Gene Acquisition by a Distinct Phyletic Group within Streptococcus pneumoniae Promotes Adhesion to the Ocular Epithelium.

Streptococcus pneumoniae (pneumococcus) displays broad tissue tropism and infects multiple body sites in the human host. However, infections of the conjunctiva are limited to strains within a distinct phyletic group with multilocus sequence types ST448, ST344, ST1186, ST1270, and ST2315. In this study, we sequenced the genomes of six pneumococcal strains isolated from eye infections. The conjunctivitis isolates are grouped in a distinct phyletic group together with a subset of nasopharyngeal isolates. The keratitis (infection of the cornea) and endophthalmitis (infection of the vitreous body) isolates are grouped with the remainder of pneumococcal strains. Phenotypic characterization is consistent with morphological differences associated with the distinct phyletic group. Specifically, isolates from the distinct phyletic group form aggregates in planktonic cultures and chain-like structures in biofilms grown on abiotic surfaces. To begin to investigate the association between genotype and epidemiology, we focused on a predicted surface-exposed adhesin (SspB) encoded exclusively by this distinct phyletic group. Phylogenetic analysis of the gene encoding SspB in the context of a streptococcal species tree suggests that sspB was acquired by lateral gene transfer from Streptococcus suis. Furthermore, an sspB deletion mutant displays decreased adherence to cultured cells from the ocular epithelium compared to the isogenic wild-type and complemented strains. Together these findings suggest that acquisition of genes from outside the species has contributed to pneumococcal tissue tropism by enhancing the ability of a subset of strains to infect the ocular epithelium causing conjunctivitis. IMPORTANCE Changes in the gene content of pathogens can modify their ability to colonize and/or survive in different body sites in the human host. In this study, we investigate a gene acquisition event and its role in the pathogenesis of Streptococccus pneumoniae (pneumococcus). Our findings suggest that the gene encoding the predicted surface protein SspB has been transferred from Streptococcus suis (a distantly related streptococcal species) into a distinct set of pneumococcal strains. This group of strains distinguishes itself from the remainder of pneumococcal strains by extensive differences in genomic composition and by the ability to cause conjunctivitis. We find that the presence of sspB increases adherence of pneumococcus to the ocular epithelium. Thus, our data support the hypothesis that a subset of pneumococcal strains has gained genes from neighboring species that enhance their ability to colonize the epithelium of the eye, thus expanding into a new niche.

Site-specific processing of Ras and Rap1 Switch I by a MARTX toxin effector domain.

Ras (Rat sarcoma) protein is a central regulator of cell growth and proliferation. Mutations in the RAS gene are known to occur in human cancers and have been shown to contribute to carcinogenesis. In this study, we show that the multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin-effector domain DUF5(Vv) from Vibrio vulnificus to be a site-specific endopeptidase that cleaves within the Switch 1 region of Ras and Rap1. DUF5(Vv) processing of Ras, which occurs both biochemically and in mammalian cell culture, inactivates ERK1/2, thereby inhibiting cell proliferation. The ability to cleave Ras and Rap1 is shared by DUF5(Vv) homologues found in other bacteria. In addition, DUF5(Vv )can cleave all Ras isoforms and KRas with mutations commonly implicated in malignancies. Therefore, we speculate that this new family of Ras/Rap1-specific endopeptidases (RRSPs) has potential to inactivate both wild-type and mutant Ras proteins expressed in malignancies.

Cytotoxicity of the Vibrio vulnificus MARTX toxin effector DUF5 is linked to the C2A subdomain.

The multifunctional-autoprocessing repeats-in-toxin (MARTX) toxins are bacterial protein toxins that serve as delivery platforms for cytotoxic effector domains. The domain of unknown function in position 5 (DUF5) effector domain is present in at least six different species' MARTX toxins and as a hypothetical protein in Photorhabdus spp. Its presence increases the potency of the Vibrio vulnificus MARTX toxin in mouse virulence studies, indicating DUF5 directly contributes to pathogenesis. In this work, DUF5 is shown to be cytotoxic when transiently expressed in HeLa cells. DUF5 localized to the plasma membrane dependent upon its C1 domain and the cells become rounded dependent upon its C2 domain. Both full-length DUF5 and the C2 domain caused growth inhibition when expressed in Saccharomyces cerevisiae. A structural model of DUF5 was generated based on the structure of Pasteurella multocida toxin facilitating localization of the cytotoxic activity to a 186 amino acid subdomain termed C2A. Within this subdomain, an alanine scanning mutagenesis revealed aspartate-3721 and arginine-3841 as residues critical for cytotoxicity. These residues were also essential for HeLa cell intoxication when purified DUF5 fused to anthrax toxin lethal factor was delivered cytosolically. Thermal shift experiments indicated that these conserved residues are important to maintain protein structure, rather than for catalysis. The Aeromonas hydrophila MARTX toxin DUF5(Ah) domain was also cytotoxic, while the weakly conserved C1-C2 domains from P. multocida toxin were not. Overall, this study is the first demonstration that DUF5 as found in MARTX toxins has cytotoxic activity that depends on conserved residues in the C2A subdomain.