MOLECULAR AND IMMUNOLOGICAL DETECTION OF GLIOTOXIN GENES IN ASPERGILLUS FUMIGATUS ISOLATED FROM PATIENTS WITH INVASIVE PULMONARY ASPERGILLOSIS
DOI:
https://doi.org/10.21272/eumj.2025;13(1):189-197Keywords:
Aspergillus fumigatus, immunity, invasive pulmonary aspergillosis, diagnosticsAbstract
Introduction: Aspergillus fumigatus, a filamentous fungus, is a leading cause of invasive pulmonary aspergillosis (IPA), a severe condition predominantly affecting immunocompromised patients. Gliotoxin, a secondary metabolite crucial to A. fumigatus virulence, is produced through a biosynthetic pathway regulated by several genes, including GliP, GliA, and GliJ. It plays a significant role in its pathogenicity, affecting host immune responses and contributing to disease progression. This study aimed to molecularly and immunologically detect gliotoxin-related genes in A. fumigatus isolates from IPA patients.
Methods: Forty patients diagnosed with IPA were recruited, and sputum and plasma samples were collected to identify fungal isolates and assess plasma interleukin-17 (IL-17) levels. Molecular detection of A. fumigatus involved extracting genomic DNA from isolates, followed by polymerase chain reaction (PCR) using primers for the gliotoxin biosynthetic genes GliP, GliZ, and GliA. Additionally, plasma IL-17 levels were measured in IPA patients to assess their immunological response and gliotoxin production was confirmed using thin-layer chromatography.
Results: Of the 40 isolates, 20 were confirmed to be A. fumigatus. PCR analysis revealed that 85% of these isolates harbored the GliP gene, while 75% carried the GliZ gene. Also, 40% of the isolates demonstrated gliotoxin production. The IL-17 levels in patients with IPA (1660.10±103.65 pg/mL) were significantly higher compared to the control group (807.92±101.51 pg/mL). The elevated IL-17 levels observed in IPA patients underscores the role of this cytokine in the immune response against A. fumigatus. The detection of gliotoxin-producing isolates varied, with genetic and environmental factors influencing gliotoxin biosynthesis. Isolates lacking either GliP or GliZ failed to produce gliotoxin, while some isolates with both genes exhibited reduced toxin production, likely due to regulatory influences.
Conclusion: The study found that a combination of molecular and immunological methods offers a promising diagnostic tool for detecting IPA and understanding A. fumigatus virulence. Future research could enhance diagnostic tools' sensitivity and specificity, apply them in clinical settings, and develop non-invasive methods like biomarkers for improved patient outcomes.
Downloads
References
Earle K, Valero C, Conn DP, Vere G, Cook PC, Bromley MJ, Bowyer P, Gago S. Pathogenicity and virulence of Aspergillus fumigatus. Virulence. 2023;14(1):2172264. https://doi.org/10.1080/21505594.2023.2172264.
Lamoth F, Calandra T. Pulmonary aspergillosis: diagnosis and treatment. Eur Respir Rev. 2022;31(166):220114. https://doi.org/10.1183/16000617.0114-2022.
Machado M, Fortún J, Muñoz P. Invasive aspergillosis: A comprehensive review. Med Clin (Barc). 2024;163(4):189-198. https://doi.org/10.1016/j.medcli.2024.01.045.
Hashim SS, Mahmood ZF, Abdulateef SF and Dheeb BI. Evaluation Cytotoxicity Effects of Centaurea Cineraria Extracts Against some of Cancer Cell Lines, Biomed Pharmacol J, 16(1), 177 (2023) https://doi.org/10.1093/ofid/ofy213.
Ben-Ami R, Lewis RE, Leventakos K, Kontoyiannis DP. Aspergillus fumigatus inhibits angiogenesis through the production of gliotoxin and other secondary metabolites. Blood. 2009;114(26):5393-9. https://doi.org/10.1182/blood-2009-07-231209.
Alonso V, Aminahuel C, Díaz Vergara L, Pereyra C, Poloni V, Dalcero A, Cavaglieri L. Ecophysiology of environmental Aspergillus fumigatus and comparison with clinical strains on gliotoxin production and elastase activity. Lett Appl Microbiol. 2016;62(2):160-8. https://doi.org/10.1111/lam.12523.
Dheeb BI, Hashim SS, Hussein HS & Hamada TA (2022). Study of TGF- β Immune Marker Expression in Mice Exposed to Candida Spp. AIP Conference Proceedings.1230,234-237. https://doi.org/10.1063/5.0121945
Dheeb BI, Abdulla SN, Shanter ALQaysi SA, Al-Sarraj BM & Farhan MS (2023). Extraction of Klebsiella pneumoniae and Candida albicans Biofilm and studying their Cytotoxic Effects on Human Lymphocytes. Jordan Journal of Biological Sciences, 16(4). https://doi.org/10.54319/jjbs/160418
Nielsen JB, Gren T, Mohite OS, Jørgensen TS, Klitgaard AK, Mourched AS, et al. Identification of the biosynthetic gene cluster for pyracrimycin A, an antibiotic produced by Streptomyces sp. ACS Chem Biol. 2022;17(9):2411-2417. https://doi.org/10.1021/acschembio.2c00480.
Adrover-Castellano ML, Schmidt JJ, Sherman DH. Biosynthetic cyclization catalysts for the assembly of peptide and polyketide natural products. Chem Cat Chem. 2021;13(9):2095-2116. https://doi.org/10.1002/cctc.202001886.
Wang DN, Toyotome T, Muraosa Y, Watanabe A, Wuren T, Bunsupa S, et al. GliA in Aspergillus fumigatus is required for its tolerance to gliotoxin and affects the amount of extracellular and intracellular gliotoxin. Med Mycol. 2014;52(5):506-18. https://doi.org/10.1093/mmy/myu007.
Hussain AF, Sulaiman GM, Dheeb BI and Hashim AJ; Abd Alrahman ES; Seddiq SH; Khashman BM. Histopathological changes and expression of transforming growth factor beta (TGF-β3) in mice exposed to gliotoxin, Journal of King Saud University – Science, 27, 193–197 (2020) https://doi.org/10.1016/j.jksus.2018.10.013
Seyedmousavi S, Zaas DW, Rubenstein E, Bollinger LE, Patterson TF, Thompson GR, et al. Aspergillus fumigatus in invasive aspergillosis: Pathogenesis and virulence factors. Clin Microb Rev. 2018;31(4), e00001-18. https://doi.org/10.1128/CMR.00001-18.
Herbrecht R, Bories P, Moulin JC, Ledoux MP, Letscher-Bru V. Risk stratification for invasive aspergillosis in immunocompromised patients. Ann New York Acad Sci. 2012; https://doi.org/10.1111/j.1749-6632.2012.06829.x
Ben-Ami R, Lewis RE, O'Brien M, Kontoyiannis DP. Aspergillus fumigatus in lung infections: A focus on gliotoxin. Antimicrobial Agents and Chemotherapy, 53(6), 2166–2172. 2009; https://doi.org/10.1128/AAC.01495-08.
Nielsen KF, Asplund SB, Høiberg HK, Møller J. The gliotoxin biosynthetic cluster in Aspergillus fumigatus. Fung Biol. 2022; 126(5), 306–318. https://doi.org/10.1016/j.funbio.2022.03.003
Abed SM, Farhan MG, Madhloom NK & Dheeb DI (2022). Magnetic Field Exposure to Clinical Isolates of Acinitobacter baumanii. Biomedical and Pharmacology Journal,15(4), 12-16. https://doi.org/10.13005/bpj/2550
Abdulateef SM, Shaima Ibraheem SM, Hussein HS, Dheeb BI, Khashman BM, Ahmed DM, Abu-Elteen KH. & Abu-Qatouseh L (2024). MMP-1 and MMP-7 expression is influenced by ginsenosides in mice exposed to aflatoxin B1: In vivo study. Jordan Journal of Pharmacceutical Science, 17 (1), 135-1139. https://doi.org/10.35516/jjps.v17i2.1989
Jonathan SG, Adeniyi MA, Asemoloye MD. Fungal biodeterioration, aflatoxin contamination, and nutrient value of suya spices. Scientifica. 2016:4602036. https://doi.org/10.1155/2016/4602036.
Mohammed SH, Hussein AS, Dheeb BI, Jafar NB. Detection of anti-mannan antibodies and TLR9 as alternative methods for diagnosis of candidiasis in immunocompromised patients with vulvovaginitis. J Appl Nat Sci. 2024; 16(3): 1183-1188. https://doi.org/10.31018/jans.v16i3.5460
Dheeb BI. Study on the pathogenicity, cytotoxicity, and virulence factors of some clinical Aspergillus fumigatus isolates and the use of RAPD markers to distinguish them [dissertation]. Tikrit: College of Science, Tikrit University; 2013
Mahmood MN, Mahal MH, Mohammed HE, Tariq TA, Mohammed AJ & Dheeb BI (2019). Evaluation of enzymes liver and kidney function in serum people the exposures at risk of chemicals volatile in the lab pharmaceutical samarra. Journal of Global Pharma Technology,11(7), 331–336 . https://doi.org/10.1007/s00253-023-12952-z.
Sael Hussein A. Molecular effect of aflatoxin B1 in some gene. Plant Arch. 2019;19(2):2148–52. https://www.plantarchives.org/SPL%20ISSUE%20SUPP%202,2019/371%20(2148-2152).pdf
Salih II, Seddiq SH, Hashim SS, Dheeb BI (2022). Application of Omics and Proteomics in Fungi. AIP Conference2394 (1). https://doi.org/10.1063/5.0121901
Maikan HK, Jabbar S, Al-Haishawi H. Isolation and identification of Candida tropicalis as a cause of cutaneous candidiasis in Kalar district, Iraq. Arch Razi Inst. 2022;77(4):1377-1382. https://doi.org/10.22092/ARI.2022.357613.2066.
Lewis RE, Wiederhold NP, Lionakis MS, Prince RA, Kontoyiannis DP. Frequency and species distribution of gliotoxin-producing Aspergillus isolates recovered from patients at a tertiary-care cancer center. J Clin Microbiol. 2005;43(12):6120-2. https://doi.org/10.1128/JCM.43.12.6120-6122.2005.
Fan Y, Wang Y, Xu J. Comparative Genome Sequence Analyses of Geographic Samples of Aspergillus fumigatus-Relevance for Amphotericin B Resistance. Microorganisms. 2020;8(11):1673. https://doi.org/10.3390/microorganisms8111673.
Abdullah HI, Hammadi SY, Hussein AS & Dheeb BI (2019). Investigation of genetic diversity and relationships among the clinical candida species using random amplified polymorphic DNA (RAPD) analysis. Research Journal of Biotechnology, 14(Special Issue 1), 6–13.
Dhingra S, Andes D, Calvo AM. VeA regulates conidiation, gliotoxin production, and protease activity in the opportunistic human pathogen Aspergillus fumigatus. Eukaryot Cell. 2012;11(12):1531-43. https://doi.org/10.1128/EC.00222-12.
Kupfahl C, Michalka A, Lass-Flörl C, Fischer G, Haase G, Ruppert T, Geginat G, Hof H. Gliotoxin production by clinical and environmental Aspergillus fumigatus strains. Int J Med Microbiol. 2008;298(3-4):319-27. https://doi.org/10.1016/j.ijmm.2007.04.006.
Awad FM, Al-Samarrai AHM & Dheeb BI (2020). Study of the inhibitory effects of rheum ribes extracts on a pathogenic fungi and cancer cell line. Plant Archives , 20(1), 3161–3168.
Sugui JA, Pardo J, Chang YC, Müllbacher A, Zarember KA, Galvez EM, et al. Role of laeA in the Regulation of alb1, gliP, Conidial Morphology, and Virulence in Aspergillus fumigatus. Eukaryot Cell. 2007; 6(9):1552-61. https://doi.org/10.1128/EC.00140-07.
Downloads
Published
How to Cite
Issue
Section
License
Copyright (c) 2025 Anmar S. Hussein, Sanaa H. Mohammed, Bari L. Mohammed
This work is licensed under a Creative Commons Attribution 4.0 International License.
ISSN: 
