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Differential transcript expression between the microfilariae of the filarial nematodes, Brugia malayi and B. pahangi.

ABSTRACT: Brugia malayi and B. pahangi are two closely related nematodes that cause filariasis in humans and animals. However, B. pahangi microfilariae are able to develop in and be transmitted by the mosquito, Armigeres subalbatus, whereas most B. malayi are rapidly melanized and destroyed within the mosquito hemocoel. A cross-species microarray analysis employing the B. malayi V2 array was carried out to determine the transcriptional differences between B. malayi and B. pahangi microfilariae with similar age distribution.Following microarray data analysis, a list of preferentially expressed genes in both microfilariae species was generated with a false discovery rate estimate of 5% and a signal intensity ratio of 2 or higher in either species. A total of 308 probes were preferentially expressed in both species with 149 probes, representing 123 genes, in B. pahangi microfilariae and 159 probes, representing 107 genes, in B. malayi microfilariae. In B. pahangi, there were 76 (62%) up-regulated transcripts that coded for known proteins that mapped into the KEGG pathway compared to 61 (57%) transcripts in B. malayi microfilariae. The remaining 47 (38%) transcripts in B. pahangi and 46 (43%) transcripts in B. malayi microfilariae were comprised almost entirely of hypothetical genes of unknown function. Twenty-seven of the transcripts in B. pahangi microfilariae coded for proteins that associate with the secretory pathway compared to thirty-nine in B. malayi microfilariae. The data obtained from real-time PCR analysis of ten genes selected from the microarray list of preferentially expressed genes showed good concordance with the microarray data, indicating that the microarray data were reproducible.In this study, we identified gene transcripts that were preferentially expressed in the microfilariae of B. pahangi and B. malayi, some of which coded for known immunomodulatory proteins. These comparative transcriptome data will be of interest to researchers keen on understanding the inherent differences, at the molecular level, between B. malayi and B. pahangi microfilariae especially because these microfilariae are capable of surviving in the same vertebrate host but elicit different immune response outcomes in the mosquito, Ar. subalbatus.

SUBMITTER: Kariuki MM

PROVIDER: S-EPMC2874553 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Differential transcript expression between the microfilariae of the filarial nematodes, Brugia malayi and B. pahangi.

Kariuki Michael M MM Hearne Leonard B LB Beerntsen Brenda T BT

BMC genomics 20100407

<h4>Background</h4>Brugia malayi and B. pahangi are two closely related nematodes that cause filariasis in humans and animals. However, B. pahangi microfilariae are able to develop in and be transmitted by the mosquito, Armigeres subalbatus, whereas most B. malayi are rapidly melanized and destroyed within the mosquito hemocoel. A cross-species microarray analysis employing the B. malayi V2 array was carried out to determine the transcriptional differences between B. malayi and B. pahangi microf ...[more]

PMID: 20370932

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Project description:Filarial nematodes are arthropod-borne nematodes that cause a variety of economically important diseases such as onchocerciasis (river blindness), lymphatic filariasis, and heartworm disease. The most widespread filarial disease of humans is lymphatic filariasis, caused by worms in the genera Wuchereria and Brugia. Lymphatic filariasis is an economic and social burden in endemic countries and affects approximately 119 million people worldwide (Michael, 1997). In humans, the worms live in and block the lymph vessels, causing improper flow of lymph, and inflammation of the lymphatic system. The symptoms are fever, swollen limbs and genitals, generalized malaise, and can progress to a debilitating condition known as elephantiasis This research focuses on the transmission of these worms to the disseminating mosquito host, and it is based on the interesting observation that mf must be at least 7 days old to successfully infect the mosquito (de Hollanda, 1982). Newborn mf that have not â??maturedâ?? cannot successfully penetrate the midgut of the mosquito, and subsequently cannot develop to the L3 stage (Fuhrman, 1987). Previous work done by another group 20 years ago suggests that the molecular makeup of the worm surface changes during this maturation process (Furman, 1983 a and b). We used microarray analysis to characterize changes in gene expression that take place during the mf maturation process. Understanding the gene expression changes that occur as the mf mature will allow us to understand the nature of the philological transition that allows mf to move from the human to the mosquito host. With this information in hand, we can eventually identify parasite molecules that could be targeted to either stop parasite reproduction or prevent transmission of the mf to the mosquito. This would stop parasite transmission in endemic areas. Brugia pahangi mature mf (30 days and older) RNA was compared to immature mf (3 days and younger). Three biological replicates were performed each with two technical replicates

Project description:BackgroundA granulomatous inflammatory response develops in jirds infected subcutaneously or intraperitoneally with filarial nematodes namely Brugia pahangi and B. malayi. Previous studies by light and electron microscopy have shown cellular inflammatory responses in and around these granulomas. Furthermore, the cellular inflammatory responses of granulomas found in the lymphatics and peritoneal cavity appear to be similar. The purpose of this study was to determine the cytokine profiles of granulomas in the peritoneal cavity of B. pahangi-infected jirds and to determine whether the granulomas release any proinflammatory cytokines ex vivo.MethodsA semiautomated quantitative polymerase chain reaction (Q-PCR) was performed on cDNA prepared from the granulomas of infected jirds to study the species-specific mRNA expression of IL-2, IL-4, IFN-gamma, IL-5, and IL-10. Genomic DNA was extracted from the granulomas, and parasite DNA was detected by Q-PCR by amplifying the HhaI repeat sequence. The levels of the inflammation-causing cytokines IL-6 and TNFalpha that were secreted by the granulomas were measured by cell-based assays.ResultsFlorid granulomas showed higher levels of IFN-gamma than other cytokines, linking this Th1 cytokine to the granulomatous inflammation that develops in jirds and humans. IL-4 expression was much lower than that of IFN-gamma but higher than that of IL-10. A low level of IL-5 mRNA expression was detectable in all granulomas as was the level of IL-2 expression. The levels of the inflammatory cytokines IL-6 and TNFalpha, secreted by intact granulomas, spontaneously increased by 48 h after culture. Parasite antigen stimulation and subsequent release of IL-6 and TNFalpha by the granulomas indicated a moderate increase in the levels of these two cytokines. The amplification of the Brugia HhaI repeat DNA and Wolbachia 16S rDNA indicated worm components and bacterial components in the granulomatous tissue.ConclusionGranuloma development in filarial infections is a complex process involving cellular reactions responding to parasite/bacteria and their components. The interactions between worm-derived granulomas and their hosts are dynamic and multifaceted. The data collected thus far suggest that the expression profiles of many of the measured cytokines in the lymphoid tissues of Brugia-infected jirds are different from those of the cytokines in granulomas. Moreover, granulomas have the ability to secrete the inflammatory cytokines IL-6 and TNFalpha.

Project description:Enzymes in the thiol redox systems of microbial pathogens are promising targets for drug development. In this study we characterized the thioredoxin reductase (TrxR) selenoproteins from Brugia malayi and Onchocerca volvulus, filarial nematode parasites and causative agents of lymphatic filariasis and onchocerciasis, respectively. The two filarial enzymes showed similar turnover numbers and affinities for different thioredoxin (Trx) proteins, but with a clear preference for the autologous Trx. Human TrxR1 (hTrxR1) had a high and similar specific activity versus the human and filarial Trxs, suggesting that, in vivo, hTrxR1 could possibly be the reducing agent of parasite Trxs once they are released into the host. Both filarial TrxRs were efficiently inhibited by auranofin and by a recently described inhibitor of human TrxR1 (TRi-1), but not as efficiently by the alternative compound TRi-2. The enzyme from B. malayi was structurally characterized also in complex with NADPH and auranofin, producing the first crystallographic structure of a nematode TrxR. The protein represents an unusual fusion of a mammalian-type TrxR protein architecture with an N-terminal glutaredoxin-like (Grx) domain lacking typical Grx motifs. Unlike thioredoxin glutathione reductases (TGRs) found in platyhelminths and mammals, which are also Grx-TrxR domain fusion proteins, the TrxRs from the filarial nematodes lacked glutathione disulfide reductase and Grx activities. The structural determinations revealed that the Grx domain of TrxR from B. malayi contains a cysteine (C22), conserved in TrxRs from clade IIIc nematodes, that directly interacts with the C-terminal cysteine-selenocysteine motif of the homo-dimeric subunit. Interestingly, despite this finding we found that altering C22 by mutation to serine did not affect enzyme catalysis. Thus, although the function of the Grx domain in these filarial TrxRs remains to be determined, the results obtained provide insights on key properties of this important family of selenoprotein flavoenzymes that are potential drug targets for treatment of filariasis.

Dataset Information

Differential transcript expression between the microfilariae of the filarial nematodes, Brugia malayi and B. pahangi.

Publications

Differential transcript expression between the microfilariae of the filarial nematodes, Brugia malayi and B. pahangi.

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