Uncovering the transcriptional control of "Bartonella henselae" host adaptation factors

A recurrent theme in bacterial pathogenicity is the understanding of the regulatory events necessary for a given pathogen to progress through its infection cycle while resisting the host defense mechanisms. This progression typically requires the coordinated expression of defined sub-portions of the virulence repertoire at the same time as others need to be tightly repressed or degraded. This so-called adaptive response is ultimately linked to the ability of the pathogen to sense its direct environment and to transduce this information into the appropriate cellular response. Bacteria have evolved numerous dedicated mechanisms for perception and signal transduction that are characterized by a wide range of signal specificity. Not surprisingly, most of these systems have been adopted by pathogenic bacteria to modulate the expression of their virulence factors. In this work, we present the results of our investigations on the mounting and the regulation of the adaptive response of the zoonotic bacterial pathogen Bartonella henselae to its eukaryotic host. The VirB/D4 type IV secretion system (T4SS) is an essential machinery for the host adaptation of this stealthy pathogen. Using the regulation of this pathogenicity factor as a red thread we uncovered two critical signal transductions pathways that enable B. henselae to coordinate the expression of its virulence factors through its infection cycle. In the research article I, we describe the adaptive response of B. henselae during host cell infection and reveal the central role of the BatR/BatS two component system (TCS) for the coordination of this response. We demonstrate that this TCS is activated at the physiological pH of blood (pH7.4) and is required for the up-regulation of a critical cluster of genes that includes the genes encoding the VirB/D4 T4SS and its cognate secreted effectors (Beps). In the research article II, we present the near complete expressed proteome of B. henselae under conditions that mimic host-interaction, using a combination of saturated transcriptome profiling by RNA-seq and directed shotgun proteomics. Of particular interest, the complete membrane proteome coverage achieved reveals the dramatic re-organization taking place in this compartment during the infection process, with the differential regulation of a large panel of autotransporters, adhesins and hemin binding proteins as well as all components of the VirB/D4 T4SS. In the research article III, we describe how a dual regulatory input controls the expression of the VirB/D4 T4SS and its secreted effector proteins. We demonstrate that additionally to the BatR/BatS TCS, the expression of this host adaptation factors requires the alternative sigma factor RpoH1, which is itself controlled by the stringent response (SR) components SpoT and DksA. In contrast to the VirB/D4 T4SS, which is needed at the early stage of mammalian host colonization and require the SR components for its full expression, we show that SpoT and DksA negatively regulate the Trw T4SS, which is required for erythrocyte invasion at a later stage of the host infection. In the research article IV, we demonstrate the possible use of B. henselae to deliver DNA into human cells through its VirB/D4 T4SS and to generate stable transgenic cell lines. We propose that due to its ancestral abilities as conjugation system, this specialized transkingdom secretion system has potential for the development of new in vivo gene therapy approaches in humans. Together, these results constitute the first comprehensive analysis of B. henselae pathogenicity factors during host cell infection. Besides the elucidation of very specific regulatory aspects for the expression of the VirB/D4 T4SS and its secreted substrates, this work allows us to propose a general model for B. henselae host adaptation strategy throughout its infection cycle. In our model, the BatR/BatS pH-dependent signaling is used to distinguish between the arthropod and the mammalian host environment whereas the SR signaling allows the modulation of the bacterial response between the early and the late colonization stages of the mammalian host.