Cholerae Cells Research Articles

Modification of The Bacterial Abundance Properties of Water by Immersed Non-Activated Charcoal

The present studies aim at reducing the planktonic bacterial load in drinking water using non-activated charcoal. The evaluation is dependent on the incubation time and the different physiological states of the bacterial cells. Three bacteria (Vibrio cholerae, Escherichia coli and Staphylococcus aureus) and two charcoal substrates (Okan and Tali) were used for the experimentation. The Bacteria adhered to charcoal fragments in varying degrees. Overall, the concentrations of fixed Vibrio cholerae reached 53.4x107 CFU/cm2 and 39.6x107 CFU/cm2 after 9 hours of contact in exponential phase on the Okan and Tali respectively. Those of fixed Staphylococcus aureus reached 5.8x106 CFU/cm2 after 3 hours of contact in exponential phase and 3.4x106 CFU/cm2 after 3 and 6 hours of contact in the same phase, on the Okan and the Tali, respectively. The highest abundances of Escherichia coli adhered to the charcoal fragments were 50.4x107 CFU/cm² on Okan and 53.2x107 CFU/cm² on Tali after 9 hours of contact in the exponential phase. The highest adsorption coefficient (639.06 adhered cells/cm2) was noted on Okan in the exponential phase with Vibrio cholerae cells; and the lowest (1.02 adhered cells/cm2) on Tali in stationary phase with Staphylococcus aureus. The incubation time significantly (P<0.01) influenced the adhesion of bacterial cells to charcoal substrates. Although the adsorption capacity and intensity of Okan were relatively higher, the comparison of the adsorption potential of the two substrates considered did not reveal any significant difference (P>0.05), reflecting the absence of the influence of the physical properties of these substrates on cell retention.

Cell-lysis sensing drives biofilm formation in Vibrio cholerae

Matrix-encapsulated communities of bacteria, called biofilms, are ubiquitous in the environment and are notoriously difficult to eliminate in clinical and industrial settings. Biofilm formation likely evolved as a mechanism to protect resident cells from environmental challenges, yet how bacteria undergo threat assessment to inform biofilm development remains unclear. Here we find that population-level cell lysis events induce the formation of biofilms by surviving Vibrio cholerae cells. Survivors detect threats by sensing a cellular component released through cell lysis, which we identify as norspermidine. Lysis sensing occurs via the MbaA receptor with genus-level specificity, and responsive biofilm cells are shielded from phage infection and attacks from other bacteria. Thus, our work uncovers a connection between bacterial lysis and biofilm formation that may be broadly conserved among microorganisms.

Genomic and functional insights into antibiotic resistance genes floR and strA linked with the SXT element of Vibrio cholerae non-O1/non-O139.

The emergence and spread of antibiotic-resistant bacterial pathogens are a critical public health concern across the globe. Mobile genetic elements (MGEs) play an important role in the horizontal acquisition of antimicrobial resistance genes (ARGs) in bacteria. In this study, we have decoded the whole genome sequences of multidrug-resistant Vibrio cholerae clinical isolates carrying the ARG-linked SXT, an integrative and conjugative element, in their large chromosomes. As in others, the SXT element has been found integrated into the 5'-end of the prfC gene (which encodes peptide chain release factor 3 involved in translational regulation) on the large chromosome of V. cholerae non-O1/non-O139 strains. Further, we demonstrate the functionality of SXT-linked floR and strAB genes, which confer resistance to chloramphenicol and streptomycin, respectively. The floR gene-encoded protein FloR belongs to the major facilitator superfamily efflux transporter containing 12 transmembrane domains (TMDs). Deletion analysis confirmed that even a single TMD of FloR is critical for the export function of chloramphenicol. The floR gene has two putative promoters, P1 and P2. Sequential deletions reveal that P2 is responsible for the expression of the floR. Deletion analysis of the N- and/or C-terminal coding regions of strA established their importance for conferring resistance against streptomycin. Interestingly, qPCR analysis of the floR and strA genes indicated that both of the genes are constitutively expressed in V. cholerae cells. Further, whole genome-based global phylogeography confirmed the presence of the integrative and conjugative element SXT in non-O1/non-O139 strains despite being non-multidrug resistant by lacking antimicrobial resistance (AMR) gene cassettes, which needs monitoring.

Characteristics of Vibrio cholerae isolates obtained from shrimp supply chains and inhibitory activities of rambutan (Nephelium lappaceum L. cv. Rong Rian) peel aqueous extract

Vibrio cholerae is a significant seafood-borne pathogen that carries multiple virulence genes. Its ability to form biofilm on surfaces enables this pathogen to persist in the environments and resistant to chemicals and antibiotics. This study investigated the virulence properties of V. cholerae isolates obtained from shrimp supply chains and evaluated the inhibitory activities of rambutan (Nephelium lappaceum L. cv. Rong Rian) peel extract (RPE) against V. cholerae and its biofilm. A total of 46 V. cholerae were isolated and most isolates harbored vasH (T6SS) (96%) and hlyA El Tor (91%) genes. Other virulence genes including stn/sto, vcsV2 (T3SS), and tcp classical genes were detected in 9, 9, and 4% of the isolates, respectively. More than 50% of the isolates produced strong biofilms and exhibited resistant against 2 or more antibiotics. RPE exhibited good activity against all isolates with the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values ranging between 2048 and 4096 and 4096 to 8192 μg/ml, respectively. Killing effect of RPE against a representative isolate V. cholerae PSU6072 was dose− and time−dependent. Transmission electron microscopy (TEM) showed that RPE effect V. cholerae cell morphology, as well as loss of cell wall were observed. At sub−MIC, RPE could reduce the swimming motility, decrease biofilm formation and disperse pre−formed biofilm of V. cholerae PSU6072. A significant down regulation of genes involved in V. cholerae quorum sensing regulator (luxO), swimming motility (flaA), and biofilm formation (vpsL, vpsR, bap1, and rbmA) was observed. In addition, a marked decrease in total aerobic bacteria and Vibrio spp. was found in shrimp meat after treatment with the RPE for short period (3 min). Thus, the results of present study strengthen the potential applications of RPE as anti−bacterial and anti−biofilm agents against V. cholerae and other bacteria contaminated in shrimp.

Recovery of Vibrio cholerae polarized cellular organization after exit from a non-proliferating spheroplast state.

Vibrio cholerae, the causative agent of cholera epidemics, is a rod-shaped bacterium with a highly polarized cellular organization. It can survive harmful growth conditions by entering a non-proliferating spheroplast state, which involves loss of the cell envelope and polarity. How polarized rod organization cells are formed when the spheroplasts exit the non-proliferating state remains largely uncharacterized. To address this question, we investigated how L-arabinose-induced V. cholerae spheroplasts return to growth. We found that de novo morphogenesis started with the elimination of an excess of periplasm, which was immediately followed by cell elongation and the formation of cell branches with a diameter similar to that of normal V. cholerae cells. Periplasm elimination was driven by bifunctional peptidoglycan synthases involved in cell-wall maintenance, the aPBPs. Elongation and branching relied on the MreB-associated monofunctional peptidoglycan synthase PBP2. The cell division monofunctional peptidoglycan synthase FtsI was not involved in any of these processes. However, the FtsK cell division protein specifically targeted the sites of vesicle extrusion. Genetic material was amplified by synchronous waves of DNA replication as periplasmic elimination began. The HubP polarity factor targeted the tip of the branches as they began to form. However, HubP-mediated polarization was not involved in the efficiency of the recovery process. Finally, our results suggest that the positioning of HubP and the activities of the replication terminus organizer of the two V. cholerae chromosomes, MatP, are independent of cell division. Taken together, these results confirm the interest of L-arabinose-induced V. cholerae spheroplasts to study how cell shape is generated and shed light on the de novo establishment of the intracellular organization and cell polarization in V. cholerae.

Adaptation to bile and anaerobicity limits Vibrio cholerae phage adsorption.

Vibrio cholerae is the bacterial pathogen responsible for cholera, a diarrheal disease that impacts people in areas without access to potable water. In regions that lack such infrastructure, cholera represents a large proportion of disease outbreaks. Bacteriophages (phages, viruses that infect bacteria) have recently been examined as potential therapeutic and prophylactic agents to treat and prevent bacterial disease outbreaks like cholera due to their specificity and stability. This work examines the interaction between V. cholerae and vibriophages in consideration for a cholera prophylaxis regimen (M. Yen, L. S. Cairns, and A. Camilli, Nat Commun 8:14187, 2017, https://doi.org/10.1038/ncomms14187) in the context of stimuli found in the intestinal environment. We discover that common signals in the intestinal environment induce cell surface modifications in V. cholerae that also restrict some phages from binding and initiating infection. These findings could impact considerations for the design of phage-based treatments, as phage infection appears to be limited by bacterial adaptations to the intestinal environment.

Increased intracellular persulfide levels attenuate HlyU-mediated hemolysin transcriptional activation in Vibrio cholerae

The vertebrate host's immune system and resident commensal bacteria deploy a range of highly reactive small molecules that provide a barrier against infections by microbial pathogens. Gut pathogens, such as Vibrio cholerae, sense and respond to these stressors by modulating the expression of exotoxins that are crucial for colonization. Here, we employ mass-spectrometry-based profiling, metabolomics, expression assays and biophysical approaches to show that transcriptional activation of the hemolysin gene hlyA in V. cholerae is regulated by intracellular forms of sulfur with sulfur-sulfur bonds termed reactive sulfur species (RSS). We first present a comprehensive sequence similarity network analysis of the arsenic repressor (ArsR) superfamily of transcriptional regulators where RSS and H2O2 sensors segregate into distinct clusters. We show that HlyU, transcriptional activator of hlyA in V. cholerae, belongs to the RSS-sensing cluster and readily reacts with organic persulfides, showing no reactivity and or DNA-dissociation following treatment with GSSG or H2O2. Surprisingly, in V. cholerae cell cultures, both sulfide and peroxide treatment downregulate HlyU-dependent transcriptional activation of hlyA. However, RSS metabolite profiling shows that both sulfide and peroxide treatment raise the endogenous inorganic sulfide and disulfide levels to a similar extent, accounting for this crosstalk, and confirming that V. cholerae attenuates HlyU-mediated activation of hlyA in a specific response to intracellular RSS. These findings provide new evidence that gut pathogens may harness RSS-sensing as an evolutionary adaptation that allows them to overcome the gut inflammatory response by modulating the expression of exotoxins.

Antibiotic sensitivity of cholera vibrions in biofilms formed on various substrates

Introduction. Evaluation of antibiotic sensitivity of biofilms formed by Vibrio cholerae can help in the search for effective drugs to combat cholera.
 The aim of the work is to evaluate the effectiveness of antibacterial drugs against V. cholerae cells being a part of a polymicrobial biofilm formed on various substrates.
 Materials and methods. Mono- and polymicrobial biofilms were simulated in vitro on chitin and plastic in flasks containing tap autoclaved water contaminated with a suspension of 104 microbial cells of three strains of V. cholerae O1 El Tor separately or in combination with Escherichia coli QD 5003.
 Results. When being a part of mono- or polymicrobial bacterial communities formed by the studied strains on chitin or plastic, the V. cholerae and E. coli cells were less susceptible to the action of antibacterial drugs.
 Conclusion. The increased antibiotic resistance of V. cholerae biofilms formed on biotic and abiotic surfaces highlights the danger of their spread and preservation in the environment, creates additional problems regarding the use of antibiotics and requires the development of alternative strategies to reduce resistance.

Kinetic principles of ParA2-ATP cycling guide dynamic subcellular localizations in Vibrio cholerae.

Dynamic protein gradients are exploited for the spatial organization and segregation of replicated chromosomes. However, mechanisms of protein gradient formation and how that spatially organizes chromosomes remain poorly understood. Here, we have determined the kinetic principles of subcellular localizations of ParA2 ATPase, an essential spatial regulator of chromosome 2 segregation in the multichromosome bacterium, Vibrio cholerae. We found that ParA2 gradients self-organize in V. cholerae cells into dynamic pole-to-pole oscillations. We examined the ParA2 ATPase cycle and ParA2 interactions with ParB2 and DNA. In vitro, ParA2-ATP dimers undergo a rate-limiting conformational switch, catalysed by DNA to achieve DNA-binding competence. This active ParA2 state loads onto DNA cooperatively as higher order oligomers. Our results indicate that the midcell localization of ParB2-parS2 complexes stimulate ATP hydrolysis and ParA2 release from the nucleoid, generating an asymmetric ParA2 gradient with maximal concentration toward the poles. This rapid dissociation coupled with slow nucleotide exchange and conformational switch provides for a temporal lag that allows the redistribution of ParA2 to the opposite pole for nucleoid reattachment. Based on our data, we propose a 'Tug-of-war' model that uses dynamic oscillations of ParA2 to spatially regulate symmetric segregation and positioning of bacterial chromosomes.

Electrochemical Detection of Vibrio cholerae by Amine Functionalized Biocompatible Gadolinium Oxide Nanoparticles.

Herein, we report the biocompatible amine-functionalized gadolinium oxide nanoparticles (Gd2O3 NPs) for the possibility of electrochemical detection of Vibrio cholerae (Vc) cells. The microwave irradiation process is applied to synthesize Gd2O3 NPs. The amine (NH2) functionalization is carried out via overnight stirring with 3(Aminopropyl)triethoxysilane (APTES) at 55 °C. The size of NPs amine functionalized APETS@Gd2O3 NPs are determined by transmission electron microscopy (TEM). APETS@Gd2O3 NPs are further electrophoretically deposited onto indium tin oxide (ITO) coated glass substrate to obtain working electrode surface. The monoclonal antibodies (anti-CT) specific to cholera toxin associated to Vc cells are covalently immobilized onto the above electrodes using EDC-NHS chemistry and further BSA is added to obtain the BSA/anti-CT/APETS@Gd2O3/ITO immunoelectrode. Further, this immunoelectrode shows the response for cells in CFU range from 3.125 × 106 to 30 × 106 and is very selective with sensitivity and LOD 5.07 mA CFUs mL cm-2 and 0.9375 × 106 CFU respectively. To establish a future potential for APTES@Gd2O3 NPs in field of biomedical applications and cytosensing, the effect of APTES@Gd2O3 NPs on mammalian cells is also observed using in vitro cytotoxicity assay and cell cycle analysis.

Single-cell segmentation in bacterial biofilms with an optimized deep learning method enables tracking of cell lineages and measurements of growth rates.

Bacteria often grow into matrix-encased three-dimensional (3D) biofilm communities, which can be imaged at cellular resolution using confocal microscopy. From these 3D images, measurements of single-cell properties with high spatiotemporal resolution are required to investigate cellular heterogeneity and dynamical processes inside biofilms. However, the required measurements rely on the automated segmentation of bacterial cells in 3D images, which is a technical challenge. To improve the accuracy of single-cell segmentation in 3D biofilms, we first evaluated recent classical and deep learning segmentation algorithms. We then extended StarDist, a state-of-the-art deep learning algorithm, by optimizing the post-processing for bacteria, which resulted in the most accurate segmentation results for biofilms among all investigated algorithms. To generate the large 3D training dataset required for deep learning, we developed an iterative process of automated segmentation followed by semi-manual correction, resulting in >18,000 annotated Vibrio cholerae cells in 3D images. We demonstrate that this large training dataset and the neural network with optimized post-processing yield accurate segmentation results for biofilms of different species and on biofilm images from different microscopes. Finally, we used the accurate single-cell segmentation results to track cell lineages in biofilms and to perform spatiotemporal measurements of single-cell growth rates during biofilm development.

A Dueling-Competent Signal-Sensing Module Guides Precise Delivery of Cargo Proteins into Target Cells by Engineered Pseudomonas aeruginosa.

To recognize and manipulate a specific microbe of a crowded community is a highly challenging task in synthetic biology. Here we introduce a highly selective protein delivery platform, termed DUEC, which responds to direct contact of attacking cells by engineering the tit-for-tat/dueling response of H1-T6SS (type VI secretion system) in Pseudomonas aeruginosa. Using a Cre-recombinase-dependent reporter, we screened H1-T6SS-secreted substrates and developed Tse6N as the most effective secretion tag for Cre delivery. DUEC cells can discriminately deliver the Tse6N-Cre cargo into the cytosol of T6SS+ but not T6SS- Vibrio cholerae cells. DUEC could also deliver a nuclease cargo, Tse6N-NucSe1, to selectively kill provoking cells in a mixed community. These data demonstrate that the DUEC cell not only is a prototypical physical-contact sensor and delivery platform but also may be coupled with recombination-based circuits with the potential for complex tasks in mixed microbial communities.

In-vivo protein nitration facilitates Vibrio cholerae cell survival under anaerobic, nutrient deprived conditions

Protein tyrosine nitration (PTN), a highly selective post translational modification, occurs in both prokaryotic and eukaryotic cells under nitrosative stress. However, its physiological function is not yet clear. Like many gut pathogens, Vibrio cholerae also faces nitrosative stress, which makes its proteome more vulnerable to PTN. Here, we report for the first time in-vivo PTN in V. cholerae by immunoblotting and LC-ESI-MS/MS proteomic analysis. Our results indicated that in-vivo PTN in V. cholerae was culture media independent. Surprisingly, in-vivo PTN was reduced in V. cholerae proteome under anaerobic or hypoxic condition in a nutrient deprived state. Interestingly, intracellular nitrate content was more than the nitrite content in V. cholerae under anaerobic conditions. Additionally, biochemical measurement of GSH/GSSG ratio, activities of catalase and SOD, ROS and RNS imaging by confocal microscopy confirmed a relative intracellular oxidizing environment in V. cholerae under anaerobic conditions. This altered redox environment favors the oxidation of nitrite which may be generated from protein denitration enriching the intracellular nitrate pool. The cell survival of V. cholerae can finally be facilitated by nitrate reductase (NapA) utilizing that nitrate pool. Our cell viability study using wild type and ΔnapA strain of V. cholerae also supported the role of NapA mediated cell survival under nutrient deprived anaerobic conditions. In spite of having nitrate reductase (NapA), V. cholerae lacks any nitrite reductase (NiR). Hence, in-vivo nitration may provide an avenue for toxic nitrite storage and also may help in nitrosative stress tolerance mechanism preventing further unnecessary protein nitration in V. cholerae proteome.

Vibrio cholerae secretion system of the type VI

The review summarizes literature data on the Vibrio cholerae secretion system of the 6th type. This system is a contact-dependent macromolecular mechanism through which bacteria translocate toxic effector proteins into target cells. It is found in many Gram-negative bacteria, including Vibrio cholerae. V. cholerae infects phagocytic amoebae, nematodes, ciliates, bacteria belonging to different species, as well as unrelated strains of V. cholerae using this system. DNA released after lysis of competing bacteria can be taken up by Vibrio cholerae cells, which leads to the acquisition of new genetic material. The type VI secretion system is involved in the infectious process. The destruction of macrophages and microbiota contributes to the active reproduction of the pathogen and colonization of host epitheliocytes, and the production of effector proteins causes the development of diarrhea and intestinal inflammation. Cholera vibrio secretion system of the 6th type has a structure similar to other gram-negative bacteria. The genes encoding the proteins of this system are located in one large region of the second chromosome and in several additional clusters. It has been shown that toxigenic strains of V. cholerae contain an identical set of secretion system genes, while their composition is variable in non-toxigenic isolates. The regulation of secretion system protein expression differs in V. cholerae strains of different toxigenicity, depends on a number of environmental signals, and is associated with other cell regulatory networks. The paper provides experimental data on the analysis of the structure of the global regulatory gene, vasH, of the type VI secretion system in toxigenic and non-toxigenic V. cholerae O1, biovar El Tor strains isolated in the Russian Federation. Thus, the type VI secretion system is an important mechanism that facilitates the survival of V. cholerae in complex communities in vitro, protects against damaging factors of the macroorganism and increases virulence in vivo, and also provides evolutionary transformations of cholera vibrio. Further study of this system will allow a better understanding of the pathogen-host interaction processes, as well as the adaptation mechanisms of V. cholerae in the external environment.

Effects of chitosan nanoparticles loaded with mesenchymal stem cell conditioned media on gene expression in Vibrio cholerae and Caco-2 cells

Vibrio (V.) cholerae forms a pellicle for self-defense in the pathological conditions in the intestine, which protects it against antibiotics and adverse conditions. Targeting biofilm genes and Toll-like receptors (TLRs) is one of the new strategies to combat multidrug-resistant bacteria. The objective of this study was to evaluate the effect of mesenchymal stem cell conditioned media (MSC CM; 1000 µg), chitosan nanoparticles incorporated with mesenchymal stem cell conditioned media (MSC CM-CS NPs; 1000 µg + 0.05%), and chitosan nanoparticles (CS NPs; 0.05%) on the expression of bap1 and rbmC biofilm genes in V. cholerae and TLR2 and TLR4 genes in Caco-2 cells. The bacteria were inoculated in the presence or absence of MSC CM, MSC CM-CS NPs, and CS NPs for 24 h at 37 °C to evaluate the expression of biofilm genes. The Caco-2 cells were also exposed to V. cholerae for 1 h and then MSC CM, MSC CM-CS NPs, and CS NPs for 18 h at 37 °C. After these times, RNA was extracted from Caco-2 cells and bacteria exposed to the compounds, and the expression of target genes was evaluated using real-time PCR. Caco-2 cell viability was also assessed by MTT assay. After adding MSC CM, MSC CM-CS NPs, and CS NPs to V. cholerae medium, the percentage reduction in gene expression of bap1 was 96, 91, and 39%, and rbmC was 93, 92, and 32%, respectively. After adding MSC CM, MSC CM-CS NPs, and CS NPs to the Caco-2 cell medium, the percentage reduction in the gene expression of TLR4 was 89, 90, and 82%, and TLR2 was 41, 43, and 32%, respectively. MTT showed that Caco-2 cell viability was high and the compounds had little toxicity on these cells. Finally, it suggests that MSC CM-CS NPs designed may be a therapeutic agent to combat inflammation and biofilm formation in multidrug-resistant V. cholerae. However, further studies in vivo are also recommended.

The VarA-CsrA regulatory pathway influences cell shape in Vibrio cholerae.

Despite extensive studies on the curve-shaped bacterium Vibrio cholerae, the causative agent of the diarrheal disease cholera, its virulence-associated regulatory two-component signal transduction system VarS/VarA is not well understood. This pathway, which mainly signals through the downstream protein CsrA, is highly conserved among gamma-proteobacteria, indicating there is likely a broader function of this system beyond virulence regulation. In this study, we investigated the VarA-CsrA signaling pathway and discovered a previously unrecognized link to the shape of the bacterium. We observed that varA-deficient V. cholerae cells showed an abnormal spherical morphology during late-stage growth. Through peptidoglycan (PG) composition analyses, we discovered that these mutant bacteria contained an increased content of disaccharide dipeptides and reduced peptide crosslinks, consistent with the atypical cellular shape. The spherical shape correlated with the CsrA-dependent overproduction of aspartate ammonia lyase (AspA) in varA mutant cells, which likely depleted the cellular aspartate pool; therefore, the synthesis of the PG precursor amino acid meso-diaminopimelic acid was impaired. Importantly, this phenotype, and the overall cell rounding, could be prevented by means of cell wall recycling. Collectively, our data provide new insights into how V. cholerae use the VarA-CsrA signaling system to adjust its morphology upon unidentified external cues in its environment.

Fermentation products of the fungus Monascus spp. impairs the physiological activities of toxin-producing Vibrio cholerae

Monascus spp. are filamentous fungi used in fermented foods. They are also natural colorants and food preservatives. Certain metabolites of Monascus spp. lower cholesterol and have other health-promoting effects in humans. In the present study, we demonstrated that the fermentation products of Monascus spp. inhibited ATP synthesis and motility in toxigenic Vibrio cholerae. Single-cell tracking and rotation assays on single flagella showed that Monascus fermentation extract (MFE) significantly impaired V. cholerae swimming by disrupting flagellar rotation. A membrane potential-sensitive carbocyanine dye revealed that MFE depolarized the V. cholerae cell membrane which, in turn, lowered the membrane potential and, by extension, restricted ATP synthesis and flagellar rotation. MFE also severely hindered the motility of other pathogenic bacteria such as V. parahaemolyticus, Pseudomonas aeruginosa, Salmonella enterica Typhimurium, and Leptospira interrogans. The foregoing findings indicate that Monascus fermentation extract could potentially preventing infection caused by multiple pathogenic bacteria as the conventional prophylaxes and slow their progression and lower mortality and morbidity.

In-vivo Protein Nitration and De-Nitration Facilitate Vibrio cholerae Cell Survival Under Anaerobic Nutrient Deprived Condition: Consequences of Nitrite Induced Protein Nitration

In-vivo Protein Nitration and De-Nitration Facilitate Vibrio cholerae Cell Survival Under Anaerobic Nutrient Deprived Condition: Consequences of Nitrite Induced Protein Nitration

Enumeration of Viable Non-Culturable Vibrio cholerae Using Droplet Digital PCR Combined With Propidium Monoazide Treatment.

Many bacterial species, including Vibrio cholerae (the pathogen that causes cholera), enter a physiologically viable but non-culturable (VBNC) state at low temperature or in conditions of low nutrition; this is a survival strategy to resist environmental stress. Identification, detection, and differentiation of VBNC cells and nonviable cells are essential for both microbiological study and disease surveillance/control. Enumeration of VBNC cells requires an accurate method. Traditional counting methods do not allow quantification of VBNC cells because they are not culturable. Morphology-based counting cannot distinguish between live and dead cells. A bacterial cell possesses one copy of the chromosome. Hence, counting single-copy genes on the chromosome is a suitable approach to count bacterial cells. In this study, we developed quantitative PCR-based methods, including real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR), to enumerate VBNC V. cholerae cells by counting the numbers of single-copy genes in samples during VBNC-state development. Propidium monoazide (PMA) treatment was incorporated to distinguish dead cells from viable cells. Both PCR methods could be used to quantify the number of DNA copies/mL and determine the proportion of dead cells (when PMA was used). The methods produced comparable counts using three single-copy genes (VC1376, thyA, and recA). However, ddPCR showed greater accuracy and sensitivity than qPCR. ddPCR also allows direct counting without the need to establish a standard curve. Our study develops a PMA-ddPCR method as a new tool to quantify VBNC cells of V. cholerae. The method can be extended to other bacterial species.

Deficiency in cytosine DNA methylation leads to high chaperonin expression and tolerance to aminoglycosides in Vibrio cholerae.

Antibiotic resistance has become a major global issue. Understanding the molecular mechanisms underlying microbial adaptation to antibiotics is of keen importance to fight Antimicrobial Resistance (AMR). Aminoglycosides are a class of antibiotics that target the small subunit of the bacterial ribosome, disrupting translational fidelity and increasing the levels of misfolded proteins in the cell. In this work, we investigated the role of VchM, a DNA methyltransferase, in the response of the human pathogen Vibrio cholerae to aminoglycosides. VchM is a V. cholerae specific orphan m5C DNA methyltransferase that generates cytosine methylation at 5’-RCCGGY-3’ motifs. We show that deletion of vchM, although causing a growth defect in absence of stress, allows V. cholerae cells to cope with aminoglycoside stress at both sub-lethal and lethal concentrations of these antibiotics. Through transcriptomic and genetic approaches, we show that groESL-2 (a specific set of chaperonin-encoding genes located on the second chromosome of V. cholerae), are upregulated in cells lacking vchM and are needed for the tolerance of vchM mutant to lethal aminoglycoside treatment, likely by fighting aminoglycoside-induced misfolded proteins. Interestingly, preventing VchM methylation of the four RCCGGY sites located in groESL-2 region, leads to a higher expression of these genes in WT cells, showing that the expression of these chaperonins is modulated in V. cholerae by DNA methylation.