Construction of a Plasmodium falciparum Rab-interactome identifies CK1 and PKA as Rab-effector kinases in malaria parasites.

BACKGROUND INFORMATION: The pathology causing stages of the human malaria parasite Plasmodium falciparum reside within red blood cells that are devoid of any regulated transport system. The parasite, therefore, is entirely responsible for mediating vesicular transport within itself and in the infected erythrocyte cytoplasm, and it does so in part via its family of 11 Rab GTPases. Putative functions have been ascribed to Plasmodium Rabs due to their homology with Rabs of yeast, particularly with Saccharomyces that has an equivalent number of rab/ypt genes and where analyses of Ypt function is well characterized. RESULTS: Rabs are important regulators of vesicular traffic due to their capacity to recruit specific effectors. In order to identify P. falciparum Rab (PfRab) effectors, we first built a Ypt-interactome by exploiting genetic and physical binding data available at the Saccharomyces genome database (SGD). We then constructed a PfRab-interactome using putative parasite Rab-effectors identified by homology to Ypt-effectors. We demonstrate its potential by wet-bench testing three predictions; that casein kinase-1 (PfCK1) is a specific Rab5B interacting protein and that the catalytic subunit of cAMP-dependent protein kinase A (PfPKA-C) is a PfRab5A and PfRab7 effector. CONCLUSIONS: The establishment of a shared set of physical Ypt/PfRab-effector proteins sheds light on a core set Plasmodium Rab-interactants shared with yeast. The PfRab-interactome should benefit vesicular trafficking studies in malaria parasites. The recruitment of PfCK1 to PfRab5B+ and PfPKA-C to PfRab5A+ and PfRab7+ vesicles, respectively, suggests that PfRab-recruited kinases potentially play a role in early and late endosome function in malaria parasites.

The Plasmodium falciparum schizont phosphoproteome reveals extensive phosphatidylinositol and cAMP-protein kinase A signaling.

The asexual blood stages of Plasmodium falciparum cause the most lethal form of human malaria. During growth within an infected red blood cell, parasite multiplication and formation of invasive merozoites is called schizogony. Here, we present a detailed analysis of the phosphoproteome of P. falciparum schizonts revealing 2541 unique phosphorylation sites, including 871 novel sites. Prominent roles for cAMP-dependent protein kinase A- and phosphatidylinositol-signaling were identified following analysis by functional enrichment, phosphoprotein interaction network clustering and phospho-motif identification tools. We observed that most key enzymes in the inositol pathway are phosphorylated, which strongly suggests additional levels of regulation and crosstalk with other protein kinases that coregulate different biological processes. A distinct pattern of phosphorylation of proteins involved in merozoite egress and red blood cell invasion was noted. The analyses also revealed that cAMP-PKA signaling is implicated in a wide variety of processes including motility. We verified this finding experimentally using an in vitro kinase assay and identified three novel PKA substrates associated with the glideosome motor complex: myosin A, GAP45 and CDPK1. Therefore, in addition to an established role for CDPK1 in the motor complex, this study reveals the coinvolvement of PKA, further implicating cAMP as an important regulator of host cell invasion.

Disseminated infection with a new genovar of Encephalitozoon cuniculi in a renal transplant recipient.

Disseminated microsporidiosis is a life-threatening opportunistic infection. Here, we report about a previously undescribed genovar of Encephalitozoon cuniculi causing disseminated infection in a non-HIV-infected renal transplant recipient. Disseminated microsporidiosis must be considered in the differential diagnosis of chronic fever in renal allograft recipients, even those without urinary symptoms.

[Trichinellosis]. / Trichinellose une zoonose parasitaire bien contrôlée en France.

Trichinellosis is a parasitic zoonosis caused by several species of the viviparous nematode of the genus Trichinella. Trichinellosis is a food borne zoonosis and can be contracted by eating raw or rare meat. This disease can be prevented by domestic and public health measures. France has thus considerably reinforced its system of prevention at slaughterhouses and in 2002 a Trichinella National Reference Centre was implemented. Trichinellosis is considered as a re-emergent disease throughout the world and more than 2500 cases have been reported in France since 1975. The possible severe neurological and cardiovascular complications, the risk of death highlight the need for a better knowledge of the disease and of its impact on public health.

Exploring the Plasmodium falciparum cyclic-adenosine monophosphate (cAMP)-dependent protein kinase (PfPKA) as a therapeutic target.

One of the prototype mammalian kinases is PKA and various roles have been defined for PKA in malaria pathogenesis. The recently described phospho-proteomes of Plasmodium falciparum introduced a great volume of phospho-peptide data for both basic research and identification of new anti-malaria therapeutic targets. We discuss the importance of phosphorylations detected in vivo at different sites in the parasite R and C subunits of PKA and highlight the inhibitor sites in the parasite R subunit. The N-terminus of the parasite R subunit is predicted to be very flexible and we propose that phosphorylation at multiple sites in this region likely represent docking sites for interactions with other proteins, such as 14-3-3. The most significant observation when the P. falciparum C subunit is compared to mammalian C isoforms is lack of phosphorylation at a key site tail implying that parasite kinase activity is not regulated so tightly as mammalian PKA. Phosphorylation at sites in the activation loop could be mediating a number of processes from regulating parasite kinase activity, to mediating docking of other proteins. The important differences between Plasmodium and mammalian PKA isoforms that indicate the parasite kinase is a valid anti-malaria therapeutic target.

Management of a Cryptosporidium hominis outbreak in a day-care center.

BACKGROUND: Cryptosporidium outbreaks in day-care centers (DCCs) occur commonly. However, controlling spread of infection in these settings is difficult, and data about effectiveness of different control strategies are sparse. In this study, a Cryptosporidium outbreak in a large DCC located in Brussels is described with evaluation of hygienic and therapeutic interventions. METHODS: During a 3-week period, 43 of 130 children attending the DCC developed enteric symptoms. Stools from 122 children were examined for microbial pathogens. Of them, 38 (31%) were diagnosed with Cryptosporidium, 29 of them being symptomatic (76%) and 9 (24%) asymptomatic. Diagnosis was performed by microscopy, antigen tests, and real-time polymerase chain reaction. Strict infection control measures were implemented during the first week after the start of outbreak. After 4 weeks, 27/38 children (71%) were still symptomatic and Cryptosporidium positive. Because of persisting symptoms and fear of further spread of infection, all 27 children were treated with paromomycin. Two weeks later, 18 of 27 children were asymptomatic and were parasitologically negative. The remaining 9 children, still symptomatic and Cryptosporidium positive, were treated with nitazoxanide. Three weeks later, week 9 after the start of outbreak, all 38 children involved in the outbreak were asymptomatic and Cryptosporidium negative. CONCLUSIONS: Our study underscores the need to rule out Cryptosporidium etiology in a diarrheal outbreak in a DCC. Rapid implementation of infection control measures can most likely halt the spread of infection. The role of nitazoxanide to limit duration of shedding of oocysts deserves more attention for its use in outbreaks.

Fetal anal incontinence evaluated by amniotic fluid digestive enzyme assay in myelomeningocele spina bifida.

The goals of this study were to determine whether anal sphincter dysfunction in spina bifida develops during fetal life or after birth and whether it reflects the severity of spina bifida and therefore can be used as a criterion to select the cases that could benefit from in uterosurgery. Total protein and digestive enzyme activities [gamma-glutamyl transpeptidase (GGTP), aminopeptidase M (AMP), and alkaline phosphatase isoenzymes including the intestinal form (iALP)] were assayed retrospectively in amniotic fluid from 80 myelomeningocele spina bifida cases without unrelated associated malformation (gestational age 14-33 wk). A normal enzyme activity profile was observed in 46 of the 80 cases. Two abnormal profiles were observed: 1) bilious vomiting, characterized by abnormally high GGTP and AMP activities but normal iALP, and 2) digestive enzyme leakage, characterized by abnormally high activities of GGTP, AMP, and iALP, typical of anal incontinence. No relation was observed between these enzyme activity profiles and the different secondary signs of spina bifida or the level of the damage. In conclusion, anal sphincter dysfunction in spina bifida revealed by amniotic fluid digestive enzyme activities occurred before 24 wk in fetal life in 28.7% of cases. This criterion may be indicative of the severity of spina bifida and therefore perhaps could be used to select cases that are suited to in utero surgery. It could also be used to establish the potential benefit of this surgery in fecal incontinence.