Antimicrobials-treated porphyromonas gingivalis persisters and human cells : interactive profiles, underlying mechanisms, tackling approaches & perspectives

Abstract

Microbial persisters are the tiny sub-population of microorganisms with high tolerance to multiple antimicrobials that is non-inheritable and reversible. The existence of persisters contributes to prolonged antibiotic exposure, generating antibiotic-resistant mutants and treatment failure. Further understanding on persisters is of profound importance for effective prevention and control of chronic infections/inflammation. Periodontal disease, one of the most serious health burdens worldwide, markedly affects oral health and yet links intimately to common inflammatory comorbidities (e.g., diabetes and cardiovascular disease). Porphyromonas gingivalis as the ‘keystone’ periodontopathogen, crucially drives the shift of microbe-host symbiosis (so-called eubiosis) to dysbiosis, acounting for periodontal pathogenesis. Notably, the recent identification of P. gingivalis persisters by our group may to some extent explain the relapse and recalcitrance of periodontal disease. This study extended to investigate i) the interactive profiles of metronidazole-treated P. gingivalis persisters (M-PgPs) with human gingival epithelial cells (HGECs), ii) relevant molecular mechanisms, and iii) new tackling strategies for eradicating P. gingivalis persisters and iv) novel approaches to exploring the phenotypes and functionalities of Escherichia coli persisters. The general morphology and cellular structure of M-PgPs remained unchanged after the treatment with lethal dose metronidazole (100 mg/L) for 6 h. Essentially, M-PgPs preserved the capabilities to adhere to and invade into HGECs. Furthermore, M-PgPs markedly suppressed the expression of pro-inflammatory cytokines (e.g. CXCL5, IL-6 and IL-8) in HGECs at a similar level to the untreated P. gingivalis cells, via the action of their thermo-sensitive components. RNA sequencing (RNA-seq) results confirmed that the forkhead box O (FOXO) signalling pathway was up-regulated by M-PgPs in HGECs, and FOXO1 inhibitor could rescue the inhibitory effects of these persisters on innate host responses. Next, colloidal bismuth subcitrate (CBS) combined with metronidazole enabled to effectively eradicate P. gingivalis persisters in planktonic mode, and nearly eliminated their existence in biofilm mode. Essentially, CBS exhibited negligible effects on the viability of HGECs, along with minimal cytotoxicity (< 5%) even at a high concentration (400 μM). Moreover, single-cell Raman spectroscopy (SCRS) and D2O-based Single-cell Raman microspectroscopy (D2O-Raman) identified two featured types (I and II) of E. coli persisters, and their metabolic activities were then assessed. These experiments demonstrated that E. coli persisters could be discriminated via analyzing their distinct Raman spectrum and metabolism. Notably, both types of persisters began to absorb D2O for metabolism shortly after the termination of ampicillin treatment, although these persister cells remained to be non-dividing. In conclusion, the current findings provide the first evidence that metronidazole-treated P. gingivalis persisters enable to perturb innate responses in human gingival epithelial cells, through upregulating the FOXO signalling pathway. The synergistic combination of bismuth drugs and metronidazole could be an alternative approach to controlling periodontitis and P. gingivalis-associated inflammation-driven comorbidities. Moreover, single-cell Raman spectroscopy as an advanced technique is promising for further extended investigations on various persister cells. This pioneering study enhances our understanding on the phenotypes, functionalities and survival mechanisms of persister cells as well as intervening approaches, thereby contributing to developing precision preventive and therapeutic strategies for tackling periodontitis and P. gingivalis-related inflammatory comorbidities.