13 research outputs found
Two Regulators of Vibrio parahaemolyticus Play Important Roles in Enterotoxicity by Controlling the Expression of Genes in the Vp-PAI Region
Vibrio parahaemolyticus is an important pathogen causing food-borne disease worldwide. An 80-kb pathogenicity island (Vp-PAI), which contains two tdh (thermostable direct hemolysin) genes and a set of genes for the type III secretion system (T3SS2), is closely related to the pathogenicity of this bacterium. However, the regulatory mechanisms of Vp-PAI's gene expression are poorly understood. Here we report that two novel ToxR-like transcriptional regulatory proteins (VtrA and VtrB) regulate the expression of the genes encoded within the Vp-PAI region, including those for TDH and T3SS2-related proteins. Expression of vtrB was under control of the VtrA, as vector-expressed vtrB was able to recover a functional protein secretory capacity for T3SS2, independent of VtrA. Moreover, these regulatory proteins were essential for T3SS2-dependent biological activities, such as in vitro cytotoxicity and in vivo enterotoxicity. Enterotoxic activities of vtrA and/or vtrB deletion strains derived from the wild-type strain were almost absent, showing fluid accumulation similar to non-infected control. Whole genome transcriptional profiling of vtrA or vtrB deletion strains revealed that the expression levels of over 60 genes were downregulated significantly in these deletion mutant strains and that such genes were almost exclusively located in the Vp-PAI region. These results strongly suggest that VtrA and VtrB are master regulators for virulence gene expression in the Vp-PAI and play critical roles in the pathogenicity of this bacterium
ナメクジウオmyc遺伝子の進化的解析
The proto-oncogene myc is one of the most important genes controlling cell proliferation. The vertebrate genome has four myc genes (c-, N-, L-, s-myc), whose evolutionary origin and relationship are unclear. Here, we isolated a myc gene from a protochordate, the amphioxus Branchiostoma belcheri, which is thought to be the nonvertebrate that is closest to the vertebrates. A 1480 bp cDNA sequence was determined and contains an ATGinitiated ORF consisting of 371 amino acids. The exon/intron structure was conserved. Southern blotting and degenerate PCR showed that the amphioxus genome contained only a single myc gene. A phylogenetic tree of Myc family genes based on the deduced amino acid sequences indicated that amphioxus Myc was located outside the vertebrate Myc family. These results suggest that myc gene duplication occurred after protochordate on phylogeny.がん原遺伝子myc は細胞増殖を制御する重要な遺伝子の一つである。脊椎動物では4種類のmycが存在しているが,その起源や類縁関係は明らかとなっていない。本研究で我々は,脊椎動物に最も近縁の無脊椎動物であるナメクジウオBranchiostoma belcheriからmyc遺伝子を単離した。全長1480塩基対のcDNA は371アミノ酸からなるORFを含んでいた。また,エキソン/イントロン構造は保存されていた。サザンブロット法およびdegenerate PCRの結果,ナメクジウオのゲノムは単一のmyc 遺伝子を持つことが明らかとなった。また,推定アミノ酸配列に基づいた系統解析の結果,ナメクジウオMyc は脊椎動物Mycファミリーの外側に位置していた。これらの結果から,myc ファミリーは原索動物以降に形成されたものと考えられた。東京海洋大学海洋科学部海洋生物資源学科東京海洋大学海洋科学部海洋生物資源学科コネチカット大学東京大学海洋研究所日本魚類生物科学研究所東京海洋大学海洋科学部海洋生物資源学
Microarray analysis of VtrA and VtrB regulon in <i>V. parahaemolyticus</i>.
a<p>Fold change in gene transcripts between the wild-type and <i>ΔvtrA</i> or <i>ΔvtrB</i> mutant as determined by microarray analysis. Statistically significant changes (≥2-fold difference with <i>P</i><0.05) are highlighted in bold as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008678#s4" target="_blank"><i>Materials and Methods</i></a>.</p
Whole-genome transcriptional profiling of <i>vtrA</i> and <i>vtrB</i> deletion strain.
<p>Genome-wide transcript analysis of the VtrA and VtrB regulons is shown. Gene expression was determined by comparing cDNA generated from WT<i>ΔvtrA</i> (A) or WT<i>ΔvtrB</i> (B) in exponential phase grown in LB medium with 0.5% NaCl with that from the WT strain. The Vp-PAI region is indicated by a bold line. Effect of the <i>vtrA</i> (C) or <i>vtrB</i> (D) deletion on expression of genes located within Vp-PAI (<i>vpa1309</i>-<i>vpa1396</i>). Representative gene functions are indicated at the top.</p
VtrB expression is under the control of VtrA.
<p>A. Neither <i>vtrA</i> nor <i>vtrB</i> was involved in the transcription of <i>vtrA</i>. <i>V. parahaemolyticus</i> strains carrying the <i>vtrA-lacZ</i> transcriptional fusion vector were assayed for β-galactosidase activity. The bars show the average of three separate experiments, and the standard deviations are indicated by error bars. B. Transcription of <i>vtrB</i> was decreased in <i>vtrA</i> deletion strains. <i>V. parahaemolyticus</i> strains carrying the <i>vtrB-lacZ</i> transcriptional fusion vector were assayed for β-galactosidase activity. The bars show the average of three separate experiments, and the standard deviations are indicated by error bars. C. Deletion of <i>vtrA</i> caused a decrease in the production of VtrB. Immunoblot analysis of VtrA and VtrB protein expression in <i>vtrA</i> and <i>vtrB</i> mutant strains are shown. Lane 1, wild-type <i>V. parahaemolyticus</i> (WT); lane 2, <i>vtrA</i> mutant strain (WT<i>ΔvtrA</i>); lane 3, <i>vtrB</i> mutant strain (WT<i>ΔvtrB</i>); lane 4, <i>vtrA</i> and <i>vtrB</i> double mutant strain (WT<i>ΔvtrAΔvtrB</i>). Blots were probed with anti-VtrA (upper panel) and anti-VtrB (lower panel) polyclonal antibodies. D. Effects of <i>vtrA</i> and <i>vtrB</i> expression on <i>vtrB</i> transcription in <i>E. coli</i>. <i>E. coli</i> MC4100 carrying <i>vtrB-lacZ</i> transcriptional fusion vector were assayed for β-galactosidase activity. The bars show the average of three separate experiments, and the standard deviations are indicated by error bars. E. Binding of purified VtrA DNA binding domain to the upstream region of <i>vtrB</i> is shown by an electrophoretic mobility shift assay. Each lane contains the same amount of upstream region of <i>vtrB</i> (30 nM) and various concentrations (0, 1.5, 2.25, 3.0, 4 µM) of VtrA DNA binding domain (upper panel) or VtrB DNA binding domain (lower panel). The molecular ratios are indicated in the top line.</p
VtrA and VtrB are not necessary for T3SS1-dependent cytotoxicity but necessary for T3SS2-dependent cytotoxicity.
<p>A. <i>vtrA</i> and <i>vtrB</i> are not necessary for T3SS1-dependent cytotoxicity. Caco-2 cells were infected for 6 h with isogenic strains of POR-3 (<i>ΔtdhASΔvcrD2</i>). Bar 1: POR-3 (<i>ΔtdhASΔvcrD2</i>); bar 2: POR-3<i>ΔvtrA</i>; bar 3: POR-3<i>ΔvtrB</i>; bar 4: <i>ΔvcrD1ΔvcrD2</i> (<i>ΔtdhASΔvcrD1ΔvcrD2</i>). Cytotoxicity was evaluated by the amount of LDH released. Error bars represent standard deviations for results from triplicate experiments. B. <i>vtrA</i> and <i>vtrB</i> are essential for T3SS2-dependent cytotoxicity. Caco-2 cells were infected for 6 h with isogenic mutant strains of POR-2 (<i>ΔtdhASΔvcrD1</i>). Bar 1: POR-2 (<i>ΔtdhASΔvcrD1</i>); bar 2: POR-2<i>ΔvtrA</i> (<i>ΔtdhASΔvcrD1ΔvtrA</i>); bar 3: POR-2<i>ΔvtrA</i> expressing <i>vtrA</i> (POR-2<i>ΔvtrA</i>+p<i>vtrA</i>); bar 4: POR-2<i>ΔvtrA</i> expressing <i>vtrB</i> (POR-2<i>ΔvtrA</i>+p<i>vtrB</i>); bar 5: POR-2<i>ΔvtrB</i> (<i>ΔtdhASΔvcrD1ΔvtrB</i>); bar 6; POR-2<i>ΔvtrB</i> expressing <i>vtrA</i> (POR-2<i>ΔvtrB</i>+p<i>vtrA</i>); bar 7: POR-2<i>ΔvtrB</i> expressing <i>vtrB</i> (POR-2<i>ΔvtrB</i>+p<i>vtrB</i>); bar 8: POR-2<i>ΔvtrAΔvtrB</i> (<i>ΔtdhASΔvcrD1ΔvtrAΔvtrB</i>); bar 9: POR-2<i>ΔvtrAΔvtrB</i> expressing <i>vtrA</i> (POR-2<i>ΔvtrAΔvtrB</i>+p<i>vtrA</i>); bar 10: POR-2<i>ΔvtrAΔvtrB</i> expressing <i>vtrB</i> (POR-2<i>ΔvtrAΔvtrB</i>+p<i>vtrB</i>); bar 11: <i>ΔvcrD1ΔvcrD2</i> (<i>ΔtdhASΔvcrD1ΔvcrD2</i>). Cytotoxicity was evaluated by the amount of LDH released. Error bars represent standard deviations for results from triplicate experiments. Asterisks indicate significant differences from the results obtained with the parent strain (*<i>P</i><0.05). C. Overexpressing of <i>vtrA</i> and <i>vtrB</i> promoted T3SS2-dependent cytotoxicity. Caco-2 cells were infected for 1.5–6 h with <i>V. parahaemolyticus</i>. Cytotoxicity was evaluated by the amount of LDH released. POR-2 (<i>ΔtdhASΔvcrD1</i>) with control vector (pSA19CP-MCS) (filled squares, solid line), POR-2 expressing <i>vtrA</i> (filled circles, solid line), POR-2 expressing <i>vtrB</i> (filled triangles, solid line), <i>ΔvcrD1ΔvcrD2</i> (<i>ΔtdhASΔvcrD1ΔvcrD2</i>) with control vector (pSA19CP-MCS) (open squares, dashed line), <i>ΔvcrD1ΔvcrD2</i> expressing <i>vtrA</i> (open circles, dashed line), and <i>ΔvcrD1ΔvcrD2</i> expressing <i>vtrB</i> (open triangles, dashed line). Error bars represent standard deviations for results from triplicate experiments. Asterisks indicate significant differences from the results obtained with the parent strain (*<i>P</i><0.05).</p
VPA1332 (VtrA) and VPA1348 (VtrB) have a winged-helix-turn-helix DNA-binding domain of OmpR family.
<p>Multiple sequence alignment and secondary structure assignments of DNA-binding and trans-activation domains of OmpR, PhoB, ToxR, VtrA, and VtrB proteins are shown. The amino acids that form the hydrophobic cores are highlighted with boxes. Highly conserved amino acids are highlighted with gray boxes.</p
VtrA and VtrB have a critical role in <i>V. parahaemolyticus</i>-induced enterotoxicity.
<p>A. VtrA and VtrB are essential for T3SS2-dependent enterotoxicity. The enterotoxic activity levels of isogenic mutants of POR-2 (<i>ΔtdhASΔvcrD1</i>) and complemented strains in rabbit ileal loops were examined. Bar 1, POR-2 (<i>ΔtdhASΔvcrD1</i>); bar 2, POR-2<i>ΔvtrA</i> (<i>ΔtdhASΔvcrD1ΔvtrA</i>); bar 3, POR-2<i>ΔvtrA</i> expressing <i>vtrA</i> (POR-2<i>ΔvtrA</i>+p<i>vtrA</i>); bar 4, POR-2<i>ΔvtrA</i> expressing <i>vtrB</i> (POR-2<i>ΔvtrA</i>+p<i>vtrB</i>); bar 5, POR-2<i>ΔvtrB</i> (<i>ΔtdhASΔvcrD1ΔvtrB</i>); bar 6, POR-2<i>ΔvtrB</i> expressing <i>vtrA</i> (POR-2<i>ΔvtrB</i>+p<i>vtrA</i>); bar 7, POR-2<i>ΔvtrB</i> expressing <i>vtrB</i> (POR-2<i>ΔvtrB</i>+p<i>vtrB</i>); bar 8, POR-2<i>ΔvtrAΔvtrB</i> (<i>ΔtdhASΔvcrD1ΔvtrAΔvtrB</i>); bar 9, POR-2<i>ΔvtrAΔvtrB</i> expressing <i>vtrA</i> (POR-2<i>ΔvtrAΔvtrB</i>+p<i>vtrA</i>); bar 10, POR-2<i>ΔvtrAΔvtrB</i> expressing <i>vtrB</i> (POR-2<i>ΔvtrAΔvtrB</i>+p<i>vtrB</i>); bar 11, <i>ΔvcrD1ΔvcrD2</i> (<i>ΔtdhASΔvcrD1ΔvcrD2</i>); bar 12, non-infected (NI) control. Results were measured as the amount of accumulated fluid (in milliliters) per length (in centimeters) of ligated rabbit small intestine. Error bars represent standard deviations for results from triplicate experiments. Asterisks indicate significant differences from the results obtained with the parental strain (<i>P</i><0.05). B. VtrA and VtrB are essential for <i>V. parahaemolyticus</i>-induced enterotoxicity. The enterotoxic activity of isogenic mutants of wild-type <i>V. parahaemolyticus</i> (WT) and complemented strains in rabbit ileal loops were examined. Bar 1, wild-type (WT); bar 2, WT<i>ΔvtrA</i>; bar 3, WT<i>ΔvtrA</i> expressing <i>vtrA</i> (WT<i>ΔvtrA</i>+p<i>vtrA</i>); bar 4, WT<i>ΔvtrA</i> expressing <i>vtrB</i> (WT<i>ΔvtrA</i>+p<i>vtrB</i>); bar 5, WT<i>ΔvtrB</i>; bar 6, WT<i>ΔvtrB</i> expressing <i>vtrA</i> (WT<i>ΔvtrB</i>+p<i>vtrA</i>); bar 7, WT<i>ΔvtrB</i> expressing <i>vtrB</i> (WT<i>ΔvtrB</i>+p<i>vtrB</i>); bar 8, WT<i>ΔvtrAΔvtrB</i>; bar 9, WT<i>ΔvtrAΔvtrB</i> expressing <i>vtrA</i> (WT<i>ΔvtrAΔvtrB</i>+p<i>vtrA</i>); bar 10, WT<i>ΔvtrAΔvtrB</i> expressing <i>vtrB</i> (WT<i>ΔvtrAΔvtrB</i>+p<i>vtrB</i>); bar 11, NI control. Error bars represent standard deviations for results from triplicate experiments. Asterisks indicate significant differences from the results obtained with the parental strain (<i>P</i><0.05).</p