ABSTRACT CRISPR–Cas systems are primarily recognized for their defensive role against foreign DNA. Recent studies, however, indicate involvement in regulatory functions. The persistence of a large spacer array without apparent phage targets in Porphyromonas gingivalis ATCC 33277 remains unexplained. This study demonstrates that deletion of the non-coding CRISPR array 30.1 in P. gingivalis ATCC 33277 results in increased biofilm formation, elevated virulence in a Galleria mellonella infection model, and significant alterations in the macrophage transcriptomic response. The ΔCRISPR-30.1 mutant forms twice as much biofilm as the wild type, induces 50% mortality in Galleria larvae within 130 h compared to 200 h for the wild type ( P < 0.0001), and elicits a cytokine profile characterized by increased IL-6, CXCL1, CXCL2, and CXCL9 secretion. Dual RNA sequencing of THP-1 macrophages infected with wild-type and ΔCRISPR 30.1 strains reveals that the loss of the array activates bacterial metabolic and secretion pathways while suppressing host innate and adaptive immune signaling. Single-primer amplification (SPA) identifies numerous self-genome loci bound by individual 30.1 spacers, supporting a direct, spacer-mediated regulatory mechanism. These findings establish CRISPR array 30.1 as a previously unrecognized regulator of P. gingivalis physiology and host-pathogen interactions. IMPORTANCE CRISPR–Cas systems are established as adaptive immune elements, yet spacer arrays without known targets are frequently observed in bacteria and often lack a defined function. In P. gingivalis , a keystone periodontal pathogen, a non-coding CRISPR array has been shown to regulate biofilm formation, virulence in an invertebrate model, and the macrophage transcriptional response. This expands the recognized functions of CRISPR arrays to include the direct regulation of bacterial physiology and the modulation of host immune responses, identifying CRISPR spacers as potential targets for antimicrobial interventions. Furthermore, elucidating the role of CRISPR arrays in P. gingivalis may have broader clinical implications, given the established associations between periodontal health and systemic inflammatory diseases. Targeting spacer arrays to modulate bacterial virulence could influence the management of these conditions and enhance the translational relevance of such therapeutic strategies.
Irfan et al. (Wed,) studied this question.