Respiratory epithelial cell invasion by group B streptococci.

Group B streptococci (GBS) are the most common cause of pneumonia and sepsis during the neonatal period; however, the pathogenesis of this infection is poorly understood. We investigated the ability of GBS to enter epithelial cells in culture. Two strains of GBS were capable of invading immortalized respiratory epithelial cell lines in vitro at different levels, suggesting strain differences in invasiveness. Intracellular replication was not observed. Invasion required actin microfilaments but not microtubular cytoskeletal elements. Active bacterial protein, DNA, and RNA syntheses were required for invasion. These findings are consistent with our previous observation of intracellular GBS in the lungs of infected primates. We hypothesize that this organism may access the bloodstream by direct invasion of the epithelial cell barrier.     

Influence of serotype of group B streptococci on C3 degradation.

Serotype III strains of group B streptococci (GBS) are isolated from the majority of young infants with bacteremia or meningitis. We hypothesized that serotype-associated differences in structure of the type-specific capsular polysaccharide or the presence of c protein would influence the extent to which C3 degradation occurs on GBS and that type-specific antibody would alter C3 deposition or degradation patterns. When clinical isolates of GBS representing serotypes Ia, Ib/c, II (with or without c protein) and III were employed with hypogammaglobulinemic serum as an opsonic source, a remarkable similarity was observed in patterns of C3 deposition and degradation for each of the four GBS serotypes and between strains with or without c protein. Both C3b and iC3b were detected by 5 min and throughout a 90-min opsonization interval. Less deposition occurred at 5 min on serotypes Ia and Ib/c than on types II and III GBS. Minimal degradation to C3d or smaller fragments was observed. Type-specific antibody facilitated C3b deposition on GBS and C3b degradation to iC3b early in opsonization. Possibly, accessibility of C3 fragments to neutrophil receptors, rather than the extent to which the surface permits C3 degradation, accounts for differential virulence among GBS serotypes.     

Complement component C3 secretion by mouse macrophage-like cell lines

Secretion of complement component C3 by the mouse macrophage-like cell lines PU5-1.8, J774A.1, RAW264.7, and P388D1 was measured using an enzyme-linked immunosorbent assay for mouse C3. All cell lines secreted antigenically detectable C3 with the relative secreted C3/10(6) cells/24 h ranked as J774A.1 greater than P388D1 greater than or equal to PU5-1.8 much greater than RAW264.7. C3 secretion was enhanced two- to fourfold in cultures of all cell lines when treated with lipopolysaccharide, streptococcal cell walls, or lymphokine-containing supernatant fluids of mitogen-stimulated spleen cells. A differential induction of C3 synthesis and secretion was indicated since secreted lysozyme and total cellular protein were not elevated in a manner comparable to C3. The relative inducibility of cell lines for C3 secretion in either lipopolysaccharide- or cell wall-treated cells could be ranked as PU5-1.8 greater than P388D1 greater than J774A.1 greater than RAW264.7. C3 secretion was inhibited by cycloheximide or hydrocortisone. Mouse macrophage-like cell lines retain baseline and inducible C3 synthetic activities as do normal macrophages and can serve as homogeneous cultures in which to study regulation of complement biosynthesis.     

Role of complement component C1q in phagocytosis of Listeria monocytogenes by murine macrophage-like cell lines.

Listeria monocytogenes is a facultative intracellular pathogen of a great variety of cells. Among them, macrophages constitute the major effector cells of listerial immunity during the course of an infection. Although the molecular bases of L. monocytogenes attachment and entry to phagocytes are not completely understood, it has been demonstrated that C3b significantly increases L. monocytogenes uptake by macrophages via complement receptor type 3. The first component of complement, C1q, is present in organic fluids at a relatively high concentration, and C1q receptor sites in macrophages are also abundant. In the present report, results of studies on the role of C1q in the internalization and infectivity of L. monocytogenes by macrophages are presented. L. monocytogenes uptake is enhanced by prior treatment of bacteria with normal sera. Heated serum or C1q-deficient serum abrogates this enhancement. Purified C1q specifically restored uptake. This effect was blocked by the addition of F(ab')2 anti-C1q antibody but not by an irrelevant matched antibody. Direct binding of C1q to L. monocytogenes was specific, saturable, and dose dependent with both fluorescent and radiolabeled C1q. N-Acetyl-D-alanyl-L-isoglutamine, diaminopimelic acid, and L-rhamnose caused a significant dose-dependent inhibition of C1q binding to bacteria, suggesting that these molecules, at least, are involved in the attachment of C1q to L. monocytogenes cell wall. When C1q binding structures on macrophage-like cells were blocked with saturating concentrations of C1q, the uptake of C1q-opsonized bacteria was less than in untreated cells. These experiments demonstrate that, in addition to other reported mechanisms, L. monocytogenes binds C1q, which mediates enhanced uptake by macrophages through C1q binding structures.     

Deposition and degradation of C3 on type III group B streptococci.

Antibody to the polysaccharide capsule of type III group B streptococci (GBS) and complement are essential to host defense against systemic infection in neonates. Interactions between C3 degradation products and specific neutrophil receptors mediate the attachment and ingestion of these organisms. To evaluate the influence of capsule on C3 disposition, we compared the C3 fragments released from a highly encapsulated clinical isolate (M861) with those from an unencapsulated mutant (COH 31-15) and an asialo mutant (COH 31-21) of type III GBS after opsonization with hypogammaglobulinemic serum. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, the three strains displayed similar patterns of C3 degradation; both C3b and iC3b were detectable. However, as the duration of opsonization increased, C3 fragment bands became more prominent on the encapsulated strain. The capsule, and specifically sialylation of the capsular polysaccharide of type III GBS, promotes C3 fragment deposition. However, C3 was deposited and degraded to iC3b in the absence of capsule. Opsonization of strain M861 with serum containing antibody specific for the polysaccharide capsule facilitated C3 fragment deposition in the early phases of opsonization. Because iC3b is one of the C3 fragments on an encapsulated strain of type III GBS, the relative deficiency of neonatal neutrophil receptors for this ligand may contribute to the virulence of this organism. Sufficient concentrations of antibody may enhance opsonization by facilitating C3 deposition as well as by interacting with Fc receptors on neutrophils.     

Cell-associated collagenolytic activity by group B streptococci.

Group B streptococci (GBS) are important pathogens in neonatal sepsis, pneumonia, and meningitis. The ability of GBS to invade the collagen-rich amniotic membrane of the placenta has been shown in vitro. In the presence of GBS, the collagen fibrils of the amnion appear disordered, suggesting a role for GBS in premature rupture of membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Sephadex G-200 column chromatography, and gelatin zymograms were used in this study to characterize cell-associated collagenolytic activities of GBS. The synthetic peptide 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), which mimics the primary structure of collagen, was degraded by GBS USF704, a clinical isolate from the placenta of a septic newborn. Cells of GBS USF704 (9 x 10(7) CFU/ml) hydrolyzed 902 nmol of FALGPA over a 24-h period. As reported for zinc metalloenzymes such as collagenase, the hydrolysis of FALGPA by GBS was inhibited by addition of EDTA or 1,10-phenanthroline. Boiling of the cells resulted in loss of activity, while higher activity was observed with crude GBS cell lysates (hydrolysis of 970 nmol of FALGPA in 1.5 h). Antiserum raised against collagenase from Clostridium histolyticum was found to cross-react with cell-associated proteins produced by GBS and to inhibit GBS FALGPA hydrolysis. Twenty-five additional GBS clinical isolates were screened and found to have various levels of FALGPA hydrolytic activity. These observations suggest a cell-associated collagenolytic activity by GBS which may be involved in premature rupture of membranes and neonatal disease.     

Extracellular neuraminidase production by group B streptococci.

Neuraminidase (sialidase) activity in concentrated culture filtrates of group B streptococci was measured with bovine submaxillary mucin as substrate. Group B streptococcal neuraminidase was not active on human alpha-1 acid glycoprotein and did not show increased activity on bovine submaxillary mucin that had been O-deacetylated by alkaline treatment. The enzyme was produced in a variety of media, including a chemically defined medium (FMC; Terleckyj et al., Infect. Immun. 11:649-655, 1975) supplemented with bovine serum albumin or human serum albumin. Maximal levels of activity were present in filtrates from cells grown in a dialyzable fraction of Todd-Hewitt broth harvested during the late exponential phase of growth. Dramatic decreases were seen when filtrates from the late stationary phase were assayed. The decrease in specific activity during the stationary phase was shown to be due to proteolytic digestion of neuraminidase and not to the elaboration of an extracellular neuraminic acid aldolase.     

Serious infection caused by group C streptococci.

Group C streptococci commonly cause infection in animals but only occasionally give rise to severe infection in man. We report here three cases of serious human infection due to this organism and discuss its pathogenicity in relation to the clinical manifestations of the disease.     

Phosphoglycerate kinase inhibits epithelial cell invasion by group B streptococci

Group B streptococci (GBS) are opportunistic human pathogens that cause infection and invasive disease in newborns, pregnant women and non-pregnant adults. The internalization of GBS into eukaryotic cells occurs in an actin-microfilament dependent process. The objective of our study was to understand what host cell and/or bacterial factors may be involved in this process. We focused on alpha-actinin, an actin binding protein closely associated with cytoplasmic F-actin in the eukaryotic cell, to determine if it is involved in actin recruitment upon GBS internalization. Initial work revealed that GBS does not recruit alpha-actinin. However, it was found that alpha-actinin antibodies bound to the surface of the GBS, suggesting GBS possess surface-exposed actin binding protein(s). Slide agglutination experiments revealed that when the bacteria were emulsified with F-actin, visible agglutination occurred, further suggesting the presence of an actin binding protein on the GBS cell. Western blot analysis found that anti-alpha-actinin antibodies bound to a 42 kDa protein; mass spectra analysis identified this protein as GBS phosphoglycerate kinase (PGK). Competitive binding assays suggest that the PGK-actin interaction is not a factor in the initial binding of GBS to epithelial cells, however, treating epithelial cells with PGK prior to performing an invasion assay inhibited GBS internalization. This occurred in a dose dependent manner with 10 microg/mL of PGK inhibiting invasion by over 70%, and 50 microg/mL PGK inhibits GBS invasion completely.     

Phenotypic diversity in the alpha C protein of group B streptococci.

Group B streptococci (GBS) is the leading cause of neonatal sepsis and meningitis. C proteins are an immunologically important group of surface-associated antigens in GBS that remain incompletely characterized. Two C proteins have been designated alpha and beta on the basis of protease susceptibility. We recently used a monoclonal antibody to describe a protective epitope of the GBS alpha (or trypsin-resistant) C protein in the prototype Ia/c GBS strain. In the present study, we examined 51 GBS isolates for expression of C-protein alpha and beta antigens. The alpha antigen, as detected with monoclonal antibody in sodium dodecyl sulfate (SDS) extracts, appears as a heterogeneous series of proteins spaced 8 kDa apart on SDS-polyacrylamide gel electrophoresis, but has a maximum molecular mass that varies among strains from 62.5 to 167 kDa. By immunoblotting with human immunoglobulin A, polyclonal antiserum, or monoclonal antibody, the beta antigen, in contrast, appears as a single protein of molecular mass between 124 and 134 kDa. The amount of alpha antigen expressed by each strain was quantified by enzyme immunoassay inhibition and was found to vary markedly from strain to strain. The susceptibility of strains of GBS to opsonization and killing by human polymorphonuclear leukocytes in the presence of either complement alone or complement with alpha-specific monoclonal antibody was examined. Strains expressing the alpha antigen were less readily killed in the absence of specific antibody than were alpha-negative strains. Killing in the presence of alpha-specific monoclonal antibody was found to correlate directly with the maximum molecular mass of the alpha antigen and with the quantity of antigen on the bacterial cell surface. Isolates of GBS that express the alpha C protein vary widely in the quantity and molecular mass of the alpha antigen produced, and this heterogeneity appears to have biologic importance.     

Opsonin-independent phagocytosis of group B streptococci: role of complement receptor type three.

The role of complement receptor type 3 (CR3) in nonopsonic recognition of group B streptococci (GBS) by macrophages was investigated. Monoclonal anti-CR3 (anti-Mac-1) inhibited phagocytosis of GBS strains by as much as 50% in serum-free cultures of both mouse peritoneal macrophages and the macrophage cell line PU5-1.8. GBS uptake was unaffected by the presence of anti-C3 or salicylhydroxamate, an inhibitor of the covalent binding reaction of C3. Soluble antibodies to LFA-1 or to the common beta-chain (CD18) of the LFA-1/CR3/p150,95 family of cell adhesion molecules did not inhibit GBS uptake. Down-modulation of surface Mac-1 on macrophages following adherence to anti-Mac-1- or anti-CD18-coated surfaces also inhibited uptake of GBS. Further evidence for GBS interaction with CR3 was demonstrated by reduction of EC3bi rosette formation in macrophages adherent to GBS-coated plates. These studies suggest that GBS can interact with macrophage CR3, promoting phagocytosis in a C3-independent fashion. In the absence of specific immunity in neonates, this recognition mechanism may be a significant virulence determinant for GBS which poorly activate the alternate complement pathway.     

Rapid identification of group B streptococci by counterimmunoelectrophoresis.

Beta-hemolytic streptococcal isolates have been examined by counterimmunoelectrophoresis (CIE) with group B antiserum to determine whether this techinque is of value in the rapid identification of group B strains. Ninety stock cultures and 100 clinical isolates of beta-hemolytic streptococci including representatives of groups A, D, C, G, and B were inoculated into Todd-Hewitt broth; after incubation at 37 C for 1, 2, 3, and 4 h, aliquots of the whole broth cultures were removed and tested by CIE. Antigen was not regularly detected in the 1-, 2-, and 3-h samples, but after 4 h all 126 group B streptococcal strains identified by the capillary precipitin reaction gave CIE precipitin bands with group B antiserum. None of the 58 non-group B strains gave precipitin reactions with this antiserum. Cerebrospinal fluid from an infant with group B streptococcal meningitis and peritoneal fluid from a patient with group B streptococcal peritonitis had free group B antigen detected by the CIE technique. CIE of broth cultures and direct body fluids appears to be a rapid and sensitive method for the identification of group B streptococcal strains.     

Capsular polysaccharide regulates neutrophil complement receptor interactions with type III group B streptococci.

The capsular polysaccharide of type III group B streptococci contributes substantially to the virulence of this organism. We explored the extent to which capsular polysaccharide influences neutrophil complement receptor interactions by using a poorly encapsulated strain (COH 31r/s), two well-encapsulated strains (M732 and M912), and strains produced from COH 31r/s by transposon mutagenesis that lacked capsule (COH 31-15) or had capsular polysaccharide lacking terminal sialic acid residues (COH 31-21). When tested with normal human serum, each strain had initially high bactericidal indices (85 to 96%). Monoclonal antibody blockade of neutrophil complement receptor 3 (CD11b/CD18) inhibited opsonophagocytosis to a significantly greater extent for the well-encapsulated strain than for the poorly encapsulated, asialo, or unencapsulated mutant strain. The addition of antibody with specificity for capsular polysaccharide reduced the inhibitory effect significantly for the encapsulated but not for the mutant strains. Blockade of neutrophil complement receptor 1 (CD35) effected only low-level inhibition. However, simultaneous blockade of complement receptors 1 and 3 augmented the inhibitory effect. When hypogammaglobulinemic serum was used as an antibody-free complement source, the initial bactericidal index was low (30% +/- 15%) for an encapsulated strain and was not affected for the mutant strains. Blockade of either neutrophil complement receptor 1 or 3 or the combination fully inhibited killing of the encapsulated strain. These results demonstrate that the type III group B streptococcal capsular polysaccharide regulates interactions with neutrophil complement receptors. We conclude that efficient phagocytic killing of encapsulated group streptococci in nonimmune serum requires ligation of complement receptors 1 and 3.     

Regulation of IgG antibody synthesis by a B cell component.

An immunoglobulin-G recruiting component (GRC) prepared from splenic B cells of antigen-primed mice was shown to be effective in recruiting more IgG plaque forming cells than normally appear among splenic cells experiencing a primary immune response. GRC caused increases in all classes of IgG PFC except perhaps IgG3, and the largest improvements were in IgG1 and IgG2a. GRC is synthesized by IgG2a-bearing cells and is effective at 96-120 h after spleen cells have been exposed to antigen. It is incapable of substituting for allogeneic effect factor, and the latter apparently must have its input on antibody producing cells before GRC can act. Together the data suggest that during a primary immune response a definite number of splenic B cells become poised for synthesizing IgG antibody, but only a portion of them are able to secrete. Apparently, the quiescent cells among them can be activated to secrete by exposure to GRC, A B cell product.     

Prevention of C3 deposition by capsular polysaccharide is a virulence mechanism of type III group B streptococci.

Strains of type III group B streptococci isolated from patients with neonatal sepsis are generally resistant to complement-mediated phagocytic killing in the absence of specific antibody. It has been suggested that the resistance of type III group B streptococci to phagocytosis results from inhibition of alternative-complement-pathway activation by sialic acid residues of the type III polysaccharide. To better define the relationship between structural features of the type III capsule and resistance of type III group B streptococci to complement-mediated phagocytic killing, we measured deposition of human C3 on group B streptococcal strains with altered capsule phenotypes. C3 binding was quantified by incubating bacteria with purified human 125I-C3 in 10% serum. Wild-type group B Streptococcus sp. strain COH1 bound eightfold fewer C3 molecules than did either of two isogenic mutant strains, one expressing a sialic acid-deficient capsule and the other lacking capsule completely. Similar results were obtained when the incubation with 125I-C3 was performed in serum chelated with Mg-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'- tetraacetic acid (MgEGTA), suggesting that the majority of C3 deposition occurred via the alternative pathway. In contrast to the wild-type strain, which was relatively resistant, both mutant strains were killed by human leukocytes in 10% serum with or without MgEGTA. We also measured C3 binding to 14 wild-type strains of type III group B streptococci expressing various amounts of capsule. Comparison of degree of encapsulation with C3 binding revealed a significant inverse correlation (r = -0.72; P less than 0.01). C3 fragments released by methylamine treatment of wild-type strain COH1 were predominantly in the form of C3bi, while those released from the acapsular mutant were predominantly C3b and those from the asialo mutant represented approximately equal amounts of C3b and C3bi. We conclude from these studies that the sialylated type III capsular polysaccharide inhibits alternative-pathway activation, prevents C3 deposition on group B streptococci, and protects the organisms from phagocytic killing.     

Regulation of B lymphocyte activation by complement C3 and the B cell coreceptor complex

Complement is an essential innate immune mechanism that recognizes and eradicates microbes and associated toxins. In addition, complement receptors (CD21 and CD35) on B cells cooperate with the B-cell antigen receptor (BCR) to efficiently recognize and respond to antigens bearing complement C3d(g). Fixation of C3d(g) to antigen confers adjuvant properties and therefore its deposition may need to be carefully regulated to avoid autoreactivity. CD21 and/or CD35 engagement is nonmitogenic, and B-cell activation via BCR-CD21 coligation is enhanced through the recruitment of CD19. Recent efforts have sought a better understanding of the topological and biochemical properties of BCR and coreceptor (CD19-CD21-CD81) signaling, as well as the context for complement activation in the response to foreign and self antigens.     

Faecal carriage of group B streptococci.

Consecutive stool samples from 116 female and 98 male patients (both adults and children), and rectal and vaginal swabs from 28 and 53 cases respectively, were quantitatively cultured for group B streptococci using Islam's medium. Group B streptococcus was recovered from 5% and 2% of faeces in female and male patients respectively, and the colony counts ranged from 10(2) to 10(3)/g. In women, the faecal carriage rate was 6%, which was significantly lower than the rectal carriage rate (p 0.02), suggesting that the higher recovery rate (27%) from rectal specimens may be due to contamination of swabs by perianal skin flora. Type II group B streptococcus was the only faecal isolate in adults (numbers involved are small for statistical significance), and we suspect that this type strain may be the only resident gut flora in adults, and the gastrointestinal tract is unlikely to serve as the main reservoir of all group B streptococci.     

Enhancement of monocyte complement component synthesis by antigen--antibody complexes.

Antigen--antibody complexes were found to enhance the synthesis of the complement components C2, C4, C3, C5, factor B, properdin, C3b inactivator and beta 1H by human monocytes in tissue culture. The synthesis of all components was increased by complexes in a dose-dependent fashion. Insoluble complexes formed at equivalence (antigen--antibody ratio 2:1) were more effective than complexes formed at eight times antigen excess (antigen--antibody ratio 16:1), two times antigen excess (antigen--antibody ratio 4:1) or four times antibody excess (antigen--antibody ratio 1:2). The latter three species of complexes each consist of a mixture of soluble and insoluble complexes. It was shown that total complexes (soluble and insoluble) were more potent than soluble complexes at stimulating complement component synthesis. Soluble complexes of different molecular sizes were prepared by gel-filtration chromatography; larger complexes enhance C2 synthesis to a greater extent than small complexes. The enhanced synthesis of the functionally active complement components by mononuclear phagocytes induced by antigen--antibody complexes probably facilitates the handling of complexes by promoting their solubilization and degradation.     

Alternate complement pathway activation by group A streptococci: role of M-protein.

Avirulent strains of group A streptococci readily activate the complement system in normal human serum via the alternate complement pathway (ACP). Virulent M-positive group A streptococci are much less potent as activators of the ACP. The ability of M-positive streptococci to activate the ACP is enhanced by trypsinization or mild peptic digestion. The latter treatment removes the serologically active and antiphagocytic type-specific moieties of M protein, but retains the surface fuzzy layer. The phagocytosis of avirulent streptococci is markedly enhanced by preopsonization in serum chelated with Mg-ethylene glycol tetraacetic acid (classic complement pathway blocked) but not in serum devoid of heat-labile factors. These studies suggest that the function of M protein as a virulence factor may be mediated, at least in part, by its ability to retard interaction of ACP components with structures present on the streptococcal cell surface.