Regulation of cell-cell adhesion in keratinocytes: The reciprocal relationship between cadherins and Rho family GTPases

Cadherins are transmembrane receptors, which mediate adhesion between adjacent cells. They play an essential role in development and maintenance of tissue architecture. The RHO GTPases modulate actin cytoskeletal architecture in different cell types. Two family members in particular, RhoA and Rad, have been found to regulate the stability of cadherin receptors at intercellular junctions. In this thesis I set out to study in more detail the relationship between cadherin-mediated adhesion and the small GTPase Rac in keratinocytes. Previous work has shown that while Rac is necessary for cell-cell contact formation, it can also disrupt cadherin-dependent adhesion when its activity is sustained. I have mapped the domain of Rac that is required for breakdown of intercellular junctions. In addition, I have shown that Rac mediates perturbation of cell-cell adhesion induced by oncogenic H-Ras. I have also investigated which Rac effectors may mediate Rac-induced disassembly of cell-cell contacts. To look for new Rac targets that may regulate cadherin-mediated adhesion, I performed a yeast two-hybrid screen of a keratinocyte library and identified a novel Rac-binding clone. Ligand binding to a variety of cell surface receptors has been found to induce Rac activation. I have found that calcium-induced cell-cell contact formation can also activate Rac. Cadherin function is necessary for Rac activation, and clustering of the cadherin receptors is sufficient to activate Rac. Initial activation of Rac is dependent on signalling from the EGF receptor but not PI 3-kinase activity. At later time-points, Rac is activated by an alternative EGF-receptor-independent mechanism. In conclusion, this work focuses on the relationship between Rac and cadherin-mediated adhesion in epithelia. This could shed light on the regulation of epithelial morphogenesis under normal conditions and potentially lead to an understanding of how this is subverted during tumorigenesis

A reverse vaccinology approach identifies putative vaccination targets in the zoonotic nematode Ascaris

Ascariasis is the most prevalent helminthic disease affecting both humans and pigs and is caused by the roundworms Ascaris lumbricoides and Ascaris suum. While preventive chemotherapy continues to be the most common control method, recent reports of anthelminthic resistance highlight the need for development of a vaccine against ascariasis. The aim of this study was to use a reverse vaccinology approach to identify potential vaccine candidates for Ascaris. Three Ascaris proteomes predicted from whole-genome sequences were analyzed. Candidate proteins were identified using open-access bioinformatic tools (e.g., Vacceed, VaxiJen, Bepipred 2.0) which test for different characteristics such as sub-cellular location, T-cell and B-cell molecular binding, antigenicity, allergenicity and phylogenetic relationship with other nematode proteins. From over 100,000 protein sequences analyzed, four transmembrane proteins were predicted to be non-allergen antigens and potential vaccine candidates. The four proteins are a Piezo protein, two voltage-dependent calcium channels and a protocadherin-like protein, are all expressed in either the muscle or ovaries of both Ascaris species, and all contained high affinity epitopes for T-cells and B-cells. The use of a reverse vaccinology approach allowed the prediction of four new potential vaccination targets against ascariasis in humans and pigs. These targets can now be further tested in in vitro and in vivo assays to prove efficacy in both pigs and humans

One Health: parasites and beyond

The field of parasitism is broad, encompassing relationships between organisms where one benefits at the expense of another. Traditionally the discipline focuses on eukaryotes, with the study of bacteria and viruses complementary but distinct. Nonetheless, parasites vary in size and complexity from single celled protozoa, to enormous plants like those in the genus Rafflesia. Lifecycles range from obligate intracellular to extensive exoparasitism. Examples of parasites include high profile medical and zoonotic pathogens such as Plasmodium, veterinary pathogens of wild and captive animals and many of the agents which cause neglected tropical diseases, stretching to parasites which infect plants and other parasites (e.g. (Blake et al., 2015; Hemingway, 2015; Hotez et al., 2014; Kikuchi et al., 2011; Meekums et al., 2015; Sandlund et al., 2015). The breadth of parasitology has been matched by the variety of ways in which parasites are studied, drawing upon biological, chemical, molecular, epidemiological and other expertise. Despite such breadth bridging between disciplines has commonly been problematic, regardless of extensive encouragement from government agencies, peer audiences and funding bodies promoting multi-disciplinary research. Now, progress in understanding and collaboration can benefit from establishment of the One Health concept (Stark et al., 2015; Zinsstag et al., 2012). One Health draws upon biological, environmental, medical, veterinary and social science disciplines in order to improve human, animal and environmental health, although it remains tantalizingly difficult to engage many relevant parties. For infectious diseases traditional divides have been exacerbated as the importance of wildlife reservoirs, climate change, food production systems and socio-economic diversity have been recognised but often not addressed in a multi-disciplinary manner. In response the 2015 Autumn Symposium organized by the British Society for Parasitology (BSP; https://www.bsp.uk.net/home/) was focused on One Health, running under the title ‘One Health: parasites and beyond…’. The meeting, held at the Royal Veterinary College (RVC) in Camden, London from September 14th to 15th, drew upon a blend of specialist parasitology reinforced with additional complementary expertise. Scientists, advocates, policy makers and industry representatives were invited to present at the meeting, promoting and developing One Health understanding with relevance to parasitology. The decision to widen the scope of the meeting to non-parasitological, but informative topics, is reflected in the diversity of the articles included in this special issue. A key feature of the meeting was encouragement of early career scientists, with more than 35% of the delegates registered as students and 25 posters

Detection of persistent Plasmodium spp. infections in Ugandan children after artemether-lumefantrine treatment

During a longitudinal study investigating the dynamics of malaria in Ugandan lakeshore communities, a consistently high malaria prevalence was observed in young children despite regular treatment. To explore the short-term performance of artemether-lumefantrine (AL), a pilot investigation into parasite carriage after treatment(s) was conducted in Bukoba village. A total of 163 children (aged 2–7 years) with a positive blood film and rapid antigen test were treated with AL; only 8·7% of these had elevated axillary temperatures. On day 7 and then on day 17, 40 children (26·3%) and 33 (22·3%) were positive by microscopy, respectively. Real-time PCR analysis demonstrated that multi-species Plasmodium infections were common at baseline, with 41·1% of children positive for Plasmodium falciparum/Plasmodium malariae, 9·2% for P. falciparum/ Plasmodium ovale spp. and 8·0% for all three species. Moreover, on day 17, 39·9% of children infected with falciparum malaria at baseline were again positive for the same species, and 9·2% of those infected with P. malariae at baseline were positive for P. malariae. Here, chronic multi-species malaria infections persisted in children after AL treatment(s). Better point-of-care diagnostics for non-falciparum infections are needed, as well as further investigation of AL performance in asymptomatic individuals

A genetic analysis of <i>Trichuris trichiura</i> and <i>Trichuris suis f</i>rom Ecuador

BACKGROUND: Since the nematodes Trichuris trichiura and T. suis are morphologically indistinguishable, genetic analysis is required to assess epidemiological cross-over between people and pigs. This study aimed to clarify the transmission biology of trichuriasis in Ecuador. FINDINGS: Adult Trichuris worms were collected during a parasitological survey of 132 people and 46 pigs in Esmeraldas Province, Ecuador. Morphometric analysis of 49 pig worms and 64 human worms revealed significant variation. In discriminant analysis morphometric characteristics correctly classified male worms according to host species. In PCR-RFLP analysis of the ribosomal Internal Transcribed Spacer (ITS-2) and 18S DNA (59 pig worms and 82 human worms), nearly all Trichuris exhibited expected restriction patterns. However, two pig-derived worms showed a "heterozygous-type" ITS-2 pattern, with one also having a "heterozygous-type" 18S pattern. Phylogenetic analysis of the mitochondrial large ribosomal subunit partitioned worms by host species. Notably, some Ecuadorian T. suis clustered with porcine Trichuris from USA and Denmark and some with Chinese T. suis. CONCLUSION: This is the first study in Latin America to genetically analyse Trichuris parasites. Although T. trichiura does not appear to be zoonotic in Ecuador, there is evidence of genetic exchange between T. trichiura and T. suis warranting more detailed genetic sampling

In Silico Docking of Nematode β-Tubulins With Benzimidazoles Points to Gene Expression and Orthologue Variation as Factors in Anthelmintic Resistance

The efficacy of benzimidazole anthelmintics can vary depending on the target parasite, with Ascaris nematodes being highly responsive, and whipworms being less responsive. Anthelmintic resistance has become widespread, particularly in strongyle nematodes such as Haemonchus contortus in ruminants, and resistance has recently been detected in hookworms of humans and dogs. Past work has shown that there are multiple β-tubulin isotypes in helminths, yet only a few of these contribute to benzimidazole interactions and resistance. The β-tubulin isotypes of ascarids and soil-transmitted helminths were identified by mining available genome data, and phylogenetic analysis showed that the ascarids share a similar repertoire of seven β-tubulin isotypes. Strongyles also have a consistent pattern of four β-tubulin isotypes. In contrast, the whipworms only have two isotypes, with one of these clustering more basally and distinct from any other group. Key β-tubulin isotypes selected based on previous studies were the focus of in silico molecular docking simulations to look at the interactions with benzimidazoles. These showed that all β-tubulins had similar interactions with benzimidazoles and maintained the key bond with residue E198 in all species, indicating similar mechanisms of action. However, the interaction was stronger and more consistent in the strongyles and whipworms than it was in the ascarids. Alteration of β-tubulin isotypes with the common resistance-associated mutations originally identified in H. contortus resulted in similar interaction modeling for all species. In conclusion, ascarids, strongyles, and whipworms all have their own unique repertoire of β-tubulins, which could explain why benzimidazole resistance and susceptibility varies between these groups of parasites. These data complement recent work that has highlighted the roles of essential residues in benzimidazole drug binding and shows that there is a separation between strongyle parasites that frequently develop resistance and ascarid parasites, which have been much less prone to developing resistance