The activity of bacterial peptidylarginine deiminase is important during formation of dual-species biofilm by periodontal pathogen Porphyromonas gingivalis and opportunistic fungus Candida albicans

Abstract

Porphyromonas gingivalis, an anaerobic Gram-negative bacterium critically involved in the development of human periodontitis, belongs to the late colonizers of the oral cavity. The success of this pathogen in the host colonization and infection results from the presence of several virulence factors, including extracellular peptidylarginine deiminase (PPAD), an enzyme that converts protein arginine residues to citrullines. A common opportunistic fungal pathogen of humans, Candida albicans, is also frequently identified among microorganisms that reside at subgingival sites. The aim of the current work was to verify if protein citrullination can influence the formation of mixed biofilms by both microorganisms under hypoxic and normoxic conditions. Quantitative estimations of the bacterial adhesion to fungal cells demonstrated the importance of PPAD activity in this process, since the level of binding of P. gingivalis mutant strain deprived of PPAD was significantly lower than that observed for the wild-type strain. These results were consistent with mass spectrometric detection of the citrullination of selected surface-exposed C. albicans proteins. Furthermore, a viability of P. gingivalis cells under normoxia increased in the presence of fungal biofilm compared with the bacteria that formed single-species biofilm. These findings suggest a possible protection of these strict anaerobes under unfavorable aerobic conditions by C. albicans during mixed biofilm formation.

Peptidylarginine Deiminase of Porphyromonas gingivalis Modulates the Interactions between Candida albicans Biofilm and Human Plasminogen and High-Molecular-Mass Kininogen

“…A list of modified fungal proteins including their mass spectrometric identification criteria is presented in Table S3 (Supplementary File 3). Eight surface-exposed C. albicans proteins were indicated as citrullinated, including Eno1, Mp65, and Eng1 that were shown to be modified after fungal growth in bacterial supernatants (Table S2) and in mixed biofilms in our previous report [16]. In addition, the citrullination of Tdh3 was identified in our present analysis under both conditions used.…”

“…The modifications that PPAD introduces to diverse molecules play a role not only in relation to the functionality of bacterial proteins, and modulation of biofilm development, but also in the interactions with host proteins and systems [25,[66][67][68]. Under the conditions used in this study citrullination of such C. albicans proteins as glucan 1,3-beta-glucosidase Bgl2, elongation factor 1-alpha 1, Tdh3, heat shock proteins Ssa1 and Ssa2, yeast-form wall protein 1, plasma membrane ATPase 1, agglutinin-like sequence protein 3, 1,3-beta-glucanosyltransferase Pga4 and pH-responsive protein 1 was newly identified, whereas Eno1, Mp65, Adh1 and Eng1 were previously identified to be citrullinated [16]. Our current study findings demonstrate that the modifications of the surface proteins of C. albicans by bacterial enzymes, mainly PPAD and gingipains, can affect the interaction of fungal cells with HPG, reducing the level of its binding to the fungal cell surface.…”

“…In addition, the citrullination of Tdh3 was identified in our present analysis under both conditions used. In the case of alcohol dehydrogenase 1 (Adh1), citrullination was detected after treatment with PPAD and in mixed biofilm in our earlier study [16], whereas the citrullination of agglutinin-like sequence protein 3 (Als3), 1,3-beta-glucanosyltransferase Pga4 and pH-responsive protein 1 was detected only after treatment with PPAD. Quantitatively, the similarities and differences in the approaches used, namely the treatment of C. albicans cells with culture supernatants or with purified PPAD, with respect to the number of fungal proteins identified as citrullinated are shown in Figure 3.…”

“…This occurs in the oral cavity-in subgingival biofilm, in the gingival pockets or root canal system-where C. albicans might form a mixed-species biofilm together with different bacteria, including strict anaerobes like P. gingivalis, providing them protection under aerobic conditions, and also during aggressive and chronic periodontitis [63][64][65]. It was indicated in our previous study that the activity of citrullinating bacterial virulence factor-PPAD-had a certain importance during the formation of dual-species bacterial-fungal biofilm [16]. The modifications that PPAD introduces to diverse molecules play a role not only in relation to the functionality of bacterial proteins, and modulation of biofilm development, but also in the interactions with host proteins and systems [25,[66][67][68].…”

“…The full list of citrullinated fungal proteins identified by LC-MS/MS in this experiment, including mass spectrometric identification criteria, is provided in Table S2 in Supplementary File 2. In a previous study [16] in which the citrullination of C. albicans proteins exposed at the surface of hyphae that formed a mixed biofilm with P. gingivalis cells of wild strain W83 was tested, the modification of three proteins from this set, i.e., Eng1, Eno1 and Mp65, was also described.…”