New Results

Comprehension of Antimicrobial Peptides Modulation of the Type VI Secretion System in Vibrio cholerae

Annabelle Mathieu-Denoncourt, Marylise Duperthuy
doi: https://doi.org/10.1101/2022.11.18.517169

Abstract

The Type VI secretion System (T6SS) is a versatile weapon used by bacteria for virulence, resistance to grazing and competition with other bacteria. We previously demonstrated that the role of the T6SS in interbacterial competition and in resistance to grazing is enhanced in Vibrio cholerae in the presence of subinhibitory concentrations of polymyxin B (PmB). In this study, we performed a global quantitative proteomic analysis by liquid chromatography coupled to mass spectrometry and a transcriptomic analysis by quantitative PCR of the T6SS known regulators in V. cholerae grown with and without PmB. The proteome of V. cholerae is greatly modified in the presence of PmB at subinhibitory concentrations with more than 39 % of the identified cellular proteins displaying a difference in their abundance, including T6SS-related proteins (Hcp, VasC, TsaB and ClpV). We identified a regulator whose abundance and expression are increased in the presence of PmB, vxrB, the response regulator of the two-component system VxrAB. In a vxrAB deficient mutant, the expression of hcp measured by quantitative PCR, although globally reduced, was not modified in the presence of PmB, confirming its role in hcp upregulation with PmB. The upregulation of the T6SS in the presence of PmB appears to be, at least in part, due to the two-component system VxrAB.

Importance The type VI secretion system is important for bacterial competition, virulence and resistance to grazing by predators. In this study, we investigated the regulation leading to the type VI secretion system activation in the presence of polymyxin B (PmB), an antimicrobial used in veterinary and human health to treat infection caused by multi-resistant Gram-negative bacteria, in V. cholerae. In addition to making an overall portrait of the modifications to the proteome, we identified the VxrAB two-component system as the main regulator responsible for this activation. Our results provide evidence that subinhibitory concentrations of antimicrobials are responsible for important modifications of the proteome of pathogenic bacteria, inducing the production of proteins involved in virulence, host colonisation, resistance and environmental survival.

Introduction

Vibrio cholerae is a ubiquitous Gram-negative bacterium found in brackish rivers, coastal water and estuaries, or associated with fishes, shellfishes and zooplankton (1). There are more than 200 serotypes of V. cholerae, the O1 and O139 causing the cholera disease. The O1 El Tor biotype is responsible for the 7th ongoing pandemic (2). Cholera is acquired by the consumption of contaminated water or food and is characterized by an acute diarrhea, leading to dehydration and eventually to death if not treated (1). V. cholerae is endemic in regions where water sanitation facilities are inadequate and access to drinking water is precarious (2, 3). Many virulence and competition effectors allow V. cholerae to cause infections, the most important being the cholera toxin and the toxin co-regulated pilus (4, 5).

The type VI secretion system (T6SS), found in more than 25% of Gram-negative bacteria, has been discovered a decade ago in V. cholerae (6). The T6SS is a molecular syringe analogous to T4 bacteriophage. It allows the translocation of various toxic effectors into target neighbouring cells. A contraction event of the VipA/B sheath propels a hemolysin-coregulated protein (Hcp) nanotube that punctures the target cell’s envelope (7–9). The effectors have many intracellular targets and modes of action in both eukaryotic and prokaryotic cells, such as disruption of the cytoskeleton by actin cross-linking (10, 11), disruption of the cell membrane (12, 13) and peptidoglycan degradation (14, 15). It makes the T6SS an important weapon against competitor bacteria and predators (12, 13, 16, 17). The T6SS genes are divided in multiple gene clusters, i.e. a large cluster (VCA0105-VCA0124) that encodes for the major structural proteins, a protease and the internal regulator vasH (VCA0117), and at least 2 auxiliary clusters (VCA0017-VCA0022 and VC1415-VC1421), harbouring hcp, vgrG, adaptor and effector/immunity proteins (6, 12, 18). The expression of the T6SS genes is complex and involves different regulators (reviewed in (7)), including vasH. VasH orchestrates, along with the alternative sigma factor rpoN, the expression of the large and auxiliary clusters (7, 19). Most studies on the T6SS in V. cholerae were done with non-O1/non-O139 environmental strains that constitutively secrete through their T6SS. However, the regulation appears to vary between strains and seems to differ in clinical isolates as the secretion needs to be triggered. In clinical isolates, high osmolarity and high cell density, involving the regulators oscR and hapR respectively, are both required for secretion, even though the T6SS is constitutively expressed and produced (20, 21). Globally, the T6SS regulation also depends on other environmental signals, such as bile (22), nucleosides levels (23) and chitin (24–27) through the chitin competence pathway. The global regulators LonA and TsrA also play a role in T6SS regulation. Cold shocks lead to the production of the regulator cspV, which modulates the expression of many genes involved in cold shock response, biofilm formation and to the up-regulation of the T6SS (28, 29). The T6SS is also up-regulated by cell wall damages through the two-component system (TCS) VxrAB (30). Bacteria use TCS, a signal transduction device, to regulate the expression of diverse stress or virulence factors in response to variations in their environment and throughout infection (31). TCS are generally composed of a membrane-bound sensor histidine kinase (HK) and a response regulator (RR), which are activated through subsequent phosphorylation. In most TCS, the detection of stimuli by HK leads to the autophosphorylation of its histidine residue, then to the transphosphorylation of the aspartate residue of the RR (32). The RR acts afterwards as a transcriptional regulator. TCS are implicated in various processes such as virulence, stress response, environmental adaptation, and quorum sensing, among others (33). In V. cholerae, vxrA and vxrB are encoded on the conserved vxrABCDE (VCA0565 - VCA0569) (34). VxrC appears to have an inhibitory role in biofìlm formation (30). As for VxrDE, their role is still unknown although it has been suggested that VxrDE have a minor role in vpsL expression without affecting the biofilm formation (30). While VxrAB are essential for the colonization of infant mouse model, the contribution of VxrCDE is minor (34).

Antimicrobial peptides (AMPs) are ubiquitous, small, and mostly cationic peptides produced by bacteria and host cells for competition, prevention of infections, or to control the microbiota (35). AMPs are produced in the gut by the microbiota and host cells, and are effective against a broad range of microorganisms such as viruses, fungi and bacteria (36). AMPs have many cellular targets, but the most common mechanism of action is an electrostatic interaction with the negative bacterial membrane that leads to pore formation, the loss of cytoplasmic content and eventually, cell death (37). Polymyxin B (PmB), a cationic AMP produced by Paenibacillus polymyxa, binds to the lipid A portion of the LPS at the outer membrane (38). Polymyxins are commonly used in prevention of infections in veterinary medicine and in the treatment of multidrug resistant infections in human health (39). Since the absorption of polymyxins is low, a high percentage is excreted through urine and can accumulate in the environment, including soil and water (40). Polymyxins can thus be found in subinhibitory concentrations in water (41, 42). Subinhibitory concentrations of AMPs are known to have a modulatory effect on virulence, persistence and resistance factors in Gram-negative bacteria (43, 44), including on V. cholerae (45–48). El Tor strains are resistant to PmB conversely to the classical strains they replaced (38).

We previously demonstrated that subinhibitory concentrations of PmB increase the expression of Hcp, the structural component of the T6SS syringe in a pandemic strain of V. cholerae (46). PmB alone was not sufficient to induce the secretion through the T6SS in an O1 El Tor strain in low osmolarity conditions, but increased its production and secretion in a dose dependant manner. Although it did not increase the resistance of V. cholerae to antimicrobial peptides or antibiotics, the increased secretion through the T6SS due to the PmB led to a more efficient elimination of the competitor bacteria E. coli and an increased cytotoxicity towards amoebas in a T6SS-dependant manner. Furthermore, the increased expression of both hcp genes lets us believe that PmB could act as an activating signal for T6SS production and secretion (44, 46). In this study, we aim to identify the regulatory pathways involved in the over expression and over secretion of Hcp in the presence of subinhibitory concentrations of PmB. A proteomic approach of the cell fraction coupled with a transcriptomic approach using quantitative PCR, allowed to determine that the presence of subinhibitory concentrations of PmB has a complex impact on the proteome of V. cholerae and greatly modulates the production of proteins implicated in cellular and metabolic processes, regulation and binding and catalytic activities. Among the proteins that were more abundant in the presence of PmB, we identified VxrB, part of the VxrAB TCS and involved in the T6SS regulation. We further constructed a deficient mutant of vxrAB and its Hcp production and expression (hcp) were assessed by western blot and quantitative PCR. Our results suggest that vxrAB is, at least partly, responsible for the increased expression and secretion of Hcp in the presence of PmB in V. cholerae.

Material & methods

Strains used in this study

Vibrio cholerae O1 El Tor strain A1552, an Inaba clinical strain isolated in 1992 from a Peruvian tourist, as well as its T6SS defective isogenic mutant A1552Δhcp1-2 were used for this study (20, 49). Bacterial strains obtained by mutagenesis by natural competence as previously described (see below) (50) and plasmids used in this study are listed in Table I. V. cholerae strains were grown on LB agar plates at 37°C. Isolated colonies were inoculated in 5 mL LB broth and incubated 16 h at 37°C with agitation. To obtain the final cultures, 100 μL of the bacterial suspension were transferred into 5 mL of LB broth with 2% NaCl (LB-2%NaCl), with or without 3 μg/mL of PmB (Sigma-Aldrich) and incubated at 37°C with agitation until they reached an optical density at 600nm (OD600nm) of 2. When needed, L-arabinose (0.05 % w/v) or antibiotics (chloramphenicol (2 μg/mL), carbenicillin (50 μg/mL)) were added to the media.

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Table I Bacterial strains and plasmids used in this study

Proteomic analysis

Proteomic analysis of the bacterial cells grown with or without PmB (3 μg/mL) was performed by liquid chromatography coupled with tandem mass spectrometry, as described before (46). The cellular fraction of V. cholerae A1552 cultures at OD600nm of 2 in LB2%NaCl in the presence or in absence of PmB were analyzed. The experiment was conducted in two biological replicates. The proteins were identified using the genome of V. cholerae N16961, a strain very closely related to A1552. The data were analyzed using the Scaffold V.5 software (protein threshold: 99%, with at least two peptides identified and a false discovery rate of 1%) and we removed the proteins identified in only one of the replicates. The proteins with differential abundance were then subjected to a Gene Ontology (GO) term enrichment (51).

Mutant construction and complementation

A1552ΔvxrAB::CmR and A1552ΔcspV::CmR defective mutants were obtained using the optimized natural competence protocol by Marvig & Blokesch (50). All the PCR amplicons were obtained with Thermo Fisher™ oligonucleotide primers listed in Table II, using Taq DNA Polymerase with Standard buffer from New England Biolabs®, according to the manufacturer’s instructions. The chloramphenicol resistance cassette (CmR), with its promoter and terminator from pCas9 CR4 (52) (Table I), was amplified by PCR. Up and downstream regions of target genes of about 1000 bp were amplified from A1552 genomic DNA by PCR using primers adding 15 bp homology to CmR 5’ and 3’ extremities (Table II). The upstream region, the CmR cassette and the downstream region were bound together by overlap PCR and purified using Monarch DNA Gel extraction kit (New England Biolabs®).

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Table II Primers used in this study

For the natural transformation, briefly, V. cholerae wild type strain A1552 was grown over night in LB at 30°C with agitation. The culture was diluted 1:50 in fresh LB and incubated at 30°C until it reached an OD600nm of 0.5. Bacteria were washed by centrifugation at 5500 x g for 5 min and suspended in M9 minimal media (Sigma) supplemented with 32 mL of MgSO4 1 M and 5.1 mL of CaCl2 1 M per liter (50). The bacteria were diluted 1:2 in fresh M9 media. Chitin powder (Sigma-Aldrich) was added, and the culture was grown overnight at 30°C with agitation. Two hundred ng of purified PCR amplicon were added to the culture, and the bacteria were further incubated 24 h at 30°C. To detach the bacteria from chitin, fresh M9 medium was added to the culture and the bacteria were vortexed vigorously for 2 min. The chitin was pelleted by quick centrifugation and the supernatant containing free bacteria was centrifuged at 5000 x g for 5 min. The bacteria were suspended in 500 μL of PBS and plated on LB agar supplemented with 2 μg/mL chloramphenicol (Fisher). Plates were incubated at 30°C for 3 days. Colonies grown on agar supplemented with chloramphenicol were further isolated on agar plates with chloramphenicol to confirm the phenotype. Deficient mutants were confirmed with PCR using either cspV – verif or vxrAB – verif primers (Table II) and sequencing of the insertion region.

For complementation, the complete open reading frame of cspV or vxrAB was amplified by PCR from purified A1552 genomic DNA using the Thermo Fisher™ oligonucleotide primers adding restriction sites listed in Table II. The amplicons and pBAD24 vector (Table I) were digested using HindIII and EcoRI (cspV) or PstI and KpnI (vxrAB) from New England Biolabs® according to the manufacturer’s instructions. Plasmids and amplicons were purified from agarose gel using Monarch Gel purification kit (New England Biolabs®) and ligated using T4 ligase from New England Biolabs®. The final construction was introduced by heat shock into thermocompetent E. coli DH5α at 42°C for 30 s (53). The bacteria were then suspended in fresh LB and incubated 1.5 h at 37°C. Bacteria were selected on LB agar supplemented with carbenicillin (50 μg/mL) (Fisher). Plasmids were extracted from E. coli using PureYield™ Plasmid Miniprep System (Promega) and suspended in milliQ water (Thermo Fisher).

V. cholerae was grown in LB for 16 h at 37°C and were made electrocompetent by successive washes in sterile water supplemented with 10% glycerol. The constructions were electroporated in the corresponding strains at 1.275 kV, 25 Ω in 1 mm electroporation cuvettes (Thermo Fisher). The bacteria were incubated 1.5 h at 37°C in LB, then plated on LB agar containing 50 μg/mL carbenicillin. Colonies were screened by PCR using pBAD24 – verif primers (Table II) to verify the construction.

Growth curves

V. cholerae was grown 16 h at 37 °C with agitation in LB, with carbenicillin when needed. A 1:50 dilution in fresh LB was done, and the bacteria were grown at 37°C to an OD600nm of 0.2. Then, 0.05 % L-arabinose was added, and the bacteria were grown until they reached OD600nm = 0.3. They were further diluted 1:50 in LB2%NaCl - 0.05 % L-arabinose, with or without 3 μg/mL of PmB, in 50 ml Falcon tubes, or 1:3000 in 96 wells plates. The bacterial growth was followed by reading the OD600nm every 15 min, at 37°C with agitation. Data were obtained from at least three independent experiments, in technical triplicates.

RNA extraction, cDNA synthesis and quantitative PCR (RT-qPCR)

V. cholerae was grown to an OD600nm of 0.5 at 37°C in 10 mL of LB2%NaCl, with or without 3 μg/mL of PmB. The bacteria were suspended in 1 mL TRIzol solution (Invitrogen) and the total RNA was extracted according to the manufacturer’s instructions. Five hundred nanograms of RNA were retrotranscribed to cDNA using QuantiNova Reverse Transcription Kit (Qiagen). RNA and cDNA purity and quality were assessed by nanodrop and migration on 2% agarose gel, respectively. Quantitative PCR analysis was done as described before (46) with primers listed in Table II. The relative hcp and regulators’ expression was calculated in PmB treated bacteria in comparison to non-treated cells using QuantStudio™ Desing and Analysis Software (Thermo Fisher) v1.5.1 and normalized using recA. The results were obtained from at least 3 independent experiments, in technical triplicates.

Western blot

Bacteria were grown to an OD600nm of 2 in LB2%NaCl, a condition that activates the T6SS secretion in V. cholerae A1552, supplemented with - 0.05 % L-arabinose and 3 μg/mL of PmB. Samples were treated and western blot conducted as described before (46). Results are representative of at least three independent experiments.

Statistical Analysis

All data are expressed as mean ± SD and were analyzed for significance using the SigmaPlot (version). Student’s t-tests were used to compare conditions between 2 groups. Single way ANOVA was used for multiple groups comparison. A result was considered as significant when p value < 0.05 (*).

Results

Proteomic analysis of V. cholerae’s cells grown with subinhibitory concentrations of PmB

To identify the cellular proteins whose abundance is modified in the presence of PmB, a quantitative proteomic analysis by liquid chromatography coupled to mass spectrometry was performed on V. cholerae A1552 bacterial cells grown in LB2%NaCl with or without 3 μg/ml of PmB, a concentration that does not affect bacterial growth but significantly increases the expression and secretion of Hcp (46). Our proteomic analysis identified a total of 22,819 peptides corresponding to 473 proteins of 7 to 184 kDa. After data curation, 454 proteins were obtained. We calculated the relative abundance of each protein in the presence of PmB in comparison with the control without PmB and determined that 177 proteins (39%) showed a modified abundance in the presence of PmB (Tables IV to VII). Among them, 130 were more abundant in the presence of PmB (Table IV), 17 were less abundant in its presence (Table V), 26 were only found in its presence (Table VI), and 4 were found only in its absence (Table VII). The proteins showing no modulation are presented in Table SI. The correlation of the biological replicates was analyzed using scatter plots and demonstrated a good correlation with R-squared of more than 0.9 (Figure 1AB).

Figure 1.
Figure 1. Polymyxin B modifies the proteome of V. cholerae.

A quantitative proteomic analysis by liquid chromatography coupled to mass spectrometry of V. cholerae A1552 grown in Type VI Secretion System activating conditions, with or without 3 μg/ml of Polymyxin B (PmB) was performed. Scatter plot analysis using the total number of spectra per protein was performed to determine the correlation between the biological duplicates of the sample without PmB (A) and with 3 μg/ml of PmB (B). A GoAnnotation analysis of the proteins identified determined the molecular functions, implication in biological processes and localization of the proteins (C).

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Table III Known regulators of the Type VI secretion system of V. cholerae identified in our proteomic analysis.
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Table IV Cellular proteins that are more abundant in the presence of polymyxin B in V. cholerae A1552.
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Table V. Cellular proteins that are less abundant in the presence of polymyxin B in V.cholerae A1552.
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Table VI. Cellular proteins that are only found in the presence of polymyxin B in V.cholerae A1552.
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Table VII. Cellular proteins that are only found in absence of polymyxin B in V.cholerae A1552.

An analysis of the proteins that are modulated by PmB using Go Annotation showed that most modulated proteins have catalytic and/or binding function, with 99 and 75 proteins with these functions, respectively (Figure 1C). Regulatory proteins, transporters, transducers and sensor proteins were also modulated by PmB. The analysis showed that the proteins that are more abundant in the presence of PmB were mainly implicated in biological (59) and cellular (75) processes (Figure 1C). Regulation, response to stimulus and locomotion were the biological processes that were greatly modified by PmB.

Among the proteins with an increased abundance in the presence of PmB, many have a role in AMPs or PmB resistance (54). For example, VexB, which is part of the efflux system VexAB (55), was 2.7 times more abundant in the presence of PmB, and AlmG and AlmE, which are directly responsible for PmB resistance by LPS modification (56), were 15 and 3.8 times more abundant with PmB, respectively (Table IV). VprB, only present in the presence of PmB (Table VI), is part of the TCS VprAB that activates the expression of AlmGE (57). The protease DegS was also 2.3 times more abundant in the presence of PmB (Table IV). It is responsible for the activation of the alternate sigma factor RpoE, which activates the repair of AMPs induced damages in the bacterial envelope (58). VarF is part of an antibiotic efflux pump (59) and is only found in the presence of PmB (Table VI). OmpR, the RR to the EnvR-OmpR TCS, is a repressor of virulence factors in response to stress envelope, was also more abundant in the presence of PmB (Table IV) (60).

As for the T6SS related proteins, many showed an increased abundance in the presence of PmB, although a lot of the structural components were not identified in our analysis. As expected, the main component of the T6SS syringe, Hcp (VC_1415, VC_A0017), was also more abundant (2.3 times) in the presence of PmB (46). VasC (VC_A0112), a cytoplasmic protein with an FHA domain essential for secretion (61), and ClpV (VC_A0116), the ATPase responsible for recycling of the contractile sheath, presented abundances that were 2.7 and 3.0 times higher in the presence of PmB, respectively. The abundance of VasK (VC_A0120), a part of the inner-membrane complex (62) was increased by 5.5 times by PmB (Table IV). TsaB (VC_1989), the immunity protein to the VgrG-3 that targets the peptidoglycan, had an increased abundance (2.0 times) in the presence of PmB (Table IV). The abundance of VipA (VC_A0107) and VipB (VC_A0108), the small and large subunits of the contractile sheath, was not modified by PmB (Table S1).

We paid a particular attention to the effect of PmB on the T6SS known regulators. As shown in the table III, our proteomic analysis identified many of them. The abundance of most of these regulators was not significantly affected by the presence of PmB, except for VxrB that was 2.7 times more abundant in the presence of PmB (Table III).

Transcriptomic analysis of known T6SS regulators in the presence of PmB

To confirm the results obtained in the proteomic analysis and to include regulators that were not identified in this analysis, we performed a transcriptomic analysis of all the known T6SS regulators of V. cholerae A1552 grown in LB2%NaCl supplemented or not with 3 μg/ml of PmB. To do so, a quantitative PCR approach was used and the relative expression of known T6SS regulators was calculated by comparing the number of transcripts from bacterial cells grown with and without PmB and normalized with recA (Figure 2A). Although statistically significant, the expression of cyclic AMP receptor protein (CRP) (1.23x), tfoY (1.15x), tsrA (1.17x) and osrC (1.26x) was only slightly higher in the presence of PmB, and was not considered for further analysis. Interestingly, the expression of vxrB was 2.1 times higher in the presence of PmB than in its absence, while the expression of cspV was decreased by 2.43 times in its presence.

Figure 2.
Figure 2. Normalized relative expression of known T6SS regulators (A) and structural components (B) in V. cholerae grown with polymyxin B.

V. cholerae A1552 was grown to midlog phase in LB2%NaCl supplemented or not with 3 μg/ml of polymyxin B (PmB). Total RNA was extracted from cultures and retrotranscribed. The expression of selected regulators and structural components was measured by quantitative PCR in the presence of PmB in comparison to the non-treated cells, and normalized using recA. Data represent mean ± SD of three independent experiments conducted in triplicates. Asterisk represents a significant difference in expression between treated and non-treated cells (P<0.05). CRP, cyclic AMP receptor protein.

We also assessed the expression of 2 genes encoding structural proteins of the T6SS, the main component of the contractile sheath, VipB, and ClpV, the ATPase protein responsible for the recycling of the sheath after a contraction event (Figure 2B) (63, 64). Our results showed that the expression of vipB is not modified by PmB (Figure 2B). This result is in line with our previous work and the proteomic analysis of this study that showed no difference in protein abundance in the bacterial cell (46). The expression of clpV is 1.54 times higher in the presence of PmB (Figure 2B), which is also in line with our proteomic analysis.

Based on the results of both proteomic and transcriptomic analysis showing an increased abundance and expression of VxrB and vxrB, respectively, in the presence of PmB, and a decreased expression of cspV in the presence of PmB, we constructed defective mutants of the regulators vxrAB and cspV using natural competence (50) and complemented strains using pBAD24 vector (65). The deletion of vxrAB (Figure S1A) or cspV (Figure S2A) had no major consequence on bacterial growth. The growth was slightly impaired by complementation and PmB for A1552ΔvxrAB::CmR (Figure S1B), but not for A1552ΔcspV::CmR (Figure S2B). Like A1552, A1552ΔcspV::CmR and A1552ΔvxrAB::CmR grew in the presence of PmB at a concentration up to 100 ug/ml, although the growth of A1552ΔvxrAB::CmR was impaired in all PmB conditions (result not shown), an observation in line with previous studies (66).

Quantitative PCR analysis of hcp shows that vxrAB, but not cspV, is responsible for its upregulation in the presence of PmB

To determine the effect of the vxrAB and cspV deletion on the regulation of hcp in the presence of PmB, we performed a quantitative PCR analysis. V. cholerae A1552, A1552ΔcspV::CmR and A1552ΔvxrAB::CmR were grown in LB2%NaCl with or without PmB. We quantified the expression of hcp1 or hcp2 by quantitative PCR in the presence of PmB, in comparison to the non-treated cells, and normalized using recA (Figure 3 and S3). The expression of hcp1 and hcp2 in A1552 treated with PmB was 3.03 and 2.16 times higher, respectively, similarly to what we observed before (46) (Figure 3). For A1552ΔcspV::CmR, the expression of hcp1 and hcp2 is similar to the wildtype strain, and an upregulation of both hcp1 and hcp2 is still observed in the presence of PmB (Figure S3)., indicating that, upon cspV mutation, the T6SS is activated by PmB. However, upon vxrAB mutation, the expression of hcp1 and -2 was significantly reduced, as expected (34), but was not increased in the presence of PmB (Figure 3), indicating that the upregulation due to the PmB is no longer effective.

Figure 3.
Figure 3. The two-component system VxrAB plays a role in hcp upregulation in V. cholerae in the presence of subinhibitory concentrations of polymyxin B.

V. cholerae A1552 and A1552ΔvxrAB::CmR (A1552ΔvxrAB) were grown to midlog phase in LB2%NaCl with or without 3 μg/ml of polymyxin B (PmB). Total RNA was extracted from cultures and retrotranscribed. The expression of hcp1 (A) or hcp2 (B) was measured by quantitative PCR in the presence of PmB in comparison to the non-treated cells, and normalized using recA. Data represent mean ± SD of three independent experiments conducted in triplicates. Asterisk represents a significant difference in expression between treated and non-treated cells (P<0.05). ns, non-significative difference. C) The complementation of vxrAB restores the level of Hcp in the supernatant of A1552ΔvxrAB::CmR pBAD24-vxrAB. A1552 pBAD24, A1552ΔvxrAB::CmR pBAD24 and A1552ΔvxrAB::CmR pBAD24-vxrAB were grown to late exponential phase in LB-2%NaCl in the presence of PmB.

The secretion of Hcp in A1552 pBAD24, A1552ΔvxrAB::CmR pBAD24 and the complemented strain A1552ΔvxrAB::CmR pBAD24-vxrAB in the presence of PmB was assessed by western blot (Figure 3C). We previously demonstrated that Hcp secretion by V. cholerae A1552 is active in the presence of PmB (46). As expected, the secretion of Hcp is greatly reduced upon vxrAB mutation (Figure 3C). However, a vxrAB complementation completely restored, or even slightly increased, the secretion of Hcp to the level of the WT in the presence of PmB (Figure 3C).

Discussion

In this study, we aim to determine the impact of subinhibitory concentrations of PmB on the proteome of V. cholerae and to identify the regulatory pathways involved in the over expression and over secretion of Hcp in the presence of subinhibitory concentrations of PmB. To do so, we performed a proteomic analysis of the cellular fraction of V. cholerae O1 El Tor strain A1552 in the presence and in absence of PmB. We identified a total of 22,819 peptides corresponding to 454 proteins of 7 to 184 kDa, of which 177 had a modified abundance in the presence of PmB.

Several proteins were identified in both our last and current proteomic analysis. It is the case of Hcp that is more abundant in supernatant and bacterial cells in the presence of PmB, and of the proteases VesC and DegP, which was expected since they are secreted components. VesC is secreted through the Type II Secretion System (67), while DegP is secreted by membrane vesicles (68). A putative hydrolase (VC_1485) and an immunogenic protein (VC_0430) were also more abundant in both analysis in the presence of PmB. While the abundance of the lipoprotein Lpp and SucD (α subunit of succinyl-CoA synthetase) was decreased by PmB in the secretome, they were more abundant in the cell fraction. Two proteins were only present in the supernatant in the presence of PmB, and more abundant in the cellular fraction, the amino-acid transporter VC_0010 and the uncharacterized protein VC_0483. One protein, VxrD, was less abundant in both supernatant and bacterial cells in the presence of PmB. Our results show that V. cholerae modulates a large proportion of its components in response to PmB, as 39% of the identified proteins had their abundance modified in its presence. Most modulated proteins with increased abundance are implicated in cellular and metabolic processes, with binding, catalytic or transporter activities, that suggests an adaptation to survive the presence of the toxic AMP. This is further supported by the upregulation of efflux systems (VexAB, VprAB, VarF), LPS modifying enzymes (AlmGE), proteases (DegS) and RpoE activating proteins, all known mechanisms of antimicrobials resistance. Numerous proteins displaying a modified abundance in the presence of PmB are yet to be described.

Our previous study has shown that Hcp, the T6SS syringe major component, was more abundant in the secretome of V. cholerae under PmB activation (46). The Hcp syringe is wrapped by the contractile sheath composed of multiple helical polymers of VipAB, assembled in an extended form (69–71). This extended form provides enough energy, upon a contraction signal, for a contraction and rotation of VipAB helical polymers that propel the Hcp syringe, along with the effectors, through the bacterial envelope. Based on the fact that, conversely to the Hcp secreted protein, the abundance of VipB, a structural recycled component of the T6SS, was not modified, we hypothesized that PmB might increase the secretion through the T6SS rather than the number of assembled systems at the bacterial surface. Our current proteomic analysis of bacterial cells confirms that the contractile sheath components VipA and VipB are not more abundant in the presence of PmB. ClpV is a AAA+ (ATPases associated with various cellular activities) that hydrolyzes ATP to reshape, or recycle, various substrates (71). In V. cholerae, ClpV recognizes the contracted state of the T6SS sheath and forces its disassembly by unfolding VipB (70). To our knowledge, VipA/B are the only recycled proteins through ClpV activity. The recycling of the contractile sheath is essential and facilitates the effectors’ secretion because it makes VipA/B available for further secretion (70, 72). The presence of more ClpV could help the contractile sheath recycling in a cell with a T6SS activated by PmB.

Our proteomic analysis also revealed that the immunity protein TsaB (14, 73), the inner-membrane complex subunit VasK (74), and the FHA protein VasC (TagH), required for secretion (61), are more abundant in the presence of PmB. VasK (TssM) is part of the inner-membrane complex (74) along with VasF (TssL) and VasD (TssJ), a complex randomly distributed in the membrane. Once the inner-membrane complex is formed, it recruits the base plate components, then the Hcp syringe along with the VipA/B sheath (75). It has been suggested that the inner-membrane complexes are pre-assembled in the membrane, more abundant than the syringe portion and that they are reused for further secretion events using new syringe complexes (74). Altogether, our results suggest that more T6SS anchoring complexes might be formed and ready for secretion, while the recycled components abundance remains constant, and the secreted effectors are more produced to increase the number of contraction events and the global T6SS activity in the presence of PmB.

Our proteomic analysis identified many known regulators of the T6SS in V. cholerae, i.e. CytR, CRP, TfoY, RpoN, TsrA, OscR and VxrB, but only VxrB had a modified abundance in the presence of PmB. The expression of crp, tfoY, tsrA and oscR, although significantly higher, was only slightly modified by PmB, while the expression of cytR and rpoN was similar to the control. OscR is a transcriptional regulator that represses T6SS genes expression at low osmolarity (21). CytR is activated upon nucleosides starvation, that further activates the expression of T6SS (23). RpoN, along with the internal regulator VasH, coordinates the expression of the different T6SS genes clusters (6). TsrA is a global regulator (76), while TfoY and CRP are produced in response to c-di-GMP and cyclic-AMP, respectively. While CspV was not identified in our proteomic analysis, our transcriptomic results showed that PmB decreases the expression of cspV. However, since its expression is reduced in the presence of PmB and known to be produced upon cold shock (29), it is not surprising that it was not identified in the proteomic analysis. We constructed a mutant in which cspV is deleted and compared the expression of hcp1 and hcp2 in absence and in the presence of PmB for the wild-type strain and the cspV mutant. The expression pattern of both hcp genes was similar for the two strains, thus suggesting that cspV is not responsible for the increased hcp expression in the presence of PmB.

Our proteomic and transcriptomic analysis showed that the presence of PmB increased the production and expression of VxrB, the response regulator of the two-component system VxrAB (34). VxrB is a known regulator of V. cholerae T6SS, as its deletion decreases the expression of many T6SS related genes, including Hcp (34). In this study, we constructed a A1552ΔvxrAB::CmR mutant, the only essential components of the vxrABCDE locus, and a complemented strain. As previously reported, the deletion of vxrAB strongly diminished hcp expression (34). The complementation of vxrAB using pBAD24 restored the secretion of Hcp in the presence of PmB. Conversely to the wild type strain, the presence of PmB did not increase hcp expression in A1552ΔvxrAB::CmR, demonstrating that it is implicated in the upregulation of hcp in the presence of PmB. VxrAB (also known as WigKR) was first identified for its implication in colonization in the infant mouse model and T6SS regulation (34). VxrAB has already been described to regulate other systems implicated in antimicrobial resistance, such as biofilm formation, motility and cell shape maintenance and homeostasis in the presence of cell wall targeting antimicrobials (30, 66, 77). In the later, they demonstrated that the expression of vxrAB is increased by antibiotic-induced cell wall damage, and that VxrAB regulates the entire cell wall synthesis pathway, leading to V. cholerae’s tolerance to antibiotics (66). Even though we detected no major damages in the cell wall of V. cholerae A1552 at a concentration of PmB as high as 25 μg/mL (47), we observed, in this study, a significant upregulation of vxrB in the presence of 3 μg/mL of PmB. Taken together, our results suggest that in V. cholerae O1 El Tor the T6SS is up regulated by PmB, and that the upregulation involves the TCS VxrAB. The over production of ClpV and VasK also suggests that the T6SS are more efficiently recycled to facilitate the secretion of effectors.

Acknowledgment

The authors would like to thank Dre Wai from Laboratory for Molecular Infection Medicine Sweden (MIMS) at Umeå University for bacterial strains and the RAQ (Ressources Aquatiques Québec), an inter-institutional group supported financially by the Fonds de recherche du Québec – Nature et technologies (FRQNT) for their support. The proteomic analysis was performed by the Center for Advanced Proteomics Analyses, a Node of the Canadian Genomic Innovation Network supported by the Canadian Government through Genome Canada. This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC; http://www.nserc-crsng.gc.ca/index_eng.asp) Discovery grant number RGPIN-2017-05322. AM-D received financial support from the NSERC scholarship program (BESC D3 – 558624 – 2021).

Bibliography

  1. 1.↵
    Harris JB, LaRocque RC, Qadri F, Ryan ET, Calderwood SB. Cholera. Lancet. 2012;379(9835):2466–76.
    OpenUrlCrossRefPubMedWeb of Science
  2. 2.↵
    Deen J, Mengel MA, Clemens JD. Epidemiology of cholera. Vaccine. 2020;38 Suppl 1:A31–A40.
    OpenUrl
  3. 3.↵
    Jutla A, Whitcombe E, Hasan N, Haley B, Akanda A, Huq A, et al. Environmental factors influencing epidemic cholera. Am J Trop Med Hyg. 2013;89(3):597–607.
    OpenUrlAbstract/FREE Full Text
  4. 4.↵
    Vanden Broeck D, Horvath C, De Wolf MJ. Vibrio cholerae: cholera toxin. Int J Biochem Cell Biol. 2007;39(10):1771–5.
    OpenUrlCrossRefPubMedWeb of Science
  5. 5.↵
    Mathieu-Denoncourt A, Giacomucci S, Duperthuy M. The Secretome of Vibrio cholerae. In: Huang L, Li J, editors. Vibrios2021.
  6. 6.↵
    Pukatzki S, Ma AT, Sturtevant D, Krastins B, Sarracino D, Nelson WC, et al. Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc Natl Acad Sci U S A. 2006;103(5):1528–33.
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    Joshi A, Kostiuk B, Rogers A, Teschler J, Pukatzki S, Yildiz FH. Rules of Engagement: The Type VI Secretion System in Vibrio cholerae. Trends Microbiol. 2017;25(4):267–79.
    OpenUrlCrossRefPubMed
  8. 8.
    Crisan CV, Hammer BK. The Vibrio cholerae type VI secretion system: toxins, regulators and consequences. Environ Microbiol. 2020;22(10):4112–22.
    OpenUrl
  9. 9.↵
    Unterweger D, Kostiuk B, Otjengerdes R, Wilton A, Diaz-Satizabal L, Pukatzki S. Chimeric adaptor proteins translocate diverse type VI secretion system effectors in Vibrio cholerae. EMBO J. 2015;34(16):2198–210.
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    Pukatzki S, Ma AT, Revel AT, Sturtevant D, Mekalanos JJ. Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin. Proc Natl Acad Sci U S A. 2007;104(39):15508–13.
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    Durand E, Derrez E, Audoly G, Spinelli S, Ortiz-Lombardia M, Raoult D, et al. Crystal structure of the VgrG1 actin cross-linking domain of the Vibrio cholerae type VI secretion system. J Biol Chem. 2012;287(45):38190–9.
    OpenUrlAbstract/FREE Full Text
  12. 12.↵
    Zheng J, Ho B, Mekalanos JJ. Genetic analysis of anti-amoebae and anti-bacterial activities of the type VI secretion system in Vibrio cholerae. PLoS One. 2011;6(8):e23876.
    OpenUrlCrossRefPubMed
  13. 13.↵
    Miyata ST, Kitaoka M, Brooks TM, McAuley SB, Pukatzki S. Vibrio cholerae requires the type VI secretion system virulence factor VasX to kill Dictyostelium discoideum. Infect Immun. 2011;79(7):2941–9.
    OpenUrlAbstract/FREE Full Text
  14. 14.↵
    Brooks TM, Unterweger D, Bachmann V, Kostiuk B, Pukatzki S. Lytic activity of the Vibrio cholerae type VI secretion toxin VgrG-3 is inhibited by the antitoxin TsaB. J Biol Chem. 2013;288(11):7618–25.
    OpenUrlAbstract/FREE Full Text
  15. 15.↵
    Altindis E, Dong T, Catalano C, Mekalanos J. Secretome analysis of Vibrio cholerae type VI secretion system reveals a new effector-immunity pair. mBio. 2015;6(2):e00075.
    OpenUrlCrossRefPubMed
  16. 16.↵
    MacIntyre DL, Miyata ST, Kitaoka M, Pukatzki S. The Vibrio cholerae type VI secretion system displays antimicrobial properties. Proc Natl Acad Sci U S A. 2010;107(45):19520–4.
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    Drebes Dorr NC, Blokesch M. Interbacterial competition and anti-predatory behaviour of environmental Vibrio cholerae strains. Environ Microbiol. 2020;22(10):4485–504.
    OpenUrlCrossRef
  18. 18.↵
    Crisan CV, Chande AT, Williams K, Raghuram V, Rishishwar L, Steinbach G, et al. Analysis of Vibrio cholerae genomes identifies new type VI secretion system gene clusters. Genome Biol. 2019;20(1):163.
    OpenUrlCrossRefPubMed
  19. 19.↵
    Kitaoka M, Miyata ST, Brooks TM, Unterweger D, Pukatzki S. VasH is a transcriptional regulator of the type VI secretion system functional in endemic and pandemic Vibrio cholerae. J Bacteriol. 2011;193(23):6471–82.
    OpenUrlAbstract/FREE Full Text
  20. 20.↵
    Ishikawa T, Rompikuntal PK, Lindmark B, Milton DL, Wai SN. Quorum sensing regulation of the two hcp alleles in Vibrio cholerae O1 strains. PLoS One. 2009;4(8):e6734.
    OpenUrlCrossRefPubMed
  21. 21.↵
    Ishikawa T, Sabharwal D, Bröms J, Milton DL, Sjöstedt A, Uhlin BE, et al. Pathoadaptive conditional regulation of the type VI secretion system in Vibrio cholerae O1 strains. Infect Immun. 2012;80(2):575–84.
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    Bachmann V, Kostiuk B, Unterweger D, Diaz-Satizabal L, Ogg S, Pukatzki S. Bile Salts Modulate the Mucin-Activated Type VI Secretion System of Pandemic Vibrio cholerae. PLoS Negl Trop Dis. 2015;9(8):e0004031.
    OpenUrlCrossRefPubMed
  23. 23.↵
    Watve SS, Thomas J, Hammer BK. CytR Is a Global Positive Regulator of Competence, Type VI Secretion, and Chitinases in Vibrio cholerae. PLoS One. 2015;10(9):e0138834.
    OpenUrlCrossRefPubMed
  24. 24.↵
    Borgeaud S, Metzger LC, Scrignari T, Blokesch M. The type VI secretion system of Vibrio cholerae fosters horizontal gene transfer. Science. 2015;347(6217):63–7.
    OpenUrlAbstract/FREE Full Text
  25. 25.
    Lo Scrudato M, Blokesch M. A transcriptional regulator linking quorum sensing and chitin induction to render Vibrio cholerae naturally transformable. Nucleic Acids Res. 2013;41(6):3644–58.
    OpenUrlCrossRefPubMedWeb of Science
  26. 26.
    Meibom KL, Blokesch M, Dolganov NA, Wu CY, Schoolnik GK. Chitin induces natural competence in Vibrio cholerae. Science. 2005;310(5755):1824–7.
    OpenUrlAbstract/FREE Full Text
  27. 27.↵
    Metzger LC, Stutzmann S, Scrignari T, Van der Henst C, Matthey N, Blokesch M. Independent Regulation of Type VI Secretion in Vibrio cholerae by TfoX and TfoY. Cell Rep. 2016;15(5):951–8.
    OpenUrlCrossRefPubMed
  28. 28.↵
    Townsley L, Sison Mangus MP, Mehic S, Yildiz FH. Response of Vibrio cholerae to Low-Temperature Shifts: CspV Regulation of Type VI Secretion, Biofilm Formation, and Association with Zooplankton. Appl Environ Microbiol. 2016;82(14):4441–52.
    OpenUrlAbstract/FREE Full Text
  29. 29.↵
    Datta PP, Bhadra RK. Cold shock response and major cold shock proteins of Vibrio cholerae. Appl Environ Microbiol. 2003;69(11):6361–9.
    OpenUrlAbstract/FREE Full Text
  30. 30.↵
    Teschler JK, Cheng AT, Yildiz FH. The Two-Component Signal Transduction System VxrAB Positively Regulates Vibrio cholerae Biofilm Formation. J Bacteriol. 2017;199(18).
  31. 31.↵
    . !!! INVALID CITATION !!! (73, 74).
  32. 32.↵
    Kamal F, Liang X, Manera K, Pei TT, Kim H, Lam LG, et al. Differential Cellular Response to Translocated Toxic Effectors and Physical Penetration by the Type VI Secretion System. Cell Rep. 2020;31(11):107766.
    OpenUrlCrossRef
  33. 33.↵
    Krell T, Lacal J, Busch A, Silva-Jimenez H, Guazzaroni ME, Ramos JL. Bacterial sensor kinases: diversity in the recognition of environmental signals. Annu Rev Microbiol. 2010;64:539–59.
    OpenUrlCrossRefPubMedWeb of Science
  34. 34.↵
    Cheng AT, Ottemann KM, Yildiz FH. Vibrio cholerae Response Regulator VxrB Controls Colonization and Regulates the Type VI Secretion System. PLoS Pathog. 2015;11(5):e1004933.
    OpenUrlCrossRefPubMed
  35. 35.↵
    Boparai JK, Sharma PK. Mini Review on Antimicrobial Peptides, Sources, Mechanism and Recent Applications. Protein Pept Lett. 2020;27(1):4–16.
    OpenUrl
  36. 36.↵
    Bevins CL, Salzman NH. Paneth cells, antimicrobial peptides and maintenance of intestinal homeostasis. Nat Rev Microbiol. 2011;9(5):356–68.
    OpenUrlCrossRefPubMed
  37. 37.↵
    Le CF, Fang CM, Sekaran SD. Intracellular Targeting Mechanisms by Antimicrobial Peptides. Antimicrob Agents Chemother. 2017;61(4).
  38. 38.↵
    Matson JS, Yoo HJ, Hakansson K, Dirita VJ. Polymyxin B resistance in El Tor Vibrio cholerae requires lipid acylation catalyzed by MsbB. J Bacteriol. 2010;192(8):2044–52.
    OpenUrlAbstract/FREE Full Text
  39. 39.↵
    Trimble MJ, Mlynarcik P, Kolar M, Hancock RE. Polymyxin: Alternative Mechanisms of Action and Resistance. Cold Spring Harb Perspect Med. 2016;6(10).
  40. 40.↵
    Couet W, Gregoire N, Gobin P, Saulnier PJ, Frasca D, Marchand S, et al. Pharmacokinetics of colistin and colistimethate sodium after a single 80-mg intravenous dose of CMS in young healthy volunteers. Clin Pharmacol Ther. 2011;89(6):875–9.
    OpenUrlCrossRefPubMed
  41. 41.↵
    Serwecinska L. Antimicrobials and Antibiotic-Resistant Bacteria: A Risk to the Environment and to Public Health. Water. 2020;12(12):3313.
    OpenUrl
  42. 42.↵
    Davis CA, Janssen EM. Environmental fate processes of antimicrobial peptides daptomycin, bacitracins, and polymyxins. Environ Int. 2020;134:105271.
    OpenUrl
  43. 43.↵
    Duperthuy M. Antimicrobial Peptides: Virulence and Resistance Modulation in Gram-Negative Bacteria. Microorganisms. 2020;8(2).
  44. 44.↵
    Linares JF, Gustafsson I, Baquero F, Martinez JL. Antibiotics as intermicrobial signaling agents instead of weapons. Proc Natl Acad Sci U S A. 2006;103(51):19484–9.
    OpenUrlAbstract/FREE Full Text
  45. 45.↵
    Duperthuy M, Sjöström AE, Sabharwal D, Damghani F, Uhlin BE, Wai SN. Role of the Vibrio cholerae matrix protein Bap1 in cross-resistance to antimicrobial peptides. PLoS Pathog. 2013;9(10):e1003620.
    OpenUrlCrossRefPubMed
  46. 46.↵
    Mathieu-Denoncourt A, Duperthuy M. Secretome analysis reveals a role of subinhibitory concentrations of polymyxin B in the survival of Vibrio cholerae mediated by the type VI secretion system. Environ Microbiol. 2022;24(3):1133–49.
    OpenUrl
  47. 47.↵
    Giacomucci S, Cros CD, Perron X, Mathieu-Denoncourt A, Duperthuy M. Flagella-dependent inhibition of biofilm formation by sub-inhibitory concentration of polymyxin B in Vibrio cholerae. PLoS One. 2019;14(8):e0221431.
    OpenUrlCrossRefPubMed
  48. 48.↵
    Giacomucci S, Mathieu-Denoncourt A, Vincent AT, Jannadi H, Duperthuy M. Experimental evolution of Vibrio cholerae identifies hypervesiculation as a way to increase motility in the presence of polymyxin B. Front Microbiol. 2022.
  49. 49.↵
    Allue-Guardia A, Echazarreta M, Koenig SSK, Klose KE, Eppinger M. Closed Genome Sequence of Vibrio cholerae O1 El Tor Inaba Strain A1552. Genome Announc. 2018;6(9).
  50. 50.↵
    Marvig RL, Blokesch M. Natural transformation of Vibrio cholerae as a tool--optimizing the procedure. BMC Microbiol. 2010;10:155.
    OpenUrlCrossRefPubMed
  51. 51.↵
    Thomas PD, Campbell MJ, Kejariwal A, Mi H, Karlak B, Daverman R, et al. PANTHER: a library of protein families and subfamilies indexed by function. Genome Res. 2003;13(9):2129–41.
    OpenUrlAbstract/FREE Full Text
  52. 52.↵
    Reisch CR, Prather KL. The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli. Sci Rep. 2015;5:15096.
    OpenUrlCrossRefPubMed
  53. 53.↵
    Chang AY, Chau VW, Landas JA, Pang Y. Preparation of calcium competent Escherichia coli and heat-shock transformation UJEMI. 2017;1:22–5.
    OpenUrl
  54. 54.↵
    Conner JG, Teschler JK, Jones CJ, Yildiz FH. Staying Alive: Vibrio cholerae’s Cycle of Environmental Survival, Transmission, and Dissemination. Microbiol Spectr. 2016;4(2).
  55. 55.↵
    Bina JE, Provenzano D, Wang C, Bina XR, Mekalanos JJ. Characterization of the Vibrio cholerae vexAB and vexCD efflux systems. Arch Microbiol. 2006;186(3):171–81.
    OpenUrlCrossRefPubMedWeb of Science
  56. 56.↵
    Henderson JC, Herrera CM, Trent MS. AlmG, responsible for polymyxin resistance in pandemic Vibrio cholerae, is a glycyltransferase distantly related to lipid A late acyltransferases. J Biol Chem. 2017;292(51):21205–15.
    OpenUrlAbstract/FREE Full Text
  57. 57.↵
    Herrera CM, Crofts AA, Henderson JC, Pingali SC, Davies BW, Trent MS. The Vibrio cholerae VprA-VprB two-component system controls virulence through endotoxin modification. mBio. 2014;5(6).
  58. 58.↵
    Mathur J, Waldor MK. The Vibrio cholerae ToxR-regulated porin OmpU confers resistance to antimicrobial peptides. Infect Immun. 2004;72(6):3577–83.
    OpenUrlAbstract/FREE Full Text
  59. 59.↵
    Lin HV, Massam-Wu T, Lin CP, Wang YA, Shen YC, Lu WJ, et al. The Vibrio cholerae var regulon encodes a metallo-beta-lactamase and an antibiotic efflux pump, which are regulated by VarR, a LysR-type transcription factor. PLoS One. 2017;12(9):e0184255.
    OpenUrl
  60. 60.↵
    Kunkle DE, Bina TF, Bina XR, Bina JE. Vibrio cholerae OmpR Represses the ToxR Regulon in Response to Membrane Intercalating Agents That Are Prevalent in the Human Gastrointestinal Tract. Infect Immun. 2020;88(3).
  61. 61.↵
    Wang G, Fan C, Wang H, Jia C, Li X, Yang J, et al. Type VI secretion system-associated FHA domain protein TagH regulates the hemolytic activity and virulence of Vibrio cholerae. Gut Microbes. 2022;14(1):2055440.
    OpenUrl
  62. 62.↵
    Nazarov S, Schneider JP, Brackmann M, Goldie KN, Stahlberg H, Basler M. Cryo-EM reconstruction of Type VI secretion system baseplate and sheath distal end. EMBO J. 2018;37(4).
  63. 63.↵
    Kapitein N, Bonemann G, Pietrosiuk A, Seyffer F, Hausser I, Locker JK, et al. ClpV recycles VipA/VipB tubules and prevents non-productive tubule formation to ensure efficient type VI protein secretion. Mol Microbiol. 2013;87(5):1013–28.
    OpenUrlCrossRefPubMed
  64. 64.↵
    Kube S, Kapitein N, Zimniak T, Herzog F, Mogk A, Wendler P. Structure of the VipA/B type VI secretion complex suggests a contraction-state-specific recycling mechanism. Cell Rep. 2014;8(1):20–30.
    OpenUrlCrossRefPubMed
  65. 65.↵
    Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995;177(14):4121–30.
    OpenUrlAbstract/FREE Full Text
  66. 66.↵
    Dorr T, Alvarez L, Delgado F, Davis BM, Cava F, Waldor MK. A cell wall damage response mediated by a sensor kinase/response regulator pair enables beta-lactam tolerance. Proc Natl Acad Sci U S A. 2016;113(2):404–9.
    OpenUrlAbstract/FREE Full Text
  67. 67.↵
    Sikora AE, Zielke RA, Lawrence DA, Andrews PC, Sandkvist M. Proteomic analysis of the Vibrio cholerae type II secretome reveals new proteins, including three related serine proteases. J Biol Chem. 2011;286(19):16555–66.
    OpenUrlAbstract/FREE Full Text
  68. 68.↵
    Altindis E, Fu Y, Mekalanos JJ. Proteomic analysis of Vibrio cholerae outer membrane vesicles. Proc Natl Acad Sci U S A. 2014;111(15):e1548–56.
    OpenUrl
  69. 69.↵
    Broms JE, Ishikawa T, Wai SN, Sjostedt A. A functional VipA-VipB interaction is required for the type VI secretion system activity of Vibrio cholerae O1 strain A1552. BMC Microbiol. 2013;13:96.
    OpenUrlCrossRefPubMed
  70. 70.↵
    Kudryashev M, Wang RY, Brackmann M, Scherer S, Maier T, Baker D, et al. Structure of the type VI secretion system contractile sheath. Cell. 2015;160(5):952–62.
    OpenUrlCrossRefPubMed
  71. 71.↵
    Bonemann G, Pietrosiuk A, Diemand A, Zentgraf H, Mogk A. Remodelling of VipA/VipB tubules by ClpV-mediated threading is crucial for type VI protein secretion. EMBO J. 2009;28(4):315–25.
    OpenUrlAbstract/FREE Full Text
  72. 72.↵
    Pietrosiuk A, Lenherr ED, Falk S, Bonemann G, Kopp J, Zentgraf H, et al. Molecular basis for the unique role of the AAA+ chaperone ClpV in type VI protein secretion. J Biol Chem. 2011;286(34):30010–21.
    OpenUrlAbstract/FREE Full Text
  73. 73.↵
    Miyata ST, Unterweger D, Rudko SP, Pukatzki S. Dual expression profile of type VI secretion system immunity genes protects pandemic Vibrio cholerae. PLoS Pathog. 2013;9(12):e1003752.
    OpenUrlCrossRefPubMed
  74. 74.↵
    Durand E, Nguyen VS, Zoued A, Logger L, Pehau-Arnaudet G, Aschtgen MS, et al. Biogenesis and structure of a type VI secretion membrane core complex. Nature. 2015;523(7562):555–60.
    OpenUrlCrossRefPubMed
  75. 75.↵
    Stietz MS, Liang X, Li H, Zhang X, Dong TG. TssA-TssM-TagA interaction modulates type VI secretion system sheath-tube assembly in Vibrio cholerae. Nat Commun. 2020;11(1):5065.
    OpenUrl
  76. 76.↵
    Zheng J, Shin OS, Cameron DE, Mekalanos JJ. Quorum sensing and a global regulator TsrA control expression of type VI secretion and virulence in Vibrio cholerae. Proc Natl Acad Sci U S A. 2010;107(49):21128–33.
    OpenUrlAbstract/FREE Full Text
  77. 77.↵
    Peschek N, Herzog R, Singh PK, Sprenger M, Meyer F, Frohlich KS, et al. RNA-mediated control of cell shape modulates antibiotic resistance in Vibrio cholerae. Nat Commun. 2020;11(1):6067.
    OpenUrl
  78. 78.
    Liu J, Chang W, Pan L, Liu X, Su L, Zhang W, et al. An Improved Method of Preparing High Efficiency Transformation Escherichia coli with Both Plasmids and Larger DNA Fragments. Indian J Microbiol. 2018;58(4):448–56.
    OpenUrl
  79. 79.
    Rogers A, Townsley L, Gallego-Hernandez AL, Beyhan S, Kwuan L, Yildiz FH. The LonA Protease Regulates Biofilm Formation, Motility, Virulence, and the Type VI Secretion System in Vibrio cholerae. J Bacteriol. 2016;198(6):973–85.
    OpenUrlAbstract/FREE Full Text
  80. 80.
    Joshi A, Mahmoud SA, Kim SK, Ogdahl JL, Lee VT, Chien P, et al. c-di-GMP inhibits LonA-dependent proteolysis of TfoY in Vibrio cholerae. PLoS Genet. 2020;16(6):e1008897.
    OpenUrlCrossRefPubMed
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Comprehension of Antimicrobial Peptides Modulation of the Type VI Secretion System in Vibrio cholerae
Annabelle Mathieu-Denoncourt, Marylise Duperthuy
bioRxiv 2022.11.18.517169; doi: https://doi.org/10.1101/2022.11.18.517169
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