**3. Multifunctional small RNA couples QS to virulence**

Regulation of virulence factors through quorum sensing mechanism involves the agr mediated pathway and the two-component system. RNAIII plays a major role in regulating the agr dependent transcriptional regulation in MRSA (Methicillin-Resistant *Staphylococcus aureus*). The significance of agr mediated regulations of *S. aureus* pathogenesis is the situation of an obvious paradox. By comparing with different *S. aureus* sRNAs, that has been discovered through bioinformatic strategies or RNA sequencing, RNAIII was the first predicted sRNA in transposon mutagenesis, which defines the epistasis outcomes for a point insertion [23]. The primary factor for virulence is agr and RNAIII, its effector molecule involved in producing virulence factors. Clinically isolated strains from acute infections of *S.aureus* have both virulence factor regulation through agr mediated along with RNAIII involvement. However, mutated strains of agr mediated pathway, which arose at some point of infection, also have been isolated from patients [24].

RNAIII as an effector regulates the expression of important virulence genes, including proteins associated with cell wall metabolism and exotoxins. Also involved in the expression of two-component systems, different global regulators such as *arl*, *sae*, *srr*, *rot* and other mechanisms in the formation of biofilm, synthesis of amino acid and peptidoglycan [25]. These factors vary quantitatively but not qualitatively in different *staphylococcal* strains.

Compared to UAMS-I (Virulent oxacillin susceptible clinical isolates) strain, the agr inactivation effect was observed more in the transcriptome of the *NCTC 8325* strain [26], but whether it exerts direct or indirect effect was studied only in certain genes from the structural prediction of RNAIII. Structurally *RNAIII* comprises 14 loop and two long helices aligned through the long-range base pairing, which blocked off self-reliant structural domains [27]. Some particular site-defined *RNAIII* domains are responsible for the regulation of various targets. The secondary structure of intramolecular RNA removes the hla ribosomal binding sites upon directly competing with the 59 ends of *RNAIII,* which positively induces translation of hla and alpha-hemolysin (**Figure 2**) [29, 30].

Production of various virulence determinants such as coagulase, protein A, and the rot (repressor of toxins) are repressed with minor variations by conserved regions or domains at the post-transcriptional level. These are repressed either individually or in combination by the H13 *RNAIII* hairpin and H14 terminator of the 39 domain, and central domain hairpin H7. The mechanism behind the repression of these virulence factors by the *RNAIII* is mediated through repression of the initiation process in translation mechanisms wherein the degradation of mRNA is initiated by *RNAIII* [31].

Staphylococcus aureus *and Virulence-Related Small RNA DOI: http://dx.doi.org/10.5772/intechopen.98520*

#### **Figure 2.**

*Integration of sRNAs into gene cascades regulation. The "agr-RNAIII" auto activation circuits is indicated with two feed-forward loops involving RNAIII. The autoinducing peptide (AIP) activates the agr autocatalytic circuit, leading to RNAIII transcription on attaining optimal cell density. RNAIII represses the expression of rot, which activates spa transcription and represses that of hla. RNAIII also activates hla mRNA translation and represses spa mRNA translation. The white and broken lines indicate the direct or indirect gene activations. The red lines represent the down regulations through different RNAs. The black question marks above the see-sawing triangles point to the unknown triggering factors. The transcriptional regulatory proteins are in blue [28].*

Complex structure was dependent on their target mRNA and included two factors (i) presence of an extended duplex between the mRNA of Ribosomal Binding Site (RBS) and *RNAIII* and (ii) an imperfect duplex which removes the finished RBS by the interaction between the loops in the coding region [32]. In the above two factors, an individual interaction between the loops is not enough for complete repression, accordingly proscribing the capability of *RNAIII* to behave as a repressor to the mRNA targets. Hence it will not have Shine Dalgarno (SD) series complementary to H7, H13, or H14 of RNAIII, however, it still show a further vicinity of communication or the potential to produce prolonged duplication. *Hfq* is an RNA binding protein and an important chaperone present in different staphylococcus species, but it does not play a role in the *RNAIII* dependent regulatory mechanisms. Whereas in the in-vitro assay, it binds to the *RNAIII* [33, 34].

The repression of all the target mRNA is carried out by the direct effects of *RNAIII* except the translational initiation of hla protein. The repression of Rot (a transcriptional regulatory protein) by RNAIII leads to indirectly regulating transcription for several genes, particularly the protein A repression and the alpha-toxin activation [35].

#### **4. sRNA dependent mechanism of antibiotic resistance**

Small RNAs play a major role in altering bacterial cell wall and hence would contribute to the antimicrobial-resistance mechanism. Small RNAs are present prominently on mobile genetic elements on which the resistance pattern for the AMR pathogens is found. SmallRNAs do not exert direct regulation on the resistance gene expression [36]. For example, Fudoh, a regulatory RNA present in *Staphylococcal* species is encoded by the SCC mec family of methicillin resistance cassettes. SCCmec is a mobile element that is responsible for the antimicrobial resistivity of methicillin-resistant *Staphylococcus aureus* (MRSA). It also involves regulating the cell distribution process and the expression of alpha phenol soluble modulins, a catalytic peptide [28].

However, the resistance pattern of methicillin through fudoh is still not known. Regulatory small RNA is responsible for the expression of intrinsic antibiotic resistance and tolerance in different bacterial species. Since only some of the small RNA related research has been performed on the clinical strains, whereas most of the studies for RNA-dependent intrinsic antibiotic resistance were performed on the AMR-related pathogens [37].

## **5. sRNA and stress responses**

Specific mechanisms and certain sRNAs involvement regulate the expression pattern of virulence factors under different stress conditions. Small RNA regulation can produce an immediate action to regulatory networks adapted to the acute stress induced by antibiotics. Emergency responders are referred to as Class I small RNAs because they enhance rapid stress responses and aids co-operative degradation of different mRNA targets. Class I sRNAs act in direct mode on the pre-existing mRNA clusters to alter the translation process or deterioration for the acute stress response. Mostly they are involved in disassociating the regulation of transcriptional responses and half-life kinetics of mRNA [38].

It has been reported that during the host infection, variations of temperature and pH, oxidative stress, quorum sensing, biofilm formation and nutrient starvation were related to the functional regulation of small RNAs in *Staphylococcus aureus* [39]. Such responses were controlled by alternative sB (sigma B factor). sB factor regulates several genes that regulate stress-mediated responses, biofilm formation, virulence factor expression, antibiotic resistance, and membrane transport mechanism [40].

Sigma B factor also represses several genes expression by an indirect pathway with the involvement of small RNA or sB-induced regulatory protein. RsaA has a typical sigma B factor promoter which detects its corresponding genes [41]. RsaA base pair with mRNAs repressed by sB like citM and involves in the encoding of Mg-citrate transport systems. sB-dependent sRNAs are the most conserved regions in *S.aureus*. It has been reported that among the three dependent sRNAs, two of them are expected to involve in the regulation of small, highly basic peptides [42].

Production of virulence factors has been regulated by sigma B factor under the stress-dependent activation process. SigB gets activated in the normal stress conditions, also during the growth phase transitions and in different physiological and biochemical changes in *S.aureus* [43]. Thus playing a major role for regulating several others downstream genes. Whereas rsaA are also regulated by a Sigma B-dependent promoter [44].

#### **6. Regulatory sRNA network**

Several sRNAs uses Hfq or ProQ chaperones to anneal with their respective mRNAs targets. *Hfq*, a RNA chaperone comprises a six-ring hexamer fostering annealing of RNAs by aligning to their distal and proximal surfaces [45]. The major function of small RNAs regulation is the suppression by base-pairing with the mRNA RBS to inhibit the initiation of translation. sRNA binding blocks binding of small ribosomal subunit [46]. They also regulate both positively and negatively

various mechanisms involved in regulating gene expression [47]. The different mechanism includes the processing and stability of transcript process [48], transporting and localization of ribosomes, antisense sponging interactions and termination of transcription process [49, 50].

It has been reported that both small RNA and transcriptional mechanisms work together within interleaved feedback and feed-forward loops and regulate the expression of genes. 108 sRNAs were identified using RNA-seq analysis in the model organism *E.coli* [51], and similarly, around 1600–1900 sRNA-mRNA interactions were identified using interactome profiling analysis [52].

Therefore, it is hardly comparable with the 3446 sRNA-mRNA interactions being regulated by the 217 transcription factors with the chromosome [53]. Several transcriptional regulatory networks have an sRNA that integrates with the extra post-transcriptional networks. Small RNAs act similar to transcription factors as a regulatory centre and unevenly controls various RNAs targets. sRNAs are involved in antibiotic sensitivity by mRNAs interactions which take place in drug import, efflux pump regulation, cell membrane synthesis and enhancing antibiotic resistance pattern [54, 55].
