Mutation in flrA and mshA Genes of Vibrio cholerae Inversely Involved in vps-Independent Biofilm Driving Bacterium Toward Nutrients in Lake Water

Abstract

Many bacterial pathogens promote biofilms that confer resistance against stressful survival conditions. Likewise Vibrio cholerae O1, the causative agent of cholera, and ubiquitous in aquatic environments, produces vps-dependent biofilm conferring resistance to environmental stressors and predators. Here we show that a 49-bp deletion mutation in the flrA gene of V. cholerae N16961S strain resulted in promotion of vps-independent biofilm in filter sterilized lake water (FSLW), but not in nutrient-rich L-broth. Complementation of flrA mutant with the wild-type flrA gene inhibited vps-independent biofilm formation. Our data demonstrate that mutation in the flrA gene positively contributed to vps-independent biofilm production in FSLW. Furthermore, inactivation of mshA gene, encoding the main pilin of mannose sensitive hemagglutinin (MSHA pilus) in the background of a ΔflrA mutant, inhibited vps-independent biofilm formation. Complementation of ΔflrAΔmshA double mutant with wild-type mshA gene restored biofilm formation, suggesting that mshA mutation inhibited ΔflrA-driven biofilm. Taken together, our data suggest that V. cholerae flrA and mshA act inversely in promoting vps-independent biofilm formation in FSLW. Using a standard chemotactic assay, we demonstrated that vps-independent biofilm of V. cholerae, in contrast to vps-dependent biofilm, promoted bacterial movement toward chitin and phosphate in FSLW. A ΔflrAΔmshA double mutant inhibited the bacterium from moving toward nutrients; this phenomenon was reversed with reverted mutants (complemented with wild-type mshA gene). Movement to nutrients was blocked by mutation in a key chemotaxis gene, cheY-3, although, cheY-3 had no effect on vps-independent biofilm. We propose that in fresh water reservoirs, V. cholerae, on repression of flagella, enhances vps-independent biofilm that aids the bacterium in acquiring nutrients, including chitin and phosphate; by doing so, the microorganism enhances its ability to persist under nutrient-limited conditions.

Virulence, Resistance, and Genomic Fingerprint Traits ofVibrio choleraeIsolated from 12 Species of Aquatic Products in Shanghai, China

“…50 The MSHA is very important for surface colonization of V. cholerae. 51 In this study, the mshA gene was detected positive in about 6.0% of the V. cholerae isolates, which was higher than that (0.8%) reported by Xu et al, 18 but lower than that (98.8%) by Rahman et al 52 The PilA, belonging to the putative Type IV pilus of V. cholerae, is a virulence factor for Vibrio vulnificus. 53 In this study, the pilA gene was absent from all the 370 V. cholerae isolates tested, but this gene was present in 0.8% of the V. cholerae isolates in a previous report.…”

Surveillance and Genomics of Toxigenic Vibrio cholerae O1 From Fish, Phytoplankton and Water in Lake Victoria, Tanzania

“…The symbiotic relationship between phytoplankton and V. cholerae is well documented and known to enhance the survival and maintenance of the pathogenicity in these strains even after cholera outbreaks have ceased (Haque et al, 2012; Mitchell et al, 2017). Attachment to aquatic organisms to take advantage of chitin and other nutrients is dependent on the MSHA (mannose - sensitive hemagglutinin) pili (Meibom et al, 2004; Sinha-Ray and Ali, 2017), which were present in our V. cholerae strains and whose expression by the msh A gene increases in warm tropical water with temperatures above 15°C (Lutz et al, 2013). The conductivity measurements and the surface water temperature in the lake averaging 25°C with an alkaline pH averaging 8.5, provides optimum conditions for survival and growth of V. cholerae (Lugomela et al, 2014; Dalusi et al, 2015a; Plisnier et al, 2015).…”

“…Moreover, in a complex environment like Lake Victoria, V. cholerae may be subjected to a number of other environmental stresses. The presence of autoinducers, AI-2 LuxP and LuxQ (Joelsson et al, 2007) in the genetic makeup of our strains favors their persistence since they are capable of quorum sensing and forming biofilm to survive and thrive (Kamruzzaman et al, 2010; Bari et al, 2013; Sinha-Ray and Ali, 2017). The strains also contained various stress response regulator proteins like the Vie SAB transduction system, the two-component response regulator proteins, and Vibrio polysaccharides (VPS) biosynthesis proteins, which are essential for environmental fitness and persistence in V. cholerae (Fong et al, 2010; Vesth et al, 2010; Lutz et al, 2013).…”

Toxigenic Vibrio cholerae evolution and establishment of reservoirs in aquatic ecosystems

“…cholerae is capable of surviving in aquatic environments due to its varied adaptive responses to stressors, including nutrient deprivation (19), changes in salinity, and temperature and predation by bacteriophages and protists (20). Persistence in the environment has been linked to a variety of phenotypes and mechanisms (21,22), including vps-dependent or vps-independent biofilm formation (23). To explore the rate at which toxigenic V. cholerae isolates replicated, we calculated the weighted average of synonymous substitution rates (K S s) as a proxy for bacterial replication rate.…”