Effects of ClpP protease on biofilm formation of Enterococcus faecalis

Abstract Enterococcus faecalis (E. faecalis), one of the main pathogens responsible for refractory periapical periodontitis and nosocomial infections, exhibits markedly higher pathogenicity in biofilms. Objectives Studies have shown that caseinolytic protease P (ClpP) is involved in biofilm formation. However, to date, few studies have investigated the role of ClpP in the survival of E. faecalis, and in enhancing biofilm formation. Therefore, we investigated the role of ClpP in the formation of E. faecalis biofilms. Methodology In our study, we used homologous recombination to construct clpP deleted and clpP complement strains of E. faecalis ATCC 29212. A viable colony counting method was used to analyze the growth patterns of E. faecalis. Crystal violet staining (CV) and confocal scanning laser microscopy (CLSM) were used to characterize biofilm mass formation and scanning electron microscopy (SEM) was used to observe the biofilm microstructure. Data was statistically analyzed via Student’s t-test or one-way analysis of variance (ANOVA). Results The results exhibited altered growth patterns for the clpP deletion strains and depleted polysaccharide matrix, resulting in reduced biofilm formation capacity compared to the standard strains. Moreover, ClpP was observed to increase bioﬁlm formation in E. faecalis. Conclusion Our study shows that ClpP can increase bioﬁlm formation in E. faecalis and emphasizes the importance of ClpP as a potential target against E. faecalis.


Introduction
Enterococcus faecalis (E. faecalis) is one of the primary etiologic pathogens of refractory periapical periodontitis, secondary root canal infections, and pulp infection. It is also the main pathogen of nosocomial infections, such as bacterial endocarditis and meningitis. [1][2] The ability of E. faecalis to form biofilms directly determines its survival, adaptability to the environment and pathogenicity. It was reported that more than 40% of E. faecalis clinical isolates can form biofilms. 3 E. faecalis is 100-1000 times more pathogenic in biofilms than in the plankton state, 4 since biofilms provide protection against environmental stress, including lacks of nutrients, high alkalinity and antibiotics. 5 In the field of Endodontics, the medicaments used to remove bacteria from the root canal system primarily include calcium hydroxide and antibiotics. However, these agents are associated with poor efficiency and have been shown to cause adverse effects. 6 Hence, there is currently a lack of effective medication capable of treating E. faecalis.
Therefore, investigating the mechanism associated with E. faecalis biofilm formation is essential to inform the development of effective treatment methods.
Accordingly, the stress response of E. faecalis has become the subject of intensive research.
The adaptability of E. faecalis to stress and crossimmunoprotectivity is associated with increased protein synthesis. However, except for a few stressrelated proteins, such as Gls24, and the molecular chaperones, GroEL and DnaK, most proteins involved in the stress response remain unknown. [7][8] Caseinolytic protease P (ClpP) is an ATP-dependent proteolytic enzyme that plays important roles in bacteria, parasites, and human mitochondria. Specifically, ClpP participates in the hydrolysis of misfolded and defective proteins, whereas ClpP is an important gene involved in biofilm formation. [9][10][11][12][13][14][15][16][17][18] Recent studies have reported that ClpP exerts different effects on bacterial biofilm formation.  16 In fact, researchers hold different views on the role of clpP in the formation of biofilm for same bacteria. Shanks, et al. 17 (2006) found that when clpP was mutated, the biofilm of Pseudomonas aeruginosa increased; however, Hall, et al. 18 (2017) stated that clpP enhanced its ability to form biofilms.
To date, few studies have investigated the role of ClpP in the survival of E. faecalis or its ability to influence biofilm formation. E. faecalis has a unique ability to tolerate hypoxia, starvation, and high alkalinity in reinfected root canals making it challenging to treat with current antibiotics. Hence, identifying effective antibiotic targets is an urgent concern for this pathogen. In our study, we investigated the role of ClpP in the formation of E. faecalis biofilms. These to identify the accuracy of the sequence, and highexpression strains were selected using media.    biofilm structure (magnification ×1,000, ×5,000, and ×10,000) comprising grainy secretions and filaceous links with no significant difference observed between the strains. Meanwhile, the ∆clpP mutant strains had a lower biofilm volume and a more regular shape. are not only necessary for bacterial adhesion, but also serve as a cytoskeleton in biofilm formation, which is beneficial for the survival of bacteria in this malnourished environment. [24][25][26] The results show that the polysaccharide matrix mass in the clpP deleted strains was decreased compared with standard and complement strains.

Growth analysis of E. faecalis
The changes in growth pattern and the decrease in polysaccharide matrix may explain the CV and fluorescence staining results for the clpP deletion strains, which were examined using SEM and CLSM.
These methods are effective for analyzing biofilms both quantitatively and qualitatively. The standard and complemented strains were observed to contain a large number of live bacteria during the quiescent stage (24 h). With an increased time, the biofilm area increased slowly, but the proportion of dead bacteria also significantly increased. In contrast, growth of the clpP deletion strains was inhibited. After the quiescent stage, the biofilm also gradually increased, but the general level was much lower than that of the standard and complement strains. It was also confirmed morphologically by SEM that mature biofilms were The role of ClpP in E. faecalis has been rarely studied. Zheng, et al. 29 (2020) found that clpP inhibits the formation of E. faecalis biofilms, although it has no effect on the growth of E. faecalis. This differs from our results, which showed that clpP expression was also closely related to growth. Studies have also found that clpP influenced growth and filament formation and biofilm formation in E. faecalis by autolysis.
Considering that E. faecalis is one of the main etiologic pathogens for root canal reinfection and persistent periapical periodontitis, whereas also exhibiting a certain resistance to most root canal therapy drugs, cleaning and disinfection methods, 34 the results of the current study may provide insights into novel targets to better control E. faecalis infections.
Thus, we showed that ClpP can increase the biofilm formation of E. faecalis. We also demonstrated that ClpP may serve as a potential therapeutic target for E. faecalis. Recently, acyldepsipeptides have been identified as a new antibiotic that targets ClpP to elicit a bactericidal role. Hence, application of acyldepsipeptides as a targeted drug for ClpP in root canal therapy could also be effective. Nevertheless, elucidation of the mechanism by which ClpP affects biofilm formation and growth of E. faecalis is necessary in future studies. 35

Conclusions
Our studied shows that ClpP can increase biofilm formation in E. faecalis and emphasizes the importance of ClpP as a potential target against E. faecalis.