Conference Agenda

Experimental and naturally acquired human helminth infections have been shown to impart varying degrees of protection against a suite of inflammatory diseases. The proclivity of helminths to regulate their host immune response and suppress inflammation is attributed to the active release of excretory/secretory proteins (ESP) into the host tissues. Experimental infection of humans with helminths presents significant complications as a therapeutic modality due to their complex lifecycles, likely poor adoption, and unavoidable side effects in some subjects. As such, there is now considerable interest in identifying bioactive ESPs and making them more drug-like. We therefore created a recombinant library of N. americanus ESPs from both the adult and larval stage secretomes and are screening the library in a range of in vitro and in vivo assays to identify proteins with potent immunoregulatory properties. Thus far, we have identified proteins that could form the basis of novel therapeutics for treating type 2 diabetes, inflammatory bowel disease and rheumatoid arthritis based on their in vitro and/or in vivo bioactivities.

Individuals infected with Plasmodium spp. parasites or other pathogens exhibit a range of disease outcomes from asymptomatic to fatal. Current understanding of molecular mechanisms underlying immune heterogeneity is limited. This study aimed to investigate immune heterogeneity in malaria to identify immune cells and molecules associated with disease outcomes following Plasmodium infection. A particular focus was on Natural Killer (NK) cells positioned at the innate-adaptive immune interface, previously implicated in parasite control. PBMCs from individuals experimentally infected with blood-stage P. falciparum were subjected to comprehensive single-cell proteomics, bulk proteomics, and single-cell sequencing to identify cells or molecules predictive of infection outcomes. Individuals were classified based on Parasite Multiplication Rate (PMR) at 48 hours post-infection as “good” (PMR<20) or “bad” (PMR>20) responders. Single-cell-RNAseq identified transcriptional signatures showing upregulation of NKG7 and GZMB and downregulation of IL-12RB across multiple NK cell clusters, suggesting immunoregulatory phenotypes in good responders are mediated through the regulation of cytokine production and activation. Single-cell proteomics further emphasised highly inflammatory NK populations in bad responders via increased CD8a expression across NK and other immune cell subsets. These findings underscore the critical role of NK cells during P. falciparum infection, and identify unique molecular signatures associated with improved infection outcomes.

Malaria parasites have complicated lifecycles involving both human and mosquito hosts. In ~8 days, a single parasite can form an oocyst in the mosquito midgut produces thousands of daughter sporozoites. These sporozoites then egress from the oocyst and undergo a complex series of translocation and invasion events to reach the mosquito salivary gland. To facilitate host cell invasion, sporozoites use specialised secretory organelles known as rhoptries. The small size of these parasites and their rhoptries, however, has made it challenging to interrogate sporozoite rhoptry biology. To overcome this, we developed mosquito-tissue ultrastructure expansion microscopy (MoTissU-ExM), a technique that physically expands parasites and their hosts ~4.5-fold. Using MoTissU-ExM, we developed a timeline for sporozoite rhoptry biogenesis in the mosquito midgut and observed the changes they undergo during salivary gland invasion. We leverage these findings to characterise the role of rhoptry neck protein (RON) 11 in sporozoites, showing that RON11 disrupted sporozoites only have half the required number of rhoptries and that therefore RON11 is the only protein known to be involved in sporozoite rhoptry biogenesis. Further, we show that while these RON11-disrupted sporozoites can anatomically enter the salivary gland, they fail to invade salivary gland epithelial cells or the secretory cavity.

Recent breakthroughs in mRNA modifications have enabled RNA therapeutics with stabilised mRNA to improve vaccine efficacy. Despite their importance in biotechnology, the impact of mRNA modifications on stability and translation are poorly understood. N⁶-methyladenosine (m⁶A) is an mRNA modification thought to either improve translation efficacy or reduce stability of transcripts. We studied this modification in Plasmodium falciparum by disrupting the methyltransferase that makes m⁶A. Sequencing of m⁶A in mRNA has only recently become possible with Oxford Nanopore Technologies direct RNA sequencing. However, it is unclear how robust and accurate this detection of m⁶A is. We therefore compared four open-source tools that have been independently developed to identify m⁶A sites in Nanopore RNA-seq from P. falciparum trophozoites. Two of these tools detect m⁶A through machine learning systems trained to artificial RNA synthesized with m⁶A, whereas two work by detecting statistical differences between transcripts that possess or lack m⁶A. We found the former were less correlated in site prediction than the latter category. For transcripts with the most m⁶A sites predicted by each tool, six highly methylated transcripts were common to three tools. Future work involves time-series analysis of P. falciparum transcriptome using updated Nanopore flow cells with proprietary m⁶A detection.

Gastrointestinal nematodes (GINs) severely affect animals, including humans, by causing serious morbidity whilst also exacerbating cycles of poverty. Commonly used diagnostics for GIN detection are often insensitive, e.g. microscopy, whilst more sensitive molecular techniques, e.g. quantitative PCR (qPCR), may only target a few species. More recently, attempts to characterise all GIN species simultaneously, i.e. the “nemabiome” have been developed using next-generation sequencing (NGS). To date, these methods have relied on short-read NGS platforms that can only detect limited genera and for some taxa cannot classify to a species level. Improving upon this, we demonstrate how a nanopore sequencing approach can accurately characterise the complete nemabiome from animals and humans. Through targeting of both internal transcribed spacer regions of the clade V nematodes and 18S ribosomal RNA gene of other key taxa in clades I, II and IV we have shown our assay can provide a comprehensive characterisation of GIN diversity. Moreover, the performance of our assay was found to be equivalent to highly sensitive and specific qPCRs for key GIN species. Overall, long-read nanopore sequencing provides unprecedented species classification accuracy and more comprehensive characterisation of GIN communities in a way previously unachievable using older technologies.