Pathogenicity islands and other virulence elements in Listeria.
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Animals↗
Biomedical subjects
J A Vázquez-Boland
Publications and source records attributed to J A Vázquez-Boland.
Comparative genomics of Listeria species.
Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.
Adaptation, Physiological↗
Pathogenicity islands and virulence evolution in Listeria.
As in other bacterial pathogens, the virulence determinants of Listeria species are clustered in genomic islands scattered along the chromosome. This review summarizes current knowledge about the structure, distribution and role in pathogenesis of Listeria virulence loci. Hypotheses about the mode of acquisition and evolution of these loci in this group of Gram-positive bacteria are presented and discussed.
Amino Acid Sequence↗
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 DeltaactA 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 DeltaactA 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* DeltaactA 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.
Amino Acid Sequence↗
Regulation of virulence genes in Listeria.
As in all pathogenic bacteria, virulence of the facultative intracellular Listeria species is a multifactorial trait. The expression of the bacterial genes involved in the different steps of the infectious process--invasion, intracellular multiplication and spreading--is temporally and spatially controlled, thus ensuring the presence of the respective gene products at the right moment and place. So far, one network which is involved in the regulation of listerial virulence, the PrfA regulon, has been characterized rather well. The key element of this regulon, PrfA, belongs to the Crp/Fnr family of transcriptional regulators. Its synthesis and activity are influenced by a variety of physico-chemical signals outside and inside of eukaryotic host cells. The analysis of virulence gene expression in vivo, i.e. in infected host cells, indicates that yet uncharacterized bacterial factors other than PrfA, and possibly also host factors, modulate the expression of the PrfA regulon. Essentially nothing is known about the signal transduction pathways involved in the observed differential expression of virulence genes. Fermentable carbon sources seem to have a particular role in virulence gene regulation. In addition to the PrfA regulon, the Clp stress proteins have an impact on Listeria virulence. These two regulons interact with each other by an unknown mechanism.
Bacterial Proteins↗
Listeria pathogenesis and molecular virulence determinants.
The gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a highly fatal opportunistic foodborne infection. Pregnant women, neonates, the elderly, and debilitated or immunocompromised patients in general are predominantly affected, although the disease can also develop in normal individuals. Clinical manifestations of invasive listeriosis are usually severe and include abortion, sepsis, and meningoencephalitis. Listeriosis can also manifest as a febrile gastroenteritis syndrome. In addition to humans, L. monocytogenes affects many vertebrate species, including birds. Listeria ivanovii, a second pathogenic species of the genus, is specific for ruminants. Our current view of the pathophysiology of listeriosis derives largely from studies with the mouse infection model. Pathogenic listeriae enter the host primarily through the intestine. The liver is thought to be their first target organ after intestinal translocation. In the liver, listeriae actively multiply until the infection is controlled by a cell-mediated immune response. This initial, subclinical step of listeriosis is thought to be common due to the frequent presence of pathogenic L. monocytogenes in food. In normal individuals, the continual exposure to listerial antigens probably contributes to the maintenance of anti-Listeria memory T cells. However, in debilitated and immunocompromised patients, the unrestricted proliferation of listeriae in the liver may result in prolonged low-level bacteremia, leading to invasion of the preferred secondary target organs (the brain and the gravid uterus) and to overt clinical disease. L. monocytogenes and L. ivanovii are facultative intracellular parasites able to survive in macrophages and to invade a variety of normally nonphagocytic cells, such as epithelial cells, hepatocytes, and endothelial cells. In all these cell types, pathogenic listeriae go through an intracellular life cycle involving early escape from the phagocytic vacuole, rapid intracytoplasmic multiplication, bacterially induced actin-based motility, and direct spread to neighboring cells, in which they reinitiate the cycle. In this way, listeriae disseminate in host tissues sheltered from the humoral arm of the immune system. Over the last 15 years, a number of virulence factors involved in key steps of this intracellular life cycle have been identified. This review describes in detail the molecular determinants of Listeria virulence and their mechanism of action and summarizes the current knowledge on the pathophysiology of listeriosis and the cell biology and host cell responses to Listeria infection. This article provides an updated perspective of the development of our understanding of Listeria pathogenesis from the first molecular genetic analyses of virulence mechanisms reported in 1985 until the start of the genomic era of Listeria research.
Animals↗
Identification and mutagenesis by allelic exchange of choE, encoding a cholesterol oxidase from the intracellular pathogen Rhodococcus equi.
The virulence mechanisms of the facultative intracellular parasite Rhodococcus equi remain largely unknown. Among the candidate virulence factors of this pathogenic actinomycete is a secreted cholesterol oxidase, a putative membrane-damaging toxin. We identified and characterized the gene encoding this enzyme, the choE monocistron. Its protein product, ChoE, is homologous to other secreted cholesterol oxidases identified in Brevibacterium sterolicum and Streptomyces spp. ChoE also exhibits significant similarities to putative cholesterol oxidases encoded by Mycobacterium tuberculosis and Mycobacterium leprae. Genetic tools for use with R. equi are poorly developed. Here we describe the first targeted mutagenesis system available for this bacterium. It is based on a suicide plasmid, a selectable marker (the aacC4 apramycin resistance gene from Salmonella), and homologous recombination. The choE allele was disrupted by insertion of the aacC4 gene, cloned in pUC19 and introduced by electroporation in R. equi. choE recombinants were isolated at frequencies between 10(-2) and 10(-3). Twelve percent of the recombinants were double-crossover choE mutants. The choE mutation was associated with loss of cooperative (CAMP-like) hemolysis with sphingomyelinase-producing bacteria (Listeria ivanovii). Functional complementation was achieved by expression of choE from pVK173-T, a pAL5000 derivative conferring hygromycin resistance. Our data demonstrate that ChoE is an important cytolytic factor for R. equi. The highly efficient targeted mutagenesis procedure that we used to generate choE isogenic mutants will be a valuable tool for the molecular analysis of R. equi virulence.
Alleles↗
SmcL, a novel membrane-damaging virulence factor in Listeria.
We describe here the fourth listerial membrane-damaging virulence factor, a sphingomyelinase C (SMase) that is produced specifically by the ruminant pathogen Listeria ivanovii. Its coding gene, smcL, is a monocistron expressed independently of PrfA. The smcL product, SmcL, is highly similar to the staphylococcal beta-toxin and is responsible for the differential hemolytic properties of L. ivanovii (bizonal hemolysis and CAMP-like reaction with R. equi). The role of SmcL in virulence was assessed by gene disruption and complementation. Our data show that SmcL mediates disruption of the membrane of primary phagosomes, thereby promoting bacterial intracellular proliferation. They also suggest that SmcL may play a role in host tropism. smcL is located in LIPI-2, a novel 18-kb pathogenicity island which also contains a cluster of internalin genes. LIPI-2 is unstable, L. ivanovii-specific and required for full virulence in mice and lambs.
Amino Acid Sequence↗
Associations of veterinary services and farmer characteristics with the prevalences of brucellosis and border disease in small ruminants in Spain.
We investigated the farm factors associated with the prevalences of brucellosis and border disease (BD) in small-ruminant herds in the Madrid region of Spain. These infections were used as models of diseases of well-known and totally unknown distribution, respectively, to assess the association between the perception of the importance of a given disease on the relative contributions of veterinary services and the farmer's attitudes to its prevention. Sera, farming-management information and data concerning veterinary assistance and farmer characteristics were collected from 60 sheep or goat herds. The overall sero-prevalence of brucellosis was 5.7% (complement fixation) and for BD was 17.9% (ELISA test). The relationship between sero-positivity and the variables in the questionnaire was assessed by multivariable analysis using random-effects logistic-normal regression. 'Availability of veterinary services' was a major protective factor for brucellosis. In contrast, no association with veterinary services was observed for BD, whereas 'membership in a farmers' organization' (a variable associated with good farming practice and animal care) was a protective factor. 'Membership of a farmers' organisation' and two other farmer variables indicative of good husbandry ('youth' and 'schooling') were associated with a lower sero-prevalence of brucellosis in univariable analysis but they did not remain significant in the multivariable model. Our observations suggest that veterinary-activity variables predominate over non-specific protective farm factors related to good husbandry in the case the disease is subject to disease surveillance. This underscores the importance of organized control programs for veterinary services to be effective in terms of animal disease prevention.
Animal Husbandry↗
The smcL gene of Listeria ivanovii encodes a sphingomyelinase C that mediates bacterial escape from the phagocytic vacuole.
The ruminant pathogen Listeria ivanovii differs from Listeria monocytogenes in that it causes strong, bizonal haemolysis and a characteristic shovel-shaped co-operative haemolytic ('CAMP-like') reaction with Rhodococcus equi. We cloned the gene responsible for the differential haemolytic properties of L. ivanovii, smcL. It encodes a sphingomyelinase C (SMase) highly similar (> 50% identity) to the SMases from Staphylococcus aureus (beta-toxin), Bacillus cereus and Leptospira interrogans. smcL was transcribed monocistronically and was expressed independently of PrfA. Low-stringency Southern blots demonstrated that, within the genus Listeria, smcL was present only in L. ivanovii. We constructed an smcL knock-out mutant. Its phenotype on blood agar was identical to that of L. monocytogenes (i.e. weak haemolysis and no shovel-shaped CAMP-like reaction with R. equi ). This mutant was less virulent for mice, and its intracellular proliferation was impaired in the bovine epithelial-like cell line MDBK. The role of SmcL in intracellular survival was investigated using an L. monocytogenes mutant lacking the membrane-damaging determinants hly, plcA and plcB, being thus unable to grow intracellularly. Complementation of this mutant with smcL on a plasmid was sufficient to promote bacterial intracellular proliferation in MDBK cells. Transmission electron microscopy showed that SmcL mediates the disruption of the phagocytic vacuole and the release of bacteria into the cytosol. Therefore, L. ivanovii possesses a third phospholipase with membrane-damaging activity that, together with PlcA and PlcB, may act in concert with the pore-forming toxin Hly to mediate efficient escape from the vacuolar compartment. The 5' end of smcL is contiguous with the internalin locus i-inlFE, which is also specific to L. ivanovii and is required for full virulence in mice. Thus, smcL forms part of a novel virulence gene cluster in Listeria that is species specific.
Amino Acid Sequence↗
The bvr locus of Listeria monocytogenes mediates virulence gene repression by beta-glucosides.
The beta-glucoside cellobiose has been reported to specifically repress the PrfA-dependent virulence genes hly and plcA in Listeria monocytogenes NCTC 7973. This led to the hypothesis that beta-glucosides, sugars of plant origin, may act as signal molecules, preventing the expression of virulence genes if L. monocytogenes is living in its natural habitat (soil). In three other laboratory strains (EGD, L028, and 10403S), however, the effect of cellobiose was not unique, and all fermentable carbohydrates repressed hly. This suggested that the downregulation of virulence genes by beta-glucosides is not a specific phenomenon but, rather, an aspect of a global regulatory mechanism of catabolite repression (CR). We assessed the effect of carbohydrates on virulence gene expression in a panel of wild-type isolates of L. monocytogenes by using the PrfA-dependent phospholipase C gene plcB as a reporter. Utilization of any fermentable sugar caused plcB repression in wild-type L. monocytogenes. However, an EGD variant was identified in which, as in NCTC 7973, plcB was only repressed by beta-glucosides. Thus, the regulation of L. monocytogenes virulence genes by sugars appears to be mediated by two separate mechanisms, one presumably involving a CR pathway and another specifically responding to beta-glucosides. We have identified in L. monocytogenes a 4-kb operon, bvrABC, encoding an antiterminator of the BglG family (bvrA), a beta-glucoside-specific enzyme II permease component of the phosphoenolpyruvate-sugar phosphotransferase system (bvrB), and a putative ADP-ribosylglycohydrolase (bvrC). Low-stringency Southern blots showed that this locus is absent from other Listeria spp. Transcription of bvrB was induced by cellobiose and salicin but not by arbutin. Disruption of the bvr operon by replacing part of bvrAB with an interposon abolished the repression by cellobiose and salicin but not that by arbutin. Our data indicate that the bvr locus encodes a beta-glucoside-specific sensor that mediates virulence gene repression upon detection of cellobiose and salicin. Bvr is the first sensory system found in L. monocytogenes that is involved in environmental regulation of virulence genes.
Bacterial Proteins↗
Factors associated with seroprevalence to Mycobacterium paratuberculosis in small-ruminant farms in the Madrid region (Spain).
A cross-sectional study was conducted in a population of small ruminants in the Madrid region (Spain) to determine the Mycobacterium paratuberculosis seroprevalence and to identify farm factors possibly associated with paratuberculosis (PTB). Farming-management information and sera were collected from 60 sheep or goat flocks. The relationship between seropositivity and the variables in the questionnaire was assessed by unconditional logistic regression, followed by random-effects logistic regression analysis to adjust for overdispersion between herds. The seroprevalence to M. paratuberculosis was 11.7% (64 out of 546) using agar-gel immunodiffusion assay (AGID). According to the sensitivity and specificity of the AGID test the true prevalence could be as high as 44%. A herd size of between 200-400 head and the presence of foreign breeds and their crosses were significantly associated with seropositivity (OR = 4.05 and OR = 4.32, respectively). A higher replacement rate was also associated with seroprevalence to M. paratuberculosis (24.2% in positive herds vs. 18.1% in negative). All these three factors were related to more intensive management in the surveyed area. In contrast, membership of a professional livestock association appeared to be a protective factor against PTB (OR = 0.28). No variables related to veterinary assistance were associated with seroprevalence, probably reflecting the current lack of interest in PTB on the part of the animal-health administration and veterinary services in Spain.
Animal Husbandry↗
Evidence for expressional crosstalk between the central virulence regulator PrfA and the stress response mediator ClpC in Listeria monocytogenes.
Virulence is a multifactorial trait which depends on the coordinated expression of many bacterial products, hence it is to be expected that the regulatory circuits that control the relevant genetic determinants are somehow interconnected. Two pleiotropic regulatory elements acting at different levels, the transcription factor PrfA which controls virulence gene expression and the potential chaperone ClpC which is involved in tolerance to environmental stress, are required for Listeria monocytogenes survival within the host. We analyzed the influence of PrfA on clpC expression in L. monocytogenes. clpC transcription is maximal under heat-shock conditions, i.e. at 42 degrees C, and is very weak or undetectable at 37 degrees C. In a prfA* mutant which constitutively overexpresses PrfA and PrfA-dependent virulence genes, clpC transcription dropped to basal levels during exponential growth at 42 degrees C. This repression was not observed during stationary phase, indicating growth phase-dependent regulation of clpC. Culture in charcoal-treated medium, which triggers in wild-type strains the transcriptional activation of the PrfA regulon, also caused a strong downregulation of clpC. Moreover, in a prfA deletion mutant, clpC transcription during exponential growth at 37 degrees C was clearly enhanced, reaching the same high levels of the wild-type at 42 degrees C. Overall, our results indicate that clpC expression is negatively controlled at the transcriptional level, directly or indirectly, by the central virulence regulator PrfA.
Bacterial Proteins↗
A novel PrfA-regulated chromosomal locus, which is specific for Listeria ivanovii, encodes two small, secreted internalins and contributes to virulence in mice.
Several large, cell wall-associated internalins and one small, secreted internalin (InlC) have been described previously in Listeria monocytogenes. Using degenerate primers derived from sequenced peptides of an L. ivanovii major secreted protein, we identified a new 4.25 kb internalin locus of L. ivanovii, termed i-inlFE. The two proteins encoded by this locus, i-InlE and i-InlF, belong to the group of small, secreted internalins. Southern blot analyses show that the i-inlFE locus does not occur in L. monocytogenes. These data also indicate that six genes encoding small, secreted internalins are present in L. ivanovii, in contrast to L. monocytogenes, in which inlC encodes the only small internalin. The mature i-InlE protein (198 amino acids) is secreted in large amounts into the brain-heart infusion (BHI) culture medium in the stationary growth phase. In minimum essential medium (MEM), which has been used previously to induce PrfA-dependent gene transcription, i-inlE mRNA and i-InlE protein are expressed at high levels. As shown by Northern blot analysis and primer extension, transcription of the tandemly arranged i-inlF and i-inlE genes is dependent on the virulence regulator PrfA, and characteristic palindromic sequences ('PrfA-boxes') were identified in the promoter regions of i-inlF and i-inlE. Non-polar i-inlE and i-inlF deletion mutants and an i-inlFE double deletion mutant were constructed and tested in the mouse infection model. After intravenous infection, all three mutants entirely failed to kill C57BL/6 mice even at high infectious doses of 109 bacteria per mouse, whereas the LD50 for the parental strain was determined as 4 x 107 bacteria per mouse. These data suggest an important role for i-InlE and i-InlF in L. ivanovii virulence.
Amino Acid Sequence↗
Functional similarities between the Listeria monocytogenes virulence regulator PrfA and cyclic AMP receptor protein: the PrfA* (Gly145Ser) mutation increases binding affinity for target DNA.
Most Listeria monocytogenes virulence genes are positively regulated by the PrfA protein, a transcription factor sharing sequence similarities with cyclic AMP (cAMP) receptor protein (CRP). Its coding gene, prfA, is regulated by PrfA itself via an autoregulatory loop mediated by the upstream PrfA-dependent plcA promoter. We have recently characterized prfA* mutants from L. monocytogenes which, as a result of a single amino acid substitution in PrfA, Gly145Ser, constitutively overexpress prfA and the genes of the PrfA virulence regulon. Here, we show that about 10 times more PrfA protein is produced in a prfA* strain than in the wild type. Thus, the phenotype of prfA* mutants is presumably due to the synthesis of a PrfA protein with higher promoter-activating activity (PrfA*), which keeps its intracellular levels constantly elevated by positive feedback. We investigated the interaction of PrfA and PrfA* (Gly145Ser) with target DNA. Gel retardation assays performed with a DNA fragment carrying the PrfA binding site of the plcA promoter demonstrated that the PrfA* mutant form is much more efficient than wild-type PrfA at forming specific DNA-protein complexes. In footprinting experiments, the two purified PrfA forms interacted with the same nucleotides at the target site, although the minimum amount required for protection was 6 to 7 times lower with PrfA*. These results show that the primary functional consequence of the Gly145Ser mutation is an increase in the affinity of PrfA for its target sequence. Interestingly, similar mutations at the equivalent position in CRP result in a transcriptionally active, CRP* mutant form which binds with high affinity to target DNA in the absence of the activating cofactor, cAMP. Our observations suggest that the structural similarities between PrfA and CRP are also functionally relevant and support a model in which the PrfA protein, like CRP, shifts from transcriptionally inactive to active conformations by interaction with a cofactor.
Bacterial Proteins↗
Actinobacillus pleuropneumoniae does not require urease activity to produce acute swine pleuropneumonia.
The role in virulence of Actinobacillus pleuropneumoniae urease activity was investigated. A urease-negative mutant was isolated following transposon mutagenesis with a mini-Tn10 derivative. Both the parent strain and the urease-negative mutant exhibited identical LD50 values in a murine infection model. Pig challenge confirmed that the urease-negative mutant was fully virulent, since experimental inoculation with 5 x 10(7) colony forming units resulted in an acute disease indistinguishable from that produced by the wild-type strain at the same dose. Our results demonstrate that urease activity is not required for the development of acute pleuropneumonia.
Actinobacillus pleuropneumoniae↗
Host cell heparan sulfate proteoglycans mediate attachment and entry of Listeria monocytogenes, and the listerial surface protein ActA is involved in heparan sulfate receptor recognition.
The mechanisms by which the intracellular pathogen Listeria monocytogenes interacts with the host cell surface remain largely unknown. In this study, we investigated the role of heparan sulfate proteoglycans (HSPG) in listerial infection. Pretreatment of bacteria with heparin or heparan sulfate (HS), but not with other glycosaminoglycans, inhibited attachment and subsequent uptake by IC-21 murine macrophages and CHO epithelial-like cells. Specific removal of HS from target cells with heparinase III significantly impaired listerial adhesion and invasion. Mutant CHO cells deficient in HS synthesis bound and internalized significantly fewer bacteria than wild-type cells did. Pretreatment of target cells with the HS-binding proteins fibronectin and platelet factor 4, or with heparinase III, impaired listerial infectivity only in those cells expressing HS. Moreover, a synthetic peptide corresponding to the HS-binding ligand in Plasmodium falciparum circumsporozoite protein (pepPf1) inhibited listerial attachment to IC-21 and CHO cells. A motif very similar to the HS-binding site of pepPf1 was found in the N-terminal region of ActA, the L. monocytogenes surface protein responsible for actin-based bacterial motility and cell-to-cell spread. In the same region of ActA, several clusters of positively charged amino acids which could function as HS-binding domains were identified. An ActA-deficient mutant was significantly impaired in attachment and entry due to altered HS recognition functions. This work shows that specific interaction with an HSPG receptor present on the surface of both professional and nonprofessional phagocytes is involved in L. monocytogenes cytoadhesion and invasion and strongly suggests that the bacterial surface protein ActA may be a ligand mediating HSPG receptor recognition.
Amino Acid Sequence↗