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ABSTRACT Infection by the fungal pathogen _Cryptococcus neoformans_ causes lethal meningitis, primarily in immune-compromised individuals. Colonization of the brain by _C. neoformans_ is

dependent on copper (Cu) acquisition from the host, which drives critical virulence mechanisms. While _C. neoformans_ Cu+ import and virulence are dependent on the Ctr1 and Ctr4 proteins,

little is known concerning extracellular Cu ligands that participate in this process. We identified a _C. neoformans_ gene, _BIM1_, that is strongly induced during Cu limitation and which

encodes a protein related to lytic polysaccharide monooxygenases (LPMOs). Surprisingly, _bim1_ mutants are Cu deficient, and Bim1 function in Cu accumulation depends on Cu2+ coordination and

cell-surface association via a glycophosphatidyl inositol anchor. Bim1 participates in Cu uptake in concert with Ctr1 and expression of this pathway drives brain colonization in mouse

infection models. These studies demonstrate a role for LPMO-like proteins as a critical factor for Cu acquisition in fungal meningitis. Access through your institution Buy or subscribe This

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ASSEMBLY OF A TIGHT CAPSULAR POLYSACCHARIDE LAYER ON THE _ACINETOBACTER BAUMANNII_ CELL SURFACE Article Open access 05 November 2021 RECIPROCAL MODULATION OF AMMONIA AND MELANIN PRODUCTION

HAS IMPLICATIONS FOR CRYPTOCOCCAL VIRULENCE Article Open access 15 February 2023 _CRYPTOCOCCUS NEOFORMANS_ ADAPTS TO THE HOST ENVIRONMENT THROUGH TOR-MEDIATED REMODELING OF PHOSPHOLIPID

ASYMMETRY Article Open access 18 October 2023 DATA AVAILABILITY The data that support the findings of this study are available from the corresponding author upon reasonable request.

REFERENCES * Köhler, J. R., Casadevall, A. & Perfect, J. The spectrum of fungi that infects humans. _Cold Spring Harb. Persp. Med._ 5, ao19273 (2015). Google Scholar  * Maret, W. The

metals in the biological periodic system of the elements: concepts and conjectures. _Int. J. Mol. Sci._ 17, 66 (2016). PubMed Central  Google Scholar  * Hood, M. I. & Skaar, E. P.

Nutritional immunity: transition metals at the pathogen-host interface. _Nat. Rev. Microbiol_ 10, 525–537 (2012). CAS  PubMed  Google Scholar  * Palmer, L. D. & Skaar, E. P. Transition

metals and virulence in bacteria. _Annu. Rev. Genet._ 50, 67–91 (2016). CAS  PubMed  PubMed Central  Google Scholar  * Gerwien, F., Skrahina, V., Kasper, L., Hube, B. & Brunke, S. Metals

in fungal virulence. _FEMS Microbiol. Rev._ 42, fux050 (2018). Google Scholar  * Rajasingham, R. et al. Global burden of disease of HIV-associated cryptococcal meningitis: an updated

analysis. _Lancet Infect. Dis._ 17, 873–881 (2017). PubMed  PubMed Central  Google Scholar  * Heitman, J., Kozel, T. R., Kwon-Chung, K. J., Perfect, J. R. & Casadevall, A. (eds)

_Cryptococcus: from Human Pathogen to Model Yeast_ (American Society of Microbiology, 2011). * Ding, C. et al. Cryptococcus neoformans copper detoxification machinery is critical for fungal

virulence. _Cell Host Microbe_ 13, 265–276 (2013). CAS  PubMed  PubMed Central  Google Scholar  * Sun, T. S. et al. Reciprocal functions of _Cryptococcus neoformans_ copper homeostasis

machinery during pulmonary infection and meningoencephalitis. _Nat. Commun._ 5, 55500 (2014). Google Scholar  * Garcia-Santamarina, S. et al. Genome-wide analysis of the regulation of Cu

metabolism in _Cryptococcus neoformans_. _Mol. Microbiol._ 108, 473–494 (2018). CAS  PubMed  PubMed Central  Google Scholar  * Ding, C. et al. The copper regulon of the human fungal pathogen

_Cryptococcus neoformans_ H99. _Mol. Microbiol._ 81, 1560–1576 (2011). CAS  PubMed  PubMed Central  Google Scholar  * Waterman, S. R. et al. Role of a CUF1/CTR4 copper regulatory axis in

the virulence of _Cryptococcus neoformans_. _J. Clin. Invest._ 117, 794–802 (2007). CAS  PubMed  PubMed Central  Google Scholar  * Ladomersky, E. et al. Host and pathogen copper-transporting

P-Type ATPases function antagonistically during salmonella infection. _Infect. Immun._ 85, e00351-17 (2017). PubMed  PubMed Central  Google Scholar  * Wagner, D. et al. Elemental analysis

of _Mycobacterium avium_-, _Mycobacterium tuberculosis_-, and _Mycobacterium smegmatis_-containing phagosomes indicates pathogen-induced microenvironments within the host cell’s endosomal

system. _J. Immunol._ 174, 1491–1500 (2005). CAS  PubMed  Google Scholar  * Garcia-Santamarina, S., Uzarska, M. A., Festa, R. A., Lill, R. & Thiele, D. J. Iron-sulfur protein biogenesis

machinery is a novel layer of protection against Cu stress. _MBio_ 8, e01742-17 (2017). PubMed  PubMed Central  Google Scholar  * Smith, A. D., Logeman, B. L. & Thiele, D. J. Copper

acquisition and utilization in fungi. _Annu. Rev. Microbiol._ 71, 597–623 (2017). CAS  PubMed  PubMed Central  Google Scholar  * Forsberg, Z. et al. Polysaccharide degradation by lytic

polysaccharide monooxygenases. _Curr. Opin. Struct. Biol._ 59, 54–64 (2019). CAS  PubMed  Google Scholar  * Arnesano, F., Banci, L., Bertini, I., Mangani, S. & Thompsett, A. R. A redox

switch in CopC: an intriguing copper trafficking protein that binds copper(I) and copper(II) at different sites. _Proc. Natl Acad. Sci. USA_ 100, 3814–3819 (2003). CAS  PubMed  Google

Scholar  * Yang, J. et al. The I-TASSER Suite: protein structure and function prediction. _Nat. Methods_ 12, 7–8 (2015). CAS  PubMed  PubMed Central  Google Scholar  * Vaaje-Kolstad, G. et

al. An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides. _Science_ 330, 219–222 (2010). CAS  PubMed  Google Scholar  * Quinlan, R. J. et al. Insights into

the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components. _Proc. Natl Acad. Sci. USA_ 108, 15079–15084 (2011). CAS  PubMed  Google Scholar  * Horn,

S. J., Vaaje-Kolstad, G., Westereng, B. & Eijsink, V. G. Novel enzymes for the degradation of cellulose. _Biotechnol. Biofuels_ 5, 45 (2012). CAS  PubMed  PubMed Central  Google Scholar

  * Johansen, K. S. Lytic polysaccharide monooxygenases: the microbial power tool for lignocellulose degradation. _Trends Plant Sci._ 21, 926–936 (2016). CAS  PubMed  Google Scholar  *

Forsberg, Z. et al. Structural and functional analysis of a lytic polysaccharide monooxygenase important for efficient utilization of chitin in cellvibrio japonicus. _J. Biol. Chem._ 291,

7300–7312 (2016). CAS  PubMed  PubMed Central  Google Scholar  * Tsukihara, T. et al. The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 Å. _Science_ 272, 1136–1144

(1996). CAS  PubMed  Google Scholar  * Walton, F. J., Idnurm, A. & Heitman, J. Novel gene functions required for melanization of the human pathogen _Cryptococcus neoformans_. _Mol.

Microbiol._ 57, 1381–1396 (2005). CAS  PubMed  Google Scholar  * Askwith, C. et al. The FET3 gene of _S. cerevisiae_ encodes a multicopper oxidase required for ferrous iron uptake. _Cell_

76, 403–410 (1994). CAS  PubMed  Google Scholar  * Dancis, A. et al. Molecular characterization of a copper transport protein in _S. cerevisiae_: an unexpected role for copper in iron

transport. _Cell_ 76, 393–402 (1994). CAS  PubMed  Google Scholar  * Culotta, V. C. et al. The copper chaperone for superoxide dismutase. _J. Biol. Chem._ 272, 23469–23472 (1997). CAS 

PubMed  Google Scholar  * Saikia, S., Oliveira, D., Hu, G. & Kronstad, J. Role of ferric reductases in iron acquisition and virulence in the fungal pathogen _Cryptococcus neoformans_.

_Infect. Immun._ 82, 839–850 (2014). PubMed  PubMed Central  Google Scholar  * Ferguson, M. A. J., Hart, G. W. & Kinoshita, T. in _Essentials of Glycobiology_ (eds Varki, A. et al.) Ch.

12 (Cold Spring Harbor Laboratory Press, 2017). * Labourel, A. et al. A fungal family of lytic polysaccharide monooxygenase-like copper proteins. _Nat. Chem. Biol._

https://doi.org/10.1038/s41589-019-0438-8 (2019). * Peisach, J. & Blumberg, W. E. Structural implications derived from the analysis of electron paramagnetic resonance spectra of natural

and artificial copper proteins. _Arch. Biochem. Biophys._ 165, 691–708 (1974). CAS  PubMed  Google Scholar  * Kau, L. S., Spira-Solomon, D. J., Penner-Hahn, J. E., Hodgson, K. O. &

Solomon, E. I. X-ray absorption edge determination of the oxidation state and coordination number of copper. Application to the type 3 site in _Rhus vernicifera_ laccase and its reaction

with oxygen. _J. Am. Chem. Soc._ 109, 6433–6442 (1987). CAS  Google Scholar  * Vu, V. V., Beeson, W. T., Span, E. A., Farquhar, E. R. & Marletta, M. A. A family of starch-active

polysaccharide monooxygenases. _Proc. Natl Acad. Sci. USA_ 111, 13822–13827 (2014). CAS  PubMed  Google Scholar  * Hansson, H. et al. High-resolution structure of a lytic polysaccharide

monooxygenase from. _J. Biol. Chem._ 292, 19099–19109 (2017). CAS  PubMed  PubMed Central  Google Scholar  * Westereng, B., Arntzen, M., Agger, J. W., Vaaje-Kolstad, G. & Eijsink, V. G.

H. Analyzing activities of lytic polysaccharide monooxygenases by liquid chromatography and mass spectrometry. _Methods Mol. Biol._ 1588, 71–92 (2017). CAS  PubMed  Google Scholar  * Pope,

C. R., Flores, A. G., Kaplan, J. H. & Unger, V. M. Structure and function of copper uptake transporters. _Curr. Top. Membr._ 69, 97–112 (2012). CAS  PubMed  Google Scholar  * Ramos, D.

et al. Mechanism of copper uptake from blood plasma ceruloplasmin by mammalian cells. _PLoS ONE_ 11, e0149516 (2016). PubMed  PubMed Central  Google Scholar  * Stefaniak, E. et al. The

N-terminal 14-mer model peptide of human Ctr1 can collect Cu(ii) from albumin. Implications for copper uptake by Ctr1. _Metallomics_ 10, 1723–1727 (2018). CAS  PubMed  Google Scholar  *

Lawton, T. J., Kenney, G. E., Hurley, J. D. & Rosenzweig, A. C. The CopC family: structural and bioinformatic insights into a diverse group of periplasmic copper binding proteins.

_Biochemistry_ 55, 2278–2290 (2016). CAS  PubMed  PubMed Central  Google Scholar  * Nimrichter, L. et al. Self-aggregation of _Cryptococcus neoformans_ capsular glucuronoxylomannan is

dependent on divalent cations. _Eukaryot. Cell_ 6, 1400–1410 (2007). CAS  PubMed  PubMed Central  Google Scholar  * Brady, D., Stoll, A. D., Starke, L. & Duncan, J. R. Chemical and

enzymatic extraction of heavy metal binding polymers from isolated cell walls of _Saccharomyces cerevisiae_. _Biotechnol. Bioeng._ 44, 297–302 (1994). CAS  PubMed  Google Scholar  * Citiulo,

F. et al. _Candida albicans_ scavenges host zinc via Pra1 during endothelial invasion. _PLoS Pathog._ 8, e1002777 (2012). CAS  PubMed  PubMed Central  Google Scholar  * Lamb, A. L., Torres,

A. S., O’Halloran, T. V. & Rosenzweig, A. C. Heterodimeric structure of superoxide dismutase in complex with its metallochaperone. _Nat. Struct. Mol. Biol._ 8, 751–755 (2001). CAS 

Google Scholar  * Loose, J. S., Forsberg, Z., Fraaije, M. W., Eijsink, V. G. & Vaaje-Kolstad, G. A rapid quantitative activity assay shows that the _Vibrio cholerae_ colonization factor

GbpA is an active lytic polysaccharide monooxygenase. _FEBS Lett._ 588, 3435–3440 (2014). CAS  PubMed  Google Scholar  * Chaudhuri, S. et al. Contribution of chitinases to _Listeria

monocytogenes_ pathogenesis. _Appl. Environ. Microbiol._ 76, 7302–7305 (2010). CAS  PubMed  PubMed Central  Google Scholar  * O’Connell, R. J. et al. Lifestyle transitions in plant

pathogenic _Colletotrichum_ fungi deciphered by genome and transcriptome analyses. _Nat. Genet._ 44, 1060–1065 (2012). PubMed  Google Scholar  * Bailey, T. L. et al. MEME Suite: tools for

motif discovery and searching. _Nucleic Acids Res._ 37, W202–W208 (2009). CAS  PubMed  PubMed Central  Google Scholar  * Petersen, T. N., Brunak, S., von Heijne, G. & Nielsen, H. SignalP

4.0: discriminating signal peptides from transmembrane regions. _Nat. Methods_ 8, 785–786 (2011). CAS  PubMed  Google Scholar  * Eisenhaber, B., Bork, P. & Eisenhaber, F. Prediction of

potential GPI-modification sites in proprotein sequences. _J. Mol. Biol._ 292, 741–758 (1999). CAS  PubMed  Google Scholar  * Steentoft, C. et al. Precision mapping of the human O-GalNAc

glycoproteome through SimpleCell technology. _EMBO J._ 32, 1478–1488 (2013). CAS  PubMed  PubMed Central  Google Scholar  * Gilbert, N. M. et al. _KRE_ genes are required for β-1,6-glucan

synthesis, maintenance of capsule architecture and cell wall protein anchoring in _Cryptococcus neoformans_. _Mol. Microbiol._ 76, 517–534 (2010). CAS  PubMed  PubMed Central  Google Scholar

  * Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. The Phyre2 web portal for protein modeling, prediction and analysis. _Nat. Protoc._ 10, 845–858 (2015). CAS 

PubMed  PubMed Central  Google Scholar  * Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. _Acta Crystallogr. D_ 66, 486–501 (2010). CAS  PubMed 

Google Scholar  * Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. _Acta Crystallogr. D_ 67, 355–367 (2011). CAS  PubMed  Google Scholar  * Chen, V.

B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. _Acta Crystallogr. D_ 66, 12–21 (2010). CAS  PubMed  Google Scholar  * Ankudinov, A. L. & Rehr, J.

J. Relativistic calculations of spin-dependent x-ray-absorption spectra. _Phys. Rev. B_ 56, R1712–R1716 (1997). CAS  Google Scholar  * Westereng, B. et al. Efficient separation of oxidized

cello-oligosaccharides generated by cellulose degrading lytic polysaccharide monooxygenases. _J. Chromatogr. A_ 1271, 144–152 (2013). CAS  PubMed  Google Scholar  Download references

ACKNOWLEDGEMENTS This work was partially supported by funds from the United States National Institutes of Health (NIH) (grant nos. GM041840 to D.J.T.; GM084176 to K.J.F.; GM127390 to

N.V.G.), the Welch Foundation (grant no. I-1505 to N.V.G.), a postdoctoral fellowship from German Research Foundation grant PR 1727/1-1 (to C.P.), fellowship support from NIH (no.

GM100678-02 to R.A.F.), NIH Molecular Mycology and Pathogenesis Training program (5T32a1052080 to A.D.S.), the Novo Nordisk Foundation grant (no. NNF17SA0027704 to K.S.J.), travel support

from the School of Science and Math at the College of Charleston (to P.R.G.) and fellowship support from NIH (no. GM084146-S1) and Duke University BioCoRE (R25-GM103765) (to S.E.C.). We

thank J. Lodge (Department of Molecular Microbiology, Washington University School of Medicine) for providing the anti-Cda2 antibody, Y. Song and M. Hoy for technical assistance and J.-G.

Berrin for sharing information before publication. Use of the Stanford Synchrotron Radiation Light source, SLAC National Accelerator Laboratory, is supported by the US Department of Energy,

Office of Science, Office of Basic Energy Sciences (contract no. DE-AC02-76SF00515). The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and

Environmental Research, and by the NIH, National Institute of General Medical Sciences (including P41GM103393). We thank authors of works that could not be appropriately cited in this work

due to space-limiting constrictions. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH. AUTHOR

INFORMATION Author notes * Sarela Garcia-Santamarina Present address: Genome Biology Unit, Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg,

Germany * Richard A. Festa Present address: Irvine Scientific, Santa Ana, CA, USA * Chen Ding Present address: College of Life and Health Sciences, Northeastern University, Shenyang, China *

Steven E. Conklin Present address: Division of Clinical Chemistry, Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA AUTHORS AND AFFILIATIONS *

Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC, USA Sarela Garcia-Santamarina, Corinna Probst, Richard A. Festa, Chen Ding, Aaron D. Smith 

& Dennis J. Thiele * Department of Chemistry, Duke University, Durham, NC, USA Steven E. Conklin & Katherine J. Franz * Department of Geoscience and Natural Resource, University of

Copenhagen, Copenhagen, Denmark Søren Brander & Katja Salomon Johansen * Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA Lisa N. Kinch 

& Nick V. Grishin * Departments of Biophysics and Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA Nick V. Grishin * Department of Chemistry, College of

Charleston, Charleston, SC, USA Pamela Riggs-Gelasco * Department of Chemistry, University of Copenhagen, Copenhagen, Denmark Leila Lo Leggio * Department of Biochemistry, Duke University

School of Medicine, Durham, NC, USA Dennis J. Thiele * Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, USA Dennis J. Thiele Authors *

Sarela Garcia-Santamarina View author publications You can also search for this author inPubMed Google Scholar * Corinna Probst View author publications You can also search for this author

inPubMed Google Scholar * Richard A. Festa View author publications You can also search for this author inPubMed Google Scholar * Chen Ding View author publications You can also search for

this author inPubMed Google Scholar * Aaron D. Smith View author publications You can also search for this author inPubMed Google Scholar * Steven E. Conklin View author publications You can

also search for this author inPubMed Google Scholar * Søren Brander View author publications You can also search for this author inPubMed Google Scholar * Lisa N. Kinch View author

publications You can also search for this author inPubMed Google Scholar * Nick V. Grishin View author publications You can also search for this author inPubMed Google Scholar * Katherine J.

Franz View author publications You can also search for this author inPubMed Google Scholar * Pamela Riggs-Gelasco View author publications You can also search for this author inPubMed 

Google Scholar * Leila Lo Leggio View author publications You can also search for this author inPubMed Google Scholar * Katja Salomon Johansen View author publications You can also search

for this author inPubMed Google Scholar * Dennis J. Thiele View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS All authors of the manuscript,

S.G.-S., C.P., R.A.F., C.D., A.D.S, P.R.-G., S.E.C., S.B., L.N.K., L.L.L., K.J.F., N.V.G., K.S.J. and D.J.T., conducted and/or planned and interpreted experiments. C.P. generated strains and

conducted experiments in Fig. 4c,d, and Supplementary Fig. 5c. R.A.F. conducted experiments in Fig. 1c,d. C.D. initiated the project and generated strains and initial results. A.D.S.

performed all mouse retro-orbital injections and participated in all mouse experiments. P.R.-G. planned, conducted and interpreted the results of all XAS experiments. S.E.C. and K.J.F.

conducted and/or planned and interpreted EPR experiments. S.B. and K.S.J. performed and/or planned and interpreted Bim1 activity experiments. L.N.K. and N.V.G. did bioinformatics analysis

that led to the identifying Bim1 as an LPMO-like protein. L.L.L. performed Bim1 homology modeling. S.G.-S. performed the rest of the experiments. S.G.-S. and D.J.T. planned and interpreted

all experiments. All authors contributed to the writing and editing of the manuscript. CORRESPONDING AUTHOR Correspondence to Dennis J. Thiele. ETHICS DECLARATIONS COMPETING INTERESTS The

authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional

affiliations. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figs. 1–9 and Tables 1–3. REPORTING SUMMARY SUPPLEMENTARY DATASET 1 Strains, oligonucleotides and plasmids,

numbers and descriptions. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Garcia-Santamarina, S., Probst, C., Festa, R.A. _et al._ A lytic polysaccharide

monooxygenase-like protein functions in fungal copper import and meningitis. _Nat Chem Biol_ 16, 337–344 (2020). https://doi.org/10.1038/s41589-019-0437-9 Download citation * Received: 15

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