The accessory gene regulator (agr) controls Staphylococcus aureus virulence in a murine arthritis model.

We have studied the role of the accessory gene regulator (agr) of Staphylococcus aureus as a virulence determinant in the pathogenesis of septic arthritis. At least 15 genes coding for potential virulence factors in Staphylococcus aureus are regulated by a putative multicomponent signal transduction system encoded by the agr/hld locus. agr and hld mutants show a decreased synthesis of extracellular toxins and enzymes, such as alpha-, beta-, and delta-hemolysin, leucocidin, lipase, hyaluronate lyase, and proteases, and at the same time an increased synthesis of coagulase and protein A as compared with the wild-type counterpart. We have used a recently described murine model of S. aureus-induced arthritis to study the virulence of S. aureus 8325-4 and two agr/hld mutants derived from it. Sixty percent of the mice injected with the wild-type strain developed arthritis, whereas agrA and hld mutants displayed joint involvement in only 10 and 30%, respectively. In addition, 40% of the mice inoculated with the wild-type strain displayed an erosive arthropathy; such changes were not detectable at all in mice inoculated with the agrA mutant. Serum levels of interleukin-6, a potent B-cell differentiation factor, were significantly higher (P < 0.001) in the mice inoculated with the wild-type strain than in those inoculated with the agrA mutant counterpart. Overall, our results suggest that the agr system of S. aureus is an important virulence determinant in the induction and progression of septic arthritis in mice.     

Ribonucleotide reductase class III, an essential enzyme for the anaerobic growth of Staphylococcus aureus, is a virulence determinant in septic arthritis

Staphylococcus aureus is the most common cause of joint infections. It also contributes to several other diseases such as pneumonia, osteomyelitis, endocarditis, and sepsis. Bearing in mind that S. aureus becomes rapidly resistant to new antibiotics, many studies survey the virulence factors, with the aim to find alternative prophylaxis/treatment regimens. One potential virulence factor is the bacterial ability to survive at different oxygen tensions. S. aureus expresses ribonucleotide reductases (RNRs), which help it to grow under both aerobic and anaerobic conditions, by reducing ribonucleotides to deoxyribonucleotides. In this study, we investigated the role of RNR class III, which is required for anaerobic growth, as a virulence determinant in the pathogenesis of staphylococcal arthritis. The wild-type S. aureus strain and its isogenic mutant nrdDG mutant were inoculated intravenously into mice. Mice inoculated with the wild-type strain displayed significantly more severe arthritis, with significantly more synovitis and destruction of the bone and cartilage versus mutant strain inoculated mice. Further, the persistence of bacteria in the kidneys was significantly more pronounced in the group inoculated with the wild-type strain. Together these results indicate that RNR class III is an important virulence factor for the establishment of septic arthritis.     

Alpha-toxin and gamma-toxin jointly promote Staphylococcus aureus virulence in murine septic arthritis

Septic arthritis is a common and feared complication of staphylococcal infections. Staphylococcus aureus produces a number of potential virulence factors including certain adhesins and enterotoxins. In this study we have assessed the roles of cytolytic toxins in the development of septic arthritis by inoculating mice with S. aureus wild-type strain 8325-4 or isogenic mutants differing in the expression of alpha-, beta-, and gamma-toxin production patterns. Mice inoculated with either an alpha- or beta-toxin mutant showed degrees of inflammation, joint damage, and weight decrease similar to wild-type-inoculated mice. In contrast, mice inoculated with either double (alpha- and gamma-toxin-deficient)- or triple (alpha-, beta-, and gamma-toxin-deficient)-mutant S. aureus strains showed lower frequency and severity of arthritis, measured both clinically and histologically, than mice inoculated with the wild-type strain. We conclude that simultaneous production of alpha- and gamma-toxin is a virulence factor in S. aureus arthritis.     

Virulence of a hemB mutant displaying the phenotype of a Staphylococcus aureus small colony variant in a murine model of septic arthritis

Persistence of Staphylococcus aureus during invasive infections has been associated with a small-colony variant (SCV) phenotype. SCVs are frequently auxotrophic for menadione or hemin, two compounds involved in the biosynthesis of the electron transport chain. SCVs have been shown to be more resistant to antibiotics such as aminoglycosides, grow slowly and persist intracellularly. The aim of this study was to assess the virulence of an hemB mutant, which has been shown to display the typical characteristics of clinical SCVs, in a murine model of septic arthritis. NMRI mice were inoculated intravenously with either the wild type strain Newman or with its mutant displaying the SCV phenotype. The clinical, bacteriological, and histopathological progression of the disease was studied. Mice inoculated with the hemB mutant displayed a higher frequency and a significantly higher severity of arthritis than mice inoculated with the wild type Newman strain. Despite that, the mutant inoculated mice displayed significantly lower bacterial burden in their kidneys and joints compared with mice exposed to the wild parental strain. Notably, the hemB mutant produced almost 20 times more protease in vitro than the parental strain. We conclude that the small colony variants of S. aureus are more virulent on a per organism basis than its isogenic parental strain in the model of septic arthritis. This can at least in part be explained by the ability of SCV to produce high amounts of destructive proteases.     

The role of staphylococcal polysaccharide microcapsule expression in septicemia and septic arthritis.

Staphylococcus aureus arthritis is a rapidly progressive and highly erosive disease of the joints in which both host and bacterial factors are of pathogenic importance. One potential bacterial virulence factor is the ability to express a polysaccharide capsule (CP). Among 11 reported capsular serotypes, CP type 5 (CP5) and CP8 comprise 80 to 85% of all clinical blood isolates. The aim of this study was to assess the role of CP5 as a virulence factor in staphylococcal septicemia and septic arthritis with a recently established murine model of hematogenously spread S. aureus arthritis. NMRI mice were inoculated intravenously with S. aureus strains isogenic for expression of CP5, and clinical, bacteriological, serological, and histopathological progression of disease was studied. Inoculation of 7 x 10(6) CFU of S. aureus per mouse induced 55% mortality in the group inoculated with the CP-expressing bacteria, compared to 18% in the group inoculated with CP- mutants. A lower dose of inoculum (3 x 10[6] per mouse) did not give rise to mortality in mice inoculated with CP mutant strains, whereas 18% of the mice inoculated with the CP5-expressing S. aureus died. Importantly, mice inoculated with S. aureus expressing CP5 had a significantly higher frequency of arthritis and a more severe form of the disease. In vitro assays suggested that macrophages were not able to phagocytize CP5+ staphylococci as efficiently as they were CP5- strains. In addition, once phagocytized, CP5+ bacteria were less efficiently killed than CP- mutants. In summary, CP5 leads to a higher frequency of arthritis and a more severe course of the disease. This seems to be related to the effects of the downregulatory properties of CP on the ingestion and intracellular killing capacity of phagocytes. Our results clearly indicate that the expression of CP5 is a determinant of the virulence of S. aureus in arthritis and septicemia.     

The surface protein Pls of methicillin-resistant Staphylococcus aureus is a virulence factor in septic arthritis

Pls, a surface protein of certain methicillin-resistant Staphylococcus aureus strains, is associated with poor bacterial adherence to solid-phase fibronectin and immunoglobulin G, as well as with reduced invasion of cultured epithelial cells. Here the importance of Pls for the development of septic arthritis and sepsis was investigated by using a mouse model. Mice inoculated with a pls knockout mutant developed a much milder arthritis and showed less grave weight reduction than mice infected with the wild-type Pls(+) clinical isolate. Also, the pls mutant induced a significantly lower frequency of mortality than the wild-type strain. The bacterial load of the kidneys was larger in mice infected with the Pls(+) strain than in animals challenged with the pls mutant. However, there was no evident inflammatory effect due to the Pls molecule alone, as indicated by knee injection of purified Pls. In conclusion, the results show that Pls is a virulence factor for septic arthritis and sepsis.     

Ribonucleotide reductase class III,an essential enzyme for the anaerobic growth of Staphylococcus aureus,is a virulence determinant in septic arthritis

Staphylococcus aureus is the most common cause of joint infections. It also contributes to several other diseases such as pneumonia, osteomyelitis, endocarditis, and sepsis. Bearing in mind that S. aureus becomes rapidly resistant to new antibiotics, many studies survey the virulence factors, with the aim to find alternative prophylaxis/treatment regimens. One potential virulence factor is the bacterial ability to survive at different oxygen tensions. S. aureus expresses ribonucleotide reductases (RNRs), which help it to grow under both aerobic and anaerobic conditions, by reducing ribonucleotides to deoxyribonucleotides. In this study, we investigated the role of RNR class III, which is required for anaerobic growth, as a virulence determinant in the pathogenesis of staphylococcal arthritis. The wild-type S. aureus strain and its isogenic mutant nrdDG mutant were inoculated intravenously into mice. Mice inoculated with the wild-type strain displayed significantly more severe arthritis, with significantly more synovitis and destruction of the bone and cartilage versus mutant strain inoculated mice. Further, the persistence of bacteria in the kidneys was significantly more pronounced in the group inoculated with the wild-type strain. Together these results indicate that RNR class III is an important virulence factor for the establishment of septic arthritis.     

Virulence of Staphylococcus aureus in a mouse mastitis model: studies of alpha hemolysin, coagulase, and protein A as possible virulence determinants with protoplast fusion and gene cloning.

Mutants of a genetically well-characterized strain of Staphylococcus aureus [SA113(83A)] were isolated after mutagenization. Alpha-hemolysin- (hla), coagulase- (coa), and protein A- (spa) negative mutants were characterized by more than 90 biochemical tests for production of extracellular proteins and biochemical profile to exclude pleiotropy. Protoplast fusion was then used to isolate double-defective (hla and coa) recombinants and recombinants with regained properties, i.e., production of alpha-hemolysin and coagulase. Studies of such mutants and recombinants in the mouse mastitis model showed that one alpha-hemolysin [SA113(83A) hla-5] and one coagulase-negative [SA113(83A) coa-147] mutant were lower in virulence compared with the wild-type strain SA113(83A). The double-negative mutant SA113(83A) hla-5 coa-147 showed a drastic decline in virulence and only induced very mild changes, as determined by microscopic examinations of infected mammary gland tissue. The recombinant with regained properties, however, was as virulent as the wild-type strain. This suggests that alpha-hemolysin and coagulase are virulence determinants of S. aureus. A high-level protein A-producing mutant (U300) showed the same virulence as the parent strain SA113(83A) in this model. One low virulence protein A-negative mutant (U320) did not markedly increase in virulence when a plasmid containing the cloned gene for protein A (pSPA15) was introduced into this mutant. By these and earlier observations, it seems likely that protein A is not an important virulence determinant in mastitis of mice. The reduced virulence of the protein A-negative mutant U320 compared with the wild-type SA113(83A) may be due to pleiotropic loss of some other unknown virulence determinant(s). Our data confirm earlier findings that pleiotropic changes are common in protein A-negative mutants.     

Study of the role of the htrB gene in Salmonella typhimurium virulence.

We have undertaken a study to investigate the contribution of the htrB gene to the virulence of pathogenic Salmonella typhimurium. An htrB::mini-Tn10 mutation from Escherichia coli was transferred by transduction to the mouse-virulent strain S. typhimurium SL1344 to create an htrB mutant. The S. typhimurium htrB mutant was inoculated into mice and found to be severely limited in its ability to colonize organs of the lymphatic system and to cause systemic disease in mice. A variety of experiments were performed to determine the possible reasons for this loss of virulence. Serum killing assays revealed that the S. typhimurium htrB mutant was as resistant to killing by complement as the wild-type strain. However, macrophage survival assays revealed that the S. typhimurium htrB mutant was more sensitive to the intracellular environment of murine macrophages than the wild-type strain. In addition, the bioactivity of the lipopolysaccharide (LPS) of the htrB mutant was reduced compared to that of the LPS from the parent strain as measured by both a Limulus amoebocyte lysate endotoxin quantitation assay and a tumor necrosis factor alpha bioassay. These results indicate that the htrB gene plays a role in the virulence of S. typhimurium.     

Role of sigma factor RpoS in initial stages of Salmonella typhimurium infection.

The sigma factor RpoS mediates the stationary-phase expression of a large group of genes, including those involved in resistance to a variety of environmental stresses, such as starvation, oxidation, and low pH. In addition, RpoS has been shown to regulate Salmonella virulence. In Salmonella typhimurium, RpoS controls the expression of the Salmonella plasmid virulence (spv) genes, which are required for systemic infection. However, the mechanism by which RpoS affects the pathogenicity of Salmonella remains incompletely defined. In this study, we focused on the ability of rpoS to affect the early stages of the infection process of S. typhimurium. An rpoS mutant of S. typhimurium exhibited wild-type abilities to attach to and invade Int-407 cells and J774 macrophage-like cells. In addition, rpoS did not affect the intracellular survival of S. typhimurium in either J774 macrophage-like cells or rat bone marrow-derived macrophages. However, the rpoS mutant demonstrated a decreased ability to colonize murine Peyer's patches after oral inoculation than its wild-type virulent parent strain showed. In addition, virulence plasmid-cured derivatives of the rpoS mutant were recovered in lower numbers from murine Peyer's patches than were plasmid-cured derivatives of the isogenic wild-type S. typhimurium. This indicates that RpoS regulation of chromosomally encoded genes is important for colonization of the gut-associated lymphoid tissue (GALT) by S. typhimurium. Microscopic analysis of histological sections taken from Peyer's patches after peroral infection of mice showed that, unlike its wild-type virulent parent strain, the isogenic rpoS mutant did not destroy the follicle-associated epithelium of the GALT. Furthermore, the rpoS mutant demonstrated a decreased ability to adhere to histological sections of murine Peyer's patches than its wild-type parent showed. Our data provide evidence for a role of RpoS in the interaction of Salmonella with cells of the GALT, specifically the Peyer's patches. This implicates the involvement of rpoS in the initial stages of systemic infection by Salmonella as opposed to infection leading to gastroenteritis.     

Virulence of non-type 1-fimbriated and nonfimbriated nonflagellated Salmonella typhimurium mutants in murine typhoid fever.

The virulence of Salmonella typhimurium mutants that were unable to synthesize type 1 fimbriae was tested in a murine typhoid fever model. Nonfimbriated mutants (fim) exhibited a lower 50% lethal dose than a wild-type (fim+) strain and produced significantly higher mortality (fim, 55%; fim+, 37% [P less than 0.002]) in mice that were challenged orally. There was no difference in virulence when the wild-type and mutant strains were injected intraperitoneally into mice. The progress of a short-term lethal infection was monitored after oral inoculation of mice with a mixture containing equivalent numbers of fim+ wild-type and fim mutant bacteria. The results indicated that while both strains colonized the intestinal tract equally well and invaded internal organs, the S. typhimurium fim mutant proliferated in the blood of the mice faster than the fim+ strain. The results of the mixed oral challenge suggested that bacteremia caused by fim+ S. typhimurium was reduced or delayed by the sequestration of the fimbriated bacteria in the spleen, liver, and kidneys. Thus, type 1 fimbriae were not virulence factors for S. typhimurium in this model, and the fimbriae may be an impediment to the pathogen in this setting. An S. typhimurium double mutant lacking type 1 fimbriae and flagella (fla) also was tested in mice. The virulence of the fim fla mutant was greatly reduced compared with that of the wild-type strain (mortality from fim fla challenge, 11% [P less than 0.0005]). The significance of this latter result is discussed in relation to host adaptation by pathogenic salmonellae.     

Metalloprotease is not essential for Vibrio vulnificus virulence in mice

Previous work suggested that a metalloprotease, Vvp, may be a virulence factor of Vibrio vulnificus, which causes severe wound infection and septicemia in humans. To determine the role of Vvp in pathogenesis, we isolated an isogenic protease-deficient (PD) mutant of Vibrio vulnificus by in vivo allelic exchange. This PD mutant was as virulent as its parental strain in mice infected intraperitoneally and was 10-fold more virulent in mice infected via the oral route. Furthermore, the PD mutant was indistinguishable from its parental strain in invasion from peritoneal cavity into blood stream, enhancement of vascular permeability, growth in murine blood, and utilization of hemoglobin and transferrin. These data suggest that Vvp is not essential for virulence in the mouse. However, the cytolysin activity in the culture supernatant of the PD mutant was found to be twofold higher than that of the wild-type strain and remained for a much longer period. The higher cytolysin activity of the PD mutant may be associated with the enhanced virulence in mice infected via the oral route.     

Staphylococcal accessory regulator (sar) in conjunction with agr contributes to Staphylococcus aureus virulence in endophthalmitis.

Previous studies showed that an agr mutant strain of Staphylococcus aureus was partially attenuated in virulence compared to a parental strain in experimental endophthalmitis. The purpose of this study was to determine whether the sar locus, either alone or through interactions with agr, contributes to the regulation of virulence in S. aureus endophthalmitis. Experimental endophthalmitis was established by the midvitreous injection of approximately 30 CFU of S. aureus RN6390 or the isogenic attenuated strains RN6911 (agr mutant), ALC136 (sar mutant), and ALC135 (agr sar double mutant). Unexpectedly, the rate of reduction in electroretinographic B-wave amplitude in eyes infected with strain ALC136 (sar mutant) was not significantly different from the parental strain on postinfection day (PID) 5 (10% retention). In contrast, ALC135 (agr sar double mutant)-infected eyes retained 73% of preoperative B-wave amplitude on PID 5. Therefore, unlike agr, a mutation in the sar locus alone does not alter the overall virulence of wild-type S. aureus in experimental endophthalmitis. However, the combined effect of insertional mutations in both the sar and agr global regulators leads to near-complete attenuation of virulence.     

Capsule-negative Staphylococcus aureus induces chronic experimental mastitis in mice

Staphylococcus aureus capsular polysaccharides (CP) have been shown to enhance staphylococcal virulence in numerous animal models of infection. Although serotype 5 CP (CP5) and CP8 predominate among S. aureus isolates from humans, most staphylococcal isolates from bovines with mastitis in Argentina are capsule negative. This study was designed to evaluate the effects of CP5 and CP8 expression on the pathogenesis of experimental murine mastitis. Lactating mice were challenged by the intramammary route with one of three isogenic S. aureus strains producing CP5, CP8, or no capsule. Significantly greater numbers of acapsular mutant cells were recovered from the infected glands 12 days after bacterial challenge compared with the encapsulated strains. Histopathological analyses revealed greater polymorphonuclear and mononuclear leukocyte infiltration and congestion in the mammary glands of mice infected with the encapsulated strains compared with the acapsular mutant, and the serotype 5 strain elicited more inflammation than the serotype 8 strain. In vitro experiments revealed that the acapsular S. aureus strain was internalized by MAC-T bovine epithelial cells in significantly greater numbers than the CP5- or CP8-producing strain. Taken together, the results suggest that S. aureus lacking a capsule was able to persist in the murine mammary gland, whereas encapsulated strains elicited more inflammation and were eliminated faster. Loss of CP5 or CP8 expression may enhance the persistence of staphylococci in the mammary glands of chronically infected hosts.     

Inhibition of cell surface export of group A streptococcal anchorless surface dehydrogenase affects bacterial adherence and antiphagocytic properties

Surface dehydrogenase (SDH) is an anchorless, multifunctional protein displayed on the surfaces of group A Streptococcus (GAS) organisms. SDH is encoded by a single gene, sdh (gap or plr) that is essential for bacterial survival. Hence, the resulting nonfeasibility of creating a knockout mutant is a major limiting factor in studying its role in GAS pathogenesis. An insertion mutagenesis strategy was devised in which a nucleotide sequence encoding a hydrophobic tail of 12 amino acids ((337)IVLVGLVMLLLS(348)) was added at the 3' end of the sdh gene, successfully creating a viable mutant strain (M1-SDH(HBtail)). In this mutant strain, the SDH(HBtail) protein was not secreted in the medium but was retained in the cytoplasm and to some extent trapped within the cell wall. Hence, SDH(HBtail) was not displayed on the GAS surface. The mutant strain, M1-SDH(HBtail), grew at the same rate as the wild-type strain. The SDH(HBtail) protein displayed the same GAPDH activity as the wild-type SDH protein. Although the whole-cell extracts of the wild-type and mutant strains showed similar GAPDH activities, cell wall extracts of the mutant strain showed 5.5-fold less GAPDH activity than the wild-type strain. The mutant strain, M1-SDH(HBtail), bound significantly less human plasminogen, adhered poorly to human pharyngeal cells, and lost its innate antiphagocytic activity. These results indicate that the prevention of the cell surface export of SDH affects the virulence properties of GAS. The anchorless SDH protein, thus, is an important virulence factor.     

Protective efficacy of protein A-specific antibody against bacteremic infection due to Staphylococcus aureus in an infant rat model.

Staphylococcal protein A (SpA) is a potent antiphagocytic component of the cell wall of most pathogenic Staphylococcus aureus strains. We studied the in vitro opsonophagocytic and in vivo protective activities of rabbit immunoglobulin G (IgG) antibody to purified SpA obtained from two unencapsulated S. aureus strains (Cowan I and 17A). Postimmune serum contained high titers of specific IgG to SpA, as measured by a modified enzyme-linked immunosorbent assay that blocked nonspecific binding of IgG to SpA. In vitro, both S. aureus strains were efficiently phagocytosed and killed by polymorphonuclear leukocytes in the presence of nonimmune sera and complement. With one strain (Cowan I), opsonophagocytosis was significantly enhanced in the presence of SpA antibody, but with the other strain (17A), killing was significantly decreased with immune serum. We then evaluated the potential protective benefit of SpA antibody in preventing S. aureus bacteremia in infant rats. Two-day-old rats received saline or various doses of SpA antiserum and were challenged subcutaneously 1 day later, but even the highest levels of antibody did not significantly reduce mortality, bacteremia or metastatic infection to lungs or liver (frequency or magnitude). This lack of protective efficacy was not related to a failure of SpA F(ab')2 to bind to cell surface-exposed epitopes, since F(ab')2 fragments prepared from hyperimmune serum bound avidly to the whole organism in an enzyme-linked immunosorbent assay.     

Staphylococcus aureus Rbf activates biofilm formation in vitro and promotes virulence in a murine foreign body infection model

We previously identified Rbf as an activator for biofilm formation on polystyrene surfaces in Staphylococcus aureus strain 8325-4. However, strain 8325-4 contains genetic mutations that may affect biofilm formation. To extend the observation to other strains, we used strain Newman, a weak biofilm producer, and strain UAMS-1, an osteomyelitis clinical strain, in this study. We found that mutations in the chromosomal rbf gene did not affect biofilm formation on polystyrene surfaces in these strains, but transformants of these strains carrying a multiple-copy plasmid containing the rbf gene formed stronger biofilms than the wild-type strains and the mutant strains. Using the flow cell method, we found that the chromosomal mutation in the rbf gene delayed biofilm formation, whereas strains with a plasmid containing the rbf gene accelerated biofilm formation in strains Newman and UAMS-1. These results led us to conclude that rbf is an activator of biofilm formation in different strains of S. aureus, although the degree of activation varies among strains. In a murine model of foreign body infection, the rbf mutations in strain Newman, but not in strain UAMS-1, reduced the bacterial survival rate in catheter lumen. However, UAMS-1 carrying multiple copies of rbf in a plasmid increased the bacterial survival rate. The animal studies therefore suggest that Rbf has a role in S. aureus virulence.     

Characterization of a protective monoclonal antibody recognizing Staphylococcus aureus MSCRAMM protein clumping factor A

The Staphylococcus aureus MSCRAMM (microbial surface components recognizing adhesive matrix molecules) protein clumping factor A (ClfA) has been shown to be a critical virulence factor in several experimental models of infection. This report describes the generation, characterization, and in vivo evaluation of a murine monoclonal antibody (MAb) against ClfA. Flow cytometric analysis revealed that MAb 12-9 recognized ClfA protein expressed by all of the clinical S. aureus strains obtained from a variety of sources. In assays measuring whole-cell S. aureus binding to human fibrinogen, MAb 12-9 inhibited S. aureus binding by over 90% and displaced up to 35% of the previously adherent S. aureus bacteria. Furthermore, a single infusion of MAb 12-9 was protective against an intravenous challenge with a methicillin-resistant strain of S. aureus in a murine sepsis model (P < 0.0001). These data suggest that anti-ClfA MAb 12-9 should be further investigated as a novel immunotherapy for the treatment and prevention of life-threatening S. aureus infections.