Molecular and Functional Analysis of the Type IV Pilus Gene Cluster in Streptococcus sanguinis SK36

Abstract

Streptococcus sanguinis, dominant in the oral microbiome, is the only known streptococcal species possessing a pil gene cluster for the biosynthesis of type IV pili (Tfp). Although this cluster is commonly present in the genome of S. sanguinis, most of the strains do not express Tfp-mediated twitching motility. Thus, this study was designed to investigate the biological functions encoded by the cluster in the twitching-negative strain S. sanguinis SK36. We found that the cluster was transcribed as an operon, with three promoters located 5= to the cluster and one in the intergenic region between SSA_2307 and SSA_2305. Studies using promoter-cat fusion strains revealed that the transcription of the cluster was mainly driven by the distal 5= promoter, which is located more than 800 bases 5= to the first gene of the cluster, SSA_2318. Optimal expression of the cluster occurred at the early stationary growth phase in a CcpA-dependent manner, although a CcpA-binding consensus is absent in the promoter region. Expression of the cluster resulted in a short hairlike surface structure under transmission electron microscopy. Deletion of the putative pilin genes (SSA_2313 to SSA_2315) abolished the biosynthesis of this structure and significantly reduced the adherence of SK36 to HeLa and SCC-4 cells. Mutations in the pil genes downregulated biofilm formation by S. sanguinis SK36. Taken together, the results demonstrate that Tfp of SK36 are important for host cell adherence, but not for motility, and that expression of the pil cluster is subject to complex regulation. IMPORTANCE The proteins and assembly machinery of the type IV pili (Tfp) are conserved throughout bacteria and archaea, and yet the function of this surface structure differs from species to species and even from strain to strain. As seen in Streptococcus sanguinis SK36, the expression of the Tfp gene cluster results in a hairlike surface structure that is much shorter than the typical Tfp. This pilus is essential for the adherence of SK36 but is not involved in motility. Being a member of the highly diverse dental biofilm, perhaps S. sanguinis could more effectively utilize this structure to adhere to host cells and to interact with other microbes within the same niche.

Manganese Depletion Leads to Multisystem Changes in the Transcriptome of the Opportunistic Pathogen Streptococcus sanguinis

“…Many of these genes were differentially expressed ( Supplementary Figure S6), which may be due to the intertwined nature of amino acid and carbohydrate metabolism (Radin et al, 2016). We also observed decreased expression of large, contiguous loci encoding ethanolamine utilization (Fox et al, 2009;Kaval and Garsin, 2018;Kaval et al, 2019), a type IV pilus system (Chen et al, 2019), and CRISPR-associated proteins (Makarova et al, 2015;Gong et al, 2020; Supplementary Figure S7), which are all also tied to CcpA-regulation (Bai et al, 2019).…”

Manganese depletion leads to multisystem changes in the transcriptome of the opportunistic pathogenStreptococcus sanguinis

“…S6). S. sanguinis is unusual in that it is the only streptococcal species to encode a gene cluster for the biosynthesis of a type IV pilus system (T4P) (Gurung et al, 2016) that is distinct from the T4P competence pilus (Sheppard et al, 2020), and yet few strains exhibit the twitching motility normally mediated by T4P (Chen et al, 2019). In SK36, T4P appears to be important for adherence to host cells and may be regulated by CcpA, despite the lack of a cre site in the promoter region (Bai et al, 2019;Chen et al, 2019).…”

“…S. sanguinis is unusual in that it is the only streptococcal species to encode a gene cluster for the biosynthesis of a type IV pilus system (T4P) (Gurung et al, 2016) that is distinct from the T4P competence pilus (Sheppard et al, 2020), and yet few strains exhibit the twitching motility normally mediated by T4P (Chen et al, 2019). In SK36, T4P appears to be important for adherence to host cells and may be regulated by CcpA, despite the lack of a cre site in the promoter region (Bai et al, 2019;Chen et al, 2019). In S. pneumoniae, the SsaR ortholog, PsaR, negatively regulates the expression of type I pilus genes in the presence of Mn (Johnston et al, 2006) whereas a Δ mntE mutant containing excess intracellular Mn showed increased expression of the same genes (Rosch et al, 2009).…”

Caldicellulosiruptor besciiregulates pilus expression in response to the polysaccharide, xylan

“…Initial bioinformatics analyses identified five putative pilin genes located in the C. bescii T4P locus ( Table 1). Pathogenic Firmicutes, such as Streptococcus sanguinis (47,48) and C. perfringens (28), also encode for multiple pilins in their respective T4P loci, and a hypervirulent C. difficile strain encodes for as many as nine pilins distributed across five genomic loci (30). Additionally, based on amino acid 295 sequence homology, concatenated C. bescii pilin sequences clusters with other T4P loci from the majority of strongly cellulolytic Caldicellulosiruptor species (Fig.…”