Auto-aggregation in Streptococcus intermedius is driven by the Pel polysaccharide

The Streptococcus milleri group (SMG), comprising Streptococcus intermedius, anginosus, and constellatus, can asymptomatically colonize various mucosal sites of healthy individuals. These bacteria are opportunistic pathogens that cause different types of infections across various anatomical sites. Although the pathogenic mechanisms leading to infections are not well defined in the SMG, auto-aggregation is a key driver of biofilm adhesion and cohesion in many Streptococci and Staphylococci. Here, we identify a S. intermedius gene cluster with significant homology to the Bacillus cereus and Pseudomonas aeruginosa pel operons, which are required for Pel exopolysaccharide production and biofilm formation in these species. This cluster contains five genes that are homologous to pelDEA_DAFG in other gram-positive species and four additional genes of unknown function. Characterization of a panel of clinical S. intermedius isolates identified a range of adherent biofilm and aggregation phenotypes , and aggregation in strain C1365 was dependent on each of the pelDEA_DAFG genes. Deletion of two of the additional genes, SIR_1592 and SIR_1594, reduced but did not abolish the aggregation phenotype. Furthermore, we demonstrate that SIR_1591 is a glycoside hydrolase and that C1365 produces a GalNAc-rich polymer as aggregates were disrupted by theα-1,4-N-acetylgalactosaminidases PelA and Sph3, but not the α-1,4-N-galactosaminidase Ega3. Using an abscess model of mouse infection, we show that loss of Pel production in a C1365 ΔpelF mutant allows for more effective bacterial clearance. The polymer also affects how S. intermedius interacts with the host immune system. Collectively, our data suggest that Pel biosynthesis contributes to S. intermedius pathogenicity.IMPORTANCESMG species are increasingly being recognized as pathogens. Despite their clinical relevance, little is known about how SMG members transition between asymptomatic colonization and infection. Herein, we show that clinical isolates of S. intermedius can be classified into four groups based on their aggregation and adherent biofilm phenotypes. We demonstrate that aggregation is dependent on the Pel polysaccharide and that Pel production allows bacteria not only to persist longer during infection but also modulates the immune responses of the host. Pel production requires the canonical pelDEA_DAFG genes. We also identified four additional genes in the S. intermedius pel cluster and found that under the conditions tested, two of these genes play a role in aggregation and Pel production. Functional homologs of the additional genes play major roles in host-pathogen interactions and stress responses in other bacteria, suggesting that these additional genes could play a role in Pel-related infections.