Decrease of Staphylococcus aureus Virulence by Helcococcus kunzii in a Caenorhabditis elegans Model

Abstract

Social bacterial interactions are considered essential in numerous infectious diseases, particularly in wounds. Foot ulcers are a common complication in diabetic patients and these ulcers become frequently infected. This infection is usually polymicrobial promoting cell-to-cell communications. Staphylococcus aureus is the most prevalent pathogen isolated. Its association with Helcococcus kunzii, commensal Gram-positive cocci, is frequently described. The aim of this study was to assess the impact of co-infection on virulence of both H. kunzii and S. aureus strains in a Caenorhabditis elegans model. To study the host response, qRT-PCRs targeting host defense genes were performed. We observed that H. kunzii strains harbored a very low (LT50: 5.7 days ± 0.4) or an absence of virulence (LT50: 6.9 days ± 0.5). In contrast, S. aureus strains (LT50: 2.9 days ± 0.4) were significantly more virulent than all H. kunzii (P < 0.001). When H. kunzii and S. aureus strains were associated, H. kunzii significantly reduced the virulence of the S. aureus strain in nematodes (LT50 between 4.4 and 5.2 days; P < 0.001). To evaluate the impact of these strains on host response, transcriptomic analysis showed that the ingestion of S. aureus led to a strong induction of defense genes (lys-5, sodh-1, and cyp-37B1) while H. kunzii did not. No statistical difference of host response genes expression was observed when C. elegans were infected with either S. aureus alone or with S. aureus + H. kunzii. Moreover, two well-characterized virulence factors (hla and agr) present in S. aureus were down-regulated when S. aureus were co-infected with H. kunzii. This study showed that H. kunzii decreased the virulence of S. aureus without modifying directly the host defense response. Factor(s) produced by this bacterium modulating the staphylococci virulence must be investigated.

Keywords: Caenorhabditis elegans; Helcococcus kunzii; Staphylococcus aureus; attenuation; co-infection; virulence.

Figure 1

In vivo kinetics of killing of C. elegans infected by S. aureus NSA739, H. kunzii H13, and co-infected by the two strains. OP50 represents the survival curve for worms fed on non-pathogenic E. coli. In all cases, worms were grown on NGM plates at 25°C and ≈ 30 Fer-15 were used in each test. The curves are representative of at least three independent trials for each group of strains.

Figure 2

Evaluation of feeding behavior by measuring bacterial content of C. elegans. Each graph represents the median of CFU count per worm found in each species alone (the H. kunzii H13, the S. aureus NSA739, and the E. coli OP50) or in co-infection (H. kunzii H13 + S. aureus NSA739). Three replicates were performed for each strain alone or in association. Differences in CFU rates were tested by a Pearson normality test. These results are representative of the data obtained for all the strains evaluated in this study.

Figure 3

C. elegans L4 host response to S. aureus and H. kunzii infections alone or in association at 12 h post infection. The figure represents the expression of six main genes involved in C. elegans immune response: cyp-37B1 (A), hlh-30 (B), lys-5 (C), sodh-1 (D), clec-7 (E), lgg-1 (F). Data are expressed as the mean ± sd of three biological replicates of qRT-PCR results. All Ct-values are normalized against the housekeeping gene snb-1. Data analysis was performed with the Pfaffl method (Pfaffl, 2001). The P-value represents the comparison between genes expression found in H. kunzii strains alone and the other combination (S. aureus alone, co-infection H. kunzii+S. aureus) ^*P < 0.05; ^**P < 0.01; ^***P < 0.001.

Figure 4

Relative mRNA expression level of genes implicated in virulence (hla, spa) and virulence regulation (agr) for S. aureus NSA739 co-infected with H. kunzii H13. The log-transformed averages of relative fold change of S. aureus+H. kunzii co-infection compared to S. aureus alone are presented. The error bars represent the standard deviation from three different RNA preparations. Significant differences from S. aureus co-infected with H. kunzii using Dunnett's test are indicated by ^**(p < 0.01) and ^*** (p < 0.001).