Group A streptococcal cysteine protease cleaves epithelial junctions and contributes to bacterial translocation.

BACKGROUND: Group A Streptococcus (GAS) translocates across the host epithelial barrier. RESULTS: Streptococcal pyrogenic exotoxin B (SpeB) directly cleaves junctional proteins. CONCLUSION: The proteolytic efficacy of SpeB allows GAS to translocate across the epithelial barrier. SIGNIFICANCE: SpeB-mediated dysfunction of the epithelial barrier may have important implications for not only bacterial invasion but also dissemination of other virulence factors throughout intercellular spaces. Group A Streptococcus (GAS) is an important human pathogen that possesses an ability to translocate across the epithelial barrier. In this study, culture supernatants of tested GAS strains showed proteolytic activity against human occludin and E-cadherin. Utilizing various types of protease inhibitors and amino acid sequence analysis, we identified SpeB (streptococcal pyrogenic exotoxin B) as the proteolytic factor that cleaves E-cadherin in the region neighboring the calcium-binding sites within the extracellular domain. The cleaving activities of culture supernatants from several GAS isolates were correlated with the amount of active SpeB, whereas culture supernatants from an speB mutant showed no such activities. Of note, the wild type strain efficiently translocated across the epithelial monolayer along with cleavage of occludin and E-cadherin, whereas deletion of the speB gene compromised those activities. Moreover, destabilization of the junctional proteins was apparently relieved in cells infected with the speB mutant, as compared with those infected with the wild type. Taken together, our findings indicate that the proteolytic efficacy of SpeB in junctional degradation allows GAS to invade deeper into tissues.

Streptolysin S contributes to group A streptococcal translocation across an epithelial barrier.

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.

Assembly mechanism of FCT region type 1 pili in serotype M6 Streptococcus pyogenes.

The human pathogen Streptococcus pyogenes produces diverse pili depending on the serotype. We investigated the assembly mechanism of FCT type 1 pili in a serotype M6 strain. The pili were found to be assembled from two precursor proteins, the backbone protein T6 and ancillary protein FctX, and anchored to the cell wall in a manner that requires both a housekeeping sortase enzyme (SrtA) and pilus-associated sortase enzyme (SrtB). SrtB is primarily required for efficient formation of the T6 and FctX complex and subsequent polymerization of T6, whereas proper anchoring of the pili to the cell wall is mainly mediated by SrtA. Because motifs essential for polymerization of pilus backbone proteins in other Gram-positive bacteria are not present in T6, we sought to identify the functional residues involved in this process. Our results showed that T6 encompasses the novel VAKS pilin motif conserved in streptococcal T6 homologues and that the lysine residue (Lys-175) within the motif and cell wall sorting signal of T6 are prerequisites for isopeptide linkage of T6 molecules. Because Lys-175 and the cell wall sorting signal of FctX are indispensable for substantial incorporation of FctX into the T6 pilus shaft, FctX is suggested to be located at the pilus tip, which was also implied by immunogold electron microscopy findings. Thus, the elaborate assembly of FCT type 1 pili is potentially organized by sortase-mediated cross-linking between sorting signals and the amino group of Lys-175 positioned in the VAKS motif of T6, thereby displaying T6 and FctX in a temporospatial manner.

Erratum: Group A Streptococcus exploits human plasminogen for bacterial translocation across epithelial barrier via tricellular tight junctions.

This corrects the article DOI: 10.1038/srep20069.

Group A Streptococcus exploits human plasminogen for bacterial translocation across epithelial barrier via tricellular tight junctions.

Group A Streptococcus (GAS) is a human-specific pathogen responsible for local suppurative and life-threatening invasive systemic diseases. Interaction of GAS with human plasminogen (PLG) is a salient characteristic for promoting their systemic dissemination. In the present study, a serotype M28 strain was found predominantly localized in tricellular tight junctions of epithelial cells cultured in the presence of PLG. Several lines of evidence indicated that interaction of PLG with tricellulin, a major component of tricellular tight junctions, is crucial for bacterial localization. A site-directed mutagenesis approach revealed that lysine residues at positions 217 and 252 within the extracellular loop of tricellulin play important roles in PLG-binding activity. Additionally, we demonstrated that PLG functions as a molecular bridge between tricellulin and streptococcal surface enolase (SEN). The wild type strain efficiently translocated across the epithelial monolayer, accompanied by cleavage of transmembrane junctional proteins. In contrast, amino acid substitutions in the PLG-binding motif of SEN markedly compromised those activities. Notably, the interaction of PLG with SEN was dependent on PLG species specificity, which influenced the efficiency of bacterial penetration. Our findings provide insight into the mechanism by which GAS exploits host PLG for acceleration of bacterial invasion into deeper tissues via tricellular tight junctions.