Introduction

Staphylococcus aureus Pathogenesis

Multi-drug resistant (MDR) S. aureus is associated with higher morbidity and mortality in both nosocomial as well as community settings [1]. Infections caused by MDR S. aureus especially in hospital settings is evidently challenging to treat due to the development of resistance to multiple antimicrobial agents as a result of both intrinsic and acquired mechanisms [2, 3]. Furthermore, the emergence of vancomycin resistance among S. aureus absence of new antibacterials have made treatment of these isolates very difficult. Hence, the knowledge about the regulation of virulence gene expression and host pathogen interaction can open new avenues towards the development of novel antivirulent agents to conquer the problem of drug resistance among bacteria. The interaction between S. aureus and the host in the course of infection is a dynamic battleground where the ingenious approaches of S. aureus for existence and localization run into head on with the challenging defenses of the host immune system. S. aureus expresses an extensive range of virulence factors that act in a synchronized manner and increases its capability to inhabit and cause various diseases in the hosts [4–6]. These virulence factors facilitate the biofilm formation, invasion, and colonization to overcome the host defense mechanisms [7]. Approximately all the S. aureus strains produce a set of toxins and enzymes like α, β, γ and δ-hemolysins, proteases, nucleases and lipases to transform the host tissues into nutrients vital for their cell growth [8]. Staphylococcal pathogenicity ensue in a phase dependent fashion involving multiple virulence factors, each phase linking one or numerous explicit virulence factors with the exception of toxin-interceded infections for instance the toxic shock syndrome (TSS) [9].

Key developments using genetic approach have recognized that the global regulators often coordinately regulate the expression of these divergent virulence determinants. The virulence factors of S. aureus can be broadly categorized into exoproteins which majorly includes hemolysins and extracellular proteases and cell wall associated proteins such as adhesin. Numerous studies have been undertaken in the past to demonstrate that the S. aureus has developed a highly advance signal transduction systems which regulates the synchronized expression of multiple virulence genes. Earlier studies have revealed that the multiple drug resistant S. aureus is well equipped to resist antibiotics and also several regulatory loci is a key determinant to establish pathogenicity [10]. Understanding the sequential auto-induction of the genetic circuits regulating the virulence gene expression can provide critical information for specific inhibition of the loci that are accountable for the S. aureus pathogenicity and could possibly evolve an exciting therapeutic prospects. Several loci including agr, sar, sae and rot are important constituents for the expression of various toxins and the cell wall associated proteins in response to various stimuli to help the S. aureus to infect and inhabit host tissues. Hence, enhanced knowledge of roles and mechanism of actions of every locus is significant for the successful prognosis of individuals suffering from the staphylococcal infections. This review highlights the role and molecular basis of well-characterized loci in S. aureus responsible for the virulence gene expression (Fig. 1; Table 1). A comprehensive study of each locus could open a way to identify inhibitors as antivirulent agents rather antimicrobials that could negatively regulate the expression of various virulence genes and may be supportive for the expansion of unique therapies for MDR S. aureus caused infections [11].

Fig. 1
figure 1

The intricate molecular mechanism for the virulence gene expressions in Staphylococcus aureus. Positive and negative symbols depict the up-regulation and down-regulation of virulence determinants

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Table 1 The pathogenic islands regulate virulence determinants of S. aureus
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agr: The Global Regulator

The accessory gene regulator (agr) locus is extensively studied in S. aureus and controlled by two distinct promoters P2 and P3 which encodes a two-component histidine kinase signal-transduction system comprising of two different transcription elements [12]. The hld RNA transcript act as a regulatory molecule of the agr system is encoded by the P3 promoter, while the divergent four ORF labeled as agrB, agrD, agrC, and agrA is driven by the P2 promoter [13]. The signal transduction is mediated by an auto-inducing peptide produced and matured by agrB and agrD that activates AgrC which is a two component histidine kinase receptor [14, 15]. The key virulence regulator, agr prompts distinct alterations in the gene expression which is the cell density dependent mechanism termed as quorum sensing [16, 17]. The accessory gene regulator (agr) is known to modulate an extensive array of virulence factors such as nucleases, proteases, lipases and expression of surface binding proteins [18, 19]. The adaptation to fine tuning of the gene expressions is thought to determine the production of explicit arrays of virulence factors at various phases to establish infection. When the bacterial population is low, the expression of adherence proteins are triggered for the attachment to host tissue and toxins are produced once the infection is established [20–22]. Sensible initiation of agr in vivo and its prominence for pathogenicity of S. aureus has been validated. Decrease in the secretion of extracellular toxins and an increased production of fibronectin binding protein (fnb), protein A and other cell-surface associated proteins were observed in agr null strains [23, 24]. The expression of agr locus also provides a simultaneous impact on colony spreading and virulent determinants and are regulated by the environmental cues [25]. The colony spreading in S. aureus is negatively regulated by the δ-hemolysin and the experimental data also demonstrated that hld mutant strains confirm the prominent colony spreading than the parental level [26]. An amino-swapping in vitro analysis revealed that virulence gene expression is suppressed by the phenylalanine whereas the hemolytic and protease activity are being repressed by the aspartic acid [27].

sarA: Pleiotropic Regulator

The synthesis of polysaccharide intercellular adhesion (PIA) and the subsequent formation of the S. aureus biofilm is governed by a pleiotropic regulator known as SarA. The sar operon comprises of three overlapping regions specifically sarA, sarC and sarB with a size of 0.58, 0.85 and 1.15 kb respectively. These transcripts encode a 14.5 kDa SarA protein and governed by three diverse promoters namely P1, P3 and P2 respectively [28, 29]. It is evident from the crystal structure of SarA that binds as homodimers to the AT rich conserved region of the regulatory domain (promoter) in the DNA. The SarA is a DNA binding protein where each monomer is a 124 residues polypeptide chain that comprises of four α-helices, a C-terminal loop and a short β-hairpin [30]. SarA, a well-characterized regulator alter the expression of the various virulence genes and also shows effective interactions on the intergenic region of P2 and P3 promoter to activate the agr operon [31, 32]. In addition to the agr intergenic promoter SarA can also directly interact to the conserved regions of the various other factors those are independent of agr locus including the cell wall associated proteins like fibronectin-binding protein, adhesins, protein-A and exo-proteins are known as sar boxes. It was observed that sarA is expressed in all the growth phases of the S. aureus and the expression is modulated by various other factors [33]. A pleotropic effect was observed on the expression of the cell wall associated and several other virulence proteins when a transposon (Tn91-LTV1) inserted into the sarA region which was distinct from the agr locus in the S. aureus chromosome. It also modulates the synthesis of several other virulence factors including extracellular proteases [34]. The study on 105 strains of S. aureus has revealed that the agr mutant strains increased the biofilm formation and failed to synthesize δ-toxin than sarA mutated strains [35, 36]. The study also demonstrated the limitation of those strains to form form biofilm due to the synthesis of δ-toxin to act as a surfactant. Significantly, it is also evident that the hld gene for the δ-toxins encoded within the agr dependent RNAIII regulatory molecule. There are limited data exist to explore the anti-biofilm potential of various SarA based inhibitors [37, 38].

sae: S. aureus Accessory Element

The sae operon involves four distinct ORFs known as sae PQRS where a response regulator and a sensor histidine kinase are encoded by saeR and saeS respectively and together they forms a two component regulatory system [39]. The operon functions are significantly regulated by another two open reading frames, saeP and saeQ which are located upstream to saeRS but the activation of these two component regulatory system is not extensively studied. Similar to the other two component signal transduction system, SaeS act as a trans membrane histidine kinase sensor protein which auto-phosphorylates in the presence of environmental signals and consecutively triggers saeR, a response regulator. The triggered saeR identify the promoter sequences of target genes and hence, may work as a transcriptional regulator. Sae is a vital global regulator and studies have shown that the deletion of 3.4 kb sae regions resulted in the decreased expression of many virulence factors including bacterial adhesion and serine proteases. It is also established from various studies that the mutation in sae results for the synthesis of extreme suppression of coagulase and to some extent decreased levels of protein A [40, 41]. Varied environmental cues, pH, salinity and other global regulators of S. aureus such as agr has an impact on the pattern of the sae expression [42, 43]. It was observed that the sub-inhibitory concentrations of antibiotics such as glycopeptides and β-lactams activate the expression of sae while antibiotics such as clindamycin impede the expression. Hence, sae seems to be an essential modulator that regulates the expression of key virulence factors for instance hla, coding for coagulase (coa) and fnbA. In vivo neonatal mouse model and mutational analysis strongly suggested that saeRS is a major regulator for the exfoliation activity [44]. However, the influence of other global regulators on sae is partially conflicting, which may be possibly due to differences in the strains used in the various studies.

lyt Regulon: The Two Component Histidine Kinase System

To understand the LytSR function, the S. aureus chromosome was cloned and the structural and functional similiarity was compared with the two component histidine kinase receptor, lytR and lytS [45]. The sequencing also revealed the presence of two additional ORFs downstream from lytR, termed as lrgA and lrgB but the functionality is still unclear [46]. But, it was observed that during the stationary growth phase of S. aureus, the LytRS governs lrgAB operon and explicitly intricate in negative regulation of the activity of murein hydrolase and lysis induced by penicillin. The data from mutation studies have revealed that the mutation in the lytS gene results in an increased production of autolysis.

A difference in the murein hydrolase activity was detected in the lytS mutant as apparent in the parental strain and hence, the integrity of the cell wall is linked with the lytRS regulon to control the activity via lrgAB [47]. The amino acid sequencing revealed that the lrgA is a 16.3 kDa protein that contain 148 residues whereas the additional ORF, lrgB that encodes a 25.1 kDa protein (LrgB) comprised of 233 residues [48]. Earlier reports demonstrated that lytSR locus play an imperative role during biofilm formation by controlling cell lysis and mediated by the lrgAB and eDNA [49].

arlRS: The Autolysis-Related Locus

The two component regulatory system, arlRS is responsible for the virulence gene expression including adhesion and autolysis [50]. The ArlRS system has identified as a first regulator to influence the biofilm formation, extracellular proteolytic activity, autolysis and the ArlR, also positively modulate the agr system [51]. Mutation analysis has revealed that the arlR-arlS two components syste are also responsible for the production of several secreted proteins in S. aureus. The arl locus is transcribed during the exponential growth phase of S. aureus and it consists of two overlapping ORFs and transcribed together into a 2.7 kb mRNA. The locus comprises a 52.4 kDa sensor protein, ArlS and a 25.5 kDa ArlR regulator protein have a higher sequence similarity with PhoB-OmpR family proteins. Studies have shown that the mutation in arlS, a DNA binding protein increases the activity of peptidoglycan hydrolase and leads to its autolysis. It encompasses a conserved C-terminal domain that binds to the upstream of the target gene promoters and most likely regulates the cell adherence and division. The arl regulon is multifaceted regulatory system to either directly or circuitously via agr and sar operon which modulate the expression of various virulence genes including the serine proteases, protein A, coagulase etc. These results indicate that the inactivation of agr operon and hence, down-regulation of the genes activated by the RNAIII during cell adherence and growth is influenced by the Arl two component regulatory system. It was also observed that the Arl also regulate genes through mgrA-dependent pathway and up-regulates the production of capsule in the S. aureus at the transcriptional level [52]. The exfoliative toxins are positively regulated by the arlRS system and the ArlRS also down regulate the autolysis in the methicillin-sensitive S. aureus strains, but when the arlRS was inactivated it had no impact on autolysis of methicillin-resistant S. aureus [53].

KdpDE: An Universal Communication Network

Autoinducer 2 (AI-2) is a small diffusible molecule widely present in the gram positive and gram negative bacteria and it is also referred as the universal language for the interspecies communication [54] AI-2 is synthesized by the LuxS enzyme during the metabolic pathway. AI-2 is an interconverting compound and derived from the 4,5-dihydroxy-2,3-pentanedione (DPD) to further cyclizes and form two epimeric furanones (2R,4S) and (2S,4S)-2,4-dihydroxy-2-methyldihydrofuran-3-one (R-and S-DHMF). LuxS/AI-2 system is more evidently investigated in the Staphylococcus epidermidis where the luxS gene was functional to regulate the transcription of polysaccharide intercellular adhesion gene ica for the biofilm formation and it also modulates the virulence gene expression [55, 56].

AI-2 quorum sensing in S. aureus is associated with the two components KdpDE system to regulate the virulence determinants as well as capsular polysaccharide synthesis, an important cell wall constituent [57], during the invasive process it interacts with the host immune system and allows the microbes to resist from phagocytosis. Moreover, 11 types of capsular polysaccharides are reported, most of the S. aureus isolates belongs to CP5 or CP8. Earlier experiments revealed that cap operon transcription in the S. aureus NCTC8325 is regulated by the array of regulatory loci they are sarA, agr, sae, arlRS, sbcDC, ccpA, mgr, yabJ-spoVG [58, 59]. Furthermore, it has also demonstrated that the diverse environmental factors also regulate the CP expression. Research data revealed that CP5 and CP8 used as a vaccine development and generated specific antibodies and showed protection against S. aureus infections. KdpDE system was first described in E. coli where the KdpE and KdpD proteins regulate the production of Kdp-ATPase, it is a high affinity K+ Transporter [60]. KdpDE comprise of two proteins, a response regulator and its sensor histidine kinase. The histidine kinase sensor perceives the environmental signals and initiates the phosphorylation mechanism. The recent data demonstrated that the inactivation of KdpDE showed the variation in the virulence gene transcription comprises spa, cap, hla, aur, geh, and hlgB and electromobility shift assay (EMSA) also revealed that KdpE directly binds to these genes promoter to modulate the transcription [61, 62]. Allelic replacement experiment demonstrated that the S. aureus luxS regulate the CP5 gene transcription via AI-2 quorum sensing pathway but the exact mechanism that how LuxS/AI-2 interact with KdpDE and activate the pathway is still require further studies. Accessory gene regulator (agr) also plays an important role to trigger the kdpDE by RNAIII, transcriptional experiment revealed that kdpD and kdpE level were increased when the cell were grown to the post exponential growth phase as it suggests that the Agr quorum sensing influence the transcription of kdpDE. Furthermore electromobility shift assay also demonstrated that the rot protein specifically binds to the promoter of kdpD (Fig. 2).

Fig. 2
figure 2

The Agr quorum sensing circuit coordinates with the KdpDE two component networks to regulate the capsular polysaccharide production in S. aureus

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Repressor of Protein (rot)

The repressor of protein (rot) is a transcription repressor to negatively regulate the production of the virulence factors such α-toxin and protease via the agr operon. It has a 498 bp open reading frame (ORF) that encodes a predicted 161 amino acids protein with a molecular mass of 15.6 kDa [63] and has a partial sequence resemblance with AgrA and SarA. Rot is a global regulator of various genes and encode exotoxins for instance proteases and lipases to play a vital role in the invasion of bacterial cells in the host tissues. The experiment has revealed that the Rot modulates the expression of at least 146 genes where some genes such as spa, sspB and sspC are up-regulated while hla and γ-hemolysin gene is suppressed by Rot [64, 65]. These results clearly indicate that the Rot has a suppressive effect on the expression of agr and it probably inhibits the expression of RNAIII, however the regulatory pathway intricate remains unrevealed. The Rot inhibits the expression of the target genes by impeding their transcription during the exponential phase of S. aureus growth by interacting with genes within the promoter region. It also proposed that Rot up-regulates the expression of adhesins and hence promotes the S. aureus cell proliferation. The study on the production of α-toxin showed that re-establishment of the rot expression totally blocks the expression of this gene and disruption of rot in the agr mutant strains partially reinstates the activity. Since, the expression of various virulence genes in S. aureus is modulated by Rot, it is very essential to comprehend the regulatory pathway and elucidate the linking of Rot with other key virulence determinants.

Conclusion

Sustained occurrence of the MDR S. aureus in the community is a serious problem that deserves enhanced attention in the diagnosis and to identify novel therapeutic targets to curb staphylococcal infections. As discussed above, the S. aureus has developed advanced signaling systems that plays an imperative role in staphylococcal infections and pathogenesis. The virulence gene expression in S. aureus is subjected to constraints at various levels by these global regulatory architectures (two-component systems) that that sense several environmental cues to configure optimal rates of toxin production. The complexity in the fine tuning of the genetic circuits support the viability and pathogenicity of multi-drug resistant S. aureus in disease progression. The disease progression involves various virulence factors that provide a discrete benefit for the cell to cell proliferate and establish cell-density dependent inter-/or intra- species communication to mediate biofilm and pathogenesis [66]. Also, the bacterial cells have shown tremendous advances and evolution in response to their environment such as development of antibacterial resistant mechanism. So our understanding to those genetic circuits with overlapping responses to dynamically control the expression of various factors, regulons and stimulators will probably lead to develop new strategies to combat staphylococcal infections.