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Abstract

The trimeric autotransporter adhesin Bartonella adhesin A (BadA) plays a decisive role in infections with Bartonella henselae. Expression of BadA is crucial for bacterial autoagglutination, adhesion to host cells, binding to extracellular matrix proteins and the induction of proangiogenic reprogramming via activation of hypoxia inducible factor (HIF)-1. BadA is constructed modularly consisting of a head domain, a long and repetitive neck-stalk module and a membrane anchor domain. To analyze the function of particular BadA domains in detail, the generation of BadA deletion mutants would be highly desirable. However, because of the slow growth of B. henselae and limited tools for genetic manipulation, we established a recombinant expression system for BadA mutants in E. coli to allow functional analysis of certain BadA domains. Therefore, we used (i) a truncated BadA mutant lacking the neck-stalk module (BadA HN23) and exchanged additionally (ii) the BadA HN23 signal sequence with the E. coli OmpA signal sequence, (iii) the BadA HN23 membrane anchor with the Yersinia adhesin A (YadA) membrane anchor or (iv) exchanged both of these modules. Using a set of expression vectors, these constructs were cloned in several E. coli expression strains to analyze the biological functions of such BadA-hybrid proteins. Expression of BadA HN23 hybrids was detected via immunoblotting and fluorescence microscopy. All BadA HN23 hybrids were expressed on the surface of E. coli strains although quantitative differences were observed. No differences in collagen-binding between E. coli expressing Bad HN23 hybrids and controls were detectable. However, we have preliminary evidence that E. coli expressing BadA hybrid-proteins adheres significantly more to endothelial cells than control strains although the total amount of bound bacteria is less than B. henselae wildtype. Further experiments using recombinantly expressed BadA-hybrids should allow to investigate BadA-mediated bacteria-host cell interactions in greater detail.