**Abstract**

*Staphylococcus aureus* causes a wide range of diseases, including both communityassociated and hospital-acquired infections such as abscesses, wound infections, osteomyelitis, endocarditis and septicemia. Regulation of the expression of various virulence factors is initiated through complex coordination between two-component systems, transcriptional regulatory proteins and regulatory small RNAs (sRNAs). *S.aureus* uses many sRNA and RNA–RNA interactions mediated the regulation of the expression of genes post-transcriptionally, but it uses few sigma factors to initiate the transcription function. sRNA transcripts are encoded within intergenic regions or in antisense orientation to mRNA transcripts, and sRNA regulation plays a central role in the response to stress stimuli encountered by pathogens during infection. One of the most intriguing examples of sRNA-mediated post-transcriptional regulation is RNAIII from *S.aureus*, which interacts with and regulates various RNA targets involved in virulence. Several genes known to be regulated by RNAIII have been demonstrated to be regulated by the *sarA* locus, independent of its effect on the expression of RNAIII. We discuss the potential role of small RNA (sRNA) in the pathogenesis and virulence factors production of *Staphylococcus aureus*.

**Keywords:** *Staphylococcus aureus*, regulatory small RNA (sRNA), virulence factors, RNA–RNA interactions

## **1. Introduction**

*Staphylococcus aureus* is a human symbiotic microorganism that commonly colonizes in the anterior nasal regions and on the skin surface for 20–25% of the world population [1–3]. The distribution of multi-drug resistant strains among asymptomatic individuals is responsible for spreading the infections among the population very quickly [4]. Among the human populations, the carriage percentages of *Staphylococcus aureus* vary based on different factors. Broadly human carriers are classified into three categories: 20% non-carriers, 25% persistent carriers, and 60% population are intermediate carriers [5]. Usually, *Staphylococcus aureus* forms colonization in the nasal passage and axillae in humans and found its occurrence as flora in vaginal tracts and digestive tracts [6].

Among the various factors responsible for the regulation of virulence, small RNA (sRNA) has a major role in determining the virulence of the bacteria. sRNA are short 50–250 nucleotide long transcripts involving bacterial gene expression

*Insights Into Drug Resistance in* Staphylococcus aureus

#### **Figure 1.**

*Virulence factors regulation by RNAIII. RNAIII is regulated positively by the quorum-sensing agr operon. Post-transcriptional regulation is marked with colored lines, whereas up-regulation represented by green arrows and down-regulation by red cross bars. RNAIII in turn, positively regulates MapW and hla at a posttranscriptional level. MapW and hla prevents leucocyte attachment and promote dissemination by lysing host cells. RNAIII also negatively regulates LytM, rot, Spa and SarT, which will promote autolysis via LytM, blood cell toxicity by rot and expression of an IgG binding protein via SarT.*

for rapid adaption to stress conditions. Small RNAs play a major role by pairing with bases of target mRNA or by interacting with the modulating proteins for both the positive and negative mechanism of biofilm formation. *Staphylococcus aureus* becomes more adherent resulting in increased biofilm formation when *agr* mediated mechanism is inhibited. Regulation of gene expression mediated by sRNAs is more beneficial when compared to proteins during a rapid response because it takes a short time for sRNAs to either synthesize or degrade. Various regulatory mechanism of sRNAs is similar to the regulation of quorum sensing in the bacteria. The quorum-sensing mechanism regulates the expression of virulence-related genes. Since the quorum sensing mechanism controls bacteria's virulence factor, it is considered a major target for finding out the new therapeutic methods [7]. We discuss the potential role of small RNA (sRNA) in the pathogenesis and virulence factors production of *Staphylococcus aureus* (**Figure 1**).

#### **2. Bacterial small RNA**

sRNAs mediate the regulation of mRNAs through direct binding interactions between the sRNA and the target. The sRNA usually binds to the 5′ end of the mRNA and blocks ribosomes binding. Although sRNAs often stimulate degradation of the target as well [8]. The interaction is initiated by a short sequence of perfect complementarity between the sRNA and target termed the seed region. Seed regions are generally 6–8 nt long, and a single sRNA can have one seed region that regulates all of its targets or multiple seed regions that each regulate a subset of targets. Additionally, seed regions are highly conserved, and mutations to the seed region lead to complete abrogation of target regulation. In order to facilitate intermolecular interactions with target mRNAs, seed regions are usually single-stranded in the folded sRNA and disruption of the sRNA secondary structure can drastically

reduce sRNA function. However, seed regions alone are generally not sufficient to mediate target binding, most sRNA characterized to date rely on the assistance of an sRNA chaperone protein [9, 10].

The regulation mechanism for the SpoVG and SprX and their targets with interactions have been discussed below, which involves regulating virulence factors. Production of capsule, virulence factors, and the cell wall's metabolism is regulated by a transcription factor *SpoVG* also called a master regulator. It is also responsible for resistance against methicillin and glycopeptide antibiotics [11]. Synthesis of pentaglycine crosslinks between peptidoglycan strands carried out by *lytSR* operon and glycine glycyl transferases is positively regulated by SpoVG, whereas murein hydrolysate lytN regulated negatively. Base pairing of *sprX* (Highly conserved RNA) with the *SpoVG* mRNA during the translation process prevents loading of the ribosome. Small RNA *sprX* negatively regulated *SpoVG*, in four phases of exponential growth (lag, log, linear and late phase), but it decreases during the stationary growth phase. The SpoVG dependent process increases glycopeptides susceptibility and disrupts the cell membrane metabolism and other independent *SpoVG* mechanisms [12].

*SpoVG* and *SprX* both seem to contain extra regulatory targets, and antibiotic susceptibility through the *SprX*-dependant mechanism can be bound to susceptibility tied to extra phenotypic advantages that continue to be studied [13]. Various strains of *S.aureus* additionally show an antibiotic determination-related phenotypic variation called as small colony variants (SCVs), which stimulates the biofilm formation and reduced sensitivity to aminoglycosides [14]. The different variation form of small colony variants (SCV) of *S. aureus* has disturbed the expression of 18 sRNAs and increased regulation of the *RsaA*, which is a *sigB* dependent small RNA [15]. However, the network link among small colony variants, antibiotic resistance, and expression profiling of sRNAs has now no longer, but been delineated, those outcomes infer that regulatory sRNAs make contributions to antibiotic resistance through phenotypic maintenance of small colony variants [16].

## **2.1 RNAIII**

RNAIII regulates the expression of genes encoding exoproteins and cell wall associated in *Staphylococcus aureus*. In the quorum sensing mechanism, RNAIII transcribed from the P3 operon, acts as an initiator for agr system in *Staphylococcus aureus*. It also contains the hld gene (delta haemolysin), which is 26 amino acid long sequences. By blocking the translation of transcription factor rot, it regulates its expression. It has been reported that binding of RNAIII with mRNA of transcriptional factor rot in an antisense manner, thus blocking the Shine-Dalgarno sequence [17, 18].

#### **2.2 Teg49**

It is a small RNA found in the extended promoter region of sarA, which is an accessory regulator of Staphylococcus bacteria. Confirmation and identification were performed by Northern blotting and RNA-sequencing method. Modulation of the expression of SarA it regulates the virulence factor of *Staphylococcus aureus* [19].

#### **2.3 SprF1-SprG1**

There are two types of Toxin-Antitoxin system, whereas Type I has two sub-type types, and Type II has three sub-types but remains uncharacterized. The first type I TA system was SprA1-SprAAS in, which the former denotes the toxin and the latter denotes the antitoxin in *Staphylococcus aureus.* Inhibition of translation and cell wall

damage is done by toxin SprA1, a short peptide. txpA-ratA family, also referred to as SprF1-SprG1, is the second type I TA system, whereas SprG1 belongs to pore-forming toxin and antitoxin are SprF1. SprG1 consists of 44 amino acid sequence long peptide and 31 amino acid sequence of short peptide and they have cytotoxic properties [20].

It has been reported that a small RNA, which expresses from pathogenicity islands of SprD upon binding with antisense base pairing of sbi mRNA (encoding an immunoglobulin binding protein) will lead to an impaired host immune response [21]. Besides direct base-pairing with target mRNA, several other mechanisms, including dual-function sRNA that acts as an antisense molecule and codes for a small peptide (e.g., Hld in RNAIII), have been proposed to act on the same or other pathway genes, and also riboswitches that exhibit a structured receptor domain specifically recognized by a small molecule or metabolite [22].
