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Vibrio cholerae, a facultative human pathogen and causative agent of the severe diarrheal disease cholera, transits between the human intestinal tract and aquatic reservoirs. Like other bacterial species, V. cholerae continuously releases bacterial extracellular vesicles (BEVs) from its surface, which have been recently characterised for their role during in vivo colonisation. However, between epidemic outbreaks, V. cholerae persists in the biofilm mode for extended periods in aquatic reservoirs, which enhances environmental fitness and host transition. In this study, we investigated the effect of V. cholerae BEVs on biofilm formation, a critical feature for ex vivo survival. In contrast to BEVs from planktonic cultures, our results show that physiological concentrations of BEVs from dynamic biofilm cultures facilitate V. cholerae biofilm formation, which could be linked to a proteinaceous factor. Comparative proteomic analyses of planktonic- and biofilm-derived BEVs identified a previously uncharacterised outer membrane protein as an abundant component of dynamic biofilm-derived BEVs, which was found to be responsible for the BEV-dependent enhancement of biofilm production. Consequently, this protein was named outer membrane-associated biofilm facilitating protein A (ObfA). Comprehensive molecular studies unravelled ObfA as a negative modulator of HapR activity. HapR is a key transcriptional regulator of the V. cholerae quorum sensing (QS) cascade acting as a potent repressor of biofilm formation and virulence. Consistently, obfA mutants not only exhibited reduced biofilm production but also reduced colonisation fitness. Surprisingly, our results demonstrate that ObfA does not affect HapR through the canonical QS system but via the Csr-cascade altering the expression of the small regulatory RNAs CsrC and CsrD. In summary, this study elucidates a novel intraspecies BEV-based communication in V. cholerae that influences biofilm formation and colonisation fitness via a new regulatory pathway involving HapR, Csr-cascade and the BEV-associated protein ObfA.

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AbstractThe type VI secretion system (T6SS) is essential for Gram-negative bacteria to antagonize a wide variety of prokaryotic and eukaryotic competitors and thus gain survival advantages. Two sets of T6SS have been found in Vibrio fluvialis, namely VflT6SS1 and VflT6SS2, among which VflT6SS2 is functionally expressed. The CqsA/LuxS-HapR quorum sensing (QS) system with CAI-1 and AI-2 as signal molecules can regulate VflT6SS2 by regulators LuxO and HapR, with LuxO repressing while HapR activating VflT6SS2. Quorum regulatory small RNAs (Qrr sRNAs) are Hfq-dependent trans-encoded sRNAs that control Vibrio quorum sensing. In V. fluvialis, Qrr sRNAs have not been characterized and their regulatory function were unknown. In this study, we first identified four Qrr sRNAs in V. fluvialis and demonstrated that these Qrr sRNAs are regulated by LuxO and involved in the modulation of VflT6SS2 function. On the one hand, Qrr sRNAs act on HapR, the activator of both the major and the auxiliary clusters of VflT6SS2, and then indirectly repress VflT6SS2. On the other hand, they directly repress VflT6SS2 by acting on tssB2 and tssD2_a, the first gene of the major cluster and the highly transcriptional one among the two units of the first auxiliary cluster, respectively. Our results give insights into the role of Qrr sRNAs in CAI-1/AI-2 based QS and VflT6SS2 modulation in V. fluvialis and further enhance understandings of the network between QS and T6SS regulation in Vibrio species.

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Vibrio cholerae is a waterborne bacterium that primarily infects the human intestine and causes cholera fatality. Quorum sensing (QS) negatively regulates the expression of V. cholerae virulence gene. However, the primary associated mechanisms remain undetermined. This investigation identified a new QS regulator from the TetR family, LuxT, which increases V. cholerae virulence by directly inhibiting hapR expression. HapR is a master QS regulator that suppresses virulence cascade expression. The expression of luxT increased 4.8-fold in the small intestine of infant mice than in Luria-Bertani broth. ΔluxT mutant strain revealed a substantial defect in the colonizing ability of the small intestines. At low cell densities, the expression level of hapR was upregulated by luxT deletion, suggesting that LuxT can suppress hapR transcription. The electrophoretic mobility shift analysis revealed that LuxT directly binds to the hapR promoter region. Furthermore, luxT expression was upregulated by the two-component system ArcB/ArcA, which responses to changes in oxygen levels in response to the host's small intestine's anaerobic signals. In conclusion, this research reveals a novel cell density-mediated virulence regulation pathway and contributes to understanding the complex association between V. cholerae virulence and QS signals. This evidence furnishes new insights for future studies on cholerae's pathogenic mechanisms.

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Objective: This study aimed to investigate whether the VCA0560 gene acts as an active diguanylate cyclase (DGC) in Vibrio cholerae and how its transcription is regulated by Fur and HapR. Methods: The roles of VCA0560 was investigated by utilizing various phenotypic assays, including colony morphological characterization, crystal violet staining, Cyclic di-GMP (c-di-GMP) quantification, and swimming motility assay. The regulation of the VCA0560 gene by Fur and HapR was analyzed by luminescence assay, electrophoretic mobility shift assay, and DNase I footprinting. Results: VCA0560 gene mutation did not affect biofilm formation, motility, and c-di-GMP synthesis in V. cholerae, and its overexpression remarkably enhanced biofilm formation and intracellular c-di-GMP level but reduced motility capacity. The transcription of the VCA0560 gene was directly repressed by Fur and the master quorum sensing regulator HapR. Conclusion: Overexpressed VCA0560 functions as an active DGC in V. cholerae, and its transcription is repressed by Fur and HapR.

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OBJECTIVE@#This study aimed to investigate whether the VCA0560 gene acts as an active diguanylate cyclase (DGC) in Vibrio cholerae and how its transcription is regulated by Fur and HapR.@*METHODS@#The roles of VCA0560 was investigated by utilizing various phenotypic assays, including colony morphological characterization, crystal violet staining, Cyclic di-GMP (c-di-GMP) quantification, and swimming motility assay. The regulation of the VCA0560 gene by Fur and HapR was analyzed by luminescence assay, electrophoretic mobility shift assay, and DNase I footprinting.@*RESULTS@#VCA0560 gene mutation did not affect biofilm formation, motility, and c-di-GMP synthesis in V. cholerae, and its overexpression remarkably enhanced biofilm formation and intracellular c-di-GMP level but reduced motility capacity. The transcription of the VCA0560 gene was directly repressed by Fur and the master quorum sensing regulator HapR.@*CONCLUSION@#Overexpressed VCA0560 functions as an active DGC in V. cholerae, and its transcription is repressed by Fur and HapR.

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We investigated the influence of hapR sequence mutations on the biofilm formation of Vibrio cholerae. In this study, hapR sequences from 85 V. cholerae strains belonging to both pandemic and nonpandemic serogroup were investigated through phylogenetic and sequence analyses. Biofilm formation assays under aerobic and anaerobic conditions were also performed. Sequence variations include single point mutations and insertions/deletions (indels) leading to either truncated or frameshifted HapR. Population structure analysis revealed two major hapR haplogroups, hapR1 and hapR2. Phylogenetic reconstruction displayed a hypothetical ancestral hapR sequence located within the hapR1 haplogroup. Higher numbers of single nucleotide polymorphisms and genetic diversity indices were observed in hapR1, while indels occurred dominantly in hapR2. Aerobic conditions supported more robust biofilms compared to anaerobic conditions. Strains with frameshifted HapR produced the largest amount of biofilm under both oxygen conditions. Quantitative real-time PCR assay confirmed that strains with truncated and frameshifted HapR resulted in a nonfunctional regulator as exhibited by the significantly low hapA gene expression. The present study shows that HapR mutations had a strong influence on biofilm formation and that sequence polymorphisms leading to the disruption of DNA-binding sites or dimerization of the HapR will result in more-robust V. cholerae biofilms. IMPORTANCE Our study revealed an ancestral hapR sequence from a phylogenetic reconstruction that displayed the evolutionary lineage of the nonpandemic to the pandemic strains. Here, we established hapR1 and hapR2 as major hapR haplogroups. The association of the O1 and O139 serogroups with the hapR2 haplogroup demonstrated the distinction of hapR2 in causing cholera infection. Moreover, mutations in this regulator that could lead to the disruption of transcription factor-binding sites or dimerization of the HapR can significantly affect the biofilm formation of V. cholerae. These observations on the relationship of the hapR polymorphism and V. cholerae biofilm formation will provide additional considerations for future biofilm studies and insights into the epidemiology of the pathogen that could ultimately help in the surveillance and mitigation of future cholera disease outbreaks.

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Cholera is a severe small intestine bacterial disease caused by consumption of water and food contaminated with Vibrio cholera. The disease causes watery diarrhea leading to severe dehydration and even death if left untreated. In the past few decades, V. cholerae has emerged as multidrug-resistant enteric pathogen due to its rapid ability to adapt in detrimental environmental conditions. This research study aimed to design inhibitors of a master virulence gene expression regulator, HapR. HapR is critical in regulating the expression of several set of V. cholera virulence genes, quorum-sensing circuits and biofilm formation. A blind docking strategy was employed to infer the natural binding tendency of diverse phytochemicals extracted from medicinal plants by exposing the whole HapR structure to the screening library. Scoring function criteria was applied to prioritize molecules with strong binding affinity (binding energy < -11 kcal/mol) and as such two compounds: Strychnogucine A and Galluflavanone were filtered. Both the compounds were found favourably binding to the conserved dimerization interface of HapR. One rare binding conformation of Strychnogucine A was noticed docked at the elongated cavity formed by α1, α4 and α6 (binding energy of -12.5 kcal/mol). The binding stability of both top leads at dimer interface and elongated cavity was further estimated using long run of molecular dynamics simulations, followed by MMGB/PBSA binding free energy calculations to define the dominance of different binding energies. In a nutshell, this study presents computational evidence on antibacterial potential of phytochemicals capable of directly targeting bacterial virulence and highlight their great capacity to be utilized in the future experimental studies to stop the evolution of antibiotic resistance evolution.

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V. cholerae, the causative agent of cholera epidemic, and V. fluvialis, the emerging foodborne pathogen, share highly homologous T6SS consisting of one large cluster and two small orphan or auxiliary clusters, and each of which was generally recognized as one operon. Here, we showed that the genes in each of the small clusters are organized into two transcriptional units. Specifically, the inner tube coding gene hcp/tssD is highly transcribed as one monocistron, while the tip component vgrG/tssI and its downstream effector and immunity genes are in one polycistron with very low transcriptional level. This conclusion is supported by qPCR analysis of mRNA abundance, reporter fusion analysis and transcriptional unit definition with RT-PCR analysis. Taking tssI2_a of V. fluvialis as an example, we further demonstrated that quorum sensing (QS) regulator HapR and global regulator IHF activate vgrG/tssI transcription by directly binding to its promoter region. Taken together, current studies deepen our understanding of T6SS system, highlighting its regulatory complexity during functional execution process.

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Vibrio fluvialis is an emerging enteric pathogen of increasing public health threat. Two quorum sensing (QS) systems, VfqI-VfqR and CqsA/LuxS-HapR, and two type VI secretion systems (T6SSs), VflT6SS1 and VflT6SS2, have been identified in V. fluvialis. Whether there exists any correlation between the two systems is unclear. In this study, we found that CqsA/LuxS-HapR circuit regulator LuxO represses while HapR activates VflT6SS2. The effect of LuxO is more pronounced at low cell density and is HapR-dependent. Deletion of hapR abolished Hcp expression and alleviated antibacterial virulence. However, these effects were rescued by HapR-expressing plasmid. Reporter fusion analyses showed that HapR is required for the promoter activities of VflT6SS2. Sequence inspection of the major cluster promoter revealed two potential Motif 1 HapR binding sites, and their bindings to HapR were confirmed by both electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay. Meanwhile, two single Motif 2 sites were identified in tssD2_a (hcpA) and tssD2_b (hcpB) promoter regions of the orphan cluster which are less conserved and displayed lower affinities to HapR. Together, our study demonstrated that CqsA/LuxS-HapR QS manipulate VflT6SS2 in V. fluvialis, and this finding will enhance our understanding of possible crosstalk between T6SS and QS in microbes.

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Adaptation to shifting temperatures is crucial for the survival of the bacterial pathogen Vibrio cholerae. Here, we show that colony rugosity, a biofilm-associated phenotype, is regulated by temperature in V. cholerae strains that naturally lack the master biofilm transcriptional regulator HapR. Using transposon-insertion mutagenesis, we found the V. cholerae ortholog of BipA, a conserved ribosome-associated GTPase, is critical for this temperature-dependent phenomenon. Proteomic analyses revealed that loss of BipA alters the synthesis of >300 proteins in V. cholerae at 22°C, increasing the production of biofilm-related proteins including the key transcriptional activators VpsR and VpsT, as well as proteins important for diverse cellular processes. At low temperatures, BipA protein levels increase and are required for optimal ribosome assembly in V. cholerae, suggesting that control of BipA abundance is a mechanism by which bacteria can remodel their proteomes. Our study reveals a remarkable new facet of V. cholerae's complex biofilm regulatory network.

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Vibrio species, including the squid symbiont Vibrio fischeri, become competent to take up DNA under specific conditions. For example, V. fischeri becomes competent when grown in the presence of chitin oligosaccharides or upon overproduction of the competence regulatory factor TfoX. While little is known about the regulatory pathway(s) that controls V. fischeri competence, this microbe encodes homologs of factors that control competence in the well-studied V. cholerae To further develop V. fischeri as a genetically tractable organism, we evaluated the roles of some of these competence homologs. Using TfoX-overproducing cells, we found that competence depends upon LitR, the homolog of V. cholerae master quorum-sensing and competence regulator HapR, and upon homologs of putative pilus genes that in V. cholerae facilitate DNA uptake. Disruption of genes for negative regulators upstream of LitR, namely, the LuxO protein and the small RNA (sRNA) Qrr1, resulted in increased transformation frequencies. Unlike LitR-controlled light production, however, competence did not vary with cell density under tfoX overexpression conditions. Analogous to the case with V. cholerae, the requirement for LitR could be suppressed by loss of the Dns nuclease. We also found a role for the putative competence regulator CytR. Finally, we determined that transformation frequencies varied depending on the TfoX-encoding plasmid, and we developed a new dual tfoX and litR overexpression construct that substantially increased the transformation frequency of a less genetically tractable strain. By advancing the ease of genetic manipulation of V. fischeri, these findings will facilitate the rapid discovery of genes involved in physiologically relevant processes, such as biofilm formation and host colonization.IMPORTANCE The ability of bacteria to take up DNA (competence) and incorporate foreign DNA into their genomes (transformation) permits them to rapidly evolve and gain new traits and/or acquire antibiotic resistances. It also facilitates laboratory-based investigations into mechanisms of specific phenotypes, such as those involved in host colonization. Vibrio fischeri has long been a model for symbiotic bacterium-host interactions as well as for other aspects of its physiology, such as bioluminescence and biofilm formation. Competence of V. fischeri can be readily induced upon overexpression of the competence factor TfoX. Relatively little is known about the V. fischeri competence pathway, although homologs of factors known to be important in V. cholerae competence exist. By probing the importance of putative competence factors that control transformation of V. fischeri, this work deepens our understanding of the competence process and advances our ability to genetically manipulate this important model organism.

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Quorum sensing is a cell density-dependent form of cellular communication among bacteria. This signaling process has been heavily studied in vibrios due to their diverse and complex phenotypes and relevance to human and aquaculture disease. Mechanistic studies of Vibrio quorum sensing have required optimization of protein purification techniques to examine the role of key proteins, such as the LuxR/HapR family of transcription factors that control quorum-sensing gene expression. Protein purification is the cornerstone of biochemistry, and it is crucial to consistently produce batches of protein that are pure, active, and concentrated to perform various assays. The methods described here are optimized for purification of the Vibrio master quorum-sensing regulators, LuxR (Vibrio harveyi), HapR (Vibrio cholerae), and SmcR (Vibrio vulnificus). We anticipate that these methods can be applied to other proteins in this family of transcription factors.

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HapR is designated as a high cell density quorum sensing master regulatory protein of Vibrio cholerae. It is a member of the TetR family protein and functions both as an activator and a repressor by directly communicating with cognate promoters, thus controlling the expression of a plethora of genes in a density-dependent manner. Molecular insights reveal the domain architecture and further unveil the significance of a cross talk between the DNA binding domain and the dimerization domain for the functionality of the wild-type protein. The DNA binding domain is made up of three α-helices, where a helix-turn-helix motif spans between the helices α2 and α3. The essentiality of the glycine-rich linker linking helices α1 and α2 came into prominence while unraveling the molecular basis of a natural non-functional variant of HapR. Subsequently, the importance of linker length was demonstrated. The present study, involving a series of biochemical analyses coupled with molecular dynamics simulation, has illustrated the indispensability of a critical arginine within the linker at position 37 contributing to HapR-DNA binding activity.

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Cholera toxin (CT) and toxin coregulated pilus (TCP, TcpA is the major subunit) are two major virulence factors of Vibrio cholerae, both of which play critical roles in developing severe diarrhea in human. Expression of CT and TCP is under the tight control of the regulatory cascade known as the ToxR virulence regulon, which is composed of three regulators ToxR, TcpP, and ToxT. Besides, their expression is also regulated by the quorum sensing (QS) master regulator HapR and the regulatory protein Fur. Though transcription of tcpP, toxT, and/or tcpA are reported to be regulated by HapR and Fur, to date there are no studies to verify their direct regulations. In the present study, we showed that HapR directly repress the transcription of tcpP and tcpA by binding to their promoter regions, and possibly repress toxT transcription in an indirect manner. Fur directly activated the transcription of tcpP, toxT, and tcpA by binding to their promoters. Taking account of the sequential expression of hapR, fur, tcpP, toxT, and tcpA in the different growth phases of V. cholerae, we deduce that at the early mid-logarithmic growth phase, Fur binds to the promoters of tcpP, toxT, and tcpA to activate their transcription; while at the later mid-logarithmic growth phase, HapR can bind to the promoters of tcpP and tcpA to repress their transcription. Our study reveals the new recognition in the virulence regulatory pathways in V. cholerae and suggests the complicated and subtle regulation network with the growth density dependence.

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Expression of cholera toxin (CT), the principal virulence factor of the cholera pathogen Vibrio cholerae, is positively modulated by the RNA polymerase binding unusual transcription factor DksA (DksAVc) of the stringent response pathway. Here we report that even though CT (encoded by the genes ctxAB) production is downregulated in the V. cholerae ΔdksA (ΔdksAVc) mutant, the expression of the ctxA gene as well as the genes encoding different virulence regulators, namely, AphA, TcpP and ToxT, were also upregulated. Since DksAVc positively regulates HapR, a known negative regulator of CT production, the increased expression of different virulence genes in ΔdksAVc was due most probably to downregulation of HapR. There was no secretion/transport-related defect in ΔdksAVc cells because whole cell lysates of the mutant showed a negligible amount of CT accumulation similar to WT cells. To understand further, the hapR gene was deleted in ΔdksAVc background, however, the double mutant failed to rescue the CT production defect suggesting strongly towards post-transcriptional/translational regulation by DksAVc. This hypothesis was further confirmed when the site-directed mutagenesis of each or both of the conserved aspartic acid residues at positions 68 and 71 of DksAVc, which are essential for transcription initiation during the stringent response, had no effect in the regulation of CT expression. Interestingly, progressive deletion analysis indicated that the C4-type Zn finger motif present in the C-terminus of DksAVc is essential for optimal CT production. Since this motif plays important roles in DNA/RNA binding, the present study indicates a novel complex post-transcriptional regulation of CT expression by DksAVc.

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The biotype El Tor of serogroup O1 and most of the non-O1/non-O139 strains of Vibrio cholerae can produce an extracellular pore-forming toxin known as cholera hemolysin (HlyA). Expression of HlyA has been previously reported to be regulated by the quorum sensing (QS) and the regulatory proteins HlyU and Fur, but lacks the direct evidence for their binding to the promoter of hlyA. In the present work, we showed that the QS regulator HapR, along with Fur and HlyU, regulates the transcription of hlyA in V. cholerae El Tor biotype. At the late mid-logarithmic growth phase, HapR binds to the three promoters of fur, hlyU, and hlyA to repress their transcription. At the early mid-logarithmic growth phase, Fur binds to the promoters of hlyU and hlyA to repress their transcription; meanwhile, HlyU binds to the promoter of hlyA to activate its transcription, but it manifests direct inhibition of its own gene. The highest transcriptional level of hlyA occurs at an OD600 value of around 0.6-0.7, which may be due to the subtle regulation of HapR, Fur, and HlyU. The complex regulation of HapR, Fur, and HlyU on hlyA would be beneficial to the invasion and pathogenesis of V. cholerae during the different infection stages.

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HapR is a TetR-family transcriptional regulator that controls quorum sensing in Vibrio cholerae, the causative agent of cholera. HapR regulates the expression of hemagglutinin protease, virulence and biofilm genes. The crystal structure of wild-type HapR from V. cholerae strain O1 El Tor C6706 has previously been solved. In this study, the structure of a DNA-binding-deficient variant of HapR (HapRV2) derived from the protease-deficient V. cholerae serotype O37 strain V2 is reported. The structure reveals no structural differences compared with wild-type HapR. However, structural alignment of HapRV2 with the TetR-family member QacR in complex with its operator DNA suggests that the aspartate residue located between the regulatory and DNA-binding domains may clash with and electrostatically repel the phosphate backbone of DNA to prevent binding.

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Bacteria can generate benefits for themselves and their kin by living in multicellular, matrix-enclosed communities, termed biofilms, which are fundamental to microbial ecology and the impact bacteria have on the environment, infections, and industry [1-6]. The advantages of the biofilm mode of life include increased stress resistance and access to concentrated nutrient sources [3, 7, 8]. However, there are also costs associated with biofilm growth, including the metabolic burden of biofilm matrix production, increased resource competition, and limited mobility inside the community [9-11]. The decision-making strategies used by bacteria to weigh the costs between remaining in a biofilm or actively dispersing are largely unclear, even though the dispersal transition is a central aspect of the biofilm life cycle and critical for infection transmission [12-14]. Using a combination of genetic and novel single-cell imaging approaches, we show that Vibrio cholerae integrates dual sensory inputs to control the dispersal response: cells use the general stress response, which can be induced via starvation, and they also integrate information about the local cell density and molecular transport conditions in the environment via the quorum sensing apparatus. By combining information from individual (stress response) and collective (quorum sensing) avenues of sensory input, biofilm-dwelling bacteria can make robust decisions to disperse from large biofilms under distress, while preventing premature dispersal when biofilm populations are small. These insights into triggers and regulators of biofilm dispersal are a key step toward actively inducing biofilm dispersal for technological and medical applications, and for environmental control of biofilms.

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The coordination of group behaviors in bacteria is accomplished via the cell-cell signaling process called quorum sensing. Vibrios have historically been models for studying bacterial communication due to the diverse and remarkable behaviors controlled by quorum sensing in these bacteria, including bioluminescence, type III and type VI secretion, biofilm formation, and motility. Here, we discuss the Vibrio LuxR/HapR family of proteins, the master global transcription factors that direct downstream gene expression in response to changes in cell density. These proteins are structurally similar to TetR transcription factors but exhibit distinct biochemical and genetic features from TetR that determine their regulatory influence on the quorum sensing gene network. We review here the gene groups regulated by LuxR/HapR and quorum sensing and explore the targets that are common and unique among Vibrio species.

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High on-aspirin platelet reactivity (HAPR) has been associated with compromised aspirin efficacy in patients with diabetes suffering from acute cardiovascular events, but the key mechanisms remain elusive. The objective of this study was to uncover the potential link between pathogenic accumulation of salicylic acid (SA), the major metabolite of aspirin, and HAPR in diabetic state. Aspirin failed to inhibit platelet CD62P expression and thromboxane (TX) B2/6-keto-prostaglandin（PG）F1α ratio in a type 2 diabetes mellitus (T2DM) mice model, particularly in the female, which were unanimously accompanied by significantly higher plasma SA concentrations. Pre-administration with SA increased both platelet CD62P expression and TXB2/6-keto-PGF1α ratio in female T2DM mice, while pretreatment with NaHCO3 caused the opposite effect. On the in vitro human umbilical vein endothelial cells (HUVECs)-platelet interaction assay, SA suppressed inflammation-induced cyclooxygenase-2 upregulation on HUVECs and attenuated their inhibitory effect on platelet aggregation in a dose-dependent manner. The prolonged retention of SA in diabetes may be partially explained by the downregulation of various SA efflux transporters in the kidney and the decreased urine pH. Importantly, in female aspirin non-responsive patients, the trough plasma concentration of SA are markedly increased with T2DM treated with long-term aspirin, and TXB2/6-keto-PGF1α ratio and uric acid level in plasma are positively correlated with SA concentration. Our findings support that the accumulation of SA represents an important factor in causing HAPR in diabetes, and that targeting impaired SA excretion may become a novel intervention strategy to diabetes-associated HAPR.

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