SarZ negatively regulates the lipase activity in Staphylococcus epidermidis

doi:10.21203/rs.3.rs-7510667/v1

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SarZ negatively regulates the lipase activity in Staphylococcus epidermidis

https://doi.org/10.21203/rs.3.rs-7510667/v1

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S. epidermidis plays a crucial role in maintaining the skin immunity barrier. However, when host immunity is compromised, it can also lead to skin infections and bloodstream infections. Staphylococcal lipases contribute to bacterial growth, detoxification, and immune evasion, while their esterification capabilities also give them potential biotechnological applications. S. epidermidis secretes at least two lipases, GehC and GehD, which are indirectly regulated by the global regulators Agr and SarA. SarZ, a transcription factor of the SarA family, regulates the expression of various exoproteins, but its role in regulation of lipase synthesis remains unknown. A sarZ gene knockout strain of S epidermidis previously constructed was utilized in this study. First, lipase activity was found to be significantly elevated in the sarZ mutant relative to the wild-type strain, as determined by both the olive oil agar plate assay and the p-nitrophenol assay. Subsequently, qRT-PCR experiments revealed that SarZ controls the transcription of gehC and gehD divergently. Furthermore, EMSA experiments demonstrated that the recombinant SarZ protein can directly bind to the promoter regions of gehC and gehD. These findings demonstrate that SarZ negatively regulates lipase activity by directly modulating expression of lipase genes, providing a basis for further understanding the regulatory mechanism of lipase production in S. epidermidis.

Staphylococcus epidermidis

SarZ

transcription factor

lipase

The skin harbors a rich symbiotic microbial community, including bacteria and fungi, which play a crucial role in maintaining the skin's immune barrier function.[1–4]. Whether through traditional isolation and culture methods or metagenomic sequencing, S. epidermidis has been identified as one of the most prevalent species[5, 6], Studies revealed that early colonization of S. epidermidis on the skin can foster immune tolerance to commensal microbiota through activation of regulatory T cells and mucosal-associated invariant T cells, while maintaining pathogen-specific immune responses, thereby facilitating the development and maturation of the skin immune system[7]. It can also enhance skin barrier homeostasis by secreting sphingomyelinase, which increases ceramide content on the skin and strengthens tight junctions between keratinocytes[8]. Additionally, studies have proven that it can accelerate skin wound healing by stimulating the secretion of type II cytokines and recruiting plasmacytoid dendritic cells, which produce type I interferon[9, 10]. Interestingly, S. epidermidis even has an effect against skin neoplasia[11].

However, in addition to its role as a probiotic bacterium, S. epidermidis often leads a dual lifestyle as an opportunistic pathogen due to its strain diversity (more aggressive isolates) and encounters with immunocompromised hosts. Fluctuations in the levels of S. epidermidis colonization correlate with a variety of skin diseases, including atopic dermatitis[12, 13], dandruff[14, 15], seborrheic dermatitis[16, 17] and rosacea[18, 19]. Moreover, S. epidermidis is the leading pathogen responsible for implant-associated infections[20]. These infections often necessitate surgical removal of the implant as the feasible treatment, thereby posing an additional burden to healthcare systems[21]. S. epidermidis also causes 30%-40% of hospital-acquired bloodstream infections, most of which originate from catheter-related infections that subsequently disseminate into the bloodstream[22]. Bloodstream infection caused by S. epidermidis is also the primary cause of late-onset sepsis in preterm neonates[23].

Regardless of its role, S. epidermidis must colonize the skin first. Give that the skin is rich in lipids, primarily consisting of sebum-derived triglycerides[6, 24], it is reasonable to infer that S. epidermidis is capable of producing lipase, which can liberate free fatty acids by hydrolyzing sebum to fulfill its nutritional requirement. Furthermore, lipases can be utilized by Staphylococcus to penetrate the hydrophobic barrier of sebum to settle into hair follicles[25]. It can also exert a detoxification function by catalyzing the esterification reaction between antibacterial fatty acids[26] and cholesterol[27]. Although the role of lipase in infection and dissemination remains incompletely elucidated, clinical study has revealed its participation in the pathogenicity of Staphylococcus. It has been disclosed that the lipase produced by Staphylococcus aureus can evade the recognition of the host's innate immune system and mitigate subsequent pro-inflammatory responses by hydrolyzing its own lipoprotein, a principal extracellular pathogen-associated molecular pattern[28]. Recent studies have shown that lipase can significantly decelerate the skin wound healing process in mice infected with S. aureus, which corroborates the aforementioned conclusion. Moreover, it has been demonstrated that some lipases also possess collagen-binding properties[29], which can facilitate persistent colonization on the surface of host skin and medical devices, thereby promoting biofilm formation in both S. epidermidis and S. aureus.

S. epidermidis expresses at least two lipases, encoded by the gehC and gehD genes[30]. Lipase is highly conserved in Staphylococcus, with similar protein sequences that typically comprising three main parts: signal peptide (35 ~ 38 amino acids), pro-peptide (207 ~ 321 amino acids) and mature peptide (383 ~ 396 amino acids)[31]. The lipase precursor requires processing by the metalloprotease (aureolysin) prior to its secretion and conversion into a functionally mature form[32]. Currently, molecular mechanism underlying the regulation of lipase gene expression in Staphylococcus remains elusive. It was suggested that the expression of lipase was modulated by the global regulators Agr and SarA[33, 34], and the mutant strains lacking Agr or SarA exhibited obvious defects in lipase activity. Further investigations have indicated that the regulation of Agr and SarA on lipase activity is achieved by regulating the expression of aureolysin and thus affecting the maturation of lipase, rather than directly impacting the transcription of lipase genes.

The transcription factor SarZ, a member of the SarA protein family in Staphylococcus, has been reported to serves as a key regulator of tissue colonization and dissemination of infection[35]. In S. aureus, SarZ modulates the responses to oxidative stress[36] and regulates the expression of virulence factors[37], particularly exoproteins such as hemolysins and proteases. In S. epidermidis, it has been revealed that SarZ inhibits the expression of phenol-soluble modulins (PSMs) while enhancing protease secretion[38]. However, it remains unclear whether SarZ affects the expression of lipase genes. Therefore, based on the sarZ knockout mutant of S. epidermidis constructed in our laboratory, the present study further explored whether SarZ exerts a regulatory effect on the exoprotein-lipase via enzymatic activity and molecular biology experiments, providing a new perspective for in-depth comprehension of the symbiotic or pathogenic mechanism of S. epidermidis.

Bacterial strains and media

S. epidermidis RP62A (WT) was preserved in our laboratory, and its derivative sarZ knockout strain (ΔsarZ), complementation strain (pCNcat-sarZ) and empty vector strain (pCNcat) were generated by our laboratory previously. All strains were conventionally cultured in Tryptic Soy Broth (TSB) at 37°C with shaking at 220 rpm. Chloramphenicol was added to a final concentration of 10 µg·ml^− 1 when necessary. The nucleotide sequence of the lip gene from S. epidermidis strain RP62A analyzed in this study is available under Ref-Seq genome accession number NC_002976.3 in the GenBank database at the National Center for Biotechnology Information (NCBI). The lipases gene can be located within the sequence using the annotated gene locus tag SERP2297, SERP2388, SERP0018.

https://www.ncbi.nlm.nih.gov/nuccore/NC_002976.3

Olive oil agar plate assay

The arabic gum solution was formulated as follows: 10% Arabic gum (w/v); 200 mM NaCl; and 50 mM CaCl₂. The olive oil agar plate[39] was prepared according to the following composition: 10% arabic gum solution (v/v); 1% tryptone (w/v); 0.5% yeast extract (w/v); 0.5% NaCl (w/v); 1.5% agar (w/v); and 1% (v/v) olive oil. Olive oil and the other components in the medium should be autoclaved separately and then fully emulsified using a homogenizer. S. epidermidis RP62A and its sarZ mutant derivatives were spread onto olive oil agar plates and cultured at 37°C for 24 h to observe whether clear zones of hydrolysis were formed around the colonies.

p-nitrophenol assay

Single colonies of S. epidermidis RP62A and its sarZ mutant derivatives were inoculated respectively into SPM medium (3% tryptone; 1% yeast extract; 0.5% NaCl; 0.1% (v/v) tributyrin; 0.01% CaCl₂·2H₂O)[40] for overnight culture. The overnight cultures were adjusted to an initial OD₆₀₀ of 0.1 in fresh SPM medium containing 4 µM CdCl₂ (for the induction of expression of sarZ gene in the complementation strain), and then incubated at 37°C with shaking at 220 rpm for 24 h. The bacterial cultures were centrifuged and the supernatants were collected as the crude enzyme extract. Lipase activity was quantified using p-nitrophenyl palmitate (pNPP) or p-nitrophenyl butyrate (pNPB) as substrate according to the procedure described by Gupta[41] with minor modifications. Briefly, 900 µl of 50 mM Tris-HCl buffer (pH 8.0) containing 0.5 mM PEG 8000 was pipetted into a 2 ml centrifuge tube, and then 100 µl of 6 mM pNPP / pNPB (prepared in isopropanol) was added and mixed thoroughly. Subsequently, 100 µl of crude enzyme extract was introduced to initiate the reaction. After incubation at 37°C for 5 minutes, the reaction was terminated by adding 100 µl of absolute ethanol. The reaction mixture was centrifuged, and the supernatant was collected for determination of absorbance at 410 nm. Enzyme activity was calculated based on the p-nitrophenol (pNP) standard curve. One unit of enzymatic activity (U) is defined as the amount of enzyme that liberate 1 µmol of p-nitrophenol (pNP) per minute. The experiment consisted of three independent biological replicates.

RNA extraction and qRT-PCR

The extraction of total RNA from S. epidermidis RP62A and its isogenic sarZ knockout mutant was carried out as previously described in our laboratory. Overnight cultures of S. epidermidis were diluted 1:100 in TSB medium and cultivated at 37°C with shaking at 220 rpm. Bacterial cells were harvested at the exponential (4 h) and stationary phases (10 h), respectively, and immediately stabilized through mixing with RNAprotect Bacteria Reagent (Qiagen, cat #76,506) at a 2:1 (v/v) ratio. Mechanical disruption of bacteria cells was performed using a Mini-Beadbeater (Biospec) at maximum speed, and subsequent total RNA was extracted using the RNeasy® Mini kit (Qiagen, cat#74,104) in accordance with the manufacturer's instructions. To eliminate DNA contamination, on-column DNase digestion was performed using the RNase-Free DNase Set (Qiagen, cat #79,254). The concentration and purity of the extracted RNA were assessed both quantitatively and qualitatively using a BioDrop spectrophotometer and 1% agarose gel electrophoresis, respectively. Two micrograms of total RNA were reversely transcribed into cDNA using GoScript™ Reverse Transcription kit (Promega, cat#A2800) following the manufacturer's instructions. Afterward, 5 µl of cDNA was employed to conduct the qRT-PCR reactions using FastStart Essential DNA Green Master (Roche, cat #06924204001). The qRT-PCR reactions were run on the LightCycler® 96 instrument under the following conditions: 95°C for 30 s; 95°C for 15 s; 60°C for 25 s; 45 cycles. The gyrB gene was used as the internal reference gene for normalization, and the relative expression of the target gene was calculated by the 2^−ΔΔct method. Specific primers were designed using the software Primer Premier 5.0 (Table. 1). Each experiment was conducted with three independent biological replicates.

Electrophoretic mobility shift assay

The promoter regions of gehC, gehD and serp0018 were amplified using 5'-biotin-labeled primers (Table. 1). And the biotin-labeled DNA fragments were incubated with the gradually increasing concentrations of recombinant SarZ protein in binding buffer (Shanghai Beyotime Biotech, cat#GS005). For competition reactions, unlabeled competitor fragments were pre-incubated with SarZ at 100-fold or 200-fold molar excess relative to the labeled probe. Following the binding reaction, protein-DNA complexes were resolved by electrophoresis on a 5% native polyacrylamide gel. Then the biotin-labeled DNA was transferred to the positively charged nylon membrane (Shanghai Beyotime Biotech, cat#FFN10). Subsequently, the membrane was UV-irradiated for 30 minutes to immobilize the DNA. Finally, Chemiluminescent EMSA Kit (Shanghai Beyotime Biotech, cat#GS009) was used for detection and analysis. The rpsJ (encoding 30S ribosomal protein S10) was used as a negative control for SarZ-DNA binding. The primers of gehC, gehD, serp0018 and rpsJ were synthesized by Shanghai Sangon Biotech.

Table 1
Primers used in this study
Primer name	Sequence (5'→3')	Product size (bp)
Primers for qRT-PCR
serp 0018-F	GTTAATGATCTGACAACGCAAGGTG	94
serp 0018-R	TCGCTGACCCTGTATAAGTAGTGTAG	94
gehD-F	TTCTGCCATAACGCTTGTGACC	104
gehD-R	CAATTATGACCGTGCTGTTGAACTG	104
gehC-F	TTGTCGTCATCGTTCTTAGCAGTC	80
gehC-R	GATAGCCAATCAACAGAAGAGGTAAAC	80
gyrB-F	CACCGTGAAGACCGCCAGATAC	99
gyrB-R	AGATGGGACGCCCTGCTGTC	99
Primers for EMSA
P_{serp 0018} -F-biotin	CGCATGCTACTTTTTTCATTTAATGATAC	291
P_{serp 0018}-R-biotin	CAAAGAAATCACCTCTATAAGATTTTTCCC	291
P_gehD -F-biotin	ATCACCTCTATAAGTTTTTTCCCCT	232
P_gehD -R-biotin	CATGCTTCTTCATCATGTGTAATGA	232
P_gehC -F-biotin	GTCTTGTCTTCACAATTAGCACC	184
P_gehC -R-biotin	GATTATGGAAGCGTTTACCTTTGG	184
P_rpsJ -F-biotin	GATTATGGAAGCGTTTACCTTTGG	119
P_rpsJ -R-biotin	AAGATTCTCGTGAACAATTC	119

Statistical analysis

Experimental data obtained were analyzed using GraphPad Prism 8. The data between the two groups were compared by t test, and the data above the two groups were analyzed by one-way analysis of variance (ANOVA). All data are expressed as means ± the SEM, and P < 0.05 indicates that the difference is statistically significant.

Inactivation of sarZ gene led to increased lipase activity in S. epidermidis

Lipase plays an important role in bacterial growth and metabolism[42], colonization and adhesion[43], immune evasion[44] and dissemination of infection[45, 46] in Staphylococcus. Moreover, lipase can address the global shortage of Influenza A virus vaccines by enhancing Influenza A Virus Replication[47], which contributes to resolution of a major public health concern. And it can catalyze esterification, transesterification and transesterification in non-aqueous media, possesses favorable adaptability to pH and temperature, thereby presenting broad application potential in many industrial fields[48]. Considering its biological significance and its increasing role played in the field of biotechnology, it is imperative to investigate the regulatory mechanism governing expression of staphylococcal lipase. Since SarZ has been proven to regulate the expression of exoproteins in Staphylococcus[49] to explore whether it can also impact the expression of lipase, the olive oil agar plate assay was employed to preliminarily detect changes in lipase activity between the sarZ knockout strain and its parent strain. As shown in Fig. 1, the clear zone of hydrolysis surrounding the mutant strain on the olive oil agar plates was significantly larger than that of the wild-type strain, indicating that the lipase activity of the mutant strain was remarkably elevated.

To further validate this observation, the lipase activity in the culture supernatants of S. epidermidis was determined by p-nitrophenol assay using pNPP (representing long-chain fatty acid esters) and pNPB (representing short-chain fatty acid esters) as the substrates, respectively. In the presence of lipase, p-nitrophenyl ester can be hydrolyzed to release yellow p-nitrophenol. The compound exhibits maximum absorption at a wavelength of 410 nm. Consequently, the lipase activity can be calculated based on variation in the absorption value. As depicted in Fig. 2, whether pNPP (Fig. 2a and 2c) or pNPB (Fig. 2b and 2d) was used as the reaction substrate, the supernatants of the mutant strain showed remarkably higher lipase activity compared to that of the wild-type strain, whereas the lipase activity of the complementation strain was comparable to that of the wild strain. The introduction of empty vector had no effect on the lipase activity for the mutant strain, which further suggested that inactivation of SarZ could notably increase the lipase activity of S. epidermidis. To our knowledge, this is the first report demonstrating that SarZ can regulate the lipase activity in Staphylococcus, which enriches the understanding of the regulatory mechanism underlying lipase expression in Staphylococcus.

SarZ regulates the transcription of gehC and gehD genes in S. epidermidis

To elucidate whether the regulation of lipase activity by SarZ is achieved through the regulation of lipase genes expression, qRT-PCR was performed to compare the transcription levels of lipase genes between the mutant strain and the wild-type strain. Three lipase genes, namely two known genes (gehC and gehD) [50]and one putative gene (serp0018)[51] were chosen for measurement during both the exponential and stationary phases. As illustrated in Fig. 3a-c, during the exponential phase, the transcription levels of gehC and serp0018 in the mutant strain were 23-fold and 3.75-fold higher respectively, than those in the wild-type strain, Conversely, the transcription of gehD was downregulated by 2.5-fold. When the bacteria entered the stationary phase, as shown in Fig. 3d-e, the transcription levels of gehC and serp0018 in the mutant strain remained higher than those in the wild-type strain, with up-regulation of 35-fold and 4-fold, respectively. In contrast, the transcription level of gehD in the mutant strain was almost equivalent to that in the wild-type strain. This finding is consistent with a prior transcriptional analysis of a sarZ insertion mutant in S. epidermidis strain 1457 using gene microarrays[51]. These observations imply that SarZ modulates lipase activity through transcriptional regulation of lipase gene expression. Specifically, it represses the transcription of gehC and serp0018 and activate the transcription of gehD.

The gehC gene (SERP2297) is composed of 2067 nucleotides, encoding a lipase precursor protein with a molecular weight of about 77-kDa, which is converted into a 43-kDa[52, 53] mature lipase after processing. Biochemical characterization of the lipase revealed that the optimal pH for the enzyme is 6, and it exhibits high stability under low pH conditions. Its activity depends on the presence of calcium ions and prefers to decompose short-chain fatty acid esters[54]. The gehD gene (SERP2388) consists of 1932 nucleotides and encodes a lipase precursor with a molecular weight of approximately 72.2-kDa. Unlike GehC, GehD exhibits broader substrate specificity and is capable of hydrolyzing both long-chain and short-chain fatty acid esters, with a marked preference for long-chain substrates[55]. Although they display 52% amino acid identity in their mature parts, phylogenetic analysis indicated that they occupy distinct branches within the staphylococcal lipase family. GehC is evolutionarily closer to GehA[56] from S. aureus, whereas GehD exhibits a higher homology with GehB [25] in S. aureus.

It is noteworthy that the divergent regulation of lipase gene expression by SarZ contradicts the overall negative effect of SarZ on lipase activity observed in our experiments. We propose that this discrepancy may be attributed to GehC functioning as the primary lipase in S. epidermidis, with its transcription being subject to the most pronounced regulation by SarZ. This speculation is further supported by qRT-PCR analysis, indicating higher mRNA abundance of gehC compared to gehD, as well as lipase activity assays demonstrating significantly stronger hydrolysis of short-chain fatty acid esters than of long-chain substrates by the culture supernatants of S. epidermidis. Nonetheless, the physiological significance of SarZ-mediated upregulation of gehD transcription during the exponential phase remains to be clarified. The serp0018 gene (SERP0018) comprises 2046 nucleotides and encodes a putative lipase with a molecular weight of approximately 76-kDa. Therefore, the significant increase in lipase activity observed in the sarZ mutant strain may also be ascribed to the enhanced transcription of the serp0018 gene.

SarZ can directly bind to the promoter region of the lipase genes.

The SarZ protein possesses DNA-binding activity. In S. aureus, it can directly bind to the promoters of the hla and hlb genes, thereby modulating the expression of α-hemolysin and β-hemolysin[37]. This let us speculated that SarZ might directly regulate the expression of lipase genes through its role as a transcription factor in S. epidermidis. Therefore, EMSA experiment was conducted to clarify whether SarZ directly regulates the expression of lipase genes. Recombinant His-tagged SarZ protein was incubated with biotin-labeled promoter fragments of the lipase genes, followed by non-denaturing gel electrophoresis. As shown in Fig. 4, the promoter fragments of gehC, gehD and serp0018 each formed DNA-protein complexes with SarZ in a dose-dependent manner. The mobility of these complexes was significantly reduced compared to that of the free DNA probes without bound protein, and a clear band shift was observed. (Fig. 4a-c, lane 2 to lane 4). The addition of a 200-fold excess of the same unlabeled DNA fragment as a competitive probe completely blocked the formation of a complex between the labeled probe and the SarZ protein (Fig. 4a-c, lane 6). As a negative control, the DNA fragment containing the rpsJ gene promoter did not form a complex with SarZ under the same conditions (Fig. 4d), indicating that SarZ could specifically bind to the promoter region of the lipase gene.

In conclusion, SarZ modulates the lipase activity of S. epidermidis by affecting the transcription of lipase genes as a transcription factor. It suppresses the expression of gehC and serp0018 genes while promoting the expression of gehD gene by directly binding to their promoter regions. These findings provide a theoretical foundation for the future development of new antibacterial drugs and the industrial application of staphylococcal lipase.

Funding

This work was supported by the Key Program of Educational Commission of Anhui Province (2023AH051737, KJ2020A0602), the Support Program for University Outstanding Youth Talent of Anhui Province (gxyq2019043), and Fudan University (FDMV-2020005).

Competing Interests

The authors declare that they have no competing interests.

Author Contributions

Tao Zhu and Runan Tan were responsible for the conception and design of the study. Nannan Zheng conducted the olive oil agar plate assay. Xiao Chen constructed SarZ protein expression strain, Wenjun Xie and Wanyang Xu carried out the qRT-PCR experiments. Runan Tan performed EMSA experiment. Tao Zhu and Runan Tan carried out the analysis and interpretation of data. Runan Tan and Tao Zhu wrote the manuscript. All authors read and approved the final manuscript.

Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval

This article did not contain any studies with animals performed by any of the authors.

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SarZ negatively regulates the lipase activity in Staphylococcus epidermidis

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Introduction

Materials and Methods

Bacterial strains and media

Olive oil agar plate assay

p-nitrophenol assay

RNA extraction and qRT-PCR

Electrophoretic mobility shift assay

Statistical analysis

Results and Discussion

Conclusion

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Funding

Competing Interests

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Data Availability

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References

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