The antigenically variant M protein of Streptococcus pyogenes enhances virulence by promoting resistance to phagocytosis. The serum opacity factor (OF), produced by a subset of M serotypes, is also antigenically variant, and its antigenic variability exactly parallels that of M protein. OF-positive and OF-negative streptococci are also phenotypically distinguishable by a number of other criteria. In order to study the differences between OF-positive and OF-negative streptococci, we cloned and sequenced the type 49 M protein gene (emm49), the first to be cloned from an OF-positive strain. This gene showed evolutionary divergence from the OF-negative M protein genes studied previously. Furthermore, emm49 was part of a gene family, in contrast to the single-copy nature of previously characterized M protein genes.The M protein, a major virulence factor of Streptococcus pyogenes (group A streptococci), enhances the pathogenicity of the organism by blocking phagocytosis in the nonimmunized host (26,27). Antibodies to M protein develop in infected individuals and confer immunity (34). Streptococcal M protein, however, is antigenically variant, and opsonic antibodies to M protein are largely type specific. Thus, with 70 or more M protein serotypes (35), group A streptococci can successfully avoid immune surveillance.Molecular biological techniques have increased our knowledge of the structure of M protein and its antigenic variability. Five M protein genes have been cloned, and all have been at least partially sequenced (16,24,40,42,46). The M proteins encoded by these sequenced genes have a common framework which includes a conserved signal peptide, a hypervariable amino terminus, and a highly conserved C-terminus. Each gene also appears to contain some internal sequence repetitions, although the extent and degree of similarity of these repeats vary among the genes. Furthermore, regions preceding and following these M protein genes are conserved (16,42). This suggests that the genes are situated in a common expression site on the streptococcal chromosome.Our understanding of how the M protein hypervariable regions evolved is still incomplete. Duplication and deletion of intragenic repeats present in the variable regions of M molecules is a possible mechanism for the introduction of variant amino acids into the M protein sequences (25). Recent evidence shows that intragenic recombination can indeed introduce variant opsonic determinants into the repetitive regions of the M6 protein (28). Not all of the opsonic antigenic determinants of M proteins, however, are located in the repetitive regions of the molecules (25, 32). Thus, additional mechanisms must be involved in the generation of M protein antigenic diversity.A model explaining the generation of M protein antigenic diversity must account for the fact that other streptococcal proteins exhibit antigenic diversity in a nonrandom manner relative to the M type. Among these are the T antigen, a * Corresponding author. (18) and by genetic means (8). Clearly, the processe...

SummaryEntry of Streptococcus pyogenes or group A streptococcus (GAS) into host cells is mediated by fibronectin bound to surface proteins, M1 or PrtF1, forming a bridge to α α α α 5 β β β β 1 integrins. This interaction leads to cytoskeletal rearrangement and uptake of streptococci. We postulated that integrin-linked kinase (ILK), which directly associates with integrins, is the universal link between integrins and several bacterial pathogens. We showed that inhibition of ILK expression by siRNA silencing, or ILK kinase activity by chemical inhibitors or expression of a dominant negative form of ILK reduced M1-mediated invasion of epithelial cells up to 80%. To evaluate the ILK requirement for PrtF1-mediated GAS invasion, a M1 -PrtF1 + + + + recombinant strain within the M1 background was constructed. Inhibition of ILK kinase activity also significantly reduced invasion of epithelial cells by this recombinant and wild-type strain JRS4 that expresses PrtF1. In addition, impaired ILK kinase activity results in significant reduction of integrin-dependent invasion mediated by invasins of two other important pathogens, Staphylococcus aureus and Yersinia spp . This study suggests that bacterial pathogens evolved different molecules and strategies to exploit the host integrin signalling pathway for their survival.

The availability of throat culture results, GAS isolates, and serial serum samples collected prospectively over a 2-year period of observation provided a unique opportunity for us to assess the serologic status of pediatric subjects before and after new pharyngeal acquisitions of GAS. With the exception of antibody responses to the homologous M peptides, no clear pattern of immune responses against the remaining GAS antigens was seen. There were no new immunologically significant acquisitions of emm types of GAS against which the subjects had preexisting elevated levels of antibodies against the homologous M peptide. The observation that 65% of new GAS acquisitions caused no symptoms yet were immunologically significant suggests that the majority of infections are not detected, which would result in missed opportunities for primary prevention of rheumatic fever and rheumatic heart disease with appropriate antimicrobial therapy.

Group A streptococci (GAS) are associated with a variety of mucosal and invasive human infections. Recurrent infections by highly heterologous serotypes indicate that cross-serotype immunity is critical for prevention of GAS infections; however, mechanisms underlying serotype-independent protection are poorly understood. Here we report that intranasal vaccination of mice with Sortase A (SrtA), a conserved cell wall bound protein, reduced colonization of nasal-associated lymphoid tissue (NALT) by heterologous serotypes of GAS. Vaccination significantly increased CD4+ IL-17A+ cells in NALT and depletion of IL-17A by neutralizing antibody prevented GAS clearance from NALT which was dependent on immunization with SrtA. Vaccination also induced high levels of SrtA-specific antibodies; however, immunized, B cell-deficient mice cleared streptococcal challenges as efficiently as wild type mice, indicating that the cross-serotype protection is Th17-biased and antibody-independent. Furthermore, efficient GAS clearance from NALT was associated with a rapid neutrophil influx into NALT of immunized mice. These results suggest that serotype independent immune protection against GAS mucosal infection can be achieved by intranasal vaccination with SrtA and enhanced neutrophil function is critical for anti-GAS defense and might be a target for prevention of GAS infections.

Nontypeable Haemophilus influenzae (NTHI) is an important etiological agent of otitis media (OM) and of exacerbated chronic obstructive pulmonary diseases (COPD). Inflammation is a hallmark of both diseases.Interleukin-8 (IL-8), one of the important inflammatory mediators, is induced by NTHI and may play a significant role in the pathogenesis of these diseases. Our studies demonstrated that a soluble cytoplasmic fraction (SCF) from NTHI induced much greater IL-8 expression by human epithelial cells than did NTHI lipooligosaccharides and envelope proteins. The IL-8-inducing activity was associated with molecules of <3 kDa from SCF and was peptidase and lipase sensitive, suggesting that small lipopeptides are responsible for the strong IL-8 induction. Moreover, multiple intracellular signaling pathways were activated in response to cytoplasmic molecules. The results indicated that the p38 mitogen-activated protein kinase (MAPK) and Src-dependent Raf-1-Mek1/2-extracellular signal-regulated kinase mitogen-activated protein kinase (ERK MAPK) pathways are required for NTHI-induced IL-8 production. In contrast, the phosphatidylinositol 3-kinase (PI3K)-Akt pathway did not affect IL-8 expression, although this pathway was concomitantly activated upon exposure to NTHI SCF. The PI3K-Akt pathway was also directly activated by IL-8 and significantly inhibited by an antagonist of IL-8 receptors during NTHI stimulation. These results indicated that the PI3K-Akt pathway is activated in response to IL-8 that is induced by NTHI and may lead to other important epithelial cell responses. This work provides insight into essential molecular and cellular events that may impact on the pathogenesis of OM and COPD and identifies rational targets for anti-inflammatory intervention.Nontypeable Haemophilus influenzae (NTHI) is a gram-negative common commensal of the human respiratory tract. It continues to be an important pathogen of the majority of mucosal diseases including otitis media (OM) (32) in children and lower respiratory infections in adults with chronic obstructive pulmonary diseases (COPD) (19). Both diseases are characterized by inflammatory responses with neutrophil infiltration in the infected tissue (30), which mainly depends on the secretion of chemokines such as interleukin-8 (IL-8). The resulting inflammation response leads to release of neutrophil-generated mediators such as elastase and reactive oxygen species to eradicate the bacteria. However, uncontrolled inflammation due to overproduction of proinflammatory cytokines and chemokines can cause indirect host-mediated tissue damage. Emerging studies revealed that higher-than-normal levels of IL-8 were detected in most middle ear effusions of OM (17, 23) and sputum of COPD (27), suggesting that IL-8 contributes to this pathology. This is further supported by an animal model in which direct injection of IL-8 into the murine middle ear induced leukocyte accumulation at that site (13). Many in vitro and in vivo studies have demonstrated that NTHI significantly induces IL-8...