Catherine Hyams

The Streptococcus pneumoniae capsule inhibits complement activity and neutrophil phagocytosis by multiple mechanisms.

ABSTRACT The Streptococcuspneumoniae capsule is vital for virulence and may inhibit complement activity and phagocytosis. However, there are only limited data on the mechanisms by which the capsule affects complement and the consequences for S. pneumoniae interactions with phagocytes. Using unencapsulated serotype 2 and 4 S. pneumoniae mutants, we have confirmed that the capsule has several effects on complement activity. The capsule impaired bacterial opsonization with C3b/iC3b by both the alternative and classical complement pathways and also inhibited conversion of C3b bound to the bacterial surface to iC3b. There was increased binding of the classical pathway mediators immunoglobulin G (IgG) and C-reactive protein (CRP) to unencapsulated S. pneumoniae, indicating that the capsule could inhibit classical pathway complement activity by masking antibody recognition of subcapsular antigens, as well as by inhibiting CRP binding. Cleavage of serum IgG by the enzyme IdeS reduced C3b/iC3b deposition on all of the strains, but there were still marked increases in C3b/iC3b deposition on unencapsulated TIGR4 and D39 strains compared to encapsulated strains, suggesting that the capsule inhibits both IgG-mediated and IgG-independent complement activity against S. pneumoniae. Unencapsulated strains were more susceptible to neutrophil phagocytosis after incubation in normal serum, normal serum treated with IdeS, complement-deficient serum, and complement-deficient serum treated with IdeS or in buffer alone, suggesting that the capsule inhibits phagocytosis mediated by Fcγ receptors, complement receptors, and nonopsonic receptors. Overall, these data show that the S. pneumoniae capsule affects multiple aspects of complement- and neutrophil-mediated immunity, resulting in a profound inhibition of opsonophagocytosis.

Streptococcus pneumoniae Resistance to Complement-Mediated Immunity Is Dependent on the Capsular Serotype

ABSTRACT Streptococcus pneumoniae strains vary considerably in the ability to cause invasive disease in humans, and this is partially associated with the capsular serotype. The S. pneumoniae capsule inhibits complement- and phagocyte-mediated immunity, and differences between serotypes in these effects on host immunity may cause some of the variation in virulence between strains. However, the considerable genetic differences between S. pneumoniae strains independent of the capsular serotype prevent an unambiguous assessment of the effects of the capsular serotype on immunity using clinical isolates. We have therefore used capsular serotype-switched TIGR4 mutant strains to investigate the effects of the capsular serotype on S. pneumoniae interactions with complement. Flow cytometry assays demonstrated large differences in C3b/iC3b deposition on opaque-phase variants of TIGR4(−)+4, +6A, +7F, and +23F strains even though the thicknesses of the capsule layers were similar. There was increased C3b/iC3b deposition on TIGR4(−)+6A and +23F strains compared to +7F and +4 strains, and these differences persisted even in serum depleted of immunoglobulin G. Neutrophil phagocytosis of the TIGR4(−)+6A and +23F strains was also increased, but only in the presence of complement, showing that the effects of the capsular serotype on C3b/iC3b deposition are functionally significant. In addition, the virulence of the TIGR4(−)+6A and +23F strains was reduced in a mouse model of sepsis. These data demonstrate that resistance to complement-mediated immunity can vary with the capsular serotype independently of antibody and of other genetic differences between strains. This might be one mechanism by which the capsular serotype can affect the relative invasiveness of different S. pneumoniae strains.

Impaired Opsonization with C3b and Phagocytosis of Streptococcus pneumoniae in Sera from Subjects with Defects in the Classical Complement Pathway

ABSTRACT Results from studies using mice deficient in specific complement factors and clinical data on patients with an inherited deficiency of the classical complement pathway component C2 suggest that the classical pathway is vital for immunity to Streptococcus pneumoniae. However, the consequences of defects in classical pathway activity for opsonization with C3b and the phagocytosis of different S. pneumoniae serotypes in human serum are not known, and there has not been a systematic analysis of the abilities of sera from subjects with a C2 deficiency to opsonize S. pneumoniae. Hence, to investigate the role of the classical pathway in immunity to S. pneumoniae in more detail, flow cytometry assays of opsonization with C3b and the phagocytosis of three capsular serotypes of S. pneumoniae were performed using human sera depleted of the complement factor C1q or B or sera obtained from C2-deficient subjects. The results demonstrate that, in human serum, the classical pathway is vital for C3b-iC3b deposition onto cells of all three serotypes of S. pneumoniae and seems to be more important than the alternative pathway for phagocytosis. Compared to the results for sera from normal subjects, C3b-iC3b deposition and total anti-S. pneumoniae antibody activity levels in sera obtained from C2−/− subjects were reduced and the efficiency of phagocytosis of all three S. pneumoniae strains was impaired. Anticapsular antibody levels did not correlate with phagocytosis or C3b-iC3b deposition. These data confirm that the classical pathway is vital for complement-mediated phagocytosis of S. pneumoniae and demonstrate why subjects with a C2 deficiency have a marked increase in susceptibility to S. pneumoniae infections.

Protective Contributions against Invasive Streptococcus pneumoniae Pneumonia of Antibody and Th17-Cell Responses to Nasopharyngeal Colonisation

The nasopharyngeal commensal bacteria Streptococcus pneumoniae is also a frequent cause of serious infections. Nasopharyngeal colonisation with S. pneumoniae inhibits subsequent re-colonisation by inducing Th17-cell adaptive responses, whereas vaccination prevents invasive infections by inducing antibodies to S. pneumoniae capsular polysaccharides. In contrast, protection against invasive infection after nasopharyngeal colonisation with mutant S. pneumoniae strains was associated with antibody responses to protein antigens. The role of colonisation-induced Th17-cell responses during subsequent invasive infections is unknown. Using mouse models, we show that previous colonisation with S. pneumoniae protects against subsequent lethal pneumonia mainly by preventing bacteraemia with a more modest effect on local control of infection within the lung. Previous colonisation resulted in CD4-dependent increased levels of Th17-cell cytokines during subsequent infectious challenge. However, mice depleted of CD4 cells prior to challenge remained protected against bacteraemia, whereas no protection was seen in antibody deficient mice and similar protection could be achieved through passive transfer of serum. Serum from colonised mice but not antibody deficient mice promoted phagocytosis of S. pneumoniae, and previously colonised mice were able to rapidly clear S. pneumoniae from the blood after intravenous inoculation. Thus, despite priming for a Th17-cell response during subsequent infection, the protective effects of prior colonisation in this model was not dependent on CD4 cells but on rapid clearance of bacteria from the blood by antibody-mediated phagocytosis. These data suggest that whilst nasopharyngeal colonisation induces a range of immune responses, the effective protective responses depend upon the site of subsequent infection.

Streptococcus pneumoniae Capsular Serotype Invasiveness Correlates with the Degree of Factor H Binding and Opsonization with C3b/iC3b

ABSTRACT Different capsular serotypes of Streptococcus pneumoniae vary markedly in their ability to cause invasive infection, but the reasons why are not known. As immunity to S. pneumoniae infection is highly complement dependent, variations in sensitivity to complement between S. pneumoniae capsular serotypes could affect invasiveness. We have used 20 capsule-switched variants of strain TIGR4 to investigate whether differences in the binding of the alternative pathway inhibitor factor H (FH) could be one mechanism causing variations in complement resistance and invasive potential between capsular serotypes. Flow cytometry assays were used to assess complement factor binding and complement-dependent neutrophil association for the TIGR4 capsule-switched strains. FH binding varied with the serotype and inversely correlated with the results of factor B binding, C3b/iC3b deposition, and neutrophil association. Differences between strains in FH binding were lost when assays were repeated with pspC mutant strains, and loss of PspC also reduced differences in C3b/iC3b deposition between strains. Median FH binding was high in capsule-switched mutant strains expressing more invasive serotypes, and a principal component analysis demonstrated a strong correlation between serotype invasiveness, high FH binding, and resistance to complement and neutrophil association. Further data obtained with 33 clinical strains also demonstrated that FH binding negatively correlated with C3b/iC3b deposition and that median FH binding was high in strains expressing more invasive serotypes. These data suggest that variations in complement resistance between S. pneumoniae strains and the association of a serotype with invasiveness could be related to capsular serotype effects on FH binding.

Changes in capsular serotype alter the surface exposure of pneumococcal adhesins and impact virulence

We examined the contribution of serotype on Streptococcus pneumoniae adhesion and virulence during respiratory tract infection using a panel of isogenic TIGR4 (serotype 4) mutants expressing the capsule types 6A (+6A), 7F (+7F) and 23F (+23F) as well as a deleted and restored serotype 4 (+4) control strain. Immunoblots, bacterial capture assays with immobilized antibody, and measurement of mean fluorescent intensity by flow cytometry following incubation of bacteria with antibody, all determined that the surface accessibility, but not total protein levels, of the virulence determinants Pneumococcal surface protein A (PspA), Choline binding protein A (CbpA), and Pneumococcal serine-rich repeat protein (PsrP) changed with serotype. In vitro, bacterial adhesion to Detroit 562 pharyngeal or A549 lung epithelial cells was modestly but significantly altered for +6A, +7F and +23F. In a mouse model of nasopharyngeal colonization, the number of +6A, +7F, and +23F pneumococci in the nasopharynx was reduced 10 to 100-fold versus +4; notably, only mice challenged with +4 developed bacteremia. Intratracheal challenge of mice confirmed that capsule switch strains were highly attenuated for virulence. Compared to +4, the +6A, +7F, and +23F strains were rapidly cleared from the lungs and were not detected in the blood. In mice challenged intraperitoneally, a marked reduction in bacterial blood titers was observed for those challenged with +6A and +7F versus +4 and +23F was undetectable. These findings show that serotype impacts the accessibility of surface adhesins and, in particular, affects virulence within the respiratory tract. They highlight the complex interplay between capsule and protein virulence determinants.

The Effects of PspC on Complement-Mediated Immunity to Streptococcus pneumoniae Vary with Strain Background and Capsular Serotype

ABSTRACT Streptococcus pneumoniae may evade complement activity by binding of factor H (FH), a negative regulator of the alternative pathway, to the surface protein PspC. However, existing data on the effects of FH binding to PspC on complement activity are conflicting, and there is also considerable allelic variation in PspC structure between S. pneumoniae strains that may influence PspC-dependent effects on complement. We have investigated interactions with complement for several S. pneumoniae strains in which the gene encoding PspC has been deleted. The degree of FH binding varied between strains and was entirely dependent on PspC for seven strains. Data obtained with TIGR4 strains expressing different capsular serotypes suggest that FH binding is affected by capsular serotype. Results of immunoblot analysis for C3 degradation products and iC3b deposition assays suggested that FH bound to PspC retained functional activity, but loss of PspC had strikingly varied effects on C3b/iC3b deposition on S. pneumoniae, with large increases on serotype 4, 6A, 6B, and 9V strains but only small increases or even decreases on serotype 2, 3, 17, and 23F strains. Repeating C3b/iC3b assays with TIGR4 strains expressing different capsular serotypes suggested that differences in the effect of PspC on C3b/iC3b deposition were largely independent of capsular serotype and depend on strain background. However, data obtained from infection in complement-deficient mice demonstrated that differences between strains in the effects of PspC on complement surprisingly did not influence the development of septicemia.

Priming innate immune responses to infection by cyclooxygenase inhibition kills antibiotic-susceptible and -resistant bacteria

Inhibition of cyclooxygenase (COX)-derived prostaglandins (PGs) by nonsteroidal anti-inflammatory drugs (NSAIDs) mediates leukocyte killing of bacteria. However, the relative contribution of COX1 versus COX2 to this process, as well as the mechanisms controlling it in mouse and humans, are unknown. Indeed, the potential of NSAIDs to facilitate leukocyte killing of drug-resistant bacteria warrants investigation. Therefore, we carried out a series of experiments in mice and humans, finding that COX1 is the predominant isoform active in PG synthesis during infection and that its prophylactic or therapeutic inhibition primes leukocytes to kill bacteria by increasing phagocytic uptake and reactive oxygen intermediate-mediated killing in a cyclic adenosine monophosphate (cAMP)-dependent manner. Moreover, NSAIDs enhance bacterial killing in humans, exerting an additive effect when used in combination with antibiotics. Finally, NSAIDs, through the inhibition of COX prime the innate immune system to mediate bacterial clearance of penicillin-resistant Streptococcus pneumoniae serotype 19A, a well-recognized vaccine escape serotype of particular concern given its increasing prevalence and multi-antibiotic resistance. Therefore, these data underline the importance of lipid mediators in host responses to infection and the potential of inhibitors of PG signaling pathways as adjunctive therapies, particularly in the con-text of antibiotic resistance.

Roles of the Alternative Complement Pathway and C1q during Innate Immunity to Streptococcus pyogenes

Complement is important for innate immunity to the common bacterial pathogen Streptococcus pyogenes, but the relative importance of the alternative and classical pathways has not been investigated. Using mice and human serum deficient in either C1q, the first component of the classical pathway, or factor B, an important component of the alternative pathway, we have investigated the role of both pathways for innate immunity to S. pyogenes. C3b deposition on four different strains of S. pyogenes was mainly dependent on factor B. As a consequence opsonophagocytosis of S. pyogenes was reduced in serum from factor B-deficient mice, and these mice were very susceptible to S. pyogenes infection. In contrast, C3b deposition was not dependent on C1q for two of the strains investigated, H372 and H305, yet opsonophagocytosis of all four S. pyogenes strains was impaired in serum deficient in C1q. Furthermore, infection in C1q-deficient mice with strain H372 resulted in a rapidly progressive disease associated with large numbers of bacteria in target organs. These results demonstrate the important role of the alternative pathway and C1q for innate immunity to S. pyogenes and suggest that C1q-mediated innate immunity to at least some strains of S. pyogenes may involve mechanisms that are independent of C3b on the bacteria.

Characterization of a New Mouse Model of Empyema and the Mechanisms of Pleural Invasion by Streptococcus pneumoniae

Although empyema affects more than 65,000 people each year in the United States and in the United Kingdom, there are limited data on the pathogenesis of pleural infection. We investigated the pathogenesis of empyema using animal and cell culture models of Streptococcus pneumoniae infection. The pathological processes during the development of empyema associated with murine pneumonia due to S. pneumoniae (strain D39) were investigated. Lungs were examined using histology, and pleural fluid and blood bacterial colony-forming units, cytokine levels, and cellular infiltrate were determined over time. Bacterial migration across mesothelial monolayers was investigated using cell culture techniques, flow cytometry, and confocal microscopy. After intranasal inoculation with 10(7) S. pneumoniae D39 strain, mice developed pneumonia associated with rapid bacterial invasion of the pleural space; raised intrapleural IL-8, VEGF, MCP-1, and TNF-α levels; and caused significant intrapleural neutrophilia followed by the development of fibrinous pleural adhesions. Bacterial clearance from the pleural space was poor, and in vitro assays demonstrated that S. pneumoniae crossed mesothelial layers by translocation through cells rather than by a paracellular route. This study describes key events during the development of S. pneumoniae empyema using a novel murine model of pneumonia-associated empyema that closely mimics human disease. The model allows for future assessment of molecular mechanisms involved in the development of empyema and evaluation of potential new therapies. The data suggest that transmigration of bacteria through mesothelial cells could be important in empyema development. Furthermore, upon entry the pleural cavity offers a protected compartment for the bacteria.