Genome-Scale Identification of Essential Metabolic Processes for Targeting the Plasmodium Liver Stage.

Plasmodium gene functions in mosquito and liver stages remain poorly characterized due to limitations in the throughput of phenotyping at these stages. To fill this gap, we followed more than 1,300 barcoded P. berghei mutants through the life cycle. We discover 461 genes required for efficient parasite transmission to mosquitoes through the liver stage and back into the bloodstream of mice. We analyze the screen in the context of genomic, transcriptomic, and metabolomic data by building a thermodynamic model of P. berghei liver-stage metabolism, which shows a major reprogramming of parasite metabolism to achieve rapid growth in the liver. We identify seven metabolic subsystems that become essential at the liver stages compared with asexual blood stages: type II fatty acid synthesis and elongation (FAE), tricarboxylic acid, amino sugar, heme, lipoate, and shikimate metabolism. Selected predictions from the model are individually validated in single mutants to provide future targets for drug development.

N-acetylation of secreted proteins in Apicomplexa is widespread and is independent of the ER acetyl-CoA transporter AT1.

Acetyl-CoA participates in post-translational modification of proteins and in central carbon and lipid metabolism in several cell compartments. In mammals, acetyl-CoA transporter 1 (AT1, also known as SLC33A1) facilitates the flux of cytosolic acetyl-CoA into the endoplasmic reticulum (ER), enabling the acetylation of proteins of the secretory pathway, in concert with the activity of dedicated acetyltransferases such as NAT8. However, the involvement of the ER acetyl-CoA pool in acetylation of ER-transiting proteins in Apicomplexa is unknown. Here, we identified homologs of AT1 and NAT8 in Toxoplasma gondii and Plasmodium berghei parasites. Proteome-wide analyses revealed widespread N-terminal acetylation of secreted proteins in both species. Such extensive acetylation of N-terminally processed proteins has not been observed previously in any other organism. Deletion of AT1 homologs in both T. gondii and P. berghei resulted in considerable reductions in parasite fitness. In P. berghei, AT1 was found to be important for growth of asexual blood stages, production of female gametocytes and male gametocytogenesis, implying its requirement for parasite transmission. In the absence of AT1, lysine acetylation and N-terminal acetylation in T. gondii remained globally unaltered, suggesting an uncoupling between the role of AT1 in development and active acetylation occurring along the secretory pathway.

Toxoplasma gondii GRA60 is an effector protein that modulates host cell autonomous immunity and contributes to virulence.

Toxoplasma gondii infects virtually any nucleated cell and resides inside a non-phagocytic vacuole surrounded by a parasitophorous vacuolar membrane (PVM). Pivotal to the restriction of T. gondii dissemination upon infection in murine cells is the recruitment of immunity regulated GTPases (IRGs) and guanylate binding proteins (GBPs) to the PVM that leads to pathogen elimination. The virulent T. gondii type I RH strain secretes a handful of effectors including the dense granule protein GRA7, the serine-threonine kinases ROP17 and ROP18, and a pseudo-kinase ROP5, that synergistically inhibit the recruitment of IRGs to the PVM. Here, we characterise GRA60, a novel dense granule effector, which localises to the vacuolar space and PVM and contributes to virulence of RH in mice, suggesting a role in the subversion of host cell defence mechanisms. Members of the host cell IRG defence system Irgb10 and Irga6 are recruited to the PVM of RH parasites lacking GRA60 as observed previously for the avirulent RHΔrop5 mutant, with RH preventing such recruitment. Deletion of GRA60 in RHΔrop5 leads to a recruitment of IRGs comparable to the single knockouts. GRA60 therefore represents a novel parasite effector conferring resistance to IRGs in type I parasites, and found associated to ROP18, a member of the virulence complex.

Gene copy number variation in natural populations of Plasmodium falciparum in Eastern Africa.

BACKGROUND: Gene copy number variants (CNVs), which consist of deletions and amplifications of single or sets of contiguous genes, contribute to the great diversity in the Plasmodium falciparum genome. In vitro studies in the laboratory have revealed their important role in parasite fitness phenotypes such as red cell invasion, transmissibility and cytoadherence. Studies of natural parasite populations indicate that CNVs are also common in the field and thus may facilitate adaptation of the parasite to its local environment. RESULTS: In a survey of 183 fresh field isolates from three populations in Eastern Africa with different malaria transmission intensities, we identified 94 CNV loci using microarrays. All CNVs had low population frequencies (minor allele frequency < 5%) but each parasite isolate carried an average of 8 CNVs. Nine CNVs showed high levels of population differentiation (FST > 0.3) and nine exhibited significant clines in population frequency across a gradient in transmission intensity. The clearest example of this was a large deletion on chromosome 9 previously reported only in laboratory-adapted isolates. This deletion was present in 33% of isolates from a population with low and highly seasonal malaria transmission, and in < 9% of isolates from populations with higher transmission. Subsets of CNVs were strongly correlated in their population frequencies, implying co-selection. CONCLUSIONS: These results support the hypothesis that CNVs are the target of selection in natural populations of P. falciparum. Their environment-specific patterns observed here imply an important role for them in conferring adaptability to the parasite thus enabling it to persist in its highly diverse ecological environment.

Memory B cells are a more reliable archive for historical antimalarial responses than plasma antibodies in no-longer exposed children.

Humans respond to foreign antigen by generating plasma Abs and memory B cells (MBCs). The Ab response then declines, sometimes to below the limit of detection. In contrast, MBCs are generally thought to be long-lived. We tested and compared Plasmodium falciparum (Pf)-specific Ab and MBC responses in two populations of children: (i) previously exposed children who had documented Pf infections several years ago, but minimal exposure since then; and (ii) persistently exposed children living in a separate but nearby endemic area. We found that although Pf-specific plasma Abs were lower in previously exposed children compared with persistently exposed children, their cognate MBCs were maintained at similar frequencies. We conclude that serological analysis by itself would greatly underestimate the true memory of Pf-specific Ab responses in previously exposed children living in areas where Pf transmission has been reduced or eliminated.

Predictors of outcome in routine care for Cryptococcal meningitis in Western Kenya: lessons for HIV outpatient care in resource-limited settings.

PURPOSE: Cryptococcal meningitis is a leading cause of mortality among HIV-infected individuals in sub-Saharan Africa but little is known about its treatment and outcomes in decentralised HIV outpatient settings. We assessed adherence to treatment guidelines and determined predictors of survival. DESIGN: A computerised laboratory database identified HIV-infected adults with cryptococcal meningitis at Family AIDS Care and Education Services in Nyanza Province, Kenya, between 2005-2009. Medical records were reviewed. Kaplan-Meier survival curves were generated. Bivariate and multivariate Cox proportional hazards models were used to determine associations between key clinical characteristics and survival. RESULTS: Medical records were located for 79% (71/90). Mortality was 38% (27/71) over a median follow-up period of 201 days (IQR: 10-705 days). Adherence to local guidelines for treatment of cryptococcal meningitis was 48% (34/71). Higher body mass index was associated with improved survival (HR: 0.82, 95% CI (0.68 to 0.99)) even after controlling for factors such as age, CD4 cell count, receipt of highly active anti-retroviral therapy, and treatment with any anti-fungal therapy. CONCLUSIONS: Cryptococcal meningitis diagnosed in routine HIV outpatient settings is largely treated as an outpatient and adherence to treatment guidelines is poor. Body mass index is a critical independent predictor of outcome. Additional research to determine the most effective strategies to reduce premature mortality is urgently needed.

Ceramide biosynthesis is critical for establishment of the intracellular niche of Toxoplasma gondii.

Toxoplasma gondii possesses sphingolipid synthesis capabilities and is equipped to salvage lipids from its host. The contribution of these two routes of lipid acquisition during parasite development is unclear. As part of a complete ceramide synthesis pathway, T. gondii expresses two serine palmitoyltransferases (TgSPT1 and TgSPT2) and a dihydroceramide desaturase. After deletion of these genes, we determine their role in parasite development in vitro and in vivo during acute and chronic infection. Detailed phenotyping through lipidomic approaches reveal a perturbed sphingolipidome in these mutants, characterized by a drastic reduction in ceramides and ceramide phosphoethanolamines but not sphingomyelins. Critically, parasites lacking TgSPT1 display decreased fitness, marked by reduced growth rates and a selective defect in rhoptry discharge in the form of secretory vesicles, causing an invasion defect. Disruption of de novo ceramide synthesis modestly affects acute infection in vivo but severely reduces cyst burden in the brain of chronically infected mice.

Establishing the extent of malaria transmission and challenges facing pre-elimination in the Republic of Djibouti.

BACKGROUND: Countries aiming for malaria elimination require a detailed understanding of the current intensity of malaria transmission within their national borders. National household sample surveys are now being used to define infection prevalence but these are less efficient in areas of exceptionally low endemicity. Here we present the results of a national malaria indicator survey in the Republic of Djibouti, the first in sub-Saharan Africa to combine parasitological and serological markers of malaria, to evaluate the extent of transmission in the country and explore the potential for elimination. METHODS: A national cross-sectional household survey was undertaken from December 2008 to January 2009. A finger prick blood sample was taken from randomly selected participants of all ages to examine for parasitaemia using rapid diagnostic tests (RDTs) and confirmed using Polymerase Chain Reaction (PCR). Blood spots were also collected on filter paper and subsequently used to evaluate the presence of serological markers (combined AMA-1 and MSP-119) of Plasmodium falciparum exposure. Multivariate regression analysis was used to determine the risk factors for P. falciparum infection and/or exposure. The Getis-Ord G-statistic was used to assess spatial heterogeneity of combined infections and serological markers. RESULTS: A total of 7151 individuals were tested using RDTs of which only 42 (0.5%) were positive for P. falciparum infections and confirmed by PCR. Filter paper blood spots were collected for 5605 individuals. Of these 4769 showed concordant optical density results and were retained in subsequent analysis. Overall P. falciparum sero-prevalence was 9.9% (517/4769) for all ages; 6.9% (46/649) in children under the age of five years; and 14.2% (76/510) in the oldest age group (≥50 years). The combined infection and/or antibody prevalence was 10.5% (550/4769) and varied from 8.1% to 14.1% but overall regional differences were not statistically significant (χ2=33.98, p=0.3144). Increasing age (p<0.001) and decreasing household wealth status (p<0.001) were significantly associated with increasing combined P. falciparum infection and/or antibody prevalence. Significant P. falciparum hot spots were observed in Dikhil region. CONCLUSION: Malaria transmission in the Republic of Djibouti is very low across all regions with evidence of micro-epidemiological heterogeneity and limited recent transmission. It would seem that the Republic of Djibouti has a biologically feasible set of pre-conditions for elimination, however, the operational feasibility and the potential risks to elimination posed by P. vivax and human population movement across the sub-region remain to be properly established.

Adaptation of Plasmodium falciparum to its transmission environment.

Success in eliminating malaria will depend on whether parasite evolution outpaces control efforts. Here, we show that Plasmodium falciparum parasites (the deadliest of the species causing human malaria) found in low-transmission-intensity areas have evolved to invest more in transmission to new hosts (reproduction) and less in within-host replication (growth) than parasites found in high-transmission areas. At the cellular level, this adaptation manifests as increased production of reproductive forms (gametocytes) early in the infection at the expense of processes associated with multiplication inside red blood cells, especially membrane transport and protein trafficking. At the molecular level, this manifests as changes in the expression levels of genes encoding epigenetic and translational machinery. Specifically, expression levels of the gene encoding AP2-G-the transcription factor that initiates reproduction-increase as transmission intensity decreases. This is accompanied by downregulation and upregulation of genes encoding HDAC1 and HDA1-two histone deacetylases that epigenetically regulate the parasite's replicative and reproductive life-stage programmes, respectively. Parasites in reproductive mode show increased reliance on the prokaryotic translation machinery found inside the plastid-derived organelles. Thus, our dissection of the parasite's adaptive regulatory architecture has identified new potential molecular targets for malaria control.

Increased Cytomegalovirus Secretion and Risks of Infant Infection by Breastfeeding Duration From Maternal Human Immunodeficiency Virus Positive Compared to Negative Mothers in Sub-Saharan Africa.

BACKGROUND: Breastfeeding imparts beneficial immune protection and nutrition to infants for healthy growth, but it is also a route for human immunodeficiency virus (HIV) and human cytomegalovirus (HCMV) infection. In previous studies, we showed that HCMV adversely affects infant development in Africa, particularly with maternal HIV exposure. In this study, we analyzed infants risks for acquisition of HCMV infection from breastfeeding and compared HIV-positive and HIV-negative mothers. METHODS: Two cohorts were studied in Zambia. (1) Two hundred sixty-one HIV-infected and HIV-uninfected mothers were compared for HCMV deoxyribonucleic acid (DNA) loads and genotypes (glycoprotein gO) in milk from birth to 4 months postpartum. (2) Maternally HIV-exposed and HIV-unexposed infants were compared for HCMV infection risk factors. The second cohort of 460 infants, from a trial of micronutrient-fortified complementary-food to breastfeeding, were studied between 6 and 18 months of age. Human cytomegalovirus seroprevalence was assayed, and logistic regression was used to calculate risk factors for HCMV infection, including maternal HIV exposure and breastfeeding duration. RESULTS: Human cytomegalovirus was detected in breast milk from 3 days to 4 months postpartum, with significantly raised levels in HIV-positive women and independent of genotype. In infants, HCMV antibody seroprevalence was 83% by 18 months age. Longer breastfeeding duration increased infection risk in maternally HIV-unexposed (odds ratio [OR] = 2.69 for 18 months vs <12 months; 95% confidence interval [CI], 0.84-8.59; P = .03) and HIV-exposed infants (OR = 20.37 for >6 months vs never; 95% CI, 3.71-111.70; P < .001). CONCLUSIONS: Prolonged breastfeeding, which is common in Africa, increased risk of HCMV infection in infants. Both HIV-positive and HIV-negative women had extended milk HCMV secretion. Women who were HIV-positive secreted higher HCMV levels, and for longer duration, with their children at increased infection risk. Human cytomegalovirus control is required to maintain health benefits of breastfeeding.

Wanted Plasmodium falciparum, dead or alive.

Mechanisms of cell death in unicellular parasites have been subjects of debate for the last decade, with studies demonstrating evidence of apoptosis or non-apoptosis like mechanisms, including necrosis, and autophagy. Recent clarifications on the definition of regulated or accidental cell death by The Nomenclature Committee on Cell Death provides an opportunity to reanalyze some data, re-evaluate conclusions in the light of parasite diversity, and to propose alternative arguments in the context of malaria drug resistance, considering lack of really new drugs in the pipeline. Deciphering the mechanisms of death may help in detection of new drug targets and the design of innovative drugs. However, classifications have been evolving rapidly since initial description of "programmed cell death", leading to some uncertainty as to whether Plasmodium cell death is accidental or regulated.