Heterologous Expression of Bartonella Adhesin A in Escherichia coli by Exchange of Trimeric Autotransporter Adhesin Domains Results in Enhanced Adhesion Properties and a Pathogenic Phenotype

Abstract

c Human-pathogenic Bartonella henselae causes cat scratch disease and vasculoproliferative disorders. An important pathogenicity factor of B. henselae is the trimeric autotransporter adhesin (TAA) Bartonella adhesin A (BadA), which is modularly constructed, consisting of a head, a long and repetitive neck-stalk module, and a membrane anchor. BadA is involved in bacterial autoagglutination, binding to extracellular matrix proteins and host cells, and in proangiogenic reprogramming. The slow growth of B. henselae and limited tools for genetic manipulation are obstacles for detailed examination of BadA and its domains. Here, we established a recombinant expression system for BadA mutants in Escherichia coli allowing functional analysis of particular BadA domains. Using a BadA mutant lacking 21 neck-stalk repeats (BadA HN23), the BadA HN23 signal sequence was exchanged with that of E. coli OmpA, and the BadA membrane anchor was additionally replaced with that of Yersinia adhesin A (YadA). Constructs were cloned in E. coli, and hybrid protein expression was detected by immunoblotting, fluorescence microscopy, and flow cytometry. Functional analysis revealed that BadA hybrid proteins mediate autoagglutination and binding to collagen and endothelial cells. In vivo, expression of this BadA construct correlated with higher pathogenicity of E. coli in a Galleria mellonella infection model.

Sequential Translocation of Polypeptides across the Bacterial Outer Membrane through the Trimeric Autotransporter Pathway

“…On the basis of first principles, it would certainly be expected that the exceptionally strong network of interactions that define the native state would be required to prevent the three subunits from dissociating under such harsh conditions. Indeed, the presence of stable TAA trimers has been taken as evidence that TAAs can assemble correctly in unnatural environments (e.g., mitochondria) and that two different TAAs can form completely assembled heterotrimers (64)(65)(66). We cannot determine if partially assembled derivatives of UpaG migrated as heat-and SDS-resistant trimers because ␤ barrel domains that contain one or more unresolved hairpins are unexpectedly stable or because structural changes occurred during sample preparation.…”

Adh enhances Actinobacillus pleuropneumoniae pathogenicity by binding to OR5M11 and activating p38 which induces apoptosis of PAMs and IL-8 release

“…Adhesion and invasion are key processes during various bacterial infections 29 . Trimeric autotransporter adhesion (TAA) is an important virulence factor that enhances bacterial adhesion to host cells and mediates biofilm formation 30 31 . Current studies suggest that excessive inflammation is an important pathogenic mechanism in many diseases.…”

Interaction with the host: the role of fibronectin and extracellular matrix proteins in the adhesion of Gram-negative bacteria

“…This suggests that this neck-stalk module might mediate host cell adherence via interactions with, e.g., beta-1 integrins [45]. The head and a short part of the neck-stalk BadA module was found to be sufficient for collagen binding after expression of a truncated BadA hybrid in E. coli resulting in significantly higher adherence rates to endothelial cells than in E. coli controls [122]. These and other data demonstrate that the BadA protein is a major pathogenicity factor of B. henselae [44] and that the head and stalk domains of the protein have overlapping functions in the adhesion process [45].…”