Distinct pathways of adaptive evolution in Cryptococcus neoformans reveal a mutation in adenylyl cyclase with trade-offs for pathogenicity

. Author manuscript; available in PMC: 2023 Oct 23.

Published in final edited form as: Curr Biol. 2023 Sep 13;33(19):4136–4149.e9. doi: 10.1016/j.cub.2023.08.054

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Mouse Anti-Glucuronoxylomannan (GXM) Antibody, clone 18B7	Sigma	Cat#MABF2069

Chemicals, peptides, and recombinant proteins

cAMP	Cayman Chemical	Cat#18820
Phorbol myristate acetate (PMA)	Sigma	Cat#P1585
Phusion Flash DNA Polymerase	Thermo Fisher	Cat# F548S
HiFi DNA Master Mix	New England Biolabs	Cat#E2621
Fluconazole	Sigma	Cat#F8929
Hexadecyltrimethylammonium bromide (CTAB)	Sigma	Cat#H9151
3,4-dihydroxy-L-phenylalanine	Sigma	Cat#D9628
L-Asparagine monohydrate	Sigma	Cat#A7094
Thiamine hydrochloride	Sigma	Cat#T4625
Biotin	Sigma	Cat#B4639
Yeast Extract	Gibco	Cat#212750
Peptone	Gibco	Cat#211677
Yeast nitrogen base without amino acids	Sigma	Cat#Y0626
Sabouraud Dextrose Broth	Difco	Cat#238230
DMEM with high glucose and L-glutamine	VWR	Cat#16777-129
Fetal Bovine Serum	Thermo Fisher	Cat#26140079
RPMI 1640	VWR	Cat#16777-145
RPMI 1640 with L-glutamine w/o sodium bicarbonate	Gibco	Cat#31800089
Dexdomitor	Zoetis	Cat#122692-5
Antisedan	Zoetis	Cat#87219-02296-2
Dexamethasone	Sigma	Cat#D4902

Critical commercial assays

NEBNext Ultra II DNA Library Prep	New England Biolabs	Cat#E7634L
Ligation Sequencing Kit	Oxford Nanopore	Cat#SQK-LSK110
Fluconazole Etest Strips	BioMerieux	Cat#412349
ChromaSpin 1000	Takara	Cat#636093

Deposited data

Nanopore and Illumina sequencing data	Sequence Read Archive	NCBI BioProject: PRJNA941895
Histopathology images from mice infected with Ftc555-1 Parental and M1 strains	HistoWiz	HistoWiz Data:https://app.histowiz.com/shared_orders/897b4aa6-bf6c-49da-9009-7113f766aee9/slides/
M. sympodalis adenylyl cyclase sequence	NCBI Genbank	GenBank: SHO76694.1
U. maydis adenylyl cyclase sequence	NCBI Genbank	GenBank: XP_011388269
C. neoformans adenylyl cylase sequence	NCBI Genbank	GenBank: XP_012050872
C. gattii adenylyl cyclase sequence	NCBI Genbank	GenBank: XP_003195134.1
S. pombe adenylyl cyclase sequence	NCBI Genbank	GenBank: NP_596159.1
N. crassa adenylyl cyclase sequence	NCBI Genbank	GenBank: BAA00755.1
B. cinerea adenylyl cyclase sequence	NCBI Genbank	GenBank: XP_024553314.1
T. rubrum adenylyl cyclase sequence	NCBI Genbank	GenBank: KDB32528.1
A. niger adenylyl cyclase sequence	NCBI Genbank	GenBank:GAq42063.1
C. albicans adenylyl cyclase sequence	NCBI Genbank	GenBank:XP_716974.2
S. cerevisiae adenylyl cyclase sequence	NCBI Genbank	GenBank:NP_012529
CAC1 sequences from all C. neoformans isolates	FungiDB	FungiDB: https://fungidb.org/fungidb/app/record/gene/CNAG_03202#category:dna-polymorphism

Experimental models: Cell lines

J774A.1	ATCC	Cat# TIB-67; RRID: CVCL_0358

Experimental models: Organisms/strains

Mice: C57BL/6NJ	Jackson Laboratory	RRID: IMSR_JAX:005304
Amoeba: Acanthamoeba castellanii	ATCC	Cat# 30234
C. neoformans: H99O	Gift from J. Perfect	N/A
C. neoformans: KN99α	Fungal Genetics Stock Center	Cat#10369
C. neoformans: KN99α cac1Δ::NAT	Fungal Genetics Stock Center, Madhani Knockout Collection (2020 Plates)	N/A
C. neoformans: Bt46	Gift from J. Perfect, Litvintseva et al.⁶¹	N/A
C. neoformans: Ze90-1	Gift from J. Perfect, Litvintseva et al.⁶²	N/A
C. neoformans: Ftc217-1	Gift from J. Perfect, Chen et al.⁵	N/A
C. neoformans: Ftc239-1	Gift from J. Perfect, Chen et al.⁵	N/A
C. neoformans: Ftc209-1	Gift from J. Perfect, Chen et al.⁵	N/A
C. neoformans: Ftc209-3	Gift from J. Perfect, Chen et al.⁵	N/A
C. neoformans: NRHc5004.ENR	Gift from J. Perfect, Chen et al.⁵	N/A
C. neoformans: Muc458-2	Gift from J. Perfect, Chen et al.⁵	N/A
C. neoformans: NRHc5026.ENR.CLIN.ISO	Gift from J. Perfect, Chen et al.⁵	N/A
C. neoformans: Ftc555-1	Gift from J. Perfect, Chen et al.⁵	N/A
C. neoformans: Ftc158-2	Gift from J. Perfect, Chen et al.⁵	N/A
C. neoformans: NRHc5029.ENR.CLIN.ISO	Gift from J. Perfect, Chen et al.⁵	N/A
C. neoformans: Gbc39-1	Gift from J. Perfect, Chen et al.⁵	N/A
C. neoformans: Ftc555-1 M1-1	This study	N/A
C. neoformans: Ftc555-1 M2-1	This study	N/A
C. neoformans: Ftc555-1 M3-2	This study	N/A
C. neoformans: Ftc555-1 A1-1	This study	N/A
C. neoformans: Ftc555-1 A2-1	This study	N/A
C. neoformans: Ftc555-1 A3-1	This study	N/A
C. neoformans: Ftc555-1 SH2-NAT	This study	ZAH_CN01
C. neoformans: Ftc555-1 Parental cac1-evo(R1227P); SH2-NAT isolate H1	This study	ZAH_CN03
C. neoformans: Ftc555-1 Parental cac1-evo(R1227P); SH2-NAT isolate H7	This study	ZAH_CN04
C. neoformans: Ftc555-1 M1 CAC1-parent (P1227R); SH2-NAT isolate M2	This study	ZAH_CN05
C. neoformans: Ftc555-1 M1 CAC1-parent (P1227R); SH2-NAT isolate H11	This study	ZAH_CN06
C. neoformans: Ftc555-1 Parental cac1Δ::HYG	This study	ZAH_CN07
C. neoformans: Ftc555-1 M1 cac1Δ::HYG; cac1-evo(R1227P)	This study	ZAH_CN08
C. neoformans: Ftc555-1 Parental aca1Δ::NAT	This study	ZAH_CN09
C. neoformans: Ftc555-1 Parental gpa1Δ::NAT	This study	ZAH_CN10
C. neoformans: Ftc555-1 Parental gpr4Δ::NAT	This study	ZAH_CN11
C. neoformans: Ftc555-1 M1 aca1Δ::NAT; cac1-evo(R1227P)	This study	ZAH_CN12
C. neoformans: Ftc555-1 M1 gpa1Δ::NAT; cac1-evo(R1227P)	This study	ZAH_CN13
C. neoformans: Ftc555-1 M1 gpr4Δ::NAT; cac1-evo(R1227P)	This study	ZAH_CN14

Oligonucleotides

See Table S1	N/A	N/A

Recombinant DNA

SH2::NAT plasmid for CRISPR repair amplification	This study	pZAH31
cac1Δ::HYG plasmid for CRISPR KO repair template	This study	pZAH40
aca1Δ::NAT plasmid for CRISPR KO repair template	This study	pMWS05
gpa1Δ::NAT plasmid for CRISPR KO repair template	This study	pMWS06
gpr4Δ::NAT plasmid for CRISPR KO repair template	This study	pMWS07
pRS316-PCnU6-scaffold for sgRNA Fusion PCRs	Huang et al.⁶³	AddGene Cat#171686; pBHM2329
pRS316-PTEF1-CnoCas9 Codon Optimized C. neoformans Cas9 for CRISPR	Huang et al.⁶³	AddGene Cat#171687; pBHM2403

Software and algorithms

Guppy-gpu basecaller (v 6.0.1)	Oxford Nanopore	RRID: SCR_023196
Canu (v 2.1.1)	Koren et al.⁶⁴	RRID: SCR_015880; https://github.com/marbl/canu
Medaka (v 1.5.0)	Oxford Nanopore	https://github.com/nanoporetech/medaka
Pilon (v 1.24)	Walker et al.⁶⁵	RRID: SCR_014731; https://github.com/broadinstitute/pilon/
GetOrganelle (v 1.7.5.3)	Jin et al.⁶⁶	https://github.com/Kinggerm/GetOrganelle
Liftoff (v 1.6.3)	Shumate and Salzberg⁶⁷	https://github.com/agshumate/Liftoff
Trimmomatic (v 0.39)	Bolger et al.⁶⁸	RRID: SCR_011848; http://www.usadellab.org/cms/index.php?page=trimmomatic
bwa-mem2 (v 2.2.1)	Vasimuddin et al.⁶⁹	RRID: SCR_022192; https://github.com/bwa-mem2/bwa-mem2
Picard	Broad Institute	RRID: SCR_006525; https://github.com/broadinstitute/picard
Genome Analysis Tool Kit (GATK, v 3.8)	McKenna et al.⁷⁰	RRID: SCR_001876; https://software.broadinstitute.org/gatk/
SAMtools (v 1.15.1)	Danecek et al.⁷¹	RRID: SCR_002105; https://www.htslib.org/
SnpEff (v 4.3t)	Cingolani et al.⁷²	RRID: SCR_005191; https://pcingola.github.io/SnpEff/
Integrated Genomics Viewer (IGV)	Thorvaldsdóttir et al.⁷³	RRID: SCR_011793; http://www.broadinstitute.org/igv/
AlphaFold (v 2.1.2)	Jumper et al.⁷⁴	https://github.com/deepmind/alphafold
Geneious	Geneious	RRID: SCR_010519; http://www.geneious.com/
IQ-Tree	Nguyen et al.⁷⁵	RRID: SCR_017254; http://www.iqtree.org/
FIJI	Schindelin et al.⁷⁶	RRID: SCR_002285; https://fiji.sc/
QCA-Quigly’s Circle App	Dragotakes and Casadevall⁷⁷	https://github.com/quigly555/QCA
Prism 9	GraphPad	RRID: SCR_002798; https://www.graphpad.com/
WebSNAPER	Drenkard et al.⁷⁸	https://pga.mgh.harvard.edu/cgi-bin/snap3/websnaper3.cgi