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.

Fig. 1.

Protein sequence of arginine deiminase. clustal 2.1 multiple sequence alignment of S. intermedius, P. aeruginosa and M. arginini ADI. Underlined amino acids are conserved and are known to be essential for full enzymic activity of the protein, either by binding substrate and/or by participating in catalysis (Das et al., 2004). Amino acid 399 (cysteine highlighted in grey) of S. intermedius ADI was mutated to a serine to produce a catalytic mutant.

Fig. 2.

Arginine deiminase activity of the native and mutant proteins. Preparations of the S. intermedius ADI and the catalytic mutant (ADIC399S) were tested for their ability to hydrolyse arginine. Shown on the left is the reaction catalysed by ADI. The assay detects the end product citrulline. No activity was detected with ADIC399S. Error bars represent the sd of technical replicates (run three times per biological replicate).

Fig. 3.

Effect of ADI on P. gingivalis biofilm formation. (a) Biofilm assays were performed with the addition of buffer control, ADI, or the catalytic mutant ADIC399S. Biofilm was quantified with safranin staining (reported as A₄₉₂). The addition of ADI reduced the amount of biofilm biomass; however ADIC399S had no effect on biofilm formation. This shows that enzymic activity is required for inhibition. The graph represents one biological replicate. Error bars represent the sd of technical replicates. (b) Effect on biofilm formation of 0.2 mg M. arginini ADI ml⁻¹ was compared with buffer control. A reduction in biofilm biomass was detected in wells containing M. arginini ADI. This shows that M. arginini ADI, a commercially available enzyme, also inhibits biofilm formation. Error bars represent the sd of technical replicates (run three times per biological replicate).

Fig. 4.

Biofilm formation by P. gingivalis in the absence and presence of l-arginine. Overnight cultures of P. gingivalis were pelleted, the supernatant was removed and the cells were resuspended in arginine-free RPMI-S or arginine-free RPMI-S with the addition of 0.04 or 0.4 % l-arginine and allowed to incubate for an additional 24 h. Biofilm was quantified with safranin staining (reported as A₄₉₂). The addition of arginine promoted biofilm formation at both concentrations tested. The graph represents three biological replicates. Error bars represent the sd of technical replicates.

Fig. 5.

Images showing the effect of l-arginine addition on P. gingivalis biofilm formation. Overnight cultures of P. gingivalis were pelleted, the supernatant removed and the cells resuspended in arginine-free RPMI-S or arginine-free RPMI-S with the addition of 0.04 or 0.4 % l-arginine. Cell resuspensions were added to Millicell EZ Slide wells and slides were incubated for 24 h. The wells were washed and stained with BacLight live/dead stain. The results show that biofilm formation was promoted by addition of arginine and the cells were viable.