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.

Thesis (PhD (Botany and Zoology))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The Rattus sensu lato complex, which harbours the important biomedical model species Rattus norvegicus, represents a group of rodents that are of clinical, agricultural and epidemiological importance. This study presents a comprehensive comparative molecular cytogenetic investigation of this complex. Karyotypes of 11 Rattus s. l. species and Hapalomys delacouri were analysed using conventional cytogenetic techniques. These data suggest that, contrary to previous claims, the usefulness of karyotypes for identifying these Asian murid species is limited, as few species-specific chromosomal characters could be identified. In order to understand the events that shaped the morphology and composition of the extant karyotypes, nine out of the 11 Rattus s. l. species were analysed by chromosome painting. This allowed the detection of a rare convergent sex-autosome translocation present in H. delacouri and B. savilei, whose establishment is hypothesised to have been favoured by the presence of interstitial heterochromatic blocks (IHBs) at the boundaries of the translocated segments. These results indicate that Rattus s. l. is characterised by slow to moderate rates of chromosome evolution in contrast to the extensive chromosome restructuring identified in most other murid rodents. Based on these data the first comprehensive putative Rattini ancestral karyotype was constructed. Their integration with published comparative maps enabled a revision of the previously postulated ancestral murid karyotype. BAC-mapping unequivocally demonstrated that the widely reported polymorphisms affecting chromosome pairs 1, 12 and 13 in the Rattus karyotypes are due to pericentric inversions. The analysis of genomic features, such as telomeres, Ag-NORs and satellite DNA suggest a constrained pattern of chromosome evolution. The investigated rat satellite I DNA family appears to be taxon (Rattus) specific, and of recent origin (consistent with a feedback model of satellite evolution). The comparative nature of the study led to the further analysis of the species within Rattus s. l. complex and their possible involvement as reservoirs of scrub typhus using a qPCR gene expression approach based on real-time PCR. The structure and transcription of syndecan-4, which had previously been linked to Orientia tsutsugamushi (causative agent of scrub typhus) infection, was compared among Rattini (typhus-positive) and Murini (typhus-negative) rodents. Although, it was not possible to conclusively link the structural variation observed between Rattini and Murini with carrier status, a link was identified between underexpression of syndecan-4 in Murini and seropositive Rattini rodents, compared to those that were seronegative. This suggests that the reduced levels of syndecan-4 transcription in Murini and Rattini is linked to the poor carrier status of Murini, and to increased longevity of seropositive Rattini (i.e., predominant host of Orientia), highlighting aspects that need further investigation. / AFRIKAANSE OPSOMMING: Die Rattus sensu lato kompleks, wat die belangrike biomediese model spesie Rattus norvegicus huisves, bevat ‘n groep knaagdiere wat van kliniese, landbou, en epidiomologiese belang is. Hierdie studie bevat ‘n omvattende sitogenetiese molekulêre vergelyking binne hierdie kompleks. Die kariotipes van 11 Rattus s. l. spesies en Hapalomys delacouri is ondersoek met die gebruik van konvensionele sitogenetiese tegnieke. Die data dui daarop, in kontras met vorige bevindinge, dat die bruikbaarheid van kariotipes om Asiese knaagdiere te identifiseer beperk is, aangesien min spesies-spesifieke chromosoom merkers geidentifiseer kon word. Om die gebeure wat tot die morfologie en komposisie van die huidige kariotipes gelei het, te verstaan, is nege van die 11 Rattus s. l. spesies met behulp van chromosoom fluoressente hibridisasie ondersoek. Dit het die ontdekking van ‘n rare konvergente geslagschromosoom-outosoom translokasie in H. delacouri en B. savilei tot gevolg gehad. Die vaslegging hiervan is heel moontlik bevoordeel deur die teenwoordigheid van interkalerende heterchromatien blokke (IHBs) by die grens van die translokeerde segmente. Hierdie resultate dui daarop dat Rattus s. l. deur ‘n stadige tempo van chromosoom verandering gekenmerk word, wat in skrille kontras staan met die hoë aantal chromosoom herrangskikkings wat in meeste ander murid knaagdiere geidentifiseer is. Vanuit hierdie data kon die eerste oerouer kariotipe van die Rattini bepaal word. Die gebruik van hierdie data in kombinasie met beskikbare vergelykende kaarte het dit moontlik gemaak om die vorige hipotetiese oerouer murid kariotipe te hersien. BAC-kartering het dit moontlik gemaak om sonder twyfel vas te stel dat die polimorfisme wat chromosoom pare 1, 12 en 13 in die Rattus kariotipe affekteer, die resultaat van perisentriese omsetting is. Die analise van genomiese eienskappe, soos telomere, Ag-NORs en satelliet DNA dui op ‘n beperkte patroon van chromosoom evolusie. Die bestudeerde rot satelliet DNA familie blyk takson (Rattus) spesifiek te wees met ‘n onlangse oorsprong, wat ooreenstem met die terugvoer model van satellite-evolusie. Die vergelykende aard van hierdie studie het gelei tot die verdere analise van die spesies in die Rattus s. l. kompleks in terme van hul moontlike rol as draers van “scrub typhus” deur gebruik te maak van qPCR geen uitdrukking, wat gebasseer is op “real-time” PCR. Die struktuur en transkripsie van syndecan-4, wat in die verlede aan Orientia tsutsugamushi (veroorsakende agent van scrub typhus) infeksie gekoppel is, is tussen Rattini (typhuspositief) en Murini (typhus-negatief) knaagdiere vergelyk. Ten spyte daarvan dat dit nie moontlik was om die strukturele variasie tussen Rattini en Murini met draer status te koppel nie, is daar wel ‘n skakel tussen die verlaagde uitdrukking van syndecan-4 in Murini en seropositiewe Rattini knaagdiere, in vergelyking met die wat sero-negatief was, gevind. Dit stel voor dat die verlaagde vlakke van syndecan-4 transkripsie in Murini en Rattini aan die swak draer status van Murini, asook die verhoogde langslewendheid van sero-positiewe Rattini (i.e., oorheersende gasheer van Orientia), gekoppel is. Hierdie bevindinge beklemtoon sekere belangrike aspekte vir verdere navorsing.

Thesis (M.C.T.M. (Clinical Tropical Medicine))--Mahidol University, 2003.

Thesis (M.C.T.M. (Clinical Tropical Medicine))--Mahidol University, 2002.

Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular pathogen. Recent studies show that the complete genome sequence of Orientia tsutsugamushi have been determined. However, the early signaling events involved in the entry of O.tsutsugamushi into mammalian cells remains a challenge. In this study, we demonstrate that adherence ability and comparison of three major outer membrane protein TSA56, TSA47 and TSA22 of O.tsutsugamushi. Through expression and purification of three type-specific antigen 56-kDa (include TSA56-antigen domain I, TSA56-antigen domain III), 47-kDa and 22-kDa of O. tsutsugamushi , antiserum immunoblots from 22 clinical O. tsutsugamushi-infected patients and in vitro adhesion assay of E.coli overexpression outer membrane protein of O. tsutsugamushi , the antiserum titer and adherence ability of bacterial outer membrane proteins are determined. The data show that antiserum titer against three major outer membrane proteins of O. tsutsugamushi was markedly higher in TSA56 compared to TSA47 and TSA22. In adhesion assay, adhesion of host cells by TSA56 was readily than TSA47 and TSA22. Furthermore, adhesion experiment and antiserum titer against antigen-domain I (ADI) region (19-114 aa) in the extracellular domain of TSA56 was also significantly higher than previously reported antigen-domain III(ADIII) region (237-366 aa) which facilitates the invasion of O. tsutsugamushi through interaction with fibronectin .Taken together, these results clearly indicate that O. tsutsugamushi exploits TSA56-mediated bacterial adhesion, abundant antiserum titer and ADI region of TSA56 may draw another adhesion site (except for previously reported ADIII) to invade eukaryotic host cells.