Nadira Nurxat,Lili Wang,Qichen Wang,Shujing Li,Chen Jin,Yaran Shi,Ayjiamali Wulamu,Na Zhao,Yanan Wang,Hua Wang,Min Li,Qian Liu
Commensal Staphylococcus epidermidis Defends against Staphylococcus aureus through SaeRS Two-Component System
Staphylococcus aureus is a high-virulent Gram-positive pathogen that is responsible for a serious of diseases. The emergence of antibiotic-resistant S. aureus poses a significant challenge in terms of treatment. The recent research on the human microbiome suggested that the application of commensal bacteria is a new strategy for combating pathogenic infections. Staphylococcus epidermidis, one of the most abundant species in the nasal microbiome, is able to inhibit the colonization of S. aureus. However, during bacterial competition, S. aureus undergoes evolutionary changes to adapt to the diverse environment. Our study has demonstrated that the nasal colonized S. epidermidis possesses the ability to inhibit the hemolytic activity of S. aureus. Moreover, we deciphered another layer of mechanism to inhibit S. aureus colonization by S. epidermidis. The active component present in the cell-free culture of S. epidermidis was found to significantly reduce the hemolytic activity of S. aureus in SaeRS- and Agr-dependent manner. Specifically, the hemolytic inhibition on the S. aureus Agr-I type by S. epidermidis is primarily dependent on the SaeRS two-component system. The active component is characterized as a small molecule that is heat sensitive and protease resistant. Critically, S. epidermidis significantly inhibit the virulence of S. aureus in a mouse skin abscess model, suggesting that the active compound could potentially be used as a therapeutic agent for managing S. aureus infections.
Commensal Staphylococcus epidermidis Defends against Staphylococcus aureus through SaeRS Two-Component System

Staphylococcus aureus is a high-virulent Gram-positive pathogen that is responsible for a serious of diseases. The emergence of antibiotic-resistant S. aureus poses a significant challenge in terms of treatment. The recent research on the human microbiome suggested that the application of commensal bacteria is a new strategy for combating pathogenic infections. Staphylococcus epidermidis, one of the most abundant species in the nasal microbiome, is able to inhibit the colonization of S. aureus. However, during bacterial competition, S. aureus undergoes evolutionary changes to adapt to the diverse environment. Our study has demonstrated that the nasal colonized S. epidermidis possesses the ability to inhibit the hemolytic activity of S. aureus. Moreover, we deciphered another layer of mechanism to inhibit S. aureus colonization by S. epidermidis. The active component present in the cell-free culture of S. epidermidis was found to significantly reduce the hemolytic activity of S. aureus in SaeRS- and Agr-dependent manner. Specifically, the hemolytic inhibition on the S. aureus Agr-I type by S. epidermidis is primarily dependent on the SaeRS two-component system. The active component is characterized as a small molecule that is heat sensitive and protease resistant. Critically, S. epidermidis significantly inhibit the virulence of S. aureus in a mouse skin abscess model, suggesting that the active compound could potentially be used as a therapeutic agent for managing S. aureus infections.

The extensive investigation of the human microbiome has led to the recognition of commensal bacteria as a crucial anti-infective strategy. The human commensal affect the susceptibility to host infectious diseases by several mechanisms including secreting antimicrobial peptides, promoting human immune system, etc. Coagulase-negative Staphylococci (CoNS) play a crucial role in defending against the nasal colonization of pathogens in the respiratory tract.Staphylococcus epidermidis is one of the most prevalent species found in the human nasal microbiome.S. epidermidis not only contributes to wound repair by promoting host immunity but also eradicates pathogens by inducing the production of antimicrobial peptides in nasal epithelial cells. Moreover, S. epidermidis affects the pathogens through direct interaction. Phenol-soluble modulins (PSMs) produced by S. epidermidis displayed antimicrobial activity against pathogens including Staphylococcus aureus.S. epidermidis disrupts biofilm formation of S. aureus by producing serine protease Esp or some unknown small molecules. S. aureus is a common Gram-positive pathogen causing a series of infectious diseases, ranging from skin and soft tissue infection to bacteremia. The pathogenesis of S. aureus is attributed to the production of virulence factors, which are controlled by several regulatory systems including the SaeRS two-component system (TCS), accessory gene regulator (Agr) quorum sensing system, SarA transcription factor, etc. Meanwhile, S. aureus can asymptomatically colonize in human nostrils, skin, and other mucosal surfaces, which is considered a risk factor for subsequent infections. During the interaction with commensal bacteria, S. aureus adapts to different environments through various mechanisms. The naturally occurring polymorphism of virulence regulators is advantageous for the colonization of S. aureus. Autoinducing peptide (AIP) originated from CoNS, including S. epidermidis or Staphylococcus caprae, have been reported to cross-inhibit the Agr system of S. aureus. It has been observed that Agr-defective S. aureus strains are frequently found in human nares, suggesting that S. aureus adopts a commensal lifestyle by suppressing the expression of key virulence regulators. Based on our previous findings, it appears that the activity of the SaeRS TCS contributes to nasal colonization. Specifically, we observed enhanced survival of strains with reduced SaeRS TCS activity in the nares of human subjects. SaeRS TCS promotes the pathogenesis of S. aureus by controlling the expression of over 20 virulence factors including α-hemolysin (hla). As one of the representative virulence factors, Hla is responsible for the hemolytic activity of S. aureus by disrupting red blood cells. We hypothesized that S. aureus SaeRS TCS might involve in the competition with commensal bacteria. In this study, we investigated the potential mechanisms underlying the inhibition of S. aureus by S. epidermidis in healthy individuals through in vitro screening the hemolytic activity of S. aureus and in vivo animal models.

Our previous findings have indicated that the production of antimicrobials by S. epidermidis is not the primary mechanism involved in its interaction with S. aureus. In order to investigate whether S. epidermidis promotes the transition of S. aureus to a commensal state, we collected the spent culture of 210 commensal S. epidermidis isolates and co-cultured them with S. aureus ST398-2012-3 for a period of 24 h. Subsequently, we evaluated the growth, hemolytic ability, and Agr activity of S. aureus (Figure 1A). Our in vitro co-culture experiments revealed that only one S. epidermidis isolate was able to significantly inhibit the growth of S. aureus (Figure 1B). Out of the 210 S. epidermidis isolates tested, approximately 46.7% (98/210) were found to inhibit the hemolytic activity of S. aureus by 50%. However, only 8 isolates were found to inhibit the Agr activity of S. aureus by 50% (Figure 1B), indicating that commensal S. epidermidis can inhibit the hemolytic activity of S. aureus through both Agr-dependent and Agr-independent mechanisms.

Our study demonstrated that commensal S. epidermidis can inhibit the hemolytic activity of S. aureus ST398-2012-3. To investigate whether S. epidermidis can also affect S. aureus strains isolated from the human nares, we randomly selected one commensal S. epidermidis isolate, N77-1, which exhibited moderate hemolytic activity. The sequence type for N77-1 was ST520, and it was found to inhibit the hemolytic activity of ST398-2012-3 by 50% (Figure 1B). We also randomly selected 36 commensal S. aureus isolates and evaluated their hemolytic activity. Our results showed a significant decrease in the hemolytic ability of S. aureus when co-cultured with the spent culture of S. epidermidis N77-1 (Figure 2A), suggesting that the inhibitory effect of S. epidermidis on the hemolytic activity of S. aureus is a general phenomenon. The Agr system in S. aureus has been classified into four types (I–IV) based on the polymorphism in the amino acid sequences of Agr. The hemolytic activity of all four Agr types of S. aureus strains was evaluated when co-cultured with the spent culture of S. epidermidis N77-1. Surprisingly, the hemolytic ability of S. aureus strains with Agr-I and -III was significantly reduced in the presence of S. epidermidis N77-1 (Figure 2B). Despite the significant decrease in the hemolytic activity of S. aureus Agr-I type in the presence of Staphylococcus epidermis N77-1, the transcription of RNAIII remained unaffected (Figure 2C), suggesting that the observed reduction in the hemolytic activity of S. aureus Agr-I type is not dependent on Agr activity.

In order to investigate the mechanism underlying the observed decrease in the hemolytic activity of S. aureus in response to commensal S. epidermidis, we examined the transcription levels of the main regulators, including RNAIII, rot, rsp, sarA, and saeS using RT-PCR when co-cultured with the spent culture of S. epidermis N77-1. The transcription levels of RNAIII, rot, rsp, and sarA were not affected by the presence of S. epidermidis N77-1 (Figure 3A). Interestingly, we observed a significantly decreased expression of saeS of S. aureus co-cultured with S. epidermidis (Figure 3B). Consistent with this finding, we also observed a significant inhibition of hla transcription in the presence of S. epidermidis (Figure 3B). These results suggest that the commensal S. epidermidis inhibits the hemolytic activity of S. aureus by down-regulating the activity of the SaeRS TCS.

By far, we observed that the spent culture of S. epidermidis inhibits the hemolytic activity of S. aureus by down-regulating the activity of the SaeRS TCS. To identify the active component secreted by S. epidermidis, the spent culture of S. epidermidis was fractionated into four fragments based on molecular weight using ultrafiltration spin columns (Millipore): <3, 3–10, 10–100, and >100 kDa. Surprisingly, all four fragments significantly inhibited the hemolytic activity of S. aureus. Notably, the fragment with a molecular weight of less than 3 kDa displayed the most potent inhibitory effect (Figure 4A). Next, we characterized the active small molecules secreted by S. epidermidis. Heat-killed (95 °C for 20 min) spent culture of S. epidermidis failed to inhibit the hemolytic activity of S. aureus (Figure 4B), indicating that the active molecules are likely to be heat sensitive. Interestingly, treatment with protease K did not affect the inhibitory effect of the spent culture on S. aureus (Figure 4B), suggesting that the active molecules are not proteins. In summary, these results suggested that S. epidermidis down-regulates the SaeRS TCS of S. aureus through the secretion of heat-sensitive small molecules.

The SaeRS TCS activity is essential for S. aureus virulence. To investigate whether the inhibition of SaeRS TCS activity by S. epidermidis contributes to the virulence of S. aureus in vivo, we co-infected S. epidermidis N77-1 and S. aureus ST398-2012-3 strains in a murine skin abscess model. The co-infection of S. epidermidis and S. aureus resulted in significantly smaller skin lesions. On day 2 post-infection, the average abscess area caused by the S. aureus group was 195.3 mm2, while the abscesses caused by strains from the co-infection group was 34.9 mm2 on average (Figure 5A,B). In order to verify the involvement of S. epidermidis in the decreased virulence of S. aureus, we implemented another co-infection group: S. aureus with heat-killed S. epidermidis. Notably, the co-infection of the heat-killed S. epidermidis N77-1 and S. aureus led to a significantly larger average abscess area of 169.7 mm2 compared to the co-infection of regular S. epidermidis and S. aureus (Figure 5A,B). Although the bacterial loads of S. aureus were not affected in the presence of S. epidermidis (Figure 5C), the histological analysis displayed more severe inflammation in the skin tissues of mice infected with S. aureus alone or with S. aureus and heat-killed S. epidermidis N77-1 compared to those with regular S. epidermidis and S. aureus (Figure 5D). These data suggested that S. epidermidis inhibits the virulence of S. aureus.

With the prevalence of antibiotic-resistant S. aureus, it is urgent to develop new antibacterial strategies. CoNS, especially S. epidermidis, develop an arsenal of mechanisms defending against S. aureus. In the study, we elucidated that heat-sensitive small molecules secreted by S. epidermidis effectively inhibit the hemolytic activity of S. aureus by down-regulating the SaeRS TCS. Critically, S. epidermidis is capable of efficiently inhibiting S. aureus virulence in vivo. In this study, we have demonstrated that the bioactive component secreted by S. epidermidis exerts a direct inhibitory effect on the hemolytic activity of S. aureus (Figure 2). The human nares have been identified as a reservoir of S. aureus, serving as a primary source for subsequent infection. Nasal commensals have developed several strategies to defend against the colonization of S. aureus. The antimicrobial peptides produced by Staphylococcus lugdunensis and Staphylococcus hominis prohibit S. aureus growth directly.S. caprae prevents the colonization of S. aureus by blocking Agr the quorum sensing system. As the most abundant species in human nares, S. epidermidis defends against pathogens by promoting immune defense. For example, S. epidermidis inhibits the colonization of S. aureus or Moraxella catarrhalis by inducing the production of antimicrobial peptides of host cells.S. epidermidis plays a role in antiviral response by promoting interferon-lambda-dependent immunity. Furthermore, the commensal strain of S. epidermidis is able to produce molecules with the anti-biofilm activity. All of these studies highlight the search for compounds that can aid people in the fight against S. aureus infection. In the present study, around 46.7% commensal S. epidermidis isolates inhibit the hemolytic activity of S. aureus Agr-I ST398-2012-3 (Figure 1). The cross-inhibition of the Agr system between CoNS and S. aureus has been reported extensively. Agr, one of the main regulators for the hemolytic activity of S. aureus, is deemed as the most popular mechanism during pathogen competition. By testing the RNAIII transcription of S. aureus with varying Agr types, we observed that the Agr activity in S. aureus ST398-2012-3 (Agr-I) remains unaffected in the presence of S. epidermidis (Figure 2). Most of the nasal colonized S. aureus isolates were Agr-I type (74.4%), while around 11.7 and 6.7% S. aureus belong to Agr-II and Agr-III types (data not shown). Our results suggested that the inhibition of nasal colonized S. aureus by S. epidermidis is not totally dependent on the Agr system. Our current investigation revealed that the small molecular secretion by S. epidermidis is resistant to the degradation by protease K, implying that the inhibitory effect is not derived from a proteinaceous molecule. One limitation of our study is that we did not identify the specific small molecules responsible for the inhibitory effects of the SaeRS TCS in S. aureus. SaeRS TCS is a regulatory system that has been shown to contribute to the virulence of S. aureus. SaeS, a histidine kinase sensor, autophosphorylates a conserved histidine residue upon sensing external stimuli. The phosphate group can be transferred to a conserved aspartic residue of the response regulator SaeR. The phosphorylated SaeR regulates the expression of a variety of virulence factors by binding to their promoter regions. Although the histidine kinase SaeS sense the environment by the extracellular linker peptide, the exact mechanism of how SaeS recognize the signals is still unknown. The S. aureus SaeRS system can be affected by host signals or environmental cues. SaeS is activated by human neutrophil peptides 1, which may contribute to the defense against neutrophils. The silkworm apolipophorin protein can inhibit S. aureus virulence through SaeRS TCS. The skin surface with a low pH (5.5) inhibits the Sae activity. The molecule 6-N-hydroxyaminopurine (6-HAP) produced by S. epidermidis protects against skin neoplasia by inhibiting the DNA polymerase activity. The metabolite short-chain fatty acids (SCFAs) including acetate, propionate, and butyrate existed in the fermentation of Cutibacterium acnes and S. epidermidis isolates by gas chromatography–mass spectrometry (GC–MS) analysis. The bacterial fatty acid inhibits the SaeS activity by affecting the sensing of the SaeS transmembrane domain. The human skin fatty acid cis-6-hexadecenoic acid also displays high antistaphylococcal activity by repressing the SaeRS TCS. It is possible that a particular heat-sensitive metabolite produced by S. epidermidis may contribute to the suppression of S. aureus. However, the precise identity of the molecule involved requires further elucidation. In conclusion, our study confirmed that the commensal S. epidermidis suppresses the hemolytic activity of S. aureus by suppressing the expression of hla, which is regulated by the SaeRS TCS. We observed that the SaeRS TCS also plays a role in regulating the bacterial competition. Furthermore, the inhibition of the SaeRS TCS by S. epidermidis was observed to impact the virulence of S. aureus in a mouse model, indicating a promising therapeutic approach for defending against S. aureus infections.

This study was approved by the Ethics Committee of Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. All individuals or their legal guardians were provided an informed consent. The animal experiments were performed following the Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. As reported previously, we isolated S. epidermidis and S. aureus from the nasal swabs of healthy volunteers with different ages. In total, 210 randomly selected S. epidermidis isolates and 36 randomly selected S. aureus isolates were used for the co-culture assay. For RT-PCR and animal experiments, S. aureus ST398-2012-3 and S. epidermidis N77-1 (this study) were used for the experiments. Generally, S. aureus isolates were grown in a tryptic soy broth (TSB; Oxoid) with shaking at 200 rpm at 37 °C.

S. epidermidis was cultured for 18 h. After centrifuging (4000g, 10 min), the supernatant was collected and filtered through a 0.22 μm container (spent culture). Fresh TSB containing 10% spent culture of S. epidermidis was used to culture S. aureus isolates. When necessary, the spent culture was heat-killed at 95 °C for 20 min or treated with protease K (PK 50 μg/mL) at 37 °C for 1 h.

Supernatants of S. aureus overnight cultures were collected and incubated with human erythrocytes (2% v/v in phosphate buffered saline, PBS) for 1 h at 37 °C in a 96-well round-bottom plate. After centrifugation (1500g, 10 min), the supernatant (100 μL) was pipetted into another sterile 96-well flat-bottom plate. The optical density was determined at 540 nm by using an enzyme-linked immunosorbent assay reader (Synergy; BioTek).

The report plasmid pOS1-P2-gfp for the Agr activity was constructed in our previous study.S. aureus was cultured overnight; the bacteria pellets were collected and incubated with lysostaphin (50 μg/mL) at 37 °C for 1 h. Lysates were then diluted to 1 mL PBS. 200 μL liquid was pipetted into a black 96-well microtiter plate in triplicate. Fluorescence (485 nm excitation, 538 nm emission) was measured on an enzyme-linked immunosorbent assay reader (Synergy; BioTek) and normalized by OD600.

Bacteria were cultured in TSB overnight. The overnight cultures were diluted (1:100) in fresh TSB or TSB with 10% spent culture of S. epidermidis and grown for 8 h at 37 °C with shaking. The pellets were collected by centrifuge at 4000g for 10 min and broken by FastPrep-24 (MP 116005500). After centrifugation, the supernatant was used to isolate the total RNA and further synthesize complementary DNA according to the manufacturer’s instructions (Qiagen). The cDNA was used as a template for real-time PCR using SYBR-green PCR reagents (Roche) with primers listed in Table S1. Reactions were performed in a MicroAmp Optical 96-well reaction plate using a 7500 Sequence Detector (Applied Biosystems). All qRT-PCR experiments were performed in triplicate using gyrB as an internal control gene at the mRNA level.

S. aureus and S. epidermidis were cultured overnight in TSB. The next day, the overnight culture was diluted at 1:100 in fresh TSB and grown at 37 °C for 4 h. After centrifugation (6000 rpm, 10 min), the bacteria pellets were washed twice with sterile PBS and suspended in PBS. When necessary, S. epidermidis was heat-killed at 95 °C for 20 min. Outbred, immune-competent hairless male mice (4–6 weeks old) were anesthetized with Avertin (Sigma T48402) by intraperitoneal injection and administered with S. aureus (108 cfu in 100 μL of PBS) by s.c. injection on the back. The skin lesion size was measured by length (L) and width (W). On the 2nd day, mice were killed by anesthesia overdose, and the infected skin tissue was excised and homogenized in 500 μL PBS. The homogenized skin tissue was diluted and plated on 5% sheep blood agar to determine cfu. Skin tissues were fixed in 4% paraformaldehyde, paraffin-embedded, sectioned, and stained with hematoxylin & eosin (H&E) staining.

Statistical analysis was performed with the GraphPad Prism 7.0. For the comparison between the two groups, the unpaired, two-tailed Student’s t-test was performed for statistical analysis. One-way ANOVA was used for the comparison among the three-set data groups. Error bars indicated the standard deviation (±SD), and P values < 0.050 were considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001.