Intracellular survival and replication of Erysipelothrix rhusiopathiae within murine macrophages: failure of induction of the oxidative burst of macrophages.

We investigated the ability of a virulent wild-type parent strain and acapsular avirulent transposon mutants to enter and survive intracellularly within murine peritoneal macrophages. In the presence of normal or immune serum, the parent and mutant strains were both ingested; however, the number of ingested bacteria was three- to fourfold greater in the case of mutant strains than in the case of the parent strain. The parent strain, but not the mutant strains, survived and replicated intracellularly when ingested in the presence of normal serum, whereas both the parent and the mutant strains were readily killed when ingested in the presence of immune serum. To further investigate the mechanism by which the parent strain can survive and replicate within macrophages, we studied the oxidative burst response of macrophages to these strains by measuring chemiluminescence and intracellular reduction of Nitro Blue Tetrazolium dye. Challenge exposure of macrophages with either the parent strain preopsonized with immune serum or the mutant strains preopsonized with normal or immune serum induced a strong oxidative burst, whereas the level was very low when the parent strain was preopsonized with normal serum. Phagocytosis of either the parent strain, in the presence of immune serum, or the mutant strains, in the presence of normal or immune serum, by macrophages reduced large amounts of intracellular Nitro Blue Tetrazolium, whereas minimal amounts were reduced by the parent strain in the presence of normal serum. These results suggest that virulent E. rhusiopathiae can survive and subsequently replicate within murine macrophages when ingested in the presence of normal serum and that the reduced production of reactive oxidative metabolites by macrophages may, in part, be responsible for this occurrence.     

A capsule-deficient mutant of Francisella tularensis LVS exhibits enhanced sensitivity to killing by serum but diminished sensitivity to killing by polymorphonuclear leukocytes

The live vaccine strain (LVS) of Francisella tularensis is killed by human polymorphonuclear leukocytes as a result of strictly oxygen-dependent mechanisms (S. L?fgren, A. T?rnvik, M. Thore, and J. Carlsson, Infect. Immun. 43:730-734, 1984). We now report that a capsule-deficient (Cap-) mutant of LVS survives in the leukocytes. In contrast to the encapsulated parent strain, the Cap- mutant was avirulent in mice and was susceptible to the bactericidal effect of nonimmune human serum. The mutant was killed by serum as a result of activation of the classical pathway of complement by naturally occurring immunoglobulin M. This killing by serum was mitigated by the presence of human polymorphonuclear leukocytes. After opsonization in complement component C5-deficient nonimmune serum, the Cap- mutant was ingested and survived in the leukocytes. Under these conditions, the parent strain was killed. The leukocytes responded to both the parent and the Cap- strain with a very low chemiluminescent response. Only the response to the parent strain was inhibited by superoxide dismutase. When the Cap- mutant was opsonized with immunoglobulin G, it induced a higher and superoxide dismutase-inhibitable chemiluminescent response and was killed by the leukocytes. In conclusion, the capsule of F. tularensis LVS seemed to protect this organism against the bactericidal effect of serum. When deprived of the capsule, the organism failed to induce an antimicrobial response in polymorphonuclear leukocytes and survived in the leukocytes. Survival in phagocytes is a key characteristic of intracellular parasites. The Cap- mutant of F. tularensis may become a useful tool in experiments to explain the differences between pathways of ingestion of intracellular parasites, evidenced by the death or survival of the parasite.     

Virulence factors of Haemophilus ducreyi

We investigated the susceptibility of virulent and avirulent strains of Haemophilus ducreyi to the bactericidal activity of normal human serum and to phagocytosis and killing by human polymorphonuclear leukocytes (PMNL). Strains were defined as virulent if intradermal inoculation into a rabbit produced a typical necrotic lesion. Nonvirulent strains produced no cutaneous lesions in rabbits. Virulent strains were resistant to the complement-mediated lethal action of normal human and rabbit sera, whereas avirulent strains were susceptible (greater than 95% kill, 60 min). Virulent strains were relatively resistant to phagocytosis and killing by human PMNL, in contrast to the avirulent strains. In past studies polymyxin resistance has been correlated with virulence in H. ducreyi. In our studies, polymyxin resistance could not be correlated with virulence, since polymyxin-sensitive mutants obtained from polymyxin-resistant parent strains remained virulent for rabbits and resistant to bactericidal action of normal serum and phagocytosis and killing by human PMNL. Similarly, polymyxin-resistant mutants obtained from polymyxin-sensitive parent strains remained avirulent for rabbits and susceptible to bactericidal action of normal serum and PMNL. The acquisition of polymyxin resistance was accompanied by the loss of a 47,000-molecular-weight protein. The association of serum resistance and resistance to phagocytosis and killing by human PMNL with virulent strains, as defined by the rabbit intradermal test, suggests that these factors may mediate the pathogenicity of H. ducreyi.     

Phagocytosis of virulent and avirulent leptospires by guinea-pig and human polymorphonuclear leukocytes in vitro

Chemiluminescence (CL) and electron microscopy were used to study the phagocytosis of both virulent and avirulent strains of Leptospira interrogans serovar copenhageni by guinea-pig and human polymorphonuclear leukocytes (PMN). A significant CL response was observed when guinea-pig PMN were incubated with virulent leptospires in the presence but not in the absence of specific immune serum. This response was markedly enhanced by the addition of guinea-pig complement. Phagocytosis was confirmed by the observation of intracellular leptospires in guinea-pig PMN by electron microscopy. The phagocytosis of avirulent leptospires by guinea-pig PMN and of both virulent and avirulent leptospires by human PMN required the presence of both specific immune serum and complement. Thus the ability of leptospires to resist phagocytosis by PMN in the absence of immune serum does not appear to be a major determinant of virulence.     

Identification of a genetic locus essential for capsule sialylation in type III group B streptococci.

The type III capsular polysaccharide of group B streptococci (GBS) consists of a linear backbone with short side chains ending in residues of N-acetylneuraminic acid, or sialic acid. The presence of sialic acid on the surface of the organism inhibits activation of the alternative pathway of complement and is thought to be an important element in the virulence function of the capsule. We showed previously that a mutant strain of GBS that expressed a sialic acid-deficient, or asialo, form of the type III polysaccharide was avirulent, supporting a virulence function for capsular sialic acid. We now report the derivation of an asialo capsule mutant from a highly encapsulated wild-type strain of type III GBS, strain COH1, by insertional mutagenesis with transposon Tn916 delta E. In contrast to the wild-type strain, the asialo mutant strain COH1-11 was sensitive to phagocytic killing by human leukocytes in vitro and was relatively avirulent in a neonatal rat model of GBS infection. The asialo mutant accumulated free intracellular sialic acid, suggesting a defect subsequent to sialic acid synthesis in the biosynthetic pathway leading to capsule sialylation. The specific biosynthetic defect in mutant strain COH1-11 was found to be in the activation of free sialic acid to CMP-sialic acid: CMP-sialic acid synthetase activity was present in the wild-type strain COH1 but was not detected in the asialo mutant strain COH1-11. One of the two transposon insertions in the asialo mutant COH1-11 mapped to the same chromosomal location as one of the two Tn916 insertions in the previously reported asialo mutant COH31-21, identifying this site as a genetic locus necessary for expression of CMP-sialic acid synthetase activity. These studies demonstrate that the enzymatic synthesis of CMP-sialic acid by GBS is an essential step in sialylation of the type III capsular polysaccharide.     

Characterization of a polysaccharide capsular antigen of septicemic Escherichia coli O115:K "V165" :F165 and evaluation of its role in pathogenicity.

Escherichia coli strains of serogroup O115:K(-):F165 have been associated with septicemia in calves and piglets. These strains express a capsular antigen referred to as K"V165" which inhibits agglutination of the O antigen by anti-O115 serum. We used hybrid transposon TnphoA mutants M48, 18b, and 2, and a spontaneous O-agglutinable mutant, 5131a, to evaluate the role of K"V165" in the pathogenicity of E. coli O115. Mutant M48 was as resistant to 90% rabbit serum and as virulent in day-old chickens as the parent strain 5131, mutants 18b and 5131a were less resistant to serum and less virulent in chickens, and mutant 2 was serum sensitive and avirulent. Analysis of outer membrane protein and lipopolysaccharide profiles failed to show any difference between the transposon mutants and the parent strain. In contrast, the spontaneous O-agglutinable mutant showed additional bands in the 16-kDa region of the polysaccharide ladder-like pattern. Mutants 2 and 5131a produced significantly less K"V165" capsular antigen than the parent strain, as demonstrated by a competitive enzyme-linked immunosorbent assay with adsorbed anti-K"V165" serum. In addition, electron microscopic analysis revealed that mutants 2 and 5131a had lost the capsular layer observed in the parent strain after fixation with glutaraldehyde-lysine. This capsule contained carbohydrate compounds and resembled an O-antigen capsule since it prevented O-antigen agglutination before the bacteria were heated at 100 degrees C and induced bacterial serum resistance. The capsule-defective mutants colonized the intestinal epithelium of experimentally infected gnotobiotic pigs but failed to induce clinical signs of septicemia. We concluded that E. coli strains of serogroup O115 expressed a polysaccharide capsular antigen which induced serum resistance and consequently contributed to the pathogenicity of the bacteria.     

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.     

Influence of encapsulation on staphylococcal opsonization and phagocytosis by human polymorphonuclear leukocytes

In previous studies, encapsulated Staphylococcus aureus strains have been shown to resist phagocytosis. In this investigation, the nature of the interference with phagocytosis by human polymorphonuclear leukocytes was examined by studying the opsonization of two pairs of unencapsulated (Smith compact and M variant) and encapsulated (Smith diffuse and M) S. aureus strains. The uptake of [3H]glycine-labeled bacteria by normal leukocytes was quantitatively measured after incubation of bacteria in pooled serum, C2-deficient serum, immunoglobulin-deficient serum, and serum from a rabbit immunized with S. aureus M. The presence of a capsule was found to interfere with opsonization by both the classical and alternative pathways of complement as well as by heat-stable opsonic factors in nonimmune human serum. This interference was significantly greater in the case of the S. aureus M strain than in the case of the Smith diffuse strain. The only effective opsonic source for S. aureus M was immune rabbit serum. It is proposed that encapsulation of S. aureus strains interferes with phagocytosis by preventing effective bacterial opsonization.     

Role of serotype-specific polysaccharide in the resistance of Streptococcus mutans to phagocytosis by human polymorphonuclear leukocytes

To clarify the role of cell surface components of Streptococcus mutans in resistance to phagocytosis by human polymorphonuclear leukocytes (PMNs), several isogenic mutants of S. mutans defective in cell surface components were studied with a luminol-enhanced chemiluminescence (CL) assay, a killing assay, and a transmission electron microscope. The CL responses of human PMNs to mutant Xc11 defective in a major cell surface antigen, PAc, and mutant Xc16 defective in two surface glucosyltransferases (GTF-I and GTF-SI) were the same as the response to the wild-type strain, Xc. In contrast, mutant Xc24R, which was defective in serotype c-specific polysaccharide, induced a markedly higher CL response than the other strains. The killing assay showed that human PMNs killed more Xc24R than the parent strain and the other mutants. The transmission electron microscopic observation indicated that Xc24R cells were more internalized by human PMNs than the parental strain Xc. These results may be reflected by the fact that strain Xc24R was more phagocytosed than strain Xc. The CL response of human PMNs to a mutant defective in polysaccharide serotype e or f was similar to the response to Xc24R. Furthermore, mutants defective in serotype-specific polysaccharide were markedly more hydrophobic than the wild-type strains and the other mutants, suggesting that the hydrophilic nature of polysaccharides may protect the bacterium from phagocytosis. We conclude that the serotype-specific polysaccharide, but not the cell surface proteins on the cell surface of S. mutans, may play an important role in the resistance to phagocytosis.     

Bactericidal, bacteriolytic and opsonic activity of human serum against Escherichia coli

The effect of human serum on Escherichia coli was studied with serum-sensitive and serum-resistant strains. The bactericidal effect of human serum on serum-sensitive strains of E. coli depended on the activation of the classical complement pathway. The role of activation of the alternative pathway was less important. After incubation in sub-bactericidal concentrations of serum these strains were also easily phagocytosed by polymorphonuclear leukocytes (PMNL). Strains of E. coli of certain O-types required not only an intact classical pathway but also the presence of specific antibodies for effective killing by serum and effective phagocytosis by PMNL, despite rapid activation of complement and rapid deposition of C3 on the bacterial surface in the absence of antibody. Capsulate strains O1K1 and O78K80 resisted the bactericidal effect of serum even in the presence of specific antibodies; phagocytosis by PMNL only occurred after opsonisation with specific antibodies.     

Staphylococcus aureus Serotype 5 Capsular Polysaccharide Is Antiphagocytic and Enhances Bacterial Virulence in a Murine Bacteremia Model

Controversy persists over the role that the capsular polysaccharide plays in the pathogenesis of Staphylococcus aureus infections. To address this issue, we compared the mouse virulence of S. aureus Reynolds and capsule-defective mutant strains cultivated under conditions of high or low capsule expression. Strain Reynolds cells cultivated on Columbia salt agar plates expressed ~100-fold more type 5 capsular polysaccharide than did cells cultivated in Columbia salt broth. The relative virulence of strain Reynolds and its capsule-defective mutants after growth on either solid or liquid medium was examined in mice challenged intraperitoneally or intravenously. The results indicated that agar-grown Reynolds cells were cleared from the bloodstream of mice less readily than broth-grown Reynolds cells. When the parental and mutant strains were cultivated on solid medium, strain Reynolds sustained a higher level of bacteremia than did the capsular mutants. We performed in vitro opsonophagocytic killing assays to determine whether staphylococcal virulence for mice correlated with resistance to phagocytosis. S. aureus Reynolds cultivated on solid medium was susceptible to phagocytic killing only in the presence of specific capsular antibodies and complement. Strain Reynolds grown in broth showed opsonic requirements for phagocytic killing that were similar to those of the capsular mutants (grown in broth or on agar); i.e., the bacteria were opsonized for phagocytosis by nonimmune serum with complement activity. These studies indicate that optimal expression of capsule enhances bacterial virulence in the mouse model of bacteremia, probably by rendering the organisms resistant to opsonophagocytic killing by leukocytes.     

Effects on virulence of mutations in a locus essential for hyaluronic acid capsule expression in group A streptococci.

Mucoid or highly encapsulated strains of group A streptococci have been associated both with unusually severe infections and with acute rheumatic fever. Previously, we described an acapsular mutant, TX4, derived from a mucoid M-type 18 strain of a group A streptococcus by transposon mutagenesis (M. R. Wessels, A. E. Moses, J. B. Goldberg, and T. J. DiCesare, Proc. Natl. Acad. Sci. USA 88:8317-8321, 1991). We now report studies further characterizing strain TX4 as well as an additional acapsular mutant, TX72. Strain TX4 was found to contain a 9.5-kb deletion of chromosomal DNA adjacent to the site of transposon Tn916 insertion. Cloned chromosomal DNA from TX4 flanking the transposon insertion site was used as a probe to demonstrate the presence of homologous regions in 11 of 11 wild-type group A streptococcal strains of various M protein types. A second acapsular mutant, TX72, had a single transposon insertion and had no apparent deletion of chromosomal DNA. The Tn916 insertion in TX72 was mapped to the hasA locus (encoding hyaluronate synthase), which lies within the chromosomal region deleted in TX4. Strain TX72 was avirulent in mice and sensitive to phagocytic killing in vitro. Transduction of either the insertion-deletion mutation from TX4 or the simple insertion mutation from TX72 to a type 24 group A streptococcus strain also resulted in loss of capsule expression, demonstrating that a homologous region of the chromosome controls capsule expression in another serotype of group A streptococci. We conclude that the hyaluronic acid capsule plays an important role in virulence and that a region of the chromosome essential for capsular polysaccharide expression is conserved among diverse group A streptococcal strains.     

Biological properties of the encapsulated Staphylococcus aureus M

Strain M, classified as a Staphylococcus aureus, behaves like the other rare encapsulated staphylococcal strains. It was clumping-factor negative, grew in diffuse-type colonies in serum-soft agar, and produced rapidly fatal disease in mice. Strain M was highly resistant to phagocytosis by human or mouse leukocytes and required both specific antibody and heat-labile serum factor(s) for efficient ingestion by human polymorphonuclear leukocytes. Electron micrographs confirmed the presence of a large capsule. Agglutination studies, active or passive mouse protection experiments, and opsonic studies revealed that strain M represents a new, immunologically distinct strain of encapsulated staphylococcus. Strain M differs from other known encapsulated staphylococci in several other respects: its cellular and colonial morphology is atypical; its LD(50) in the mouse peritoneal model is 100 times less than that of other mouse lethal strains; it is poorly opsonized by normal human serum; and, finally, it possesses an unusually large capsule as seen in electron micrographs.     

The capsule is a virulence determinant in the pathogenesis of Pasteurella multocida M1404 (B:2)

Capsules from a range of pathogenic bacteria are key virulence determinants, and the capsule has been implicated in virulence in Pasteurella multocida. We have previously identified and determined the nucleotide sequence of the P. multocida M1404 (B:2) capsule biosynthetic locus (J. D. Boyce, J. Y. Chung, and B. Adler, Vet. Microbiol. 72:121-134, 2000). The cap locus consists of 15 genes, which can be grouped into three functional regions. Regions 1 and 3 contain genes proposed to encode proteins involved in capsule export, and region 2 contains genes proposed to encode proteins involved in polysaccharide biosynthesis. In order to construct a mutant impaired in capsule export, the final gene of region 1, cexA, was disrupted by insertion of a tetracycline resistance cassette by allelic replacement. The genotype of the tet(M) OmegacexA mutant was confirmed by Southern hybridization and PCR. The acapsular phenotype was confirmed by immunofluorescence, and the strain could be complemented and returned to capsule production by the presence of a cloned uninterrupted copy of cexA. Wild-type, mutant, and complemented strains were tested for virulence by intraperitoneal challenge of mice; the presence of the capsule was shown to be a crucial virulence determinant. Following intraperitoneal challenge of mice, the acapsular bacteria were removed efficiently from the blood, spleen, and liver, while wild-type bacteria multiplied rapidly. Acapsular bacteria were readily taken up by murine peritoneal macrophages, but wild-type bacteria were significantly resistant to phagocytosis. Both wild-type and acapsular bacteria were resistant to complement in bovine and murine serum.     

Contribution of superoxide dismutase and catalase activities to Shigella flexneri pathogenesis.

A Shigella flexneri serotype 5 strain deficient in the production of the iron-containing superoxide dismutase FeSOD (sodB) and a catalase-negative (katFG) S. flexneri serotype 5 strain were isolated. Both strains were examined for increased sensitivity to oxygen stress by using assays involving killing by mouse peritoneal macrophages and human polymorphonuclear leukocytes as well as infection of rabbit ileal loops. The sodB mutant was extremely sensitive to killing by phagocytes when compared with the wild-type parent, M90T. The catalase mutant also showed an increased sensitivity to killing, but to a much lesser extent. Upon infection of rabbit ileal loops and subsequent histopathological examination, the sodB mutant caused very little detectable damage to intestinal villi. The pattern of infection was roughly similar to that of BS176, an avirulent plasmidless derivative of M90T. The katFG mutant, on the other hand, showed a high degree of destruction, similar to that caused by M90T. This evidence suggests that the superoxide dismutase encoded by sodB may play an important role in the pathogenesis of S. flexneri. In contrast, catalases appear to make a limited contribution to virulence.     

Localization of the third component of complement on the cell wall of encapsulated Staphylococcus aureus M: implications for the mechanism of resistance to phagocytosis.

Encapsulated Staphylococcus aureus strains are more virulent than unencapsulated staphylococci, and this phenomenon has been associated with decreased opsonization of encapsulated bacteria by normal human serum. Peptidoglycan, a major cell wall component of S. aureus, has been shown to promote opsonization of this bacterial species by certain components of the serum complement system. However, when the processes of complement activation and opsonization of encapsulated staphylococci have been studied, it has been found that encapsulated bacteria activate complement efficiently and C3 is bacteria associated, yet these organisms are not efficiently phagocytized by human polymorphonuclear leukocytes. In this study, the hypothesis was tested that opsonically active molecules are not on the true external surface of encapsulated organisms but rather are cell wall associated and thus "hidden" from human polymorphonuclear leukocytes. By using immunoelectronmicroscopy, C3 was found to be localized on the cell wall of an encapsulated S. aureus strain after incubation of the organism in normal human serum. When shrinkage of the capsule was prevented by treatment with anticapsular antibody, the C3 was again shown to be cell wall associated and located beneath the bacterial capsule. These results suggest that encapsulated S. aureus may resist phagocytosis because opsonically active C3 molecules are not exposed at the true external surface of the bacterium.     

The role of K antigens as virulence factors in Klebsiella

The importance of K antigen of Klebsiella as a virulence factor was studied in nine pairs of K+ and K- strains, each pair isogenic apart from the presence of K antigen. Loss of K antigen by nine K+ strains resulted in the reduced virulence of their K- variants in a mouse-skin model. This reduced virulence of K- strains for mice may be explained in all strains by a higher degree of phagocytosis as measured by chemiluminescence response of human polymorphonuclear leukocytes (PMNL) and in most strains by enhanced killing by either human PMNL or human serum or both. Although the protective role of the K antigen in serum-induced killing and killing by PMNL was generally evident, our results also suggested that other virulence factors were sometimes involved.     

Phagocytic killing of encapsulated and microencapsulated Staphylococcus aureus by human polymorphonuclear leukocytes.

Phagocytosis by human polymorphonuclear leukocytes (PMNs) is an important host defense against infections caused by Staphylococcus aureus. Using an in vitro assay, we compared the opsonic requirements for phagocytic killing of prototype strains of encapsulated (type 1) and microencapsulated (type 5 and type 8) S. aureus by human PMNs. More than 85% of broth-grown, logarithmic-phase type 5 and 8 S. aureus organisms were killed by PMNs incubated with fresh normal human, rabbit, or guinea pig serum with complement activity. Under similar conditions, the highly encapsulated type 1 strain was not killed. Both encapsulated and microencapsulated strains were opsonized for phagocytosis by heat-inactivated serum raised in rabbits to killed bacteria. Opsonization by homologous serum was required for phagocytosis of the type 1 strain. In contrast, microencapsulated type 5 and 8 S. aureus organisms were killed by heat-inactivated rabbit serum raised to type 5, type 8, or nonencapsulated isolates; this result suggested that antibodies to the capsule or to cell wall components other than the capsule could opsonize these organisms for phagocytosis. The specificity of the assay was confirmed with capsule type 5-specific monoclonal antibodies, which were opsonic only for the type 5 S. aureus isolate. These studies indicate that, unlike the highly encapsulated type 1 strain, broth-grown microencapsulated S. aureus strains do not resist opsonophagocytic killing in vitro by normal serum.     

Overproduction of type 8 capsular polysaccharide augments Staphylococcus aureus virulence

Type 8 capsular polysaccharide (CP8) is the most prevalent capsule type in clinical isolates of Staphylococcus aureus. However, its role in virulence has not been clearly defined. CP8 strains such as strain Becker produce a small amount of capsule on their surface in vitro. In contrast, CP1 strains such as strain M produce a large amount of capsule, which has been shown to be an important antiphagocytic virulence factor. The cap8 and cap1 operons, required for the synthesis of CP8 and CP1, respectively, have been cloned and sequenced. To test whether CP8 contributes to the pathogenesis of S. aureus, we replaced the weak native promoter of the cap8 operon in strain Becker with the strong constitutive promoter of the cap1 operon of strain M. The resultant strain, CYL770, synthesized cap8-specific mRNA at a level about sevenfold higher than that in the parent strain. Remarkably, the CYL770 strain produced about 80-fold more CP8. In a mouse infection model of bacteremia, the CP8-overproducing strain persisted longer in the bloodstream, the liver, and the spleen in mice than the parent strain. In addition, strain CYL770 was more resistant to ospsonophagocytosis in vitro by human polymorphonuclear leukocytes. These results indicate that CP8 is an antiphagocytic virulence factor of S. aureus.