Cholera, the Diarrheal Disease

Diarrheal outbreaks have hampered the normal lives of people in developing and under -developed countries. Poor sanitation and hygiene practices have been the root causes behind such outbreaks. Cholera, the diarrheal disease is an outcome of infestation of the human intestine and its milieu with Vibrio cholerae. Two serotypes of the organism viz., the O1 and the O139 have been responsible for pandemic outbreaks throughout. Cholera has been a subject of research from many centuries and has influenced people throughout in their perspective views regarding the disease, ranging from the Miasmatic theory of 19th century which made people believe that this disease was spread through miasma (bad air) [1] to the current bacterial communication (Quorum Sensing) mediated disease progression [2].

World Health Organization (WHO) reports suggest that about 1.4–4.3 million cholera cases are incident every year in the globe of which nearly 28,000–1,42,000 lives are swiped out. Fifteen discrete outbreaks of cholera were documented during 1971–2010 [3]. The mortality rates due to cholera in 2011 increased by 57.96 and 3.62 % when compared to 2009 and 2010 [4, 5]. Reports suggest that numbers of cholera cases in India are more than the ones that are submitted to WHO Weekly Epidemiological Record. Epidemic outbreaks in India were largely due to the regular pilgrims to the Ganges river [6]. According to one of the reports, about 37,783 cholera cases (84 deaths) were incidents in the country between 1997 and 2006 but only very less numbers were given for WHO Records [7]. Among Asian region, the Indian subcontinent continues to be a hub for cholera cases with an occurrence of 78 %. In the year 2013, an aggregate of 47 nations from all mainland’s reported 1,29,064 incidences of cholera to WHO, of which 43 % was accounted for from Africa and 47 % from the America where a huge episode that began in Haiti at the end of October 2010 additionally influenced the Dominican Republic.

The cholera tainted patient ought to be basically supplemented with liquids and electrolytes along with antibiotics to counteract lack of hydration formally. This was done through intravenous using sterile, pyrogen free intravenous solution or by using ORS which contains salts like NaCl (3.5 gm), KCl (1.5 gm), NaHCO3(2.5 gm) and glucose (New formula of all rehydration salt, which reduce osmolarity [8]. Conventional therapy employs the administration of antibiotics as a means of relieving the worsened diarrheal conditions. As defined by CDC (Center for Disease Control) doxycycline/tetracycline are given as a first line treatment in adults whereas azithromycin/erythromycin are given for children and pregnant women. Furazolidone, erythromycin, trimethoprim-Sulphamethoxazole, chloramphenicol, azithromycin, ciprofloxacin are preferred for adult administration [9–12]. The significant detriment of the antibiotic is that it will abbreviate the time of infection instead of counter-acting the disease. Currently used vaccines are WC-rBS and BivWC, where the WC-rBS (marketed as Dukoral) is a monovalent vaccine consist of attenuated V. cholerae O1 along with recombinant cholera toxin B subunit. BivWC (marketed as Shancol and mORCVAX), a bivalent vaccine consist of inactivated V. cholerae O139. Vibrio cholerae has a unique property so that they can exist in an autochthonous state in river, brackish water estuaries and coastal waters and also in dwarfish forms under nutrient deprived conditions as a viable but non cultivable form.

The foremost limitation with the conventional antibiotic therapy is the development of MDR strains of Vibrio cholerae make this therapy abortive [9, 13]. Clinical isolates of V. cholerae strain causative executor for various outbreaks have become impervious to numerous antibiotics including tetracycline, ampicillin, nalidixic acid, streptomycin, sulphonamides, trimethoprim, gentamicin, ciprofloxacin etc. [14, 15]. Reports say that people infected with these MDR strains exhibits features indicating more severe illness than patients affected with wild strains. The infection cause by the MDR strains are paramount not just on the grounds that they are harder to treat, additionally, on the grounds that they may prompt higher morbidity and mortality rate [16]. Therefore, it is an era to overlook the limitations of anti-bacterial approach with anti-virulent therapies on meddling the quorum sensing (Vibrio cholerae murmurs) to counteract cholera [17]. This can be achieved either by interrupting the synthesis of QS signaling molecules or by blocking the receptor molecules thus disturbing the interaction between signaling molecules and receptor [18, 19].

Quorum Sensing: The Bacterial Samvada

The discrete behavioral pattern of bacteria to the diverse milieu they face is an outcome of the cell to cell communication between them called Quorum Sensing (QS) [20]. Chemicals known as autoinducers serve as dialect for the bacterial cells [21]. These autoinducers promotes group behavior in bacteria to establish their virulence traits as they are being sensed by their own populations/different populations [22, 23]. Bacteria define a high degree of signal specificity owing to the differences in the structures of QS signals and its binding domains of the receptor protein [24]. It is also known that bacteria use three classes of highly species specific and non-species specific autoinducers to elicit QS response. The most widely used species specific signal are Acyl-homoserine lactone (AHL) in the Gram negative bacteria, whereas oligopeptide in the Gram-positive bacteria [25]. Most of the other class of autoinducer (AI-2), is known to be a derivative of 4,5-dihydroxy-2,3-pentanedione (DPD), was found to be non-species specific that mediates crosstalk between inter-specious [26]. Just like people of different regions have different dialects; prokaryotes also have cell signaling variants e.g., Streptococcus pyogenes that resides in the pharyngeal tract has peptides as the cell signaling molecule whereas, Uropathogenic E.coli (UPEC), a resident of urinary tract has Acyl Homo Serine Lactones (AHSL) as the cell signaling moiety [27, 28]. The autoinducer concentration is directly proportional to the cell density and its concentration reaches a threshold level at HCD is being sensed by surface receptors and elicits a response at the target gene level expression via activation or repression [29]. This adaptive aggressive behavior is most predominantly observed in bacteria like Pseudomonas aeruginosa, Uropathogenic Escherichia coli etc., paradoxically, the converse happens in case of Vibrio cholerae i.e., under conditions of LCD (Low Cell Density) state virulence factors are expressed and at HCD (High Cell Density) state, virulence is repressed. Additionally, at HCD activation of HapA (Haemagglutinin Protease) occurs which facilitates the dissemination of the bacterial cells from the human intestinal cells.

Quorum Sensing in Vibrio cholerae

There are three QS systems in Vibrio cholerae which converge to regulate virulence identified till date. System I is the Cholera Autoinducer-1 (CAI-1)/CqsS system responsible for inter-vibrio and intra-species communication [2, 30, 31] System II—Autoinducer-2 (AI-2)/LuxP/Q responsible for inter species communication [2, 30, 31] and the System III – Unknown signals/VarS which is yet to be explored [32]. At LCD, both the enzymes CqsA and LuxS that synthesize CAI-1 & AI-2 [30, 33] produce them in a lower concentration. CqsS is the transmembrane receptor for CAI-1 and LuxP/Q is the periplasmic/transmembrane receptor for AI-2. These receptors are bifunctional i.e. at LCD they behave as kinases transferring the phosphate group to the downstream proteins and HCD they behave as phosphatases removing the phosphate groups from the phosphorylated proteins. Lower concentration of these signals leads to an ineffective binding to their cognate receptors. Consequently, the receptors serve as kinases to mediate the phosphate flow a protein called LuxU and this, further transfers it to the regulator protein LuxO (Fig. 1). LuxO is the point of convergence of both the system I and II [2, 31, 34, 35]. System III converges with System I and System II at LuxO protein through activation of VarS/A pathway but the exact mechanism of the series of steps that occur to activate LuxO is unknown [2, 32]. Phosphorylated LuxO along with σ54 is in turn, a transcriptional activator of the Quorum regulatory RNAs (Qrr1-4). Qrr1-4 bound to the RNA chaperone Hfq, binds to mRNA transcript of HapR (the global virulence repressor protein in Vibrio cholerae) and renders the translation of HapR impossible [36].

Fig. 1
figure 1

Vibrio cholerae quorum sensing circuit at LCD state

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HapR is a transcriptional repressor of VpsT, the latter is a transcriptional activator of genes required for biofilm formation [30, 37]. HapR also represses the production of AphA, a protein needed for activation of Cholera Toxin (CT) and Toxin Co-regulated Pilus (the main virulence factors of Vibrio cholerae). HapR represses haemolysin, the major virulence factor of Vibrio cholerae Biotype Eltor both transcriptionally and post-translationally [38]. HapR is also an activator of HapA protease and RNA polymerase sigma factor (rpoS σ54). HapA protease serves as a “detachase” in detaching the Vibrio cholerae cells from the human cells, via digestion of GM-1 receptor of human intestinal cells, which serves as a bridge between bacterial and human cells [39]. σ54 increases the stress response of the bacterial cells to extreme nutrition and oxidative conditions [40]. Thus, under LCD conditions, virulence factors like Cholera toxin, Toxin co-regulated pilus, Haemolysin, Biofilm are expressed but factors like HapA protease, σ54 are repressed because of the absence of the regulatory protein of HapR. Hence, it could be inferred that “virulence and autoinducer concentration are inversely proportional” in case of Vibrio cholerae.

At HCD, the binding of the signals, CAI-1 and AI-2 to their respective cognate receptors CqsS and LuxP/Q will be achieved. This binding switches these receptors from functioning as kinase to phosphatase enzymes. Ultimately, dephosphorylation of LuxO protein, takes place which brings about repression in qrr1-4 production. Qrr1-4 repression subsequently leads to a successful translation of HapR mRNA transcript. As a result, virulence factors like haemolysin, Biofilm, Cholera Toxin, Toxin co-regulated pilus are repressed and HapA protease and σ54 are expressed at HCD conditions (Fig. 2). Thus, virulence is repressed at HCD and established at LCD reflecting on the fleeting and ephemeral life style of Vibrio cholerae i.e. they have a short stay at intestinal environment completion of LCD gives them a positive signal to switch their life style in fecal environment and get disseminated from the intestine of host cells. Therefore, probing QS systems in Vibrio cholerae is an approach to arrive at novel anti-virulent drugs for an MDR scenario free Cholera therapy. So, throughout this review the plausible QS components (signals, receptors, sensors, regulators, regulatory RNAs) have been addressed that could be exploited to disrupt or attenuate the species specific and non-specific mediated quorum sensing as potential antimicrobial targets.

Fig. 2
figure 2

Vibrio cholerae quorum sensing circuit at HCD state

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Plausible Targets

Signals/Autoinducers

CAI-1 and AI-2

The two autoinducers, cholera autoinducer-1 (CAI-1) and autoinducer-2 (AI-2) synergistically impose their control to their target gene expression. It is well documented that the species specific signal, CAI-1 is the stronger, whereas the LuxS is known to synthesize the weaker signal AI-2 (furanosyl borate diester (2S, 4S) -2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran borate). The catalytic process of LuxS involves the fragmentation of a byproduct of SAM metabolism, (S) -ribosyl-homocysteine (SRH), to produce homocysteine and the AI-2 precursor, 4,5-dihydroxy- 2,3-pentanedione (DPD). DPD spontaneously cyclizes and borate is chelated to yield AI-2, identified as (2S,4S)-2-methyl-2,3,4- tetrahydroxytetrahydrofuran-borate [41, 42]. The CAI-1 (Cholera Autoinducer-1), a stronger signal, was previously identified as (S)-3-hydroxytridecan-4-one known to require the enzyme CqsA for its biosynthesis [33, 43]. The sole substrate for CqsA substrates are (S)-2-aminobutyrate and decanoyl coenzyme A, and the product of the reaction is 3-aminotridecan-4-one, dubbed amino-CAI-1 is a pyridoxal phosphate-dependent acyl-CoA transferase reaction. Further, amino-CAI-1 is being converted to CAI-1 via a CqsA-independent mechanism and greater than or equal to100 times more CAI-1 release is observed than amino-CAI-1. V. cholerae QS response is being elicited on sensing the signals, amino-CAI-1 or CAI-1, but forseen to show any significant response to the CAI-1 variants [44]. In coherence to this, a commensal E. coli manipulated to express the cqsA gene that produced a variant CAI-1 inhibits pathogenesis of V. cholerae in an infant mouse model [45] provides an insight that targeting the stronger signal with its structural and functional mimic would probably lead to the development of an anticholera treatment.

figure a

Receptor

CqsS

Bacteria are ubiquitous with Two-component systems (TCS) systems that are used to adapt to environmental changes [46]. CqsS, a unique TCS system in Vibrio cholerae (CqsSvc) known to be highly conserved (70 %) in the transmembrane ligand sensing domains among all Vibrio species as it detects CAI-1 molecules with the extended side chains either with 10-carbon or 8-carbon [47]. The residues Cys170/Phe175 was found to be crucial in the CqsSvc as it signifies the ligand chain length [44]. The CqsSvc Sensor histidine kinases has a N-terminal transmembrane sensing domains, dimerization histidine phosphotransfer (DHp) domains and C-terminal catalytic ATP-binding (CA) domains [48]. It has been theoretically defined to have two-state model for histidine kinases with a “kinase on” and a “kinase off” mechanism [49]. In response to the CAI-1 (HCD), the binding switches the CqsS to an “kinase off” state (phosphotase) and results in the dephosphorylation of phosphorylated response regulators (LuxU ∼ P; LuxO ∼ P). Significantly at LCD condition, the switch is reversed to “kinase off” state, where the His194 is critical for the phosphate flow to the DHp domain as it is evidenced that the H194Q do affect the phosphate flow. Also mutation in the highly conserved glycine residue CqsG379A/G381A of the CA domain was also found to be incapable of autophosphorylation are known to be critical for ATP binding. In accordance to this, researchers have also evidenced to hinder the QS system on targeting those key residues of the CqsS receptor that affect the phosphate flow [50].

LuxPQ

LuxP, a factor belonging to the large protein family PBPs (Periplasmic Binding Protein) sense the weaker non species specific autoinducer, AI-2 to elicit its QS response. [51]. Notably, it interacts with AI-2 molecules in conjugation with a two-component sensor kinase, LuxQ with a hybrid of a periplasmic sensor domain and cytoplasmic histidine domains [52]. Irrespective of LuxP interaction with AI-2 or not, the unliganded apoform hold open to have specific interaction through the PAS domain with the LuxQ (LuxQp) [53]. The PAS domain is quite similar to that of PBPs protein and the LuxP will bind to the tandem PAS folds present in the LuxQ. These PAS folds have prosthetic qroups that help them to bind to ligand molecules [54]. Therefore, the ligand AI-2 interaction regulates LuxQ activity on bringing structural changes in the constitutively associated LuxP: LuxQ complex. Notably, at LCD state, the intrinsic kinase activity of LuxQ would cross phosphorylate the histidine residues within the histidine kinase domain [55] and at HCD state, a retrograde flow of phosphate group. It is also known, that the LuxQ intrinsic activity of kinase is being reduced/converted to phosphatase with AI-2 interaction via integrating the signal to the regulator, LuxR [56].

Regulators

HapR

The master regulator, HapR belong to the family members, TetR and QacR with nine α helices [57]. The C-terminal dimerization interface mediates its contact with its target DNA binding site, aphA promoter. The HapR DNA-binding domain at the N-terminal region is composed of three α helices, α 1, α 2 and α 3 and a dominant electropositive surface was noticed in the HTH motif (helices α 2 and α 3). The dimerization interface promote the HTH motif to be competent to interact to its DNA binding domain without making any significant conformational change in the kink in the α 7 helices as it results with an extended α 5 helix. The residues surrounding helix α 3, (Ser50, Val51, Ala52, Thr53, Phe55, and Asn56) were found to show either polar or hydrophobic base-specific interactions in the major groove of DNA. Also, these residues Arg10, Arg12, Arg18, Lys19, Arg33, Arg37, His40, and Arg61 was found to show significant interaction with the phosphate backbone to provide additional stability to the regulator binding to DNA. The mutational studies (F55A- defective; T53A–weak) have revealed to show higher DNA binding affinity (base-specific interaction) lies in the α 3 (Thr 53 and Phe55) to efficiently bind to aphA promoter to repress biofilm phenotype [58]. Also, HapR molecules has tunnel (C-Terminal) to help the movement of the solvent molecules that contacts an binding pocket (amphipathic cavity) for an unidentified ligand similarly present in the TetR protein molecules. The cAMP- receptor protein (CRP) was found to lower the HapR expression level via integrated with the environmental signals to enhance the growth of organisms in both the human host and the environment [59].

LuxU

LuxU a Phosphorelay protein has two independent functional domains to receive sensory signals (phosphate receiver domain) from a sensory kinase (phosphate donor domain) and integrate the signals to LuxO [60]. At LCD state, kinase switch on and phosphate flow from the sensory kinase, is being transferred to response regulatory protein, named LuxO and it is reversed at HCD state [61]. LuxU with an aminoacid sequence of length 133a.a show greater sequence similarity to other phosphorelay proteins, (HPt), BvgS, ArcB, and Ypd. Its phosphorelay function is highly defined by a conserved His residue (His 58) and it is consistent with the surrounded positively charged residues (Lys54 and Lys61) provides an ultimate binding site for the negatively charged phosphate [62]. We also speculate LuxU, a hub for signal integration as V. cholerae QS relies on two parallel sensory pathways, CAI-1/CqsS and AI-2/LuxPQ where the output converges at LuxU [2]. Similar sensory mechanism has been revealed in the related bacterium V. harveyi, where the information flow from three parallel sensors, CqsS, LuxPQ and LuxN converges at LuxU [63]. The Vibrio QS pathway ensures to have a central domain architecture (LuxU) that means to perform the enzymatic activities required for phosphotransfer to and dephosphorylation of its target proteins. Also LuxU mediated phospho-flow in both directions is well documented as a feed-back regulation [64].

LuxO

LuxO, a NtrC family regulator act as a genetic switch to influence the biofilm formation, toxin production, phosphorylation and bioluminescence in Vibrio species [31]. The LuxO has two separate N-terminal regulatory and C-terminal DNA binding domain where the oligomerization or AAA+ ATPase domain is located between the receiver domain and a helix turn helix (HTH) motif [31, 65, 66]. The HTH motif are known to recognize enhancer like sequence between 100 and 150 base pairs present in upstream of the promoter of the target genes. ATPase domain with its walker A and B motif and Arginine finger motif at active state will recruit the core RNA polymerase with the sigma factor (σ) to promote the transition from closed to open complex. The Walker A motif has consensus sequence GXXXGXGX (X means any amino acid) shows higher affinity of interaction with the target DNA binding site and the magnesium ion present in B motif co-ordinates the stability of this complex. The key residues of LuxO that increases its stability to its DNA binding site are four aliphatic amino acid residues and two negatively charged residues [62]. Also, the Arginine finger motif with the conserved Arg residues probably found to aid ATP binding and hydrolysis as it is required to remodel the closed DNA complex. The critical role of LuxO includes to accept phosphate group then activated to bind to its target site in DNA enhanced by Fis and with the hydrolysis of ATP [53]. Its efficiency of activation/deactivation entirely depends on the concentration of the signal, AI-2 and CAI-2 as it is directly proportional to the cell density state. So, LuxO is activated in the LCD state and vice versa in the HCD state. Recent studies have also shown to characterize novel inhibitors (ML366 and 5-thio-6-azauracil derivatives) to selectively inhibit the ATPase domain of LuxO (response regulatory protein) and reverse the LCD to a HCD condition [31, 67].

Qrrl

Qrrls are the small regulatory RNAs that control the transcription of target genes involved in establishing Vibrio cholerae pathogenicity. The cells at LCD state, the signals (AI-2) integrate to various regulators like, LuxO enhances the expression of the small regulatory RNAs (Qrrl), likely to antisense with the HapR mRNA most probably protecting its 5′UTR- ribosome binding site (RBS) from preventing its initiation of translation and also repress its action of regulating the target gene expression. It has also been observed that the Qrrls shows negative feedback regulation of repressing the translation of LuxO protein and their interaction to its target mRNA are being stabilized by an RNA chaperone, Hfq [68, 69]. The Qrrl protection of its target mRNA would act as signal sequence to be either degraded or to prevent them from translation. Base pairing of Qrrl RNA to its target mRNA is aided with the first two stem loops (S1) and (S2) the third stem loop (S3) stabilizes the complex and the fourth stem loop (S4) act as terminator probably it means to provide an intrinsic strength to respond to the QS response [70].

ToxR

Vibrio cholerae respond to environmental factors as it triggers the expression of membrane bound regulatory protein, ToxR and elicit QS response. The regulator, ToxR has two separate N-terminal cytoplamic domain (binds to DNA molecules) and C- terminal periplasmic domain (interact with transmembrane protein) [71]. The role of ToxS protein is very essential for the activation of ToxR gene, although its functional significance is not clear other than it is known to stabilize and dimerise the Tox R protein [71, 72]. Combined effect of ToxR-ToxS complex is well known to activate OmpU, OmpT and porins molecules [73]. The cells should have an equal balance of porin molecules to resist against bile and intestinal colonization. The main target of ToxR-ToxS is the ToxT, as it is triggered with the factor complex, TcpP-TcpH that result in the CT toxin. It has been well documented that the factor complex, TcpP-TcpH is regulated by the apha gene at the LCD state. The ToxR (control of virulence) gene expression has been related to have influenced with the environmental cues where the cAMP-CRP system was found to be critical in its regulation. When the concentration of cAMP increases, the cAMP-CRP complex is formed and thus repress the expression of the gene, tcpPH as both CRP and AphAB binding site overlap at tcpPH promoter [59]. Since the global regulator, cAMP-CRP exhibit combinatorial control and its influence over ToxR regulon under various environmental condition provides an insight to explore the involvement of multiple overlapping systems on its control (Fig. 3).

Fig. 3
figure 3

Vibrio cholerae quorum sensing circuit response to environmental cues

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Conclusion

The review has established that the signals AI-2/CAI-1/environmental cues elicit QS response via QS molecules (regulators, small regulatory RNA etc.) to establish Vibrio cholerae pathogenicity. Notably, exploiting QS molecules might be a novel strategy to discover novel anti-infectives or anti-virulent molecules for cholera therapy. Earlier reports accounted to use similar approach for the development of anti-pathogenic compounds from Terminalia chebula Retz to quench AHL mediated QS in Pseudomonas aeruginosa PAO1 and sponge derived compounds against Serratia marcescens [74, 75]. Our earlier studies have identified CAI-1 mimic molecules from Melia dubia leaves and its potency to establish QS in Vibrio cholerae has opened the roads to a new anti-virulent Cholera therapy employing QS mimic molecules [76]. We could also prove that the methanol extract of Melia dubia seed will competitively inhibit SdiA (quorum regulator) in E.coli thus inhibits the hemolysin, biofilm production and motility of the bacteria [77]. Any QS based anti-virulent therapy aims at reversal of the CqsS and LuxP/Q molecular switch from kinase to phosphatase i.e. mimics HCD conditions in an LCD state. This anti-virulent strategy is highly efficacious since it completely eliminates the possibility of development of MDR strains, making the therapy effective and also eliminates the subsequent need for strain tracking. Hence, interfering with Vibrio cholerae murmurs is a way employed currently to combat Cholera and recover Cholera victims.