NWMN2330 May Be Associated with the Virulence of Staphylococcus aureus by Increasing the Expression of hla and saeRS

Introduction

Staphylococcus aureus is an asymptomatic human nostril colonizer, permanently colonizing approximately 30% of individuals.¹ And it is one of the most notorious and widespread bacterial pathogens, causing incalculable numbers of uncomplicated skin infections and possibly hundreds of thousands to millions of more serious invasive infections worldwide each year.^2,3 S. aureus infections are particularly problematic due to the frequent emergence of antibiotic resistance among S. aureus isolates, of which methicillin-resistant S. aureus (MRSA) is the most clinically important.⁴ Resistance of S. aureus to other antibiotics is also common. For example, resistance to β-lactamase-sensitive traditional β-lactam antibiotics (penicillin and derivatives) is almost ubiquitous in S. aureus.⁵ Furthermore, S. aureus typically exhibits resistance to nearly all available antibiotics in combination.⁶ Vancomycin remains the antibiotic of last resort for MRSA infections, and highly vancomycin-resistant strains (VRSA) have emerged but have not spread, probably due to the greatly increased fitness costs imposed by vancomycin resistance genes.⁷ However, some strains have acquired moderate resistance to vancomycin (VISA).⁸ Available antibiotics are not sufficiently effective against multidrug-resistant S. aureus strains, and although several S. aureus vaccines have been tested in clinical trials, all vaccine candidates have so far failed clinical trials.^9,10 Overreliance on animal models and an incomplete understanding of S. aureus pathogenesis during human infection may explain this failure.¹¹ Based on the failure of all vaccines, people are forced to seek new treatments for S. aureus infections.

Since S. aureus produces a variety of virulence factors that play important roles in infection, including hemolysin, enterotoxins, plasma coagulase, and leukocidal toxins,^12,13 anti-virulence strategies for the treatment of S. aureus infections have received increasing attention. The anti-bacterial virulence strategy is to achieve the purpose of anti-infection by reducing the virulence of pathogens. It does not directly kill the bacteria, and it is not easy to cause drug resistance and destroy the beneficial microbiota. Current studies have found that α-hemolysin, SrtA and golden yellow pigment are good virulence targets.^14–18 In our previous study, our group found that after sub-inhibitory concentrations of resveratrol reduced the virulence of S. aureus, the expression of the NWMN2330 gene was down-regulated in its transcriptome.¹⁹ Therefore, we speculate that this gene may be closely related to the virulence of S. aureus. See Supplementary Material for NWMN2330 gene sequence.

Our study mainly focuses on the role of NWMN2330 in the virulence of S. aureus, which provides a theoretical basis for further understanding the role of NWMN2330 in S. aureus, and also provides a new target for the treatment of S. aureus infection.

Materials and Methods

Bacterial Strains, Plasmids, and Culture Conditions

The bacterial strains and plasmids used in this study were listed in Table 1. According to the manufacturer’s instructions (bioMérieux, Marcy l’Etoile, France), the VITEK-2 microbial analyzer was used to identify the isolates. S. aureus Newman and S. aureus Newman NWMN2330 mutants were grown in tryptic soy broth (TSB)(BD) medium, supplemented in tryptic soy broth (TSB)(BD, NJ, United States) containing 10 mg/l chloramphenicol Grow in a medium, shake 220 rpm at 37°C. Escherichia coli was cultured in Luria broth (LB,)(Oxoid) medium containing appropriate antibiotics (ampicillin 100 mg/L and anhydrotetracycline 50 mg/L).

Table 1 Bacterial Strains and Plasmids Used in This Study

Construction of S. aureus NWMN2330 Mutant Strain (Newman-ΔNWMN2330) and Its Complemented Strain (Newman-ΔNWMN2330-C)

The accession number of the Newman2330 gene in the NCBI GenBank is BAF68602.1.

The specific function in S. aureus Newman is unclear. We blasted other S. aureus with this gene. In common S. aureus, such as USA300, the gene expresses hypothetical protein, and in some other S. aureus such as 16,405, the gene expresses GtrA family protein. The temperature-sensitive plasmid pKOR1 was used to construct the NWMN2330 deletion mutant of S. aureus Newman through allelic replacement. Use primer sets NWMN2330-UP-F/NWMN2330-UP-R and NWMN2330-DOWN-F/NWMN2330-DOWN-R to amplify the upstream and downstream fragments of NWMN2330 from the genomic DNA of S. aureus Newman (Table 2). The resulting amplicon was digested with XhoI and then ligated with T4 DNA ligase to generate a homology arm fragment with NWMN2330 gene deletion, which was then cloned into pKOR1. The recombinant plasmid pKOR1-ΔNWMN2330 was sequentially transferred to E. coli DH5α and DC10B competent cells, and finally electroporated into S. aureus Newman competent cells. The allelic replacement mutants were selected as described previously.²⁰ Briefly, we amplified the NWMN2330 fragment using Newman genomic DNA as a template. The plasmid pLI50 and the gene fragment were digested with EcoRI and KpnI restriction enzymes, and the plasmid and fragment were ligated with T4 ligase. The ligated products were sequentially thermally transformed into E. coli DH5α and DC10B for amplification and modification. Finally, it was electroporated into Newman-ΔNWMN2330, and each step is verified by PCR and first-generation Sanger DNA sequencing.

Table 2 Primers Used in This Study

Growth Assay

The strain Newman, the deletion mutant Newman-ΔNWMN2330, and the complementary strain Newman-ΔNWMN2330-C were inoculated on blood agar plates and incubated overnight at 37 ° C incubator. Pick a single clone and inoculate in 5mL of tryptic soy broth (TSB) (BD, NJ, United States) for shaking culture, and then inoculate in 50mL TSB for shaking culture at a ratio of 1: 200. All cultures were induced at 37 °C with shaking at 220 r.p.m. and the OD₅₆₂ values were measured every hour for a total of 24 h. The assay was performed in triplicate.

RNA-seq and Identification of Differentially Expressed Genes

The wild strains of S. aureus Newman and Newman-ΔNWMN2330 were cultured in TSB for 9h, and then the bacteria were collected by centrifugation at 12,000 g for 1 minute at 4 ° C. The method recommended by the manufacturer (QIAGEN, Berlin, Germany) for RNA extraction. Illumina HiSeq X platform and pe150 (150 bp double-stranded assay) strategy were used to analyze the tested RNA, and DEGseq software was used to analyze differentially expressed genes. log2 (fold-change) |> 1, and p <0.005 represents the difference in gene expression between samples.

Quantitative Enzyme-Linked Immunosorbent Assay for α-Toxin

The bacteria were cultured in TSB with shaking for 24 hours, after which the supernatant was collected by centrifugation at 6000 g for 2 minutes and filtered with a 0.22 μm filter. We used the staphylococcus alpha-toxin enzyme-linked immunoassay kit (Sigma-Aldrich, St. Louis, Missouri, USA) to detect alpha-toxin. The above-extracted supernatant was added to the detection plate, and then horseradish peroxidase (HRP) label was added to the plate, and the above mixture became an antibody-antigenase labeled antibody complex. After washing, TMB (3,30,5,50-tetramethylbenzidine) substrate solution was added, and finally sulfuric acid solution was added to stop the reaction, and the color change was measured by spectrophotometry at 450 nm wavelength. Use the standard curve y = ax + b to calculate the concentration of alpha toxin in each sample and then multiply it by the corresponding dilution factor. Each test was repeated three times.

Determination of Hemolytic Ring

Newman and Newman-ΔNWMN2330 were inoculated on Columbia blood agar medium, Newman-ΔNWMN2330-C was inoculated on the plate containing chloramphenicol, and placed in a 37°C incubator for overnight culture. Bacteria were shaken overnight at 37°C in TSB liquid medium at 220 rpm. Centrifuge at 4000g at room temperature for 5 minutes, discard the supernatant, resuspend the bacterial solution in PBS, and centrifuge again at 4000g at room temperature for 5 minutes. After that, 10 μL of bacteria was dropped on Columbia blood agar medium, placed in a 37°C incubator for 24 hours, and the size of hemolytic ring was observed.

α-Toxin Activity Determination

The experimental method is slightly modified on the basis of the previous literature.¹⁵ Briefly, S. aureus strains were cultured in TSB at 37 ° C, and the supernatant was extracted according to the above experimental method. Then add 100 µL supernatant to 875 µL hemolysis buffer, incubate with 25 µL defibrillated rabbit blood at 37 ° C for 1 h, and centrifuge at 6000 g for 2 minutes. Take the supernatant and measure the OD value of the supernatant at 450 nm. Triton X-100 was used as a positive control and 0.9% NaCl was used as a negative control. Each test was performed independently in triplicate.

Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)

After shaking culture of S. aureus for 9h, RNA was extracted according to the above method. The primer pairs used in real-time RT-PCR are listed in Table 1. The total RNA was reversed to cDNA using Takara RNA PCR kit (Takara, Tokyo, Japan). PCR was performed in 20 µL reaction mixture using Luna Universal qPCR Master Mix (NEB, MA, United States). Each test was performed in triplicate.

Determination of Silkworms Survival Rate

This experiment mainly refers to previous studies with minor modifications.²¹ Newman and Newman-ΔNWMN2330 were inoculated on Columbia blood agar medium, Newman-ΔNWMN2330-C was inoculated on the plate containing chloramphenicol, and placed in a 37°C incubator for overnight culture. Pick a single clone and add it to 5mL TSB liquid medium, shake it at 220rpm, 37°C, and grow it in the late logarithmic growth period, about 8 hours. The bacteria were centrifuged at 5000 g for 5 min at room temperature. After discarding the supernatant, they were resuspended in PBS, centrifuged again and repeated three times. Adjust the bacterial solution to a consistent OD value, probably at OD₅₆₂=1.5. The experiment was divided into 4 groups, namely wild bacteria group (Newman), deletion mutant group (Newman-ΔNWMN2330), complementary strain group (Newman-ΔNWMN2330-C), and blank control group, which was injected with PBS. The experimental sample for each group is 12. Between the second and third gastropods of the silkworms, 8×10⁸ CFU/100 μL of bacteria were injected into the body with an insulin needle, and PBS was used as a blank control. Placed in a 37°C incubator, record the survival time of the wax moth, and draw it with drawing software.

Mouse Skin Abscess Model

Animal research was approved by the Institutional Animal Care and Use Committee.

All animal assays were carried out in accordance with The Regulation on the Management of Laboratory Animals for the welfare of the laboratory animals, and the protocol approved by the Ethics Committee of Shanghai pulmonary hospital, Tongji University School of medicine. In the study, 4 to 6 week old male BALB/c nude mice were used, divided into three groups of four. For the specific method, refer to the previously published article.²² BALB/c mice were completely anesthetized with 1% (mass/volume) pentobarbital sodium (50 mg/kg body weight). Then, 100 μL of 5×10⁸ CFU bacteria were inoculated subcutaneously on the back of the mice, and the mice injected with PBS were used as blank controls. The area evaluated by the maximum length and width of the skin damaged part is measured every day. Use the formula A = L × W to calculate the damage area, where L is the length and W is the width.

Statistical Analysis

Experimental data were analyzed using GraphPad Prism 6 software (version 6.00, La Jolla, CA, United States). A p-value less than 0.05 was considered to be statistically significant.

Result

NWMN2330 Did Not Affect the Growth of S. aureus

In order to test whether NWMN2330 had an effect on the growth of S. aureus Newman, we measured the growth curves of S. aureus Newman, Newman-ΔNWMN2330 and Newman-ΔNWMN2330-C. The results showed that the growth of the three strains was similar, with no statistical difference (Figure 1), indicating NWMN2330 had no effect on the growth of S. aureus Newman.

Figure 1 Growth curves of Newman, Newman-ΔNWMN2330, Newman-ΔNWMN2330-C. The data were analyzed by one-way ANOVA, and there was no difference in daily bacterial growth between Newman and Newman-ΔNWMN2330.

NWMN2330 Participates in the Regulation of Multiple Genes Related to Virulence

Transcriptome data showed that Newman-ΔNWMN2330 has a total of 224 gene expression changes (according to |log2(Fold Change)| >1 and q value<0.05 criteria) relative to Newman, of which 91 genes are up-regulated and 133 genes are down-regulated (Figure 2). After bioinformatics analysis, it was found that these genes are involved in multiple metabolic pathways, including S. aureus infection, cell killing, carotenoid biosynthesis, two-component regulatory system, complement binding, and amino acid anabolism (Figure 3 and Table 3).

Table 3 Newman-ΔNWMN2330 vs Newman Genes with Significantly Changed Expression

Figure 2 Volcano graph showing the difference in expression between Newman and Newman-ΔNWMN2330. The abscissa represents the fold change of gene expression in different samples; the ordinate represents the statistical significance of the difference in gene expression; the red dots in the figure indicate the up-regulated genes with significant differential expression, and the green dots indicate the down-regulation of significant differential expression gene.

Figure 3 Scatter plot of KEGG enrichment of differential genes. The vertical axis represents the name of the pathway, the horizontal axis represents the enrichment factor, the size of the dots indicates the number of differentially expressed genes in this pathway, and the color of the dots corresponds to different q value ranges.

NWMN2330 Inhibits the Expression of Virulence Gene hla

Transcriptome data showed that the expression of hla in Newman-ΔNWMN2330 was significantly down-regulated by 2^10.008-fold compared to Newman. We used RT-qPCR to verify the transcriptome data, the results showed (Figure 4) that hla expression in Newman-ΔNWMN2330 was significantly down-regulated, indicating that NWMN2330 may be linked with hla regulation.

Figure 4 RT-PCR results of hla. Newman represents the wild strain, Newman-ΔNWMN2330 represents the deletion mutant, Newman-ΔNWMN2330-C represents the reverted strain. Statistical significance was determined by the unpaired t-test, wild strain compared to deletion mutant strain, and ****Represents P<0.0001.

NWMN2330 Enhances the Hemolytic Ability of S. aureus

The production of hemolysin is an important part of the virulence of S. aureus, and alpha toxin is the most important toxin in hemolysin. We tested the hemolytic ability of S. aureus Newman, Newman-ΔNWMN2330 and Newman-ΔNWMN2330-C. The amount of α-toxin was quantitatively detected by ELISA, and it was found that the amount of Newman-ΔNWMN2330 produced was significantly less than that of Newman (Figure 5A). And the hemolytic ring produced by Newman was significantly larger than that of Newman-ΔNWMN2330, and the complementary strain reached the level of Newman (Figure 5B). Because alpha toxin is a punch protein, it has the most obvious hemolytic effect on rabbit red blood cells. We used defibrillated rabbit blood to detect hemolytic activity, and used Triton as a positive control and normal saline as a negative control. It was found that the hemolytic activity of Newman-ΔNWMN2330 was significantly lower than that of wild strain Newman (Figure 5C and D). The above results indicate that NWMN2330 can positively regulate the expression of S. aureus hemolysin.

Figure 5 (A) ELISA detects the amount of α-toxin produced by each strain; (B) Colombian blood agar medium detects the hemolytic capacity of each strain; (C and D), detects the hemolytic activity of each strain. Notes: (C) is a picture of hemolytic activity measurement, and (D) is a drawing software drawing after measuring the OD value and converting it with Triton as the 100% hemolysis rate. Newman represents a wild strain, Newman-ΔNWMN2330 represents a deletion mutant, Newman-ΔNWMN2330-C represents a reverted strain, Statistical significance was determined by the one-way ANOVA test, wild strain compared to deletion mutant strain and complementary strains compared to deletion mutants,****Represents P<0.0001.

NWMN2330 Enhances the Lethality of S. aureus to Silkworms

Because the production of toxins is closely related to the pathogenicity of S. aureus, after observing the effect of NWMN2330 on hemolysin, we speculate that NWMN2330 may be related to the virulence and pathogenicity of S. aureus. So we measured the survival rate of the silkworms after infection with the three kinds bacteria. The results showed that the survival time of silkworms infected with Newman was significantly shorter than the time of injection of Newman-ΔNWMN2330, which initially indicated that NWMN2330 could enhance the virulence of S. aureus (Figure 6).

Figure 6 Determination of silkworms survival rate (N=12). PBS injection was used as a negative control. Statistical significance was determined by the Gehan-Breslow-Wilcoxon test, wild strain compared to deletion mutant strain and **Represents P<0.01.

Effect on Infection of Subcutaneous Abscess in Mice

S. aureus infection often causes skin abscesses.^23,24 We used skin abscess model in nude mice to investigate whether NWMN2330 had an effect on the ability of S. aureus to cause skin abscesses. The results showed that the abscess area of mice infected with the mutant strain (4±0.76) was significantly reduced compared to the wild strain (130±16.16) (Figure 7). The above results indicate that NWMN2330 is associated with the virulence and pathogenicity of S. aureus and can enhance the pathogenicity of S. aureus.

Figure 7 Results of nude mouse skin abscess model. PBS injection was used as a negative control. Statistical significance was determined by the unpaired t-test, wild strain compared to deletion mutant strain, *P < 0.05, **P < 0.01.

NWMN2330 May Promote hla Expression by Up-Regulating saeRS

The saeRS two-component regulatory system is a regulator that controls the expression of multiple virulences.^25–28 According to the transcriptome data, it is speculated that saeRS is also involved in the regulation of hla expression by NWMN2330. RT-PCR results showed that when NWMN2330 was knocked out, the expression of saeRS was significantly down-regulated, and there was no downward trend in other related regulators sarA (Figure 8). The previous speculations have been further confirmed, indicating that NWMN2330 may promote hla expression by up-regulating saeRS.

Figure 8 Relative expression of saeRS and sarA at the mRNA level. Statistical significance was determined by the unpaired t-test, wild strain compared to deletion mutant strain, ****Represents P<0.0001.

Discussion

Due to the increasing number of multidrug-resistant S. aureus in recent years, there is an urgent need for new antibacterial agents that can overcome these more robust S. aureus strains. The discovery of new antibiotics has slowed in recent years, and strategies to reduce the virulence of S. aureus to reduce its lethality have attracted great interest.²⁹ In recent years, there have been many studies on the use of anti-virulence strategies for the treatment of S. aureus infections,^30–32 but not enough for the rapidly evolving multidrug-resistant bacteria. Our previous study found that NWMN2330 in S. aureus may be related to virulence,¹⁹ but the specific role was not clear. The gene annotation of NWMN2330 is “similar to functionally unknown protein”, and its specific function is worth exploring.

In the present study, we mainly focused on the virulence control of S. aureus by NWMN2330. We successfully constructed the S. aureus Newman deletion mutant Newman-ΔNWMN2330 and the complemented strain Newman-ΔNWMN2330-C. In recent years, transcriptome sequencing analysis has been frequently used to study the pathogenic and drug resistance mechanisms of S. aureus.^33,34 The results of transcriptome sequencing showed that compared with Newman, the expression of hla in the mutant strain was down-regulated very significantly. We also confirmed by real-time quantitative PCR that the expression of hla at the mRNA level was indeed significantly reduced in the mutant strain. We speculate that NWMN2330 may regulate S. aureus virulence by enhancing hla expression.

Alpha-toxin (encoded by hla) is the most studied pore-forming toxin and plays an important role in S. aureus skin and soft tissue infections.^35–37 Alpha-toxin has an obvious hemolytic effect on red blood cells of various mammals, especially rabbit red blood cells.³⁸ Using the above characteristics of α-toxin, we detected the hemolytic activity, hemolytic ring and α-toxin production of Newman, Newman-ΔNWMN2330 and Newman-ΔNWMN2330-C strains, respectively. The deletion mutant Newman-ΔNWMN2330 showed lower hemolytic activity, lower α-toxin production and smaller hemolytic ring than the wild strain in the above three experiments. We know that NWMN2330 also enhance the virulence and pathogenicity of S. aureus from survival experiments in silkworms and skin abscess model in nude mice. The production of S. aureus α-toxin is regulated by multiple regulatory systems, of which saeRS is considered to be the most important one because the sae locus is important for hla transcription.³⁹ The S. aureus sar locus also has alpha and beta hemolysins, and SarA directly increases α-toxin production.⁴⁰ We found by real-time quantitative PCR that the expression of saeRS decreased significantly in the deletion mutant, but the change of sarA was not significant. We speculate that NWMN2330 may up-regulate the expression of hla by up-regulating the expression of saeRS, thereby enhancing the virulence of S. aureus.

Our research has many limitations. First, it is only tentatively speculated that NWMN2330 up-regulates the expression of hla through saeRS to enhance the virulence of S. aureus, and has not been studied more precisely. Secondly, we only studied the regulatory effect of NWMN2330 on the α-toxin of S. aureus, and there are many other functions that are not covered in this paper. We can see in the transcriptome sequencing data that NWMN2330 also affects the expression of many other important virulence genes. Also, it has not been studied whether the gene has the same effect in other S. aureus strains.

In summary, our study shows that the gene NWMN2330 can up-regulate the expression of hla and enhance the pathogenicity in S. aureus Newman strain. The gene NWMN2330 may be an important target for limiting S. aureus infection in people colonised by this potential pathogen.