Characterization of all RND-type multidrug efflux transporters in Vibrio parahaemolyticus

Abstract

Resistance nodulation cell division (RND)-type efflux transporters play the main role in intrinsic resistance to various antimicrobial agents in many gram-negative bacteria. Here, we estimated 12 RND-type efflux transporter genes in Vibrio parahaemolyticus. Because VmeAB has already been characterized, we cloned the other 11 RND-type efflux transporter genes and characterized them in Escherichia coli KAM33 cells, a drug hypersusceptible strain. KAM33 expressing either VmeCD, VmeEF, or VmeYZ showed increased minimum inhibitory concentrations (MICs) for several antimicrobial agents. Additional four RND-type transporters were functional as efflux pumps only when co-expressed with VpoC, an outer membrane component in V. parahaemolyticus. Furthermore, VmeCD, VmeEF, and VmeYZ co-expressed with VpoC exhibited a broader substrate specificity and conferred higher resistance than that with TolC of E. coli. Deletion mutants of these transporter genes were constructed in V. parahaemolyticus. TM32 (ΔvmeAB and ΔvmeCD) had significantly decreased MICs for many antimicrobial agents and the number of viable cells after exposure to deoxycholate were markedly reduced. Strains in which 12 operons were all disrupted had very low MICs and much lower fluid accumulation in rabbit ileal loops. These results indicate that resistance nodulation cell division-type efflux transporters contribute not only to intrinsic resistance but also to exerting the virulence of V. parahaemolyticus.

Keywords: Drug resistance; RND; V. parahaemolyticus; multidrug efflux transporter.

Figure 1

RND-type efflux transporter genes encoded in the V. parahaemolyticus genome. The chromosomal positions of genes coding for the putative RND-type efflux transporters, outer membrane proteins, and membrane fusion proteins are indicated by the kb in the V. parahaemolyticus RIMD2210633 genome. Arrows correspond to the lengths and directions of the genes.

Figure 2

Active efflux of ethidium from E. coli cells expressing VmeCD-VpoC or VmeEF-VpoC. Energy-starved E. coli cells were loaded with 10 μmol/L ethidium. At the time point indicated by the arrow, potassium lactate (final concentration, 40 mmol/L) was added to energize cells. Ethidium efflux was represented by a rapid decrease in fluorescence. (A) KAM43/pSTV28/pBVT3 (vpoC only); (B) KAM43/pSVP201/pBVT3 (vmeCD+vpoC); (C) KAM43/pSVP202/pBVT3 (vmeEF+vpoC).

Figure 3

RT–PCR analyses in V. parahaemolyticus AQ3334. (A) Total RNA (0.5 μg) purified from exponential-phase-grown cells was used for RT-PCR. Amplifications for all RND-type efflux transporter genes and 16S rRNA were performed in 23 cycles. (B) Cells were grown until the exponential phase in the presence or absence of 0.2% sodium deoxycholate, (+) and (−), respectively. One nanogram of total RNA was used for RT-PCR. Amplifications for vmeD and 16S rRNA were performed in 28 cycles or 13 cycles, respectively. The absence of DNA contamination was confirmed using total RNA without reverse transcription as a template.

Figure 4

Active efflux of ethidium from V. parahaemolyticus cells. At the time point indicated by the arrow, potassium lactate (final concentration, 40 mmol/L) was added to energize cells. (A) Active efflux in a series of gene disruptants. (B) Active efflux in TM312 expressing indicated RND-type transporters. TM312 carrying pSTV28 (empty vector) was used as a control.

Figure 5

Involvement of RND-type efflux transporters in induced enterotoxicity. The enterotoxicity of the 12 RND-type efflux transporter gene-deficient strain, RTM313, was evaluated by the rabbit ileal loop test. The fluid accumulation (FA) ratio in each loop was measured 18 h after the injection. FA was measured by the amount of accumulated fluid (in mL) per length (in cm) of ligated rabbit small intestine. Error bars represent standard deviations. Three independent experiments were performed and a significant difference was observed (P < 0.005).

Figure 6

Excretion of siderophores from V. parahaemolyticus cells. Excreted siderophores from V. parahaemolyticus cells were detected with the CAS assay. An aliquot of the supernatant of the culture was mixed with the CAS assay solution and was incubated at room temperature for 2 h. Siderophore excretion was measured by the absorbance of the reaction mixture at 630 nm per O.D.₆₅₀ when cells were harvested.

Figure 7

Phylogenetic tree for the RND family of multidrug transporters. The phylogenetic tree was obtained using the CLUSTAL W system provided by the DNA Data Bank of Japan (available at the website http://clustalw.ddbj.nig.ac.jp/top-j.html). The amino acid sequences of IMP components for RND-type efflux pumps of V. parahaemolyticus, V. cholerae, V. fischeri, V. harveyi, and V. vulnificus were applied to the CLUSTAL W system.