Gene expression in Staphylococcus aureus skin infection

The success of a microbe to colonize its corresponding host depends on several factors, including the ability of the microbe to control the expression of its genes during the colonization of its host and on the signaling events initiated by the colonized host in response to the infecting microorganism. The initial signaling events are crucial to activate rapidly the host defense mechanisms, which results in the clearance of the pathogen, limiting its further spread beyond the site of infection. Transcriptional analysis of a microorganism and its corresponding host can be combined in such a way that can provide a truly comprehensive picture of the gene expression patterns induced in the microbe when it encounters the host and on the signaling pathways induced in the colonized host by the infecting bacteria. This can be done in a single experiment using the technology of DNA microarrays.

Staphylococcus aureus is an important nosocomial and community-acquired pathogen. It commonly causes local and systemic infections in mammals ranging from minor wound infections to life-threatening conditions such as endocarditis, osteomyelitis, and toxic shock syndrome (1). S. aureus responds to the host environment by altering its gene expression (2). However, the entire stimuli to which S. aureus responds to the host cells or to host factors remained undetermined. Several investigations have shown that the contact of a microbe with eukaryotic host cells results in the expression of genes that are specifically required for survival or virulence (3,4).

The lack of alternative therapies to antibiotics against S. aureus is a major problem in the treatment of staphylococcal disease, especially due to the increase in methicillin-resistant strains (MRSA). After the introduction of methicillin in the 1960s (5), the incidence of MRSA has grown significantly (6,7). The most likely explanation for this appearance and spread of MRSA is the increased and indiscriminate use of broad-spectrum antibiotics. The increasing prevalence of multidrug-resistant strains and the recent appearance of strains with reduced susceptibility to vancomycin (8), the antibiotic of last resort, raise the specter of untreatable staphylococcal infections and add urgency to the search for new anti-infective strategies. To find new strategies is important to understand the mechanisms that S. aureus uses to adapt to its host and how the host responds to the presence of staphylococcal cells. Therefore, we want to review and compare the expression profiles of the two infection models and identify important genes.

We conducted an extensive literature search using validated keyword filters to select articles related to Staphylococcus aureus gene expression in vivo or animal models. The research was conducted on articles published from 1 January 2000 to 31 January 2021, and an in-depth and selective search was performed on biomedical bibliographic databases PubMed, SCOPUS, and Google Scholar. Finally, the studies that contained tables with changes in S. aureus transcriptome during the infection were chosen. The tables with the fold-change expression were compared using Microsoft access.

Mainly two studies fit the criteria described above. Malachowa et al. (2015) monitored gene expression changes in S. aureus transcriptome and host during abscess formation in a rabbit skin infection model (9). Jacquet et al. (2019) investigated how the diabetic environment affects the S. aureus gene expression in diabetic models of S. aureus skin infection in mice (10).

The hyperglycemic mice were generated through daily injections over five days with 50 mg/kg of body weight of streptozotocin. In both studies, the animals were infected with 10^6-8 CFUs of S. aureus strain USA300 subcutaneously. In the rabbit study, bacterial RNA isolated from the abscess 24 hours post-infection was analyzed on Affymetrix GeneChip. cDNA synthesized from total RNA from days one to 14 post-infection was analyzed using RT² Profiler PCR Array Rabbit Inflammatory Cytokines and Receptors platform (11). In the mice study, it was used a dual transcriptome sequencing (RNAseq) approach to analyze the mRNA level differences between control and diabetic mice one day after infection.

The expression of 1262 genes was unaffected in either animal model. Within the first 24 h, significant changes in gene expression were detected for S. aureus genes involved in basic functions and survival. 731 genes were upregulated and 423 genes downregulated only in the rabbit model while 84 genes were upregulated and 27 downregulated only in the mouse model. 19 genes were upregulated and four genes were downregulated in both systems. 34 genes that were upregulated in the rabbit were downregulated in mice while 20 genes that were downregulated in the rabbit were upregulated in mice.

The 19 genes upregulated in both systems include chaperones (e.g., groES, groEL, grpE, dnaK9), sodM, hrcA, sbi, and the gene encoding a cadmium-exporting ATPase protein. Also, four genes were downregulated in both systems including a gene that encodes a hydrolase and three genes for hypothetical proteins.

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The functional class most frequently represented among these genes were those involved in transport and metabolism of amino acids (e.g., hutH, arcB-D, etc.), nucleotides (e.g., purA, deoD, tmk, etc.), inorganic ions (e.g., sirA-C, phnB, phnC, phnE, etc.), carbohydrate (e.g., bglA, fruA, fruB, etc.).

The transcripts of the accessory gene regulator agr system (agrA, agrB, and agrC) were downregulated (8- to 19-fold) while sarV, a member of the SarA protein family, increased 6.4-fold. The gene expression of the genes of the Sigma B operon (sigB, rsbW, and rsbV) that have been shown to respond to environmental stresses decreased 2- to 4-fold. Also, the gene expression of two different genes encoding TetR family transcription regulators was upregulated. TetRs are widely associated with antibiotic resistance and the regulation of genes encoding small-molecule exporters (12). Two genes encoding MarR family transcription regulators were upregulated 10.4-fold while the other was downregulated 2.46-fold. The members of the MarR family of transcription factors are critical for bacterial cells to respond to chemical signals and to convert such signals into changes in gene activity (13). The MsrR transcriptional regulator which affects resistance to methicillin and teicoplanin was upregulated by 2.3-fold. LexA repressor that regulated the expression of the SOS gene was downregulated by 3.4-fold. The srrA genes of the two-component regulatory system (TCRS) positively influence the transcription of genes involved in aerobic respiration in response to changes in respiratory flux were downregulated by 3-fold.

The gene expression of several S. aureus genes encoding virulence factors changed during the first 24 hours of skin infection. The expression of fnbA increased by 2.3-fold as well as the gene encoding fibronectin-binding proteins (e.g., fnbA, fnbB, sdrC, sdrD, sdrE, SAUSA300_1028, and SAUSA300_1029) that showed substantially increased mRNA levels (2- to 125-fold). The sarA locus up-regulates the expression of many cell wall proteins including FnbA. Fibronectin-binding proteins contribute to the colonization and infection of the host by S. aureus via adhesion to fibronectin present in the extracellular matrix of the tissues. They play a role in the virulence of S. aureus skin abscess infection, infective endocarditis, bacteremia, sepsis, pneumonia, and foreign body infections (14).

The gene expression of spa that had been described previously as down-regulated by sarA locus (15), increased in the skin abscess by 4.0-fold. Protein A effectively blocks the formation of IgG hexamers and subsequent complement activation (16). The gene expression of clfA, a cell surface-associated protein that binds fibrinogen promoting S. aureus colonization of host tissues and biomedical devices under physical stress (17), was downregulated by 6.5-fold. The gene expression of hemolysin gamma A gene (hlgA, SAUSA300_2365) that have been described previously to require both Sae and Agr (18), in this model increased 20.8-fold. Actually, hlgB (SAUSA300_2367) and hlgC (SAUSA300_2366) increased by 76.3- and 27.4-fold, respectively. Haemolysin gamma A, B, and C (HlgABC) displayed cytotoxicity to monocytes and natural killer cells (19).

The expression of genes encoding coagulase (Coa), and two von Willebrand factor binding proteins (vWbp) with coagulase activity that are required for septicemia and abscess formation (20) increased 12- to 34-fold. Coa is involved in the virulence of S. aureus. The fibrinogen-binding motifs also found in coagulase block neutrophil αMβ2 adherence to fibrinogen and attract fibrinogen to the bacterial surface, forming capsule-like structures that block phagocytosis (21).

The gene expression of a gene (scin) belonging to the family of S. aureus complement inhibitory proteins increased more than 57-fold. They share a common domain of three helices at the C3 binding region (22) and are implicated in the S. aureus evasion of the complement-mediated immune response. Surprising the gene efb which encodes another complement inhibitory protein was unaffected. Also, gene expression of emp that encodes a protein with a fibrous structure that binds to different extracellular matrices proteins increased 11.7-fold (23). Emp plays an important role in low-iron-induced biofilm formation (24). The expression of the gene sasC encoding the S. aureus surface protein C which is involved in cell aggregation and biofilm accumulation (25) increased by 10-fold. The gene expression of gene encoding SpoVG which is a repressor of the expression of sasC, decreased by 8.3-fold.

The gene expression of genes encoding exotoxins increased. For example, the mRNA level of genes (SAUSA300_0396, _0397,_0399, _0400, _0401,_0402, _0404, _00407 and  SAUSA300_1059 to _1061) encoding staphylococcal superantigen-like (SSL) proteins increased from 5.0- to 125-fold. SSL proteins have been shown to help S. aureus to escape from the protective adaptive immune response of the host and thus may contribute to bacterial pathogenicity (26,27). The gene expression of several multidrug resistance genes was upregulated like fmtB methicillin resistance proteins (SAUSA300_2109 and SAUSA300_2110) that increase by 42.5- to 38.2-fold, multidrug resistance protein B (SAUSA300_2298) that increased by 12.2-fold, and multidrug resistance protein A defense mechanisms/virulence (SAUSA300_2299) that increased 37.0-fold.

The accessory regulators sarA, rot, ecb, ctsR, spx, many ribosomal proteins, and hypothetical proteins increased while cap5k, lysE, rusA, and many hypothetical proteins decreased only in the mice model and not in the rabbit model.

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The gene expression of sarU, transcriptional regulators of the LysR family, Cro family, crp family, TetR family, tenA, and many hypothetical proteins were upregulated in the rabbit system but downregulated in the mouse system. The genes rps, rpl, rpm, and several others involved, for example, in translation and transcription were downregulated in the rabbit system but upregulated in the mouse system.

In the rabbit, gene expression changes of 84 genes encoding host inflammatory cytokines and receptors were analyzed using PCR arrays. 11 genes were downregulated and 52 genes were upregulated 24 h post-infection compared to untreated healthy rabbits. The expression level of many transcripts encoding proinflammatory molecules was highly up-regulated such as IL8, IL1B, oncostatin M, CCR1, CXCR1, CXCR2, CCL4, and CCL3. The expression level of some proinflammatory molecules (e.g., CCR5, CXCR4, IL1A, IL1R1, IL8, and TNF) peaked on days three to six post-infection. Moreover, there is a group of molecules, including LTA, LTB, IL21, IFNAR2, CXCR3, IL17F, and CD40LG, whose expression increased throughout staphylococcal skin infection.

          In the diabetic mice model, 231 genes were differentially expressed compared to the control group, of which 110 were upregulated while 125 were downregulated. Whatever the diabetic condition of infected mice, it increased the expression of genes encoding proteins involved in the adhesion of neutrophils, neutrophil migration, inflammation, and immune cell trafficking. Also, the genes with the major changes were those encoding Rho, Sarcospan, filamin A, IL-10, and genes associated with macrophages, fibroblasts, and endothelial cells. On the other hand, the genes associated with lipid, vitamin, and mineral metabolism, dermatological disease, and skeletal and muscular system development, EIF2 signaling, stearate signaling, and heme biosynthesis were downregulated.

The global analysis of gene expression patterns makes it possible to identify genes that could be relevant for pathogenesis and the colonization of host tissues. S. aureus encounters hostile conditions in the host, in response to which it is believed that this organism alters its gene expression to allow adaptation and colonization of the host tissue. It has been reviewed two important in vivo studies that described gene expression of S. aureus and the host during skin infection based on two different animal models. The main differences between these two systems are that they are two types of animals and the levels of sugar or glucose in the blood of these animals. Such a level of sugar probably made some differences in gene expression between the two systems. Patients with diabetes may be more sensitive to S. aureus bacteremia due to tissue hyperglycemia, reduced oxygenation, and usually reduced immunity (28). S. aureus is the most frequently isolated pathogen in diabetic wound infections.

Many genes did not show any differences in gene expression in S. aureus in skin infection in rabbits, diabetic mice, and control groups. However, few genes were affected in the same way in both systems including chaperones (e.g., groES, groEL, grpE, dnaK9, sodM, hrcA, and sbi). Besides, there were genes expressed in different ways in the two systems (e.g., sarU, lysR family, cro family, crp, tetR family, tenA, rps, rpl, rpm, genes for hypothetical proteins, involved translation and transcription). Some genes are expressed only in one of the two systems which expression was unaffected in the other system. For example, in the rabbit skin infection model increased important gene regulators, including agr and sarV, while others were downregulated such as stress response genes (e.g., sigB and lexA). The gene expression of several S. aureus genes encoding virulence factors such fibronectin-binding proteins, hemolysins, coagulases, complement inhibitory proteins, Emp, and many exotoxins were upregulated while clumping factor A was downregulated. Few genes were upregulated only in the mouse model including sarA, rot, ecb, ctsR, spx, many ribosomal proteins, and hypothetical proteins increased while cap5k, lysE, rusA, and many hypothetical proteins decreased.

The identification and characterization of genes encoding proteins implicated in bacterial pathogenesis may lead to new therapeutic drugs to treat staphylococcal infections (29). Early detection of infecting bacteria by the host is crucial for effective mobilization of innate and specific defense mechanisms. Several studies have described changes in host transcription during infection with a pathogen (30–33). In the skin infection models, the immune system of the rabbit response in the early stage of the infection by inducing the expression of genes encoding pro-inflammatory cytokines while in a later stage of the infection was induced the expression of receptors. The immune response of the diabetic mice induced the expression of genes associated with neutrophil adhesion and migration, infection, inflammation, and immune cell trafficking.

The staphylococcal genes with unclear function in infection can be mutated, and the constructed mutants can be subsequently characterized and their roles in pathogenesis determined. In this way, we can identify critical host responses during S. aureus infection. This information can make significant contributions in the field of S. aureus since many steps involved in the infection process can be targeted for antimicrobial chemotherapy, including adherence, invasion, and host defense evasion.

Ethical approval

Not required.

Author contributions

M.P. was the sole author of this article.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of competing interest

M.P. is Editor-in-Chief of Current Bioscience and a member of Current Bioscience editorial board. This article was reviewed by Current Bioscience editors and reviewers. The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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