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The FCT regions of Streptococcus pyogenes strains encode a variety of cell wall-anchored surface proteins that bind the extracellular matrix of the human host and/or give rise to pilus-like appendages. Strong linkage is evident between transcription-regulatory loci positioned within the FCT and emm regions and the emm pattern genotype marker for preferred infection of the throat or skin. These findings provide a basis for the hypothesis that FCT region gene products contribute to tissue-specific infection. In an initial series of steps to address this possibility, the FCT regions of 13 strains underwent comparative sequence analysis, the gene content of the FCT region was characterized for 113 strains via PCR, and genetic linkage was assessed. A history of extensive recombination within FCT regions was evident. The emm pattern D-defined skin specialist strains were highly homogenous in their FCT region gene contents, whereas the emm pattern A-C-defined throat specialist strains displayed a greater variety of forms. Most pattern A-C strains harbored prtF1 (75%) but lacked cpa (75%). In contrast, the majority of emm pattern D strains had cpa (92%) but lacked prtF1 (79%). Models based on FCT and emm region genotypes revealed the most parsimonious pathways of evolution. Using niche-determining candidate genes to infer phylogeny, emm pattern E strains-the so-called generalists, which lack a strong tissue site preference-occupied a transition zone separating most throat specialists from skin specialists. Overall, population genetic analysis supports the possibility that the FCT region gives rise to surface proteins that are largely necessary, but not always sufficient, to confer tissue site preference for infection.

FCT region genes of Streptococcus pyogenes encode surface proteins that include fibronectin-and collagenbinding proteins and the serological markers known as T antigens, some of which give rise to pilus-like appendages. It remains to be established whether FCT region surface proteins contribute to virulence by in vivo models of infection. In this study, a highly sensitive and ecologically relevant humanized mouse model was used to measure superficial skin infection. Three genes encoding FCT region surface proteins essential for Tserotype specificity were inactivated. Both the ⌬cpa and ⌬prtF2 mutants were highly attenuated for virulence when topically applied to the skin following exponential growth but were fully virulent when delivered in stationary phase. In contrast, the ⌬fctA mutant was virulent at the skin, regardless of its initial growth state. Immunoblots of cell extracts revealed anti-FctA-reactive, ladder-like polymers characteristic of streptococcal pili. In addition, FctA formed a heteropolymer with the putative collagen-binding protein Cpa. The ⌬fctA mutant showed a loss in anti-Cpa-reactive polymers, whereas anti-FctA-reactive polymers were reduced in the ⌬cpa mutant. The findings suggest that both FctA and Cpa are required for pilus formation, but importantly, an intact pilus is not essential for Cpa-mediated virulence. Although it is an integral part of the T-antigen complex, the fibronectin-binding protein PrtF2 is not covalently linked to the FctA-and Cpa-containing heteropolymer derived from cell extracts. The data provide direct evidence that streptococcal T antigens function as virulence factors in vivo, but they also reveal that a pilus-like structure is not essential for the most common form of streptococcal skin disease.Pili can function to mediate bacterial adherence to host surfaces and facilitate horizontal gene transfer (14,31,43). Although well studied among many gram-negative organisms, pilus-like surface structures are becoming increasingly recognized as a component of gram-positive bacteria (26,42,45). Included among pilus-bearing bacteria are at least three species of Streptococcus that are important human pathogens (3,23,24,28).Genes encoding pilus-associated proteins map to the FCT region of Streptococcus pyogenes (28), a highly diversified portion of the genome that contains several well-studied genes (cpa, prtF1, and prtF2) encoding microbial surface cell recognition adhesion matrix molecules (MSCRAMMs) (5,12,21,32). The gene encoding the T6 antigen, which is an antigenic target of a major serological typing scheme, maps to the FCT regions of some isolates, where it forms part of the pilus structure (5, 28, 34). The T6 protein and other FCT region gene products are anchored to the peptidoglycan cell wall via specialized sortases, whose genes also lie within the FCT region (1, 2). Some isolates of S. pneumoniae have a genomic region, called a pathogenicity islet, that is similar in its general layout to the FCT region of S. pyogenes (16,40); at least two of these pneumococcal ...

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Integrin adhesion receptors can signal in two directions: first, they can regulate cellular behaviors by modulating cellular signaling enzymes ("outside-in signaling"); second, cells can regulate the affinity of integrins ("inside-out signaling") by such pathways. Integrin ␤ cytoplasmic domains (tails) mediate both types of signaling, and Src family kinases (SFKs) and talin, which bind to ␤ tails, are important for integrin signaling. Here, we utilized "homology scanning" mutagenesis to identify ␤ tail mutants selectively defective in c-Src binding and found that amino acid exchanges affecting a combination of an Arg and Thr residue in the integrin ␤3 tail control the binding specificity for SFKs but have no effect on talin binding. Using ␤ tail mutants at these residues, we found that SFK binding to integrin ␤ tails is dispensable for inside-out signaling but is obligatory for cell spreading, a marker of outside-in signaling. Conversely, we found that point mutations that disrupt talin binding abolish integrin activation, but they do not inhibit SFK binding to the ␤3 tail or the initiation of outside-in signaling once the integrins are in a high affinity form. Thus, we show that inside-out and outside-in integrin signaling are mediated by distinct and separable interactions of the integrin ␤ tails. Furthermore, based on our results, it is possible to discern the relative contributions of the direction of integrin signaling on biological functions in cell culture and, ultimately, in vivo.

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Transcription of several key virulence factors of Streptococcus pyogenes is under the control of Mga and Nra/RofA. In an M serotype 49 (M49) strain, Nra is a negative regulator of pilus gene transcription; also, Nra represses mga expression, leading to downregulation of the M protein surface fibril and secreted cysteine protease SpeB. In this report, the role of Nra in the virulence of an M53 classical skin strain was investigated. In contrast to the case for the M49 strain, Nra functions as a positive regulator of pilus gene transcription in the M53 strain, and inactivation of nra leads to loss of virulence in a humanized mouse model of superficial skin infection. Furthermore, Nra has no measurable effect on mga transcription in the M53 strain; this finding is further supported by a lack of detectable Nra effects on M protein-and SpeB-dependent phenotypes. Whereas MsmR is reported to activate nra and pilus gene transcription in the M49 strain, in the M53 strain it acts as a repressor of these genes. In both strains, MsmR and Nra form a feed-forward loop network motif for pilus gene transcription, but their effects have opposite signs. The findings demonstrate key strain-specific differences in the transcriptional circuitry governing virulence gene expression in S. pyogenes and its impact on pathogenesis.Pilus surface structures were recently identified as potentially important virulence factors for several species of pathogenic streptococci and closely related organisms, such as en-