Orientia tsutsugamushi Stimulates an Original Gene Expression Program in Monocytes: Relationship with Gene Expression in Patients with Scrub Typhus

Orientia tsutsugamushi is the causal agent of scrub typhus, a public health problem in the Asia-Pacific region and a life-threatening disease. O. tsutsugamushi is an obligate intracellular bacterium that mainly infects endothelial cells. We demonstrated here that O. tsutsugamushi also replicated in monocytes isolated from healthy donors. In addition, O. tsutsugamushi altered the expression of more than 4,500 genes, as demonstrated by microarray analysis. The expression of type I interferon, interferon-stimulated genes and genes associated with the M1 polarization of macrophages was significantly upregulated. O. tsutsugamushi also induced the expression of apoptosis-related genes and promoted cell death in a small percentage of monocytes. Live organisms were indispensable to the type I interferon response and apoptosis and enhanced the expression of M1-associated cytokines. These data were related to the transcriptional changes detected in mononuclear cells isolated from patients with scrub typhus. Here, the microarray analyses revealed the upregulation of 613 genes, which included interferon-related genes, and some features of M1 polarization were observed in these patients, similar to what was observed in O. tsutsugamushi-stimulated monocytes in vitro. This is the first report demonstrating that monocytes are clearly polarized in vitro and ex vivo following exposure to O. tsutsugamushi. These results would improve our understanding of the pathogenesis of scrub typhus, during which interferon-mediated activation of monocytes and their subsequent polarization into an M1 phenotype appear critical. This study may give us a clue of new tools for the diagnosis of patients with scrub typhus

Involvement of Complexin 2 in Docking, Locking and Unlocking of Different SNARE Complexes during Sperm Capacitation and Induced Acrosomal Exocytosis

Acrosomal exocytosis (AE) is an intracellular multipoint fusion reaction of the sperm plasma membrane (PM) with the outer acrosomal membrane (OAM). This unique exocytotic event enables the penetration of the sperm through the zona pellucida of the oocyte. We previously observed a stable docking of OAM to the PM brought about by the formation of the trans-SNARE complex (syntaxin 1B, SNAP 23 and VAMP 3). By using electron microscopy, immunochemistry and immunofluorescence techniques in combination with functional studies and proteomic approaches, we here demonstrate that calcium ionophore-induced AE results in the formation of unilamellar hybrid membrane vesicles containing a mixture of components originating from the two fused membranes. These mixed vesicles (MV) do not contain the earlier reported trimeric SNARE complex but instead possess a novel trimeric SNARE complex that contained syntaxin 3, SNAP 23 and VAMP 2, with an additional SNARE interacting protein, complexin 2. Our data indicate that the earlier reported raft and capacitation-dependent docking phenomenon between the PM and OAM allows a specific rearrangement of molecules between the two docked membranes and is involved in (1) recruiting SNAREs and complexin 2 in the newly formed lipid-ordered microdomains, (2) the assembly of a fusion-driving SNARE complex which executes Ca2+-dependent AE, (3) the disassembly of the earlier reported docking SNARE complex, (4) the recruitment of secondary zona binding proteins at the zona interacting sperm surface. The possibility to study separate and dynamic interactions between SNARE proteins, complexin and Ca2+ which are all involved in AE make sperm an ideal model for studying exocytosis

Orientia tsutsugamushi in Human Scrub Typhus Eschars Shows Tropism for Dendritic Cells and Monocytes Rather than Endothelium

Scrub typhus is a common and underdiagnosed cause of febrile illness in Southeast Asia, caused by infection with Orientia tsutsugamushi. Inoculation of the organism at a cutaneous mite bite site commonly results in formation of a localized pathological skin reaction termed an eschar. The site of development of the obligate intracellular bacteria within the eschar and the mechanisms of dissemination to cause systemic infection are unclear. Previous postmortem and in vitro reports demonstrated infection of endothelial cells, but recent pathophysiological investigations of typhus patients using surrogate markers of endothelial cell and leucocyte activation indicated a more prevalent host leucocyte than endothelial cell response in vivo. We therefore examined eschar skin biopsies from patients with scrub typhus to determine and characterize the phenotypes of host cells in vivo with intracellular infection by O. tsutsugamushi, using histology, immunohistochemistry, double immunofluorescence confocal laser scanning microscopy and electron microscopy. Immunophenotyping of host leucocytes infected with O. tsutsugamushi showed a tropism for host monocytes and dendritic cells, which were spatially related to different histological zones of the eschar. Infected leucocyte subsets were characterized by expression of HLADR+, with an “inflammatory” monocyte phenotype of CD14/LSP-1/CD68 positive or dendritic cell phenotype of CD1a/DCSIGN/S100/FXIIIa and CD163 positive staining, or occasional CD3 positive T-cells. Endothelial cell infection was rare, and histology did not indicate a widespread inflammatory vasculitis as the cause of the eschar. Infection of dendritic cells and activated inflammatory monocytes offers a potential route for dissemination of O. tsutsugamushi from the initial eschar site. This newly described cellular tropism for O. tsutsugamushi may influence its interaction with local host immune responses

Etude de la réponse immunitaire innée au cours de l'infection à Orientia tsutsugamushi

Orientia tsutsugamushi, l'agent pathogène responsable du typhus des broussailles, est une bactérie cytosolique qui envahit l'endothélium et les monocytes/macrophages. La réponse immune à l'infection par O. tsutsugamushi reste à ce jour mal connue. L'objectif de cette thèse est de mieux comprendre la réponse des cellules de la réponse immune innée humaine à O. tsutsugamushi. Nous avons montré que O. tsutsugamushi se réplique dans les monocytes humains. En utilisant un microarray portant sur la totalité du génome, nous avons également montré que les bactéries vivantes induisent de profondes modifications du profil transcriptionnel des monocytes. C'est ainsi que l'expression des gènes codant l'interféron de type I et des gènes stimulés par l'interféron est fortement augmentée. Les monocytes infectés expriment plusieurs gènes codant des cytokines et des chimiokines inflammatoires, ce qui montre qu'ils sont polarisés vers un phénotype M1 (classically-activated phenotype). Les bactéries vivantes induisent également la sécrétion de l'interleukine-1β et probablement l'activation des inflammasomes et de la caspase-1. O. tsutsugamushi affecte enfin l'expression des gènes associés à l'apoptose et induit la mort d'une partie des monocytes infectés. Nous avons en outre étudié le profil transcriptionnel de patients atteints d'un typhus des broussailles et avons trouvé une signature spécifique incluant la modulation de gènes de type M1 et de gènes stimulés par l'interféron. Nous avons finalement étudié la réponse des macrophages humains dérivés des monocytes à O. tsutsugamushi.Orientia tsutsugamushi, the causative pathogen of scrub typhus, is a cytosolic bacterium that invades endothelium and monocytes/macrophages. So far, the knowledge of immune response to O. tsutsugamushi is still limited. The objective of this thesis is to better understand the response of human innate immune cells against this pathogen. We demonstrated that O. tsutsugamushi was able to replicate in human monocytes. Using whole genome microarrays, we showed that live O. tsutsugamushi induced robust changes in the transcriptional profiles of monocytes. First, type I interferons and interferon-stimulated genes were remarkably up-regulated. Second, infected monocytes expressed several inflammatory cytokine and chemokine genes, and were polarized toward the classically-activated M1 phenotype. Third, live bacteria induced interleukin-1β secretion and likely inflammasome and caspase-1 activation. We also showed that O. tsutsugamushi altered the expression of apoptosis-related genes and induced cell death in monocytes. We extended our work to the study of the transcriptional profiles of patients with scrub typhus and found a specific signature in patients that included the modulation of M1-associated genes and interferon-stimulated genes. We finally studied the response of human monocyte-derived macrophages to O. tsutsugamushi. The transcriptional and functional responses of macrophages to O. tsutsugamushi were roughly similar to those observed in circulating monocytes including type I IFN response, pro-inflammatory cytokine gene expression and IL-1β secretion

Anti-Mycoplasma Activity of Daptomycin and Its Use for Mycoplasma Elimination in Cell Cultures of Rickettsiae

Mycoplasma contamination detrimentally affects cellular functions and the growth of intracellular pathogens in cell cultures. Although several mycoplasmacidal agents are commercially available for sterile cell cultures, they are not applicable to rickettsia-infected cells. In our attempt to find an anti-mycoplasma drug for contaminated rickettsial cultures, we determined the susceptibilities of three common Mycoplasma species to daptomycin. Mycoplasma orale and M. arginini showed low-level resistance to daptomycin (minimum inhibitory concentration, MIC = 2 mg/L), whereas M. hyorhinis was high-level resistant (MIC = 32 mg/L). However, some Mycoplasma isolates developed higher resistance to daptomycin after failed treatments with inadequate doses or durations. An aminoglycoside (gentamicin) was still active against M. hyorhinis and could be used in Orientia cultures. For complete eradication of mycoplasmas in Rickettsia cultures, we recommend a 3-week treatment with daptomycin at 256 mg/L. In contaminated Orientia cultures, daptomycin at 32 mg/L was effective in eradicating M. orale, whereas either gentamicin or amikacin (100 mg/L) was effective in eradicating M. hyorhinis. Unlike each drug alone, the combinations of daptomycin plus clindamycin and/or quinupristin/dalfopristin proved effective in eradicating M. hyorhinis. In summary, our study demonstrated the in vitro anti-mycoplasma activity of daptomycin and its application as a new mycoplasma decontamination method for Rickettsia and Orientia cultures