A role for ActA in epithelial cell invasion by Listeria monocytogenes

We assessed the role of the actin-polymerizing protein, ActA, in host cell invasion by Listeria monocytogenes . An in frame Δ actA mutant was constructed in a hyperinvasive strain of prfA * genotype, in which all genes of the PrfA-dependent virulence regulon, including actA , are highly expressed in vitro . Loss of ActA production in prfA * bacteria reduced entry into Caco-2, HeLa, MDCK and Vero epithelial cells to basal levels. Reintroduction of actA into the Δ actA prfA * mutant fully restored invasiveness, demonstrating that ActA is involved in epithelial cell invasion. ActA did not contribute to internalization by COS-1 fibroblasts and Hepa 1-6 hepatocytes. Expression of actA in Listeria innocua was sufficient to promote entry of this non-invasive species into epithelial cell lines, but not into COS-1 and Hepa 1-6 cells, indicating that ActA directs an internalization pathway specific for epithelial cells. Scanning electron microscopy of infected Caco-2 human enterocytes suggested that this pathway involves microvilli. prfA * bacteria, but not wild-type bacteria (which express PrfA-dependent genes very weakly in vitro ) or prfA *Δ actA bacteria, efficiently invaded differentiated Caco-2 cells via their apical surface. Microvilli played an active role in the phagocytosis of the prfA * strain, and actA was required for their remodelling into pseudopods mediating bacterial uptake. Thus, ActA appears to be a multifunctional virulence factor involved in two important aspects of Listeria pathogenesis: actin-based motility and host cell tropism and invasion.     

A hypermutator phenotype attenuates the virulence of Listeria monocytogenes in a mouse model

The integrity of the genetic material of bacteria is guaranteed by a set of distinct repair mechanisms. The participation of these repair systems in bacterial pathogenicity has been addressed only recently. Here, we study for the first time the participation in virulence of the MutSL mismatch repair system of Listeria monocytogenes. The mutS and mutL genes, which are contiguous in the L. monocytogenes chromosome, were identified after in silico analysis. The deduced MutS shares 62% identity with MutS of Bacillus subtilis and 50% identity with HexA, its homologue in Streptococcus pneumoniae; MutL shares 59% identity with MutL of B. subtilis and 47% identity with HexB of S. pneumoniae. Functional analysis of the mutSL locus was studied by constructing a double knock-out mutant. We showed that the deletion DeltamutSL induces: (i) a 100- to 1000-fold increase in the spontaneous mutation rate; and (ii) a 10- to 15-fold increase in the frequency of transduction, thus demonstrating the role of mutSL of L. monocytogenes in both mismatch repair and homologous recombination. We found that the deletion DeltamutSL moderately affected bacterial virulence, with a 1-log increase in the lethal dose 50% (LD50) in the mouse. Strikingly, repeated passages of the mutant strain in mice reduced virulence further. Competition assays between wild-type and mutant strains showed that the deletion DeltamutSL reduced the capacity of L. monocytogenes to survive and multiply in mice. These results thus demonstrate that, for the intracellular pathogen L. monocytogenes, a hypermutator phenotype is more deleterious than profitable to its virulence.     

Tailoring host immune responses to Listeria by manipulation of virulence genes -- the interface between innate and acquired immunity

Although attenuated strains of microbial pathogens have triggered vaccine development from its origin, the role of virulence factors in determining host immunity has remained largely unexplored. Using the murine listeriosis model, we investigated whether the induction and expansion of protective and inflammatory T cell responses may be modified by selective manipulation of virulence genes. We intentionally deleted specific genes of Listeria monocytogenes, including those encoding the positive regulatory factor (prfA), hemolysin (hly), the actin nucleator (actA), and phospholipase B (plcB). The resulting strains showed decisive differences in their immunogenic properties. In particular, we identified a double-deletion mutant that retained Listeria's profound ability to induce protective CD8(+) T cells, but that is strongly attenuated and exhibits a significantly reduced ability to induce CD4(+) T cell-mediated inflammation. We conclude that this mutant, L. monocytogenes DeltaactADeltaplcB, is at present the most promising mutant for a bacterial vaccine vector and is able to safely induce potent CD8(+) T cell-mediated immunity.     

GFP用于单核细胞增生李斯特菌PrfA调控毒力基因actA转录表达的研究

PrfA是单核细胞增生李斯特菌(LM)中迄今为止发现的惟一个调控绝大多数毒力基因转录表达的蛋白因子.为了研究PrfA转录调控毒力基因表达的分子机制,将无启动子的绿色荧光蛋白(GFP)基因与毒力基因actA的启动子融合,连接到穿梭载体pLSV16质粒上,构建成表达融合载体pLSV16-PactA-gfp,然后将其电转化入LM野生株P14、PrfA高表达突变株P14a和prfA基因等位缺失突变株A42中表达.利用荧光显微镜和荧光酶标仪检测上述3株细菌中绿色荧光蛋白的不同表达强度,从而评价actA基因依赖于PrfA的转录活性强弱.结果显示,绿色荧光蛋白在P14a中发出的荧光强度最高,P14次之,A42最弱,两两比较均有显著差异(P＜0.01),表明毒力基因actA的转录水平高低与PrfA的活性成正相关,其转录表达依赖于PrfA的调控;该试验同时也显示GFP能方便、有效地用于研究PrfA调控LM不同毒力基因的转录表达水平.     

ClpE, a novel member of the HSP100 family, is involved in cell division and virulence of Listeria monocytogenes

We identified, in the facultative intracellular pathogen Listeria monocytogenes , a previously unknown Clp ATPase, unique among the HSP100 proteins because of the presence of a short N-terminal region with a potential zinc finger motif. This protein of 726 amino acids is highly homologous to ClpE of Bacillus subtilis , and is a member of a new subfamily of HSP100/Clp ATPases. The clpE gene is transcribed as a monocistronic mRNA from a typical consensus σ^A promoter. clpE is not stimulated by various stresses, but is upregulated in a clpC mutant. This is the first example of cross-regulation between Clp ATPases. By constructing a clpE mutant of L. monocytogenes , we found that ClpE is required for prolonged survival at 42°C and is involved in the virulence of this pathogen. A double mutant deficient in both ClpE and ClpC was avirulent in a mouse model and completely eliminated in the liver. Electron microscopy studies did not show any morphological alterations in clpE or clpC mutants. In the clpE–clpC double mutant, however, cell division was affected, indicating that ClpE acts synergistically with ClpC in cell septation. These results show that the Clp chaperones play a crucial role in both cell division and virulence of L. monocytogenes .     

A putative P-type ATPase required for virulence and resistance to haem toxicity in Listeria monocytogenes

Regulation of iron homeostasis in many pathogens is principally mediated by the ferric uptake regulator, Fur. Since acquisition of iron from the host is essential for the intracellular pathogen Listeria monocytogenes, we predicted the existence of Fur-regulated systems that support infection. We examined the contribution of nine Fur-regulated loci to the pathogenicity of L. monocytogenes in a murine model of infection. While mutating the majority of the genes failed to affect virulence, three mutants exhibited a significantly compromised virulence potential. Most striking was the role of the membrane protein we designate FrvA (Fur regulated virulence factor A; encoded by frvA [lmo0641]), which is absolutely required for the systemic phase of infection in mice and also for virulence in an alternative infection model, the Wax Moth Galleria mellonella. Further analysis of the ΔfrvA mutant revealed poor growth in iron deficient media and inhibition of growth by micromolar concentrations of haem or haemoglobin, a phenotype which may contribute to the attenuated growth of this mutant during infection. Uptake studies indicated that the ΔfrvA mutant is unaffected in the uptake of ferric citrate but demonstrates a significant increase in uptake of haem and haemin. The data suggest a potential role for FrvA as a haem exporter that functions, at least in part, to protect the cell against the potential toxicity of free haem.     

Population studies of methicillin-resistant and -sensitive Staphylococcus aureus strains reveal a lack of variability in the agrD gene, encoding a staphylococcal autoinducer peptide

The virulence of Staphylococcus aureus is controlled by the accessory gene regulator (agr) system, including an extracellular inducer encoded by agrD. Variable agr PCR restriction fragment length polymorphism (RFLP) patterns of unique S. aureus strains (n = 192) were determined for a region comprising agrD and parts of the neighboring agrC and agrB genes. Twelve unique RFLP patterns were identified among S. aureus strains in general; these patterns were further specified by sequencing. All sequences could be catalogued in the three current agr groups. A major proportion of the S. aureus strains belong to agr group 1, whereas only 6% of the methicillin-susceptible S. aureus strains and 5% of the methicillin-resistant S. aureus strains belong to agr groups 2 and 3, respectively. The homology between groups varied from 75 to 80%, and within groups it varied from 96 to 100%. Different levels of sequence variability were observed in the different agr genes. agr-related bacterial interference among colonizing S. aureus strains in the noses of persistent and intermittent human carriers was studied. S. aureus strains belonging to different agr groups were encountered in the same individual. This may suggest that the activity of the agrD gene product does not define colonization dynamics, which is further substantiated by the rarity of agr group 2 and 3 strains.     

Phase variation, capsular polysaccharide, pilus and flagella contribute to uptake of Vibrio vulnificus by the Eastern oyster (Crassostrea virginica)

Vibrio vulnificus infections are associated with raw oyster consumption, and disease reservoirs are determined by the ability of this bacterium to infect and persist in oysters. Surface structures, such as capsular polysaccharide (CPS), pili and flagella, function as virulence factors in mouse infection models. Furthermore, virulence is related to phase variation in colony morphology, which reflects CPS expression and includes opaque (encapsulated, virulent), translucent (reduced encapsulation, avirulent) and rugose (wrinkled, biofilm-enhanced) colony types. The role of these factors in environmental survival is unknown; therefore, mutational analysis and phase variation of V. vulnificus were examined in an oyster infection model. Oysters ( Crassostrea virginica ) were pre-treated with tetracycline to reduce background bacteria and subsequently inoculated via filter feeding with 10⁶ colony-forming units (cfu) ml⁻¹ of V. vulnificus wild-type strains and phase variants, as well as strains with deletion mutations in genes related to CPS (Δ wza ), pili (Δ pilA ), flagella (Δ flaCDE/ Δ flaFBA ) and motility (Δ motAB ). All mutants were significantly reduced in their dissemination to oyster haemolymph as compared with wild type; however, recovery of mutants from gills and intestinal tissue was generally similar to wild type. Translucent and rugose inocula showed induction of high-frequency phase variation to the opaque encapsulated phenotype (100% and 72% respectively) during oyster infections that did not occur in strains recovered from seawater. Thus, multiple bacterial factors determine uptake of V. vulnificus in oysters, and phase variation during oyster infection is a likely mechanism for environmental survival and for induction of the more virulent phenotype.     

The use of listeriolysin to identify in vivo induced genes in the gram-positive intracellular pathogen Listeria monocytogenes

Listeria monocytogenes is capable of growth within the cytoplasm of infected host cells. Escape from the host cell phagosome is mediated primarily through secretion of listeriolysin, a haemolytic factor which functions to actively lyse the phagosomal membrane. Listeriolysin negative mutants of L. monocytogenes are non-haemolytic on blood agar plates and demonstrate a significant reduction of virulence in the mouse model of infection. We have developed a system for the identification of in vivo induced genes in L. monocytogenes which utilizes the listeriolysin gene, hly, as both a reporter of gene expression and as a means of selection of promoter elements expressed in vivo. The system is analogous to in vivo expression technology (IVET) first reported for Salmonella, however, as listeriolysin functions in the environment of the host phagosome the loci identified in this study are most likely expressed during residence in the phagosome. The system was successfully tested using the promoter of the inducible virulence gene plcA. A bank was created by fusing a promoterless copy of hly to random promoter elements in a listeriolysin negative IVET host. Sequential inoculations of mice with this bank resulted in the isolation of clones with increased survival potential in the mouse model relative to a negative control, but which remained haemolysin negative on blood agar plates. Nine in vivo induced loci were identified including genes encoding a DNA topoisomerase III, a cellobiose transporter and a fumarase. Two isolates represented fusions to proteins of unknown function and three isolates contained no significant homologues in the database. A mutant in the fumarase gene demonstrated reduced virulence for mice and an inability to grow in cultured mouse phagocytes.     

An iron-dependent mutant of Listeria monocytogenes of attenuated virulence

Abstract A bank of Tn 917 -insertional mutants from the facultative intracellular pathogen Listeria monocytogenes was screened by an original method based on bacterial growth on synthetic medium under iron-limiting conditions. One mutant, whose in vitro growth in synthetic medium was specifically dependent upon the availability of iron in its environment, was isolated and characterized. The insertional event occurred in a non-coding region, upstream of a rrn operon and located within a 1100-kb Not I fragment of the physical map, where the virulence genes already identified in L. monocytogenes were also present. Protein analysis by SDS-PAGE revealed a pleiotropic effect of the insertional event on cell-associated proteins, suggesting a polar effect of the transposon on adjacent unknown gene(s). The virulence in the mouse of this mutant was strongly impaired, although it was capable in vitro of growing intracellularly and of spreading from cell to cell, as shown by the production of lytic plaques on cell culture.     

A nonribosomal peptide synthetase (Pes1) confers protection against oxidative stress in Aspergillus fumigatus

Aspergillus fumigatus is an important human fungal pathogen. The Aspergillus fumigatus genome contains 14 nonribosomal peptide synthetase genes, potentially responsible for generating metabolites that contribute to organismal virulence. Differential expression of the nonribosomal peptide synthetase gene, pes1, in four strains of Aspergillus fumigatus was observed. The pattern of pes1 expression differed from that of a putative siderophore synthetase gene, sidD, and so is unlikely to be involved in iron acquisition. The Pes1 protein (expected molecular mass 698 kDa) was partially purified and identified by immunoreactivity, peptide mass fingerprinting (36% sequence coverage) and MALDI LIFT-TOF/TOF MS (four internal peptides sequenced). A pes1 disruption mutant (delta pes1) of Aspergillus fumigatus strain 293.1 was generated and confirmed by Southern and western analysis, in addition to RT-PCR. The delta pes1 mutant also showed significantly reduced virulence in the Galleria mellonella model system (P < 0.001) and increased sensitivity to oxidative stress (P = 0.002) in culture and during neutrophil-mediated phagocytosis. In addition, the mutant exhibited altered conidial surface morphology and hydrophilicity, compared to Aspergillus fumigatus 293.1. It is concluded that pes1 contributes to improved fungal tolerance against oxidative stress, mediated by the conidial phenotype, during the infection process.     

Identification and disruption of btlA,a locus involved in bile tolerance and general stress resistance in Listeria monocytogenes

A transposon Tn917 mutant of Listeria monocytogenes L028 was isolated on the basis of reduced growth on agar adjusted to pH 5.5. The disrupted gene, designated btlA (bile tolerance locus), encodes a putative secondary transporter of the major facilitator superfamily, which has significant homology to yxiO in Bacillus subtilis (lmo1417 in L. monocytogenes EGDe). The mutant demonstrated decreased growth rates relative to the wild-type when grown in sub-lethal levels of various stressors (acid, salt, ethanol, bile, SDS, ampicillin and phosphomycin). The mutant was also more sensitive to lethal levels of bile. A pORI19 insertion mutant demonstrated similar phenotypes. Murine virulence studies indicated that disruption of btlA does not influence virulence potential. BtlA therefore represents a membrane protein essential for the maintenance of homeostasis under stress conditions, but is not involved in pathogenicity.     

The MprF protein is required for lysinylation of phospholipids in listerial membranes and confers resistance to cationic antimicrobial peptides (CAMPs) on Listeria monocytogenes

Pathogenic bacteria have to cope with defence mechanisms mediated by adaptive and innate immunity of the host cells. Cationic antimicrobial peptides (CAMPs) represent one of the most effective components of the host innate immune response. Here we establish the function of Lmo1695, a member of the VirR-dependent virulence regulon, recently identified in Listeria monocytogenes. Lmo1695 encodes a membrane protein of 98 kDa with strong homology to the multiple peptide resistance factor (MprF) of Staphylococcus aureus. Like staphylococcal MprF, we found that Lmo1695 is involved in the synthesis of the membrane phospholipid lysylphosphatidylglycerol (L-PG). In addition, Lmo1695 is also essential for lysinylation of diphosphatidylglycerol (DPG), another phospholipid widely distributed in bacterial membranes. A Deltalmo1695 mutant lacking the lysinylated phospholipids was particularly susceptible to CAMPs of human and bacterial origin. The mutant strain infected both epithelial cells and macrophages only poorly and was attenuated for virulence when tested in a mouse model of infection. Lmo1695 is a member of a growing list of survival factors which enable growth of L. monocytogenes in different environments.     

Listeria monocytogenes sigma B regulates stress response and virulence functions

While the stress-responsive alternative sigma factor sigma(B) has been identified in different species of Bacillus, Listeria, and Staphylococcus, the sigma(B) regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify sigma(B)-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidate sigma(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 sigma(B)-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and Delta 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 sigma(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-fold-higher expression in the wild type). Of the 55 genes exhibiting sigma(B)-dependent expression, 54 were preceded by a sequence resembling the sigma(B) promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the sigma(B)-dependent nature of a subset of eight selected promoter regions. Notably, the sigma(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, sigma(B) also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest that sigma(B) contributes to L. monocytogenes gene expression during infection.     

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.