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Supplementary Materials 1
TABLE S1 2
Overview on generation of isogeneic mutants and their genotypes. 3
strain c‐ID* genotype AA substitutions parentSSWT SC5314 FKS1/FKS1 None CA‐MT424 FKS1/fks1Δ::FRT None SC5314 CA‐MT432 FKS1/fks1Δ::FKS1 None CA‐MT424RRMH2 CA‐MT441 FKS1/fks1Δ::FKS1 A1939G C1946T Heterozygous R647R/G; P649P/L CA‐MT424 CA‐MT442 FKS1/fks1Δ::FKS1 A1939G C1946T Heterozygous R647R/G; P649P/L CA‐MT424 CA‐MT443 FKS1/fks1Δ::FKS1 A1939G C1946T Heterozygous R647R/G; P649P/L CA‐MT424RSMH1 CA‐MT453 FKS1/fks1Δ::FKS1 A1939G Heterozygous R647R/G CA‐MT424 CA‐MT454 FKS1/fks1Δ::FKS1 A1939G Heterozygous R647R/G CA‐MT424SRMH1 CA‐MT455 FKS1/fks1Δ::FKS1 C1946T Heterozygous P649P/L CA‐MT424 CA‐MT456 FKS1/fks1Δ::FKS1 C1946T Heterozygous P649P/L CA‐MT424 CA‐MT457 fks1Δ::FRT/fks1Δ::FKS1 A1939G Heterozygous R647R/G CA‐MT424 CA‐MT458 fks1Δ::FRT/fks1Δ::FKS1 C1946T Heterozygous P649P/L CA‐MT424 CA‐MT463 fks1Δ:: FKS1 A1939G /fks1Δ::FKS1 A1939G Heterozygous R647R/G CA‐MT457 CA‐MT464 fks1Δ:: FKS1 A1939G /fks1Δ::FKS1 A1939G Heterozygous R647R/G CA‐MT457 CA‐MT465 fks1Δ:: FKS1 C1946T /fks1Δ::FKS1 C1946T Heterozygous P649P/L CA‐MT458 CA‐MT466 fks1Δ:: FKS1 C1946T /fks1Δ::FKS1 C1946T Heterozygous P649P/L CA‐MT458RRMHO2 CA‐MT468 fks1Δ::FKS1 A1939G C1946T /fks1Δ::FKS1 A1939G C1946T Homozygous R647R/G; P649P/L CA‐MT441construction ID (c‐ID) 4
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TABLE S2 6
Plasmids used for inserting FKS1 mutations into parental strain SSWT. 7
plasmid name relevant inserts and cloning sites parent
pSFS2a
pSFS2a‐FKS1dr SacII‐FKS1 3’ region‐SacI pSFS2a
pSFS2a‐FKS1drur ApaI‐FKS1 5’ region‐XhoI‐SAT1‐FLP‐SacII‐FKS1 3’ region‐SacI pSFS2a‐FKS1dr
pSFS2a‐FKS1reint ApaI‐FKS1 coding sequence‐XhoI‐SAT1‐FLP‐SacII‐FKS1 3’ region‐SacI pSFS2a‐FKS1dr
pSFS2a‐FKS1A1939G_reint ApaI‐FKS1 A1939G coding sequence‐XhoI‐SAT1‐FLP‐SacII‐FKS1 3’ region‐SacI pSFS2a‐FKS1dr
pSFS2a‐FKS1C1946T_reint ApaI‐FKS1 C1946T coding sequence‐XhoI‐SAT1‐FLP‐SacII‐FKS1 3’ region‐SacI pSFS2a‐FKS1dr
pSFS2a‐FKS1_2mut _reint ApaI‐FKS1 A1939G C1946T coding sequence‐XhoI‐SAT1‐FLP‐SacII‐FKS1 3’ region‐SacI pSFS2a‐FKS1dr
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TABLE S3 10
Primers used for manipulation of nucleic acids and strain construction. 11
name sequence (5’ – 3’)* FKS1_55 gctagggccctagccgatcgGAACCGTGTGATCACTCCAC FKS1KO_53 gtcgctcgagCCTTTCCCCCGTTTTACTG FKS1_35 gctaccgcggATTATTTCCAAATGTAACTTTATATATTAG FKS1_33 gtcggagctccgaccgatcgCTTTTATGGTCGTGTTCGGGTG FKS1clo_53 gtcgctcgagCCTTTCCCCCGTTTTACTG FKS1_55_2 CAGATTCATCAGATCTCAAGTTTATG FKS1_33_2 CCTCTTCCTCCTATTATCAGACC FKS1_A1939G_fwd GACATTGTCTTTAgGAGATCCTATTAG FKS1_A1939G_rev CTAATAGGATCTCcTAAAGACAATGTC FKS1_C1946T_fwd GTCTTTAAGAGATCtTATTAGAAACTTG FKS1_C1946T_rev CAAGTTTCTAATAaGATCTCTTAAAGAC FKS1_A1939G_C1946T_fwd GACATTGTCTTTAgGAGATCtTATTAGAAACTTG FKS1_A1939G_C1946T_rev CAAGTTTCTAATAaGATCTCcTAAAGACAATGTC lower case and upper case letters denote exogenous and endogenous sequences, respectively 12
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Table S4. Statistical significance calculation using Kruskal‐Wallis test and Dunnett's multiple comparisons. 14
Outlier within the placebo treated control groups (P < 100 CFU/ml kidney homogenate) were excluded from calculations. Statistical significance is 15
indicated as follows: P < 0.05 (*), P < 0.01 (**), P < 0.001 (***). 16
SclANI500 SclANI100 SclCAS500 SclCAS100 SclMICA500 SclMICA100 RRclANI500 RRclANI100 RRclCAS500 RRclCAS100 RRclMICA500 RRclMICA100 RRclC SclC ** ** ** ** ** * n.s.
RRclC n.s. n.s n.s. n.s. n.s. n.s.
SclANI500 SclCAS500 SclMICA500 RRclANI500 RRclCAS500 RRclMICA500
SclANI100 n.s.
SclCAS100 n.s.
SclMICA100 n.s.
RRclANI100 n.s.
RRclCAS100 n.s.
RRclMICA100 n.s.
SclANI500 SclANI100 SclCAS500 SclCAS100 SclMICA500 SclMICA100
RRclANI500 n.s.
RRclANI100 n.s.
RRclCAS500 n.s.
RRclCAS100 n.s.
RRclMICA500 n.s.
RRclMICA100 n.s
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Table S5. Median and interquartile for mice challenged with a clinical isolate or an isogeneic mutant strain and treated with anidulafungin 19
(ANI), caspofungin (CAS), or micafungin (MICA). 20
Median CFU/mL kidney tissue homogenates Clinical isolates # Isogeneic mutant strains # SCL RRCL SSWT RRMH2 RSMH1 SRMH1 RRMHO2
ANI500 20* 100 20* 23* 20* 20* 75ANI100 20* 250 20* 75 20* 23* 150CANI ‐ ‐ 5250 8375 1203 2853 2500CAS500 20* 162 20* 20* 20* 20* 20*CAS100 20* 175 20* 85 23* 20* 20*CCAS ‐ ‐ 12300 7750 3500 2250 12650MICA500 23* 937 20* 20* 25* 20* 135MICA100 23* 865 20* 188 25* 38 113CMICA ‐ ‐ 4375 14000 7000 7500 9750CTOTAL 2625 287 ‐ ‐ ‐ ‐ ‐# clinical strains 11304 (S
cl ; no mutation in FKS1) and 110.12 (RR
cl; heterozygous double mutation in FKS1 at position R647R/G and P649P/L); isogenetic mutant 21
strains: SSwt parental strain (no mutation in FKS1), RR
MH2 heterozygous double mutation (R647R/G and P649P/L), RS
MH1 heterozygous single mutation 22
(R647R/G), SRMH1
(P649P/L), and RRMHO2
homozygous double mutation (R647G and P649L); for more detailed information see Table 3. 23
*Limit of Detection (LoD) was set at 25 CFU/mL tissue homogenate. 24
25
Table S6. Statistical significance calculation using Kruskal‐Wallis test and Dunnett's multiple comparisons. 26
Outlier within the placebo treated control groups (P < 100 CFU/mL kidney homogenate) were excluded from calculations. Statistical significance is 27
indicated as follows: P < 0.05 (*), P < 0.01 (**), P < 0.001 (***). A anidulafungin, B caspofungin, and C micafungin. 28
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A 30
SSWTANI500 SSWTANI100 SSWTC RRMH2ANI500 RRMH2ANI100 RRMH2C RSMH1ANI500 RSMH1ANI100 RSMH1C SRMH1ANI500 SRMH1ANI100 SRMH1C RRMH1ANI500 RRMHO2ANI100 RRMHO2C
SSWTC ** ** n.s. n.s. n.s n.s.
RRMH2C ** *
RSMH1C ** **
SRMH1C ** *
RRMHO2C ** *
SSWTANI500 RRMH2ANI500 RSMH1ANI500 SRMH1ANI500 RRMHO2ANI500
SSWTANI100 n.s.
RRMH2ANI100 n.s.
RSMH1ANI100 n.s.
SRMH1ANI100 n.s.
RRMHO2ANI100 n.s.
SSWTANI500 RRMH2ANI500 RSMH1ANI500 SRMH1ANI500 RRMHO2ANI500
SSWTANI500 n.s. n.s. n.s. n.s
RRMH2ANI500 n.s. n.s. n.s. n.s.
RSMH1ANI500 n.s. n.s. n.s. n.s.
SRMH1ANI500 n.s. n.s. n.s. n.s.
RRMHO2ANI500 n.s. n.s n.s. n.s.
SSWTANI100 RRMH2ANI100 RSMH1ANI100 SRMH1ANI100 RRMHO2ANI100
SSWTANI100 n.s. n.s. n.s. n.s
RRMH2ANI100 n.s. n.s. n.s. n.s.
RSMH1ANI100 n.s. n.s. n.s. n.s.
SRMH1ANI100 n.s. n.s. n.s. n.s.
RRMHO2ANI100 n.s. n.s n.s. n.s.
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B 33
SSWTCAS500 SSWTCAS100 SSWTC RRMH2CAS500 RRMH2CAS100 RRMH2C RSMH1CAS500 RSMH1CAS100 RSMH1C SRMH1CAS500 SRMH1CAS100 SRMH1C RRMH1CAS500 RRMHO2CAS100 RRMHO2C
SSWTC ** ** n.s. n.s. n.s. n.s.
RRMH2C ** *
RSMH1C *** **
SRMH1C ** **
RRMHO2C ** **
SSWTCAS500 RRMH2CAS500 RSMH1CAS500 SRMH1CAS500 RRMHO2CAS500
SSWTCAS100 n.s.
RRMH2CAS100 n.s.
RSMH1CAS100 n.s.
SRMH1CAS100 n.s.
RRMHO2CAS100 n.s.
SSWTCAS500 RRMH2CAS500 RSMH1CAS500 SRMH1CAS500 RRMHO2CAS500
SSWTCAS500 n.s. n.s. n.s. n.s.
RRMH2CAS500 n.s. n.s. n.s. n.s.
RSMH1CAS500 n.s. n.s. n.s. n.s.
SRMH1CAS500 n.s. n.s. n.s. n.s.
RRMHO2CAS500 n.s. n.s. n.s. n.s.
SSWTCAS100 RRMH2CAS100 RSMH1CAS100 SRMH1CAS100 RRMHO2CAS100
SSWTCAS100 n.s. n.s. n.s. n.s.
RRMH2CAS100 n.s. n.s. n.s. n.s.
RSMH1CAS100 n.s. n.s. n.s. n.s.
SRMH1CAS100 n.s. n.s. n.s. n.s.
RRMHO2CAS100 n.s. n.s. n.s. n.s. 34
35
C 36
SSWTMICA500 SSWTMICA100 SSWTC RRMH2MICA500 RRMH2MICA100 RRMH2C RSMH1MICA500 RSMH1MICA100 RSMH1C SRMH1MICA500 SRMH1MICA100 SRMH1C RRMH1MICA500 RRMHO2MICA100 SSWTC ** ** * n.s. n.s. RRMH2C *** * RSMH1C ** ** SRMH1C *** * RRMHO2C ** *
SSWTMICA500 RRMH2MICA500 RSMH1MICA500 SRMH1MICA500 RRMHO2MICA500 SSWTMICA100 n.s. RRMH2MICA100 n.s. RSMH1MICA100 n.s. SRMH1MICA100 n.s. RRMHO2MICA100 n.s.
SSWTMICA500 RRMH2MICA500 RSMH1MICA500 SRMH1MICA500 RRMHO2MICA500 SSWTMICA500 n.s. n.s. n.s. n.s. RRMH2MICA500 n.s. n.s. n.s. n.s. RSMH1MICA500 n.s. n.s. n.s. n.s. SRMH1MICA500 n.s. n.s. n.s. n.s. RRMHO2MICA500 n.s. n.s. n.s. n.s.
SSWTMICA100 RRMH2MICA100 RSMH1MICA100 SRMH1MICA100 RRMHO2MICA100 SSWTMICA100 n.s. n.s. n.s. n.s. RRMH2MICA100 n.s. n.s. n.s. n.s. RSMH1MICA100 n.s. n.s. n.s. n.s. SRMH1MICA100 n.s. n.s. n.s. n.s.
RRMHO2MICA100 n.s. n.s. n.s. n.s.
FIG S1 Chitin content measurement for parental strain SSWT (SC5314) and its isogenic mutants: A RRMHO2 homozygous double mutation (R647G and
P649L), B RSMH1 heterozygous single mutation (R647R/G), C SRMH1 (P649P/L), and D RRMH2 heterozygous double mutation (R647R/G and P649P/L),
using calcofluor white as fluorescent dye. Stained cells were detected in a fluorescence‐activated cell sorter (FACSverse, BD).
SSWT was used as wild type reference and is indicated in all centre plots as green population. Centre plots show cell count and fluorescence
intensity. Relative size and granularity of yeast cells is given in the left column with forward (FSC‐A) and sideward scatter (SSC‐A) values. SSC‐A and
fluorescence intensity are given in the right column. Gated cells are indicated by P1.
FIG S2 Chitin content measurement for SCL (no mutation in fks1) and RRCL (heterozygous double mutation in fks1 at position R647R/G and
P649P/L) using calcofluor white as fluorescent dye. Stained cells were detected in a fluorescence‐activated cell sorter (FACSverse, BD).
SSWT (SC5314) was used as wild type reference and is indicated in all centre plots as green population. Centre plots show cell count and
fluorescence intensity. Relative size and granularity of yeast cells is given in the left column with forward (FSC‐A) and sideward scatter (SSC‐A)
values. SSC‐A and fluorescence intensity are given in the right column. Gated cells are indicated by P1.
Cells in B were found to strongly aggregate with each other, resulting in overexposed cell clumps.