SummarySilkworms are killed by injection of pathogenic bacteria, such as Staphylococcus aureus and Streptococcus pyogenes , into the haemolymph. Gene disruption mutants of S. aureus whose open reading frames were previously uncharacterized and that are conserved among bacteria were examined for their virulence in silkworms. Of these 100 genes, three genes named cvfA , cvfB , and cvfC were required for full virulence of S. aureus in silkworms. Haemolysin production was decreased in these mutants. The cvfA and cvfC mutants also had attenuated virulence in mice. S. pyogenes cvfA -disrupted mutants produced less exotoxin and had attenuated virulence in both silkworms and mice. These results indicate that the silkworm-infection model is useful for identifying bacterial virulence genes.

Streptococcus pyogenes is an important pathogen that causes pharyngitis, sepsis, and rheumatic fever. Cell-associated streptococcal C5a peptidase (ScpA) protects S. pyogenes from phagocytosis and has been suggested to interrupt host defenses by enzymatically cleaving complement C5a, a major factor in the accumulation of neutrophils at sites of infection. How S. pyogenes recognizes and binds to C5a, however, is unclear. We detected a C5a-binding protein in 8 M urea extracts of S. pyogenes by ligand blotting using biotinylated C5a. Searching of genome databases showed that the C5a-binding protein is identical to the streptococcal plasmin receptor (Plr), also known as streptococcal surface dehydrogenase (SDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In the present study we identified a novel function of this multifunctional protein. Western blotting and immunofluorescence microscopy with anti-Plr/SDH/GAPDH showed that Plr/ SDH/GAPDH is located on the bacterial surface and released into the culture supernatant. Next, we examined whether the streptococcal Plr/ SDH/GAPDH inhibits the biological effects of C5a on human neutrophils. We found that soluble Plr/SDH/GAPDH inhibits C5a-activated chemotaxis and H 2 O 2 production. Furthermore, our results suggested that soluble Plr/SDH/GAPDH captures C5a, inhibiting its chemotactic function. Also, cell-associated Plr/SDH/GAPDH and ScpA were both necessary for the cleavage of C5a on the bacterial surface. Together, these results indicate that the multifunctional protein Plr/SDH/ GAPDH has additional functions that help S. pyogenes escape detection by the host immune system. Group A Streptococcus pyogenes is a human pathogen that causes streptococcal pharyngitis as well as more severe invasive infections, including necrotizing fasciitis, sepsis, and streptococcal toxic shock syndrome. The initial step of S. pyogenes infection is bacterial adhesion to host epithelial cells through extracellular matrix proteins (e.g. fibronectin) (1-3). Fibronectin-binding proteins of S. pyogenes have been identified as adhesins/invasins, and how S. pyogenes invade epithelial cells has been examined in several studies (1, 3, 4 -8).To cause systemic and septic infections, it is apparent that S. pyogenes must escape from detection by the host immune system and survive in plasma, after which they invade and spread into various organs via the blood stream. Recently, it was reported that infiltrating neutrophils were not observed at sites of S. pyogenes infection (9). The complement system plays an important role in the innate immune system, which acts as a protective shield in the early phases of infection as well as an effector in the acquired immune system. During activation of the complement system, complement fragments C3a, C4a, and C5a are generated, whereupon they act as anaphylatoxins. C5a is the most important anaphylatoxin because it acts as a chemoattractant for neutrophils and macrophages and because it integrates activation of the classical, alternative, and lectin binding pa...

The human pathogen Streptococcus pyogenes (group A streptococcus [GAS]) pilus components, suggested to play a role in pathogenesis, are encoded in the variable FCT (fibronectin-and collagen-binding T-antigen) region. We investigated the functions of sortase A (SrtA), sortase C2 (SrtC2), and the FctA protein of the most prevalent type 3 FCT region from a serotype M49 strain. Although it is considered a housekeeping sortase, SrtA's activity is involved in pilus formation in addition to its essentiality for GAS extracellular matrix protein binding, host cell adherence/internalization, survival in human blood, and biofilm formation. SrtC2 activity is crucial for pilus formation but dispensable for the other phenotypes tested in vitro. FctA is the major pilus backbone protein, simultaneously acting as the M49 T antigen, and requires SrtC2 and LepA, a signal peptidase I homologue, for monomeric surface expression and polymerization, respectively. Collagen-binding protein Cpa expression supports pilus formation at the pilus base. Immunofluorescence microscopy and fluorescence-activated cell sorting analysis revealed several unexpected expression patterns, as follows: (i) the monomeric pilus protein FctA was found exclusively at the old poles of GAS cells, (ii) FctA protein expression increased with lower temperatures, and (iii) FctA protein expression was restricted to 20 to 50% of a given GAS M49 population, suggesting regulation by a bistability mode. Notably, disruption of pilus assembly by sortase deletion rendered GAS serotype M49 significantly more aggressive in a dermonecrotic mouse infection model, indicating that sortase activity and, consequently, pilus expression allow a subpopulation of this GAS serotype to be less aggressive. Thus, pilus expression may not be a virulence attribute of GAS per se.Streptococcus pyogenes (group A streptococcus [GAS]) is a bacterial pathogen that is perfectly adapted to colonization, infection, and persistence in its human host (8,10,14,22). Many of the associated virulence factors expressed by this bacterium are encoded in discrete regions of the GAS genome (28). Two of these pathogenicity regions (Mga and FCT [see below]) each comprise genes for secreted and surface-exposed virulence factors and at least one stand-alone transcriptional regulator. These genes have been shown to act together in a growth phase-dependent regulatory network to coordinate GAS host cell adherence, internalization, and intracellular persistence (summarized in references 28 and 29).The FCT region (fibronectin-and collagen-binding T-antigen region) (6) is present in all GAS genomes specifically tested (26). The FCT region always contains a RALP transcriptional regulator whose type correlates with the type of emm pathogenicity region and the preferred infection site of the GAS strain, i.e., the throat or the skin. Since a similar association was also shown for the fibronectin-and collagenbinding proteins encoded by this region, the FCT region gene products could contribute to tissue-specific GAS infecti...

A relative lack of neutrophils around Streptococcus pyogenes is observed in streptococcal toxic shock syndrome (STSS). Because the bacteria spread rapidly into various organs in STSS, we speculated that S. pyogenes is equipped with molecules to evade the host innate immune system. Complement C3b opsonizes the pathogen to facilitate phagocytosis, and a complex of C3b converts C5 into anaphylatoxin. Because we found that C3 (C3b) is degraded in sera from patients with STSS, we investigated the mechanism of C3 (C3b) degradation by S. pyogenes. We incubated human C3b or serum with recombinant SpeB (rSpeB), a wild-type S. pyogenes strain isolated from an STSS patient or its isogenic ⌬speB mutant and examined the supernatant by Western blotting with anti-human C3b. Western blot and Biacore analyses revealed that rSpeB and wild-type S. pyogenes rapidly degrade C3b. Additionally, C3 (C3b) was not detected in sera collected from infected areas of STSS patients. Furthermore, the survival rate in human blood and in mice was lower for the ⌬speB mutant than the wild-type strain. Histopathological observations demonstrated that neutrophils were recruited to and phagocytosed the ⌬speB mutant, whereas with the wild-type strain, few neutrophils migrated to the site of infection, and the bacteria spread along the fascia. We observed the degradation of C3 (C3b) in sera from STSS patients and the degradation of C3 (C3b) by rSpeB. This suggests that SpeB contributes to the escape of S. pyogenes from phagocytosis at the site of initial infection, allowing it to invade host tissues during severe infections.Streptococcus pyogenes is a Gram-positive bacterium that often causes throat and skin infections such as pharyngitis and impetigo. During the past decade, it was reported that S. pyogenes causes severe infectious diseases, including acute rheumatic fever, necrotizing fasciitis, and streptococcal toxic shock syndrome (STSS) 2 (1, 2). The death toll from severe infections was estimated to be at least 500,000 each year (2). Therefore, many researchers have focused on S. pyogenes, and some aspects of the infection have been elucidated.The initial step of S. pyogenes infection is bacterial adhesion to host epithelial cells through extracellular matrix proteins, for example, fibronectin (3-5). Fibronectin-binding proteins of S. pyogenes have been identified as adhesins and invasins, and their role in the invasion of epithelial cells by S. pyogenes has been examined in several studies (6, 7); however, how the invading bacteria escape the immune system and grow in host tissues is not understood. To cause systemic and septic infections, S. pyogenes must evade the immune system of the host and survive in plasma, after which the organisms may spread into various organs via the blood stream. Histopathological studies have demonstrated that there are few or no inflammatory cells (e.g. neutrophils) at the site of infection in patients with STSS and severe streptococcal infections (8, 9).The complement system plays an important role in innate immu...

Group A Streptococcus pyogenes has surface-located fibronectin (Fn)-binding proteins known to be a major virulence factor, which adheres to and invades host cells. We present a novel Fn-binding protein of group A streptococcus serotype M3 and M18 strains isolated from patients with toxic shock-like syndrome (TSLS). By searching the whole genome sequence of an M3 strain from a TSLS patient, an open reading frame was found among the putative surface proteins. It possessed an LPXTG motif and Fn-binding repeat domains in the Cterminal region and was designated as FbaB (Fn-binding protein of group A streptococci type B). The fbaB gene was found in all M3 and M18 strains examined, although not in other M serotypes. Furthermore, FbaB protein was expressed on the cell surface of TSLS strains but not on non-TSLS ones. Enzyme-linked immunosorbent assay and ligand blotting revealed that recombinant FbaB exhibits a strong Fn-binding ability. An FbaB-deficient mutant strain showed 6-fold lower adhesion and invasion efficiencies to HEp-2 cells than the wild type. Moreover, mortality was decreased in mice infected with the mutant strain in comparison to the wild type. These data suggest that FbaB is etiologically involved in the development of invasive streptococcal diseases.

Streptococcus pneumoniae is a major causative agent of mortality throughout the world. The initial event in invasive pneumococcal disease is the attachment of pneumococci to epithelial cells in the upper respiratory tract. Several bacterial proteins can bind to host extracellular matrix proteins and function as adhesins and invasins. To identify adhesins or invasins on the pneumococcal cell surface, we searched for several proteins with an LPXTG anchoring motif in the whole-genome sequence of Streptococcus pneumoniae and identified one, which we called PfbA (plasmin-and fibronectin-binding protein A), that bound to human serum proteins. Immunofluorescence microscopy and fluorescence-activated cell sorter analysis revealed that PfbA was expressed on the pneumococcal cell surface. A ⌬pfbA mutant strain was only half as competent as the wild-type strain at adhering to and invading lung and laryngeal epithelial cells. In addition, epithelial cells infected with ⌬pfbA showed morphological changes, including cell flattening and a loss of microvilli, that did not occur in cells infected with the wild-type strain. The mutant strain also exhibited a weaker antiphagocytotic activity than wild type in human peripheral blood. Moreover, the growth of wild-type bacteria in human whole blood containing anti-PfbA antibodies was reduced by ϳ50% after 3 h compared with its growth without the antibody. These results suggest that PfbA is an important factor in the development of pneumococcal infections.

In infection by Streptococcus pyogenes, fibronectin (Fn)‐binding proteins play important roles as adhesins and invasins. Here, we present a novel Fn‐binding protein of S. pyogenes that exhibits a low similarity to other Fn‐binding proteins reported. After searching the Oklahoma Streptococcal Genome Sequencing Database for open reading frames (ORFs) with an LPXTG motif, nine ORFs were found among those recognized as putative surface proteins, and one of them was designated as Fba. The fba gene was found in M types 1, 2, 4, 22, 28 and 49 of S. pyogenes, but not in other serotypes or groups of streptococci. Fba, a 37.8 kDa protein, possesses three or four proline‐rich repeat domains and exhibits a high homology to FnBPA, the Fn‐binding protein of Staphylococcus aureus. Recombinant Fba exhibited a strong binding ability to Fn. In addition, Fba‐deficient mutants showed diminished invasive capabilities to HEp‐2 cells and low mortality in mice following skin infection. The fba gene was located downstream of the mga regulon and analysis using an mga‐inactivated mutant revealed that it was transcribed under the control of the Mga regulator. These results indicate that Fba is a novel protein and one of the important virulence factors of S. pyogenes.

Group A Streptococcus pyogenes (GAS) is a human pathogen that causes local suppurative infections and severe invasive diseases. Systemic dissemination of GAS is initiated by bacterial penetration of the epithelial barrier of the pharynx or damaged skin. To gain insight into the mechanism by which GAS penetrates the epithelial barrier, we sought to identify both bacterial and host factors involved in the process. Screening of a transposon mutant library of a clinical GAS isolate recovered from an invasive episode allowed identification of streptolysin S (SLS) as a novel factor that facilitates the translocation of GAS. Of note, the wild type strain efficiently translocated across the epithelial monolayer, accompanied by a decrease in transepithelial electrical resistance and cleavage of transmembrane junctional proteins, including occludin and E-cadherin. Loss of integrity of intercellular junctions was inhibited after infection with a deletion mutant of the sagA gene encoding SLS, as compared with those infected with the wild type strain. Interestingly, following GAS infection, calpain was recruited to the plasma membrane along with E-cadherin. Moreover, bacterial translocation and destabilization of the junctions were partially inhibited by a pharmacological calpain inhibitor or genetic interference with calpain. Our data indicate a potential function of SLS that facilitates GAS invasion into deeper tissues via degradation of epithelial intercellular junctions in concert with the host cysteine protease calpain.