Mutation in csrR global regulator reduces Streptococcus pyogenes internalization.

Transposon (Tn 916) mutagenesis was employed to identify genes in group A streptococcus (GAS) that are involved in bacterial internalization by epithelial cells. One mutant displayed significantly reduced internalization efficiency and was therefore selected for further characterization. The mutant harbored a single Tn 916 insertion in csr, a genetic locus encoding a two-component regulatory system. Mutations in csr were found to derepress hyaluronic acid (HA) capsule synthesis. Since capsule expression has been previously reported to interfere with internalization of GAS, it was possible that the transposon exerted its inhibitory effect either by derepression of capsule synthesis, or by another mechanism. To study the effect of the csr mutation on bacterial internalization, isogenic mutants deficient in either csrR, hasA or both were generated. The hasA mutant adhered to and internalized into HEp-2 cells significantly better than the parent and the csrR mutant strains. The internalization efficiency of the double mutant (csrR(-)/hasA(-)) was reduced by seven-fold compared to that of the hasA mutant. These findings suggest that csrR affects streptococcal entry by modulating capsule expression as well as by another, yet unknown, mechanism.

Protein F1 is required for efficient entry of Streptococcus pyogenes into epithelial cells.

It was recently reported that strains of Streptococcus pyogenes are capable of inducing entry of the bacterium into epithelial cells; however, nothing is known regarding the gene(s) and the underlying mechanism(s) involved. Using isogenic mutants of S. pyogenes JRS4 strain that are defective in the expression of each of the surface proteins F1 and M6, it was demonstrated that both are required for efficient internalization. Expression of F1 on the surface of a poorly invading S. pyogenes strain significantly enhances its internalization efficiency. Protein F1-mediated internalization is inhibited by UR, the nonrepetitive fibronectin-binding domain of this protein, and to a lesser extent, by the repetitive fibronectin-binding domain, RD2. Polyclonal anti-human fibronectin antibodies completely abolish F1-mediated internalization; increasing fibronectin concentrations result in a significant enhancement of bacterial uptake. The findings shown here suggest that protein F1 mediates streptococcal internalization and that the M6 protein is required for more efficient entry of the bacterium.

Insertional inactivation of streptolysin S expression is associated with altered riboflavin metabolism in Streptococcus pyogenes.

Transposon Tn916 mutagenesis was used to create a mutant of Streptococcus pyogenes M type 3, designated ISS417, in which the ability to produce streptolysin S (SLS) and several other exoproteins was impaired. Concomitantly, the mutant became dependent upon riboflavin for growth and was able to grow in Todd Hewitt broth (THB) when supplemented with riboflavin or riboflavinrich yeast extract. The parent strain was apparently able to utilize THB-derived components as a substitute for riboflavin, while the mutant was not. Although the parent strain grew well in synthetic medium, it was unable to produce SLS, except when it was supplemented with a small amount of THB. Thus, a component of THB was able to "trigger" SLS formation in the parent strain. The mutant grew well in this medium, but was unable to produce SLS even when it was supplemented with THB. Southern hybridization analysis revealed that the ISS417 mutant harbours a single transposon insertion in its chromosome. Phage transduction experiments showed that the riboflavin dependency and the inability to make SLS phenotypes are co-transducible. The pleotrophic properties of the ISS417 mutant differ from those reported for insertional inactivation of the mga locus which regulates production of a number of surface proteins in S. pyogenes and the sar locus which regulates production of a number of exoproteins in Staphylococcus aureus. In view of the possibility that there exist a genetic linkage between the riboflavin biosynthetic pathway and expression of the oxygen-stable SLS, we hypothesize that SLS has a role in the growth economy of S. pyogenes.

Proteins M6 and F1 are required for efficient invasion of group A streptococci into cultured epithelial cells.

Group A streptococci were recently shown to be capable of invading human epithelial cell monolayers. Cell invasion might be an important virulence trait of streptococci that enable the pathogen to gain entry into deeper tissues after initial binding to host cells. Nothing is known concerning the nature of streptococcal components that mediate invasion. Using isogenic mutants of strain JRS4 that are defective in the expression of either M6 protein or protein F1, or both proteins, it was demonstrated that both adhesins are required for efficient invasion. Further more, expression of protein F1 on the surface of a non-invasive strain rendered the latter invasive, suggesting that protein F1 is directly involved in the invasion process.