Cross-Talk between Staphylococcus aureus and Other Staphylococcal Species via the agr Quorum Sensing System

Abstract

Staphylococci are associated with both humans and animals. While most are non-pathogenic colonizers, Staphylococcus aureus is an opportunistic pathogen capable of causing severe infections. S. aureus virulence is controlled by the agr quorum sensing system responding to secreted auto-inducing peptides (AIPs) sensed by AgrC, a two component histidine kinase. agr loci are found also in other staphylococcal species and for Staphylococcus epidermidis, the encoded AIP represses expression of agr regulated virulence genes in S. aureus. In this study we aimed to better understand the interaction between staphylococci and S. aureus, and show that this interaction may eventually lead to the identification of new anti-virulence candidates to target S. aureus infections. Here we show that culture supernatants of 37 out of 52 staphylococcal isolates representing 17 different species inhibit S. aureus agr. The dog pathogen, Staphylococcus schleiferi, expressed the most potent inhibitory activity and was active against all four agr classes found in S. aureus. By employing a S. aureus strain encoding a constitutively active AIP receptor we show that the activity is mediated via agr. Subsequent cloning and heterologous expression of the S. schleiferi AIP in S. aureus demonstrated that this molecule was likely responsible for the inhibitory activity, and further proof was provided when pure synthetic S. schleiferi AIP was able to completely abolish agr induction of an S. aureus reporter strain. To assess impact on S. aureus virulence, we co-inoculated S. aureus and S. schleiferi in vivo in the Galleria mellonella wax moth larva, and found that expression of key S. aureus virulence factors was abrogated. Our data show that the S. aureus agr locus is highly responsive to other staphylococcal species suggesting that agr is an inter-species communication system. Based on these results we speculate that interactions between S. aureus and other colonizing staphylococci will significantly influence the ability of S. aureus to cause infection, and we propose that other staphylococci are potential sources of compounds that can be applied as anti-virulence therapy for combating S. aureus infections.

Keywords: Staphylococcus aureus; Staphylococcus schleiferi; agr; anti-virulence therapy; auto-inducing peptide; cross-talk; quorum sensing; quorum sensing inhibition.

FIGURE 1

Modulation of Staphylococcus aureus virulence gene expression by staphylococcal culture supernatants. TSA agar plates (with erythromycin and X-gal) containing (A) the hla-lacZ (PC322; Ery^r), (B) the rnaIII-lacZ (SH101F7; Ery^r), or (C) the spa-lacZ (PC203; Ery^r) reporter strain of S. aureus were exposed to 20 μL (in pre-drilled wells) of supernatants from centrifugation (8000 rpm for 60 s) of overnight cultures of strains 27472 (Staphylococcus intermedius), 28993 (Staphylococcus haemolyticus), 30755 (Staphylococcus pseudintermedius), 30743 (Staphylococcus schleiferi), 29886 (Staphylococcus delphini), 30106 (Staphylococcus warneri) and 2898 (Staphylococcus schleiferi). H₂O was used as a control. Zones appeared between 9 and 36 h of incubation at 37°C. This figure is representative of one set of screening plates.

FIGURE 2

Quantification of interference by S. schleiferi on RNAIII expression in S. aureus. (A) RT-qPCR quantitative verification of RNAIII downregulation in the S. aureus 8325-4 strain. S. aureus in 15 mL TSB was grown to an OD₆₀₀ = 0.35 and supplemented with a 1:10 volume of S. schleiferi 2898 supernatant. Samples were collected 30 and 60 min post-exposure to S. schleiferi supernatant, RNA was isolated, cDNA prepared (and RNAIII levels were quantified. Interference of S. schleiferi supernatant on S. aureus agr was monitored using the strains (B) RN10829(P2-agrA:P3-blaZ)/pagrC-I (WT) and (C) RN10829(P2-agrA:P3-blaZ)/pagrC-I-R23H (AgrC const.) Reporter strains were grown to an OD₆₀₀ of 0.4–0.5 where a 1/10 volume of AIP-I containing supernatant from strain 8325-4 and 1/10 S. schleiferi supernatant were added to the reporter strain culture. Samples obtained at 30 min time intervals after addition of test solutions were analyzed for β-lactamase activity by nitrocefin conversion (Nielsen et al., 2014). Each bar represents the average of 3 biological replicates and the error bars represent the standard deviation.)

FIGURE 3

Staphylococcus schleiferi supernatant affects the activity of all four S. aureus agr types. Activity of four S. aureus agr group reporters (agr-I, agr-II, agr-III, and agr-IV) assessed by P3 expression (P3-yfp) measured as YFP accumulation by flow cytometry. Non-fluorescent, exponential phase cells were grown with (blue) or without (red) 20% S. schleiferi 2898 supernatant until stationary phase. Depicted fluorescence histograms originate from cells analyzed at the 24 h time point.

FIGURE 4

Staphylococcus schleiferi AIP interferes with S. aureus agr. (A) RNAIII expression was recorded as β-lactamase expressed from the P3-blaZ reporter fusion in S. aureus RN10829(P2-agrA:P3-blaZ)/pagrC-I (WT) with addition of 5% AIP-I supernatant and 20% supernatant from either strain 8325-4Δagr/pRAB12-agrBD_Ss (AIP_Ss) or the control strain 8325-4Δagr/pRAB12-lacZ (vector control) that had been grown and induced (0.2 μg/ml anhydrotetracycline) overnight. Each bar represents the average of three biological replicates and the error bars represent the standard deviation. (B) P3-blaZ expression recorded from S. aureus RN10829(P2-agrA:P3-blaZ)/pagrC-I (WT) when the inducing AIP-I containing supernatant (10%) is challenged for 45 min with different concentrations of synthetic S. schleiferi AIP at indicated concentrations. No induction and AIP-I containing supernatant alone was included as controls. Each bar represents the average of three biological replicates and the error bars represent the standard deviation.

FIGURE 5

Staphylococcus schleiferi inhibits S. aureus agr during co-culture without influencing growth. (A) S. aureus rnaIII:lacZ reporter strain SH101F7 grown alone or in co-culture (1:1) with S. schleiferi strain 30743 or 2898 in TSB. β-galactosidase activity under each condition was determined to monitor agr induction. Each bar represents the average of three replicates and the error bars represent the standard error of the mean. (B) Growth of the bacterial cultures measured as colony forming units (CFUs)/mL monitored in parallel for each time point. Distinction between the two species in the co-culture was made by plating on TSA substituted with X-gal, resulting in the SH101F7 reporter strain growing as blue colonies, while the S. schleiferi growing as white.

FIGURE 6

Dual presence of S. schleiferi and S. aureus influence colonization capabilities in a Galleria mellonella infection model. Fifth-instar G. mellonella larvae were inoculated with a total of 2 × 10⁷ CFU/mL of S. aureus (SH101F7), S. schleiferi (2898) or a co-culture of the two strains (to a combined final CFU/mL of 2 × 10⁷) and split into groups for CFU counting (35 per group) and survival benefit observation (20 per group). (A) At 24, 48, and 72 h post-inoculation, the hemolymph of the larvae was collected for CFU determination. Colonies were counted after O/N incubation at 37°C on TSA containing erythromycin and X-gal, as both strains are erythromycin resistant. (B) Survival of the larvae was monitored at the same time points as hemolymph collection. This experiment was repeated three times with similar results.

FIGURE 7

agrD homology of selected Staphylococci. Isolates were analyzed for agrD homology by Illumina sequencing; (A) Table showing AIP-alignment. The AIP region is highlighted in red. S.I.G.: Staphylococcus intermedius Group; S.A.G.: S. aureus Group; Strains 8325-4, RN6607, MOZ53, and RN4580 were included as S. aureus AIP sequence references. (B) Phylogenetic analysis based on a Muscle alignment of the AIP sequences with midpoint rooting. Scale bar indicate substitutions per site.