Attachment to and biofilm formation on abiotic surfaces by Acinetobacter baumannii: involvement of a novel chaperone-usher pili assembly system

Abstract

Acinetobacter baumannii causes severe infections in compromised patients, survives on abiotic surfaces in hospital environments and colonizes different medical devices. In this study the analysis of the processes involved in surface attachment and biofilm formation by the prototype strain 19606 was initiated. This strain attaches to and forms biofilm structures on plastic and glass surfaces, particularly at the liquid-air interface of cultures incubated stagnantly. The cell aggregates, which contain cell stacks separated by water channels, formed under different culture conditions and were significantly enhanced under iron limitation. Electron and fluorescence microscopy showed that pili and exopolysaccharides are part of the cell aggregates formed by this strain. Electron microscopy of two insertion derivatives deficient in attachment and biofilm formation revealed the disappearance of pili-like structures and DNA sequencing analysis showed that the transposon insertions interrupted genes with the highest similarity to hypothetical genes found in Pseudomonas aeruginosa, Pseudomonas putida and Vibrio parahaemolyticus. Although the products of these genes, which have been named csuC and csuE, have no known functions, they are located within a polycistronic operon that includes four other genes, two of which encode proteins related to chaperones and ushers involved in pili assembly in other bacteria. Introduction of a copy of the csuE parental gene restored the adherence phenotype and the presence of pili on the cell surface of the csuE mutant, but not that of the csuC derivative. These results demonstrate that the expression of a chaperone-usher secretion system, some of whose components appear to be acquired from unrelated sources, is required for pili formation and the concomitant attachment to plastic surfaces and the ensuing formation of biofilms by A. baumannii cells.

Insights into Acinetobacter baumannii pathogenicity

“…One of the earliest studies showed the importance of exopolysaccharide production and pilus formation to biofilm development in A. baumannii (5). Using random mutagenesis and screening of mutants for biofilm formation, csuE was identified as being important in bacterial attachment and biofilm formation, as well as pili production and assembly (5). Further genetic characterization identified a csu operon, made up of six genes (csuA/BABCDE), which encodes a chaperoneusher secretion system that is important for pilus assembly (5).…”

“…For example, the mean survival time of clinical isolates on glass coverslips when subjected to dehydrating conditions was 27 days (26), and it has been shown that A. baumannii can survive on hospital bed rails for up to 9 days (27). Indeed, the pioneering studies on A. baumannii pathogenesis focused on the ability of A. baumannii to form biofilms on abiotic surfaces, as well as the organism's ability to survive in iron-limiting conditions (5,11,12). The biofilm studies were mainly performed using a 96-well polystyrene assay, as well as attachment to glass and other plastic surfaces.…”

“…Using random mutagenesis and screening of mutants for biofilm formation, csuE was identified as being important in bacterial attachment and biofilm formation, as well as pili production and assembly (5). Further genetic characterization identified a csu operon, made up of six genes (csuA/BABCDE), which encodes a chaperoneusher secretion system that is important for pilus assembly (5). Despite the importance of the csu operon in biofilm formation on abiotic surfaces, it is less important for adherence to mammalian cells (28).…”

“…medical devices and environmental surfaces (3)(4)(5)(6); (ii) its ability to adhere to, colonize and invade human epithelial cells (7,8); (iii) its repertoire of antibiotic resistance mechanisms that are able to be promptly up-regulated as required; and (iv) its ability to acquire foreign genetic material through lateral gene transfer to promote its own survival under antibiotic and host selection pressures (9,10). Specific virulence mechanisms identified to date are summarized in Table 1.…”

Persistence of Vibrio parahaemolyticus in the Pacific Oyster, Crassostrea gigas, Is a Multifactorial Process Involving Pili and Flagella but Not Type III Secretion Systems or Phase Variation

“…V. parahaemolyticus encodes a homologue of type I pili that is most similar to the CsuA/B operon from Acinetobacter baumannii (7) and homologues of two wellstudied type IV pili from vibrios, the mannose-sensitive hemagglutinin (MSHA) and the chitin-regulated pilus (PilA) (http://img .jgi.doe.gov/) (8,9). Vibrio vulnificus uses PilA for persistence in Crassostrea virginica (10,11).…”