Demonstration of the anthelmintic potency of marimastat in the Heligmosomoides polygyrus rodent model

In the course of a structure based drug discovery program the known anticancer candidate marimastat was uncovered as a potent inhibitor of an enzyme in nematode cuticle biogenesis. It was shown to kill Caenorhabditis elegans, and the sheep parasites Haemonchus contortus and Teladorsagia circumcinta via an entirely novel nematode-specific pathway, specifically by inhibiting cuticle-remodelling enzymes that the parasites require for the developmentally essential moulting process. This discovery prompted an investigation of the compound's effect on Heligmosomoides polygyrus parasites in a mouse model of helminth infection. Mice were administered the drug via oral gavage daily from day of infection for a period of 2 wk. A second group received the drug via intra-peritoneal implantation of an osmotic minipump for 4 wk. Control groups were administered identical volumes of water by oral gavage in both cases. Counts of H. polygyrus faecal egg and larval load showed that marimastat effected a consistent and significant reduction in egg laying, and a consistent but minor reduction in adult worm load when administered every day, starting on the first day of infection. However, the drug failed to have any significant effect on egg counts or worm burdens when administered to mice with established infections. Therefore, marimastat does not appear to show promise as an anthelmintic in gastrointestinal nematode infections, although other metalloproteases such as batimastat may prove more effective

Hsp21potentiates antifungal drug tolerance in Candida albicans

Peer reviewedPublisher PD

daf-7-related TGF-beta homologues from Trichostrongyloid nematodes show contrasting life-cycle expression patterns

OBJECTIVE: The transforming growth factor-β (TGF-β) gene family regulates critical processes in animal development, and plays a crucial role in regulating the mammalian immune response. We aimed to identify TGF-β homologues from two laboratory model nematodes (Heligmosomoides polygyrus and Nippostrongylus brasiliensis) and two major parasites of ruminant livestock (Haemonchus contortus and Teladorsagia circumcincta). METHODS: Parasite cDNA was used as a template for gene-specific PCR and RACE. RESULTS: Homologues of the TGH-2 subfamily were isolated, and found to differ in length (301, 152, 349 and 305 amino acids respectively), with variably truncated N-terminal pre-proteins. All contain conserved C-terminal active domains (>85% identical over 115 amino acids) containing 9 cysteine residues, as in C. elegans DAF-7, Brugia malayi TGH-2 and mammalian TGF-β. Surprisingly, only the H. contortus homologue retains a conventional signal sequence, absent from shorter proteins of other species. RT-PCR assays of transcription show that in H. contortus and N. brasiliensis expression is maximal in the infective larval stage, and very low in adult worms. In contrast, in H. polygyrus and T. circumcincta, tgh-2 transcription is higher in adults than infective larvae. CONCLUSION: The molecular evolution of this gene family in parasitic nematodes has diversified the pre-protein and life-cycle expression patterns of TGF-β homologues while conserving the structure of the active domain

Small but crucial : the novel small heat shock protein Hsp21 mediates stress adaptation and virulence in Candida albicans

Peer reviewedPublisher PD

Identification of a Novel Response Regulator, Crr1, That Is Required for Hydrogen Peroxide Resistance in Candida albicans

Candida albicans colonises numerous niches within humans and thus its success as a pathogen is dependent on its ability to adapt to diverse growth environments within the host. Two component signal transduction is a common mechanism by which bacteria respond to environmental stimuli and, although less common, two component-related pathways have also been characterised in fungi. Here we report the identification and characterisation of a novel two component response regulator protein in C. albicans which we have named CRR1 (Candida Response Regulator 1). Crr1 contains a receiver domain characteristic of response regulator proteins, including the conserved aspartate that receives phosphate from an upstream histidine kinase. Significantly, orthologues of CRR1 are present only in fungi belonging to the Candida CTG clade. Deletion of the C. albicans CRR1 gene, or mutation of the predicted phospho-aspartate, causes increased sensitivity of cells to the oxidising agent hydrogen peroxide. Crr1 is present in both the cytoplasm and nucleus, and this localisation is unaffected by oxidative stress or mutation of the predicted phospho-aspartate. Furthermore, unlike the Ssk1 response regulator, Crr1 is not required for the hydrogen peroxide-induced activation of the Hog1 stress-activated protein kinase pathway, or for the virulence of C. albicans in a mouse model of systemic disease. Taken together, our data suggest that Crr1, a novel response regulator restricted to the Candida CTG clade, regulates the response of C. albicans cells to hydrogen peroxide in a Hog1-independent manner that requires the function of the conserved phospho-aspartate

Comparative genomics of the major parasitic worms

Parasitic nematodes (roundworms) and platyhelminths (flatworms) cause debilitating chronic infections of humans and animals, decimate crop production and are a major impediment to socioeconomic development. Here we report a broad comparative study of 81 genomes of parasitic and non-parasitic worms. We have identified gene family births and hundreds of expanded gene families at key nodes in the phylogeny that are relevant to parasitism. Examples include gene families that modulate host immune responses, enable parasite migration though host tissues or allow the parasite to feed. We reveal extensive lineage-specific differences in core metabolism and protein families historically targeted for drug development. From an in silico screen, we have identified and prioritized new potential drug targets and compounds for testing. This comparative genomics resource provides a much-needed boost for the research community to understand and combat parasitic worms

A Human-Curated Annotation of the Candida albicans Genome

Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations) that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications

Hyphal Development in Candida albicans Requires Two Temporally Linked Changes in Promoter Chromatin for Initiation and Maintenance

Phenotypic plasticity is common in development. For Candida albicans, the most common cause of invasive fungal infections in humans, morphological plasticity is its defining feature and is critical for its pathogenesis. Unlike other fungal pathogens that exist primarily in either yeast or hyphal forms, C. albicans is able to switch reversibly between yeast and hyphal growth forms in response to environmental cues. Although many regulators have been found involved in hyphal development, the mechanisms of regulating hyphal development and plasticity of dimorphism remain unclear. Here we show that hyphal development involves two sequential regulations of the promoter chromatin of hypha-specific genes. Initiation requires a rapid but temporary disappearance of the Nrg1 transcriptional repressor of hyphal morphogenesis via activation of the cAMP-PKA pathway. Maintenance requires promoter recruitment of Hda1 histone deacetylase under reduced Tor1 (target of rapamycin) signaling. Hda1 deacetylates a subunit of the NuA4 histone acetyltransferase module, leading to eviction of the NuA4 acetyltransferase module and blockage of Nrg1 access to promoters of hypha-specific genes. Promoter recruitment of Hda1 for hyphal maintenance happens only during the period when Nrg1 is gone. The sequential regulation of hyphal development by the activation of the cAMP-PKA pathway and reduced Tor1 signaling provides a molecular mechanism for plasticity of dimorphism and how C. albicans adapts to the varied host environments in pathogenesis. Such temporally linked regulation of promoter chromatin by different signaling pathways provides a unique mechanism for integrating multiple signals during development and cell fate specification

Analysis of Expressed Sequence Tags of the Cyclically Parthenogenetic Rotifer Brachionus plicatilis

Background. Rotifers are among the most common non-arthropod animals and are the most experimentally tractable members of the basal assemblage of metazoan phyla known as Gnathifera. The monogonont rotifer Brachionus plicatilis is a developing model system for ecotoxicology, aquatic ecology, cryptic speciation, and the evolution of sex, and is an important food source for finfish aquaculture. However, basic knowledge of the genome and transcriptome of any rotifer species has been lacking. Methodology/Principal Findings. We generated and partially sequenced a cDNA library from B. plicatilis and constructed a database of over 2300 expressed sequence tags corresponding to more than 450 transcripts. About 20% of the transcripts had no significant similarity to database sequences by BLAST; most of these contained open reading frames of significant length but few had recognized Pfam motifs. Sixteen transcripts accounted for 25% of the ESTs; four of these had no significant similarity to BLAST or Pfam databases. Putative up- and downstream untranslated regions are relatively short and AT rich. In contrast to bdelloid rotifers, there was no evidence of a conserved trans-spliced leader sequence among the transcripts and most genes were single-copy. Conclusions/Significance. Despite the small size of this EST project it revealed several important features of the rotifer transcriptome and of individual monogonont genes. Because there is little genomic data for Gnathifera, the transcripts we found with no known function may represent genes that are species-, class-, phylum- or even superphylum-specific; the fact that some are among the most highly expressed indicates their importance. The absence of trans-spliced leader exons in this monogonont species contrasts with their abundance in bdelloid rotifers and indicates that the presence of this phenomenon can vary at the subphylum level. Our EST database provides a relatively large quantity of transcript-level data for B. plicatilis, and more generally of rotifers and other gnathiferan phyla, and can be browsed and searched at gmod.mbl.edu

Low-level regulatory T-cell activity is essential for functional type-2 effector immunity to expel gastrointestinal helminths

Helminth infection is frequently associated with the expansion of regulatory T cells (Tregs) and suppression of immune responses to bystander antigens. We show that infection of mice with the chronic gastrointestinal helminth Heligmosomoides polygyrus drives rapid polyclonal expansion of Foxp3(+)Helios(+)CD4(+) thymic (t)Tregs in the lamina propria and mesenteric lymph nodes while Foxp3(+)Helios(-)CD4(+) peripheral (p)Treg expand more slowly. Notably, in partially resistant BALB/c mice parasite survival positively correlates with Foxp3(+)Helios(+)CD4(+) tTreg numbers. Boosting of Foxp3(+)Helios(+)CD4(+) tTreg populations by administration of recombinant interleukin-2 (rIL-2):anti-IL-2 (IL-2C) complex increased worm persistence by diminishing type-2 responsiveness in vivo, including suppression of alternatively activated macrophage and granulomatous responses at the sites of infection. IL-2C also increased innate lymphoid cell (ILC) numbers, indicating that Treg functions dominate over ILC effects in this setting. Surprisingly, complete removal of Tregs in transgenic Foxp3-DTR mice also resulted in increased worm burdens, with "immunological chaos" evident in high levels of the pro-inflammatory cytokines IL-6 and interferon-γ. In contrast, worm clearance could be induced by anti-CD25 antibody-mediated partial depletion of early Treg, alongside increased T helper type 2 responses and without incurring pathology. These findings highlight the overarching importance of the early Treg response to infection and the non-linear association between inflammation and the prevailing Treg frequency