Shailja Singh

Bio: Shailja Singh is an academic researcher from Jawaharlal Nehru University. The author has contributed to research in topics: Plasmodium falciparum & Medicine. The author has an hindex of 21, co-authored 139 publications receiving 2001 citations. Previous affiliations of Shailja Singh include Shiv Nadar University & University of Delhi.

Distinct external signals trigger sequential release of apical organelles during erythrocyte invasion by malaria parasites.

Abstract: The invasion of erythrocytes by Plasmodium merozoites requires specific interactions between host receptors and parasite ligands. Parasite proteins that bind erythrocyte receptors during invasion are localized in apical organelles called micronemes and rhoptries. The regulated secretion of microneme and rhoptry proteins to the merozoite surface to enable receptor binding is a critical step in the invasion process. The sequence of these secretion events and the external signals that trigger release are not known. We have used time-lapse video microscopy to study changes in intracellular calcium levels in Plasmodium falciparum merozoites during erythrocyte invasion. In addition, we have developed flow cytometry based methods to measure relative levels of cytosolic calcium and study surface expression of apical organelle proteins in P. falciparum merozoites in response to different external signals. We demonstrate that exposure of P. falciparum merozoites to low potassium ion concentrations as found in blood plasma leads to a rise in cytosolic calcium levels through a phospholipase C mediated pathway. Rise in cytosolic calcium triggers secretion of microneme proteins such as the 175 kD erythrocyte binding antigen (EBA175) and apical membrane antigen-1 (AMA-1) to the merozoite surface. Subsequently, interaction of EBA175 with glycophorin A (glyA), its receptor on erythrocytes, restores basal cytosolic calcium levels and triggers release of rhoptry proteins. Our results identify for the first time the external signals responsible for the sequential release of microneme and rhoptry proteins during erythrocyte invasion and provide a starting point for the dissection of signal transduction pathways involved in regulated exocytosis of these key apical organelles. Signaling pathway components involved in apical organelle discharge may serve as novel targets for drug development since inhibition of microneme and rhoptry secretion can block invasion and limit blood-stage parasite growth.

Phosphoproteomics reveals malaria parasite Protein Kinase G as a signalling hub regulating egress and invasion

Abstract: Our understanding of the key phosphorylation-dependent signalling pathways in the human malaria parasite, Plasmodium falciparum, remains rudimentary. Here we address this issue for the essential cGMP-dependent protein kinase, PfPKG. By employing chemical and genetic tools in combination with quantitative global phosphoproteomics, we identify the phosphorylation sites on 69 proteins that are direct or indirect cellular targets for PfPKG. These PfPKG targets include proteins involved in cell signalling, proteolysis, gene regulation, protein export and ion and protein transport, indicating that cGMP/PfPKG acts as a signalling hub that plays a central role in a number of core parasite processes. We also show that PfPKG activity is required for parasite invasion. This correlates with the finding that the calcium-dependent protein kinase, PfCDPK1, is phosphorylated by PfPKG, as are components of the actomyosin complex, providing mechanistic insight into the essential role of PfPKG in parasite egress and invasion.

Perforin‐like protein PPLP2 permeabilizes the red blood cell membrane during egress of Plasmodium falciparum gametocytes

Abstract: Egress of malaria parasites from the host cell requires the concerted rupture of its enveloping membranes. Hence, we investigated the role of the plasmodial perforin-like protein PPLP2 in the egress of Plasmodium falciparum from erythrocytes. PPLP2 is expressed in blood stage schizonts and mature gametocytes. The protein localizes in vesicular structures, which in activated gametocytes discharge PPLP2 in a calcium-dependent manner. PPLP2 comprises a MACPF domain and recombinant PPLP2 has haemolytic activities towards erythrocytes. PPLP2-deficient [PPLP2(−)] merozoites show normal egress dynamics during the erythrocytic replication cycle, but activated PPLP2(−) gametocytes were unable to leave erythrocytes and stayed trapped within these cells. While the parasitophorous vacuole membrane ruptured normally, the activated PPLP2(−) gametocytes were unable to permeabilize the erythrocyte membrane and to release the erythrocyte cytoplasm. In consequence, transmission of PPLP2(−) parasites to the Anopheles vector was reduced. Pore-forming equinatoxin II rescued both PPLP2(−) gametocyte exflagellation and parasite transmission. The pore sealant Tetronic 90R4, on the other hand, caused trapping of activated wild-type gametocytes within the enveloping erythrocytes, thus mimicking the PPLP2(−) loss-of-function phenotype. We propose that the haemolytic activity of PPLP2 is essential for gametocyte egress due to permeabilization of the erythrocyte membrane and depletion of the erythrocyte cytoplasm.

Calcium-dependent permeabilization of erythrocytes by a perforin-like protein during egress of malaria parasites.

Abstract: Malaria parasites exit erythrocytes by triggering permeabilization and rupture of the host plasma membrane. Here, the authors identify a perforin-like protein that is secreted by the parasite in a calcium-dependent manner and mediates permeabilization through its insertion into the host membrane.

A lineage of myeloid cells independent of Myb and hematopoietic stem cells

Abstract: Macrophage Development Rewritten Macrophages provide protection against a wide variety of infections and critically shape the inflammatory environment in many tissues. These cells come in many flavors, as determined by differences in gene expression, cell surface phenotype and specific function. Schulz et al. (p. 86, published online 22 March) investigated whether adult macrophages all share a common developmental origin. Immune cells, including most macrophages, are widely thought to arise from hematopoietic stem cells (HSCs), which require the transcription factor Myb for their development. Analysis of Myb-deficient mice revealed that a population of yolk-sac–derived, tissue-resident macrophages was able to develop and persist in adult mice in the absence of HSCs. Importantly, yolk sac–derived macrophages also contributed substantially to the tissue macrophage pool even when HSCs were present. In mice, a population of tissue-resident macrophages arises independently of bone marrow–derived stem cells. Macrophages and dendritic cells (DCs) are key components of cellular immunity and are thought to originate and renew from hematopoietic stem cells (HSCs). However, some macrophages develop in the embryo before the appearance of definitive HSCs. We thus reinvestigated macrophage development. We found that the transcription factor Myb was required for development of HSCs and all CD11bhigh monocytes and macrophages, but was dispensable for yolk sac (YS) macrophages and for the development of YS-derived F4/80bright macrophages in several tissues, such as liver Kupffer cells, epidermal Langerhans cells, and microglia—cell populations that all can persist in adult mice independently of HSCs. These results define a lineage of tissue macrophages that derive from the YS and are genetically distinct from HSC progeny.

How malaria has affected the human genome and what human genetics can teach us about malaria.

Abstract: Malaria is a major killer of children worldwide and the strongest known force for evolutionary selection in the recent history of the human genome. The past decade has seen growing evidence of ethnic differences in susceptibility to malaria and of the diverse genetic adaptations to malaria that have arisen in different populations: epidemiological confirmation of the hypotheses that G6PD deficiency, α + thalassemia, and hemoglobin C protect against malaria mortality; the application of novel haplotype-based techniques demonstrating that malaria-protective genes have been subject to recent positive selection; the first genetic linkage maps of resistance to malaria in experimental murine models; and a growing number of reported associations with resistance and susceptibility to human malaria, particularly in genes involved in immunity, inflammation, and cell adhesion. The challenge for the next decade is to build the global epidemiological infrastructure required for statistically robust genomewide association analysis, as a way of discovering novel mechanisms of protective immunity that can be used in the development of an effective malaria vaccine.

MicroRNAs: Target Recognition and Regulatory Functions

Abstract: MicroRNAs (miRNAs) are endogenous ∼23 nt RNAs that play important gene-regulatory roles in animals and plants by pairing to the mRNAs of protein-coding genes to direct their posttranscriptional repression. This review outlines the current understanding of miRNA target recognition in animals and discusses the widespread impact of miRNAs on both the expression and evolution of protein-coding genes.

The perfect mix: recent progress in adjuvant research

Abstract: Developing efficient and safe adjuvants for use in human vaccines remains both a challenge and a necessity. Past approaches have been largely empirical and generally used a single type of adjuvant, such as aluminium salts or emulsions. However, new vaccine targets often require the induction of well-defined cell-mediated responses in addition to antibodies, and thus new immunostimulants are required. Recent advances in basic immunology have elucidated how early innate immune signals can shape subsequent adaptive responses and this, coupled with improvements in biochemical techniques, has led to the design and development of more specific and focused adjuvants. In this Review, I discuss the research that has made it possible for vaccinologists to now be able to choose between a large panel of adjuvants, which potentially can act synergistically, and combine them in formulations that are specifically adapted to each target and to the relevant correlate(s) of protection.