Date Published: September 25, 2015
Publisher: Public Library of Science
Author(s): Jin Hwan Park, Youmi Jo, Song Yee Jang, Haenaem Kwon, Yasuhiko Irie, Matthew R. Parsek, Myung Hee Kim, Sang Ho Choi, Karla J.F. Satchell.
A transcriptome analysis identified Vibrio vulnificus cabABC genes which were preferentially expressed in biofilms. The cabABC genes were transcribed as a single operon. The cabA gene was induced by elevated 3′,5′-cyclic diguanylic acid (c-di-GMP) and encoded a calcium-binding protein CabA. Comparison of the biofilms produced by the cabA mutant and its parent strain JN111 in microtiter plates using crystal-violet staining demonstrated that CabA contributed to biofilm formation in a calcium-dependent manner under elevated c-di-GMP conditions. Genetic and biochemical analyses revealed that CabA was secreted to the cell exterior through functional CabB and CabC, distributed throughout the biofilm matrix, and produced as the biofilm matured. These results, together with the observation that CabA also contributes to the development of rugose colony morphology, indicated that CabA is a matrix-associated protein required for maturation, rather than adhesion involved in the initial attachment, of biofilms. Microscopic comparison of the structure of biofilms produced by JN111 and the cabA mutant demonstrated that CabA is an extracellular matrix component essential for the development of the mature biofilm structures in flow cells and on oyster shells. Exogenously providing purified CabA restored the biofilm- and rugose colony-forming abilities of the cabA mutant when calcium was available. Circular dichroism and size exclusion analyses revealed that calcium binding induces CabA conformational changes which may lead to multimerization. Extracellular complementation experiments revealed that CabA can assemble a functional matrix only when exopolysaccharides coexist. Consequently, the combined results suggested that CabA is a structural protein of the extracellular matrix and multimerizes to a conformation functional in building robust biofilms, which may render V. vulnificus to survive in hostile environments and reach a concentrated infective dose.
Biofilm formation provides bacteria with protection from antimicrobial agents and host immune defense systems during infection as well as from a variety of stresses in the environment [1,2]. Therefore, biofilms of pathogenic bacteria are considered as one of the most important causes for new outbreaks and account for 65% of bacterial infections in humans . Biofilm formation can be divided into sequential developmental stages beginning from initial surface attachment of planktonic bacteria continuing to multicellular structures (microcolony), subsequent maturation to biofilms, and detachment (dispersal) of bacterial cells from mature biofilms [1,2,4]. Mature biofilms are specialized and highly differentiated three-dimensional communities of bacteria encased in an extracellular polymeric matrix (EPM), the framework contributing to the organization and maintenance of biofilm structure and stability . The major components of the EPM are polysaccharides, proteins, nucleic acids, and lipids, which are distributed between the cells in a non-homogeneous pattern . Changes in colony morphology frequently reflect variations in biofilm matrix production levels, as smooth colony morphology of bacterial cells can switch to rugose (or wrinkled) by producing increased levels of EPM .
The bacterial secondary messenger, c-di-GMP, is a global regulatory molecule that controls multiple phenotype changes in the bacterial world. Among the phenotype changes affected by c-di-GMP, life style switches from free-living planktonic cells to sessile biofilms have been most extensively studied . Although little is known about the c-di-GMP regulation of the phenotype changes of V. vulnificus, it has been reported that increased intracellular c-di-GMP levels induced production of EPS, a component of the Vibrio spp. biofilm matrix, and subsequent development of biofilm and rugose colonies [7,22]. In this study, elevated c-di-GMP induced the expression of cabA encoding a calcium-binding matrix protein CabA (Fig 3B and 3C). A mutation in cabA significantly impaired biofilm development even in the presence of elevated c-di-GMP without reducing the amount of EPS produced (Figs 4, 7, 9, and 10). The results indicated that EPS alone is not sufficient to develop the well-structured robust biofilm and that CabA is another key component of the V. vulnificus biofilm matrix.