A BCAM0223 Mutant of Burkholderia cenocepacia Is Deficient in Hemagglutination, Serum Resistance, Adhesion to Epithelial Cells and Virulence Dalila Mil-Homens1, Arsenio M. Fialho1,2* 1 IBB-Institute for Biotechnology and Bioengineering, Center for Biological and Chemical Engineering, Instituto Superior Te´cnico, Lisbon, Portugal, 2 Department of Bioengineering, Instituto Superior Te´cnico, Technical University of Lisbon, Lisbon, Portugal Abstract Burkholderia cepacia complex (Bcc) bacteria are a problematic group of microorganisms causing severe infections in patients with Cystic Fibrosis. In early stages of infection, Bcc bacteria must be able to adhere to and colonize the respiratory epithelium. Although this is not fully understood, this primary stage of infection is believed to be in part mediated by a specific type of adhesins, named trimeric autotransporter adhesins (TAAs). These homotrimeric proteins exist on the surface of many Gram negative pathogens and often mediate a number of critical functions, including biofilm formation, serum resistance and adherence to an invasion of host cells. We have previously identified in the genome of the epidemic clinical isolate B. cenocepacia J2315, a novel cluster of genes putatively encoding three TAAs (BCAM0219, BCAM0223 and BCAM0224). In this study, the genomic organization of the TAA cluster has been determined. To further address the direct role of the putative TAA BCAM0223 in B. cenocepacia pathogenicity, an isogenic mutant was constructed via insertional inactivation. The BCAM0223::Tp mutant is deficient in hemagglutination, affected in adherence to vitronectin and in biofilm formation and showed attenuated virulence in the Galleria mellonella model of infection. Moreover, the BCAM0223::Tp mutant also showed a significant reduction in its resistance to human serum as well as in adherence, but not in invasion of, cultured human bronchial epithelial cells. Altogether these results demonstrate that the BCAM0223 protein is a multifunctional virulence factor that may contribute to the pathogenicity of B. cenocepacia. Citation: Mil-Homens D, Fialho AM (2012) A BCAM0223 Mutant of Burkholderia cenocepacia Is Deficient in Hemagglutination, Serum Resistance, Adhesion to Epithelial Cells and Virulence. PLoS ONE 7(7): e41747. doi:10.1371/journal.pone.0041747 Editor: Michael Hensel, University of Osnabrueck, Germany Received February 14, 2012; Accepted June 25, 2012; Published July 25, 2012 Copyright: 2012 Mil-Homens, Fialho. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by FEDER and Fundac¸a˜o para a Cieˆncia e a Tecnologia (FCT), Portugal (grants: PTDC/BIA-MIC/71453/2006, PTDC/EBB-BIO/ 100326/2008 and PTDC/BIA-MIC/118386/2010) and a Ph.D. fellowship to D. Mil-Homens. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: afialho@ist.utl.pt Introduction The Burkholderia cepacia complex (Bcc) is a group of closely related Gram-negative bacteria found in the environment. Over the last decades, Bcc bacteria have emerged as highly problematic opportunistic pathogens causing severe respiratory infections in cystic fibrosis (CF) patients [1]. Currently there are 17 species described as members of the Bcc complex. B. cenocepacia, B. multi- vorans and B. dolosa are those species that occur at a greater frequency and are particularly problematic [2,3]. Colonized Bcc bacteria are transmitted among CF-patients and are extremely resistant to antimicrobial therapies [4]. In a subset of CF patients, lung infections with Bcc lead to declining lung function, with necrotising pneumonia and a fatal speticemia termed ‘‘cepacia syndrome’’ [5]. Many virulence determinants associated with Bcc-lung infection have been described. Overall, they enable bacteria to attach to the surface of host cells, evade host defences and replicate either extra- or intracellularly [6,7]. During the early stages of interaction, Bcc bacteria must interact with surface receptors of the epithelial cells, such as proteins, glycolipid receptors and mucins [8]. Although these binding events remain poorly understood, it is believed that several bacterial surface-bound proteins named adhesins, are key determinants of adherence and/or invasion of target host cells. Up to date, only the cable pili-associated adhesin (cbl, 22 kDa) from B. cenocepacia strains and a 55 kDa protein from the piliated B. cenocepacia strain BC7 have been described to interact with cytokeratin 13 and TNF receptor1 (TNFR1) of epithelial cells, respectively [9,10]. However, since both proteins were absent in many pathogenic Bcc isolates [11], others uncharacterized adhesins may participate in the bacterial adherence to host cells. Among such sets of proteins are the trimeric autotransporter adhesins (TAAs), a class of adhesins produced by many Gram- negative pathogens as key virulence factors. These extracellular proteins have been shown to possess adhesive properties, thereby enabling bacterial adherence to host cells [12,13]. Over the last years, increased attention has been devoted to the study of TAAs, revealing that they are often multifunctional virulence factors with decisive roles at different stages of bacterial infection. In fact, besides their role in adherence to extracellular matrix and host cells, TAAs are also involved in several other biological traits of pathogenic bacteria including hemagglutina- tion, autoaggregation, cytotoxicity, serum resistance and invasion to host cells [14,15]. TAAs are found exclusively in Gram-negative bacteria and several of them have been characterized in detail, including among others, YadA from Yersinia enterocolitica, the PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e41747