Bartonella henselae trimeric autotransporter adhesin BadA expression interferes with effector translocation by the VirB/D4 type IV secretion system Yun-Yueh Lu, 1† Bettina Franz, 2† Matthias C. Truttmann, 1† Tanja Riess, 2 Jérémie Gay-Fraret, 1 Marco Faustmann, 1‡ Volkhard A. J. Kempf 2 ** and Christoph Dehio 1 * 1 Focal Area Infection Biology, Biozentrum of the University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. 2 Institute of Medical Microbiology and Infection Control, University Hospital Johann Wolfgang Goethe University Frankfurt am Main, Paul-Ehrlich-Strasse 40, D-60596 Frankfurt/Main, Germany. Summary The Gram-negative, zoonotic pathogen Bartonella henselae is the aetiological agent of cat scratch disease, bacillary angiomatosis and peliosis hepatis in humans. Two pathogenicity factors of B. henselae – each displaying multiple functions in host cell interaction – have been characterized in greater detail: the trimeric autotransporter Bartonella adhesin A (BadA) and the type IV secre- tion system VirB/D4 (VirB/D4 T4SS). BadA medi- ates, e.g. binding to fibronectin (Fn), adherence to endothelial cells (ECs) and secretion of vascular endothelial growth factor (VEGF). VirB/D4 translo- cates several Bartonella effector proteins (Beps) into the cytoplasm of infected ECs, resulting, e.g. in uptake of bacterial aggregates via the invasome structure, inhibition of apoptosis and activation of a proangiogenic phenotype. Despite this knowl- edge of the individual activities of BadA or VirB/D4 it is unknown whether these major virulence factors affect each other in their specific activities. In this study, expression and function of BadA and VirB/D4 were analysed in a variety of clinical B. henselae isolates. Data revealed that most isolates have lost expression of either BadA or VirB/D4 during in vitro passages. However, the phenotypic effects of coexpression of both viru- lence factors was studied in one clinical isolate that was found to stably coexpress BadA and VirB/ D4, as well as by ectopic expression of BadA in a strain expressing VirB/D4 but not BadA. BadA, which forms a dense layer on the bacterial surface, negatively affected VirB/D4-dependent Bep trans- location and invasome formation by likely pre- venting close contact between the bacterial cell envelope and the host cell membrane. In contrast, BadA-dependent Fn binding, adhesion to ECs and VEGF secretion were not affected by a functional VirB/D4 T4SS. The obtained data imply that the essential virulence factors BadA and VirB/D4 are likely differentially expressed during different stages of the infection cycle of Bartonella. Introduction Bartonella henselae is a Gram-negative, arthropod-borne and facultative intracellular bacterial pathogen with world- wide distribution and is considered to be the most relevant human pathogenic Bartonella species (Anderson and Neuman, 1997; Franz and Kempf, 2011; Harms and Dehio, 2012). Cats have been identified as the primary reservoirs of B. henselae. Zoonotic transmission from infected cats to humans occurs via cat scratches or cat fleas (Rolain et al., 2001; Dehio, 2004). Human infections with B. henselae can manifest in a broad spectrum of clinical symptoms. Immunocompetent patients typically suffer from cat scratch disease (CSD), characterized by local lymph node swelling and fever (Florin et al., 2008). In contrast, immunocompromised patients (e.g. AIDS patients) can develop vasoproliferative lesions in the skin (bacillary angiomatosis) or the liver (peliosis hepatis) respectively (Relman et al., 1990; 1991). These tumour- like lesions arise from bacterial infection of vascular ECs leading to enhanced ECs migration and proliferation (Dehio, 2005). Two major pathogenicity factors of B. henselae have been identified: VirB/D4, a type IV secretion system (T4SS) translocating Bartonella effector proteins (Beps) Received 5 June, 2012; revised 7 November, 2012; accepted 9 November, 2012. For correspondence. *E-mail christoph.dehio@ unibas.ch; Tel. (+41) 61 267 2140; Fax (+41) 61 267 2118; **E-mail volkhard.kempf@kgu.de; Tel. (+49) 69 6301 5019; Fax (+49) 69 6301 83431. † These authors contributed equally. ‡ Deceased on 20 February 2010. Cellular Microbiology (2013) 15(5), 759–778 doi:10.1111/cmi.12070 First published online 6 December 2012 © 2012 Blackwell Publishing Ltd cellular microbiology