Abstract
Vibrio cholerae ATP-binding cassette transporter VcaM (V. cholerae ABC multidrug resistance pump) has previously been shown to confer resistance to a variety of medically important drugs. In this study, we set to analyse its properties both in vitro in detergent-solubilised state and in vivo to differentiate its dependency on auxiliary proteins for its function. We report the first detailed kinetic parameters of purified VcaM and the rate of phosphate (Pi) production. To determine the possible functional dependencies of VcaM on the tripartite efflux pumps we then utilized different E. coli strains lacking the principal secondary transporter AcrB (Acriflavine resistance protein), as well as cells lacking the outer membrane factor (OMF) TolC (Tolerance to colicins). Consistent with the ATPase function of VcaM we found it to be susceptible to sodium orthovanadate (NaOV), however, we also found a clear dependency of VcaM function on TolC. Inhibitors targeting secondary active transporters had no effects on either VcaM-conferred resistance or Hoechst 33342 accumulation, suggesting that VcaM might be capable of engaging with the TolC-channel without periplasmic mediation by additional transporters. Our findings are indicative of VcaM being capable of a one-step substrate translocation from cytosol to extracellular space utilising the TolC-channel, making it the only multidrug ABC-transporter outside of the MacB-family with demonstrable TolC-dependency.
Highlights
Vibrio cholerae is a Gram-negative non-invasive enteric pathogen and the causative agent of cholera—a severe diarrheal disease [1]
We present the first direct kinetic parameters of VcaM from V. cholerae based on an in vitro detergent-solubilised form and, by using a range of different E. coli genetic knock-out strains, demonstrate for the first time its functional dependency in vivo on the outer membrane factor (OMF) TolC
In order to determine the kinetic parameters of the putative ATPase transporter VcaM from V. cholerae (GenBank Q93GU0), the vcaM gene was amplified and cloned onto pET21a vector to generate the plasmid pET21a-vcaM
Summary
Vibrio cholerae is a Gram-negative non-invasive enteric pathogen and the causative agent of cholera—a severe diarrheal disease [1]. V. cholerae has been shown to develop resistance to a broad range of frontline antibiotics including tetracycline, macrolides and fluoroquinolones [4]. Multidrug efflux pumps and transporters provide a first line of defence allowing development of additional resistance mechanisms and, understanding their function is critical for addressing it. Numerous multidrug transporters have been identified and investigated in V. cholerae, e.g., VexAB and VexCD from Resistance-Nodulation-Division (RND) family [6,7]. Several different groups of active transporters (including RND, ABC and the MFS families) require a member of TolC (OMF) family form functional tripartite efflux pumps [14]. TolC orthologues are involved in the ABC-transporter-based type 1 secretion systems (T1SS) such as RTX (Repeats-in-toxins) toxin secretion in V. cholerae [24] and the well characterized HlyBD-TolC in E.coli [25]
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