Brugia malayi: In vitro effects of ivermectin and moxidectin on adults and microfilariae

The effect of ivermectin and moxidectin on the motility of Brugia malayi adults and microfilariae and on the fertility of B. malayi females was examined. Motility was reduced in adults after exposure to both drugs and worms were non-motile and dead within eight days. The motility of microfilariae was significantly reduced at all drug concentrations and ceased at concentrations of 2500 and 5000 μg/mL. The motility of microfilariae released by females was reduced after exposure to both drugs, however ivermectin had a greater effect at concentrations between 170 and 5000 μg/mL. Both drugs reduced the number of microfilariae released by females and within four days their release was inhibited. The presence of the bacterial endosymbiont Wolbachia was examined in adults and microfilariae after exposure to increasing concentrations of ivermectin and moxidectin. A decrease in wsp expression was correlated with increasing drug concentration.     

Vaccination with a genetically modified Brugia malayi cysteine protease inhibitor-2 reduces adult parasite numbers and affects the fertility of female worms following a subcutaneous challenge of Mongolian gerbils (Meriones unguiculatus) with B. malayi infective larvae

Vaccination of Mongolian gerbils with Brugia malayi cysteine protease inhibitor-2 in which the amino acid Asn66 was mutated to Lys66 (Bm-CPI-2M) resulted in reduced parasite numbers of 48.6% and 48.0% at 42 and 90 days p.i. with B. malayi L3s. Fertility of female worms was also affected at 90 days p.i. In vitro killing of L3s observed in the presence of gerbil peritoneal exudate cells and anti-Bm-CPI-2M sera suggests antibody-dependent cell-mediated cytotoxicity as a putative protective mechanism. These observations suggest that Bm-CPI-2M is a promising prophylactic and anti-fecundity vaccine candidate.     

Brugia pahangi: Antibodies and microfilaremias in Lewis rats

Male and female Lewis rats were inoculated subcutaneously in the left groin with 75 infective larvae of Brugia pahangi and microfilaremias were followed for as long as 420 days postinoculation. Patent infections developed in 64% of the female rats and 95% of the male rats. Mean prepatent periods were similar (65.9 and 63.9 days, respectively), but mean microfilaremias in males rose much higher, to a mean of 218 mf/0.25 ml blood at 270 days postinoculation. IgG titers, as measured by enzyme-linked immunosorbent assay (ELISA), to adult worm somatic antigen were higher than those to microfilariae in almost all rats. For both sexes, the most consistently microfilaremic rats had highest titers to these antigens. Granulomas with degenerating microfilaria were present in the spleen of male rats with high microfilaremias (>100–300 mf/0.25 ml blood). Ouchterlony precipitin reactions suggested that most rats with spleen granulomas responded to microfilarial antigen components to which most rats without granulomas did not. Neither spleen granulomas nor antibody responses measured in this study appeared to have protective (microfilaremia-lowering) value. As measured by microfilaremias, the male Lewis rat is not as susceptible as some conventional hosts of B. pahangi, but it does consistently become infected and remains microfilaremic for more than a year. Preferential male susceptibility indicates that this model may be useful for studying this aspect of human lymphatic filariasis.     

Brugia malayi and B. pahangi: Cultivation in vitro of infective larvae to the fourth and fifth stages

Cultivation of fourth stage Brugia pahangi and B. malayi larvae from infective larvae (stage 3) were obtained in culture medium RPMI 1640 supplemented with 10% human AB serum and an LCC-MK₂ rhesus monkey kidney continuous cell line feeder layer. This culture system kept larvae alive in excess of 7 weeks, and served as a source for collection of the worms' secretory, excretory, and moulting antigens.     

Crystal structure of Brugia malayi venom allergen-like protein-1 (BmVAL-1), a vaccine candidate for lymphatic filariasis

Brugia malayi is a causative agent of lymphatic filariasis, a major tropical disease. The infective L3 parasite stage releases immunomodulatory proteins including the venom allergen-like proteins (VALs), which are members of the SCP/TAPS (Sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. BmVAL-1 is a major target of host immunity with >90% of infected B. malayi microfilaraemic cases being seropositive for antibodies to BmVAL-1. This study is part of ongoing efforts to characterize the structures and functions of important B. malayi proteins. Recombinant BmVAL-1 was produced using a plant expression system, crystallized and the structure was solved by molecular replacement and refined to 2.1?Å, revealing the characteristic alpha/beta/alpha sandwich topology of eukaryotic SCP/TAPS proteins. The protein has more than 45% loop regions and these flexible loops connect the helices and strands, which are longer than predicted based on other parasite SCP/TAPS protein structures. The large central cavity of BmVAL-1 is a prototypical CRISP cavity with two histidines required to bind divalent cations. The caveolin-binding motif (CBM) that mediates sterol binding in SCP/TAPS proteins is large and open in BmVAL-1 and is N-glycosylated. N-glycosylation of the CBM does not affect the ability of BmVAL-1 to bind sterol in vitro. BmVAL-1 complements the in vivo sterol export phenotype of yeast mutants lacking their endogenous SCP/TAPS proteins. The in vitro sterol-binding affinity of BmVAL-1 is comparable with Pry1, a yeast sterol transporting SCP/TAPS protein. Sterol binding of BmVAL-1 is dependent on divalent cations. BmVAL-1 also has a large open palmitate-binding cavity, which binds palmitate comparably to tablysin-15, a lipid-binding SCP/TAPS protein. The central cavity, CBM and palmitate-binding cavity of BmVAL-1 are interconnected within the monomer with channels that can serve as pathways for water molecules, cations and small molecules.     

Naturally mosaic operons for secondary metabolite biosynthesis: variability and putative horizontal transfer of discrete catalytic domains of the epothilone polyketide synthase locus

A putative instance of horizontal gene transfer (HGT) involving adjacent, discrete -ketoacyl synthase (KS), acyl carrier protein (ACP) and nonribosomal peptide synthase (NRPS) domains of the epothilone Type I polyketide biosynthetic gene cluster from the myxobacterium Sorangium cellulosom was identified using molecular phylogenetics and sequence analyses. The specific KS domain of the module EPO B fails to cluster phylogenetically with other epothilone KS sequences present at this locus, in contrast to what is typically observed in many other Type I polyketide synthase (PKS) biosynthetic loci. Furthermore, the GC content of the epoB KS, epoA ACP and NRPS domains differs significantly from the base composition of other epothilone domain sequences. In addition, the putatively transferred epothilone loci are located near previously identified transposon-like sequences. Lastly, comparison with other KS loci revealed another possible case of horizontal transfer of secondary metabolite genes in the genus Pseudomonas. This study emphasizes the use of several lines of concordant evidence (phylogenetics, base composition, transposon sequences) to infer the evolutionary history of particular gene and enzyme sequences, and the results support the idea that genes coding for adaptive traits, e.g. defensive natural products, may be prone to transposition between divergent prokaryotic taxa and genomes.Communicated by W. Arber     

Brugia malayi,Brugia pahangi, and Brugia patei: Pulmonary pathology in jirds, Meriones unguiculatus

The chronological development of pulmonary lesions due to subperiodic Brugia malayi and related species was studied in the jird, Meriones unguiculatus. Major pathologic changes included (1) granulomas induced by larvae and adults, (2) obstructive endarteritis, and (3) chronic interstitial inflammation with degenerating microfilariae. The majority of pulmonary granulomas were initiated near the time of the final molt, about 30 days postinoculation, followed by involution and formation of residual vascular lesions over the next several months. A minority of granulomas arose about sexually mature adult worms and showed a characteristic sequence of development along the length of these worms. Ultrastructural observations suggested that within granulomas internal structures of the worm underwent an autolytic-like disintegration, while the cuticle remained intact. A material, presumably of parasitic origin, then appeared between the cuticle and adherent epitheloid and giant cells and was subsequently phagocytized by these cells. Obstructive endarteritis appeared to peak at about the time of the final molt, and became largely fibrotic by 125 days postinoculation; electron microscopy of the subintimal infiltrates revealed a variety of cells including inflammatory cells and others interpreted as modfied cells of vascular origin (smooth muscle cells and two types of endothelial cells).Parasitological data suggested that both larvae and adults migrated to the lungs and that mating occurred here, rather than in peripheral sites of development. In terms of development, reproduction and survival, the pulmonary arteries of the male jird offered a suitable alternative for the localization of these primarily lymphatic filariae; our results thus suggest that the pulmonary localization of these worms should not be considered indicative of an aberrant mode of development. The failure of female jirds to develop detectable peripheral microfilaremia was associated with a high rate of infertility among female worms, rather than pulmonary sequestration or destruction of microfilariae.     

A major chemotaxis gene cluster in Azospirillum brasilense and relationships between chemotaxis operons in alpha-proteobacteria

Azospirillum brasilense shows chemotaxis to a variety of nutrients and oxygen. Genes encoding the central signal transduction pathway in chemotaxis were identified by phenotypic complementation of generally non-chemotactic mutants. Sequencing of a DNA fragment, which complemented two different mutants, revealed a region of five open reading frames translated in one direction and encoding homologs of known genes comprising excitation and adaptation pathways for chemotaxis in other bacterial species. The major chemotaxis gene cluster appears to be essential for all known behavioral responses that direct swimming motility in A. brasilense. Phylogenetic and genomic analysis revealed three groups of chemotaxis operons in alpha-proteobacterial species and assigned the A. brasilense operon to one of them. Interestingly, operons that are shown to be major regulators of behavior in several alpha-proteobacterial species are not orthologous.     

Functional annotation of putative hypothetical proteins from Candida dubliniensis

An extensive analysis of C. dubliniensis proteomics data showed that ~ 22% protein are conserved hypothetical proteins (HPs) whose function is still not determined precisely. Analysis of gene sequence of HPs provides a platform to establish sequence–function relationships to a more profound understanding of the molecular machinery of organisms at systems level. Here we have combined the latest versions of bioinformatics tools including, protein family, motifs, intrinsic features from the amino acid sequence, sequence–function relationship, pathway analysis, etc. to assign a precise function to HPs for which no any experimental information is available. Our results show that 27 HPs have well defined functions and we categorized them as enzyme, nucleic acid binding, transport protein, etc. Five HPs showed adhesin character that is likely to be essential for the survival of yeast and pathogenesis. We also addressed issues related to the sub-cellular localization and signal peptide identification which provides an idea about its colocalization and function. The outcome of the present study may facilitate better understanding of mechanism of virulence, drug resistance, pathogenesis, adaptability to host, tolerance for host immune response, and drug discovery for treatment of C. dubliniensis infections.     

Brugia pahangi: Feeding and nutrient uptake in vitro and in vivo

Incubation in vitro of adult Brugia pahangi in an apparatus which permitted the separate exposure of the anterior, middle, or posterior region of the worms to medium-containing radioactively labeled d-glucose, l-leucine, and adenosine has provided evidence that these materials are taken up in physiologically significant amounts by a transcuticular route. No evidence for an oral ingestion of materials has been obtained from worms in vitro, but in vivo an oral uptake of Trypan blue has been demonstrated. The ultrastructure and cytochemical staining reactions for nonspecific esterase, acid phosphatase (EC-3.1.3.2), and leucine naphthylamidase of the gut and body wall are described.     

A novel fluorescent timer based on bicistronic expression strategy in Caenorhabditis elegans

Fluorescent timers are useful tools for studying the spatial and temporal cellular or molecular events. Based on the trans-splicing mechanism in Caenorhabditis elegans, we constructed a “fluorescent timer” through bicistronic expression of two fluorescent proteins with different maturation times. When used in vivo, this “timer” changes its color over time and therefore can be used to monitor the activity of the targeted promoters in C. elegans. Using this “timer”, we have successfully traced the time-dependent activity of myo-3 promoter which drives expression in body wall muscle and vulval muscle. We found that the myo-3 promoter started to be active about 7 h after egg-laying and sustained its activity in the following hatching process. We have also determined the myo-3 promoter activity during larval development by this “timer”. We anticipate that more new “fluorescent timers” with variable time-resolution could be designed by bicistronic expression of different fluorescent protein pairs.     

Gene replacement in Haloarcula marismortui: construction of a strain with two of its three chromosomal rRNA operons deleted

Site-directed mutagenesis were done in Haloarcula marismortui using the strategy that Khorana and coworkers devised for deleting the bacteriorhodopsin gene from Halobacterium halobium [Krebs et al. Proc Natl Acad Sci USA 90:1987–1991 (1993)]. Strains have been prepared from H. marsimortui, which normally has three rRNA operons, that are missing either its rrnB operon or both its rrnB and rrnC operons. In rich media, both strains grow at about the same rate as wild type. The G2099 in the 23S rRNA gene of the single operon strain was changed to A, and a three amino acid deletion was introduced into the gene for ribosomal protein L22 of the wild-type organism. The structural consequences of these and other such mutations can be determined with unusual accuracy because crystals of the large ribosomal subunit of H. marismortui diffract to atomic resolution.     

Combination of DEC plus aspirin induced mitochondrial mediated apoptosis in filarial parasite Setaria cervi

Diethylcarbamazine (DEC) is the main drug used against lymphatic filariasis but it is only microfilaricidal. Hence there is an urgent need for adulticidal drug. Aspirin is known nonsteroidal anti-inflammatory drugs which can inhibit prostaglandin H synthase and also induces apoptosis. Studies presented in this paper demonstrated that exposure of worms to the combination of DEC plus aspirin (DEC + A) at 100 μM concentration irreversibly paralyzed adult worms as well as microfilariae within 2 h. Some of the apoptosis markers viz; DNA fragmentation with accompanying ladder formation, upregulation of Bax expression and decrease in Bcl-2 have suggested that the parasite may be killed due to mitochondrial mediated apoptosis. The levels of several apoptosis regulating proteins and enzymes have also shown to be altered. DEC + A treated worms showed significant decrease in prostaglandin H synthase activity (PGHS) and increase in the level of nitric oxide (NO) and cysteine proteases while glutathione (GSH) and peroxidase level was found to be decreased. NO is known inducer of mitochondrial mediated apoptosis and acts by increasing the permeability of mitochondrial membrane through Bax and allowing cytochrome c to release in cytosol, inducing caspases leading to apoptosis. The DEC + A concentration used in this study is much lower than recommended dose so its intake is safe. Here we report for the first time that combination of DEC and aspirin is more effective and could be used as an adulticidal for control of human filarial infections.     

Brugia pahangi: Growth improvement with lecithin in the diet of axenically reared hosts, Aedes aegypti

Growth and development of the filarial nematode Brugia pahangi was examined in Aedes aegypti mosquitoes axenically reared on defined synthetic dietary media containing crude animal lecithin and synthetic dipalmitoyl lecithin. The first was a crude preparation containing impurities (mostly lipids) up to 40%, while the second was a highly purified preparation of lecithin (99.95%). Substantial proportions of apparently alive but undeveloped prefirst-stage parasite juveniles (presausage forms) were found in mosquitoes reared on all synthetic dietary media but not in mosquitoes reared on liver powder (control). Of the two lecithin preparations tested, parasites grew best in mosquitoes reared on a diet augmented with crude animal lecithin. Development was synchronized and the filarial parasites were able to complete two molts to become third-stage juveniles. Synthetic dl-dipalmitoyl lecithin in mosquito diets did not improve filarial development. The improvement of worm growth in terms of rate of morphogenesis and facility in molting, with the addition of crude, and not the pure lecithin preparation, suggested that the effect observed on filariae may not be directly due to the lecithin molecule, but due to a compound (probably a lipid), associated with the lecithin molecule in the crude preparation.     

Requirement of duplicated operons for maximal metabolism of phthalate by Rhodococcus sp. strain DK17

The operons encoding the transformation of phthalate to protocatechuate are duplicated and present on two different megaplasmids [pDK2 (330 kb) and pDK3 (750 kb)] in Rhodococcus sp. strain DK17. RT-PCR experiments using gene-specific primers showed that both the pDK2- and the pDK3-encoded dihydroxyphthalate decarboxylase genes are simultaneously expressed during growth on phthalate. The doubling time of the pDK2-cured mutant strain DK176 in minimal liquid medium with 5mM phthalate is 52.5% of that of the wild-type strain DK17. The data indicate that both copies of the phthalate operon are equally functional in DK17, and gene dosage is the main reason for slower growth of DK176 on phthalate.     

Cellular analysis of host cell infection by different developmental stages of Trypanosoma cruzi

Trypanosoma cruzi is an obligate intracellular parasite that infects phagocytic and non-phagocytic mammalian cells by a complex process that appears to involve several discrete steps. Even though the infection process was described many years ago, the molecular mechanisms involved remain poorly understood. As fluorescent proteins have proven to be excellent tools for live-cell imaging, we used EGFP- and DsRed1-1-transfected trypomastigotes, amastigotes and epimastigotes to study the infection process in living cells. Contrary to what has been reported, our results showed that epimastigotes are as infective as trypomastigotes and amastigotes. Besides, differences in replication, differentiation and parasite release times were observed among the stages. Our results suggest that the different developmental stages use distinct attachment and invasion mechanisms. We propose that fluorescent-based plasmid expression systems are good models for studying the infection process of intracellular microorganisms and could offers insights about the molecular mechanisms involved.     

Functional analysis of putative adhesion factors in Lactobacillus acidophilus NCFM

Lactobacilli are major inhabitants of the normal microflora of the gastrointestinal tract, and some select species have been used extensively as probiotic cultures. One potentially important property of these organisms is their ability to interact with epithelial cells in the intestinal tract, which may promote retention and host-bacterial communication. However, the mechanisms by which they attach to intestinal epithelial cells are unknown. The objective of this study was to investigate cell surface proteins in Lactobacillus acidophilus that may promote attachment to intestinal tissues. Using genome sequence data, predicted open reading frames were searched against known protein and protein motif databases to identify four proteins potentially involved in adhesion to epithelial cells. Homologous recombination was used to construct isogenic mutations in genes encoding a mucin-binding protein, a fibronectin-binding protein, a surface layer protein, and two streptococcal R28 homologs. The abilities of the mutants to adhere to intestinal epithelial cells were then evaluated in vitro. Each strain was screened on Caco-2 cells, which differentiate and express markers characteristic of normal small-intestine cells. A significant decrease in adhesion was observed in the fibronectin-binding protein mutant (76%) and the mucin-binding protein mutant (65%). A surface layer protein mutant also showed reduction in adhesion ability (84%), but the effect of this mutation is likely due to the loss of multiple surface proteins that may be embedded in the S-layer. This study demonstrated that multiple cell surface proteins in L. acidophilus NCFM can individually contribute to the organism's ability to attach to intestinal cells in vitro.