Arginine deiminase inhibits Porphyromonas gingivalis surface attachment

Abstract

The oral cavity is host to a complex microbial community whose maintenance depends on an array of cell-to-cell interactions and communication networks, with little known regarding the nature of the signals or mechanisms by which they are sensed and transmitted. Determining the signals that control attachment, biofilm development and outgrowth of oral pathogens is fundamental to understanding pathogenic biofilm development. We have previously identified a secreted arginine deiminase (ADI) produced by Streptococcus intermedius that inhibited biofilm development of the commensal pathogen Porphyromonas gingivalis through downregulation of genes encoding the major (fimA) and minor (mfa1) fimbriae, both of which are required for proper biofilm development. Here we report that this inhibitory effect is dependent on enzymic activity. We have successfully cloned, expressed and defined the conditions to ensure that ADI from S. intermedius is enzymically active. Along with the cloning of the wild-type allele, we have created a catalytic mutant (ADIC399S), in which the resulting protein is not able to catalyse the hydrolysis of L-arginine to L-citrulline. P. gingivalis is insensitive to the ADIC399S catalytic mutant, demonstrating that enzymic activity is required for the effects of ADI on biofilm formation. Biofilm formation is absent under L-arginine-deplete conditions, and can be recovered by the addition of the amino acid. Taken together, the results indicate that arginine is an important signal that directs biofilm formation by this anaerobe. Based on our findings, we postulate that ADI functions to reduce arginine levels and, by a yet to be identified mechanism, signals P. gingivalis to alter biofilm development. ADI release from the streptococcal cell and its cross-genera effects are important findings in understanding the nature of inter-bacterial signalling and biofilm-mediated diseases of the oral cavity. INTRODUCTIONPorphyromonas gingivalis is an oral pathobiont, i.e. a natural member of the human microbiota, that under certain perturbations to the host and/or microflora can cause pathology. This Gram-negative, highly proteolytic anaerobe is regarded as the primary aetiological agent of adult periodontal disease, leading to chronic inflammation and destruction of both the soft and hard tissues supporting the teeth (Choil et al., 1990;Dzink et al., 1988; Grossi et al., 1994;Lamont & Jenkinson, 2000;Moore et al., 1991). As a pathobiont, the ability to proliferate and express virulence determinants is central to its shift to pathogenicity, thus determining the mechanisms that control the emergence of its pathogenic state are of fundamental importance.P. gingivalis is a strict anaerobe that preferentially utilizes protein or peptide substrates for growth. Although studies have shown that P. gingivalis may utilize free amino acids or dipeptides, the uptake and growth rates on these substrates are limited and highly variable among strains (Seddon et al., 1988;Takahashi et al., 2000;Tang-Larsen et al., 1995). In general...

Characterization and development of SAPP as a specific peptidic inhibitor that targets Porphyromonas gingivalis

“…We found that S cristatus arginine deiminase (ArcA) can repress FimA expression in several P. gingivalis strains, including strains expressing fimA types I, II, and III . Similar results were later observed by the Davey lab, in which they reported a downregulation of P. gingivalis genes encoding the major ( fimA ) and minor ( mfa1 ) fimbriae by Streptococcus intermedius ArcA. Our data from a peptide array showed that an eleven amino acid peptide with the native sequence of streptococcal ArcA, which we designate streptococcal ArcA derived anti‐ P. gingivalis peptide (SAPP), not only repressed fimA and mfa1 expression but also repressed mfa1, rgpA/B, and kgp expression in P. gingivalis .…”

Expression and Characterization of Serotype 2 <b><i>Streptococcus suis </i></b>Arginine Deiminase

“…In addition, the difference in optimum temperature and pH of ArcA activity was observed between the assay reaction with and without DTT (data not shown). Our data confirmed the previous results reported by Cugini et al [2013] that ArcA activity was rapidly lost in a nonreducing environment and the addition of DTT greatly increased enzymatic activity. These results indicated that the SS2 ArcA was highly sensitive to oxidizing conditions and Cys399 residue could play an essential role in the SS2 ArcA catalysis.…”

Effects of Different Amino Acids on Biofilm Growth, Swimming Motility and Twitching Motility in Escherichia Coli BL21

“…This phenomenon of inhibition of twitching motility by arginine has been reported by Cugini et al (2012), in which they observed that the arginine deiminase secreted by Streptococcus intermedius inhibited biofilm formation in the commensal pathogen Porphyromonas gingivalis. This is achieved via downregulation of genes encoding the major (fimA) and minor (mfa1) fimbriae, both of which are required for proper biofilm development (Cugini et al, 2012). Arginine deiminase is an enzyme that catalyzes the hydrolysis of L-arginine to L-citrulline and ammonia gas (Gong et al, 2000).…”