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Inquiry Article

Constitutive Type Half dozen Secretion System Expression Gives Vibrio cholerae Intra- and Interspecific Competitive Advantages

• Daniel Unterweger,
• Maya Kitaoka,
• Sarah T. Miyata,
• Verena Bachmann,
• Teresa M. Brooks,
• Jessica Moloney,
• Oscar Sosa,
• David Silva,
• Jorge Duran-Gonzalez,
• Daniele Provenzano

x

• Published: October 26, 2012
• https://doi.org/10.1371/journal.pone.0048320

Figures

Abstruse

The type Half-dozen secretion system (T6SS) mediates protein translocation beyond the cell membrane of Gram-negative bacteria, including Vibrio cholerae – the causative amanuensis of cholera. All V. cholerae strains examined to date harbor cistron clusters encoding a T6SS. Structural similarity and sequence homology betwixt components of the T6SS and the T4 bacteriophage cell-puncturing device suggest that the T6SS functions as a contractile molecular syringe to inject effector molecules into prokaryotic and eukaryotic target cells. Regulation of the T6SS is critical. A subset of V. cholerae strains, including the clinical O37 serogroup strain V52, limited T6SS constitutively. In contrast, pandemic strains impose tight command that tin can be genetically disrupted: mutations in the quorum sensing cistron luxO and the newly described regulator factor tsrA pb to constitutive T6SS expression in the El Tor strain C6706. In this report, we examined environmental V. cholerae isolates from the Rio Grande with regard to T6SS regulation. Rough V. cholerae lacking O-antigen carried a nonsense mutation in the gene encoding the global T6SS regulator VasH and did not brandish virulent behavior towards Escherichia coli and other environmental bacteria. In contrast, smooth V. cholerae strains engaged constitutively in type Half-dozen-mediated secretion and displayed virulence towards prokaryotes (E. coli and other environmental bacteria) and a eukaryote (the social amoeba Dictyostelium discoideum). Furthermore, smooth V. cholerae strains were able to outcompete each other in a T6SS-dependent way. The work presented here suggests that constitutive T6SS expression provides V. cholerae with an advantage in intraspecific and interspecific competition.

Commendation: Unterweger D, Kitaoka M, Miyata ST, Bachmann V, Brooks TM, Moloney J, et al. (2012) Constitutive Type VI Secretion Organization Expression Gives Vibrio cholerae Intra- and Interspecific Competitive Advantages. PLoS 1 7(x): e48320. https://doi.org/10.1371/journal.pone.0048320

Editor: Thierry Soldati, Université de Genève, Switzerland

Received: July 19, 2012; Accepted: September 24, 2012; Published: Oct 26, 2012

Copyright: © 2012 Unterweger et al. This is an open-admission commodity distributed under the terms of the Artistic Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original writer and source are credited.

Funding: Work in S.P.'south laboratory is supported by the Canadian Institute for Health Research Operating Grant MOP-84473 and Alberta Innovates-Wellness Solutions (funded by the Alberta Heritage Foundation for Medical Research Endowment Fund). D.P. was supported by National Institutes of Wellness grants MD001091-01 and GM068855-02 and an Olegario Rana fellowship. D.U. and South.T.Grand. were supported by Graduate Studentships from Alberta Innovates-Health Solutions. The funders had no role in study pattern, data collection and assay, conclusion to publish, or training of the manuscript.

Competing interests: The authors take declared that no competing interests exist.

Introduction

The Gram-negative bacterium Vibrio cholerae is the causative agent of the astute diarrheal illness cholera and remains a serious wellness risk to humans. In addition to the two master virulence factors needed to cause massive watery diarrhea–cholera toxin [1] and the toxin coregulated hair [ii] –the bacterium utilizes accessory virulence factors as well capable of causing diarrheal disease. Accessory toxins such as hemolysin (HlyA) and actin-cantankerous-linking repeats-in-toxin (RtxA) have been reported to exist virulence mechanisms exploited by some strains [3].

Another such accessory virulence factor is the type Half-dozen secretion organisation (T6SS), which confers cytotoxic furnishings against both prokaryotic and eukaryotic cells [4]–[6]. Bacteria accept developed numerous mechanisms to consign proteins, including toxins, across their cell walls into the surrounding environment or into host cells. To date, 6 distinctive pathways, collectively called secretion systems and classified into blazon I to type Half-dozen (T1SS – T6SS), have been identified in Gram-negative bacteria [7]. The T6SS of Five. cholerae mediates cytotoxicity towards eukaryotic hosts, including murine macrophages [5], [8], [nine] and the amoeba Dictyostelium discoideum [4]. The 5. cholerae T6SS is encoded by three cistron clusters on two carve up chromosomes: ane big cluster (VCA0107 – VCA0124) [10] and ii small-scale auxiliary clusters (VCA0017 – VCA0021 and VC1415 – VC1421). Bioinformatic analyses and a series of experimental approaches have elucidated the functions of several genes belonging to the V. cholerae T6SS clusters. For example, the Hcp protein [11], secreted past bacteria with a functional T6SS, forms a nanotube structure with an internal diameter of 4 nm [12]. Three VgrG proteins were shown to interact with each other to form a trimeric complex that structurally resembles a T4-bacteriophage gp5-gp27 tail spike complex [9], just dissimilar their phage counterparts lack an internal channel [13]. The current working model of the T6SS is based on these observations and the finding that Hcp and VgrG are codependent for secretion. The model proposes that the Hcp nanotube, decorated with a VgrG trimer at its elevation, is pushed through the bacterial envelope of the predator jail cell and into the prokaryotic or eukaryotic target prison cell. It is suggested that cytoplasmic VipA and VipB (VCA0107 and VCA0108) form a contractile sheath around the Hcp tube similar to the T4 phage outer sheath; contraction of the VipAB sheath ejects the Hcp tube from the predator prison cell [14]. The VgrG cap might mediate toxicity via the C-last extensions of evolved VgrGs upon delivery into the target prison cell [5]. Alternatively, the cap might dissociate from the Hcp nanotube to allow delivery of soluble toxin(s) or effector molecule(s) through the Hcp conduit [13]. VasH (VCA0117) acts as a sigma-54 activator poly peptide and controls transcription of T6SS genes including hcp and vgrG. We recently reported that the Five. cholerae T6SS also exerts contact-dependent killing properties against other Gram-negative bacteria such every bit Escherichia coli [6]. This finding suggests that 5. cholerae may utilize the T6SS to compete with commensal bacteria in the human intestine and/or environmental reservoirs.

The environmental reservoirs of Five. cholerae (river deltas with stagnant waters, oceans, and deep seas [15]) are as various equally the genomic content of this bacterium. The V. cholerae pangenome is estimated to consist of ∼half-dozen,500 genes [16]. Because all V. cholerae genomes sequenced so far contain the three gene clusters encoding the T6SS, we conclude that the T6SS belongs to the ane,500-gene core genome. Although the T6SS appears to be conserved in 5. cholerae, the system is regulated differently between strains. While the O37 serotype V52 strain expresses T6SS genes constitutively, the O1 El Tor strain C6706 represses its T6SS under laboratory weather condition. Mutations in the genes encoding the transcriptional regulator TsrA (VC0070) and the quorum sensing system regulator LuxO (VC1021) are required for T6SS expression in C6706 under laboratory conditions [17]. Another El Tor strain, A1552, activates its T6SS when grown under loftier osmolarity conditions and/or low temperature [xviii]. A transcriptional regulator encoded within the T6SS gene–cluster is VasH (VCA0117). As a sigma-54 activator protein, VasH controls the expression of T6SS genes including hcp and vgrG [xix], [20]. These differences in T6SS regulation led the states to investigate whether V. cholerae strains employ constitutive or restricted T6SS regulation in defined ecology reservoirs. We focused on the Rio Grande, a river that empties into the Gulf of United mexican states and is considered to be a major reservoir of unique, nonpandemic O1 El Tor strains responsible for sporadic food-borne cholera in the summer [21]. Ecology V. cholerae isolates (RGVCs) collected at two locations along the Rio Grande were examined to test whether constitutive T6SS expression is prevalent in V. cholerae exposed to microbial competitors and predators.

Materials and Methods

Strains and Culture Conditions

A streptomycin-resistant Five. cholerae strain V52 (O37 serogroup) defective hapA, rtxA, and hlyA genes [4] was used as a T6SS-positive strain in all experiments presented in this study. DH5αλpir and SM10λpir were used for cloning, and mating of pWM91-based plasmids, respectively. The strains and plasmids used in this study are listed in Table 1. Unless stated otherwise, bacteria were grown in a Luria-Bertani (LB) broth at 37°C with shaking (200 rpm). Rifampicin-resistant (50 µg·mL⁻¹) Vibrio communis, Vibrio harveyi, and Pseudoalteromonas phenolica were grown in ½ YTSS broth (2.5 g·L⁻ⁱ tryptone, 4 g·L^−ane yeast extract, 20 g·L⁻¹ sea salts (Sigma)) at thirty°C. Antibiotic concentrations used to maintain the plasmids were 100 µg·mL^−ane ampicillin or 50 µg·mL⁻ⁱ kanamycin. D. discoideum AX3 cells were obtained from the Dicty Stock Center and maintained in liquid culture (HL5) with shaking (150 rpm) at 22°C [22]. Environmental bacteria were collected past submerging a Turtox tow cyberspace (Envco, New Zealand) with a 20 µm pore-size Nitex mesh spanning a 30.48 cm bore mouth in estuary water for one infinitesimal. H2o samples (200 mL) collected from estuaries of the Rio Grande delta were composite with a handheld homogenizer (PRO Scientific; Oxford, CT), and vacuum filtered through Whatman filter paper number iii (GE Healthcare, Lilliputian Chalfont, Britain). A second vacuum filtration was performed on the filtrate through 0.45 µM pore-size membranes (Millipore, Bedford, MA). Filters were incubated separately in a pocket-size volume of 0.15 K sterile NaCl for ane hour shaking at RT. The suspensions were plated on thiosulfate-citrate-bile salts-sucrose (TCBS) agar (BD, Franklin Lakes, NJ) and/or marine agar 2216 (BD, Franklin Lakes, NJ). Post-obit incubation for sixteen hours at 30°C, colony forming units (CFUs) were isolated and cultured in LB goop. A polymorphic 22-kb region was sequenced for both isolates, DL2111 and DL2112, for strain identification. Sequences were submitted to GenBank (accession number JX669612 and JX669613).

Dna Sequence Analysis and Protein Structure Prediction Analysis

Nucleotide sequence analyses and alignments were performed with MacVector software (version 11.0.2).

Figure ane. Ability of RGVC isolates to kill E. coli. Crude RGVC isolates DL2111 and DL2112, and smooth RGVC isolates DL4211 and DL4215 were tested for their ability to confer T6SS-mediated prokaryotic killing.

V52 and V52ΔvasK were used every bit virulent and avirulent controls, respectively. V. cholerae and E. coli were mixed in a 10∶1 ratio and incubated for 4 hours at 37°C. Bacterial spots were resuspended, serially diluted, and plated on East. coli-selective media to determine the number of surviving East. coli. The averages and standard deviations of two independent experiments, each performed in duplicates are shown.

https://doi.org/10.1371/journal.pone.0048320.g001

16S Ribosomal Sequencing

Primers binding to conserved 16S ribosomal cistron sequences were used to PCR-amplify the 16S ribosomal sequences from environmental bacterial isolates. Primer sequences are summarized in Tabular array 2. DNA sequencing was performed at the Academy of Alberta Practical Genomics Centre and species were identified using BLASTn.

Figure 2. RGVC isolates with a constitutive T6SS kill D. discoideum.

ten³ D. discoideum cells were plated with indicated bacteria on SM/v agar plates that support bacterial but not amoeboid growth. Plaques formed past D. discoideum were counted on the third day of incubation. The graph summarizes the results of ii contained experiments. Standard deviations are shown. KP: Klebsiella pneumoniae.

https://doi.org/10.1371/periodical.pone.0048320.g002

Protein Secretion Profiles

Overnight cultures of bacterial strains were diluted to 1∶100 in 3 mL of fresh LB containing appropriate antibiotics and incubated until they reached belatedly mid-logarithmic growth phase (OD₆₀₀ ∼0.half-dozen). 50-arabinose (0.1%) was added to induce expression of the P_BAD promoter in pBAD24 and pBAD18. Bacteria were pelleted at loftier speed in a tabletop microcentrifuge for 5 minutes. Supernatants were filtered through 0.22 µm depression poly peptide-binding polyvinylidine fluoride (PVDF) syringe filters (Millipore). Proteins were precipitated with 20% trichloroacetic acid (TCA) for 15 minutes on ice, pelleted past centrifugation at 14,000× g for 5 minutes at four°C, and washed twice with ice-common cold acetone to remove residual TCA. Protein pellets were resuspended in 40 µL SDS-Folio lysis buffer (40% glycerol; 0.24 M Tris-HCl, pH six.8; 8% SDS; 0.04% bromophenol blue; 5% β-mercaptoethanol) and boiled for 10 minutes. 300 µL of bacterial culture was centrifuged at 14,000× g for 5 minutes. Bacterial pellets were resuspended in an equal volume of lysis buffer and boiled for 10 minutes. Samples were subjected to SDS-Folio (10% acrylamide) and analyzed by western blotting using a rabbit polyclonal antibody against DnaK (Stressgen, diluted 1∶15,000), mouse anti-RNAP (Neoclone, diluted1∶thousand), mouse anti-beta-lactamase (Sigma, diluted i∶200), and polyclonal rabbit anti-Hcp [5] antiserum (diluted 1∶500). Secondary antibodies used were caprine animal anti-mouse horseradish peroxidase (HRP) and caprine animal anti-rabbit HRP (both Santa Cruz, diluted i∶3000).

Figure iii. RGVC isolates differ in T6SS regulation.

Indicated RGVC isolates and V52 (positive control) were cultured to midlogarithmic stage of growth followed by centrifugal separation of pellets and culture supernatants. Supernatant portions were concentrated by TCA precipitation and both fractions were subjected to SDS-Page followed past western blotting using the antibodies indicated. Experiments were repeated at least three times with equivalent results.

https://doi.org/10.1371/journal.pone.0048320.g003

D. discoideum Plaque Assays

100 µL of overnight bacterial civilization and 10³ D. discoideum AX3 cells were spread on SM/5 plates [22]. Arabinose (0.one%) was added to SM/v plates when indicated. Plates were incubated at 22°C for 3 days to assess the number of plaques.

Effigy iv. Complementation of a vasK aught-mutation restores T6SS-dependent secretion and virulence.

(A) VasK-mutants of shine RGVC isolates conveying a plasmid for arabinose-induced vasK expression were cultured to midlogarithmic phase of growth in the presence or absenteeism of 0.i% arabinose. V52 and the isogenic vasK mutant were used as positive and negative controls, respectively. Pellets and civilization supernatants were separated by centrifugation. The supernatant portions were concentrated past TCA precipitation and both fractions were subjected to SDS-PAGE followed by western blotting using the antibodies indicated. (B) Survival of E. coli MG1655 after mixing with V. cholerae. Five. cholerae and Eastward. coli were mixed in a ten∶1 ratio and incubated for 4 hours at 37°C before the resulting spots were resuspended, serially diluted, and plated on E. coli-selective media. Data represent the averages of three contained experiments. Standard deviations are included. (C) Survival of D. discoideum after mixing with V. cholerae. D. discoideum was plated with V. cholerae and the number of plaques formed by surviving D. discoideum were counted after a 3-24-hour interval incubation at 22°C. Data are representative of three independent experiments. Standard deviations are shown.

https://doi.org/10.1371/journal.pone.0048320.g004

Bacterial Killing Analysis

Bacterial strains were grown as lawns on LB-agar plates with appropriate antibiotics. Environmental non-Five. cholerae strains were grown on 1/2 YTSS agar plates with appropriate antibiotics. Streptomycin-resistant (rifampicin-sensitive) predator and rifampicin-resistant (streptomycin-sensitive) prey were harvested and mixed at a 10∶ane ratio with volumes normalized by OD₆₀₀ readings. 25 µL of the mixed bacterial culture was spotted onto prewarmed LB-agar (or 1/2 YTSS agar plates for mixtures containing non-5. cholerae strains) and incubated at 37°C (or 30°C for non-V. cholerae strains) for iv h. Bacterial spots were harvested and the CFU·mL⁻¹ of surviving casualty and predator were measured past serial dilution and selective growth on agar containing 50 µg·mL⁻¹ rifampicin and 100 µg·mL⁻ⁱ streptomycin, respectively. Where applicable, arabinose was added to LB plates at a final concentration of 0.one% to induce expression from the P_BAD promoter during the 4 hour incubation.

Figure 5. Alignment of VasH polypeptide sequences of RGVC isolates.

VasH of V52, N16961, and four RGVC isolates were aligned. In the rough isolates, a guanine was inserted at position 157 of vasH to restore the open reading frame. Colored bars point substitutions compared to VasH from V52.

https://doi.org/ten.1371/journal.pone.0048320.g005

Dna manipulations

iii′-Myc-tagged vasH was PCR-amplified from V. cholerae V52 chromosomal DNA with primers 5′vasH and 3′vasH::myc (Table 1). The resulting PCR product was restricted with 5′-EcoRI and iii′-XbaI, cloned into pGEM T-like shooting fish in a barrel (Promega), and subcloned into pBAD18.

Figure vi. VasH complementation restores Hcp synthesis only not secretion in rough RGVC isolates.

V. cholerae isolates were transformed with pBAD18-vasH::myc. The isolates were cultured to midlogarithmic phase of growth in the presence or absence of 0.one% arabinose. Pellets and culture supernatants were separated by centrifugation. The supernatant portions were concentrated past TCA precipitation and both fractions were subjected to SDS-Page followed by western blotting using the antibodies indicated. Information are representative of three contained experiments.

https://doi.org/ten.1371/periodical.pone.0048320.g006

In-frame deletion of vasK was performed as described past Metcalf et al. [23] using the pWM91-based vasK knockout construct [9]. During sucrose selection, sucrose concentration was increased from six% to 20% for all RGVC gene deletions because these isolates exhibited increased tolerance to sucrose compared to V52.

Effigy 7. RGVC isolates kill bacterial neighbors.

V. cholerae and prey bacteria were mixed in a ten∶1 ratio and incubated on ½ YTSS agar for iv hours at 30°C. Bacterial spots were resuspended, serially diluted, and plated on selective YTSS agar to make up one's mind the number of surviving prey. The average and standard deviations of three independent experiments, each performed in duplicates, are shown.

https://doi.org/10.1371/journal.pone.0048320.g007

For complementation, vasK was amplified from V52 chromosomal Dna using primers 5′-vasK-pBAD24 and 3′-vasK-pBAD24 (Table 1). The resulting PCR product was purified using the Qiagen PCR cleanup kit, digested with EcoRI and XbaI, and cloned into pBAD24.

Figure 8. T6SS-dependent competition among V. cholerae isolates.

(A–C) Smooth V. cholerae isolates successfully competed with each other and outcompeted the rough isolates in a T6SS-dependent manner. All combinations amongst the isolates and their isogenic vasK mutants were tested in a killing assay: Predator- and prey-V. cholerae were mixed in a 10∶1 ratio and incubated for 4 hours at 37°C. Bacterial spots were resuspended, serially diluted, and plated on selective media to decide the number of surviving prey. The number of surviving prey in the presence of T6SS⁺ or T6SS⁻ predator are shown. (D) Arrows bespeak the competitive relationship between isolates such that the arrow points from the predator towards the prey. Arrow thickness indicates relative killing efficiency. T6SS-dependence of the killing phenotype was confirmed by employing the vasK-deficient predator of each V. cholerae isolate examined. To avoid killing of the predator, vasK-scarce prey of polish T6SS⁺ isolates were used. The average and standard deviations of two independent experiments, each performed in duplicates, are shown.

https://doi.org/10.1371/journal.pone.0048320.g008

Results

RGVC Isolates Showroom T6SS-Mediated Antimicrobial Properties

We previously demonstrated that clinical V. cholerae O37 serogroup strain V52 uses its T6SS to impale E. coli and Salmonella Typhimurium [6]. To determine the part of the T6SS in ecology strains, nosotros employed two dissimilar types of V. cholerae isolated from the Rio Grande: smoothen isolates with distinct O-antigens as office of their lipopolysaccharides (LPS), and rough isolates that lack O-antigen (Table 3). Due to concerns that rough bacteria are genetically unstable because the lack of O-antigen allows the uptake of chromosomal DNA [24], we assessed the virulence potential of ii separately isolated just genetically identical crude isolates DL2111 and DL2112 (as adamant by deep sequencing (Illumina platform) of a polymorphic 22-kb fragment [Genbank accretion numbers JX669612 and JX669613]) to minimize the chance of phenotypic variation due to genetic substitution.

To determine whether ecology RGVC V. cholerae are capable of killing bacteria, we performed an E. coli killing assay (Figure one). RGVC isolates and Eastward. coli strain MG1655 were spotted on LB nutrient agar plates, and the number of surviving MG1655 cells was adamant after a 4-hour incubation at 37°C. V52 and V52ΔvasK were used as virulent and avirulent controls, respectively. The presence of V52 resulted in an average ∼5-log reduction of viable Due east. coli. Smoothen isolates DL4211 and DL4215 killed East. coli at levels comparable to V52 (Figure i). In contrast, both crude isolates, DL2111 and DL2112, were unable to kill E. coli casualty. In summary, smoothen RGVC isolates readily killed E. coli while rough RGVC isolates appeared to exist attenuated.

RGVC Isolates Display T6SS-Mediated Virulence Towards D. discoideum

The clinical V. cholerae O37 serogroup strain V52 displays T6SS-dependent cytotoxicity towards the social amoeba D. discoideum [4]. We tested whether RGVC isolates were also capable of evading amoeboid grazing by killing the eukaryotic predator. RGVC isolates were plated together with amoebae on nutrient agar plates that exclusively support bacterial growth. For amoebae to survive on agar plates, they must obtain nutrients from phagocytosed leaner. This amoeboid grazing behavior on bacteria results in the formation of plaques–articulate zones in the bacterial lawn that are devoid of bacteria [25]. The T6SS mediates bacterial virulence towards D. discoideum and abrogates plaque germination. Wild-type V52 and Klebsiella pneumoniae were used equally virulent (no plaques) and avirulent (plaque formation) controls, respectively. Smooth isolates DL4211 and DL4215 killed D. discoideum at levels comparable to V52. In contrast, rough DL2111 and DL2112 did non kill D. discoideum similar to the T6SS-nil mutant V52ΔvasK and the avirulent Klebsiella pneumoniae negative control (Effigy 2).

Expression of Hcp in RGVC Isolates

Next, we ready out to test whether RGVC isolates were able to produce and secrete the T6SS hallmark protein Hcp because experimental results presented thus far suggested that V. cholerae'southward ability to impale bacterial competitors or eukaryotic predators [6] could be mediated by the T6SS. As shown in Figure 3, smoothen isolates DL4211 and DL4215 produced Hcp at sufficient levels to be detected by western blots probed with Hcp antiserum. In dissimilarity, rough isolates did not produce or secrete Hcp. The presence of Hcp correlated with virulence every bit the smooth isolates secreted Hcp (Effigy 3) and killed E. coli (Figure i) likewise every bit D. discoideum (Figure ii), while rough isolates did not produce Hcp and appeared to be attenuated.

RGVC Isolates Engage in T6SS-Mediated Secretion and Virulence

To determine whether killing of E. coli (Figure i) and D. discoideum (Figure two) depends on a functional T6SS, nosotros performed killing assays and plaque assays with DL4211ΔvasK and DL4215ΔvasK as a predator. VasK is an inner membrane protein believed to provide the energy for T6SS-mediated secretion [26], [27]. VasK is, therefore, crucial for a functional T6SS. Equally shown in figure 4A, parental V52, DL4211, and DL4215 constitutively produced and secreted Hcp, while deletion of vasK blocked secretion but non synthesis of Hcp. To complement the vasK chromosomal deletion, vasK from V52 was cloned downstream of an arabinose-inducible promoter in the plasmid pBAD24 and introduced into DL4211ΔvasK (DL4211ΔvasK/pvasK) and DL4215ΔvasK (DL4215ΔvasK/pvasK). Trans complementation of vasK restored Hcp secretion in V52 and the 2 smooth isolates (Figure 4A). To appraise the role of T6SS in killing Due east. coli, nosotros incubated East. coli with diverse V. cholerae isolates and determined the number of surviving East. coli after a four-hour incubation at 37°C (Figure 4B). VasK mutants of V52, DL4211, and DL4215 lost their ability to kill E. coli, just providing vasK in trans restored virulence. Furthermore, amoebae were unable to course plaques in lawns of V52, DL4211, and DL4215, just did so in lawns of V52ΔvasK, DL4211ΔvasK and DL4215ΔvasK (Figure 4C). Complemented isolates, V52ΔvasK/pvasK, DL4211ΔvasK/pvasK and DL4215ΔvasK/pvasK, regained virulence towards D. discoideum (Figure 4C). Although the wild-blazon phenotype of DL4211 could not be fully complemented past episomal expression of vasK, the complemented phenotype is statistically meaning (unpaired t-test, p = 0.0116). Nosotros conclude that smooth RGVC isolates conferred T6SS-mediated virulence towards E. coli and D. discoideum, demonstrating that the virulence phenotype described in Figures 1 and 2 is T6SS-dependent.

Rough RGVC Isolates Comport Unique vasH Sequences

Nosotros previously showed that the global transcriptional activator VasH is essential for expression of hcp and other T6SS genes. Every bit the rough isolates failed to synthesize Hcp (Effigy iii), we tested whether these isolates carried a nonfunctional vasH allele. The 1594 nucleotide-long vasH sequences of V52 and RGVC isolates were PCR-amplified and their polypeptide sequences aligned. The rough RGVC isolates were missing a guanine in codon 157 (ΔG157) which resulted in a frameshift. To include vasH of the rough isolates in our comparative analysis, we restored the vasH reading frame by in-silico insertion of G157. We constitute that all RGVC VasH sequences aligned with V52 and N16961 besides as with each other (Figure five). Therefore, vasH is conserved in environmental (RGVC), pandemic (N16961), and endemic (V52) V. cholerae strains. The repaired vasH open reading frame closely resembled vasH from N16961 with simply two unique substitutions (Q278L and T456I). Smooth RGVC isolate DL4211 carried an intact VasH gene identical to N16961; DL4215 differed from N16961 and V52 by three and four residues, respectively (Tabular array three). Substitutions of histidine to aspartic acid at position 116 (H116D) and threonine to alanine at position 449 (T449A) appear to be common substitutions that are also present in N16961 (Figure five). In determination, RGVC isolates conduct a VasH gene related to the El Tor version with the feature D116 and A449 residues (Figure 5). Even so, rough V. cholerae isolates carried a nonsense mutation and are likely to produce a truncated 63 amino acid-long VasH mutant protein.

VasH Complementation in Rough RGVC Isolates

We tested whether heterologous expression of vasH in the T6SS-silent RGVC isolates DL2111 and DL2112 restored T6SS-dependent protein synthesis/secretion. Myc-tagged vasH from V52 was cloned into pBAD18 to episomally express vasH. V52ΔvasH/pBAD18-vasH::myc was used as a control for the arabinose-dependent expression of vasH. As shown in Figure 6, episomal vasH::myc expression in V52ΔvasH induced Hcp production and subsequent secretion, while but synthesis just not secretion was restored in the rough RGVC isolates.

Smooth RGVC Isolates Use Their T6SS to Compete with Natural Neighbors

Because RGVC isolates with active T6SSs kill E. coli, nosotros hypothesized that RGVC isolates utilise their T6SS to compete with other bacteria in their environmental niche. To test this hypothesis, we isolated iii environmental bacterial non-5. cholerae strains from estuaries where the Rio Grande meets the Gulf of United mexican states. Sequencing of 16S-rRNA identified these bacterial species as Vibrio communis, Vibrio harveyi, and Pseudoalteromonas phenolica (data not shown). We then tested whether DL4211 and DL4215 were able to kill these ecology bacteria in a T6SS-dependent mode. As shown in Figure seven, both DL4211 and DL4215 killed all iii environmental isolates. The observed killing required a functional T6SS, every bit isogenic vasK mutants lost their ability to kill. Killing of the environmental bacteria was restored by complementing the vasK mutant backgrounds with episomal vasK in trans. Therefore, we propose that constitutive expression of T6SS genes provides smooth RGVC isolates with the ways to kill both their bacterial neighbors and potential eukaryotic predators.

Smooth RGVC Isolates Utilize Their T6SS for Intraspecific Competition

Five. cholerae O37 strain V52 kills E. coli and S. Typhimurium, but is unable to impale other V. cholerae, including the O1 serogroup N16961 (El Tor) and O395 (classical biotype) strains [half-dozen]. Accordingly, the T6SS⁺ isolates V52, DL4211 and DL4215 also exhibited immunity, because we did not observe a decline in viable CFUs when we recovered these isolates from unmarried-isolate spots on LB agar plates subsequently a iv-60 minutes incubation (data not shown). We hypothesized that V. cholerae employs an amnesty system that provides protection against T6SS-mediated toxicity. A functional link between T6SS and toxin/antidote systems has been established in Pseudomonas aeruginosa and Burkholderia species [28], [29], which use antitoxin proteins to annul T6SS effectors [28]. VCA0124, an open reading frame downstream of the T6SS effector factor vgrG3 (VCA0123), has been implicated as an antitoxin gene in V. cholerae [30]. As RGVCs killed close relatives such as 5. harveyi (Figure 7), we wondered if the RGVC isolates have the ability to kill each other. Nosotros hypothesized that if RGVC isolates apply different toxins (and antitoxins), the T6SS might be used for intraspecific competition. We predicted that immunity of an RGVC isolate would be lost when approached past a V. cholerae bacterium with a different prepare of T6SS toxins to which the former lacks the corresponding antitoxin cistron. To test this hypothesis, nosotros mixed V52, DL4211, and DL4215 (predators) with smoothen and rough RGVC isolates as prey bacteria. To eliminate the killing activity of smooth T6SS⁺ prey, nosotros used vasK-deficient mutants with a disabled T6SS equally prey. Rough wild-type RGVC isolates were used equally prey since they do not express Hcp (Figure iii) and are thus T6SS-negative. Following a 4-hour coincubation, we adamant the number of surviving prey. T6SS-negative casualty bacteria were not killed past their isogenic T6SS⁺ parent strain, only were killed by other T6SS⁺ isolates (Figure 8A–C). Exposure to a predator with a disabled T6SS resulted in nigh 10⁸ surviving prey bacteria. Similar numbers of surviving prey were obtained when the prey was mixed with an isogenic strain that was marked with a different antibiotic resistance cassette (data not shown). Thus, killing of T6SS-negative prey required a functional T6SS. Surprisingly, the vasK mutant of DL4215 displayed virulence towards V52ΔvasK, but non against DL4211ΔvasK or a differently-marked DL4215ΔvasK sister strain (Figure 8C). Since DL4215ΔvasK does not kill 5. communis, V. harveyi, or P. phenolica (Figure 7), we hypothesize that DL4215 exhibits some degree of selective T6SS-independent antimicrobial action against V52ΔvasK.

In conclusion, V. cholerae uses its T6SS not solely for contest with bacterial neighbors (Effigy 7), but also for competition within its own species (Effigy 8D).

Discussion

We examined environmental smooth and rough V. cholerae isolates (RGVCs) collected at two locations forth the Rio Grande to written report T6SS regulation in V. cholerae exposed to microbial competitors and predators.

Our study showed that smooth RGVC isolates use their T6SS to kill other Gram-negative bacteria isolated from the Rio Grande delta. Deletion of the T6SS factor vasK resulted in a loss of bacterial killing. Importantly, the killing phenotype was restored by vasK complementation in trans. The requirement of VasK for killing implies that a constitutively agile T6SS provides smoothen RGVC isolates with a competitive advantage compared to their bacterial neighbors. By killing other bacteria, RGVC isolates might enhance their own survival in their ecology niche. In addition, we institute that Five. cholerae isolates apply their T6SS to compete against each other.

In our experiments, Hcp synthesis and secretion correlated with eukaryotic and prokaryotic host jail cell killing (Table 4). For example, smooth Hcp-secreting RGVC isolates DL4211 and DL4215 (Figure 3) displayed total virulence towards E. coli (Figure ane) and D. discoideum (Figure 2). Rough RGVC isolates with their frameshift mutations in the T6SS transcriptional activator factor vasH did non produce or secrete Hcp, and their virulence was attenuated. Sequencing and gene alignments of the T6SS transcriptional activator vasH in rough strains indicated a missing guanine at position 157 in rough isolates, resulting in a frameshift mutation. Because VasH was recently implicated in regulating both the large and auxiliary T6SS cistron clusters in V. cholerae O395 [twenty], we speculated that the vasH frameshift mutation in the rough isolates silences T6SS expression. However, trans-complementation of the vasH mutation by episomal expression of V52′s vasH restored synthesis but not secretion of the T6SS hallmark poly peptide Hcp (Figure 6). Trans-complementation with vasH from N16961, which is more than closely related to vasH from RGVC isolates DL2111 and DL2112 (Figure 5), restores Hcp synthesis and secretion in a vasH mutant of V52, but only restores Hcp synthesis (and not secretion) in a vasH mutant of N16961 [19]. Thus, we believe that the inability to restore Hcp secretion in rough strains is not a reflection of the polymorphic nature of VasH.

At this time, it is unclear whether selective pressures for T6SS regulation exist that drive constitutive T6SS expression in shine isolates and disable T6SSs in rough Five. cholerae strains. V. cholerae LPS's O-antigen has been shown to induce protective immune responses in humans and experimental animals [31]–[35]. To counteract the host immune response, V. cholerae may utilize its T6SS to kill phagocytic immune cells such as macrophages [nine]. Because rough isolates lacking O-antigen are often isolated from convalescent cholera patients [36], repression of O-antigen biosynthesis may represent an immune evasion mechanism for V. cholerae [37]. Such evasion would allow the pathogen to persist in the host, perhaps in a subclinical state as rough V. cholerae have been shown to be avirulent. In this scenario, crude 5. cholerae does not require a functional T6SS, but tolerates mutations that disable its expression. Rough isolates have been shown to revert to a smooth, virulent state [37] but it remains to be adamant whether newly reverted smooth leaner restore expression of their disabled T6SSs. We did not find restoration of the T6SS in crude isolates through uptake and homologous recombination of chromosomal DNA from a T6SS⁺ donor, because crude isolates remained T6SS-negative in the presence of polish T6SS⁺ 5. cholerae strain V52 (data not shown).

El Tor strains possess a tightly controlled T6SS [17] and thus differ from the shine RGVCs that limited the T6SS constitutively. Every bit pandemic strains are believed to originate from environmental strains, we speculate that constitutive T6SS expression is prevalent in 5. cholerae exposed to microbial competitors and predators until virulence factors such as cholera toxin and toxin-coregulated pilus genes are caused. However, how pandemic V. cholerae regulate expression of T6SS during their circuitous life cycle remains to be adamant.

It is becoming increasingly clear from our investigation and other reports [6], [28]–[30], [38] that T6SS-expressing V. cholerae deploy bactericidal effector proteins. Therefore, T6SS expression is likely tied to a protective machinery, a form of T6SS-immunity that prevents the effector proteins from harming bacteria within a clonal population. Nosotros postulate that V52, DL4211, and DL4215 employ unique sets of toxin/antidote gene products and therefore form singled-out compatibility groups. Members of a T6SS compatibility group could coexist considering they encode antitoxins that match the cognate toxins. Conversely, members of dissimilar T6SS compatibility groups kill each other since the antitoxins of ane compatibility group exercise not protect against the toxins of the other group. Hence, T6SS-mediated selective interstrain killing allows V. cholerae to distinguish self from nonself. This class of kin pick may permit the evolution of distinct lineages, including those that give rise to toxigenic strains. The observations presented in this study indicate that the T6SS contributes to V. cholerae's pathogenesis and fitness past providing an advantage in interspecific competition with eukaryotes or prokaryotes, and intraspecific competition with V. cholerae strains.

Acknowledgments

The authors give thanks Tracy Raivio for helpful discussions, Marcia Craig for critically reviewing the manuscript, and Andrea Schwarzbach for her bioinformatics support. We acknowledge the Dicty Stock Center for providing the D. discoideum strain AX3 used in this report.

Author Contributions

Conceived and designed the experiments: DU MK STM DP SP. Performed the experiments: DU MK STM VB TB JM OS DS JDG. Analyzed the data: DU MK STM VB TB JM OS DS JDG DP SP. Contributed reagents/materials/assay tools: DP. Wrote the newspaper: DU MK DP SP.

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Source: https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0048320