Antimicrobial tolerance of Porphyromonas gingivalis and Candida albicans persisters : molecular profiles, underlying mechanisms and perspectives

Abstract

Antimicrobial tolerance of microbial persisters has increasingly become a critical challenge for effective prevention and control of infectious/inflammatory diseases worldwide. It is of great importance that persisters critically account for the antimicrobial resistance of biofilms that are responsible for the majority of chronic infections/inflammation. Currently, the underlying molecular mechanisms of persister tolerance remain unclear. The present study systematically investigated the phenotypic profiles of P. gingivalis and Candida persisters, the underlying mechanisms of their antimicrobial tolerance and antifungal resistance in C. albicans biofilms.

P. gingivalis was incubated with varying concentrations of hemin and treated with metronidazole (MTZ). Notably, a small fraction of P. gingivalis persisters could survive MTZ treatment at 100 μg/ml, and such persistence was not heritable. P. gingivalis formed much higher levels of persisters at stationary phase than late exponential phase. The frequency of persisters significantly increased under hemin-limitation (1 μg/ml) condition with reference to hemin-repletion (10 μg/ml) condition in late exponentially growing P. gingivalis after 6 h MTZ exposure. Shotgun proteomic analysis of the persister fractions further demonstrated decreased expression of the proteins involved in electron transfer (e.g. PGN_1753) and heme/iron utilization (e.g. HagA) that are important for redox regulation and MTZ activation. Additionally, P. gingivalis persisters could shut down major cellular activities (e.g. translation and DNA replication) and overexpress stress proteins.

C. albicans biofilms were established and they exhibited higher resistance to caspofungin (CAS) and amphotericin B (AMB) than their planktonic counterparts. Candida biofilms harbored a subpopulation of persisters highly tolerant to both CAS and AMB, and interestingly consecutive AMB treatments failed to control them. Proteomic analysis further investigated the responses of C. albicans planktonic and biofilm cells to CAS treatments at respective MICs. The CAS-treated biofilms and planktonic cells revealed 224 and 148 differentially expressed proteins, respectively. Certain key proteins for cell wall maintenance, stress response and metabolic regulation (e.g. PIL1, LSP1 and HSP90) were exclusively involved in the biofilms, with reference to the planktonic cells. A specific proteomic signature was identified in the AMB-tolerant C. albicans biofilm persisters. The major metabolic activities were subdued, e.g. glycolysis, tricarboxylic acid cycle and protein synthesis; whereas certain metabolic pathways were activated, like the glyoxylate cycle in the persisters. A number of proteins essential for virulence and stress response were greatly upregulated such as CSP37, GRP2 and TSA1. Notably, the persisters were tolerant to oxidative stress, despite of highly induced intracellular superoxide.

This pioneering study provides the first evidence on the persisters of the keystone periodontopathogen P. gingivalis, and indicates that hemin is an important mediator of their formation and survival. It is noteworthy that the tolerance of P. gingivalis persisters to MTZ may be dominated by proactive regulation of cellular redox state. Persisters-bound C. albicans biofilms could undergo more complex regulation of cellular pathways than its planktonic cells in response to antifungals. Delicate metabolic control and coordinated stress response markedly determine Candida biofilm resistance and tolerance. The current findings may contribute to the development of novel antimicrobials for effective control and management of persistent infections/inflammation.