Prp8 intein in fungal pathogens: target for potential antifungal drugs

Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada B3H 4H7.
FEBS Letters (Impact Factor: 3.34). 09/2004; 572(1-3):46-50. DOI: 10.1016/j.febslet.2004.07.016
Source: PubMed

ABSTRACT Inteins are self-splicing intervening sequences in proteins, and inteins of pathogenic organisms can be attractive drug targets. Here, we report an intein in important fungal pathogens including Aspergillus fumigatus, Aspergillus nidulans, Histoplasma capsulatum, and different serotypes of Cryptococcus neoformans. This intein is inside the extremely conserved and functionally essential Prp8 protein, and it varies in size from 170 aa in C. neoformans to 819 aa in A. fumigatus, which is caused by the presence or absence of an endonuclease domain and a putative tongs subdomain in the intein. Prp8 inteins of these organisms were demonstrated to do protein splicing in a recombinant protein in Escherichia coli. These findings revealed Prp8 inteins as attractive targets for potential antifungal drugs to be identified using existing selection and screening methods.

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    ABSTRACT: Aspergillus fumigatus causes a wide range of diseases that include mycotoxicosis, allergic reactions and systemic diseases (invasive aspergillosis) with high mortality rates. Pathogenicity depends on immune status of patients and fungal strain. There is no unique essential virulence factor for development of this fungus in the patient and its virulence appears to be under polygenetic control. The group of molecules and genes associated with the virulence of this fungus includes many cell wall components, such as ß(1-3)-glucan, galactomannan, galactomannanproteins (Afmp1 and Afmp2), and the chitin synthetases (Chs; chsE and chsG), as well as others. Some genes and molecules have been implicated in evasion from the immune response, such as the rodlets layer (rodA/hyp1 gene) and the conidial melanin-DHN (pksP/alb1 gene). The detoxifying systems for Reactive Oxygen Species (ROS) by catalases (Cat1p and Cat2p) and superoxide dismutases (MnSOD and Cu,ZnSOD), had also been pointed out as essential for virulence. In addition, this fungus produces toxins (14 kDa diffusible substance from conidia, fumigaclavin C, aurasperon C, gliotoxin, helvolic acid, fumagilin, Asp-hemolysin, and ribotoxin Asp fI/mitogilin F/restrictocin), allergens (Asp f1 to Asp f23), and enzymatic proteins as alkaline serin proteases (Alp and Alp2), metalloproteases (Mep), aspartic proteases (Pep and Pep2), dipeptidyl-peptidases (DppIV and DppV), phospholipase C and phospholipase B (Plb1 and Plb2). These toxic substances and enzymes seems to be additive and/or synergistic, decreasing the survival rates of the infected animals due to their direct action on cells or supporting microbial invasion during infection. Adaptation ability to different trophic situations is an essential attribute of most pathogens. To maintain its virulence attributes A. fumigatus requires iron obtaining by hydroxamate type siderophores (ornitin monooxigenase/SidA), phosphorous obtaining (fos1, fos2, and fos3), signal transductional falls that regulate morphogenesis and/or usage of nutrients as nitrogen (rasA, rasB, rhbA), mitogen activated kinases (sakA codified MAP-kinase), AMPc-Pka signal transductional route, as well as others. In addition, they seem to be essential in this field the amino acid biosynthesis (cpcA and homoaconitase/lysF), the activation and expression of some genes at 37 °C (Hsp1/Asp f12, cgrA), some molecules and genes that maintain cellular viability (smcA, Prp8, anexins), etc. Conversely, knowledge about relationship between pathogen and immune response of the host has been improved, opening new research possibilities. The involvement of non-professional cells (endothelial, and tracheal and alveolar epithelial cells) and professional cells (natural killer or NK, and dendritic cells) in infection has been also observed. Pathogen Associated Molecular Patterns (PAMP) and Patterns Recognizing Receptors (PRR; as Toll like receptors TLR-2 and TLR-4) could influence inflammatory response and dominant cytokine profile, and consequently Th response to infection. Superficial components of fungus and host cell surface receptors driving these phenomena are still unknown, although some molecules already associated with its virulence could also be involved. Sequencing of A. fumigatus genome and study of gene expression during their infective process by using DNA microarray and biochips, promises to improve the knowledge of virulence of this fungus.
    Revista Iberoamericana de Micología 03/2005; 22(1):1-23. DOI:10.1016/S1130-1406(05)70001-2 · 0.97 Impact Factor
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    ABSTRACT: The PRP8 intein is the most widespread intein among the Kingdom Fungi. This genetic element occurs within the prp8 gene, and is transcribed and translated simultaneously with the gene. After translation, the intein excises itself from the Prp8 protein by an autocatalytic splicing reaction, subsequently joining the N and C terminals of the host protein, which retains its functional conformation. Besides the splicing domain, some PRP8 inteins also have a homing endonuclease (HE) domain which, if functional, makes the intein a mobile element capable of becoming fixed in a population. This work aimed to study 1) The occurrence of this intein in Histoplasma capsulatum isolates (n = 99) belonging to different cryptic species collected in diverse geographical locations, and 2) The functionality of the endonuclease domains of H. capsulatum PRP8 inteins and their phylogenetic relationship among the cryptic species. Our results suggest that the PRP8 intein is fixed in H. capsulatum populations and that an admixture or a probable ancestral polymorphism of the PRP8 intein sequences is responsible for the apparent paraphyletic pattern of the LAmA clade which, in the intein phylogeny, also encompasses sequences from LAmB isolates. The PRP8 intein sequences clearly separate the different cryptic species, and may serve as an additional molecular typing tool, as previously proposed for other fungi genus, such as Cryptococcus and Paracoccidioides.
    Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 05/2013; 18. DOI:10.1016/j.meegid.2013.05.001 · 3.26 Impact Factor
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    ABSTRACT: Protein sequences are diversified on the DNA level by recombination and mutation and can be further increased on the RNA level by alternative RNA splicing, involving introns that have important roles in many biological processes. The protein version of introns (inteins), which catalyze protein splicing, were first reported in the 1990s. The biological roles of protein splicing still remain elusive because inteins neither provide any clear benefits nor have an essential role in their host organisms. We now report protein alternative splicing, in which new protein sequences can be produced by protein recombination by intermolecular domain swapping of inteins, as elucidated by NMR spectroscopy and crystal structures. We demonstrate that intein-mediated protein alternative splicing could be a new strategy to increase protein diversity (that is, functions) without any modification in genetic backgrounds. We also exploited it as a post-translational protein conformation-driven switch of protein functions (for example, as highly specific protein interference).
    Nature Chemical Biology 08/2013; DOI:10.1038/nchembio.1320 · 13.22 Impact Factor


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