T-cell epitope polymorphisms of the Plasmodium falciparum circumsporozoite protein among field isolates from Sierra Leone: age-dependent haplotype distribution?

<p>Abstract</p> <p>Background</p> <p>In the context of the development of a successful malaria vaccine, understanding the polymorphisms exhibited by malaria antigens in natural parasite populations is crucial for proper vaccine design. Recent observations have indicated that sequence polymorphisms in the C-terminal T-cell epitopes of the <it>Plasmodium falciparum </it>circumsporozoite protein (Pf<it>csp</it>) are rather low and apparently stable in low endemic areas. This study sought to assess the pattern in a malaria endemic setting in Africa, using samples from Freetown, Sierra Leone.</p> <p>Methods</p> <p>Filter-paper blood samples were collected from subjects at a teaching hospital in Freetown during September–October 2006 and in April–May 2007. The C-terminal portion of the Pf<it>csp </it>gene spanning the Th2R and Th3R epitopes was amplified and directly sequenced; sequences were analysed with subject parameters and polymorphism patterns in Freetown were compared to that in other malaria endemic areas.</p> <p>Results and Discussion</p> <p>Overall, the genetic diversity in Freetown was high. From a total of 99 sequences, 42 haplotypes were identified with at least three accounting for 44.4% (44/99): the 3D7-type (19.2%), a novel type, P-01 (17.2%), and E12 (8.1%). Interestingly, all were unique to the African sub-region and there appeared to be predilection for certain haplotypes to distribute in certain age-groups: the 3D7 type was detected mainly in hospitalized children under 15 years of age, while the P-01 type was common in adult antenatal females (Pearson Chi-square = 48.750, degrees of freedom = 34, <it>P </it>= 0.049). In contrast, the single-haplotype predominance (proportion > 50%) pattern previously identified in Asia was not detected in Freetown.</p> <p>Conclusion</p> <p>Haplotype distribution of the T-cell epitopes of Pf<it>csp </it>in Freetown appeared to vary with age in the study population, and the polymorphism patterns were similar to that observed in neighbouring Gambia, but differed significantly at the sequence level from that observed in Asia. The findings further emphasize the role of local factors in generating polymorphisms in the T-cell epitopes of the <it>P. falciparum </it>circumsporozoite protein.</p

Long-Range Chromosome Organization in E. coli: A Site-Specific System Isolates the Ter Macrodomain

The organization of the Escherichia coli chromosome into a ring composed of four macrodomains and two less-structured regions influences the segregation of sister chromatids and the mobility of chromosomal DNA. The structuring of the terminus region (Ter) into a macrodomain relies on the interaction of the protein MatP with a 13-bp target called matS repeated 23 times in the 800-kb-long domain. Here, by using a new method that allows the transposition of any chromosomal segment at a defined position on the genetic map, we reveal a site-specific system that restricts to the Ter region a constraining process that reduces DNA mobility and delays loci segregation. Remarkably, the constraining process is regulated during the cell cycle and occurs only when the Ter MD is associated with the division machinery at mid-cell. The change of DNA properties does not rely on the presence of a trans-acting mechanism but rather involves a cis-effect acting at a long distance from the Ter region. Two specific 12-bp sequences located in the flanking Left and Right macrodomains and a newly identified protein designated YfbV conserved with MatP through evolution are required to impede the spreading of the constraining process to the rest of the chromosome. Our results unravel a site-specific system required to restrict to the Ter region the consequences of anchoring the Ter MD to the division machinery

Fresh-blood-free diet for rearing malaria mosquito vectors

Mosquito breeding depends on the supply of fresh vertebrate blood, a major bottleneck for large-scale production of Anopheles spp. Feeding alternatives to fresh blood are thus a priority for research, outdoor large-cage trials and control interventions. Several artificial meal compositions were tested and Anopheles oogenesis, egg laying and development into the next generation of adult mosquitoes were followed. We identified blood-substitute-diets that supported ovarian development, egg maturation and fertility as well as, low progeny larval mortality, and normal development of offspring into adult mosquitoes. The formulated diet is an effective artificial meal, free of fresh blood that mimics a vertebrate blood meal and represents an important advance for the sustainability of Anopheles mosquito rearing in captivity.Agência financiadora / Número do subsídio Bill and Melinda Gates Foundation OPP1138841 Fundacao para a Ciencia e Tecnologia GHTM - UID/Multi/04413/201 CCMAR - UID/Multi/04326/2013 UID/Multi/04326/2013 RF SFRH/BPD/89811/2012 FAPEAM, Brazil 19716.UNI472.2459.20022014info:eu-repo/semantics/publishedVersio

P-Type ATPase TAT-2 Negatively Regulates Monomethyl Branched-Chain Fatty Acid Mediated Function in Post-Embryonic Growth and Development in C. elegans

Monomethyl branched-chain fatty acids (mmBCFAs) are essential for Caenorhabditis elegans growth and development. To identify factors acting downstream of mmBCFAs for their function in growth regulation, we conducted a genetic screen for suppressors of the L1 arrest that occurs in animals depleted of the 17-carbon mmBCFA C17ISO. Three of the suppressor mutations defined an unexpected player, the P-type ATPase TAT-2, which belongs to the flippase family of proteins that are implicated in mediating phospholipid bilayer asymmetry. We provide evidence that TAT-2, but not other TAT genes, has a specific role in antagonizing the regulatory activity of mmBCFAs in intestinal cells. Interestingly, we found that mutations in tat-2 also suppress the lethality caused by inhibition of the first step in sphingolipid biosynthesis. We further showed that the fatty acid side-chains of glycosylceramides contain 20%–30% mmBCFAs and that this fraction is greatly diminished in the absence of mmBCFA biosynthesis. These results suggest a model in which a C17ISO-containing sphingolipid may mediate the regulatory functions of mmBCFAs and is negatively regulated by TAT-2 in intestinal cells. This work indicates a novel connection between a P-type ATPase and the critical regulatory function of a specific fatty acid

IL-6 is constitutively expressed during lung morphogenesis and enhances fetal lung explant branching

Previous studies have shown that chorioamnionitis, with increased IL-6, promotes fetal lung maturation and decreases the incidence of respiratory distress syndrome in premature neonates. However, the expression pattern and the effects of IL-6 on fetal lung growth mechanisms remain unknown. IL-6 expression was assessed by in situ hybridization and by real-time PCR between 14.5 and 21.5 d postconception. Normal and nitrofen-induced hypoplastic lung explants were cultured with increasing IL-6 doses or IL-6 neutralizing antibodies. Branching, cellular proliferation (Ki-67) and MAPK phosphorylation in fetal lung explants were analyzed. Pulmonary primitive epithelium expressed IL-6 constitutively throughout all gestational ages, displaying highest levels during earliest stages. In normal and hypoplastic lung explants, IL-6 neutralizing antibodies significantly reduced, whereas IL-6 supplementation induced a biphasic effect (lower doses increased, while the highest dose did not accomplish additional effect) on branching and cellular proliferation. IL-6 enhanced p38-MAPK phosphorylation without changing MEK1/2 and JNK pathways. The present study suggests a physiological role for IL-6 on pulmonary branching mechanisms most likely involving p38-MAPK intracellular signalling pathway

Characterization of immunoglobulin G antibodies to Plasmodium falciparum sporozoite surface antigen MB2 in malaria exposed individuals

<p>Abstract</p> <p>Background</p> <p>MB2 protein is a sporozoite surface antigen on the human malaria parasite <it>Plasmodium falciparum</it>. MB2 was identified by screening a <it>P. falciparum </it>sporozoite cDNA expression library using immune sera from a protected donor immunized via the bites of <it>P. falciparum</it>-infected irradiated mosquitoes. It is not known whether natural exposure to <it>P. falciparum </it>also induces the anti-MB2 response and if this response differs from that in protected individuals immunized via the bites of <it>P. falciparum </it>infected irradiated mosquitoes. The anti-MB2 antibody response may be part of a robust protective response against the sporozoite.</p> <p>Methods</p> <p>Fragments of polypeptide regions of MB2 were constructed as recombinant fusions sandwiched between glutathione S-transferase and a hexa histidine tag for bacterial expression. The hexa histidine tag affinity purified proteins were used to immunize rabbits and the polyclonal sera evaluated in an <it>in vitro </it>inhibition of sporozoite invasion assay. The proteins were also used in immunoblots with sera from a limited number of donors immunized via the bites of <it>P. falciparum </it>infected irradiated mosquitoes and plasma and serum obtained from naturally exposed individuals in Kenya.</p> <p>Results</p> <p>Rabbit polyclonal antibodies targeting the non-repeat region of the basic domain of MB2 inhibited sporozoites entry into HepG2-A16 cells <it>in vitro</it>. Analysis of serum from five human volunteers that were immunized via the bites of <it>P. falciparum </it>infected irradiated mosquitoes that developed immunity and were completely protected against subsequent challenge with non-irradiated parasite also had detectable levels of antibody against MB2 basic domain. In contrast, in three volunteers not protected, anti-MB2 antibodies were below the level of detection. Sera from protected volunteers preferentially recognized a non-repeat region of the basic domain of MB2, whereas plasma from naturally-infected individuals also had antibodies that recognize regions of MB2 that contain a repeat motif in immunoblots. Sequence analysis of eleven field isolates and four laboratory strains showed that these antigenic regions of the basic domain of the <it>MB2 </it>gene are highly conserved in parasites obtained from different parts of the world. Moreover, anti-MB2 antibodies also were detected in the plasma of 83% of the individuals living in a malaria endemic area of Kenya (n = 41).</p> <p>Conclusion</p> <p>A preliminary analysis of the human humoral response against MB2 indicates that it may be an additional highly conserved target for immune intervention at the pre-erythrocytic stage of <it>P. falciparum </it>life cycle.</p

Biofilm Induced Tolerance towards Antimicrobial Peptides

Increased tolerance to antimicrobial agents is thought to be an important feature of microbes growing in biofilms. We address the question of how biofilm organization affects antibiotic susceptibility. We established Escherichia coli biofilms with differential structural organization due to the presence of IncF plasmids expressing altered forms of the transfer pili in two different biofilm model systems. The mature biofilms were subsequently treated with two antibiotics with different molecular targets, the peptide antibiotic colistin and the fluoroquinolone ciprofloxacin. The dynamics of microbial killing were monitored by viable count determination, and confocal laser microscopy. Strains forming structurally organized biofilms show an increased bacterial survival when challenged with colistin, compared to strains forming unstructured biofilms. The increased survival is due to genetically regulated tolerant subpopulation formation and not caused by a general biofilm property. No significant difference in survival was detected when the strains were challenged with ciprofloxacin. Our data show that biofilm formation confers increased colistin tolerance to cells within the biofilm structure, but the protection is conditional being dependent on the structural organization of the biofilm, and the induction of specific tolerance mechanisms

Biochemical Properties of a Novel Cysteine Protease of Plasmodium vivax, Vivapain-4

Plasmodium vivax affects hundreds of millions each year and results in severe morbidity and mortality. Plasmodial cysteine proteases (CPs) play crucial roles during the progression of malaria since inhibition of these molecules impairs parasite growth. These CPs might be targeted for new antimalarial drugs. We characterized a novel P. vivax CP, vivapain-4 (VX-4), which appeared to evolve differentially among primate Plasmodium species. VX-4 showed highly unique substrate preference depending on surrounding micro-environmental pH. It effectively hydrolyzed benzyloxycarbonyl-Leu-Arg-4-methyl-coumaryl-7-amide (Z-Leu-Arg-MCA) and Z-Phe-Arg-MCA at acidic pH and Z-Arg-Arg-MCA at neutral pH. Three amino acids (Ala90, Gly157 and Glu180) that delineate the S2 pocket were found to be substituted in VX-4. Alteration of Glu180 abolished hydrolytic activity against Z-Arg-Arg-MCA at neutral pH, indicating Glu180 is intimately involved in the pH-dependent substrate preference. VX-4 hydrolyzed actin at neutral pH and hemoglobin at acidic pH, and participated in plasmepsin 4 activation at neutral/acidic pH. VX-4 was localized in the food vacuoles and cytoplasm of the erythrocytic stage of P. vivax. The differential substrate preferences depending on pH suggested a highly efficient mechanism to enlarge biological implications of VX-4, including hemoglobin degradation, maturation of plasmepsin, and remodeling of the parasite architecture during growth and development of P. vivax

Scabies Mite Peritrophins Are Potential Targets of Human Host Innate Immunity

The gut of most invertebrates is lined by a protective layer of chitin and glycoproteins, often designated as a peritrophic matrix. Previous research suggests that it forms a barrier that may protect the midgut epithelium from abrasive food particles and pathogens. Parasitic invertebrates ingesting vertebrate plasma have evolved additional strategies to protect themselves from hazardous host molecules consumed during feeding. An important part of the immediate defense in vertebrate plasma is complement-mediated killing. The Complement system is a complex network of more than 35 proteins present in human plasma that results in killing of foreign cells including the gut epithelial cells of a feeding parasite. Recently we found that scabies mites, who feed on skin containing plasma, produce several proteins that inhibit human complement within the mite gut. The mites excrete these molecules into the upper epidermis where they presumably also inhibit complement activity. Mite gut antigens that initially trigger the complement cascade have not been identified previously. Obvious possible targets of complement attack within the mite gut could be peritrophins. Our study describes the first peritrophin identified in scabies mites and indicates a possible role in complement activation