Effects of Polyamines on Vibrio cholerae Virulence Properties

Citation: Goforth JB, Walter NE, Karatan E ( Effects of Polyamines on Vibrio cholerae Virulence Properties John Bradley Goforth 0 Nicholas Emmanuel Walter 0 Ece Karatan 0 Roy Martin Roop II, East Carolina University School of Medicine, United States of America 0 Department of Biology, Appalachian State University , Boone, North Carolina , United States of America Vibrio cholerae is the causative agent of the severe enteric disease cholera. To cause cholera the bacterium must be able to synthesize both cholera toxin (CT) and toxin-coregulated pilus (TCP) which mediates autoagglutination and is required for colonization of the small intestine. Only a few environmental signals have been shown to regulate V. cholerae virulence gene expression. Polyamines, which are ubiquitous in nature, and have been implicated in regulating virulence gene expression in other bacteria, have not been extensively studied for their effect on V. cholerae virulence properties. The objective of this study was to test the effect of several polyamines that are abundant in the human intestine on V. cholerae virulence properties. All of the polyamines tested inhibited autoagglutination of V. cholerae O1 classical strain in a concentration dependent manner. Putrescine and cadaverine decreased the synthesis of the major pilin subunit, TcpA, spermidine increased its production, and spermine had no effect. Putrescine and spermidine led to a decrease and increase, respectively, on the relative abundance of TCP found on the cell surface. Spermine led to a small reduction in cholera toxin synthesis whereas none of the other polyamines had an effect. The polyamines did not affect pili bundling morphology, but caused a small reduction in CTXw transduction, indicating that the TCP present on the cell surface may not be fully functional. We hypothesize the inhibition of autoagglutination is likely to be caused by the positively charged amine groups on the polyamines electrostatically disrupting the pili-pili interactions which mediate autoagglutination. Our results implicate that polyamines may have a protective function against colonization of the small intestine by V. cholerae. - Funding: Funding for this work was provided by the following sources: Appalachian State University Department of Biology, the Office of Student Research and the Graduate Student Association Senate at Appalachian State University and and by NIH (Grant number AI096358 to E.K.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Vibrio cholerae, a Gram-negative, enteropathogenic organism, is the causative agent of the disease cholera. Cholera is a severe and life threatening diarrheal disease, with a high mortality rate in regions without potable drinking water. Areas with poor sanitation and access to potable drinking water are at high risk for epidemic outbreaks due to the oral-fecal transmission route of the bacterium. Currently, V. cholerae is classified into over 200 distinct serogroups based upon the differences in the sugar composition of the O antigen present on the bacterial surface [1,2]. Of these only O1, subdivided into classical and El Tor biotypes, and O139 are capable of causing epidemic cholera. Cholera occurs when an infectious dose of V. cholerae is orally ingested, and the bacteria subsequently colonize the small intestine [3,4,5]. Inside the host, V. cholerae synthesizes two main virulence factors, cholera toxin (CT) and toxin-coregulated pilus (TCP). CT is an enterotoxin secreted by the bacterium that causes the characteristic voluminous diarrhea [6,7] and TCP aids in the colonization of the small intestine [6,8]. In addition to TCP and CT, V. cholerae synthesizes other virulence factors which contribute to pathogenesis; however, when either TCP and/or CT synthesis is reduced, or absent, colonization and virulence are markedly attenuated [6]. TCP belongs to the Type IVb class of pili, which are involved in pathogenesis of other organisms as well, including Escherichia coli and Neiserria gonorrhoeae [9,10,11]. TCP is composed of a repeating homopolymer of the major pilin subunit, TcpA, which forms a helical arrangement originating in the inner membrane and protruding outward from the cell surface [12,13]. The genes which code for the proteins making up the TCP biogenesis apparatus, and the major pilin subunit are located within the tcp operon found on a segment of the chromosome referred to as the Vibrio pathogenicity island [14]. Colonization and autoagglutination resulting in microcolony formation within the small intestine has been shown to be mediated by TCP [6,9,12,15,16,17]. Autoagglutination can be assessed in vitro by culturing bacteria under optimal TCP expressing conditions [8,15]. In vitro autoagglutination has been shown to correlate well with colonization in the infant mouse model of cholera, indicating the reliability of in vitro autoagglutination as a positive indicator of V. cholerae to effectively colonize the host [15]. The regulation of V. cholerae virulence genes is controlled through a complex pathway consisting of multiple proteins, each playing an integral role in the regulation of both ctxAB genes encoding the two subunits of CT, and the tcp operon [18]. Virulence gene expression has been shown to respond to specific environmental stimuli in vitro, including temperature, pH, bile salts, osmolarity, bicarbonate and the presence of certain amino acids [19,20,21,22]. Interestingly, the conditions required to promote maximal virulence gene expression in vitro are different than those encountered within the small intestine. Conditions for maximal virulence gene expression in vitro for V. cholerae O1 classical biotype were found to be a temperature of 30uC, pH 6.5 and a salt concentration of approximately 66 mM NaCl [23]. Conversely, environmental conditions within the small intestine are a temperature of 37uC, a higher pH and an osmolarity equivalent to approximately 300 mM NaCl [23]. Within the intestinal environment only a few signals have been identified which regulate virulence gene expression. Animal studies have shown that wild-type V. cholerae preferentially colonize the middle/ distal portion of the small intestine in a TCP-dependent manner, indicating a potential gradient of either an attractant or repellent leading to the area of colonization [24,25]. The purpose for this temporal/proximal colonization and virulence gene expression at the more distal portion of the small intestine has not been elucidated. There are a plethora of potential signals within the small intestine which have yet to be studied for their effect on V. cholerae virulence properties. One group of molecules present in this environment is polyamines, flexible hydrocarbon chains with interspersed amine groups that are positively charged at neutral pH ( (...truncated)