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

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;

Uncovering the principles of membrane effector translocation in Legionella pneumophila

The type III and type IV secretion systems are a major factor of pathogenicity for many bacteria. Effector proteins translocated by the secretion machinery are used to hijack and subvert host cell functions in order to colonize and live within the host cell. Next to soluble effectors, transmembrane domain effectors can successfully be secreted by the type III and type IV secretion system and carry out their function within a host cell membrane. Before being translocated, effector proteins must be targeted to the respective secretion system. Generally, protein targeting starts in the bacterial cytoplasm where protein biosynthesis takes place, and depends on various signals residing within the protein. Type III effectors harbor a N-terminal secretion signal followed by a chaperone binding domain. In contrast, the translocation signal for effectors of the T4SS resides at the C-terminal end of the protein. Additionally, TMD-effectors of both secretion systems contain hydrophobic segments which are essential for their proper localization within the host cell. The presence of these two incompatible signals within the same protein poses a possible targeting conflict as transmembrane segments can be recognized by the Signal Recognition Particle (SRP) and result in subsequent inner membrane insertion. For transmembrane domain effectors of the T3SS it was shown that inner membrane (mis-)targeting is avoided by a balanced hydrophobicity of the TMS (passive avoidance) as well as protection by chaperone binding (active avoidance). In contrast, some transmembrane domain effectors of the Dot/Icm system in L. pneumophila are predicted to possess a sufficiently hydrophobic signal for targeting to the bacterial inner membrane by SRP. The aim of this study was to investigate if transmembrane domain effectors of the type IV secretion system can, similarly to effectors of the type III secretion system, avoid SRP targeting or uses a hypothetical two-step secretion pathway through the Dot/Icm machinery with an inner membrane intermediate. Using membrane fractionation by a sucrose gradient centrifugation protocol as well as urea extraction, I could show that transmembrane domain effectors in L. pneumophila can indeed be found properly integrated in the bacterial inner membrane. The same results were obtained when the type IV chaperones IcmSW were overexpressed suggesting that there is no active avoidance of SRP targeting and no direct delivery to the secretion machinery by chaperone binding. Investigation of the membrane topology of transmembrane domain effectors showed that transmembrane domain effectors are “anchored” in the bacterial inner membrane with a Nin-Cin topology and only small loops of few amino acids located in the periplasm. Furthermore, I could show that, similarly to soluble effectors, the C-terminal translocation signal located in the cytoplasm and possible internal signals as well as the presence of the chaperones IcmSW are crucial for the successful translocation of transmembrane domain effectors into host cells. Based on these results, I propose that transmembrane domain effectors in L. pneumophila follow a two-step secretion pathway with SRP XI targeting as the first step. Once “anchored” in the bacterial inner membrane, transmembrane domain effectors are recognized as substrates of the Dot/Icm system by IcmSW, resulting in their extraction towards the cytoplasmic side before being translocated into host cells.Dissertation ist gesperrt bis 16.12.202

Screening for eukaryotic motifs in <i>Legionella pneumophila</i> reveals Smh1 as bacterial deacetylase of host histones

Legionella pneumophila (L.p.) is a bacterial pathogen which is a common causative agent of pneumonia. In humans, it infects alveolar macrophages and transfers hundreds of virulence factors that interfere with cellular signalling pathways and the transcriptomic landscape to sustain its own replication. By this interaction, it has acquired eukaryote-like protein motifs by gene transfer events that partake in the pathogenicity of Legionella. In a computational screening approach for eukaryotic motifs in the transcriptome of Legionella, we identified the L.p. strain Corby protein ABQ55614 as putative histone-deacetylase and named it “suppressing modifier of histones 1” (Smh1). During infection, Smh1 translocated from the Legionella vacuole into the host cytosol. When expressed in human macrophage THP-1 cells, Smh1 was localized predominantly in the nucleus, led to broad histone H3 and H4 deacetylation, blunted expression of a large number of genes (e.g. IL-1β and IL-8), and fostered intracellular bacterial replication. L.p. with a Smh1 knockdown grew normally in media but showed a slight growth defect inside the host cell. Furthermore, Smh1 showed a very potent histone deacetylation activity in vitro, e.g. at H3K14, that could be inhibited by targeted mutation of the putative catalytic centre inferred by analogy with eukaryotic HDAC8, and with the deacetylase inhibitor trichostatin A. In summary, Smh1 displays functional homology with class I/II type HDACs. We identified Smh1 as a new Legionella virulence factor with a eukaryote-like histone-deacetylase activity that moderates host gene expression and might pave the way for further histone modifications. Legionella pneumophila (L.p.) is a prominent bacterial pathogen which is a common causative agent of pneumonia. In order to survive inside the host cell, the human macrophage, it profoundly interacts with host cell processes to advance its own replication. In this study, we identify a bacterial factor, Smh1, with yet unknown function as a host histone deacetylase. The activity of this factor in the host cell leads to attenuated gene expression and increased intracellular bacterial replication.</p