Biophysics of Malarial Parasite Exit from Infected Erythrocytes

Upon infection and development within human erythrocytes, P. falciparum induces alterations to the infected RBC morphology and bio-mechanical properties to eventually rupture the host cells through parasitic and host derived proteases of cysteine and serine families. We used previously reported broad-spectrum inhibitors (E64d, EGTA-AM and chymostatin) to inhibit these proteases and impede rupture to analyze mechanical signatures associated with parasite escape. Treatment of late-stage iRBCs with E64d and EGTA-AM prevented rupture, resulted in no major RBC cytoskeletal reconfiguration but altered schizont morphology followed by dramatic re-distribution of three-dimensional refractive index (3D-RI) within the iRBC. These phenotypes demonstrated several-fold increased iRBC membrane flickering. In contrast, chymostatin treatment showed no 3D-RI changes and caused elevated fluctuations solely within the parasitophorous vacuole. We show that E64d and EGTA-AM supported PV breakdown and the resulting elevated fluctuations followed non-Gaussian pattern that resulted from direct merozoite impingement against the iRBC membrane. Optical trapping experiments highlighted reduced deformability of the iRBC membranes upon rupture-arrest, more specifically in the treatments that facilitated PV breakdown. Taken together, our experiments provide novel mechanistic interpretations on the role of parasitophorous vacuole in maintaining the spherical schizont morphology, the impact of PV breakdown on iRBC membrane fluctuations leading to eventual parasite escape and the evolution of membrane stiffness properties of host cells in which merozoites were irreversibly trapped, recourse to protease inhibitors. These findings provide a comprehensive, previously unavailable, body of information on the combined effects of biochemical and biophysical factors on parasite egress from iRBCs.Singapore. Agency for Science, Technology and ResearchSingapore-MIT AllianceGlobal Enterprise for Micro-Mechanics and Molecular MedicineNational University of SingaporeNational Institutes of Health (U.S.) (Grant R01 HL094270-01A1)National Institutes of Health (U.S.) (Grant 1-R01-GM076689-01)National Institutes of Health (U.S.) (P41-RR02594-18-24

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2–4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

Characterization of differentially expressed human gene transcripts in uninfected versus dengue virus-infected ECV304 endothelial-like cells

Master'sMASTER OF SCIENC

Global genomics analysis of the rodent malaria parasites Plasmodium Berghei, Plasmodium Chabaudi and Plasmodium Yoelii with emphasis on the identification of novel virulence factors in Plasmodium Yoelii

The incompletely sequenced and annotated genomes of the rodent malaria parasite (RMP) species were re-annotated via complementary genomic DNA hybridizations and bioinformatics tools. A common ’core’ set of orthologous Plasmodia genes was then elucidated. Similarly, a RMP specific ortholog gene set was also elucidated. Comparative genomics of different P. yoelii lines confirmed the presence of polymorphic genes under immune selection pressure and revealed potential variant hypothetical genes containing transmembrane domains that may be involved in novel parasite-host interactions. Next, transcriptomic differences between the virulent YM and avirulent YA lines of P. yoelii were determined and genes that are linked to virulence were identified. Two of these genes (PySERA 1 & 2) were further characterized using knockout strategies. While disruption of PySERA1 appears to reduce parasite virulence, there is no apparent effect in the case of disruption of PySERA2, thus suggesting a certain degree of redundancy of this multigene family.DOCTOR OF PHILOSOPHY (SBS

The Role of Serine-Type Serine Repeat Antigen in <i>Plasmodium yoelii</i> Blood Stage Development

<div><p></p><p>A key step for the survival of the malaria parasite is the release from and subsequent invasion of erythrocytes by the merozoite. Differences in the efficiency of these two linked processes have a direct impact on overall parasite burden in the host and thereby virulence. A number of parasite proteases have recently been shown to play important roles during both merozoite egress as well as merozoite invasion. The rodent malaria parasite <i>Plasmodium yoelii</i> has been extensively used to investigate the mechanisms of parasite virulence in vivo and a number of important proteins have been identified as being key contributors to pathology. Here we have utilized transcriptional comparisons to identify two protease-like SERAs as playing a potential role in virulence. We show that both SERAs are non-essential for blood stage development of the parasite though they provide a subtle but important growth advantage in vivo. In particular SERA2 appears to be an important factor in enabling the parasite to fully utilize the whole age repertoire of circulating erythrocytes. This work for the first time demonstrates the subtle contributions different protease-like SERAs make to provide the parasite with a maximal capacity to successfully maintain an infection in the host.</p></div

Growth competition between two different parasite lines.

<p>Parasitemia of Balb/C mice with 10⁴ parasites of each parasite line were mixed and injected by iv. on day 0 and recorded daily. The average parasitemia and percentage of gene expression calculated through real-time PCR of 4 mice in each group were presented. A. growth competition of YM and SERA1-ko; B. growth competition of YM and SERA2-ko; C. growth competition of SERA1-ko and SERA2-ko.</p

Merozoite proteins identified with significant difference between YM and SERA2-ko line.

<p>Protein spots with selection numbers and the corresponding gene information are indicated and up-regulation (+) or down-regulation (−) in the fold changes of average ratio are presented.</p

Comparison of growth characteristics between wildtype YM and SERA knock-out lines.

<p>A. Parasitemia of Balb/C mice with 10³ parasites injected by iv. on day 0 and recorded daily. The average parasitemia of 5 mice for each group were presented.† denotes the death of animals; B. Survival of Balb/C mice infected with YM or KO lines. Note that there was one outlier mouse infected with SERA1-ko C6 survived beyond post-infection day9 but ultimately died two days later; C. Average selective index of 5 BALB/c mice infected with YM or KO lines. Parasite smears were analyzed when parasitaemia was in the range of 3–13%. Differences in SI between YM and SERA1-ko C6 and SERA2-ko C1 and C2 were significant (p<0.01), indicated by **.</p