Aerosol transmission of Streptococcus pyogenes remains largely unstudied. S. pyogenes can be detected on settle plates during outbreaks, providing indirect evidence of aerosol transmission. We recently showed that S. pyogenes exhibits enhanced entry into an artificially-generated aerosol, when compared with seven other pathogenic bacterial species. Bioaerosol entry frequency was strongly correlated with high cell surface hydrophobicity. However, the genetic factors underpinning aerosolisation and hydrophobicity remain unknown.
To identify genes involved in aerosolisation and hydrophobicity, we constructed a transposon library with approximately 67,000 unique insertions in the M1UK lineage of S. pyogenes. The mutant pool was aerosolised, and mutants recovered from settle plates were compared to the pre-aerosolised pool using next-generation sequencing. Additionally, the transposon library was iteratively enriched for mutants with reduced hydrophobicity using a microbial adherence to hydrocarbon (MATH) assay.
Analysis of the pre-aerosolised transposon library predicted 502 of 1926 genes to be essential, similar to previous studies in other M1 lineages. Comparative analysis of aerosolised mutants identified insertions in 133 genes that reduced aerosolisation. Among these, disruption of srtA (sortase A, MGAS5005_Spy0875) was also associated with significantly reduced hydrophobicity, as determined by the MATH enrichment assay. Validation of these findings using an isogenic srtA mutant confirmed a ~50% reduction in hydrophobicity and a 7-fold reduction in aerosolisation compared to the wild type. These results establish srtA as a key factor linking hydrophobicity to aerosolisation and suggest a role for cell-wall anchored proteins. This study provides the first insights into genetic factors that contribute to aerosolisation of S. pyogenes.