide resistance in mycobacteria: importance of lack of uptake in addition to lack of pyrazinamidase activity.

Mycobacteria are known to acquire resistance to the antituberculous drug pyrazinamide (PZA) through mutations in the gene encoding pyrazinamidase (PZase), an enzyme that converts PZA into pyrazinoic acid, the presumed active form of PZA against bacteria. Additional mechanisms of resistance to the drug are known to exist but have not been fully investigated. Among these is the non-uptake of the pro-drug, a possibility investigated in the present study. The uptake mechanism of PZA, a requisite step for the activation of the pro-drug, was studied in Mycobacterium tuberculosis. The incorporation of [14C]PZA by the bacilli was followed in both neutral and acidic environments since PZA activity is known to be optimal at acidic pH. By using a protonophore (carbonyl cyanide m-chlorophenylhydrazone; CCCP), valinomycin, arsenate and low temperature, it was shown that an ATP-dependent transport system is involved in the uptake of PZA. Whilst the structurally analogous compound nicotinamide inhibited the transport system of PZA, other structurally related compounds such as pyrazinoic acid, isoniazid and cytosine did not. Acidic conditions were also without effect. Based on diffusion experiments in liposomes, it was found that PZA diffuses rapidly through membrane bilayers, faster than glycerol, whilst the presence of OmpATb, the porin-like protein of M. tuberculosis, in proteoliposomes slightly increased the diffusion of the drug. This finding may explain why the cell wall mycolate hydrophobic layer does not represent the limiting step in the diffusion of PZA, as judged from comparative experiments using a M. tuberculosis strain and its isogenic mutant elaborating 40% less covalently linked mycolates. PZase activity, and PZA uptake and susceptibility in different mycobacterial species were compared. M. tuberculosis, a naturally PZA-susceptible species, was the only species that exhibited both PZase activity and PZA uptake; no such correlation was observed with the four naturally resistant species examined. Mycobacterium smegmatis possessed a functional PZase but did not take up PZA; the reverse was true for the PZase-negative strain of Mycobacterium avium used, with PZA uptake comparable to that of M. tuberculosis. Mycobacterium bovis BCG and Mycobacterium kansasii exhibited neither a PZase activity nor PZA uptake. These data clearly demonstrate that one of the mechanisms of resistance to PZA resides in the failure of strains to take up the drug, indicating that susceptibility to PZA in mycobacteria requires both the presence of a functional PZase and a PZA transport system. No correlation was observed between the occurrence and cellular location of PZase and of nicotinamidase in the strains examined, suggesting that one or both amides can be hydrolysed by other mycobacterial amidases.

Role of the wbt locus of Francisella tularensis in lipopolysaccharide O-antigen biogenesis and pathogenicity.

ABSTRACT Francisella tularensis is a highly infectious bacterial pathogen, responsible for the zoonotic disease tularemia. We screened a bank of transposon insertion mutants of F. tularensis subsp. holarctica LVS for colony morphology alterations and selected a mutant with a transposon insertion in wbtA, the first gene of the predicted lipopolysaccharide O-antigen gene cluster. Inactivation of wbtA led to the complete loss of O antigen, conferred serum sensitivity, impaired intracellular replication, and severely attenuated virulence in the mouse model. Notably, this mutant afforded protection against a challenge against virulent LVS.

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 Δfur 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 Δfur derivative and wild‐type L. monocytogenes grown in iron‐deficient media anchored a fraction of the SvpA proteins (∼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.

Role of FliF and FliI of Listeria monocytogenes in Flagellar Assembly and Pathogenicity

ABSTRACT 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°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 ΔfliF and ΔfliI 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°C. Notably, all these strains adhered less efficiently than strain EGD-e to Caco-2 cells at 37°C but showed no defect of intracellular multiplication. These data suggest that expression of the flagella at 37°C might hinder optimal adhesion to epithelial cells but has no impact on intracytosolic survival of L. monocytogenes.

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 (∼ 100‐fold). Inactivation of sipZ impaired the secretion of phospholipase C (PC‐PLC) and listeriolysin O (LLO), restricted intracellular multiplication and almost abolished virulence (LD50 of 108.3), 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 (LD50 of 107.2), 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.

Erp, an extracellular protein family specific to mycobacteria

Erp (exported repeated protein) was originally characterized as a virulence factor in Mycobacterium tuberculosis and was thought to be present only in Mycobacterium leprae and members of the TB complex. Here it is shown that Erp is a ubiquitous extracellular protein found in all of the mycobacterial species tested. Erp proteins have a modular organization and contain three domains: a highly conserved amino-terminal domain which includes a signal sequence, a central variable region containing repeats based on the motif PGLTS, and a conserved carboxy-terminal domain rich in proline and alanine. The number and fidelity of PGLTS repeats of the central region differ considerably between mycobacterial species. This region is, however, identical in all of the clinical M. tuberculosis strains tested. In addition, it is shown here that a Mycobacterium smegmatis erp::aph mutant displays altered colony morphology which is complemented by all the Erp orthologues tested. The genome sequence flanking the erp gene includes cell-wall-related ORFs and displays extensive conservation between saprophytic and pathogenic mycobacteria.