A streptococcal effector protein that inhibits Porphyromonas gingivalis biofilm development

Abstract

Dental plaque formation is a developmental process involving cooperation and competition within a diverse microbial community, approximately 70 % of which is composed of an array of streptococci during the early stages of supragingival plaque formation. In this study, 79 cell-free culture supernatants from a variety of oral streptococci were screened to identify extracellular compounds that inhibit biofilm formation by the oral anaerobe Porphyromonas gingivalis strain 381. The majority of the streptococcal supernatants (61 isolates) resulted in lysis of P. gingivalis cells, and some (17 isolates) had no effect on cell viability, growth or biofilm formation. One strain, however, produced a supernatant that abolished biofilm formation without affecting growth rate. Analysis of this activity led to the discovery that a 48 kDa protein was responsible for the inhibition. Protein sequence identification and enzyme activity assays identified the effector protein as an arginine deiminase. To identify the mechanism(s) by which this protein inhibits biofilm formation, we began by examining the expression levels of genes encoding fimbrial subunits; surface structures known to be involved in biofilm development. Quantitative RT-PCR analysis revealed that exposure of P. gingivalis cells to this protein for 1 h resulted in the downregulation of genes encoding proteins that are the major subunits of two distinct types of thin, single-stranded fimbriae (fimA and mfa1). Furthermore, this downregulation occurred in the absence of arginine deiminase enzymic activity. Hence, our data indicate that P. gingivalis can sense this extracellular protein, produced by an oral streptococcus (Streptococcus intermedius), and respond by downregulating expression of cell-surface appendages required for attachment and biofilm development.

Fig. 1.

Detection of biofilm inhibitory activity in the spent supernatant of the clinical strain of S. intermedius grown in BHI broth at 37 °C for 24 h. (a) Inhibitory activity (*) was detected in mid- and late-exponential growth and during stationary phase. (b) Effect of 18 h spent supernatant on biofilm formation. Supernatant obtained after 18 h anaerobic growth significantly inhibited P. gingivalis biofilm formation at all dilutions tested. Results are means ±sd (n=3).

Fig. 2.

Growth curves of P. gingivalis in the presence or absence of biofilm inhibitor. The dilution of partially purified biofilm inhibitor (DEAE fraction; Fig. 3b, lane B) was 20-fold diluted (•) compared to the control buffer (▴). The addition of the arginine deiminase had no effect on the growth rate of P. gingivalis.

Fig. 3.

(a) Biofilm assays were used to monitor the biofilm inhibitory activity during purification from the spent supernatant. Samples of the fraction eluted from the DEAE column using 500 mM NaCl (DEAE) and fractions eluted from a Mono Q column using between 0 and 600 mM NaCl (Mono Q 1–5) were tested in the biofilm assay, in triplicate, at dilutions of 1 : 10 and 1 : 20. Note: the active fraction applied to Mono Q2 eluted from the Mono Q column within the 200 to 240 mM NaCl range. Results are means ±sd (n=3). (b) Samples of the active fraction from the DEAE column (lane B) and of the subsequent fractions from the Mono Q columns (lanes C–H) were submitted to SDS-PAGE analysis. Proteins were detected with silver staining. The material applied to lane B corresponds to the fraction tested in the biofilm assay in (a) (DEAE). Lane C contains an active fraction of all material eluted from 200 to 240 mM NaCl [corresponding to material applied in (a) Mono Q 2], while lanes D–I contain material eluted serially within the 200 to 240 mM NaCl range, at a flatter gradient. Of these, the greatest activity was observed in the material applied to lane G, which contained a prominent ∼48 kDa band (marked with arrow). (c) Effect of a 1 : 50 dilution (final concentration ≤3.8 μg arginine deiminase ml⁻¹) of the purified fraction (lane G) on P. gingivalis biofilm formation. The results show a significant inhibition (P<0.0001) of biofilm formation when compared to the untreated control. Results are means ±sd (n=3).

Fig. 4.

Arginine deiminase activity of purified protein. Serial twofold dilutions (highest concentration 300 μg ml⁻¹) of the S. intermedius purified protein were tested in a colorimetric assay for arginine deiminase activity. The enzymic activity of the purified protein demonstrated a linear dependence on the protein concentration.

Fig. 5.

qPCR analysis of the fimA (0.41±0.2-fold change, P-value 0.006) and mfa1 (0.50±0.2-fold change, P-value 0.011) genes in P. gingivalis cells exposed to the purified arginine deiminase. A 1 : 50 dilution of the protein corresponds to ≤3.8 μg arginine deiminase ml⁻¹. The data presented are means of three separate datasets. Error bars represent sem.