Streptolysin S Contributes to Group A Streptococcal Translocation across an Epithelial Barrier

Abstract

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.

Group A Streptococcal Cysteine Protease Cleaves Epithelial Junctions and Contributes to Bacterial Translocation

“…At the end of the infection period, the infected cells were lysed with Laemmli gel loading buffer containing 6% 2-mercaptoethanol. Cleavage of intercellular junctions was detected by Western blot analysis (11). Western blot signals were quantified using Scion Image 4.0.3.2 software (Scion).…”

“…An in-frame speB deletion mutant, its complemented strain, and sagA-speB double mutant were constructed using pSET4s, as previously reported (11,17). Primers speBkoF1 (5Ј-GCGGATCCTGTTTAATCGAAATGTTTT-TTGAATGC-3Ј), speBkoR1 (5Ј-ACTTTGGTAACCGTTGA-AGCCCATTTTTTTTATACCTCTTTC-3Ј), speBkoF2 (5Ј-GAAAGAGGTATAAAAAAAATGGGCTTCAACGGTTAC-CAAAGT-3Ј), and speBkoR2 (5Ј-AACTGCAGGTCTTAAA-GGATGTACCGTATTGG-3Ј) were used for deletion of speB gene.…”

“…Polarized monolayers were infected with GAS at an multiplicity of infection (MOI) of 10. The ability of GAS strains to translocate into monolayers was assessed by quantitative cultures of media obtained from the lower chambers at various times after infection as described previously (11).…”

“…For translocation assays, Caco-2 cells were seeded at 2 ϫ 10 5 cells/well onto polycarbonate Millicell culture plate inserts (12-mm diameter, 3-m pore size; Millipore) and cultured for 5 days at 37°C under a 5% CO 2 atmosphere, as described previously (11). Transepithelial electrical resistance (TER) of the filter-grown monolayers was measured using a Millicell-ERS device (Millipore), and monolayers exhibiting TER values of 450 -500 ⍀⅐cm 2 were used in the experiments.…”

“…Meanwhile, some studies that investigated the direct interactions of bacteria with epithelial junctions also elucidated the underlying mechanisms of GAS pathogenesis, with interaction of the hyaluronic acid capsule with CD44 implicated in this process (10). Furthermore, our recent study identified streptolysin S (SLS) as a novel factor that facilitates GAS translocation via degradation of intercellular junctions in concert with the host cysteine protease calpain (11). However, the precise mechanism by which GAS disrupts the epithelial barrier has yet to be completely elucidated.…”