Virulence of the Pathogen Porphyromonas gingivalis Is Controlled by the CRISPR-Cas Protein Cas3

摘要

The CRISPR ( c lustered r egularly i nterspaced s hort p alindromic r epeat)-Cas system is a unique genomic entity that provides prokaryotic cells with adaptive and heritable immunity. Initial studies identified CRISPRs as central elements used by bacteria to protect against foreign nucleic acids; however, emerging evidence points to CRISPR involvement in bacterial virulence. The present study aimed to identify the participation of one CRISPR-Cas protein, Cas3, in the virulence of the oral pathogen Porphyromonas gingivalis , an organism highly associated with periodontitis. Our results show that compared to the wild type, a mutant with a deletion of the Cas3 gene, an essential nuclease part of the class 1 type I CRISPR-Cas system, increased the virulence of P. gingivalis . In vitro infection modeling revealed only mildly enhanced production of proinflammatory cytokines by THP-1 cells when infected with the mutant strain. Dual transcriptome sequencing (RNA-seq) analysis of infected THP-1 cells showed an increase in expression of genes associated with pathogenesis in response to Δ cas3 mutant infection, with the target of Cas3 activities in neutrophil chemotaxis and gene silencing. The importance of cas 3 in controlling virulence was corroborated in a Galleria mellonella infection model, where the presence of the Δ cas 3 mutant resulted in a statistically significant increase in mortality of G. mellonella . A time-series analysis of transcription patterning during infection showed that G. mellonella elicited very different immune responses to the wild-type and the Δ cas 3 mutant strains and revealed a rearrangement of association in coexpression networks. Together, these observations show for the first time that Cas3 plays a significant role in regulating the virulence of P. gingivalis . IMPORTANCE Porphyromonas gingivalis is a key pathogen of periodontitis, a polymicrobial disease characterized by a chronic inflammation that destroys the tissues supporting the teeth. Thus, understanding the virulence potential of P. gingivalis is essential to maintaining a healthy oral microbiome. In nonoral organisms, CRISPR-Cas systems have been shown to modulate a variety of microbial processes, including protection from exogenous nucleic acids, and, more recently, have been implicated in bacterial virulence. Previously, our clinical findings identified activation of the CRISPR-Cas system in patient samples at the transition to disease; however, the mechanism of contribution to disease remained unknown. The importance of the present study resides in that it is becoming increasingly clear that CRISPR-associated proteins have broader functions than initially thought and that those functions now include their role in the virulence of periodontal pathogens. Studying a P. gingivalis cas 3 mutant, we demonstrate that at least one of the CRISPR-Cas systems is involved in the regulation of virulence during infection.