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

Abstract

Microorganisms that create mixed-species biofilms in the human oral cavity include, among others, the opportunistic fungus Candida albicans and the key bacterial pathogen in periodontitis, Porphyromonas gingivalis. Both species use arsenals of virulence factors to invade the host organism and evade its immune system including peptidylarginine deiminase that citrullinates microbial and host proteins, altering their function. We assessed the effects of this modification on the interactions between the C. albicans cell surface and human plasminogen and kininogen, key components of plasma proteolytic cascades related to the maintenance of hemostasis and innate immunity. Mass spectrometry was used to identify protein citrullination, and microplate tests to quantify the binding of modified plasminogen and kininogen to C. albicans cells. Competitive radioreceptor assays tested the affinity of citrullinated kinins to their specific cellular receptors. The citrullination of surface-exposed fungal proteins reduced the level of unmodified plasminogen binding but did not affect unmodified kininogen binding. However, the modification of human proteins did not disrupt their adsorption to the unmodified fungal cells. In contrast, the citrullination of kinins exerted a significant impact on their interactions with cellular receptors reducing their affinity and thus affecting the role of kinin peptides in the development of inflammation.

Moonlighting Proteins at the Candidal Cell Surface

“…In addition to host endogenous PADs, enzymes synthesized and released by an anaerobic periodontal bacterium P. gingivalis (PPAD) may also appear in the host organism. For C. albicans , it was shown that seven moonlighting proteins Eno1, Adh1, elongation factor 1-alpha 1, Tdh3, heat shock proteins Ssa1 and Ssa2 and ATPase 1 of the plasma membrane could be citrullinated by PPAD [ 133 , 134 ]. The ability to citrullinate fungal moonlighting proteins may affect the disclosure of epitopes that have no immune tolerance, which consequently induces antigen production and autoimmunity.…”

The Role of Candida albicans Virulence Factors in the Formation of Multispecies Biofilms With Bacterial Periodontal Pathogens

“…Moreover, these three fungal proteins, Als3, Mp65, and Eno1, were identified as citrullinated by the bacterial enzyme—peptidylarginine deiminase (PPAD)—during the interactions of C. albicans ATCC 10231 with P. gingivalis at a ratio of 1:10 in mixed biofilm formed for 24 h in the simultaneous model of interaction in RPMI 1640 medium. Additionally, also other surface-exposed candidal moonlighting proteins were prone to this modification, including Hxk2, Pgk1, Adh1, pyruvate decarboxylase (Pdc1), and glyceraldehyde-3-phosphate dehydrogenase (Tdh3) ( Karkowska-Kuleta et al., 2018 ; Karkowska-Kuleta et al., 2020 ). This modification may influence their role in biofilm, as it was pointed out that citrullination by PPAD is important in the process of mixed biofilm formation because the adhesion of the P. gingivalis mutant strain deprived of PPAD was significantly lower than that observed for the wild-type strain ( Karkowska-Kuleta et al., 2018 ).…”

“…This modification may influence their role in biofilm, as it was pointed out that citrullination by PPAD is important in the process of mixed biofilm formation because the adhesion of the P. gingivalis mutant strain deprived of PPAD was significantly lower than that observed for the wild-type strain ( Karkowska-Kuleta et al., 2018 ). In addition, also the interaction with the host proteins may be altered since the citrullination of surface-exposed fungal proteins resulted also in the reduced binding of human plasminogen ( Karkowska-Kuleta et al., 2020 ).…”

“…Moreover, changes were also noticed for protein Bgl2, a cell wall-associated 1,3-β-glucosyltransferase involved in cell wall remodeling, whose level increased in normoxia in biofilm formed with mutant strain, while for Phr1, cell surface glycosidase involved in glucan cross-linking also in normoxia, but for wild-type strain ( Sarthy et al., 1997 ; Fonzi, 1999 ; Bartnicka et al., 2019 ). Moreover, some of these proteins were also indicated as susceptible for modifications by P. gingivalis PPAD, as the citrullination at most two places was identified with mass spectrometry analysis for Eng1, Bgl2, Phr1, and Mp65 ( Karkowska-Kuleta et al., 2020 ). Importantly, in the case of the bacterial deiminase, the citrullination also requires a pre-hydrolysis of the protein by R gingipain to expose the C-terminal arginine ( Goulas et al., 2015 ); however, the impact of these enzymatic modifications on the structure and activity of fungal enzymes still needs to be elucidated.…”

Kinins and Their Receptors in Infectious Diseases

“…It was proposed that gingipains might also play a part for the increased risk of oral cancer in patients with periodontitis—this was associated with the inactivation of SPINK6, an inhibitor of kallikrein activation in the epithelium, probably resulting in augmentation of kinins, which are recognized mitotic mediators [ 67 ]. Remarkably, it was found that formation of complex oral biofilms between P. gingivalis and Candida albicans is dependent on the action of gingipains and peptidylarginine deiminase, which in turn leads to kinin citrullination, changing the affinity for kinin receptors, what finally contributes for pathogen escaping mechanisms [ 68 ]. Altogether, literature evidence clearly highlights a relevant role for kinins and their receptors in the pathogenesis of periodontitis.…”