Plasmodium parasites of mammals, including the species that cause malaria in humans, infect the liver first and develop there into clinically silent liver stages. Liver stages grow and ultimately produce thousands of first-generation merozoites, which initiate the erythrocytic cycles causing malaria pathology. Here, we present a Plasmodium protein with a critical function for complete liver stage development. UIS4 (up-regulated in infective sporozoites gene 4) is expressed exclusively in infective sporozoites and developing liver stages, where it localizes to the parasitophorous vacuole membrane. Targeted gene disruption of UIS4 in the rodent model malaria parasite Plasmodium berghei generated knockout parasites that progress through the malaria life cycle until after hepatocyte invasion but are severely impaired in further liver stage development. Immunization with UIS4 knockout sporozoites completely protects mice against subsequent infectious WT sporozoite challenge. Genetically attenuated liver stages may thus induce immune responses, which inhibit subsequent infection of the liver with WT parasites. attenuated parasite ͉ malaria ͉ parasitophorous vacuole ͉ stage-specific gene expression

Injection of Plasmodium salivary gland sporozoites into the vertebrate host by Anopheles mosquitoes initiates malaria infection. Sporozoites develop within oocysts in the mosquito midgut and then enter and mature in the salivary glands. Although morphologically similar, oocyst sporozoites and salivary gland sporozoites differ strikingly in their infectivity to the mammalian host, ability to elicit protective immune responses, and cell motility. Here, we show that differential gene expression coincides with these dramatic phenotypic differences. Using suppression subtractive cDNA hybridization we identified highly up-regulated mRNAs transcribed from 30 distinct genes in salivary gland sporozoites. Of those genes, 29 are not significantly expressed in the parasite's blood stages. The most frequently recovered transcript encodes a protein kinase. Developmental up-regulation of specific mRNAs in the infectious transmission stage of Plasmodium indicates that their translation products may have unique roles in hepatocyte infection and/or development of liver stages.Malaria transmission occurs by mosquito bite when Plasmodium sporozoites located in the salivary glands of anopheline mosquitoes enter the vertebrate host. Sporozoites invade hepatocytes and differentiate into exo-erythrocytic forms (EEFs) 1 that after a few days contain several thousand merozoites. After exiting the hepatocyte, merozoites invade erythrocytes and start the blood stage cycle that causes malaria disease. Salivary gland sporozoites and EEFs are rational targets for immunoprophylaxis and drug prophylaxis because they precede the development of the pathogenic blood stages. One important limitation for drug discovery and vaccine development is the lack of candidate target molecules specifically expressed in the pre-erythrocytic stages.To date, only a few sporozoite-expressed proteins have been identified, mainly due to the difficulty of obtaining sporozoites in large quantities. Of those proteins, the sporozoite-specific coat protein CS (1, 2) and the sporozoite-specific invasin TRAP (3, 4) have been identified in a range of different Plasmodium species, and both proteins are lead malaria vaccine candidates either in single formulations or as components of multi-subunit vaccines (5, 6). Sporozoite biology provides a unique opportunity to identify candidate virulence factors that contribute to the successful transmission of Plasmodium. We refer to the observations that oocyst sporozoites and salivary gland sporozoites display dramatically different phenotypes. Sporozoites isolated from the mosquito salivary glands are highly infectious to the mammalian host, migrate in a typical circular gliding pattern, and can elicit strong protective immune responses (7-10). In marked contrast, oocyst sporozoites are ϳ10,000-fold less infective to the mammalian host, do not exhibit circular gliding, and fail to produce protective immunity (Fig. 1). Furthermore, sporozoites that entered the salivary glands are no longer capable of reentering them, suggesting tha...

Apicomplexan host cell invasion and gliding motility depend on the parasite's actomyosin system located beneath the plasma membrane of invasive stages. Myosin A (MyoA), a class XIV unconventional myosin, is the motor protein. A model has been proposed to explain how the actomyosin motor operates but little is known about the components, topology and connectivity of the motor complex. Using the MyoA neck and tail domain as bait in a yeast two-hybrid screen we identified MTIP, a novel 24 kDa protein that interacts with MyoA. Deletion analysis shows that the 15 amino-acid C-terminal tail domain of MyoA, rather than the neck domain, specifically interacts with MTIP. In Plasmodium sporozoites MTIP localizes to the inner membrane complex (IMC), where it is found clustered with MyoA. The data support a model for apicomplexan motility and invasion in which the MyoA motor protein is associated via its tail domain with MTIP, immobilizing it at the outer IMC membrane. The head domain of the immobilized MyoA moves actin filaments that,directly or via a bridging protein, connect to the cytoplasmic domain of a transmembrane protein of the TRAP family. The actin/TRAP complex is then redistributed by the stationary MyoA from the anterior to the posterior end of the zoite, leading to its forward movement on a substrate or to penetration of a host cell.

Whole blood transcriptional signatures distinguishing active tuberculosis patients from asymptomatic latently infected individuals exist. Consensus has not been achieved regarding the optimal reduced gene sets as diagnostic biomarkers that also achieve discrimination from other diseases. Here we show a blood transcriptional signature of active tuberculosis using RNA-Seq, confirming microarray results, that discriminates active tuberculosis from latently infected and healthy individuals, validating this signature in an independent cohort. Using an advanced modular approach, we utilise the information from the entire transcriptome, which includes overabundance of type I interferon-inducible genes and underabundance of IFNG and TBX21, to develop a signature that discriminates active tuberculosis patients from latently infected individuals or those with acute viral and bacterial infections. We suggest that methods targeting gene selection across multiple discriminant modules can improve the development of diagnostic biomarkers with improved performance. Finally, utilising the modular approach, we demonstrate dynamic heterogeneity in a longitudinal study of recent tuberculosis contacts.

SummaryInvasive sporozoite and merozoite stages of malaria parasites that infect mammals enter and subsequently reside in hepatocytes and red blood cells respectively. Each invasive stage may exhibit unique adaptations that allow it to interact with and survive in its distinct host cell environment, and these adaptations are likely to be controlled by differential gene expression. We used suppression subtractive hybridization (SSH) of Plasmodium yoelii salivary gland sporozoites versus merozoites to identify stage-specific pre-erythrocytic transcripts. Sequencing of the SSH library and matching the cDNA sequences to the P. yoelii genome yielded 25 redundantly tagged genes including the only two previously characterized sporozoite-specific genes encoding the circumsporozoite protein (CSP) and thrombospondin-related anonymous protein (TRAP). Twelve novel genes encode predicted proteins with signal peptides, indicating that they enter the secretory pathway of the sporozoite. We show that one novel protein bearing a thrombospondin type 1 repeat (TSR) exhibits an expression pattern that suggests localization in the sporozoite secretory rhoptry organelles. In addition, we identified a group of four genes encoding putative low-molecular-mass proteins. Two proteins in this group exhibit an expression pattern similar to TRAP, and thus possibly localize in the sporozoite secretory micronemes. Proteins encoded by the differentially expressed genes identified here probably mediate specific interactions of the sporozoite with the mosquito vector salivary glands or the mammalian host hepatocyte and are not used during merozoite-red blood cell interactions.

The ability to undergo apoptosis, previously thought to be exclusive to multicellular organisms, has been demonstrated in unicellular parasites. On the basis of an observation that Plasmodium "crisis forms" were seen in vitro after cultivation in media containing an antimalarial drug, we attempted to determine whether Plasmodium falciparum has the ability to undergo apoptosis. By use of either the apoptosis-inducer etoposide or the antimalarial chloroquine, apoptosis in Plasmodium asexual stages was evident by the observation of DNA fragmentation and disruption of transmembrane mitochondrial potential. Next, we sought to determine whether Plasmodium produces specific cysteine proteases that can induce apoptosis. We hypothesized that the 2 metacaspase-like proteins present in the Plasmodium genome contained features typical of downstream execution steps and upstream signaling pathways such caspase activation and domain recruitment. We report that one of the metacaspase genes, PF13_0289, in addition to a universally conserved catalytic cysteine and histidine dyad required for catalysis activity, contains a putative caspase recruitment domain in the N-terminal amino acid sequence. This putative P. falciparum metacaspase protein has been designated PfMCA1. Our findings offer important insights into parasite survival strategies that could open new ways for therapeutic alternatives to drug resistance.

Whole blood transcriptional signatures distinguishing patients with active tuberculosis from asymptomatic latently infected individuals have been described but, no consensus exists for the composition of optimal reduced gene sets as diagnostic biomarkers that also achieve discrimination from other diseases. We have recapitulated a blood transcriptional signature of active tuberculosis using RNA-Seq, previously reported by microarray that discriminates active tuberculosis from latently infected and healthy individuals, also validated in an independent cohort. We show that an advanced modular approach, which preserves and presents a signature of the entire transcriptome, can better discriminate patients with active tuberculosis from both latently infected and acute viral and bacterial infections. We suggest a method of targeted gene selection across modules for constructing diagnostic biomarkers, more representative of the transcriptome that overcomes some limitations of existing techniques. Finally, we utilise the modular approach to demonstrate dynamic heterogeneity in a longitudinal study of recent tuberculosis contacts.Tuberculosis (TB) is the leading cause of global mortality from an infectious disease. In 2016, there were 6.3 million new cases of TB disease and 1.67 million deaths and its diagnosis is problematic 1 . However, clinical disease represents one end of a spectrum of infection states. It is estimated that up to one third of all individuals worldwide have been infected with the causative pathogen, Mycobacterium tuberculosis, but the vast majority remain clinically asymptomatic with no radiological or microbiological evidence for active infection. This is termed latent TB infection (LTBI) and conceptually denotes a state in which M. tuberculosis persists within its host, while maintaining viability with the potential to replicate and cause symptomatic disease. Indeed, LTBI represents the primary reservoir for future incident TB, with 90% of all TB cases estimated to arise from reactivation of existing infection 1,2 . The risk of incident TB arising from existing LTBI is heterogeneous, poorly characterised and modifiable with anti-tuberculous treatment. Modelling studies indicate effective TB prevention to significantly reduce future TB incidence requires policies directed at the identification and treatment of LTBI 3 . However, implementation of mass screening programmes for this purpose are severely constrained by the size of the target population. Transformative advances in diagnostic tools that can effectively stratify TB risk in the LTBI population are therefore implicit to the realisation of systematic screening.The basis for LTBI heterogeneity rests with the limited scope of the tools we have available to identify the state. LTBI is inferred solely through evidence that immune sensitization has occurred, by the tuberculin skin test (TST) or the M. tuberculosis antigen-specific interferon-gamma (IFN-g) release assay (IGRA). Although these tests are both sensitive and specific for identi...

Malaria parasite species that infect mammals, including humans, must first take up residence in hepatic host cells as exoerythrocytic forms (EEF) before initiating infection of red blood cells that leads to malaria disease. Despite the importance of hepatic stages for immunity against malaria, little is known about their biology and antigenic composition. Here, we show that sporozoites, the parasites' transmission stage that resides in the mosquito vector salivary glands, can transform into early EEF without intracellular residence in host hepatocytes. The morphological sequence of transformation and the expression of proteins in the EEF appear indistinguishable from parasites that develop within host cells. Transformation depends on temperature elevation to 37°C and serum. Our findings demonstrate that residence in a host hepatocyte or specific host cell–derived factors are not necessary to bring about the profound morphological and biochemical changes of the parasite that occur after its transmission from vector to mammalian host.