Impact of M protein loss and release of extracellular M protein on streptococcal disease

Abstract

Background: The streptococcal cell surface M protein is arguably the most studied virulence factor of S. pyogenes and is postulated to be essential for S. pyogenes survival in the human host through mechanisms which culminate in evasion of opsonophagocytic killing. Despite the central role ascribed to M protein in combatting bacterial clearance, we identified selection and emergence of M protein defective S. pyogenes which was related to a premature stop codon and secretion during experimental infection. We therefore set out to determine the impact of M protein loss and the release of extracellular M protein on streptococcal disease.

Methods: Isogenic strains with mutations in emm1 were constructed using standard techniques. Adhesion to host factors was measured using solid phase adherence assays, and bacterial survival and uptake were assessed in human whole blood and neutrophil phagocytosis assays. Bacterial survival and dissemination was investigated using in vivo models of intramuscular and intranasal infections, including coinfections to determine competitive advantage of strains. The prevalence of M protein mutations in whole genome sequences was analysed and the effect of vaccination using M1 on bacterial clearance was assessed.

Results: M1 protein mutants were unable to bind fibrinogen, fibronectin and collagen, and were vulnerable to immune killing and neutrophil uptake in comparison to the WT parent strain in vitro. Surprisingly, and in contrast to in vitro clearance studies, M protein mutations did not affect bacterial survival 3 h after intramuscular infection of mice. The strain shedding truncated M protein was recovered in greater numbers than the parent strain expressing full length anchored M protein from certain niches 24 hours post infection, such as lymph nodes. This advantage was confirmed using coinfection experiments. In contrast, M protein mutants were unable to establish intranasal infection in mice. While no other truncating mutations were found in M protein when analysing whole genome sequences, it was shown that all M1 S. pyogenes tested secrete soluble M protein during normal culture. Conclusion: M protein is not required for survival of S. pyogenes during invasive infection, and some M protein mutations seem to provide an advantage in the lymphatic niche. Expression of soluble M protein is an important characteristic of all S. pyogenes strains and may affect immune evasion and pathogenesis.