Browsing by Subject "Plasmodium falciparum"

Hydroxyurea, a mainstay of sickle cell management in the developed world, offers a range of potential benefits to children with sickle cell disease. There is strong evidence that hydroxyurea induces production of fetal hemoglobin (HbF) in red blood cells, which is generally associated with reduced morbidity and fewer incidents of clinical events in sickle cell patients. Based on literature from in vitro investigations, it has also been suggested that hydroxyurea may confer some protection against malaria parasitemia by inhibiting proliferation of the malaria-causing parasite - Plasmodium falciparum.

The purpose of this study was to examine the effects hydroxyurea use on P. falciparum infection, parasite density, HbF and morbidity among children with sickle cell disease living in a malaria endemic setting. We analyzed baseline data of 95 children (aged 1 – 10 years) enrolled in the EPiTOMISE clinical trial (Enhancing Preventive Therapy of Malaria in children with Sickle cell anemia in East Africa) between January 2018 and September 2018.

Our analyses showed no significant difference in the prevalence of P. falciparum infection between hydroxyurea users and non-hydroxyurea users, prevalence ratio [95% CI] = 1.14 [0.76, 1.71]. Among infected children, median (IQR) log parasites densities were also similar between hydroxyurea users, -0.96 (-1.67, 0.41) and hydroxyurea non-users, -0.12 (-1.32, 3.48), p-value=0.146.

We did observe substantial hematological benefits among hydroxyurea users including an approximate 1 unit increase in median hemoglobin concentration and a 2.7-fold increase in median percentage HbF. However, this observation did not translate to any meaningful difference in the prevalence of morbidity events.

In agreement with the few studies on hydroxyurea use in malaria endemic settings, these results suggest that there may be no added risk of P. falciparum infection to sickle cell patients who routinely use hydroxyurea. Furthermore, our results reflect that hydroxyurea use is associated with increased HbF and a better hematological profile in this population. There is need for more research on hydroxyurea use in sub-Saharan Africa to help resolve any existing concerns and conflicting data in the current body of literature.

Biological systems are highly dynamic, yet our temporal resolution of such

dynamical processes is often limited or difficult to test in the laboratory. The 24-hour

circadian rhythm and the approximately 75-minute cell cycle of a budding yeast cell are

both examples of dynamical processes that contain precisely ordered events, repeating

over each cycle. Organisms utilize such biological clock processes to time a particular

function. Dynamic cellular events are ordered, in part, by coordinated programs of

periodic gene expression. Up to 40% of all mouse genes are periodically expressed with

respect to the circadian cycle, and almost 20% of all yeast genes are periodic during the

cell cycle. Furthermore, more than half of the most frequently prescribed drugs in human

patients target an effector whose expression is under circadian control. Given the large

proportion of genes that are periodically expressed across different biological processes,

it is critically important to understand mechanisms that regulate dynamics in biology.

In this dissertation, I focus on two biological processes that are dynamic and are

not yet fully understood: the eukaryotic cell cycle and malaria parasite development.

Large programs of periodic genes emerge when these biological clock processes are

synchronized and profiled over time. Gene regulatory networks composed of

transcription factors, kinases, and other transcriptional regulators play a critical role in

generating periodicity in gene expression programs, ordering clock events, and

maintaining oscillations in subsequent cycles.

Many previous studies have profiled gene expression during the cell cycle in the

budding yeast Saccharomyces cerevisiae. I have added to this detailed body of work by

demonstrating that regulatory motifs involving negative feedback are required to

maintain normal gene expression levels. Additionally, I showed that many periodic

mRNAs are also periodically abundant at the protein level during the cell cycle. Both

projects provide evidence for the hypothesis that cell-cycle dynamics are driven by a

network of transcription factors with complex protein dynamics and with negative

feedback motifs. Using this ground truth cell-cycle network in S. cerevisiae, I next

performed a comparative transcriptomics study on cell-cycle genes in the less studied,

but more human health relevant fungal pathogen, Cryptococcus neoformans. This work

not only begins to identify a cell-cycle network in C. neoformans but also has

implications for future antifungal drug development, as some genes that are important

for fungal virulence were found to be expressed periodically during the cell cycle.

During infection, the human malaria parasite Plasmodium falciparum cyclically

develops and re-infects red blood cells. Many groups have shown that a very large

program of gene expression occurs during this red blood cell developmental cycle. In

this dissertation, I deploy the experimental and analysis tools that I used to characterize

the fungal cell cycle to ask if a network of transcription factors can explain

developmental gene expression dynamics and cycle period control in malaria.

Biological systems are highly dynamic to respond to environmental signals, grow,

and survive. As the application of genetics and genomics has moved toward

characterizing complex diseases, host-pathogen interactions, or even the cell cycle of a

single yeast cell, it has become increasingly clear that networks of interacting genes are

required to explain biological mechanisms. Results from this dissertation where I

investigate dynamic gene regulatory networks are broadly applicable to our

understanding of both basic molecular biology and of human infectious diseases.

The sickle-cell trait hemoglobin genotype (HbAS) is known to protect against severe malaria caused by Plasmodium falciparum. However, the biological mechanisms behind this protection are not well understood. Cyclophilin 19B (PfCyP-19B) is a parasitic gene that produces the protein cyclophilin 19B, a member of the unfolded protein response that is important in parasitic protein folding and trafficking. We set out to measure the transcript expression level of PfCyP-19B to investigate its potential role in the mechanisms that confer protection for HbAS individuals. RNA was extracted from both in vivo samples collected from Malian children as well as in vitro samples harvested throughout a 48-hour incubation period. RNA extracts were reverse transcribed and transcript expression was measured via qPCR. Wilcoxon rank sum and bootstrapping methods were used to analyze transcript units between parasites grown in normal (HbAA) and HbAS red blood cells. The results from our cross-sectional in vivo data revealed under expression of PfCyP-19B among individuals with the HbAS genotype compared to those with the HbAA genotype (Wilcoxon rank sum p=0.006). In vitro time series results showed no significant difference in PfCyP-19B transcript expression levels between genotypes but did display a 24-hour pattern of peak expression for both HbAA and HbAS genotypes. The under expression of PfCyP-19B among HbAS individuals could be linked to impaired protein trafficking, interfering with the parasite’s ability to display surface proteins vital for cytoadherence and severe disease manifestation. The 24-hour peak transcript expression displayed in vitro roughly aligns with the P. falciparum parasite stage transition states, suggesting cyclophilin 19B may aid in parasitic transitions.

Malaria persists as a global health problem, with 212 million cases and 429,000 deaths worldwide in 2015 alone. It is caused by the apicomplexan parasite Plasmodium, which follows a complex life cycle that consists of multiple stages spanning from the human host to the mosquito vector. Among the Plasmodium parasites causing human malaria, the deadliest species is Plasmodium falciparum. Most P. falciparum parasites follow an asexual cycle in human erythrocytes that is characterized by a tightly synchronized continuous cascade of gene expression, although a small proportion commits to a sexual fate. Parasites committed to the sexual stage develop into male and female gametocytes over 9-12 days, with mature gametocytes being the only form of the parasite transmissible to the mosquito vector.

This commitment to a sexual fate is rare, and little is known about the transcriptional programs related to sexual commitment and mating-type determination. Furthermore, discrete changes that occur in these cells are largely undetectable in traditional bulk-cell analyses. Bulk-cell analyses were used to establish models for synchronous stage-specific transcriptional programming during the asexual intraerythrocytic developmental cycle (IDC) but left little resolved in terms of cellular heterogeneity and cell-fate decisions. Due to these limitations, we developed unbiased single-cell approaches on a microfluidic platform to analyze single parasites during late asexual and sexual stages. This work was divided into two main parts. The first focused on single-cell gene expression in male and female mid-to-late stage gametocytes. We captured 90 single parasites and compared the expression of 91 genes, including 87 gametocyte-specific genes. Our analysis identified distinct gene clusters whose expression associated with male, female, or all gametocytes. In addition, a small number of male gametocytes clustered separately from female gametocytes based on sex-specific expression independent of stage. RNA fluorescent in situ hybridization (RNA-FISH) validated the mutually exclusive expression pattern of male and female transcripts in gametocytes. These analyses uncovered novel male and female markers that are expressed as early as stage III gametocytogenesis, providing further insight into Plasmodium sex-specific differentiation previously masked in population analyses.

The second part of this work centered on single-cell RNA sequencing (scRNA-seq) of P. falciparum late asexual and sexual stages. First, we uncovered a large number of previously undefined gametocyte-specific genes. 46 asexual cells were then segregated into three separate clusters based on the differential expression of SERAs, rhoptries, and EXP2 plus transporters. RNA-FISH of cluster-specific genes validated this distinct stage-specific expression during the IDC and defined the highly variable transcriptional pattern of EXP2. Additionally, these analyses indicated huge variations in the stage-specific transcript levels among parasites. Overall, scRNA-seq and RNA-FISH of P. falciparum revealed distinct stage transitions and unexpected degrees of heterogeneity with potential impact on transcriptional regulation during the IDC and adaptive responses to the host.