Calcium promotes exopolysaccharide phase variation and biofilm formation of the resulting phase variants in the human pathogen Vibrio vulnificus

Abstract

Vibrio vulnificus is a Gram-negative bacterium found in estuaries and coastal waters and is associated with human disease caused by ingestion of raw shellfish. Pathogenesis is directly related to the presence of capsular polysaccharide (CPS). Encapsulated virulent strains exhibit an opaque colony phenotype, while unencapsulated attenuated strains appear translucent. A third colony type, rugose, is caused by expression of rugose extracellular polysaccharide (rEPS) and forms robust biofilms. Vibrio vulnificus undergoes phase variation associated with altered levels of CPS and rEPS, and we show here that calcium (Ca²(+) ) significantly increases the rate of CPS and rEPS phase variation in this species. Interestingly, multiple phenotypic responses to increased [Ca²(+) ] were observed among strains, which suggests the existence of underlying cognate genetic or epigenetic differences. Certain translucent isolates contained deletions at the group I CPS operon, inferring increased [Ca²(+) ] upregulates existing phase variation mechanisms. Expanding on a previous observation (Kierek and Watnick, Proc. Natl. Acad. Sci. USA 100: 14357-14362, 2003), increased [Ca²(+) ] also enhanced biofilm formation for all phase variants. Our results show that Ca²(+) promotes both polysaccharide phase variation and biofilm formation of the resulting phase variants, thereby likely serving a dual role in persistence of V. vulnificus in the environment.

Calcium Causes Multimerization of the Large Adhesin LapF and Modulates Biofilm Formation by Pseudomonas putida

“…Previous research has shown that increasing amounts of calcium disable Bap-mediated biofilm formation and intercellular adhesion of Staphylococcus aureus (1). However, recent reports indicate that calcium promotes biofilm formation in other microorganisms, such as Xyllela fastidiosa or Vibrio vulnificus (4,7). Therefore, calcium may modulate biofilm formation in opposite ways in different bacteria, but the molecular basis for its role remains unclear.…”

“…Calcium has been related to a variety of biological processes in bacteria (4,7,10), but its role in biofilm development is somewhat controversial. Previous research has shown that increasing amounts of calcium disable Bap-mediated biofilm formation and intercellular adhesion of Staphylococcus aureus (1).…”

Bacterial Genome Instability

“…Environmental or intracellular stress conditions that result in DNA supercoiling can modify phase variation switching frequency (445). Some systems are regulated by information about the cell's location, outside or within a host or a tissue, that can be given by the temperature (446,447) or the composition of the environment (pH [448], carbon sources [449], oxygen [450] and iron [451] levels, amino acid concentrations [446], or the presence of specific elements [452]). Certain mechanisms answer to eukaryotic host-specific signal molecules, such as the presence of sialic acid, which is released by the host as a defense mechanism (453).…”

Role of Anaerobiosis in Capsule Production and Biofilm Formation in Vibrio vulnificus

“…A correlation between polysaccharide production and biofilm formation has been demonstrated in V. vulnificus, V. cholerae, V. parahaemolyticus, and V. fischeri under aerobic conditions; however, this relationship has yet to be investigated anaerobically (40,(43)(44)(45)(46). The genome of V. vulnificus contains multiple highly conserved polysaccharide loci, including those that produce exopolysaccharides (EPS), lipopolysaccharides (LPS), and capsular polysaccharides (CPS) (31,33,34,36,(47)(48)(49). EPS has been shown to correlate positively with biofilm formation in V. vulnificus (50), but previously a negative relationship between CPS and biofilm formation aerobically in this bacterium, as well as other Vibrio species, has been described (43,44,51).…”

“…Encoded in these operons are three highly conserved genes, wza wzb and wzc, involved in transport of polymers across the outer membrane of the cell (31,33,34). Environmental conditions have been previously shown to initiate differential regulation of group 1 CPS genes and play a role in phase variation in this bacterium as well as other human pathogens (29,32,35,36). It is important to understand the regulation of CPS genes in V. vulnificus during anaerobiosis, a condition it encounters in its natural environment and in the human host (14,15,37).…”