Role of plasmodium translationally repressed gene products in malaria transmission

Tese de doutoramento, Ciências Biomédicas (Microbiologia e Parasitologia), Universidade de Lisboa, Faculdade de Medicina, 2015

Malaria is caused by parasite species of the genus Plasmodium belonging to the phylum Apicomplexa. Plasmodium parasites cause disease when they infect red blood cells of the mammalian host and present a very complex life cycle that includes transmission by a female anopheline mosquito vector. The infection of this vector depends on sexual precursor cells called gametocytes that develop in the blood stream of the vertebrate host. At this stage, parasites display sexual dimorphism that is evident at both morphological and molecular levels. In order to successfully colonise the mosquito midgut, female gametocytes quiescently store specific messenger ribonucleic acids (mRNAs) in messenger ribonucleoproteins (mRNPs) by a molecular mechanism designated translational repression (TR). Following a blood meal, these transcripts are translated in the developing zygote, thereby initiating the morphological and functional changes that allow the parasites to form the motile ookinete and establish a replicating oocyst underneath the midgut epithelium cell layer. Prior to the zygote-to-ookinete transformation (ZOT), TR of selected mRNAs depends on a multiprotein complex where DOZI and CITH are core components. When absent, the otherwise silenced mRNAs are downregulated, preventing mistimed protein expression in the mammalian host’s blood stream that could allow for the generation of malaria transmission blocking antibodies. Parasites lacking DOZI or CITH take part in fertilisation but cannot develop further. The first Plasmodium translationally repressed genes to be identified were p25 and p28; they encode for the two major ookinete surface proteins involved in ookinete survival, penetration of midgut epithelium and development into oocysts. TR of these mRNAs depends on uridine-rich regions in their untranslated regions (UTRs), namely in the 5’ UTR of p25 and in the 3’ UTR of p28. Additionally, these have also been the gold standard and leading target candidates for malaria transmission blocking vaccines (MTBVs). These vaccines rely on the presence of antibodies against Plasmodium antigens that, when taken up by a mosquito during a blood meal, bind to the developing parasite preventing infection of the vector. It is then evident that TR is paramount for the timely production of parasite molecules that otherwise could raise transmission blocking antibodies with deleterious consequences for Plasmodium transmission. TR is also believed to take place in salivary gland sporozoites, the mammalian-infective parasite form that is responsible for disease transmission from the mosquito to the human or rodent host. Deletion of puf2 in the rodent malaria parasite species P. berghei results in transcriptional and translational alterations that normally take place in hepatic stages. These molecular modifications precede morphological changes reminiscent of sporozoite-to-liver stage transformation while still in the mosquito salivary glands, suggesting that sporozoite quiescence relies on post-transcriptional control of certain mRNAs and on the function of the protein Pumilio 2 or Puf2. We are interested in the factors that drive parasite life cycle progression in the mosquito vector, from the onset of sexual development in the erythrocyte to sporozoite formation and liver cell invasion. In the present study we characterised the function of novel putative translationally repressed gene products of P. berghei and determine their contribution to parasite development within the mosquito and consequently to malaria transmission. Three main genes (and corresponding proteins) are addressed herein – epsf, dhhc10 and limp – all of which were found downregulated in the Δdozi parasite line. We show here that they are translationally repressed in female gametocytes, are bound by DOZI and CITH and are targeted to the ookinete crystalloid bodies (CBs) upon translation, a short-lived and enigmatic organelle with unclear function. EPSF (Essential Protein for Sporozoite Formation) is conserved among apicomplexans but its function was so far unknown. This protein contains, although with low probability, a bioinformatically annotated TPM-similar domain that in plants has been implicated in the synthesis/degradation cycle of photosystem II proteins in the chloroplast. DHHC10 is a member of an evolutionarily conserved family of palmitoyl-S-acyl-transferases (PATs) characterised by the presence of an Asp-His-His-Cys (DHHC) motif (sometimes DHYC) within a cysteine-rich domain. These proteins are responsible for the addition of palmitate (C-16-long chain fatty acids) to cysteine residues of other proteins, a reversible post-translational modification (PTM) that tethers its targets to lipid membranes. On the other hand, LIMP is a small (110 amino acids-long) protein unique to Plasmodium spp. and exhibits no identifiable functional domains. Δepsf mutants establish normal oocyst numbers but fail to develop sporozoites, remain vacuolated and continue to increase in size without concomitant deoxyribonucleic acid (DNA) replication or circumsporozoite protein expression. Consequently, Δepsf parasites are completely blocked in transmission. Δdhhc10 mutants show a very similar oocyst phenotype despite the fact that DNA replication does not seem to be affected in this case. Although protein palmitoylation and the relevance of DHHC-PATs in Plasmodium biology have recently been the focus of much interest in blood stage parasites, nothing is know about the importance of such PTM in parasite development within its mosquito vector. Using drug inhibitor and gene deletion studies we show for the first time that palmitoylation is crucial for the execution of developmental and cell biological processes during several phases of mosquito stage infection: it is indispensable for ZOT and defines the efficiency of sporozoite invasion of the host hepatocyte while being redundant for rapid protein turn-over during gliding motility and cell traversal. By deleting dhhc10 we demonstrate that this DHHC-PAT is key to the correct targeting of other CB components and to the formation of the ookinete CB itself. Similar to other CB-associated proteins (the members of the LCCL protein family of adhesins), both EPSF and DHHC10 have an essential role for P. berghei sporogony (formation of sporozoites within oocysts) and therefore for progression of the parasite life cycle in the mosquito. Our data thus further establishes the vital function of CB-resident proteins in oocyst sporulation. LIMP is also localised to the ookinete CBs but, on the contrary to EPSF and DHHC10, is not involved in the formation of sporozoites. In fact, Δlimp oocysts are equally efficient as wild-type (WT) parasites in producing midgut sporozoites. We establish LIMP as a novel malaria-specific gliding motility factor that is important for efficient ookinete infectivity and essential for sporozoite motility and capability to adhere, traverse and invade host liver cells. The absence of this protein reduces transmission to the mosquito vector by half, salivary gland invasion 10-fold and renders parasites unable to infect naïve mice by mosquito bite or when injected intravenously. While LIMP-depleted parasites display no gliding motility, in situ green fluorescent protein (GFP) tagging of this protein resulted in a limping movement characterised by reduced speed and frequent stretching, which has been correlated with reduced turnover of parasite-substrate adhesion sites. Together with the plasma membrane localisation of LIMP in sporozoites, our results suggest that LIMP plays a critical role in regulating the attachment/detachment of adhesion sites during gliding motility and invasion of host target cells. Overall, this dissertation highlights the relevance of TR and its target mRNAs for Plasmodium biology and demonstrates that translationally repressed gene products are crucial for parasite transmission and survival at multiple steps of its development within the mosquito. This work raises new conceptual approaches to combat malaria, namely by inhibiting the formation of CBs and interfering with the parasite gliding motility machinery.

Fundação para a Ciência e a Tecnologia (FCT)