Immunization with FimA protects against Streptococcus parasanguis endocarditis in rats.

FimA, a surface-associated protein of Streptococcus parasanguis, is associated with initial colonization of damaged heart tissue in an endocarditis model (D. Burnette-Curley, V. Wells, H. Viscount, C. Munro, J. Fenno, P. Fives-Taylor, and F. Macrina, Infect. Immun. 63:4669-4674, 1995). We have evaluated the efficacy of recombinant FimA as a vaccine in the rat model of endocarditis and investigated in vitro the mechanism for the protective role of immunization. FimA-immunized and nonimmunized control animals were catheterized to induce heart valve damage and infected intravenously with 10(7) CFU of wild-type S. parasanguis FW213 bacteria. The presence of bacteria associated with platelet-fibrin vegetations 24 h postchallenge was evaluated. Immunized rats were significantly less susceptible to endocarditis (2 cases among 34 animals) than the control group (21 cases among 33 animals) (P < 0.001). Incubation of S. parasanguis FW213 with rabbit anti-FimA immune serum decreased the mean percent adherence (0.34% of added cells) to platelet-fibrin matrix in vitro compared with that of preimmune normal serum (5.04% of added cells; P < 0.001). Adsorption of immune serum with FimA-positive S. parasanguis FW213 yielded antiserum that failed to block adherence to the platelet-fibrin matrix. We assessed the vaccine potential of FimA as a common immunogen able to provide cross-protection in streptococcal endocarditis by determining the occurrence and expression of fimA in the viridans group streptococci and enterococci. We detected the presence of fimA homologs by Southern hybridization and PCR amplification analyses and determined by immunoblotting the expression of FimA-like proteins among a variety of streptococci and enterococci that frequently cause endocarditis. Eighty-one percent (26 of 32) of streptococcal and enterococcal strains isolated from bacteremic patients expressed proteins that comigrated with FimA and were reactive with polyclonal anti-FimA serum. Streptococcal DNA from strains that were positive by Western blot (immunoblot) analysis hybridized to the full-length fimA probe. Our studies suggest that FimA immunization results in antibody-mediated inhibition of bacterial adherence, a critical early event in the pathogenesis of endocarditis. Our data demonstrate that a majority of streptococcal strains associated with endocarditis have genes that encode FimA-like proteins. Taken together, these results suggest that FimA is a promising candidate for an endocarditis vaccine.     

Vaccination with FimA from Streptococcus parasanguis Protects Rats from Endocarditis Caused by Other Viridans Streptococci

The FimA protein of Streptococcus parasanguis is a virulence factor in the rat model of endocarditis, and immunization with FimA protects rats against homologous bacterial challenge. Because FimA-like proteins are widespread among the oral streptococci, the leading cause of native valve endocarditis, we evaluated the ability of this vaccinogen to protect rats when challenged by other streptococcal species. Here we report that FimA vaccination produced antibodies that cross-reacted with and protected against challenge by the oral streptococci S. mitis, S. mutans, and S. salivarius. FimA thus has promise as a vaccinogen to control infective endocarditis caused by oral streptococci.     

Phenotypic characterization of Streptococcus sanguis virulence factors associated with bacterial endocarditis.

Certain strains of Streptococcus sanguis adhere (Adh+) selectively to human platelets and, in plasma, induce them to aggregate (Agg+) into in vitro thrombi. In this study, we examined 18 recent endocarditis and dental plaque isolates of microorganisms that were biotyped as S. sanguis for coexpression of platelet interactivity phenotypes with another possible virulence factor in bacterial endocarditis, dextran synthesis. Detectable production of extracellular glucosyltransferase ranged from 0.2 to 66 mU/mg of culture fluid for 10 representative strains tested. Production of extracellular or cell-associated glucosyltransferase, fructosyltransferase, and soluble or insoluble dextrans was not necessarily coexpressed with platelet interactivity phenotypes, since the levels of production of soluble and insoluble dextrans varied among representative Adh+ Agg+ and Adh- Agg- strains. Analysis of a second panel of 38 fresh dental plaque isolates showed that S. sanguis distributes in a reproducible manner into the possible phenotype groups. Strains with different platelet interactivity phenotypes were distinguished with a panel of four murine monoclonal antibodies (MAbs) raised against Adh+ Agg+ strain 133-79 and screened to rule out artifactual reactions with antigenic components in culture media. The MAbs reacted selectively with Adh+ Agg+ strains in a direct-binding, whole-cell, enzyme-linked immunosorbent assay and also inhibited their interactions with platelets. Analysis of minimal tryptic digests of many strains, including variants that failed to bind the MAbs, suggested that some noninteractivity phenotypes possess cryptic surface determinants. Since the ability to adhere to platelets and induce them to aggregate is relatively stable, these traits may be useful in a phenotyping scheme for these Lancefield nontypeable streptococci.     

Neuraminidase production by a Streptococcus sanguis strain associated with subacute bacterial endocarditis.

The properties of an extracellular neuraminidase produced by a Streptococcus sanguis strain (isolated from a confirmed case of subacute bacterial endocarditis) during growth in a defined medium was examined in this investigation. This enzyme, isolated from concentrated culture supernatants of S. sanguis biotype II, was active against human alpha-1 acid glycoprotein, N-acetylneuramin lactose, bovine submaxillary mucin, and fetuin. Neuraminidase production paralleled bacterial growth in defined medium and was maximal in the early stationary phase of growth but decreased dramatically, probably owing to protease production, during the late stationary phase. The enzyme was purified to near homogeneity by a combination of salt fractionation, ion-exchanged chromatography on DEAE-Sephacel, and gel filtration on Sephadex G-200. These procedures yielded an enzyme preparation that possessed a specific activity of 174.4 mumol of sialic acid released per min per mg of protein against human alpha-1 acid glycoprotein. The Km value for this enzyme with human alpha-1 acid glycoprotein as substrate was 2.5 X 10(-3) M, and the enzyme possessed a pH optimum of 6.5. The S. sanguis neuraminidase had a molecular weight of approximately 85,000 as estimated by gel filtration and approximately 90,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was stable at temperatures of 4 and 37 degrees C for 3 h, but approximately 50% of the enzymatic activity was lost within 30 min at 50 degrees C, with 100% of the enzymatic activity being destroyed within 10 min at temperatures of greater than or equal to 65 degrees C.     

Streptococcus iniae virulence is associated with a distinct genetic profile

Streptococcus iniae causes meningoencephalitis and death in commercial fish species and has recently been identified as an emerging human pathogen producing fulminant soft tissue infection. As identified by pulsed-field gel electrophoresis (PFGE), strains causing disease in either fish or humans belong to a single clone, whereas isolates from nondiseased fish are genetically diverse. In this study, we used in vivo and in vitro models to examine the pathogenicity of disease-associated isolates. Strains with the clonal (disease-associated) PFGE profile were found to cause significant weight loss and bacteremia in a mouse model of subcutaneous infection. As little as 10(2) CFU of a disease-associated strain was sufficient to establish bacteremia, with higher inocula (10(7)) resulting in increased mortality. In contrast, non-disease-associated (commensal) strains failed to cause bacteremia and weight loss, even at inocula of 10(8) CFU. In addition, disease-associated strains were more resistant to phagocytic clearance in a human whole blood killing assay compared to commensal strains, which were almost entirely eradicated. Disease-associated strains were also cytotoxic to human endothelial cells as measured by lactate dehydrogenase release from host cells. However, both disease-associated and commensal strains adhered to and invaded cultured human epithelial and endothelial cells equally well. While cellular invasion may still contribute to the pathogenesis of invasive S. iniae disease, resistance to phagocytic clearance and direct cytotoxicity appear to be discriminating virulence attributes of the disease-associated clone.     

Overexpression and purification of a fimbria-associated adhesin of Streptococcus parasanguis.

A Streptococcus parasanguis adhesin that blocks the attachment of S. parasanguis FW213 to saliva-coated hydroxyapatite (SCHA) has been purified. Previous work demonstrated that the attachment of FW213 to SCHA is mediated by fimbriae and that one component associated with fimbriae is a 36-kDa protein (FimA) that reacts with antifimbria serum in Western blots (immunoblots) and is not present in afimbriated mutants. To obtain amounts of FimA sufficient for adhesion blocking assays, we cloned the gene coding for FimA into an Escherichia coli T7 overexpression system. The resulting strain produced large amounts of FimA, as much as 50% of the total cell protein. FimA was purified by elution from sodium dodecyl sulfate-polyacrylamide gels, and its native conformation was reestablished by sodium dodecyl sulfate removal, resolubilization in guanidine hydrochloride, and 50-fold dilution. Some refolded FimA aggregated into dimers and trimers. Preincubation of SCHA with 100 micrograms of purified, renatured FimA per ml blocked 85% of the binding of FW213. The FimA-SCHA complex was quite stable and could be washed continuously for at least 2 h with only a slight loss of FimA blocking activity. When FimA was added to preformed bacterium-SCHA complexes, it displaced 40% of the bacteria already bound to SCHA. The results suggest that FimA is an adhesin with a high substrate affinity and may prove useful in the development of a therapeutic agent for the prevention of plaque formation and endocarditis initiated by the sanguis streptococci.     

Protease production by Streptococcus sanguis associated with subacute bacterial endocarditis.

A viridans streptococcus (Streptococcus sanguis biotype II) isolated from the blood of a patient with subacute bacterial endocarditis was examined for protease production. In broth culture, extracellular proteolytic enzymes were not produced by this organism until after the early exponential phase of growth, with maximal protease production occurring during the stationary phase. Four distinct proteases were isolated and purified from the supernatant fluids of stationary-phase cultures, employing a combination of ion-exchange column chromatography, gel filtration column chromatography, and polyacrylamide gel electrophoresis. All four proteases could be eluted from a diethylaminoethyl cellulose column at a sodium chloride gradient concentration of 0.25 M but were separable by gel filtration chromatography on a Sephadex G-100 column. They varied in molecular weights as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis from approximately 13,000 to 230,000. All four proteases had pH optima of between 8.0 and 9.0, and two of the proteases were active against casein, human serum albumin, and gelatin but were not active against elastin and collagen. The remaining two proteases were able to degrade only casein and gelatin. These results show that S. sanguis is able to excrete maximal levels of potentially destructive enzymes when the organisms are not actively multiplying. This finding may explain some of the damage caused in heart tissue by these organisms during subacute bacterial endocarditis.     

Peptidoglycan N-acetylglucosamine deacetylase,a putative virulence factor in Streptococcus pneumoniae

Many glucosamine residues of the pneumococcal peptidoglycan (PG) are not acetylated, which makes the PG resistant to lysozyme. A capsular type III mutant with an inactivated pgdA gene (encoding the peptidoglycan N-acetylglucosamine deacetylase A) became hypersensitive to exogenous lysozyme and showed reduced virulence in the intraperitoneal mouse model.     

Identification of two proteins associated with virulence of Streptococcus suis type 2.

The protein profiles of various cell fractions of 180 strains of Streptococcus suis type 2, which were isolated from diseased pigs, from healthy pigs when they were slaughtered, and from human patients, were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The isolates from diseased pigs contained two proteins that were absent in most of the isolates from healthy pigs. One of these proteins was a 136-kDa protein that was previously identified as the muramidase-released protein (MRP). This protein was predominantly detected in protoplast supernatants and culture supernatants. The second protein was a 110-kDa protein that was detected only in culture supernatants and therefore was provisionally called extracellular factor (EF). Three phenotypes of S. suis type 2 strains were recognized. Isolates from organs of diseased pigs mainly belonged to the MRP+ EF+ phenotype (77%), while isolates from tonsils of healthy pigs mainly had the MRP- EF- phenotype (86%). Most of the isolates from human patients contained MRP (89%); 74% had the MRP+ EF- phenotype. These findings confirm the results of previous investigations which demonstrated that S. suis type 2 strains differ in virulence. Monoclonal antibodies raised against the 110-kDa EF recognized proteins with higher molecular weights in culture supernatants of all of the strains with the MRP+ EF- phenotype. However, none of the strains with the MRP+ EF+ phenotype produced these high-molecular-weight proteins. Our results demonstrate that MRP and EF are associated with virulence. This suggests that one or both of these proteins are virulence factors that play a role in the pathogenesis of S. suis type 2 infections in pigs and human patients.     

Oral immunization with recombinant Streptococcus gordonii expressing porphyromonas gingivalis FimA domains

Porphyromonas gingivalis, a gram-negative anaerobe, is implicated in the etiology of adult periodontitis. P. gingivalis fimbriae are one of several critical surface virulence factors involved in both bacterial adherence and inflammation. P. gingivalis fimbrillin (FimA), the major subunit protein of fimbriae, is considered an important antigen for vaccine development against P. gingivalis-associated periodontitis. We have previously shown that biologically active domains of P. gingivalis fimbrillin can be expressed on the surface of the human commensal bacterium Streptococcus gordonii. In this study, we examined the effects of oral coimmunization of germfree rats with two S. gordonii recombinants expressing N (residues 55 to 145)- and C (residues 226 to 337)-terminal epitopes of P. gingivalis FimA to elicit FimA-specific immune responses. The effectiveness of immunization in protecting against alveolar bone loss following P. gingivalis infection was also evaluated. The results of this study show that the oral delivery of P. gingivalis FimA epitopes via S. gordonii vectors resulted in the induction of FimA-specific serum (immunoglobulin G [IgG] and IgA) and salivary (IgA) antibody responses and that the immune responses were protective against subsequent P. gingivalis-induced alveolar bone loss. These results support the potential usefulness of the S. gordonii vectors expressing P. gingivalis fimbrillin as a mucosal vaccine against adult periodontitis.     

Identification of virulence determinants for endocarditis in Streptococcus sanguinis by signature-tagged mutagenesis

Streptococcus sanguinis is a gram-positive, facultative anaerobe and a normal inhabitant of the human oral cavity. It is also one of the most common agents of infective endocarditis, a serious endovascular infection. To identify virulence factors for infective endocarditis, signature-tagged mutagenesis (STM) was applied to the SK36 strain of S. sanguinis, whose genome is being sequenced. STM allows the large-scale creation, in vivo screening, and recovery of a series of mutants with altered virulence. Screening of 800 mutants by STM identified 38 putative avirulent and 5 putative hypervirulent mutants. Subsequent molecular analysis of a subset of these mutants identified genes encoding undecaprenol kinase, homoserine kinase, anaerobic ribonucleotide reductase, adenylosuccinate lyase, and a hypothetical protein. Virulence reductions ranging from 2-to 150-fold were confirmed by competitive index assays. One putatively hypervirulent strain with a transposon insertion in an intergenic region was identified, though increased virulence was not confirmed in competitive index assays. All mutants grew comparably to SK36 in aerobic broth culture except for the homoserine kinase mutant. Growth of this mutant was restored by the addition of threonine to the medium. Mutants containing an insertion or in-frame deletion in the anaerobic ribonucleotide reductase gene failed to grow under strictly anaerobic conditions. The results suggest that housekeeping functions such as cell wall synthesis, amino acid and nucleic acid synthesis, and the ability to survive under anaerobic conditions are important virulence factors in S. sanguinis endocarditis.     

Streptococcus iniae phosphoglucomutase is a virulence factor and a target for vaccine development

Streptococcus iniae represents a major health and economic problem in fish species worldwide. Random Tn917 mutagenesis and high-throughput screening in a hybrid striped bass (HSB) model of meningoencephalitis identified attenuated S. iniae mutants. The Tn917 insertion in one mutant disrupted an S. iniae homologue of a phosphoglucomutase (pgm) gene. Electron microscopy revealed a decrease in capsule thickness and cell wall rigidity, with DeltaPGM mutant cells reaching sizes approximately 3-fold larger than those of the wild type (WT). The DeltaPGM mutant was cleared more rapidly in HSB blood and was more sensitive to killing by cationic antimicrobial peptides including moronecidin from HSB. In vivo, the DeltaPGM mutant was severely attenuated in HSB, as intraperitoneal challenge with 1,000 times the WT lethal dose produced only 2.5% mortality. Reintroduction of an intact copy of the S. iniae pgm gene on a plasmid vector restored antimicrobial peptide resistance and virulence to the DeltaPGM mutant. In analysis of the aborted infectious process, we found that DeltaPGM mutant organisms initially disseminated to the blood, brain, and spleen but were eliminated by 24 h without end organ damage. Ninety to 100% of fish injected with the DeltaPGM mutant and later challenged with a lethal dose of WT S. iniae survived. We conclude that the pgm gene is required for virulence in S. iniae, playing a role in normal cell wall morphology, surface capsule expression, and resistance to innate immune clearance mechanisms. An S. iniae DeltaPGM mutant is able to stimulate a protective immune response and may have value as a live attenuated vaccine for aquaculture.     

Resistance of Streptococcus gordonii to polymorphonuclear leukocyte killing is a potential virulence determinant of infective endocarditis

Significant differences in virulence among seven representative Streptococcus gordonii strains were observed by using the rat model of infective endocarditis. Five strains, including S. gordonii DL1, caused severe disease, while the other two strains, including S. gordonii SK12, caused minimal or no disease. The differences in virulence were evident from the visible presence of streptococci in the vegetations present on the aortic valves of catheterized rats that were challenged with individual strains and also from the much greater recovery of rifampin-resistant S. gordonii DLl than of streptomycin-resistant S. gordonii SK12 from the hearts of animals coinfected with both organisms. Each S. gordonii strain aggregated with human platelets and bound to polymorphonuclear leukocytes (PMNs), as shown by the stimulation of PMN superoxide anion production. These interactions were reduced or abolished by pretreatment of the platelets or PMNs with sialidase, indicating that there was bacterial recognition of host sialic acid-containing receptors. Adhesin-mediated binding of each S. gordonii strain to PMNs also triggered phagocytosis. However, the subsequent PMN-dependent killing differed significantly for the seven strains. The five virulent strains included three strains that were not killed and two strains whose numbers were reduced by approximately 50%. In contrast, the level of killing of each avirulent strain under the same conditions was significantly greater and approached 90% of the bacteria added. Parallel studies performed with rat PMNs revealed comparable differences in the resistance or susceptibility of representative virulent and avirulent strains. Thus, the ability of S. gordonii to survive in PMNs following adhesin-mediated phagocytosis may be an important virulence determinant of infective endocarditis.     

Diffuse glomerulonephritis in rabbits with Streptococcus viridans endocarditis.

Intravenous administration of live microorganisms to rabitts with cardiac catheters produces an experimental model of infective endocarditis. Despite the development of infected valvular vegetations, positive blood cultures, splenomegaly, and focal embolic renallesions, glomerulonephritis has not been found in these animals. In the present study, acute diffuse proliferative glomerulonephritis, featuring endothelial and mesangial proliferation, capillary occlusion, and leukocytic infiltration was produced in rabbits immunized withthe infecting agent prior to the establishment of left sided alpha-streptococcal endocarditis. Controls receiving immunization alone, immunization and sterileendocarditis, or infective endocarditis alone did not develop diffuse glomerulonephritis.Electron microscopic findings of occasional subendothelial electron-dense deposits and immunofluorescence deposition of IgG and C3 in a peripheral granular capillary pattern were consistent with an immune complex type nephritis. Decreased serum complement levels were demonstrated in those animals developing diffuse glomerulonephritis, and some animals developed circulating rheumatoid factor. In view of the morphologic findings and the necessity of preimmunization for development of glomerular changes, it is concluded that immune mechanisms play a role in the diffuse glomerulonephritis associated with this model of infective endocarditis.     

Diminished platelet binding in vitro by Staphylococcus aureus is associated with reduced virulence in a rabbit model of infective endocarditis.

The direct binding of platelets by bacteria is a postulated central mechanism in the pathogenesis of endocarditis. To address the role of binding more definitively, we employed Tn551 insertional mutagenesis of Staphylococcus aureus parental strain ISP479 to generate an isogenic variant (strain PS12) that bound platelets minimally. As compared with the binding of ISP479, the binding of PS12 to platelet monolayers was reduced by 67.2%. Similarly, the binding of PS12 to platelets in suspension was reduced by 71.3%, as measured by flow cytometry. The low-binding phenotype was transducible into both ISP479 and S. aureus Newman. Southern blotting indicated that a single copy of Tn551 was inserted within the chromosomes of PS12 and the transductants. When tested in a rabbit model, animals inoculated with PS12 were significantly less likely to develop endocarditis and had lower densities of organisms (CFU per gram) within vegetations and a decreased incidence of renal abscess formation, as compared with animals inoculated with the parental strain. The diminished virulence of PS12 was not attributable to a reduction in the initial attachment of organisms to the damaged endocardium, since 30 min after inoculation, PS12-infected animals had microbial densities on the valve surface comparable to those seen with the parental strain. These results indicate that the direct binding of Staphylococcus aureus to platelets is a major determinant of virulence in the pathogenesis of endocarditis. Staphylococcus-platelet binding appears to be critical for pathogenetic events occurring after the initial colonization of the valve surface, such as vegetation formation and septic embolization.     

ZmpB,a novel virulence factor of Streptococcus pneumoniae that induces tumor necrosis factor alpha production in the respiratory tract

Inflammation is a prominent feature of Streptococcus pneumoniae infection in both humans and animal models. Indeed, an intense host immune response to infection is thought to contribute significantly to the pathology of pneumococcal pneumonia and meningitis. Previously, induction of the inflammatory response following infection with S. pneumoniae has been attributed to certain cell wall constituents and the toxin pneumolysin. Here we present data implicating a putative zinc metalloprotease, ZmpB, as having a role in inflammation. Null mutations were created in the zmpB gene of the virulent serotype 2 strain D39 and analyzed in a murine model of infection. Isogenic mutants were attenuated in pneumonia and septicemia models of infection, as determined by levels of bacteremia and murine survival. Mutants were not attenuated in colonization of murine airways or lung tissue. Examination of cytokine profiles within the lung tissue revealed significantly lower levels of the proinflammatory cytokine tumor necrosis factor alpha following challenge with the Delta zmpB mutant (Delta 739). These data identify ZmpB as a novel virulence factor capable of inducing inflammation in the lower respiratory tract. The possibility that ZmpB was involved in inhibition of complement activity was examined, but the data indicated that ZmpB does not have a significant effect on this important host defense. The regulation of ZmpB by a two-component system (TCS09) located immediately upstream of the zmpB gene was examined. TCS09 was not required for the expression of zmpB during exponential growth in vitro.     

Fhb, a novel factor H-binding surface protein, contributes to the antiphagocytic ability and virulence of Streptococcus suis

Streptococcus suis serotype 2 is a Gram-positive bacterium that causes sepsis and meningitis in piglets and humans. The mechanisms of S. suis serotype 2 invasive disease are not well understood. The surface proteins of pathogens usually play important roles in infection and bacterium-host interactions. Here, we identified a novel surface protein that contributed significantly to the virulence of S. suis serotype 2 in a piglet infection model. This protein showed little similarity to other reported proteins and exhibited strong binding activity to human factor H (hFH). It was designated Fhb (factor H-binding protein). The fhb genes found in S. suis serotypes 1, 2, 4, 7, and 9 exhibited molecular polymorphism. Fhb possessed two proline-rich repeat sequences and XPZ domains, and one repeat sequence exhibited a high homology to Bac, an IgA-binding protein of Streptococcus agalactiae. Evidence strongly indicated that fhb-deficient mutants had diminished phagocytosis resistance in bactericidal assays. In addition, Fhb plays important roles in complement-mediated immunity by interacting with hFH. These findings indicated that Fhb is a crucial surface protein contributing to the virulence of S. suis, with important functions in evading innate immune defenses by interaction with host complement regulatory factor hFH.     

Identification of a disulfide isomerase protein of Leishmania major as a putative virulence factor

Several approaches have been previously used to elucidate the genetic basis of Leishmania virulence. In general, they were based on laboratory Leishmania clones genetically modified or grown in the presence of selecting agents. In a previous study, we demonstrated that Leishmania major freshly isolated from human cutaneous lesions showed significant differences in the severity of the experimental disease induced in BALB/c mice. Here, using the mRNA differential display technique, we analyzed gene expression in L. major promastigotes showing different levels of virulence. We have identified a novel Leishmania gene encoding a 477-amino-acid protein exhibiting two distinct regions that are identical to the putative active-site sequence (CGHC) of the eukaryotic protein disulfide isomerase (PDI). The recombinant protein displayed a specific PDI enzymatic activity. This L. major disulfide isomerase protein (LmPDI) is predominantly expressed, at both the mRNA and protein levels, in highly virulent strains. Specific PDI inhibitors abolished the enzymatic activity of the recombinant protein and profoundly affected parasite growth. These findings suggest that LmPDI may play an important role in Leishmania natural pathogenicity and may constitute a new target for anti-Leishmania chemotherapy.