Temperature Shift and Host Cell Contact Up-Regulate Sporozoite Expression of Plasmodium falciparum Genes Involved in Hepatocyte Infection

Plasmodium sporozoites are deposited in the skin by Anopheles mosquitoes. They then find their way to the liver, where they specifically invade hepatocytes in which they develop to yield merozoites infective to red blood cells. Relatively little is known of the molecular interactions during these initial obligatory phases of the infection. Recent data suggested that many of the inoculated sporozoites invade hepatocytes an hour or more after the infective bite. We hypothesised that this pre-invasive period in the mammalian host prepares sporozoites for successful hepatocyte infection. Therefore, the genes whose expression becomes modified prior to hepatocyte invasion would be those likely to code for proteins implicated in the subsequent events of invasion and development. We have used P. falciparum sporozoites and their natural host cells, primary human hepatocytes, in in vitro co-culture system as a model for the pre-invasive period. We first established that under co-culture conditions, sporozoites maintain infectivity for an hour or more, in contrast to a drastic loss in infectivity when hepatocytes were not included. Thus, a differential transcriptome of salivary gland sporozoites versus sporozoites co-cultured with hepatocytes was established using a pan-genomic P. falciparum microarray. The expression of 532 genes was found to have been up-regulated following co-culture. A fifth of these genes had no orthologues in the genomes of Plasmodium species used in rodent models of malaria. Quantitative RT-PCR analysis of a selection of 21 genes confirmed the reliability of the microarray data. Time-course analysis further indicated two patterns of up-regulation following sporozoite co-culture, one transient and the other sustained, suggesting roles in hepatocyte invasion and liver stage development, respectively. This was supported by functional studies of four hitherto uncharacterized proteins of which two were shown to be sporozoite surface proteins involved in hepatocyte invasion, while the other two were predominantly expressed during hepatic parasite development. The genome-wide up-regulation of expression observed supports the hypothesis that the shift from the mosquito to the mammalian host contributes to activate quiescent salivary gland sporozoites into a state of readiness for the hepatic stages. Functional studies on four of the up-regulated genes validated our approach as one means to determine the repertoire of proteins implicated during the early events of the Plasmodium infection, and in this case that of P. falciparum, the species responsible for the severest forms of malaria

Identification and Genome-Wide Prediction of DNA Binding Specificities for the ApiAP2 Family of Regulators from the Malaria Parasite

The molecular mechanisms underlying transcriptional regulation in apicomplexan parasites remain poorly understood. Recently, the Apicomplexan AP2 (ApiAP2) family of DNA binding proteins was identified as a major class of transcriptional regulators that are found across all Apicomplexa. To gain insight into the regulatory role of these proteins in the malaria parasite, we have comprehensively surveyed the DNA-binding specificities of all 27 members of the ApiAP2 protein family from Plasmodium falciparum revealing unique binding preferences for the majority of these DNA binding proteins. In addition to high affinity primary motif interactions, we also observe interactions with secondary motifs. The ability of a number of ApiAP2 proteins to bind multiple, distinct motifs significantly increases the potential complexity of the transcriptional regulatory networks governed by the ApiAP2 family. Using these newly identified sequence motifs, we infer the trans-factors associated with previously reported plasmodial cis-elements and provide evidence that ApiAP2 proteins modulate key regulatory decisions at all stages of parasite development. Our results offer a detailed view of ApiAP2 DNA binding specificity and take the first step toward inferring comprehensive gene regulatory networks for P. falciparum

Plasmodium Protease ROM1 Is Important for Proper Formation of the Parasitophorous Vacuole

Apicomplexans are obligate intracellular parasites that invade host cells by an active process leading to the formation of a non-fusogenic parasitophorous vacuole (PV) where the parasite replicates within the host cell. The rhomboid family of proteases cleaves substrates within their transmembrane domains and has been implicated in the invasion process. Although its exact function is unknown, Plasmodium ROM1 is hypothesized to play a role during invasion based on its microneme localization and its ability to cleave essential invasion adhesins. Using the rodent malaria model, Plasmodium yoelii, we carried out detailed quantitative analysis of pyrom1 deficient parasites during the Plasmodium lifecycle. Pyrom1(-) parasites are attenuated during erythrocytic and hepatic stages but progress normally through the mosquito vector with normal counts of oocyst and salivary gland sporozoites. Pyrom1 steady state mRNA levels are upregulated 20-fold in salivary gland sporozoites compared to blood stages. We show that pyrom1(-) sporozoites are capable of gliding motility and traversing host cells normally. Wildtype and pyrom1(-) sporozoites do not differ in the rate of entry into Hepa1–6 hepatocytes. Within the first twelve hours of hepatic development, however, only 50% pyrom1(-) parasites have developed into exoerythrocytic forms. Immunofluorescence microscopy using the PVM marker UIS4 and transmission electron microscopy reveal that the PV of a significant fraction of pyrom1(-) parasites are morphologically aberrant shortly after invasion. We propose a novel function for PyROM1 as a protease that promotes proper PV modification to allow parasite development and replication in a suitable environment within the mammalian host

Why Functional Pre-Erythrocytic and Bloodstage Malaria Vaccines Fail: A Meta-Analysis of Fully Protective Immunizations and Novel Immunological Model

Background: Clinically protective malaria vaccines consistently fail to protect adults and children in endemic settings, and at best only partially protect infants. Methodology/Principal Findings: We identify and evaluate 1916 immunization studies between 1965-February 2010, and exclude partially or nonprotective results to find 177 completely protective immunization experiments. Detailed reexamination reveals an unexpectedly mundane basis for selective vaccine failure: live malaria parasites in the skin inhibit vaccine function. We next show published molecular and cellular data support a testable, novel model where parasite-host interactions in the skin induce malaria-specific regulatory T cells, and subvert early antigen-specific immunity to parasite-specific immunotolerance. This ensures infection and tolerance to reinfection. Exposure to Plasmodium-infected mosquito bites therefore systematically triggers immunosuppression of endemic vaccine-elicited responses. The extensive vaccine trial data solidly substantiate this model experimentally. Conclusions/Significance: We conclude skinstage-initiated immunosuppression, unassociated with bloodstage parasites, systematically blocks vaccine function in the field. Our model exposes novel molecular and procedural strategies to significantly and quickly increase protective efficacy in both pipeline and currently ineffective malaria vaccines, and forces fundamental reassessment of central precepts determining vaccine development. This has major implications fo

Comparative gustatory responses in four species of gerbilline rodents

Integrated taste responses to chemical stimulation of the tongue were recorded from the intact chorda tympani nerve in four species of gerbils ( Meriones libycus, M. shawi, M. unguiculatus and Psammomys obesus ).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47116/1/359_2004_Article_BF00618177.pd

Wplyw roslin motylkowatych na wartosc pokarmowa kostrzewy trzcinowej Festuca arundinacea Schreb.

W warunkach polowych badano wpływ mieszanek motylkowo-trawiastych na wielkość plonu, udział roślin motylkowatych w mieszankach i zawartość N, P i Ca w odmianach trawy (Festuca arundinacea Schreb.) pochodzących z mieszanek. Stwierdzono dodatnie działanie Trifolium pratense L. i Medicago media Pers. na wielkość plonu mieszanek (10,7-12,3 t/ha) i samych traw (5,5-7,5 t/ha) pochodzących z mieszanek. Już w drugim roku zbioru pod wpływem Trifolium pratense L. zwiększyła się znacznie zawartość azotu w trawach, a fosforu w trzecim roku wegetacji. Dodatnie działanie Medicago media Pers. na zawartość azotu, fosforu i wapnia wzrastała w kolejnych latach wegetacji traw.Influence of grass-papilionaceous mixtures on the yield, percentage of papilionaceous plants in mixtures and contents of N, P and Ca in grass varieties (Festuca arundinacea Schreb.) in these mixtures was studied in field experiments. Positive influence of Trifolium pratense L. and Medicago media Pers. on yields of the mixtures (10.7-12.3 t/ha) and grass varieties being components of these mixtures(5.5-7.5 t/ha) was found. It was found also that the N content in grass increased significantly in second year yielding under the influence of Trifolium pratense L. while P content increased in the third year of vegetation. Positive influence of Medicago media Pers. on the N, P and Ca contents was increasing in subsequent years of grass vegetation

Niektóre cechy jakościowe traw pochodzących z mieszanek trawiasto-motylkowatych

W doświadczeniu polowym badano wpływ koniczyny czerwonej (Trifolium pratense L.) i lucerny mieszańcowej (Medicago media Pers.) na plon i zawartość azotu w mieszankach z odmianami kostrzewy trzcinowej (Festuca arundinacea Schreb.) W pierwszym roku zbioru, a drugim wegetacji roślin, stwierdzono znaczne działanie plonotwórcze T. pratense (9,7- 11,4 t/ha) w porównaniu do M. media (7,5-8,1 t/ha) jako komponentów mieszanek z trawami. Dopiero w drugim roku zbioru wyraźnie wzrosły plony mieszanek na obiektach z M. media do 11,3 t/ha. W trzecim roku zbioru plony na obiektach z T. pratense spadły do poziomu 7,5 t/ha, a mieszanki z M. media utrzymywały się w granicach 10,5 t/ha. Zawartość azotu ogólnego była niska na obiektach bez roślin motylkowatych. Pod wpływem T. pratense zawartość tego składnika wzrosła o 50% w pierwszym roku zbioru, a w latach następnych o 20% w porównaniu do monokultur F. arundinacea. Dodatnie działanie lucerny łąkowej na zawartość azotu w trawach zaznaczyła się dopiero w drugim roku użytkowania, w miarę zwiększania się ilości tego gatunku w runi.Influence of the mixtures of Trifolium pratense L. and Medicago media Pers. with Festuca arundinacea Schreb. on yield and nitrogen content was studied in field experiments. First year of yielding, i.e. the second year of vegetation showed that the mixtures containing red clover significantly increased the yields (9.7-11.5 t/ha) in comparison with these obtained for M. media containing mixtures (7.5-8.1 t/ha). In last case the yields increased significantly in second year - up to 11.3 t/ha. Third year yields from T. pratense fields decreased to 7.5 t/ha, whereas fields sown with M. media gave the yields of about 10.5 t/ha. Total nitrogen content was low on the fields without papilionaceous plant. The N content increased by 50% in the first year yielding under the influence of T. pratense and by 20% in next years yields in comparison to yields obtained from F. arundinacea monoculture fields. Positive influence of M. media on grass nitrogen content was observed only in second year of studies and was following by increased quantity of that species in yields of subsequent years