Rhizopus spp. are the most common etiological agents of mucormycosis, causing over 90% mortality in disseminated infections. The diagnosis relies on histopathology, culture, and/or polymerase chain reaction. For the first time, the glycosylation of rhizoferrin (RHF) was described in a Rhizopus microsporus clinical isolate by liquid chromatography and accurate tandem mass spectrometry. The fermentation broth lyophilizate contained 345.3 ± 13.5, 1.2 ± 0.03, and 0.03 ± 0.002 mg/g of RHF, imido-RHF, and bis-imido-RHF, respectively. Despite a considerable RHF secretion rate, we did not obtain conclusive RHF detection from a patient with disseminated mucormycosis caused by the same R. microsporus strain. We hypothesize that parallel antimycotic therapy, RHF biotransformation, and metabolism compromised the analysis. On the other hand, the full profile of posaconazole metabolites was retrieved by our in house software CycloBranch.

Department of Analytical Chemistry Faculty of Science Palacký University 771 46 Olomouc Czech Republic

Department of Medical Microbiology 2nd Faculty of Medicine Charles University and Motol University Hospital 150 06 Prague Czech Republic

Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 142 20 Prague Czech Republic

Public Health Institute in Ostrava 702 00 Ostrava Czech Republic

See more in PubMed

Morin-Sardin S., Nodet P., Coton E., Jany J.-L. Mucor: A Janus-faced fungal genus with human health impact and industrial applications. Fungal Biol. Rev. 2017;31:12–32. doi: 10.1016/j.fbr.2016.11.002. DOI

Voigt K., Wolf T., Ochsenreiter K., Nagy G., Kaerger K., Shelest E., Papp T. Genetic and metabolic aspects of primary and secondary metabolism of the zygomycetes. In: Hoffmeister D., editor. Biochemistry and Molecular Biology. Springer International Publishing; Cham, Switzerland: 2016. pp. 361–385.

Müller C., Neugebauer T., Zill P., Lass-Flörl C., Bracher F., Binder U. Sterol composition of clinically relevant mucorales and changes resulting from posaconazole treatment. Molecules. 2018;23:1218. doi: 10.3390/molecules23051218. PubMed DOI PMC

Gooday G.W., Carlile M.J. The discovery of fungal sex hormones: III. Trisporic acid and its precursors. Mycologist. 1997;11:126–130. doi: 10.1016/S0269-915X(97)80017-1. DOI

Hollmann M., Razzazi-Fazeli E., Grajewski J., Twaruzek M., Sulyok M., Böhm J. Detection of 3-nitropropionic acid and cytotoxicity in Mucor circinelloides. Mycotoxin Res. 2008;24:140–150. doi: 10.1007/BF03032341. PubMed DOI

Jennessen J., Nielsen K.F., Houbraken J., Lyhne E.K., Schnürer J., Frisvad J.C., Samson R.A. Secondary metabolite and mycotoxin production by the Rhizopus microsporus group. J. Agric. Food Chem. 2005;53:1833–1840. doi: 10.1021/jf048147n. PubMed DOI

Partida-Martinez L.P., Flores de Looß C., Ishida K., Ishida M., Roth M., Buder K., Hertweck C. Rhizonin, the first mycotoxin isolated from the zygomycota, is not a fungal metabolite but is produced by bacterial endosymbionts. Appl. Environ. Microbiol. 2007;73:793–797. doi: 10.1128/AEM.01784-06. PubMed DOI PMC

Winkelmann G. A search for glomuferrin: A potential siderophore of arbuscular mycorrhizal fungi of the genus Glomus. Biometals. 2017;30:559–564. doi: 10.1007/s10534-017-0026-x. PubMed DOI

Carroll C.S., Grieve C.L., Murugathasan I., Bennet A.J., Czekster C.M., Lui H., Naismith J., Moore M.M. The rhizoferrin biosynthetic gene in the fungal pathogen Rhizopus delemar is a novel member of the NIS gene family. Int. J. Biochem. Cell Biol. 2017;89:136–146. doi: 10.1016/j.biocel.2017.06.005. PubMed DOI

Polaino S., Gonzalez-Delgado J.A., Arteaga P., Herrador M.M., Barrero A.F., Cerdá-Olmedo E. Apocarotenoids in the sexual interaction of Phycomyces blakesleeanus. Org. Biomol. Chem. 2012;10:3002–3009. doi: 10.1039/c2ob07147a. PubMed DOI

Lee S.C., Heitman J. Sex in the Mucoralean fungi. Mycoses. 2014;57(Suppl. 3):18–24. doi: 10.1111/myc.12244. PubMed DOI PMC

Sutter R.P., Capage D.A., Harrison T.L., Keen W.A. Trisporic acid biosynthesis in separate plus and minus cultures of Blakeslea trispora: Identification by Mucor assay of two mating-type-specific components. J. Bacteriol. 1973;114:1074–1082. doi: 10.1128/JB.114.3.1074-1082.1973. PubMed DOI PMC

Smith M.J., Shoolery J.N., Schwyn B., Holden I., Neilands J.B. Rhizobactin, a structurally novel siderophore from Rhizobium meliloti. J. Am. Chem. Soc. 1985;107:1739–1743. doi: 10.1021/ja00292a047. DOI

Drechsel H., Tschierske M., Thieken A., Jung G., Zähner H., Winkelmann G. The carboxylate type siderophore rhizoferrin and its analogs produced by directed fermentation. J. Ind. Microbiol. 1995;14:105–112. doi: 10.1007/BF01569891. DOI

Haselwandter K., Haas H., Häninger G., Winkelmann G. Siderophores in plant root tissue: Tagetes patula nana colonized by the arbuscular mycorrhizal fungus Gigaspora margarita. Biometals. 2020 doi: 10.1007/s10534-020-00238-0. PubMed DOI

Gao S.-S., Li X.-M., Williams K., Proksch P., Ji N.-Y., Wang B.-G. Rhizovarins A–F, indole-diterpenes from the mangrove-derived endophytic fungus Mucor irregularis QEN-189. J. Nat. Prod. 2016;79:2066–2074. doi: 10.1021/acs.jnatprod.6b00403. PubMed DOI

Schwartze V.U., Winter S., Shelest E., Marcet-Houben M., Horn F., Wehner S., Linde J., Valiante V., Sammeth M., Riege K., et al. Gene expansion shapes genome architecture in the human pathogen Lichtheimia corymbifera: An evolutionary genomics analysis in the ancient terrestrial Mucorales (Mucoromycotina) PLoS Genet. 2014;10:e1004496. doi: 10.1371/journal.pgen.1004496. PubMed DOI PMC

Drechsel H., Metzger J., Freund S., Jung G., Boelaert J.R., Winkelmann G. Rhizoferrin—A novel siderophore from the fungus Rhizopus microsporus var. rhizopodiformis. Biol. Met. 1991;4:238–243. doi: 10.1007/BF01141187. DOI

Sullivan J.T., Jeffery E.F., Shannon J.D., Ramakrishnan G. Characterization of the siderophore of Francisella tularensis and role of fslA in siderophore production. J. Bacteriol. 2006;188:3785–3795. doi: 10.1128/JB.00027-06. PubMed DOI PMC

Carroll C.S., Moore M.M. Ironing out siderophore biosynthesis: A review of non-ribosomal peptide synthetase (NRPS)-independent siderophore synthetases. Crit. Rev. Biochem. Mol. Biol. 2018;53:356–381. doi: 10.1080/10409238.2018.1476449. PubMed DOI

Pluhacek T., Lemr K., Ghosh D., Milde D., Novak J., Havlicek V. Characterization of microbial siderophores by mass spectrometry. Mass Spectrom. Rev. 2016;35:35–47. doi: 10.1002/mas.21461. PubMed DOI

Hider R.C., Kong X. Chemistry and biology of siderophores. Nat. Prod. Rep. 2010;27:637–657. doi: 10.1039/b906679a. PubMed DOI

Felsenfeld A.J., Rodriguez M., Coleman M., Ross D., Llach F. Desferrioxamine therapy in hemodialysis patients with aluminum-associated bone disease. Kidney Int. 1989;35:1371–1378. doi: 10.1038/ki.1989.136. PubMed DOI

Bonomo R.A. Cefiderocol: A novel siderophore cephalosporin defeating carbapenem-resistant pathogens. Clin. Infect. Dis. 2019;69:S519–S520. doi: 10.1093/cid/ciz823. PubMed DOI PMC

Dobias R., Havlicek V. Microbial siderophores: Markers of infectious diseases. In: Das S., Das H.R., editors. Microbial and Natural Macromolecules: Synthesis and Applications. Academic Press, Elsevier; Cambridge, MA, USA: 2020.

Hoenigl M., Orasch T., Faserl K., Prattes J., Loeffler J., Springer J., Gsaller F., Reischies F., Duettmann W., Raggam R.B., et al. Triacetylfusarinine C: A urine biomarker for diagnosis of invasive aspergillosis. J. Infect. 2019;78:150–157. doi: 10.1016/j.jinf.2018.09.006. PubMed DOI PMC

Pluhacek T., Skriba A., Novak J., Luptakova D., Havlicek V. Analysis of microbial siderophores by mass spectrometry. In: Bhattacharya S.K., editor. Methods Molecular Biology. Springer Nature; New York, NY, USA: 2019. PubMed

Devireddy L.R., Hart D.O., Goetz D.H., Green M.R. A mammalian siderophore synthesized by an enzyme with a bacterial homolog involved in enterobactin production. Cell. 2010;141:1006–1017. doi: 10.1016/j.cell.2010.04.040. PubMed DOI PMC

Bao G., Clifton M., Hoette T.M., Mori K., Deng S.-X., Qiu A., Viltard M., Williams D., Paragas N., Leete T., et al. Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex. Nat. Chem. Biol. 2010;6:602–609. doi: 10.1038/nchembio.402. PubMed DOI PMC

Münzinger M., Taraz K., Budzikiewicz H., Drechsel H., Heymann P., Winkelmann G., Meyer J.-M. S, S-rhizoferrin (enantio-rhizoferrin)–a siderophore of Ralstonia (Pseudomonas) pickettii DSM 6297–the optical antipode of R, R-rhizoferrin isolated from fungi. Biometals. 1999;12:189–193. doi: 10.1023/A:1009259118034. DOI

Burnside D.M., Wu Y., Shafaie S., Cianciotto N.P. The Legionella pneumophila siderophore legiobactin is a polycarboxylate that is identical in structure to rhizoferrin. Infect. Immun. 2015;83:3937–3945. doi: 10.1128/IAI.00808-15. PubMed DOI PMC

Kühn S., Braun V., Köster W. Ferric rhizoferrin uptake into Morganella morganii: Characterization of genes involved in the uptake of a polyhydroxycarboxylate siderophore. J. Bacteriol. 1996;178:496–504. doi: 10.1128/JB.178.2.496-504.1996. PubMed DOI PMC

Matzanke B.F., Böhnke R., Möllmann U., Schünemann V., Schumann G., Trautwein A.X., Winkelmann G. Transport and utilization of rhizoferrin bound iron in Mycobacterium smegmatis. Biometals. 1999;12:315–321. doi: 10.1023/A:1009274415607. PubMed DOI

Dadwal S.S., Kontoyiannis D.P. Recent advances in the molecular diagnosis of mucormycosis. Expert Rev. Mol. Diagn. 2018;18:845–854. doi: 10.1080/14737159.2018.1522250. PubMed DOI

Reid G., Lynch J.P., Fishbein M.C., Clark N.M. Mucormycosis. Semin Respir. Crit. Care Med. 2020;41:99–114. doi: 10.1055/s-0039-3401992. PubMed DOI

Houšť J., Spížek J., Havlíček V. Antifungal drugs. Metabolites. 2020;10:106. doi: 10.3390/metabo10030106. PubMed DOI PMC

Larcher G., Dias M., Razafimandimby B., Bomal D., Bouchara J.-P. Siderophore production by pathogenic mucorales and uptake of deferoxamine B. Mycopathologia. 2013;176:319–328. doi: 10.1007/s11046-013-9693-5. PubMed DOI

Kousser C., Clark C., Sherrington S., Voelz K., Hall R.A. Pseudomonas aeruginosa inhibits Rhizopus microsporus germination through sequestration of free environmental iron. Sci. Rep. 2019;9 doi: 10.1038/s41598-019-42175-0. PubMed DOI PMC

Boukhalfa H., Reilly S.D., Michalczyk R., Iyer S., Neu M.P. Iron(III) coordination properties of a pyoverdin siderophore produced by Pseudomonas putida ATCC 33015. Inorg. Chem. 2006;45:5607–5616. doi: 10.1021/ic060196p. PubMed DOI

Novák J., Škríba A., Havlíček V. CycloBranch 2: Molecular formula annotations applied to imzML data sets in bimodal fusion and LC-MS data files. Anal. Chem. 2020;92:6844–6849. doi: 10.1021/acs.analchem.0c00170. PubMed DOI

Donnelly J.P., Chen S.C., Kauffman C.A., Steinbach W.J., Baddley J.W., Verweij P.E., Clancy C.J., Wingard J.R., Lockhart S.R., Groll A.H., et al. Revision and update of the consensus definitions of invasive fungal disease from the European organization for research and treatment of cancer and the mycoses study group education and research consortium. Clin. Infect. Dis. 2019 doi: 10.1093/cid/ciz1008. PubMed DOI PMC

Kamimura H. Conversion of zearalenone to zearalenone glycoside by Rhizopus sp. Appl. Environ. Microbiol. 1986;52:515–519. doi: 10.1128/AEM.52.3.515-519.1986. PubMed DOI PMC

Li P., Sun C., Wang Y., Wang S., Yan C., Deng S., Huo X., Feng L., Wang C., Tian Y., et al. Efficiently regioselective glucosylation of estrogen analogues mediated by fungus Rhizopus oryzae AS 3.2380. Catal. Commun. 2017;97:106–110. doi: 10.1016/j.catcom.2017.04.027. DOI

Li Y., Theuretzbacher U., Clancy C.J., Nguyen M.H., Derendorf H. Pharmacokinetic/pharmacodynamic profile of posaconazole. Clin. Pharmacokinet. 2010;49:379–396. doi: 10.2165/11319340-000000000-00000. PubMed DOI

Hamilos G., Samonis G., Kontoyiannis D.P. Pulmonary mucormycosis. Semin Respir. Crit. Care Med. 2011;32:693–702. doi: 10.1055/s-0031-1295717. PubMed DOI

Ramakrishnan G. Iron and virulence in Francisella tularensis. Front. Cell. Infect. Microbiol. 2017;7 doi: 10.3389/fcimb.2017.00107. PubMed DOI PMC

Diagnosis of Aspergillosis in Horses

Distinguishing Invasive from Chronic Pulmonary Infections: Host Pentraxin 3 and Fungal Siderophores in Bronchoalveolar Lavage Fluids

Noninvasive Combined Diagnosis and Monitoring of Aspergillus and Pseudomonas Infections: Proof of Concept