Adhesive protein-mediated cross-talk between Candida albicans and Porphyromonas gingivalis in dual species biofilm protects the anaerobic bacterium in unfavorable oxic environment.

The oral cavity contains different types of microbial species that colonize human host via extensive cell-to-cell interactions and biofilm formation. Candida albicans-a yeast-like fungus that inhabits mucosal surfaces-is also a significant colonizer of subgingival sites in patients with chronic periodontitis. It is notable however that one of the main infectious agents that causes periodontal disease is an anaerobic bacterium-Porphyromonas gingivalis. In our study, we evaluated the different strategies of both pathogens in the mutual colonization of an artificial surface and confirmed that a protective environment existed for P. gingivalis within developed fungal biofilm formed under oxic conditions where fungal cells grow mainly in their filamentous form i.e. hyphae. A direct physical contact between fungi and P. gingivalis was initiated via a modulation of gene expression for the major fungal cell surface adhesin Als3 and the aspartic proteases Sap6 and Sap9. Proteomic identification of the fungal surfaceome suggested also an involvement of the Mp65 adhesin and a "moonlighting" protein, enolase, as partners for the interaction with P. gingivalis. Using mutant strains of these bacteria that are defective in the production of the gingipains-the proteolytic enzymes that also harbor hemagglutinin domains-significant roles of these proteins in the formation of bacteria-protecting biofilm were clearly demonstrated.

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

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.

Characterization of Enzymatic Activity of MlrB and MlrC Proteins Involved in Bacterial Degradation of Cyanotoxins Microcystins.

Bacterial degradation of toxic microcystins produced by cyanobacteria is a common phenomenon. However, our understanding of the mechanisms of these processes is rudimentary. In this paper several novel discoveries regarding the action of the enzymes of the mlr cluster responsible for microcystin biodegradation are presented using recombinant proteins. In particular, the predicted active sites of the recombinant MlrB and MlrC were analyzed using functional enzymes and their inactive muteins. A new degradation intermediate, a hexapeptide derived from linearized microcystins by MlrC, was discovered. Furthermore, the involvement of MlrA and MlrB in further degradation of the hexapeptides was confirmed and a corrected biochemical pathway of microcystin biodegradation has been proposed.

Heterologous expression and characterisation of microcystinase.

The first enzyme in the microcystin (MC) degradation pathway identified in bacterial strains is coded by mlrA gene and is referred to as microcystinase. To date, there has been no biochemical characterisation of this enzyme. The results presented herein show a successful heterologous expression of MlrA as well as mutational studies, partial purification and biochemical characterisation of the enzyme. The mutation and inhibition study confirmed previous ideas that MlrA is a metalloprotease and allowed to calculate the inhibition parameters. Moreover, the kinetic parameters of MC-LR linearization were measured showing that MlrA exhibits a positive cooperativity towards MC-LR. Furthermore, in vitro experiments with Escherichia coli cells expressing MlrA indicated the potency of the heterologous host to eliminate MCs with very high efficiency. This study reports a new approach to the analysis of a microcystin degrading enzyme, extends the knowledge about MC biodegradation and opens broad scope for future study.