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Title: Interactions between pathogenic and non-pathogenic Rickettsiales and the tick host
Author: Al-Khafaji, A. L. A. A.
ISNI:       0000 0004 7656 4769
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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Background: Ticks are obligate haematophagous arthropods constituting one of the most important groups of vectors for parasitic, bacterial and viral diseases of humans and other animals. Ticks have complex microbiomes, but only a small proportion of the bacterial symbionts recorded from ticks are vertically transmitted. In this study, we focus on Rickettsiales bacteria found in ticks, some of which are vertebrate pathogens, and how they interact with their tick hosts by analysing different aspects of their ecology and molecular evolution, as well as the cellular responses they induce in host cells. Methods: Three species of ticks, Ixodes ricinus from Western Europe, Amblyomma variegatum from Cameroon, and Ixodes scapularis from the Eastern USA, were examined in this study alongside several species of Rickettsiales found in each. Bacterial densities in each tick species were quantified by qPCR. For the symbiont of I. ricinus, Candidatus (Ca.) Midichloria mitochondrii, a multi-locus sequence typing (MLST) scheme for both the symbiont and host mitochondria was used to examine potential co-evolution across the UK and mainland Europe. For the vertebrate pathogen Anaplasma phagocytophilum, in vitro culture in tick cells coupled with next-generation genome sequencing and both shotgun and targeted proteomics was used to investigate host-pathogen interactions. Finally, geLC-MS was used to quantify protein expression from the symbiont of I. scapularis, Rickettsia buchneri, in tick ovaries and salivary glands. Results: A quantitative survey of Ca. M. mitochondrii in I. ricinus was conducted in the UK for the first time, revealing a similar pattern of infection across different tick life stages as recorded for mainland Europe, except that the prevalence rate in nymphs and adult male ticks in Wales was unexpectedly high. The MLST analysis of tick and symbiont revealed that Ca. M. mitochondrii exhibits very low levels of sequence diversity, although a consistent signal of host-symbiont coevolution was apparent in Scotland. Moreover, the tick MLST scheme revealed that Scottish specimens form a clade that is partially separated from other British ticks, with almost no contribution of continental sequence types in this north-westerly border of the tick's natural range. The prevalence of two Rickettsiales bacteria in the tropical bont tick, A. variegatum, in the Adamawa Region of Cameroon was very low (3.1%) for Ehrlichia ruminantium (the causative agent of heartwater disease in ruminants), and very high (95.3%) for the vertically transmitted Rickettsia africae (the causative agent of African tick-bite fever in humans). A genome for a British isolate of A. phagocytophilum (strain Old Sourhope from sheep) was obtained for the first time. This was used to support a quantitative label-free mass spectrometry analysis to examine changes in the proteome of two tick cell lines (ISE6 and IRE/CTVM19) in response to infection at different time-points. Interestingly, we observed an apparent shared response involving cholesterol hijacking by the pathogen in both cell lines; however, the IRE/CTVM19 responses reflected the initial stages of infection, while most of the ISE6 responses were derived from the establishment phase of infection. Targeted proteomics was used to quantify expression of an A. phagocytophilum variable outer membrane protein family (Msp2/p44) in vitro. Although few significant differences in abundance between the two time-points in culture were apparent, the dominant Msp2/p44 we observed shared orthologues primarily with Anaplasma strains infecting ruminants, rather than other mammalian hosts. Finally, the presence of the non-pathogenic R. buchneri was demonstrated in salivary glands of I. scapularis for the first time, suggesting that it might generate an immune response of diagnostic significance in humans. Expression of biotin synthase from a R. buchneri plasmid was detected in tick ovaries, indicating a possible nutritional basis to the symbiosis. Conclusions: Many tick species are potentially infected with multiple species of Rickettsiales. To date, the main research focus has been on those species that cause disease in vertebrates. However, non-pathogenic Rickettsiales are likely to interact with their pathogenic relatives and possibly other tick-borne pathogens in the tick host. Here, we have contributed to our understanding of the roles of three vertically-transmitted symbionts in three of the most important tick vectors worldwide: Ca. M. mitochondrii in I. ricinus, R. africae in A. variegatum, and R. buchneri in I. scapularis. The use of tick cells to examine Rickettsiales:tick interactions in vitro, as shown here with A. phagocytophilum, could be expanded in future to include pathogen-symbiont coinfections.
Supervisor: Makepeace, Ben ; McGarry, John ; Darby, Alistair Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral