Bartonella taylorii: A Model Organism for Studying Bartonella Infection in vitro and in vivo

Abstract

Bartonella spp. are Gram-negative facultative intracellular pathogens that infect diverse mammals and cause a long-lasting intra-erythrocytic bacteremia in their natural host. These bacteria translocate Bartonella effector proteins (Beps) into host cells via their VirB/VirD4 type 4 secretion system (T4SS) in order to subvert host cellular functions, thereby leading to the downregulation of innate immune responses. Most studies on the functional analysis of the VirB/VirD4 T4SS and the Beps were performed with the major zoonotic pathogen Bartonella henselae for which efficient in vitro infection protocols have been established. However, its natural host, the cat, is unsuitable as an experimental infection model. In vivo studies were mostly confined to rodent models using rodent-specific Bartonella species, while the in vitro infection protocols devised for B. henselae are not transferable for those pathogens. The disparities of in vitro and in vivo studies in different species have hampered progress in our understanding of Bartonella pathogenesis. Here we describe the murine-specific strain Bartonella taylorii IBS296 as a new model organism facilitating the study of bacterial pathogenesis both in vitro in cell cultures and in vivo in laboratory mice. We implemented the split NanoLuc luciferase-based translocation assay to study BepD translocation through the VirB/VirD4 T4SS. We found increased effector-translocation into host cells if the bacteria were grown on tryptic soy agar (TSA) plates and experienced a temperature shift immediately before infection. The improved infectivity in vitro was correlating to an upregulation of the VirB/VirD4 T4SS. Using our adapted infection protocols, we showed BepD-dependent immunomodulatory phenotypes in vitro. In mice, the implemented growth conditions enabled infection by a massively reduced inoculum without having an impact on the course of the intra-erythrocytic bacteremia. The established model opens new avenues to study the role of the VirB/VirD4 T4SS and the translocated Bep effectors in vitro and in vivo.

Translocation of YopJ family effector proteins through the VirB/VirD4 T4SS ofBartonella

“…B. henselae and B. taylorii were shown to translocate Bartonella effector protein D (BepD) via the VirB/VirD4 T4SS resulting in downregulation of pro-inflammatory cytokine ( e.g., TNF-α) secretion (36). However, mutant analysis indicated that B. taylorii must translocate at least one additional anti-inflammatory effector via its VirB/VirD4 T4SS that is different from the known Bep effectors (BepA-I) (37). As ByeA of B. taylorii inhibits p38 and JNK signaling, which might downregulate pro-inflammatory cytokine secretion as shown for other YopJ-family effectors, ByeA represents a candidate for this elusive immune-modulatory effector.…”

“…To test for functionality of a ByeA full-length fusion in this assay we introduced plasmids encoding HiBiT-FLAG (p control , negative control), HiBiT-FLAG-BepD (p bepD , bepD sequence of B. henselae , positive control (37)) or HiBiT-FLAG-ByeA (p byeA, byeA sequence of B. taylorii ) into the B. henselae effector-less Δ bepA-G background or the T4SS-deficient Δ virB4 background.…”

“…Escherichia coli , Bartonella strains and Yersinia strains were cultured as previously described (37, 60). See supplementary material for more detailed information.…”

The T4bSS ofLegionellafeatures a two-step secretion pathway with an inner membrane intermediate for secretion of transmembrane effectors

“…HiBiT-tagged effector proteins were overexpressed in L. pneumophila using the plasmid pxDC61. One day prior to infection, RAW 264.7 macrophages expressing LgBiT [28,29] were seeded in a white clear-bottom 96 well cell culture plate (Greiner) at 8 x 10 4 cells/well. L. pneumophila strains were grown for 2-3 days on BCYE-Agar plates supplemented with “BCYE Growth Supplements” (Oxoid) and appropriate antibiotics.…”

“…To further elucidate the importance of the presence and localization of the C-terminal secretion signal of TMD-effectors for their translocation into host cells, we adapted a split-NanoLuc based translocation assay [28][29][30]. Plasmid-expressed HiBiT-effector-fusions were translocated by L. pneumophila into RAW 264.7 macrophages, stably expressing LgBiT in their cytoplasm and luminescence was assayed over time in a microtiter plate reader.…”