An Updated Meta-Analysis of the Associations Between MicroRNA Polymorphisms and Susceptibility to Rheumatoid Arthritis

Aims: Rheumatoid arthritis (RA) is characterized by cartilage and bone damage leading to disability. Here, the association between microRNA (miRNA) polymorphisms and susceptibility to RA was evaluated by performing an updated meta-analysis and systematic review. Main methods: An electronic search of databases including PubMed and Embase was performed from inception to December 8, 2017 to retrieve studies investigating the association between miRNA polymorphisms and RA risk. Two reviewers independently screened literature according to the inclusion and exclusion criteria and extracted data. The meta-analysis was conducted using Stata 14.0 software. Key findings: Thirteen case-control studies with 2660 cases and 4098 controls were screened out after a systematic search. One study from the miR-146a rs2910164 G > C polymorphism group and two from the miR-499 rs3746444 T > C polymorphism group were excluded because of deviations from Hardy-Weinberg equilibrium. Pooled analysis demonstrated that miR-146a rs2910164 G > C polymorphism was not significantly associated with susceptibility to RA. However, a significant association was observed between miR-499 rs3746444 T > C polymorphism and RA risk (C vs. T: OR = 1.22, 95% CI = 1.05–1.42, P = 0.008; TC vs. TT: OR = 1.26, 95% CI = 1.05–1.50, P = 0.011; TC/CC vs. TT: OR = 1.26, 95% CI = 1.07–1.5, P = 0.007). Subgroup analysis based on ethnicity showed no significant association between miR-499 T > C polymorphism and susceptibility to RA in the Asian population (P > 0.05). However, in Caucasian population, the C allele in the miR-499 T > C polymorphism was a contributor to RA susceptibility in some genetic models (C vs. T: OR = 1.64, 95% CI = 1.28–2.11, P < 0.001; TC vs. TT: OR = 1.95, 95% CI = 1.40–2.71, P < 0.001; TC/CC vs. TT: OR = 1.96, 95% CI = 1.43–2.69, P < 0.001). Significance: The miR-146a rs2910164 G > C polymorphism was not associated with susceptibility to RA. In the Caucasian population, the C allele in the miR-499 T > C polymorphism contributed to RA susceptibility.

INTRODUCTION
Rheumatoid arthritis (RA), one of the most prevalent chronic inflammatory diseases, is characterized by damage to cartilage and bone leading to disability (Smolen et al., 2016). It is a substantial burden for individuals and society, with up to 0.5% of the global population affected (Neogi et al., 2010), and the identification of novel therapeutic strategies to control inflammation and reduce severe consequences is increasingly imperative. The pathogenesis of RA is closely associated with genetic and environmental factors, with the major histocompatibility complex (MHC) region at 6p21 contributing to 30-50% of genetic liability to RA (Yamamoto et al., 2015;Okada et al., 2016;Terao et al., 2016). Cigarette smoking also plays an important role in RA risk (Sokolove et al., 2016).
Rheumatoid arthritis risk was recently shown to be associated with epigenetic regulation including DNA methylation, histone modification, and transcriptional/post-transcriptional regulation (Klein and Gay, 2015). MicroRNAs (miRNAs), which are the most widely studied class of non-coding RNAs, function in the post-transcriptional regulation of gene expression by controlling the translation of mRNA into proteins (He and Hannon, 2004). Moreover, miRNAs play a significant role in regulating biological processes, including proliferation, apoptosis, and development (Esteller, 2011).
A growing body of evidence indicates that single nucleotide polymorphisms (SNPs) in miRNAs may affect RA susceptibility because certain SNPs located in miRNA encoding genes can alter miRNA expression, pri-miRNA and pre-miRNA processing, or miRNA-mRNA interactions (Furer et al., 2010;Dai and Ahmed, 2011). Ciccacci et al. (2016) reported that the C allele of rs2910164 in miR-146a has a protective role in the pathogenesis of RA. However, Piatek et al. (2016) reported no significant association between miR-146a polymorphisms and RA susceptibility or progression. With regard to miR-499, the effect of C allele of rs3746444 on RA risk is also controversial (El-Shal et al., 2013;Zhang et al., 2013). The association between miRNA polymorphisms and susceptibility to RA remain unclear. Because of increasing number of case-control studies published, an updated meta-analysis is required to evaluate the relationship between miRNA polymorphisms and RA, including subgroup analyses investigating ethnicity-specific effects.

Literature Search Strategy
A comprehensive search of the PubMed and Embase databases was performed to retrieve studies investigating the association between miRNA polymorphisms and RA susceptibility published in English up to December 8, 2017. The search terms were as follows: "rheumatoid arthritis" "arthritis" "microRNA" "miRNAs" "polymorphism" and "polymorphisms." Relevant journals and the references of the included studies were also reviewed.

Inclusion and Exclusion Criteria
The inclusion criteria of our meta-analysis were as follows: (1) case-control studies; (2) investigation of miRNA polymorphisms and RA susceptibility; (3) detailed numbers of alleles and genotypes between cases and controls; (4) full text published in English; (5) control group consistent with Hardy-Weinberg Equilibrium (HWE); and (6) any specific miRNA with at least two case-control studies to evaluate the association between single nuclear polymorphism and RA susceptibility.
The exclusion criteria were as follows: (1) reviews or case reports without controls; (2) no available data reported; and (3) duplicated reports.

Data Extraction
Two investigators (Zhou and Jiang) screened the literature and extracted data independently according to the inclusion and exclusion criteria. Discrepancies and differences were resolved by a third investigator (Zhu) until consensus was reached. The following data were retrieved: author list, year of publication, ethnicity, sample size, alleles, genotype of each gene polymorphism, and HWE.

Data Synthesis and Statistical Analysis
The odds ratios (ORs) with their corresponding 95% confidence intervals (CIs) were used to assess the strength of the association between polymorphisms in miRNAs and RA. For miR-146a rs2910164 G > C, the dominant (GC + CC vs. GG), recessive (CC vs. GC + GG), heterozygous (GC vs. GG), homozygous (CC vs. GG), and allelic (C vs. G) genetic models were used to calculate pooled ORs. These models were also used to evaluate the miR-499 rs3746444 T > C polymorphism, including the dominant (TC + CC vs. TT), recessive (CC vs. TC + TT), heterozygous (TC vs. TT), homozygous (CC vs. TT), and allelic (C vs. T) models.
The Chi square-based Q-test was used to assess the betweenstudy heterogeneity and a P-value of 0.1 was considered to indicate significant heterogeneity among studies (Lau et al., 1997). The I 2 metric value was also used to describe the proportion of total variation in study estimates due to heterogeneity, with values of 25, 50, and 75% considered as evidence of low, moderate, and high heterogeneity, respectively (Higgins and Thompson, 2002). The pooled OR estimation of each study was calculated by the fixed-effects model (the Mantel-Haenszel method) when the P-value was >0.1 and I 2 was <50% (Mantel and Haenszel, 1959). Otherwise, the randomeffects model (the DerSimonian and Laird method) was used (DerSimonian and Laird, 1986). Subgroup analyses according to ethnicity were also performed to calculate ethnic-specific ORs. The HWE was tested for each study by comparing the observed and expected genotype frequencies of the control group (Chi-square test). Publication bias was evaluated with Egger's linear regression and Begg's funnel plots (Hayashino et al., 2005). Statistical analyses were performed using the Stata software (version 14.1; Stata Corp., College Station, TX, United States) with a two-sided P-value. P < 0.05 was considered significant.

Study Characteristics
The results of the systematic search and selection performed according to the inclusion and exclusion criteria are shown in Figure 1. A total of 13 articles with 2660 cases and 4098 controls were finally included in the meta-analysis (Chatzikyriakidou et al., 2010;Yang et al., 2011Yang et al., , 2016Jimenez-Morales et al., 2012;El-Shal et al., 2013;Hashemi et al., 2013;Zhang et al., 2013;Zhou X. et al., 2015;Bogunia-Kubik et al., 2016;Ciccacci et al., 2016;Toraih et al., 2016;Fattah et al., 2017;Hassine et al., 2017). The 13 articles comprised 16 studies, including 9 studies on miR-146a rs2910164 and 7 studies on miR-499 rs3746444. Table 1 shows the characteristics of the retrieved studies. Various genotyping methods were used, including PCR-RFLP, TaqMan, and PCR. The genotype distribution of the controls in all studies was consistent with HWE, except one study from the miR-146a polymorphism group (El-Shal et al., 2013) and two from the miR-499 polymorphism group (Hashemi et al., 2013;Toraih et al., 2016).

Sensitivity Analysis and Publication Bias
A leave-one-out analysis was performed to investigate the influence of each individual study on the pooled ORs (Figures 3B,  5B). The results indicated no significant alteration in the pooled ORs after exclusion of any study on miR-146a rs2910164 G > C and miR-499 rs3746444 T > C. Moreover, because of the limited number of studies included (<10), publication bias was not evaluated.

DISCUSSION
Genetic and epigenetic factors play pivotal roles in cancer and autoimmune diseases. MiRNAs are important for the regulation of the cartilage-invading phenotype of RA synovial fibroblasts, leukocyte activation, and cytokine production, and their roles contribute to our understanding of the mechanisms underlying RA and provide novel therapeutic targets for the treatment of RA (Baxter et al., 2012). MiR-146a, one of the most extensively investigated miRNAs, exerts significant effects on cancer and autoimmune diseases. MiR-146a is widely expressed and involved in the differentiation and activation of the innate and adaptive immune systems (O'Connell et al., 2010). It suppresses NF-κB signaling by downregulating the NF-κB signaling transducers interleukin-1 (IL-1) receptor associated kinase 1 and tumor necrosis factor receptor-associated factor 6. Meanwhile, miR-146a directly activates signal transducer and activator of transcription 1 to promote the activity of Treg cells in RA, which mediate the main mechanisms underlying RA (Zhou Q. et al., 2015). Polymorphisms of miR-146a are associated with various cancers and autoimmune diseases (Wang et al., 2012;Xu et al., 2012).
A single nucleotide polymorphism consisting of the G/C nucleotide substitution in miR-146a rs2910164 could result in the replacement of a G:U pair by a C:U mismatch (Hassine et al., 2017). However, the association between mir146a polymorphisms and RA remains controversial. Because of the increasing number of studies, we set strict inclusion and exclusion criteria to avoid potential bias from inadequate studies. Data from Singh et al. (2014). were not included in this meta-analysis because the patients were diagnosed as juvenile idiopathic arthritis (JIA), which includes other types of arthritis in young patients. Data from El-Shal et al. (2013) were also excluded from the pooled analysis for mir146a because of deviation from HWE in the control group. The results of the updated meta-analysis suggest that mir146a polymorphisms are not associated with RA risk, which is consistent with previous studies (Li et al., 2014;Lee and Bae, 2015).
The mechanism underlying the association between RA and miR-499 was investigated previously. One possible explanation is that mir-499 modulates the expression of regulatory factor X4, thus regulating the human leukocyte antigen DRB1, which is closely associated with RA (van Gaalen et al., 2004;Yang et al., 2011). Moreover, miR-499 targets IL-17 receptor B, IL-6, and several cytokines, which are important factors involved in RA pathogenesis (Ying et al., 2011;El-Shal et al., 2013). Although previous studies demonstrated the association between miR-499 polymorphisms and RA, the limited study number and high heterogeneity decreased the robustness of the conclusions. In the present study, two studies from the miR-499 polymorphism group were excluded from the meta-analysis because the genotype distribution of the controls in all studies was inconsistent with HWE (Hashemi et al., 2013;Toraih et al., 2016).  Frontiers in Physiology | www.frontiersin.org Fattah et al. (2017) found that miR-499 rs3746444 T > C is associated with the susceptibility to RA, and the heterozygote is characterized by higher levels of C-reactive protein, anticyclic citrullinated peptide, antibody and disease activity score 28 than those of the homozygote (Fattah et al., 2017). Hashemi et al. (2013) also reported that the C allele of the mir-499 rs3746444 polymorphism is a risk factor for RA. Although the overall results of the pooled analysis indicated that the miR-499 T > C polymorphism was associated with RA risk, the high heterogeneity suggested an insufficient robustness of the conclusion. In the subgroup analysis according to ethnicity, a strong correlation between the miR-499 T > C polymorphism and RA was observed in the Caucasian group, which suggested that the C allele in the miR-499 T > C polymorphism was an obvious contributor to RA susceptibility. However, no relationship was observed in the Asian group. The results were in consistency with previous meta-analysis (Fu et al., 2016), however, Fu et al. (2016) drew a conclusion about miR-499 polymorphism group based on three studies including two studies (El-Shal et al., 2013;Hashemi et al., 2013) from the Caucasian group and one (Yang et al., 2011) from the Asian group. As increasing number of case-control studies published, this updated meta-analysis retrieved five studies including two (El-Shal et al., 2013;Fattah et al., 2017) from the Caucasian group and three from Asian group (Yang et al., 2011(Yang et al., , 2016Zhang et al., 2013). Due to the inconsistency with HWE, two studies (Hashemi et al., 2013;Toraih et al., 2016) were excluded from the miR-499 polymorphism group, which increased the robustness of this meta-analysis.
The present study had several limitations. Firstly, the variation in sample size and in genotyping methods was a limitation of the study. Secondly, all studies included in our meta-analyses were in accordance with HWE, and because of the limited number of included studies, no publication bias could be tested. Because of the limited quantity and quality of the included studies, the above conclusion needs to be further verified in additional high quality studies.

CONCLUSION
In conclusion, the present meta-analysis showed no significant association between miR-146a and RA risk, which is consistent with previous studies. However, the present study demonstrated the contribution of the C allele in the miR-499 T > C polymorphism to RA susceptibility in the Caucasian population, and this association was not present in the Asian population.

AUTHOR CONTRIBUTIONS
XZ conceived and designed the study and contributed to statistical analyses. MZ, BJ, and MX contributed to eligible study collection and data extraction. XZ and BJ prepared the tables and figures. XZ, MZ, and MX wrote and revised the manuscript.


s2fieldsofstudy: Biology Medicine

text:
Immune Responses to Dengue and Zika Viruses—Guidance for T Cell Vaccine Development

Despite numerous efforts to identify the molecular and cellular effectors of the adaptive immunity that induce a long-lasting immunity against dengue or Zika virus infection, the specific mechanisms underlying such protective immunity remain largely unknown. One of the major challenges lies in the high level of dengue virus (DENV) seroprevalence in areas where Zika virus (ZIKV) is circulating. In the context of such a pre-existing DENV immunity that can exacerbate ZIKV infection and disease, and given the lack of appropriate treatment for ZIKV infection, there is an urgent need to develop an efficient vaccine against DENV and ZIKV. Notably, whereas several ZIKV vaccine candidates are currently in clinical trials, all these vaccine candidates have been designed to induce neutralizing antibodies as the primary mechanism of immune protection. Given the difficulty to elicit simultaneously high levels of neutralizing antibodies against the different DENV serotypes, and the potential impact of pre-existing subneutralizing antibodies induced upon DENV infection or vaccination on ZIKV infection and disease, additional or alternative strategies to enhance vaccine efficacy, through T cell immunity, are now being considered. In this review, we summarize recent discoveries about cross-reactive B and T cell responses against DENV and ZIKV and propose guidelines for the development of safe and efficient T cell vaccines targeting both viruses.

History
Zika virus (ZIKV) is a Flavivirus transmitted by Aedes species mosquitoes. It is a single positive-stranded RNA virus closely related to yellow fever virus (YFV), dengue virus (DENV) and West Nile virus (WNV) [1]. First isolated in Uganda in 1947 [2], it remained confined to several regions in Africa and Asia from that time until the early 2000s. In 2007, however, it caused an explosive outbreak for the first time outside of Africa and Asia, on Yap Island, Federated States of Micronesia [3,4], followed by subsequent outbreaks with higher numbers of cases in 2013-2014 in French Polynesia and other South Pacific Islands and more recently in the Americas [5][6][7][8][9]. Although initially believed to only cause mild disease, the 2013-2014 and 2015 outbreaks in French Polynesia and Brazil clearly revealed that ZIKV causes neurological complications, such as Guillain-Barré syndrome in adults and microcephaly in infants born to ZIKV-infected women [10][11][12][13]. Phylogenetic studies indicated the presence of two lineages of ZIKV, the African and Asian lineages, the latter being responsible for the recent major outbreaks in French Polynesia and South America [14,15]. Notably, it was suggested that the enhanced infectivity of the Asian lineage of ZIKV was due to a spontaneous mutation in the gene coding for Non-Structural Protein 1 (NS1) leading to its higher secretion in the serum and infectivity to mosquitoes [16], which could explain its recent re-emergence in the Americas [14,15] despite its relative absence in South East Asia. More strikingly, several amino acid substitutions in the proteome or more specifically in the precursor membrane (prM) protein with possible functional implications for ZIKV biology and pathogenesis have been identified from ZIKV outbreak strains in South America [17,18].
In addition to the high infectivity of the Asian lineage in the Americas, one of the most important concerns today is related to the high level of DENV seroprevalence in areas where ZIKV is circulating [19]. This is particularly important given the structural similarities between ZIKV and DENV [20][21][22], and the existence of cross-reactive immune responses associated with disease pathogenesis [23][24][25][26][27]. Nevertheless, while it is now well established that a secondary infection with a heterologous DENV serotype represents a risk factor for the development of severe dengue disease, because of serotype cross-reactive or sub-neutralizing antibodies which can mediate antibody dependent enhancement (ADE) [28], it remains to be determined whether a previous DENV infection can also increase the risk of developing a more severe ZIKV disease in humans, as suggested by studies in mice [26]. Likewise, while ZIKV-immune plasma can enhance DENV infection in immune-deficient mice [24], the role of ZIKV immunity in protection or enhancement of dengue disease in humans is still unknown.
In this review, we address the most recent findings regarding the adaptive immune response against ZIKV, focusing on the effect of DENV pre-existing immunity on ZIKV infection, the underlying idea being to identify immunological parameters predictive of increased susceptibility or protection against ZIKV infection and disease. In this respect, we will review the current state of knowledge on the impact of anti-DENV antibodies on ZIKV infection and disease, and then summarize the recent data on the potential role of T cells in DENV and ZIKV immunity, with the aim to promote a long lasting immune protection against these two viruses.

Antibody Cross-Reactivity between Zika and Dengue Viruses
The high level of cross-reactivity among flaviviruses, especially DENV and ZIKV which share 54-59% sequence identity in the E protein [20,29,30], and their co-circulation in the same endemic regions have complicated serological approaches to discriminate between these two viral infections. In most cases, reverse transcription-polymerase chain reaction (RT-PCR)-based assays within a week post-infection, in combination with serological binding assays to recombinant proteins and functional neutralization assays in vitro, either by Plaque Reduction Neutralization Test (PRNT) or Flow-Cytometry-Based Neutralization Assay have been developed to distinguish between ZIKV and DENV infections [23,24,[31][32][33][34].
Upon primary DENV infection, different types of antibodies have been found in polyclonal human sera: serotype-specific antibodies with strong neutralizing activity against the homologous DENV serotype, and cross-reactive antibodies with weak neutralizing activity and strong enhancing potential against heterologous DENV serotypes [35,36]. In secondary DENV infections, both type-specific and cross-reactive neutralizing antibodies, as well as cross-reactive enhancing antibodies to the other DENV serotypes, are elicited [37]. Following multiple DENV infections, the polyclonal response has been shown to contain mainly cross-reactive antibodies that recognize the different DENV serotypes, these antibodies being potentially associated with a more durable cross-protective immunity [37,38].
Upon ZIKV infection, it has been shown that the quality and type of antibody response depends on previous infection with DENV, with a more restricted specificity against ZIKV in DENV naïve individuals [24,25]. Using a panel of monoclonal antibodies (mAbs) derived from ZIKV-infected individuals with or without previous DENV immunity, it was shown notably that the antibody response against ZIKV includes envelope domain III (EDIII)-specific antibodies with strong neutralizing activity against ZIKV, as well as envelope domains I and II (EDI/II)-specific antibodies with high degree of cross-reactivity against the E protein of all four DENV serotypes and low neutralizing potential against ZIKV [24]. Thus, just like how EDI/II-specific and cross-reactive antibodies induced after DENV infection can enhance DENV and ZIKV infection [23,[39][40][41][42][43], EDI/II cross-reactive antibodies raised by ZIKV infection can also potently enhance DENV and ZIKV infection [24,27]. Interestingly, not all cross-reactive antibodies are enhancing, as shown in a recent study where memory B cell clones derived from different ZIKV-infected individuals could produce antibodies that neutralize both ZIKV and DENV serotype 1 (DENV1) [44]. In this case, cross-reactivity was shown to result from a previous DENV1 infection and the clonal expansion of memory B cells specific of an EDIII epitope of ZIKV and DENV1 and neutralize both types of viruses. This observation provides the first evidence for a ZIKV-neutralizing antibody response that derives from pre-existing immunity to DENV. However, the low frequency of these cross-reactive memory B cells within the polyclonal population of circulating memory B cells raises the question of the impact of such responses in the immune protection against ZIKV infection. In this sense, it would be helpful to determine whether multiple DENV infections could boost such memory B cells with a broad spectrum of neutralization, in the same manner as the DENV1-induced cross-neutralizing response [44].
At the polyclonal level, it was confirmed that while a primary ZIKV infection could induce predominantly ZIKV-specific antibodies that poorly cross-react with the four DENV serotypes, a ZIKV infection in individuals with previous DENV infections results in the production of both ZIKV-specific and DENV-cross-reactive antibodies [45]. Interestingly, depletion of these DENV-cross-reactive antibodies did not affect the level of anti-ZIKV neutralizing antibodies, suggesting that ZIKV-specific neutralizing antibodies are produced after ZIKV infection regardless of previous DENV immunity [45].
Thus, while it is clear that DENV infections can induce the production of cross-reactive antibodies that recognize the four DENV serotypes, and to a lesser extent ZIKV, in most cases, they do not induce durable, high-level ZIKV cross-neutralizing antibodies [45].
Similarly, upon primary or secondary DENV infections, it is now well established that the level of DENV-specific neutralizing antibodies decay rapidly in the absence of re-exposure, or more slowly in endemic settings [46][47][48][49], and individuals with high neutralizing antibody titers have lower probability of symptomatic infections, in comparison with individuals with low neutralizing antibody titers [49]. In this sense, a positive correlation was clearly established between the low level of pre-existing anti-DENV antibodies and the severity of secondary dengue disease in humans, with a higher risk of severe dengue with anti-DENV antibody titers of 1:21 to 1:80 and a stronger protection against symptomatic dengue disease observed with higher antibody levels [19,50]. More recently and finally, while type-specific neutralizing responses were initially thought to elicit life-long immunity against homologous reinfections [51,52], there is now clear evidence that homologous reinfection can occur in the presence of serotype-specific neutralizing antibodies, leading to symptomatic disease [53,54]. While in some cases these homotypic reinfections have been observed more than 10 years after the initial infection [53], in other cases, such reinfections were identified with a shorter time interval of 1 to 2 years between the successive infections [54]. In both situations, the specific level of pre-existing anti-DENV antibodies provides an explanation for the homologous serotype reinfection and symptomatic disease observed in individuals from different endemic regions [53,54]. Although it is too early to determine whether the same correlation applies to ZIKV infection, it seems likely that the decay in the level of ZIKV-specific neutralizing antibodies in the absence of re-exposure to this virus should affect the outcome of future ZIKV infection and disease.

T Cell Responses against DENV and Prospects for a Vaccine
Although the exact role of T cells during dengue virus infection and disease is still a matter of debate [55], there is currently a growing body of evidence supporting a protective role for T cells in dengue virus infection, both in human and mouse studies [56,57].
Briefly, similarly to the ADE phenomenon hypothesis associated to cross-reactive antibodies, it was proposed that DENV-specific T cells could play a detrimental role during secondary dengue infection. In this "original antigenic sin" scenario, an expansion of cross-reactive T cells with higher avidity to the previous infecting serotype would mask the specific T cell response against secondary infection, and would result in less efficient elimination of DENV-infected cells [58][59][60]. These cross-reactive T cells, stimulated upon a secondary infection with a different serotype, also displayed quantitative and qualitative differences in their response to the cross-reactive epitope [61], with higher ratios of Tumor Necrosis Factor alpha (TNF-α) to Interferon gamma (IFN-γ)-producing CD4 T cells [62], suboptimal degranulation but high cytokine production [63], or more specifically impaired IFN-γ production [64].
However, in spite of these studies, the direct demonstration of a pathogenic role of DENV-specific T cells in patients experiencing natural secondary dengue infection is still missing, and recent reports do not support a causative role for cross-reactive T cells in the pathogenesis of dengue hemorrhagic fever during secondary infections [65,66]. Indeed, increased frequencies of DENV-specific CD4 + and CD8 + T cells were detected in school children who subsequently experienced subclinical infection, in comparison with symptomatic secondary DENV infections [67]. In addition, protection or susceptibility to severe dengue disease has been associated with the expression of certain Human Leukocyte Antigen (HLA) molecules [68][69][70][71][72][73] and a beneficial function of CD8 + T cells against DENV infection was demonstrated after depletion of the CD8 T-cell compartment in interferon-α receptor knock-out mice (ifnar −/− ) [74]. More strikingly, a strong correlation was established between protection against severe dengue and a polyfunctional memory CD8 + T cell response with a high magnitude in healthy dengue-immune individuals [75]. Finally and more recently, we observed a higher activation of Natural Killer (NK) cells and T cells in asymptomatic dengue viral infection; different T cell populations proliferate more and have an activated phenotype, with increased pathogen recognition, signal transduction and higher cytotoxic activity in asymptomatic DENV infected individuals compared to individuals with symptoms during DENV infection [76].
While CD4 + T cell responses are mainly directed toward the structural proteins capsid (C) and envelope (E) and the non-structural protein NS1, similarly to DENV-specific B cells that target prM, E and NS1, most CD8 T cell epitopes reside in the non-structural proteins NS3, NS4B and NS5, and to a lesser extent in the structural protein E [60,75,[77][78][79][80].
Besides the identification of DENV-specific T cell epitopes recognized by activated T cells, these studies contributed to identifying correlates of protection, as the result of an efficient T cell activation by immunodominant peptides restricted by certain HLA alleles, and a robust and polyfunctional CD4 or CD8 response [75,[81][82][83][84][85]. This is true, for example, for the HLA-B*0702 and -B*3501 class I molecules, and the HLA-A*0101 and -A*2402 associated with a high and a low CD8 T-response frequency and magnitude, respectively [75]. The same goes for the HLA-DRB1*0401 or -DRB1*0802 class II alleles, which are associated with an increased resistance or susceptibility to severe dengue disease, respectively, and the phenotype of responding cytotoxic CD4 T cells [81].

Identification ZIKV-Specific and DENV/ZIKV Cross-Reactive T Cells
In contrast to a large number of studies on T cell responses to DENV infection, to date, relatively little is known regarding the analysis of T cell responses to ZIKV infection.
First predictions of ZIKV T cell antigens were conducted by modelling potential epitopes that could bind to different HLA class I or class II alleles, from the ZIKV proteome [86][87][88], and by identifying short peptides targeted by DENV-specific CD8 + T cells with conserved sequences between DENV and ZIKV [89], with the underlying assumption that these epitopes should stimulate cross-reactive T cells after sequential DENV and ZIKV infection.
While such sequence conservation between several immunogenic CD8 + T cell peptides from DENV and the corresponding ZIKV sequence was highlighted in the E, NS1, NS3 and NS5 proteins [89], a study revealed that memory T cells against NS1or E proteins were poorly cross-reactive, even in donors previously infected by DENV [24].
To identify first the dominant epitopes of ZIKV recognized by CD8 + T cells in the context of human HLA class I molecules, and to clarify the protective role of CD8 + T cells in ZIKV infection, different mouse models have been used. Type I interferon receptor-deficient mice expressing human HLA class I molecules (HLA-B*0702 or HLA-A*0101), which are susceptible to DENV and ZIKV infection, enabled identification of ZIKV peptides that are targeted by CD8 + T cells, and to show that dengue or Zika virus infections can induce the development of cross-reactive CD8 + T cells that are protective against ZIKV infection [90]. In HLA class II transgenic mice, CD4 immunodominant epitopes have also been mapped from ZIKV Envelope, and from non-structural proteins NS1, NS3 and NS5, among which several peptides in the envelope revealed cross-reactivity with other flaviviruses [91] ( Figure 1).
infection, enabled identification of ZIKV peptides that are targeted by CD8 + T cells, and to show that dengue or Zika virus infections can induce the development of cross-reactive CD8 + T cells that are protective against ZIKV infection [90]. In HLA class II transgenic mice, CD4 immunodominant epitopes have also been mapped from ZIKV Envelope, and from non-structural proteins NS1, NS3 and NS5, among which several peptides in the envelope revealed cross-reactivity with other flaviviruses [91] (Figure 1). In another mouse model, which lacks the type I interferon receptor in a subset of myeloid cells [92], adaptive transfer of ZIKV-immune CD8 + T cells was shown to inhibit primary ZIKV infection and replication [93]. Likewise, in ifnar −/− and in wild-type mice, adoptive transfer and depletion experiments have demonstrated that DENV-immune CD8 + T cells, but not DENV-immune sera can mediate cross-protective responses against ZIKV infection [94]. Although a correlation was previously established in mice and rhesus monkeys between the level of neutralizing antibodies induced upon ZIKV vaccination and the immune protection against ZIKV challenge [95,96], these studies highlight the essential role of CD8 + T cells in providing a protective immunity, in the presence or not of pre-existing DENV immunity, a finding that could help define future strategies of vaccine against DENV and ZIKV [97]. Finally, wild-type immunocompetent mice, ifnar −/− mice and Interferon Alpha Receptor (IFNAR)-depleted rag1 −/− mice, have helped to characterize the phenotype of responding T cells after ZIKV infection and to demonstrate the protective effect of CD8 + T cells against ZIKV infection in various organs, including the brain and testes [98][99][100]. However, while these studies clearly demonstrate a major role for CD8 + T cells in controlling ZIKV infection, they all relied on experiments performed in mice with an altered immune system, a model that supports DENV or ZIKV infection but does not reflect the human situation. In addition, these mouse models do not take into account the human innate immune response against these flaviviruses, more specifically the NK cells that kill infected cells through the recognition of viral peptides in the context of HLA-C*0102 [101]. In summary, even though HLA transgenic mice expressing a single HLA allele do not mimic the human situation, the identification of immunodominant epitopes in these transgenic mice constitutes an important step towards preclinical evaluation of T-cell based vaccines.
For this purpose, to formally identify ZIKV-derived peptides in humans, and to determine whether a previous DENV infection can activate cross-reactive T cells, functional studies have been performed using human Peripheral Blood Mononuclear Cells (PBMC) collected from ZIKV endemic areas. From blood donors from different countries in Central and South America, the majority of the CD8 + T cell responses in DENV seronegative individuals were shown to be directed against structural proteins, whereas in DENV seropositive donors, a large proportion of CD8 + T cell responses were directed against the non-structural proteins with cross-reactivity for peptides in NS2A, NS3, and NS5 [102] (Figure 1). Strikingly, cross-reactive CD4 + and CD8 + T cells recognized peptides with identical or highly conserved sequences between DENV and ZIKV, with a higher magnitude of response, showing that DENV-specific memory CD8 + T cells can enhance the T cell responses to ZIKV.
In light of the low protection observed in individuals DENV-seronegative at the time of vaccination with Dengvaxia, which lacks DENV non-structural proteins and fails to induce a In another mouse model, which lacks the type I interferon receptor in a subset of myeloid cells [92], adaptive transfer of ZIKV-immune CD8 + T cells was shown to inhibit primary ZIKV infection and replication [93]. Likewise, in ifnar −/− and in wild-type mice, adoptive transfer and depletion experiments have demonstrated that DENV-immune CD8 + T cells, but not DENV-immune sera can mediate cross-protective responses against ZIKV infection [94]. Although a correlation was previously established in mice and rhesus monkeys between the level of neutralizing antibodies induced upon ZIKV vaccination and the immune protection against ZIKV challenge [95,96], these studies highlight the essential role of CD8 + T cells in providing a protective immunity, in the presence or not of pre-existing DENV immunity, a finding that could help define future strategies of vaccine against DENV and ZIKV [97]. Finally, wild-type immunocompetent mice, ifnar −/− mice and Interferon Alpha Receptor (IFNAR)-depleted rag1 −/− mice, have helped to characterize the phenotype of responding T cells after ZIKV infection and to demonstrate the protective effect of CD8 + T cells against ZIKV infection in various organs, including the brain and testes [98][99][100]. However, while these studies clearly demonstrate a major role for CD8 + T cells in controlling ZIKV infection, they all relied on experiments performed in mice with an altered immune system, a model that supports DENV or ZIKV infection but does not reflect the human situation. In addition, these mouse models do not take into account the human innate immune response against these flaviviruses, more specifically the NK cells that kill infected cells through the recognition of viral peptides in the context of HLA-C*0102 [101]. In summary, even though HLA transgenic mice expressing a single HLA allele do not mimic the human situation, the identification of immunodominant epitopes in these transgenic mice constitutes an important step towards preclinical evaluation of T-cell based vaccines.
For this purpose, to formally identify ZIKV-derived peptides in humans, and to determine whether a previous DENV infection can activate cross-reactive T cells, functional studies have been performed using human Peripheral Blood Mononuclear Cells (PBMC) collected from ZIKV endemic areas. From blood donors from different countries in Central and South America, the majority of the CD8 + T cell responses in DENV seronegative individuals were shown to be directed against structural proteins, whereas in DENV seropositive donors, a large proportion of CD8 + T cell responses were directed against the non-structural proteins with cross-reactivity for peptides in NS2A, NS3, and NS5 [102] (Figure 1). Strikingly, cross-reactive CD4 + and CD8 + T cells recognized peptides with identical or highly conserved sequences between DENV and ZIKV, with a higher magnitude of response, showing that DENV-specific memory CD8 + T cells can enhance the T cell responses to ZIKV.
In light of the low protection observed in individuals DENV-seronegative at the time of vaccination with Dengvaxia, which lacks DENV non-structural proteins and fails to induce a competent T cell response [103], and taking into account the role of CD8 + T cells in preventing ADE [104,105] it is now becoming obvious that efficient vaccine candidates against DENV and ZIKV should be formulated to include CD4 and CD8 T cell epitopes, either alone, or in combination with B cell epitopes.
Based on the recent identification of these T cell epitopes derived from ZIKV, work is currently ongoing to define a minimal antigen, which includes the most immunodominant peptides recognized by cross-reactive T cells, and which can induce a long lasting immune protection against DENV and ZIKV infection and disease.

Conclusions
Our understanding of the immune response to ZIKV has dramatically increased in the last years through in vitro studies with polyclonal and monoclonal antibodies from ZIKV-infected individuals, and through the identification of the epitopes inducing a strong memory and cross-reactive T cell response. While several animal models that could mimic the human situation have been developed to clarify the beneficial or detrimental role of the different immune mediators in disease protection, further phenotypic analyses of ZIKV-specific T cells, in asymptomatic or symptomatic donors will help define correlates of protection in natural immunity and vaccination against ZIKV infection and disease.


extfieldsofstudy: Chemistry Medicine

text:
Quorum sensing intervened bacterial signaling: Pursuit of its cognizance and repression

Bacteria communicate within a system by means of a density dependent mechanism known as quorum sensing which regulate the metabolic and behavioral activities of a bacterial community. This sort of interaction occurs through a dialect of chemical signals called as autoinducers synthesized by bacteria. Bacterial quorum sensing occurs through various complex pathways depending upon specious diversity. Therefore the cognizance of quorum sensing mechanism will enable the regulation and thereby constrain bacterial communication. Inhibition strategies of quorum sensing are collectively called as quorum quenching; through which bacteria are incapacitated of its interaction with each other. Many virulence mechanism such as sporulation, biofilm formation, toxin production can be blocked by quorum quenching. Usually quorum quenching mechanisms can be broadly classified into enzymatic methods and non-enzymatic methods. Substantial understanding of bacterial communication and its inhibition enhances the development of novel antibacterial therapeutic drugs. In this review we have discussed the types and mechanisms of quorum sensing and various methods to inhibit and regulate density dependent bacterial communication.

Introduction
Communication stands crucial in progress of a fraternity and it's observed that bacteria too communicate with each, which is collectively called as Quorum sensing. This is a density dependent mechanism where communication is mediated by signaling molecules termed as autoinducers; it assists bacterial community to coordinate and work as a single unit in a population [1][2][3]. Numerous bacterial functions such as secondary metabolite production, sporulation, biofilm formation and symbiosis are regulated by quorum sensing mechanism [4,5]. All bacterial quorum sensing systems fulfills three basic precepts i. Concentration dependent response to autoinducers; where autoinducers are secreted outside the cell and later inflexed depending on autoinducer concentration [6,7], ii. Bacteria consists specialized receptors in its cell membrane or in cytoplasm that sense and respond to concentration of autoinducer, iii. Detection of autoinducers by receptors recommence quorum sensing loop thereby bacterial virulence [8,9]. Quorum sensing was first observed in a gram negative marine bacteria Vibrio fischeri [10] as studies showed that phenotype bioluminescence was regulated by quorum sensing machinery [11,12]. Since then many bacteria have been successfully scrutinized for their quorum sensing ability. Understanding of quorum sensing open up possibilities of regulating bacterial virulence as far as the immense role of quorum sensing upon it is concerned [13]. Recently many studies have been carried out to down regulate bacterial quorum sensing and this strategy is collectively called quorum quenching [14,15]. Inhibiting communication of a bacterial population disables them to initiate most of its virulence activity which help host for an effective immunological clearance. In this review we discus about the types and mechanisms of quorum sensing and different approaches of quorum quenching through which bacterial communication can be inhibited.

Autoinducers: alphabets of bacterial dialect
Specialized signaling molecule; autoinducers play a key role in quorum sensing and consequently considered as alphabets of bacterial language. Autoinducers are studied under three different classes based on their structure and specific function; they are AHLs (Acyl Homoserine Lactones), AIP (Autoinducing Peptides) and Autoinducer-2 (AI-2) [16]. AHLs are small diffusible molecules with a core lactone ring and acyl side chain which is responsible for facilitating signaling in gram negative bacteria [17]. Quorum sensing in gram positive bacteria are found to be mediated by AIPs which are short peptide chains synthesized in cell. AIP lack free transportation across the cell membrane hence requires specialized membrane transport proteins [18,19]. AI-2 are furonone derived signaling molecules found functioning in both gram negative and gram positive bacteria [20] also exhibit features of both AHLs and AIPs [21]. Signaling molecules are produced inside the bacterial cells which will be processed internally or externally hinge upon the organism. Despite of their functional and structural differences, autoinducers possess certain common characteristics such as high degree of receptor specificity and transport across cell membrane which may be active or passive.

AHL mediated bacterial communication
AHL mediated quorum sensing is intensely studied in gram negative bacteria that constitutes maximum pathogenic strains in it. Numerous virulence factors in gram negative bacteria such as bacterial adhesion, biofilm formation, exozyme secretion, pigment production are regulated by N-Acyl homoserine lactone(AHL) dependent quorum sensing [22,23]. Typical quorum sensing system in gram negative bacteria contains two integrant; an autoinducer synthase which is responsible to synthesize AHLs [24,25] and an autoinducer receptor cum transcriptional activator [26]. AHL mediated signaling is highly intra-specious specific due to peculiar receptor binding sites that recognize only precise AHLs [27][28][29] thus signals produced by one specious will not disturb the communication mechanism of other [30,31]. Modification in AHL structure is accomplished by varying number of Carbon and modification upon R-group [32] which gives advantage to bacteria in an endosymbiont environment. Some important signaling systems mediated by AHLs are discussed here.

LuxIR: typical gram negative bacterial quorum sensing system
LuxIR quorum sensing circuit stands archetypal of gram negative bacterial communication ( Fig. 1) as over hundred gram negative bacterial strains communicate by the engagement of LuxIR homologues genes [33]. SmaIR in Serratia marcescens [34,35], CviIR in Chromobacterium violaceum [36,37], hanIR in Halomonas anticariensis [38]and TraIR of Agrobacterium tumefaciens [39] all work based on the principle of LuxIR but with slight variations in AHLs. This is the first studied bacterial linguistics model and was discovered in marine bioluminescent bacteria Vibrio harveyi which is capable to lead a free and symbiotic lifestyle [40].
This system is based in the reactions mediated by LuxI and LuxR. LuxI is an autoinducer synthase that catalyze the interaction between S-adenosylmethionine and acyl carrier protein which leads into the formation of N-(3-Oxohexanoyl)-L-homoserine lactone which subsequently functions as autoinducer [25,41,42]. AHL will be dispersed out of cell until a specific threshold level is attained. In high external concentrations, AHL will be taken back into the cell which sequentially interact with LuxR. If not bound with AHL, in free state LuxR will be degraded inside bacterial cell, whereas after forming LuxR-AHL complex it will be capped from degrading [43]. LuxR-AHL complex binds upon Lux promoter region that initiate bioluminescence and other quorum sensing regulated functions [44,45].

LasIR-RhlIR: overlapping quorum sensing system
LasIR-RhlIR is serially arranged overlapping quorum sensing circuits observed in Pseudomonas aeruginosa where LasIR and RhlIR are arranged one after other in a series (Fig. 2) [46,47]. Pseudomonas aeruginosa is a widely observed human opportunistic pathogen which is mainly concerned with nosocomial infections upon patients suffering from Cancer, AIDS and cystic fibrosis [48,49]. It produces virulent factors such as elastase, protease, exotoxin A that collectively cause serious tissue damage in mammals [50]. Such mechanisms are found to be controlled by Quorum sensing circuits [51].
Signaling is initiated by the production of AHL (3OC12homoserine lactone) by LasI which act as a homologous of LuxI [52]. AHL will be diffused out of the cell and in high concentration it will be in taken and binds with LasR. Numerous activities such as production of elastase, protease and exotoxins are triggered by LasR-AHL complex [53]. Other than triggering this virulence factor production LasR-AHL also initiate the second system RhlIR. [54]. As a result RhlI produce a secondary AHL (C4-homoserine lactone) that binds with RhlR resulting in the production of subsequent products such as sidophores and pyocyanin [55,56]. All Quorum  sensing controlled mechanism in Pseudomonas aeruginosa is regulated by any one circuit but immense overlap between this systems are noted [57,58] which enables to carry on virulence by any one system [59].

ExpIR: virulence down regulating quorum sensing system
ExpIR mediated quorum sensing is observed in opportunistic plant pathogen Erwinia carotovora (Fig. 3) that frequently causes soft rot in plants [60]. Study of Exp IR system have lot of economic importance on it as soft rot disease affect lot of economic crop plants such as potato, carrot, pineapple, cucumber, onion. It is also observed that Exp IR also regulate synthesis of antibiotics such as carbapenem ß-lactum which give dominance to the bacteria to survive in a high bacterial diverse rhizosphere [61].
Exp circuit is initiated by the production of OHHL by Exp I. Unlike other QS systems the virulence factors of bacteria Erwina carotovora is such as pectate lyase, pectin lyases, cellulases and proteases are positively influenced by the OHHL alone [62] OHHL also triggers the action of Exp R by forming conjugated complex but Exp R have no significant role in bacterial virulence in fact it is found decreasing exozyme production by bacteria [63]. This stand in contrast to many other bacterial virulence mechanism whereas virulence is triggered by regulatory protein not directly by AHL. The difference possessed by Exp IR system found to help bacteria against host defense mechanism [64], Exp R bind with low density of AHL would produce only less exozyme hence low level of effect; this indeed provokes host defense mechanism which challenges the existence of bacteria. Where as in Erwina carotovora ExpR neutralizes the low AHL density which inhibits the production of exozyme. Therefore exozymes will be produced only in high level of OHHL

Peptide mediated bacterial communication
The signaling in gram positive bacteria are controlled by Oligopeptide which is commonly referred as autoinducer peptides [AIPs] [65]. AIPs are produced inside the bacterial cell as pro-AIP which will be processed and modified inside or outside the cell hinge upon the organism [66]. Unlike AHL signaling molecule AIP's are impermeable to cell membrane hence requires specialized transport proteins for the inward and outward carriage of AIP's [67]. This transport of AIP's are generally accomplished by cell membrane bound sensor kinases [68]. Competence mechanism such as sporulation in Bacillus subtilis virulence initiation by Staphylococcus aureus, Listeria monocytogenes, Clostridium perfringens, Enterococcus faecalis are regulated by Quorum sensing systems [69][70][71][72][73]. Even though gram positive Quorum sensing circuits have a general resemblance some small variation in mechanism is observed depending on specious and living environment which is discussed here

Two component system in gram positive bacteria
Most of the gram positive bacterial linguistics are depending upon membrane bound two component system that identify signaling molecule autoinducer peptides ( Fig. 4) [74,75]. A classic example for two component quorum sensing system is found in Staphylococcus aureus [76]. This is a nosocomial pathogen which is normally concerned with skin infection if neglected leads to bacteremia and sepsis [77].
Virulence and communication of this bacteria is regulated by Arg locus which is a combination of two transcripts RNA II and RNA III [78]. Pro-AIP will be produced by ArgD one among the four component of Quorum sensing system. Pro-AIP will be processed and modified by Arg B after which it will be transported out by Fig. 3. ExpIR mediated signaling in Erwinia carotovora which is homologues to LuxIR signaling circuit. the same. [79]. During the course of modification pro-AIP that of 47 amino acid residue will be derived to 9 residue peptide. In high bacterial density, AIP will accumulate extra cellular environment and attain a certain threshold level. In this stage Arg C; a trans membrane protein will get activated which in turn bind with AIP [80]. Agr C is a histidine kinase which phosphorylate by the combination of AIP. Hence available phosphate group interacts with Agr A which is the response regulator [81]. Agr C and Agr A together constitute two component system. Activation of Agr C -Agr A activates the transportation of RNA II which continues the quorum sensing circuit and RNA III that is responsible for virulence [82].

Extracellular protease processed AIP quorum sensing circuit
In some gram positive bacteria processing of pro AIP is done in the external environment of bacterial cell by extracellular protease enzymes after which AIP will be transported back to cell for regulating transcription (Fig. 5) [83]. Many virulent factors like sporulation and enzyme production in Bacillus cereus, plasmid transfer in Enterococcus faecalis are regulated by such mechanism [84,85]. Quorum sensing circuit in Bacillus cereus is a significant example for extracellular protease processed AIPs. A 48 amino acid long pap R intercellular pro-AIP is been produced by pap R gene. An amino terminal signaling peptide present in pro-AIP will initiate a secretory pathway due to which PRO-AIP is been taken out of bacterial cells and will be processed by an extracellular protease into an active AIP [86,87]. Once the concentration of processed AIP reach a threshold level it will be transported inside bacterial cell by oligopeptide permease trans membrane protein [88]. It is observed that only processed pap R could interact with oligopeptide permease system whereas during the process pro -AIP will be degraded into peptides of 5, 7, 8 and 11. This is because of the specific activity of Intercellular transcription regulator plc R upon pentapeptide and heptapeptide [89]. Interaction of AIP on transcription factor plc R brings conformational changes and initiate plc R oligomerization which subsequently induces production of virulence factors [90].

Competitive quorum-sensing system
In this type of Quorum sensing; network of different phenotype would antagonize each other based upon the desired lifestyle of bacteria [46]. This is moreover a combination of other two above mentioned systems. Bacillus subtilis stands as a perfect example of such signaling circuits (Fig. 6). In this bacteria signaling system of competence and sporulation influence each other based on necessity. Competence in bacteria is controlled by Com X peptide which is a ten amino acid [91] sized and processed and secreted by Com Q [92]. In high density: Com X is identified by an histidine kinase Com P that initiate formation of Com X -Com P complex which eventually trigger autophosphorylation that enable Com A to consume a phosphate group [93]. Com A is a DNA binding response regulator that initiate numerous competence mechanism [94].
In other hand this bacteria also produce another oligopeptide by gene phr C and called as CSF (competence and sporulation factor). From cytoplasm CSF is effluxed out by transmembrane proteins [95]. In optimum threshold level CSF is taken back into the bacterial cell by by oligopeptide permease [96]. Internalized CSF have two positive fates depending upon its internal concentrations. In low concentrations CSF bind with cytoplasmic protein Rap C and promote bacterial competence [97]. Rap C protein in free state disturbs Com A hence bacterial competence. Therefore CFS-Rap C complex leads to smooth regulation of bacterial competence. In the high internal concentration level CSF form a complex with Rap B protein hence induce sporulation [98]. In free State Rap B block sporulation by phosphorylation of SPOOF gene which is responsible for sporulation [99]. Complex formation between CSF-Rap B also ensures the availability of Rap C to inhibit competence.

Bacterial silencing: taking antibacterial strategy to a new dimension
The mechanism through which bacteria are made ''silent" by blocking quorum sensing system is called as quorum quenching.  In this era of antibiotic depletion world is searching for new remedies against bacterial infections. Quorum sensing targeted antibacterial therapy has evolved new revolution in this field. Suppression of Quorum quenching have immense value in clearing bacterial infections such as chronic lung infections in CF patients, severe wound infection [100,101]. Quorum sensing targeting drugs basically does not killing the bacteria but it is only attenuating bacterial virulence [102] and offers additional time to host defense mechanism that effect in better immunological clearance of pathogen. Quorum sensing circuit targeted treatment against Staphylococcus aureus [103], Pseudomonas aeruginosa [104], Vibrio cholera [105] were successful and comprehensive. Not only in pharmacological field but agriculture, aqua culture, industries are also found benefited by quorum quenching. There are many strategies which can be relied for blocking bacterial communication which is generally classified as enzymatic and non-enzymatic quorum quenching methods. There are many techniques to find appropriate quorum quencher such as cross streak assay, disc diffusion method, overlay assay, metagenomic analysis, microarray based screening. The choice of technique vary with the requirement. In this part we discuss some important quorum quenching methods and its applications.

Enzymatic quorum quenching
Enzymatic quorum quenching is concerned with altering conformation and structure of signaling molecule which eventually block bacterial communication. These quorum quenching enzymes are mostly derived from microorganisms which is found to give benefit to the producer in a competitive environment [106,107]. This hypothesis of bacterial benefit by producing quorum quench-ing enzymes are based on the discovery co-existence between quorum sensing and quorum quenching bacteria [108,109]. Four different types of chemical reactions are observed behind enzymatic quorum sensing they are decarboxylation, deaminisation acylase and lactonase activity [110]. So far enzymes those found degrading signal molecules are studied under three categories which is constituted by lactonase enzymes, acylase enzymes and oxydoreductase enzymes.

Lactonase mediated quorum quenching
AHL lactonanses hydrolyze lactonase ring of the signaling molecules due to which opened ring structure will be formed (Fig. 7). It is also observed that lactonase enzyme doesn't disturb anything other than lactone ring. [111,112]. It was believed that enzyme hydrolyze amide linkage between lactone and acyl side chain but recent studies on structure and function proved that ester link is affected. Based on phylogeny lactonase belongs to metallo-betalactonase superfamily and phosphotriesterase family among which maximum candidates belong to metallo-beta-lactamase superfamily [113,114]. AHL lactonase are extensively produced by bacteria those have no phylogenetic relation suggests that enzyme production is not dependent upon taxonomic classification. First analyzed bacterial AHL-lactonase is AiiA 24B1 , A product of aiiA gene possessed by Bacillus sp. 24B1 [115]. Since then many bacteria were found producing AHL lactonases which is homologous of AiiA [116]. A thermostable lactonase enzyme namely GKL was obtained from Geobacillus kaustophilus which belonged to phosphotriesterase family and showed relatively low paraoxonase activity suggesting its non-involvement of phosphate ester as substrate [117]. Geobacillus stearothermophilus was observed to produce low catalytic enzyme but with high thermo stability [118]. Thermostable lactonase enzyme were also produced by Geobacillus caldoxylosilyticus YS-8 and Geobacilius kaustophilus HTA426 [119,120].
It was also evident that some bacterial strains produce lactonase enzyme that show major deviation from aiiA gene product. For instance, AiiM lactonase enzyme was obtained from Agrobacterium tumefaciens [121] AhiD of Arthrobacter, Ochrobactrum produced Aid H Aiim of Microbacterium testaceum, Qsd A of Rhodococcus [122][123][124][125] all were examples of lactonases that showed deviation from aiiA. It was believed that AiiA hydrolyze amide linkage between lactone and acyl side chain but recent studies on structure and function of AiiA proved that ester link is the one got attacked by enzyme. Crystal structure of AiiA suggested that there are two Zn 2+ ions present in active center and these metal ions are very much essential for the catalytic activity and folding of enzyme [126]. Analysis on lactonase from Bacillus thuringiensis indicated that Zn1 binds to His 104, HiS 106 and HiS 109 however Z2 binds up on Asp 108, His 109 and His 235 [127] substitution of di-zinc by di-cobalt, di-manganese and dicadmium suppressed the lactonase activity which proves the role of Zn ion in enzyme activity [128].

Acylase mediated quorum quenching
Acylase are the group of quorum sensing enzyme that hydrolyze amide bond between homoserine lactone and acyl side chain (Fig. 8) [129]. Major number of identified AHL acylases belong to Ntn Hydrolase superfamily and are classified into two clusters referred as AAC and Qui P cluster [130]. These two cluster differ in their substrate specificity whereas AAC specifically degrade AHL's longer than C8-HSL however Qui P cluster have a varying range of catalytic activity [131] initial reports of acylase enzyme was by gram negative bacterium Variovorax paradoxus that effi-ciently degraded AHL in growth media [129]. Till date many bacterial strains have been studied for its production of acylase enzyme. Gram positive Streptomyces Sp. was found producing acylase enzyme. This stand first such example for gram positive bacteria [132] AiiD from Ralstonia sp. XJ12B effectively degraded short and long AHLs. Actinoplanes utahensis and Brevundimonas diminuta produced acylase those with high similarities to Ralstonia acylase [133] acylase in substrate selection. Bacterial strain Pseudomonas syringae strain B728a were immensely studied for the ability to produce two acylase namely Hac A and Hac B [134]. Many other bacteria such as Shewanella sp. [135] Tenacibaculum maritimum [136], Comamonas testosterone [137] were also able to produce acylase. From the structural analysis it was observed that acylase enzyme is composed of two or more sub units. Amino acid sequence usually consists of four domains those are signal peptide, Alpha-subunit, Linear spacer and Beta-subunit [138]. Pro acylase enzyme is not functional and it will be converted to activate enzyme by proteolysis.

Oxidoreductase mediated quorum quenching
Oxidreducatase mediated Quorum quenching is targeting signal receptor specificity towards AHL signals. This enzymes modify the chemical structure of AHLs that disable them to interact with receptor and hence blocking signaling pathway (Fig. 9) such reports were first observed in Rhodococcus erythropolis [139]. AHL oxydoreductase obtained from Bacillus megaterium found oxidising x-1,2,3 carbons of acyl chain [140]. There are not much reports obtained about oxydoreductases but NADH-dependent BpiBoa enzyme [141] obtained from Burkholderia GG4 give great hope to carry on more intense study in the possibility of structural remodeling of AHL signal molecules

Non enzymatic quorum quenching
This is a widely used quorum quenching method now a day. Basic principle of this mechanism is to block communication signals rather than degrading it. Blocking of signaling molecules are generally done by competitive inhibition or by structural modification [142]. However quorum sensing antagonistic molecules either bind with signal receptors which enable the interaction of receptor-signal molecule or bind with signaling molecule which disable them to interact with receptors. In the present study, some of the non-enzymatic quorum quenching components have been discussed.
One among the popular strategy is using prokaryotic byproducts to regulate the quorum se mechanism in a quorum sensing bacterial strain. Numerous reports of bacterial byproducts to be a quorum quencher have been postulated. Isobutyramide and 3-me thyl-N-(2-phenylethl)-butyramide produced by Halobacillus salinus was able to target Lux system of Vibrio harveyi [143] in the same way Bacillus cereus strain D28 effectively inhibited AHL mediated quorum sensing in Chromobacterium violaceum [144]. Toxin production in Staphylococcus Sp. found successfully reduced by cyclic dipeptides produced by Lactobacillus reuteri [145]. Yayurea A and B purified from gram positive bacteria Staphylococcus delphini showed its action against QS mediated mechanisms such as pigment production, bioluminance and biofilm formation [146]. CviR gene found inhibited by Cis-9-octadecenoic acid produced by unusual bacteria Stenotrophomonas maltophilia BJ01 which was isolated from rhizosphere [147]. In the same way many reports of Cynobacterial products inhibited bacterial quorum sensing. One well studied blue green algae for quorum quenching is Blennothrix cantharidosmum due to its abundance production of tumonic acids (E, F, G and G). These compounds were effective in regulating bioluminescence in Vibrio harveyi [148]. Lyngbya majuscula has evolved a strong individual and it was found that chemical components like lyngbic acid, lyngbyoic acid, Malyngolide, Pitinoic acid and peptides as microcolins which was produced by them have shown quorum sensing antagonistic action [149][150][151]. Leptolyngbya crosbyana found producing Honaucins that showed inhibition of quorum sensing mediated bacterial communication [152].
In this post antibiotic era fungal metabolites have evolved as important quorum quenching tools. An early report of fungal metabolites that block bacterial communication was by patulin and penicillic acid which were produced by Penicillium coprobium and Penicillium radicicola respectively. This extracts found very effective in blocking quorum sensing of Pseudomonas aeruginosa. In vivo studies conducted in mouse models indicated that there were rapid clearance of pathogens which was treated with patulin compared with the placebo group [153]. Later the quorum quenching antagonistic action of a sesquiterpine farnesol extracted from Candida albicans was reported. Addition of farnesol as well as co-culture with fungul strain decreased the pyocyanin production, a quorum sensing controlled system in Pseudomonas aeruginosa. This report suggests the advantage of quorum quenching organism in an ecosystem [154]. A secondary fungal metabolic ambuic acid purified from fungal strain KAP-21 was reported very efficient against quorum sensing in gram positive bacterial strains where it inhibited the quorum-sensing-mediated gelatinase production without influencing the growth of Enterococcus faecalis also targeted the biosynthesis of a cyclic peptide quormone called gelatinase biosynthesis-activating pheromone. Furthermore, ambuic acid also inhibited the biosynthesis of the cyclic peptide quormones of Staphylococcus aureus and Listeria innocua. These results suggest the potential use of ambuic acid as a lead compound of anti-pathogenic drugs that target the quorum sensing-mediated virulence expression of gram-positive bacteria [155].
Aspergillus sp produced Kojic acid that inhibited quorum sensing dependent biofilm formation by regulating Lux system. Kojic acid inhibited formation of microbial communities on glass slides, decreasing the densities of bacteria this study suggests that natural products with quorum sensing inhibitory properties can be used for controlling biofouling communities [156]. Polyhydroxyanthraquinones purified from distinct red guttates of the endophytic fungus Penicillium restrictum efficiently inhibited quorum sensing in clinical isolate of Methicillin-resistant Staphylococcus aureus (MRSA) [157]. Recent studies on marine endosymbiotic fungi revealed the quorum quenching activity of four different genera Sarocladium, Fusarium, Epicoccum, and Khuskia. These strains proved an abundant source of novel secondary metabolites against bacterial quorum sensing [158].
There have been some reports of quorum quenching by some invertebrate animals such as Molluscs, Arthropods and Annelida. First among them is honey a product by honey bee that efficiently reduced bacterial communication. Honey was found antagonizing LasR and RhIR quorum sensing systems [159,160]. Another report says the efficacy of solenopsin A produced by fire ant that effectively regulated rhI circuits [161]. LuxR signaling was greatly influenced by byproducts of Annelids and Molluscs as cembranoids from Pseudoplexaura flagellosa and exadates from Caenorhabditis elegans showed anti quorum sensing action [162,163]. Add on to these Poriferans also had some great deal in neutralizing bacterial quorum sensing. Luffaria variabilis inhibited the action of Lux regulatory proteins by its metabolite secomanoalide [164] and hymenialdisin an alkaloid produced by Hymeniacidon aldis also had inhibitory effect on LuxI and LasR proteins [156].
Since the ancient medical era plants and plant products have contributed a lot against disease and infections. In the same way many plant products were found antagonizing bacterial communication. L-Canavanine obtained from Medicago sativa influenced ExpR and CviR [165] and thereby suppressed the biofilm formation of pathogens. 2,5-di-O-galloyl-d-Hamamelose obtained from Hamamamelis virginiana which is commonly called as witch hazel and obacunone from grapes possessed ability to inhibit quorum sensing [166]. CviR and RhiR controlled quorum sensing was inhibited by plant derivatives Benzopyran and Catachin [167]. Malabaricone C purified from Myrstica cinnamomea as well as Curcumin from Curcuma longa regulated quorum sensing dependent biofilm development [168,169]. Along with this, many other several macromolecules such as chitosan is also found to have high degree of quorum quenching ability [170,171]. This recent observation could open up new dimensions of quorum quenching studies.

Significance and future aspects of quorum quenching mediated bacterial silencing
Strategy of quorum quenching is widely employed in various fields as a powerful weapon against bacterial caused misfortunes. In agricultural field a great loss is being occurred due to the pathogenic action of bacteria. Quorum quenching is been effectively applied to remediate such issues, among them a major breakthrough have been reported against soft rot disease [172,173]. The disease can be controlled either by introducing a gene into plants whose products block AHL synthesis or by the employment of AHL degrading bacteria [174,175]. Transgenic plants that express AiiA lactonase were pretty much effective against infection by Pectobacterium carotovorum [111]. Despite achieving this successful hypothesis, the less acceptability of transgenic food around the globe limits this method. Biofouling has grown as a major concern over years which accumulates the microorganisms on the surfaces those are in contact with water [176]. Quorum quenching agents can be successfully employed to reduce bacterial reduce biofouling [177].
In pharmacology, quorum quenching compounds have initialized a new aeon of antibacterial treatment. The main principle behind this strategy is the down regulation of quorum sensing which mediates virulence factor production by quorum quenching [178]. Many Quorum quenching compounds have been experimented for its pharmacological activity either by blocking AHL signal or by degrading AHL signals. It is observed that enzymatic quorum quencher could be of great significance in antibacterial therapy as they inactivate signaling molecule without messing with bacterial metabolism [179]. It is also observed that database of quorum quenching molecules for different bacteria are available which could aid the future research in this field. Databases such as Quorumpeps [180] which deliberately explains about quorum quenching peptides and Sigmol [181] that contains data of prokaryotic are perfect examples of quorum quenching databases. Even though numerous studies are emerging about quorum quenching molecules as antibacterial drugs, much of the components haven't reached clinical level due to their low biocompatibility. Thus the discovery of novel quorum quenching component without toxic effects could pave the way towards new pastures of antibacterial therapy.

Conclusions
Quorum sensing is a bacterial density depended mechanism which regulate virulence. It is controlled by signalling molecules termed as autoinducers. It is evident that by manipulating the signalling circuit of bacterial communication it is possible to attenuate pathogen.

Conflict of interest
We declare 'no conflict of interest'.