Identification of a Novel Membrane Transporter Mediating Resistance to Organic Arsenic in Campylobacter jejuni

Abstract

Although bacterial mechanisms involved in the resistance to inorganic arsenic are well understood, the molecular basis for organic arsenic resistance has not been described. Campylobacter jejuni, a major food-borne pathogen causing gastroenteritis in humans, is highly prevalent in poultry and is reportedly resistant to the arsenic compound roxarsone (4-hydroxy-3-nitrobenzenearsonic acid), which has been used as a feed additive in the poultry industry for growth promotion. In this study, we report the identification of a novel membrane transporter (named ArsP) that contributes to organic arsenic resistance in Campylobacter. ArsP is predicted to be a membrane permease containing eight transmembrane helices, distinct from other known arsenic transporters. Analysis of multiple C. jejuni isolates from various animal species revealed that the presence of an intact arsP gene is associated with elevated resistance to roxarsone. In addition, inactivation of arsP in C. jejuni resulted in 4-and 8-fold reductions in the MICs of roxarsone and nitarsone, respectively, compared to that for the wild-type strain. Furthermore, cloning of arsP into a C. jejuni strain lacking a functional arsP gene led to 16-and 64-fold increases in the MICs of roxarsone and nitarsone, respectively. Neither mutation nor overexpression of arsP affected the MICs of inorganic arsenic, including arsenite and arsenate, in Campylobacter. Moreover, acquisition of arsP in NCTC 11168 led to accumulation of less roxarsone than the wild-type strain lacking arsP. Together, these results indicate that ArsP functions as an efflux transporter specific for extrusion of organic arsenic and contributes to the resistance to these compounds in C. jejuni. Campylobacter, a Gram-negative microaerobic bacterium, is a leading bacterial cause of food-borne diseases, and Campylobacter infections account for 400 to 500 million cases of diarrhea each year in developed and developing countries (1). A recent estimate from the CDC indicated that campylobacteriosis accounts for 9% of food-borne illness (over 840,000 cases) every year in the United States (2). Campylobacter jejuni and Campylobacter coli are the two most important Campylobacter species that cause food-borne infections of humans, and raw poultry meat serves as the main source of infection (3). Roxarsone, an organoarsenic compound, has been extensively used as a feed additive in the poultry industry to control bacterial and coccidial infections and improve weight gain, feed utilization, and pigmentation (4). Although it was recently withdrawn from poultry use in the United States, it has been estimated that roxarsone was utilized in approximately 70% of the U.S. broiler production units (4). The concentrations of roxarsone used in feed formulations vary from 22.7 to 45.4 g/ton (5). In animals, roxarsone is excreted into fresh litter and then converted to inorganic arsenate [As(V)] in composted litter via the bioconversion processes (5, 6). The total arsenic concentration in the litter may range from 15 to 48 mg/kg (5-...

ArsP: a methylarsenite efflux permease

“…The arsP gene from C. jejuni confers resistance to trivalent organoarsenicals Cells of C. jejuni expressing the arsP gene have been shown to exhibit resistance to pentavalent roxarsone and nitarsone (Shen et al, 2014). In this study, the CjarsP gene was cloned downstream of the trc promoter in plasmid pTrcHis2A and expressed in the arsenitehypersensitive E. coli strain AW3110, in which the chromosomal arsRBC operon was deleted (Carlin et al, 1995).…”

“…Recently, Shen et al . () demonstrated that the arsP gene of C. jejuni is responsible for roxarsone resistance. They showed that ArsP is a permease that results in lowered roxarsone in the cells of C. jejuni .…”

“…Recently, a putative membrane permease (CjArsP), encoded in a four-gene ars operon in Campylobacter jejuni, was shown to confer resistance to cells exposed to roxarsone (Shen et al, 2014). Roxarsone, a pentavalent aromatic arsenical, has been widely used in poultry production to treat coccidiosis and for growth promotion (Cortinas et al, 2006).…”

The organoarsenical biocycle and the primordial antibiotic methylarsenite

“…Purified ArsH from either S. meliloti or P. putida are yellow and show a typical flavoprotein absorption spectrum. 63,66 P. putida oxidized MAs(III), PhAs(III) and Rox(III). 64 The crystal structure of S. meliloti ArsH (Figure 5) was determined at 1.8 Å resolution and is similar to other flavoproteins.…”

Current Perspectives on Campylobacter

“…It has been determined that Campylobacter mediates much of its antibiotic resistance through a multidrug efflux system, CmeABC (Lin et al, 2002). However, studies have demonstrated that these genes do not confer additional antibiotic resistance (Shen et al, 2013(Shen et al, , 2014Wang et al, 2009). However, studies have demonstrated that these genes do not confer additional antibiotic resistance (Shen et al, 2013(Shen et al, , 2014Wang et al, 2009).…”