Chapter Six
6. VIBRIO CHOLERAE MEMBRANE PROTEINS
IN ANTIMICROBIAL RESISTANCE
AND VIRULENCE
Manjusha Lekshmi1, Nicholas Wenzel2, Sanath H. Kumar1,
Manuel F. Varela2, * 1 Post Harvest Technology, ICAR-Central Institute of Fisheries Education, Seven Bungalows, Andheri (W), Mumbai, 400061, India 2
Department of Biology, Eastern New Mexico University, Portales, NM
88130, USA
ABSTRACT
Vibrio cholerae, the causative agent of cholera, is a common estuarine bacterium and a versatile human pathogen. The success of this pathogen can be attributed to the myriad of membrane proteins that contribute to its physiology of persistence in the aquatic environment and its virulence characters. Membrane proteins also contribute to antimicrobial resistance in V. cholerae. The ToxR-regulated outer membrane protein OmpU is an important virulence protein of V. cholerae, which is also essential for its survival in hostile environments. OmpU, along with another membrane protein, OmpT, mediates resistance to bile, detergents and antimicrobial peptides. In V. cholerae, antimicrobial resistance is mediated by efflux pumps belonging to the RND (resistance-nodulation-division) family such as VexABDK and to the MFS (major facilitator superfamily) such as the EmrD3 transporter. Some RND efflux pumps such as VexB, VexD, and VexK contribute to the virulence of V. cholerae, and the mutants lacking these proteins were attenuated in a mouse model. The production of cholera toxin and the toxin co-regulated protein, two important virulence proteins of V. cholerae, were significantly reduced in RND-negative mutants. The proton-driven EmrD3 efflux pump of V. cholerae confers resistance to an array of anionic detergents, dyes and antibiotics. Inhibition of efflux pumps with synthetic or natural compounds can reduce the virulence of V. cholerae and restore its susceptibility to conventional antibiotic treatment. A complex network involving quorum sensing, efflux pumps and virulence gene expression has also been elucidated in V. cholerae, suggesting that membrane proteins represent a hub of molecular activities regulating multiple physiologies and virulence of pathogenic bacteria such as V.
cholerae.
Keywords: Vibrio cholerae, virulence, efflux pump, outer membrane proteins, ToxRS, biofilm
* Direct all correspondence to Dr. Manuel Varela; Department of Biology, Eastern New Mexico University, Portales, NM 88130, USA. E-mail: manuel.varela@enmu.edu.
6.1. VIBRIO CHOLERAE - A VERSATILE HUMAN PATHOGEN
Cholera disease caused by Vibrio cholerae can affect up to 4 million people each year and, of those infected annually, between 28,000–130,000 may succumb to the disease (WHO, 2004; GBD 2015 Mortality and Causes of Death Collaborators, 2016). Increased prevalence of cholera infection occurs in underdeveloped and overcrowded regions of the world, where food and water sanitation practices are inadequate (WHO, 2004). In addition to its occurrence as an endemic disease in these underdeveloped regions, cholera can cause large-scale epidemic outbreaks in more developed middle-income countries.
Cholera disease owes its pathogenicity to toxigenic serotypes of V. cholerae (Foster, Aliabadi and Slonczewski, 2018). V. cholerae is most often spread via fecal-oral transmission of contaminated food and water sources (CDC, 2019). Infected individuals gradually begin to suffer from acute but severe watery diarrhea, which contains billions of organisms that can make their way into water systems used by others (Azman et al., 2013; NIH, 2017). Those suffering from a severe cholera infection often experience rapid dehydration and hyponatremia, both of which can be fatal if not treated (Foster, Aliabadi and Slonczewski, 2018). Accelerated oral or intravenous rehydration with isotonic liquids is the basis for treating an infected individual (Harris et al., 2012). Successful rehydration care can reduce the mortality of a severe cholera infection to less than 0.2% (Sack et al., 2004). Prevention of the disease is focused around preparedness, surveillance, and effective sanitation methods (WHO, 2004). Vaccination provides an additional level of cholera prevention, and several killed oral whole cell vaccines against cholera are in the market (Sinclair et al., 2011). Appropriate administration of these vaccines can prevent 50–60% of cholera incidents; however their adoption into the regular vaccination routines of affected countries is dependent on the cost-effectiveness as a result of the prevalence of infection and access to rehydration therapy in the area.
6.1.1. Virulence of V. cholerae
V. cholerae is a motile, Gram-negative, curved rod-shaped bacterium of the family Vibrionaceae. Based on the structure of their cell surface lipopolysaccharides, V. cholerae strains are classified into more than 200 serogroups. Of these, only the O1 and O139 serotypes can produce the cholera toxin (CT) and cause severe pandemic cholera (Kaper, Morris and Levine, 1995). The O1 serotype is further classified into the classical and El Tor biotypes based on phenotypic differences such as their susceptibility to polymyxin B and phage infection pattern (Conner et al., 2016).
V. cholerae is an opportunistic pathogen that causes human infections through contaminated food and water. The organism has to pass the gastric acid barrier of the stomach before it can reach the upper small intestine, where it typically colonizes and causes infection. To continue its life cycle, V. cholerae exits the host during excretion and then find its way back to an aquatic environment (Reidl and Klose, 2002).
CT belonging to the AB5 family of ADP-ribosyltransferase, and a toxin coregulated pilus (TCP) that mediates adherence and microcolony formation are the major factors contributing to virulence of V. cholerae (Kaper, Morris and Levine, 1995). The enterotoxin affects ion transport by intestinal epithelial cells and TCP helps the organism to colonize the small intestinal epithelium. Excessive loss of water and electrolytes following infection leads to severe diarrhea which is characteristic of cholera (Silva and Benitez, 2016). The CT subunits are encoded by the genes ctxA and ctxB. The genes required for TCP biogenesis form a large cluster known as the V. cholerae pathogenicity island (VPI). Within this cluster, tcpA encodes the major pilus subunit (Karaolis et al., 1998).
6.1.2. Life of V. cholerae outside the host, in the environment
V. cholerae spends the majority of its life cycle outside of the human host in estuarine and coastal environments. Many strains of V. cholerae including the toxigenic ones are present naturally in aquatic ecosystems and hence exist as facultative human pathogens (Colwell, Kaper and Joseph, 1977). Within the marine environment, they attach to surfaces provided by plants, copepods (zooplankton), crustaceans, and insects (Huq et al., 1983). In order to survive in the marine and estuarine environment, V. cholerae makes use of several mechanisms to tide over the adverse conditions. Some of these include biofilm formation, shifting to a viable but non-culturable state, nutrient storage and initiation of protective responses to specific physiological and biological stressors. While inhabiting aquatic environments, V. cholerae can be found as biofilms or free swimming vibrios (Sack et al., 2004).
Biofilms are mutli-cellular microbial communities that are composed of cells attached to a substratum, an interface, or to each other and are embedded in a selfproduced matrix. Biofilms can be present on a variety of surfaces such as living tissues or even plastics and metals (Varela et al., 2017). Biofilms contribute to the environmental persistence of V. cholerae and provide protection from a number of environmental stresses, including nutrient limitation and predation by protozoa and bacteriophages (Conner et al., 2016). V. cholerae biofilm formation is a wellregulated process that can be triggered by quorum sensing and the subsequent up or down regulation of Vibrio pathogenicity island-1 (VPI-1) or V. cholerae biofilm matrix cluster (VcBMC)-associated genes (Teschler et al., 2015; Lekshmi et al., 2018). Biofilm formation begins with the V. cholerae type IV pilus attaching to a surface, after which bacteria begin to form microcolonies and secrete Vibrio polysaccharide (VPS) (Watnick and Kolter, 1999). The development of highly organized, three-dimensional biofilm structures occurs following the formation of microcolonies. The extracellular matrix is composed of Vibrio polysaccharides, the biofilm matrix proteins, and extracellular nucleic acids, each playing unique roles in infection by V. cholerae (Watnick and Kolter, 1999). VPS is the structural basis for the three-dimensional lattice that is the V. cholerae biofilm (Mukherjee et al., 2016). Growth and transcription profiles of these biofilms follow an altered phenotype (Silva and Benitez, 2016). Once biofilm formation is complete, V. cholerae begin to reduce the production of both motility structures and the expression of certain virulence genes, as they become unnecessary (Moorthy and Wanick 2004; Tischler and Camilli 2004). The gene expression pattern of a mature biofilm provides resistance to environmental stressors. The formation of biofilms alone are enough to confer antimicrobial resistance to V. cholerae (Mukherjee et al., 2016). V. cholerae must be capable of adopting both motile and biofilm lifestyles during the infective process to successfully colonize the intestine and result in characteristic diarrhea (Fong and Yildiz, 2008).
6.2. STRUCTURAL AND FUNCTIONAL FEATURES OF MAJOR MEMBRANE PROTEINS OF V. CHOLERAE
The efflux pumps and porins situated in the membrane of V. cholerae are accompanied by many other additional proteins. The well-studied type II secretion system (T2SS) of V. cholerae and E. coli are a common class of such membrane proteins (Dunstan et al., 2013; Natarajan, Singh and Rapaport, 2019). Secretion systems are common in Gram-negative bacteria and in V. cholerae, a particular T2SS is responsible for the secretion of CT (Korotkov, Sandkvist and Hol, 2012). V. cholerae T2SS is a complex multiprotein system consisting of inner and outer membrane portions of the Gram-negative cell wall, which together facilitate the translocation of the CT protein through the bacterial membrane and into the exoplasm of a bacterial cell (Johnson et al., 2006). During this process, the fully formed CT protein must pass through a gated channel in the outer bacterial membrane, which is composed of many secretin proteins (Kubori, 2016; Natarajan, Singh and Rapaport, 2019). Individual V. cholerae secretin proteins called GspD, form the T2SS secretin complex–a complex that is conserved across all classes of secretion systems (DeAngelis et al., 2019). GspD has also recently been shown to associate with phage shock protein (Psp), a stress response pathway triggered by inner membrane damage (DeAngelis et al., 2019). The complex of V. cholerae membrane proteins ultimately aid in the virulence and pathogenicity of V. cholerae.
6.2.1. ToxR - A global regulator of virulence genes in V. cholerae
V. cholerae has a characteristic 3-component global regulator ToxRST, which controls the expression of genes in response to environmental signals (Pfau and Taylor, 1998). ToxR is a 32-kDA transmembrane protein belonging to the OmpR family of transcriptional regulators, with a dimeric periplasmic domain and a monomeric membrane domain (Miller, Taylor and Mekalanos, 1987; Mizuno and Tanaka, 1997; Chatterjee, Saha and Chakrabarti, 2007). ToxR regulates important virulence factors of V. cholerae such as the CT and the toxin-coregulated pilus (TCP) (Miller, Taylor and Mekalanos, 1987). ToxR is influenced by environmental stimuli such as pH, temperature, osmolarity, oxygen tension etc, which in turn regulates several physiological functions in V. cholerae (Faruque, Albert and Mekalanos, 1998; Pfau and Taylor, 1998). ToxS, a 19 kDA transmembrane protein, stimulates the activity of ToxR by interacting with its perisplasmic domain, and together, these two proteins perform co-ordinated regulation of the expression of genes (DiRita and Mekalanos, 1991). The third regulatory element in the system is a 32-kDa protein ToxT, which controls the expression of some genes within the ToxR regulon. Expression of the toxT gene is controlled by ToxR itself. ToxT is a member of the AraC family of bacterial transcriptional activators, which play very important roles in regulating the expression of virulence factors (DiRita, 1992).
6.2.2. Outer membrane porins
Several membrane proteins help V. cholerae in efficient colonization of the small intestine, such as the ToxR-regulated outer membrane porin proteins OmpU and OmpT (Miller and Mekalanos, 1988). ToxR binds to a 7-bp tandemly repeated DNA sequence 5'-TTTTGAT-3' in the cholera toxin promoter region and regulates its expression (Chatterjee, Saha and Chakrabarti, 2007). ToxR is influenced by environmental stimuli, which in turn regulates several physiological functions in V. cholerae (Faruque, Albert and Mekalanos, 1998). The expression of OmpU is increased by ToxR, while the expression of OmpT is repressed (Li et al., 2000; Provenzano and Klose, 2000). The 40-kDa OmpT, and the 38-kDa OmpU have been purified to homogeneity and their pore–forming ability has been demonstrated using liposomes (Chakrabarti et al., 1996). These porins play important roles in solute transport and respond to diverse environmental stimuli (Miller and Mekalanos, 1988; Wibbenmeyer et al., 2002). Both OmpU and OmpT are involved in bile transport, the latter being more permeable to bile salts (Provenzano and Klose 2000). ToxR-mediated overexpression of OmpT and repression of OmpU result in increased bile sensitivity and altered solute flux across the outer membrane, consequently influencing virulence gene expression (Reidl and Klose, 2002).
V. cholerae has a distinct regulator mechanism that responds to low iron concentration by stimulating the expression of genes encoding hemolysins and outer membrane proteins (Sigel and Payne, 1982; Faruque, Albert and Mekalanos, 1998). This mechanism is mediated by Fur protein, which represses the expression of iron-regulated proteins in the presence of iron by binding to the promoter region upstream of these genes (Goldberg, Boyko and Calderwood, 1990). Studies on one of the iron-regulated proteins IrgA showed that insertional inactivation of irgA resulted in the loss of a 77-kDa major outer membrane protein in V. cholerae (Goldberg, Boyko and Calderwood, 1990; 1991). Further, the transcription of irgA is regulated by a 900-bp open reading frame (irgB) upstream of the irgA promoter in an inverse orientation to irgA (Goldberg, Boyko and Calderwood, 1991). IrgB resembles the LysR family of positive transcriptional activators, and both irgA and irgB are negatively regulated by the Fur protein under iron-limiting conditions (Goldberg, Boyko and Calderwood, 1991). ViuA is a 74 kDa iron-regulated outer membrane protein of V. cholerae, which acts as the receptor for the iron-siderophore complex, ferric vibriobactin (Butterton et al., 1992). Subsequent studies have revealed that ViuA performs early steps in the biosynthesis of vibriobactin and is part a gene cluster comprising vibA, vibB, vibC, vibE and vibF (Wyckoff et al., 1997; Butterton et al., 2000).
V. cholerae is capable of living in both an aquatic environment and within the intestine of a human host (DeAngelis et al., 2019). In order to survive these very different environments, V. cholerae has evolved various methods to respond to environmental stresses that it may encounter (Saul-McBeth and Matson, 2019). One such method of response that has recently been described is centered on the SipA protein, which is found in the periplasmic space of V. cholerae membranes (Saul-McBeth and Matson, 2019). SipA is crucial for bacterial survival in different environments, namely in the presence of antimicrobial peptides (AMPs). SipA binds AMPs from the environment and interacts with outer membrane proteins, such as OmpA, to aid in removal of AMPs from the cell and promote V. cholerae survival.
6.2.3. Efflux pumps of V. cholerae – Role in antibiotic resistance and virulence
Antimicrobial efflux pumps of V. cholerae represent a crucial resistance mechanism (Kitaoka et al., 2011; Andersen et al., 2015; Varela, 2019). While such multidrug efflux resistance systems may potentially compromise the chemotherapeutic efficacy of severe cholera cases, the resistance mechanisms nevertheless make promising targets for resistance modulation in key bacterial pathogens (Varela et al., 2017). These and other antimicrobial efflux systems in bacteria consist of either secondary or primary active transporters (Konings, Poolman and van Veen, 1994). We discuss below related antimicrobial efflux systems from V. cholerae from a phylogenetic perspective.
6.2.3.1. V. cholerae and the major facilitator superfamily
The multidrug efflux pump VceB from V. cholerae consists of 14transmembrane domains and is associated with the VceCAB operon on the bacterial genome (Colmer, Fralick and Hamood, 1998). VceB is a member of the large superfamily of solute transporters (Andersen et al., 2015). Like its homologous counterpart EmrAB system in E. coli, VceB participates in a tripartite complex system in which VceB resides in the inner cytoplasmic membrane, VceA is located in the periplasm, and VceC is integral to the outer membrane of the bacterial cell wall (Woolley et al., 2005). Together, as a multicomponent transport system in the V. cholerae cell wall, resistance to the quinolone nalidixic acid, deoxycholate, phenylmercuric acetate, and carbonyl-mchlorophenylhydrazone (CCCP) is brought about (Colmer, Fralick and Hamood, 1998; Woolley et al., 2005). Expression of the VceCAB system is negatively controlled by the VceR repressor (Alatoom et al., 2007), which is structurally homologous to the TetR repressor (Cuthbertson and Nodwell, 2013).
From a toxigenic strain of V. cholerae O395, our laboratory cloned a multidrug efflux pump system referred to as EmrD-3, which is a member of the major facilitator superfamily (MFS) of transporters (Smith, Kumar and Varela, 2009). We showed that the emrD-3 determinant encodes a 397 amino acid polypeptide chain with 12 predicted transmembrane domains and the N- and Ctermini residing at the cytoplasmic side of the inner membrane. We also found that host cells containing EmrD-3 conferred resistance to linezolid, rifampin, erythromycin, chloramphenicol, rhodamine 6G, and tetraphenylphosphonium chloride (Smith, Kumar and Varela, 2009). Further, we demonstrated that host cells harboring EmrD-3 actively transported ethidium bromide, an essential multidrug efflux substrate (Smith, Kumar and Varela, 2009). More recently, we discovered that extract of garlic (Allium sativum) and one of its bioactive components called allyl sulfide specifically inhibits the growth of host cells harboring EmrD-3 (Bruns et al., 2017). We found that relatively low extract concentrations targeted the EmrD-3 multidrug efflux pump, while higher concentrations appeared to affect the respiratory chain, possibly collapsing the proton-motive force (Bruns et al., 2017).
More recently, five new related multidrug efflux pumps called MFS pumps numbered 1 through 5, were discovered in V. cholerae, and were shown to be controlled under the regulator of transcription, a protein called MfsR, which is related to the well-known LysR family of regulators (Chen et al., 2013). Mutagenic analyses of the msf determinants showed reduced resistance to tetracycline and crude bile (Chen et al., 2013).
6.2.3.2. V. cholerae and the multidrug and toxic compound extrusion superfamily
The V. cholerae non-O1 bacterium harbors the VcrM antimicrobial transporter, which confers resistance to multiple agents such as acriflavine, ethidium bromide, rhodamine 6G, and tetraphenylphosphonium chloride (Huda et al., 2003). Interestingly, VcrM is a sodium-driven transporter of the so-called multidrug and toxic compound extrusion (MATE) superfamily (Kuroda and Tsuchiya, 2009). Additional members of the MATE superfamily of transporters from non-O1 V. cholerae include VcmB, VcmD, and VcmH, which were also shown to be Na+-dependent, but VcmN was found to be independent of the sodium motive force (Begum et al., 2005).
6.2.3.3. V. cholerae and the resistance-nodulation-cell division superfamily
Members of the resistance-nodulation-cell division (RND) superfamily from V. cholerae include VexB, VexD, VexF, VexH, VexK, and VexM (Bina et al., 2006; Taylor, Bina and Bina, 2012; Kunkle, Bina and Bina, 2017). Interestingly, these RND transporters participate in forming a tripartite system composed of three components lining the cell wall, such as the outer and inner membranes and the periplasm (Destoumieux-Garzón et al., 2014). Furthermore, these RND systems are located within operons on the V. cholerae genome and are regulated (Nikaido, 2018).
6.2.3.4. V. cholerae and the ATP-binding cassette superfamily
A critical member of the sizeable ATP-binding cassette (ABC) superfamily from V. cholerae includes the VcaM transporter (Huda et al., 2003). Hydrolysis of ATP is the prime mode of energization (Orelle, Mathieu and Jault, 2019; Cui and Davidson, 2011). The ABC transporter confers resistance to ciprofloxacin, norfloxacin, tetracycline, doxorubicin, daunomycin, and dyes such as 4′,6diamidino-2-phenylindole (DAPI) and Hoechst 33342 (Huda et al., 2003). More recently, evidence was reported that VcaM relies on the outer-membrane component TolC for active extrusion from host cells (Lu et al., 2018).
6.2.4. Carbohydrate transporters of V. cholerae
Nutrient acquisition is an important physiological function of bacteria which possess a robust machinery to accomplish this goal. Many cellular activities such as motility, chemotaxis, enzymes degrading macromolecules, membrane transporters, etc are dedicated to nutrient acquisition, transport and digestion (Dills et al., 1980). Bacteria have a peptidoglycan cell wall covering the outside of their cell membrane, and in the case of Gram-negative bacteria, a relatively thin peptidoglycan cell wall is surrounded by an additional envelope called the outer membrane made of lipopolysaccharide (Silhavy, Kahne and Walker, 2010). Gram-positive bacteria have a thick cell wall that lacks the outer membrane, but in some bacteria such as Staphylococcus aureus the cell wall is surrounded by a teichoic acid layer. Cellular metabolism requires that the essential compounds are transported from the external environment into the bacterial cytoplasm. At the same time, extrusion of toxic metabolites of cellular metabolism is also important. Carbohydrate transport across the membrane is critical for survival of the bacterium and these mechanisms are either passive or active (Dills et al., 1980). The passive mechanism of sugar transport works by simple diffusion across the membrane (e.g. transport of glycerol), and this mechanism does not facilitate transport against a concentration gradient. On the other hand, the active transport mechanism is energized by a proton gradient or ATP, and this method can transport macromolecules against their concentration gradients. Bacterial metabolism generates a proton electrochemical potential difference across the membrane, which energizes several membrane-associated activities including macromolecular transport (Mitchell, 1961). This mechanism is termed secondary active transport, which is accomplished by three different ways namely symport, antiport and uniport mechanisms. Symport is a common mechanism of carbohydrate transport in bacteria which is coupled to H+ electrochemical gradient. The other equally important carbohydrate transport mechanism is mediated by phosphoenolpyruvate-dependent phospotransferase systems (PTSs) (Kundig, Ghosh and Roseman, 1964). In this mechanism, the substrate is chemically modified for it to be transported across the membrane. The PTS is a high affinity system in bacteria, which helps to sequester carbohydrate present in low concentrations in the environment. This efficient system helps bacteria to survive under nutrient limiting conditions, such as in seawater in the case of V. cholerae.
The PTS catalyzes concomitant phosphorylation and transport of a series of carbohydrates by a process known as group translocation, which involves the phosphorylation of a number of carbohydrates with phosphoenol pyruvate (PEP) as the phosphoryl donor (Postma, Lengeler and Jacobson, 1993; Lengeler, Jahreis and Wehmeier, 1994; Barabote and Saier, 2005). The multi-component PTS consist of at least three proteins, two of which are cytoplasmic soluble proteins namely the heat stable protein (HPr) and enzyme I, and the third protein is a membrane bound transporter Enzyme II (EII). Enzyme I and HPr catalyze the phosphorylation of carbohydrates with the utilization of a phosphoenol pyruvate (PEP) resulting in phosphoenzyme I and pyruvate. Phosphoenzyme I in turn phosphorylates an intermediate phospho carrier protein HPr to form phospho HPr, and the phosphoryl group is finally transferred to the carbohydrate via the PTS enzyme II complex (Postma, Lengeler, and Jacobson 1993). The phosphorylated carbohydrate is subsequently taken up by Enzyme II (Postma, Lengeler and Jacobson, 1993). Enzyme II is generally made of A, B and C components (Kumar et al., 2011). The V. cholerae mannitol operon is a 3.9-kb operon comprised of mtlADR, encoding a mannitol-specific enzyme IICBA (EIIMtl) component (MtlA), a mannitol-1-phosphate dehydrogenase (MtlD), and a repressor (MtlR) (Kumar et al., 2011). In marine bacteria such as vibrios, mannitol derived from decaying seaweeds is an important source of carbon, which also plays an important role in osmoregulation and stress tolerance (Stoop and Mooibroek, 1998; Efiuvwevwere et al., 1999).
Mannitol fermentation is an important biochemical feature of many vibrios. In V. cholerae, the rate of mannitol ferme