Abstract
Fungal infections pose a great threat to public health and there are
only four main types of antifungal drugs, which are often limited with
toxicity, drug-drug interactions and antibiotic resistance.
Streptomyces is an important source of antibiotics, represented by the
clinical drug amphotericin B. Here we report the discovery of
alligamycin A (1) as an antifungal compound from the rapamycin-producer
Streptomyces iranensis through genome-mining, genetics and natural
product chemistry approaches. Alligamycin A harbors a unique chemical
scaffold with 13 chiral centers, featuring a β-lactone moiety, a
[6,6]-spiroketal ring, and an unreported 7-oxo-octylmalonyl-CoA
extender unit incorporated by a potential crotonyl-CoA
carboxylase/reductase. It is biosynthesized by a type I polyketide
synthase which is confirmed through CRISPR-based gene editing.
Alligamycin A displayed potent antifungal effects against numerous
clinically relevant filamentous fungi, including resistant Aspergillus
and Talaromyces species. β-Lactone ring is essential for the antifungal
activity since alligamycin B (2) with disruption in the ring abolished
the antifungal effect. Proteomics analysis revealed alligamycin A
potentially disrupts the integrity of fungal cell walls and induces the
expression of stress-response proteins in Aspergillus niger. Discovery
of the potent antifungal candidate alligamycin A expands the limited
antifungal chemical space.
Subject terms: Antimicrobials, Drug discovery and development,
Antifungal agents
__________________________________________________________________
From the rapamycin-producer Streptomyces iranensis, authors report on
the discovery of the antifungal candidate alligamycin A, which
exhibited strong activities against several drug-resistant Aspergillus
and Talaromyces.
Introduction
About 1.2 billion people worldwide are estimated to suffer from a
fungal infection^[54]1. Invasive fungal diseases are prevalent among
immunocompromised populations, such as patients with HIV/AIDS, chronic
lung diseases, prior tuberculosis, cancer, diabetes, and patients
receiving immunosuppressant treatment and invasive medical
procedures^[55]2. In particular, invasive fungal diseases are
associated with unacceptably high mortality rates. A recent report
suggested an annual incidence of 6.5 million invasive fungal infections
and 3.8 million deaths^[56]3. For example, infections with Aspergillus
have emerged as one of the most common death causes in severely
immunocompromised patients, i.e. with acute leukemia and recipients of
hematopoietic stem cell transplantation with mortality rates up to 40%
to 50%^[57]1. Furthermore, new risk cohorts are emerging, including
patients with severe respiratory virus infection such as influenza or
COVID-19^[58]4. Currently, four classes of systemic antifungal
medicines (azoles, echinocandins, pyrimidines, and polyenes) are used
in clinical practice, and only a limited number of candidates are in
the clinical development pipeline^[59]5–[60]8. While existing
antifungal medications are effective in numerous instances, challenges
persist due to side effects and complications arising from drug-drug
interactions^[61]9. Moreover, the emergence of resistance had rendered
some existing medications ineffective, and this was partly driven by
the inappropriate use of antifungals^[62]10. For example, agricultural
use of azoles has been linked to rising rates of azole-resistant
Aspergillus fumigatus infections, with resistance rates of 15–20%
reported in parts of Europe and exceeding 80% in environmental samples
from Asia^[63]10–[64]12.
Streptomyces are producers of bioactive small molecules exhibiting a
broad range of structural and functional diversity called secondary or
specialized metabolites. Secondary metabolites (SMs) are of great
relevance to human health, offering pharmaceutical properties such as
antibacterial, antifungal, anticancer, and immunosuppressive
activities. Remarkably, in all new drug approvals from 1981 to 2019,
over 60% of the 1394 small molecule drugs were either secondary
metabolites or derivatives thereof^[65]13–[66]15. Nearly two-thirds of
antibiotics approved for clinical use originate from Streptomyces, and
polyketides represent a large and diverse group of Streptomyces-derived
SMs. Many possess valuable antifungal properties, among which nystatin
and amphotericin B are two active polyenes used clinically as
first-line drugs to treat fungal infections. The first example is the
antifungal drug nystatin isolated from Streptomyces noursei in 1950 by
Hazen and Brown^[67]16. Amphotericin B, isolated from Streptomyces
nodosus in 1955, remains a crucial component in the therapeutic arsenal
for combating invasive fungal diseases and leishmaniasis^[68]17. Recent
studies revealed that the mode of action of polyenes antibiotics
involves acting as sterol “sponges” and extracting ergosterol from the
fungal membrane, causing cell death, in addition to the classical model
of pore formation in the fungal cell membrane^[69]18–[70]20.
Amphotericin B has been used to treat mucormycosis, aspergillosis,
blastomycosis, candidiasis, coccidioidomycosis, and cryptococcosis.
However, adverse effects from amphotericin B treatment are common, with
nephrotoxicity being the most serious^[71]21,[72]22. There has been a
continued interest in finding new antifungal metabolites, such as
turonicin A, hygrobafilomycin, iseolides, cyphomycin, azalomycins,
niphimycins, and resistomycin^[73]23–[74]29. However, most exhibit
non-selectivity and display toxicity to human cells. Thus, it is of
clinical importance to develop effective selective antifungal drug
candidates, with a new mode of action and low cytotoxicity.
Given the significant number of biosynthetic gene clusters (BGCs)
present in Streptomyces, a plethora of their metabolites remains
undiscovered^[75]30. Access to modern analytical chemistry techniques
in combination with genetics and bioinformatics tools has enabled us to
revisit the “talented” microbes discovered decades ago^[76]31.
Streptomyces iranensis, initially identified as a rapamycin producer,
was originally isolated from soil in Isfahan City, Iran^[77]32.
Subsequent investigations revealed its great capability to produce
elaiophylin, azalomycins, nigericin, and other new metabolites yet to
be discovered^[78]33. In our recent research, we discovered that S.
iranensis produced pteridic acids F and H, which could alleviate
abiotic stresses efficiently during plant growth^[79]33.
During our pipeline search for antifungal polyketides, S. iranensis
exhibited strong antifungal activity in co-cultivation with Aspergillus
flavus, Aspergillus niger, Aspergillus fumigatus and Aspergillus
tubingensis (Supplementary Fig. [80]1), suggesting the production of
antifungal metabolites. LC-MS analysis revealed a high production of
the well-known antifungal azalomycin. However, the azalomycin-deficient
mutant of S. iranensis still exhibited strong antifungal activity
against those Aspergillus pathogens (Supplementary Fig. [81]1), which
indicated the production of other potential novel antifungal
metabolites. Genome mining of S. iranensis revealed the presence of
other uncharacterized BGCs including a type-I modular polyketide BGC
(ali) featuring sixteen modules, which could be responsible for
producing new antifungal metabolites.
In this work, we report the discovery, antifungal activity,
biosynthesis and mode of action of a new class of antifungal compound
alligamycin A by genome-mining, metabolomic analysis, large-scale
fermentation and isolation, as well as label-free quantitative
proteomics analysis.
Results and discussion
Genome mining in S. iranensis
To obtain complete genomic data for S. iranensis, we conducted
whole-genome resequencing by integrating the Oxford Nanopore
Technologies MinION and Illumina MiSeq system. The high-resolution,
full-genome sequencing of S. iranensis reveals a linear chromosome
spanning 12,213,033 nucleotides, featuring inverted terminal repeats
comprising 156,145 nucleotides (Supplementary Data [82]1). The BGC
annotation of the acquired S. iranensis genome was carried out using
antiSMASH version 7.0^[83]34, which resulted in the identification of a
cryptic BGC (ali) situated at the terminal region of the linear
chromosome (Fig. [84]1a and Supplementary Fig. [85]2). The gene cluster
family (GCF) annotation of ali biosynthetic gene cluster showed it
belongs to GCF_00315 and a total of 11 hits were detected (distance ≤
900.0) in the BiG-FAM database (Supplementary Table [86]1,
Supplementary Data [87]3)^[88]35. The distinctive architecture of the
biosynthetic genes in the ali BGC suggests its novelty as a modular
Type-I PKS BGC, setting it apart from others (Supplementary
Fig. [89]3). Furthermore, the ali BGC demonstrated its uniqueness when
compared with other BGCs in the NPDC database
([90]https://npdc.rc.ufl.edu) as well as in-house database.
Fig. 1. Organization of the alligamycin biosynthetic gene cluster (ali) and
genome editing in S. iranensis.
[91]Fig. 1
[92]Open in a new tab
a The ali BGC is situated at the end of the linear chromosome, adjacent
to the inverted terminal repeats. Open reading frames are shown as
arrows indicating the size and direction of transcription. b
Confirmation of the ali BGC through knock-out of aliA. Extracted ion
chromatogram (m/z 881.4658 [M + Na]^+) showing the abolishment of
production of alligamycin in the ΔaliA mutant compared to the wild type
strain. c) Inactivation of aliH in S. iranensis via CRISPR base
editing. Extracted ion chromatogram (m/z 881.4658 [M + Na]^+) showing
the abolishment of production of alligamycin in the mutant ΔaliH
compared to the wild strain. The production of alligamycin was resumed
in complementation mutant ΔaliH::aliH.
To identify the potential products of the ali BGC, we combined genome
editing with metabolomics approaches. Initially, we constructed PKS
knock-out mutant (ΔaliA) and crotonyl-CoA
carboxylase/reductase-inactivated mutant (ΔaliH) using the CRISPR-Cas9
method and CRISPR-based editing^[93]36, respectively (Fig. [94]1).
Comparative metabolic profiling using LC-MS and LC-UV revealed compound
1 with m/z 881.4658 ([M + Na]^+), which was absent in both mutants
(Fig. [95]1b, [96]1c). The complementation of pGM1190-based aliH in
mutant S. iranensis/ΔaliH led to the restoration of the production of 1
(Fig. [97]1c).
Structure elucidation of alligamycins
To obtain the products of the ali BGC, a 200 L fermentation was carried
out followed by a downstream processing using different chromatography
techniques, including Amberchrom 161c resin, silica gel, Sephadex
LH-20, and high-performance liquid chromatography (HPLC) to yield pure
alligamycin A (1) and alligamycin B (2). The structures were elucidated
using HR-ESI-MS, NMR spectroscopy, and X-ray crystallography.
Alligamycin A (1) was obtained as a white solid with a molecular
formula of C[47]H[70]O[14] as determined by HR-ESI-MS data (calcd m/z
881.4658 [M + Na]^+, Supplementary Fig. [98]5). The ^1H NMR spectrum
revealed signals for six methyl groups Me-39 (δ 2.15), Me-41 (δ 0.94),
Me-43 (δ 0.98), Me-44 (δ 0.93), Me-45 (δ 2.10), and OMe-8 (δ 3.37).
Additionally, four olefinic protons were observed for H-2 (δ 6.19), H-3
(δ 7.91), H-4 (δ 6.47) and H-28 (δ 6.48). The coupling constant between
H-2 and H-3 ( J = 15.4 Hz) confirmed a trans-orientation of the double
bond. Besides the presence of several oxygen-bearing methines (H-6,
H-8, H-10, H-14, H-16, and H-22), various aliphatic proton signals were
also observed (Supplementary Fig. [99]6).
The ^13C NMR spectrum showed signals for several carbonyl groups, one
carboxylic acid C-1 (δ 168.6), three keto- groups C-12 (δ 207.3), C-27
(δ 201.3) and C-38 (δ 209.6) and two ester groups C-40 (δ 161.8) and
C-46 (δ 174.9), with C-40 from the β-lactone ring. Furthermore, signals
were observed for six olefinic methines C-2 (δ 128.5), C-3 (δ 137.8),
C-4 (δ 129.1), C-5 (δ 143.2), C-28 (δ 123.0), and C-29 (δ 156.4).
Finally, signals for the methyl groups C-39 (δ 29.9), C-41 (δ 8.4),
C-42 (δ 9.2), C-43 (δ 14.3), C-44 (δ 19.5), C-45 (δ 17.4) and the
methoxy group 8-OMe (δ 59.6) were also observed (Supplementary
Fig. [100]7). The chemical shift of C-18 (δ 101.0) indicated the
presence of an acetal moiety.
COSY correlations further established five fragments including one
saturated aliphatic chain. The HMBC correlations (Fig. [101]2) observed
from H-6 and C-40 established a β-lactone moiety. The connectivity of
this β-lactone to a polyene moiety was supported by the HMBC
correlations between H-4 and C-6, as well as H-4 and C-40. A
macrolactone bridge between H-10 and C-46 was established by HMBC
correlation between them. The key HMBC correlation between H-13 and
C-18 indicated the presence of an oxane ring. Moreover, H-16 and Me-43
showed HMBC correlations with C-18. Considering the number of double
bond equivalents and the number of oxygen atoms inferred from
HR-ESI-MS, a spiroketal structure was assigned. Finally, a planar
structure with a novel carbon skeleton was proposed and named
alligamycin A. The ^1H and ^13C NMR data, as well as the HSQC, COSY,
HMBC and NOESY correlations of 1 are shown in Supplementary Data [102]4
and Supplementary Fig. [103]8-[104]11. The complex structure exhibited
no similarities to reported natural products. Meanwhile, the dispersion
of the thirteen chiral centers made it challenging to elucidate the
absolute configuration. Multiple attempts on crystallization were
carried out on compound 1 and slow evaporation from
dichloromethane/methanol solution yielded a single crystal for X-ray
crystallography analysis, which successfully elucidated the
configurations (Supplementary Fig. [105]4, Supplementary Data [106]2 &
[107]7).
Fig. 2. Structure elucidation of alligamycin A and B.
[108]Fig. 2
[109]Open in a new tab
Top: Chemical structures of alligamycin A (1) and B (2).
Bottom: Structure elucidation was carried out by 2D NMR spectroscopy.
Selected COSY, HMBC, and NOESY correlations of 1 were shown and marked
in different colors and arrows.
Alligamycin B (2) was obtained as a derivative of alligamycin A, with
an open β-lactone ring. It was assigned the molecular formula of
C[48]H[74]O[15] by HR-ESI-MS (m/z 889.4966 [M – H]^-, Supplementary
Fig. [110]14). Following 1D/2D NMR spectra, compound 2 exhibited high
similarity to 1. However, the downfield shift of the carbonyl carbon at
C-40 (δ 166.9), and the presence of an additional methoxy signal (δ
3.85) showing HMBC correlation to C-40, indicating 2 consists of an
opened β-lactone ring compared to 1. Analysis of HSQC, COSY, HMBC and
NOESY data of 2 further confirmed the structure of alligamycin B
(Supplementary Fig. [111]15-[112]20, Supplementary Data [113]4).
Proposed biosynthesis of alligamycins
The proposed ali BGC spanned 87.99 kb with 12 open reading frames
(Fig. [114]1), including five core polyketide synthases (AliA, AliC,
AliD, AliE, and AliF), a crotonyl-CoA carboxylase/reductase (AliH), an
O-methyltransferase (AliG), three cytochrome P450s (AliB, AliK and
AliL), and two hypothetical enzymes (AliI and AliJ) (Supplementary
Table [115]2). The core PKS genes are all transcribed in the same
direction as other genes within the ali BGC, which consists of one
loading module and fifteen extender modules. The detailed domains,
ketosynthase (KS) domain, acyltransferase (AT) domain, and acyl carrier
protein (ACP), with additional ketoreductase (KR), dehydratase (DH),
and enoyl reductase (ER) domains and the proposed biosynthesis is shown
in Fig. [116]3. Multiple sequence alignment revealed that most KS
domains contain conserved catalytic sites (Supplementary Fig. [117]21),
consisting of a cysteine (TACSSS motif) and two histidines (EAHGTG and
KSNIGHT motifs)^[118]37. Only the reactive cysteine in KS[1] has been
replaced by glutamine, which was observed in the platensimycin/FabF
(the type II FAS homology of KS) complex^[119]38. The conserved
fingerprint residues for extender unit selectivity, the GHSIG and HAFH
motifs are present in the nine AT domains (AT[1], AT[2], AT[5], AT[7],
AT[8], AT[9], AT[11], AT[12], and AT[14]) that are specific for binding
malonyl-CoA while GHSQG and YASH motifs are present in the six AT
domains (AT[3], AT[4], AT[6], AT[10], AT[13], and AT[15]) indicating
binding of (2S)-methylmalonyl-CoA (Supplementary Table [120]3 and
Fig. [121]22)^[122]37. The AT[16] from module 15, which was thought to
be accountable for loading distinct extender units, exhibits an unusual
IASH motif. KR domains have been previously classified as A1-, A2-,
B1-, and B2-types, which reduce ketones to their L-hydroxy or D-hydroxy
counterparts^[123]39. We determined that the KR[3], KR[4], and KR[15]
appear to belong to the B1-type: all have the fingerprint LDD motif but
with the absence of a P residue in the catalytic region. KR[5] and
KR[12], with the characteristic W residue but lacking the LDD motif and
H residue, were classified as A1-type KRs (Supplementary Fig. [124]23).
The other KR domains couldn’t be classified according to the sequence,
but they are expected to be functional based on retrobiosynthesis of
alligamycin A. Similarly, combined with structural information and
analysis of conserved sites, the five DH domains (DH[6], DH[7], DH[8],
DH[9] and DH[10]) were considered inactive during alligamycin
biosynthesis (Supplementary Fig. [125]24). ER[11] is classified as
L-type due to the presence of a tyrosine residue that donates a proton
to the enol intermediate in L-type ER, whereas ER[13] and ER[14] belong
to D-type due to the lack of tyrosine residue (Supplementary
Fig. [126]25). The final release and cyclization of the linear product
was probably accomplished by thioesterase (TE) domain in the last
module, which contains an α/β-hydrolase catalytic core and loop regions
that form a substrate-binding lid^[127]40.
Fig. 3. Proposed biosynthesis pathway of alligamycin A (1) and B (2).
[128]Fig. 3
[129]Open in a new tab
Proposed biosynthesis pathway of alligamycin A (1) and B (2). Figure
shows the type one module PKS with five core genes responsible for
alligamycin biosynthesis. Abbreviation: KS, ketosynthase; AT,
acyltransferase; DH, dehydratase; ER, enoylreductase; KR,
ketoreductase; ACP, acyl carrier protein; TE, thioesterase.
In the ali BGC, there are three cytochrome P450 enzymes (AliB, AliK,
and AliL) with conserved motifs of this family (Supplementary
Fig. [130]26)^[131]41. The cytochrome P[450] enzyme BonL from
Burkholderia gladioli was previously identified to confer the C-22
carboxyl group in the biosynthesis of bongkrekic acid via sequential
six-electron-oxidation^[132]42. In addition, there are some cytochromes
P[450] enzymes that catalyze the carboxyl group formation in microbial
SMs, such as XiaM, PimG, AmphN, NysN, and FscP^[133]43–[134]45. To
identify the cytochrome P450s responsible for carboxylation, we built
the Hidden Markov model (HMM) based on these sequences and the results
showed that AliL exhibited the closest match to these known P450s with
an E-value of 4e-117. In addition, bioinformatic analysis of XiaM
indicated that the cytochrome P450s with carboxylation function harbor
highly conserved segment AGHET, which was also presented in AliK
(Supplementary Fig. [135]26). Therefore, AliK and AliL, were considered
putative candidates for catalyzing the two-step oxidation to form
carboxyl groups at positions C-1 and C-5 during alligamycin A
biosynthesis. AliB, homologous to cytochrome P450 monooxygenase CftA in
the clifednamide biosynthetic gene cluster with 45.6%
similarity^[136]46, was likely to accomplish the oxidation at C-27 in
alligamycin A. We constructed mutant strains of S. iranensis
inactivating the AliB, AliK, and AliL enzymes (designated as ΔaliB,
ΔaliK, and ΔaliL) (Supplementary Fig. [137]27). Despite our efforts, no
expected intermediates were observed in either mutant, likely due to
their low yield.
Currently, there are only few enzymatic mechanisms corresponding to
β-lactone ring formation that have been reported^[138]47. For example,
(1) the intramolecular cyclization from seven-membered ring, catalyzed
by cyclase VibC in vibralactone biosynthesis^[139]48; (2) the tandem
aldol-lactonization bicyclization reaction to generate the
γ-lactam-β-lactone structure, catalyzed by standalone ketosynthase SalC
in salinosporamide A biosynthesis^[140]49; (3) the β-lactone formation
during the intramolecular attack of the β-hydroxyl group onto the
thioester carbonyl, catalyzed by the C-terminal TE domain of ObiF in
obafluorin biosynthesis or esterase GloD in globilactone A
biosynthesis^[141]50,[142]51; (4) the conversion of β-hydroxyl to
β-lactone, catalyzed by β-lactone synthase OleC in olefin
biosynthesis^[143]52,[144]53. We proposed that the β-lactone formation
in alligamycin A involves a two-step oxidation to form a carboxylic
acid, followed by dehydration. It is likely that either the cytochrome
P450 enzyme AliK or AliL is involved in the process, and we also
identified several genes with unknown functions, such as aliJ and aliI.
AliJ was annotated as a member of UbiD family decarboxylases, but it
exhibits very distinguished divergences from other proteins in this
family based on sequence similarity network (Supplementary
Fig. [145]28). In addition, NCBI Blastp results showed that proteins
homologous to AliI have not been reported to have any clear biological
functions. Inactivation of both genes abolished the production of
alligamycin A (Supplementary Fig. [146]27).
Phylogenetic analysis indicated that AliH is a separate branch and
clusters with other crotonyl-CoA reductase/carboxylases (CCRs) that
catalyze long-chain precursors like butylmalonyl-CoA and
hexylmalonyl-CoA^[147]54,[148]55. The previous study showed that the
large residue Phe380 in the CCR from S. coelicolor may constrain the
potential pocket to accept long-chain substrates, which were also found
in most ethylmalonyl-CoA specific crotonyl-CoA reductase/carboxylases,
while its corresponding residue in AliH is smaller cysteine
(Supplementary Fig. [149]29)^[150]56. Hence, we proposed that AliH
catalyzes the conversion leading to the formation of
7-oxo-octylmalonyl-CoA in alligamycin A, which is likely synthesized
from a precursor that has previously been isolated from other
Streptomyces species^[151]57. AliG, an O-methyltransferase homologous
to AveD in avermectin biosynthesis (40% identity and 55%
similarity)^[152]58, was proposed to catalyze the methylation of a
hydroxyl group in C-8 of alligamycin A.
Antifungal and cytotoxic activities of alligamycins
Microbial β-lactone natural products are chemically diverse and have
been employed in antimicrobial, antiviral, anticancer, and antiobesity
therapeutics^[153]47. In the initial antifungal assay against A. niger
ATCC 1015, alligamycin A exhibited the strongest antifungal effect,
while alligamycin B with an opened β-lactone ring did not show
antifungal activity (MIC > 50 µg/mL). Since both alligamycins contain
an α,β-unsaturated ester, this suggests that the electrophilic nature
of the β-lactone (and not the ability to act as a Michael acceptor) was
responsible for the antifungal activity of alligamycin A. The in vitro
antifungal susceptibility screens of alligamycin A was further
performed against 34 fungal species, represented by 38 clinical
isolates, in comparison to antifungal drugs using a microdilution assay
according to the EUCAST protocol. Satisfyingly, alligamycin A
demonstrated potent antifungal effects against several sections of the
genus Aspergillus and the genus Talaromyces with minimal inhibitory
concentration (MIC) ranging from 0.06 to 8 µg/mL (Table [154]1,
Supplementary Data [155]5). The most potent activity of alligamycin A
compared to standard drugs were found in in the Aspergillus section
Terrei with high MICs for amphotericin B and in the section Usti with
intrinsically high MICs for azoles. In Aspergillus sections Flavi,
Nidulantes, and Nigria, as well as in the section Talaromyces, a
distinctly changed morphology of the hyphae was found for all species
but low MICs were observed only in some species. Only a weak effect was
observed for the two Fusarium species studied. Aspergillus species
belong to the section Fumigati, Candida spp., Scedosporium prolificans,
Purpureocillium lilacinum, Trichoderma longibrachiatum and Mucorales
were unaffected by alligamycin A (Supplementary Data [156]5).
Table 1.
Antifungal activities of alligamycin A (1) in comparison with
amphotericin B, itraconazole and voriconazole M, the values for MIC
(MEC) are in mg/L
Species Section MIC (MEC) in mg/L
amphotericin B itraconazole voriconazole M alligamycin A
A. brasiliensis Nigri 0.5 2 2 0.5 (0.25)
A. luchuensis Nigri 0.25 n.a. 1 0.5 (0.25)
A. citrinoterreus Terrei 4 0.5 0.5 0.5 (0.25)
A. neoafricanus Terrei 2 0.5 0.5 0.5 (0.25)
A. terreus Terrei 8 0.5 1 0.125 (0.125)
A. calidoustus Usti 16 8 4 0.5 (0.25)
A. ustus Usti 1 >8 >8 0.125 (0.06)
T. marneffei 0.06 ≤0.016 ≤0.016 0.125 (0.125)
T. purpureogenes 1 >8 8 0.5 (0.25)
[157]Open in a new tab
MIC minimum inhibition concentration, MEC minimum effective
concentration.
Importantly, alligamycin A did not exhibit cytotoxicity against the
human acute promyelocytic leukemia cell line (HL-60) with
IC[50] > 10 µg/mL highlighting an important advantage over other
anti-mycotics and its potential for drug development.
Mechanism of action of alligamycin A
To obtain initial insights into the mode of action for alligamycin, we
carried out a label-free quantitative proteomics analysis of the model
organism A. niger ATCC 1015. We initially cultured A. niger in PDB for
16 h, followed by 1-h and 4-h treatments with alligamycin A. The
proteomics analysis showed a total of 4447 proteins (40.4% of the total
encoded proteins) and 3992 of these proteins (89.8% of the total
detected proteins) did not show significant differences between the
treatments (1-h or 4-h treatment with alligamycin A) and the control
(without alligamycin A treatment). Compared with the control, the
abundance of 134 proteins significantly increased, while 60 proteins
significantly decreased after 1-h of alligamycin A treatment. In
comparison, the abundance of 273 proteins significantly increased and
113 proteins significantly decreased after 4-h of alligamycin A
treatment (Fig. [158]4). The Cluster of Orthologous Groups (COGs)
functional classification of these differential proteins was performed
by eggNOG-mapper v2^[159]59. Except for (S) function unknown, these
proteins were mainly clustered into a few biological processes
including (Q) Secondary structure, (C) Energy production and
conversion, (E) Amino acid metabolism and transport, and (O)
Post-translational modifications, protein turnover, chaperone functions
(Supplementary Fig. [160]30). The Gene Ontology (GO) annotation of
these proteins was mainly involved in the metabolic process, cellular
process, and single-organism process during the biological process
(Supplementary Fig. [161]31). Cellular component analysis showed that
these proteins were mainly localized intracellularly, especially in
organelles. Meanwhile, most of these proteins participated in catalytic
activity and binding in the category of molecular function. The Kyoto
Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis
showed that these proteins mainly participate in amino acids metabolism
and purine metabolism (Supplementary Fig. [162]32).
Fig. 4. Volcano plot of the protein abundance of A. niger after 1-h and 4-h
alligamycin A treatments versus control based on proteomics analysis.
[163]Fig. 4
[164]Open in a new tab
Two-sided Wald tests based on negative binomial generalized linear
models were used and the Benjamini-Hochberg method was employed for
multiple comparison adjustment. Source data are provided as a
[165]Source Data file.
Proteomic changes in fungi induced by known antifungal agents with
well-described targets were subsequently studied. The abundance of
Erg13 ([166]EHA22977.1, involved in ergosterol biosynthesis) and
manganese-superoxide dismutase ([167]EHA27012.1, involved in oxidative
stress response), which were regarded as the polyene antibiotic (such
as amphotericin B) responsive proteins^[168]60, was not significantly
altered in the presence of alligamycin A. Allylamine antibiotics
(naftifine and terbinafine) inhibit squalene epoxidase, causing the
accumulation of squalene, and thereby damaging the intracellular
membranes of fungi^[169]61. Alligamycin A does not belong to the
allylamine family and we did not observe any changes in the squalene
epoxidase ([170]EHA21184.1). Azoles are synthetic antifungal agents
derived from non-natural origin that inhibit cytochrome P450
sterol-14α-demethylase (CYP51) to block the ergosterol biosynthesis in
fungi^[171]62. Similarly, alligamycin A lacked the azoles substructure
and we did not observe impact on the abundance of CYP51
([172]EHA19435.1). Natural echinocandins comprise cyclic hexapeptide
and lipid residues, like caspofungin, anidulafungin, and micafungin,
preventing biosynthesis of glucans of the fungal cell wall through
non-competitive inhibition of 1,3-β-D-glucan synthase^[173]63. Previous
proteomics analyses have proven that the level of chitinase ChiA1 in A.
fumigatus was significantly decreased in response to
caspofungin^[174]64. It may represent a common effect of
self-resistance development due to caspofungin increased chitin content
via induction of chitin synthases during cell wall remodeling^[175]65.
We did not observe significant changes of the abundance of
1,3-β-D-glucan synthase ([176]EHA18547.1) and ChiA1-like chitinase
([177]EHA28582.1) after 1-h and 4-h treatment of alligamycin A in
comparison to the control group (Supplementary Fig. [178]33).
The fungal cell wall constitutes a critical structural component that
maintains cell shape, protects fungi against environmental stress, and
plays roles in growth, invading ecological niches invasion, and
counteracting the host immune response^[179]66. Fungal cell walls are
composed mainly of glucans, chitin, and glycoproteins, synthesized by
glycosyltransferases, glycoside hydrolases, and
transglycosylases^[180]67. Congo Red Hypersensitivity (CRH) family
transglycosylases are usually highly expressed in multiple stages
during the conidial germination of fungi and may be involved in cell
wall synthesis and stability. The volcano plot depicting differential
proteins indicated that a CRH family transglycosylase ([181]EHA23738.1)
was significantly inhibited, suggesting alligamycin A might interfere
with the formation of fungal cell wall. Trehalose-6-phosphate
synthase/phosphatase is another important enzyme for cell wall
integrity and fungal virulence in various Aspergillus species^[182]68.
The relative abundance of trehalose 6-phosphate synthase
([183]EHA27700.1) was also significantly decreased after alligamycin A
treatment. The above data indicated that the mode of action of
alligamycin A could be through disruption of fungal cell wall
biosynthesis and stability (Fig. [184]4). Besides, we found that the
two enzymes Erg24 ([185]EHA26587.1) and Erg27 ([186]EHA20711.1), which
catalyze the biosynthesis of 4,4-dimethylcholesta-8,14,24-trienol to
fecosterol^[187]69, were remarkably upregulated after alligamycin A
treatment (Fig. [188]4). Drug efflux and resistance in fungi could be
mediated by ATP-binding cassette (ABC) transporters, such as CDR1 was
induced to high expression in A. fumigatus AF293 in the presence of
azole antibiotics^[189]70. The expression of an ABC transporter
([190]EHA25684.1) was activated after alligamycin A treatment. We noted
significant alterations in the abundance of several putative
alligamycin A-responsive proteins. For example, the relative abundance
of several proteins including inositol hexaphosphate kinase KCS1
([191]EHA27881.1), ubiquitin-protein ligase ASI3 ([192]EHA19328.1),
mitochondrial phosphate carrier protein PIC2 ([193]EHA22842.1),
pyridoxamine 5’-phosphate oxidase ([194]EHA19800.1) and glucohydrolase
([195]EHA21384.1) was significantly increased, but oxygen-dependent
FAD-linked oxidoreductase ([196]EHA22577.1) and dienelactone hydrolase
([197]EHA27638.1) was significantly decreased in alligamycin A-treated
samples. Intriguingly, a number of proteins of unknown function
displayed variations in abundance attributable to alligamycin A and
their specific roles warrant further investigation.
These data indicated that treatment with alligamycin A results in the
inhibition of transglycosylase and trehalose-6-phosphate
synthase/phosphatase, higher expression of ABC transporters, suggesting
its potential role in targeting intracellular fungal cell wall
biosynthesis. The exact intracellular targets will be further
investigated through chemical proteomics or genetic approaches.
Methods
All the protocols in this research comply with all relevant ethical
regulations in accordance with guidelines by the Technical University
of Denmark and Leibniz Institute for Natural Product Research and
Infection Biology.
Strains, plasmids, and culture conditions
All strains and plasmids used in this study were summarized in
Supplementary Table [198]4. All primers used in this study were
summarized in Supplementary Data [199]6. All constructed E. coli
strains were grown in lysogeny broth (LB) liquid or on agar medium at
37 °C. Wild-type Streptomyces strains and its mutants were cultivated
on Mannitol Soya Flour (MS) agar medium (20.0 g mannitol, 20.0 g soya
flour, 20.0 g agar, 1.0 L tap water, pH=7.0-7.5) at 30 °C. The
small-scale fermentation of Streptomyces strains was performed in
250 mL flask using 50 mL MS liquid medium, shaking at 200 rpm, 30 °C
for 7 days. The large-scale fermentation of Streptomyces strains were
carried out in medium 2 (CaCl[2]·2H[2]O, 3.0 g; citric acid/Fe III,
1.0 g; MnSO[4]·H[2]O, 0.2 g; ZnCl[2], 0.1 g; CuSO[4]·5H[2]O, 0.025 g;
Na[2]B[4]O[7]·10H[2]O, 0.02 g; Na[2]MoO[4]·2H[2]O, 0.01 g; and oatmeal,
20.0 g, in 1.0 L distilled water), at 200 L scale in a 300 L
fermentation vessel, for 6 days with aeration of 25–50 L min^−1,
stirring at 200 rpm, 28 °C, with a pH range of 5.4–6.4. Antibiotics
such as apramycin (50 mg mL^−1), kanamycin (50 mg mL^−1) or
chloramphenicol (25 mg mL^−1) were appropriately used for resistance
selection.
Genomic DNA extraction, sequencing, and assembly
The S. iranensis culture was grown in 50 mL sterilized liquid ISP2
medium (yeast extract 4.0 g; malt extract 10.0 g; and dextrose 4.0 g in
1.0 L distilled water, pH = 7.2) in 250 mL flask at 30 °C and 160 rpm
for 5 days to generate sufficient biomass. The genomic DNA of S.
iranensis was isolated using the QIAGEN Genomic-tip 20/G kit with a
modified protocol and the cell pellet was ground using a mortar and
pestle submerged in liquid nitrogen^[200]71. The genomic DNA sequencing
was performed combining Oxford Nanopore Technologies MinION and
Illumina MiSeq systems. To generate the nanopore data, both the
SQK-RBK004 and the SKQ-RBK110-96 rapid barcoding kits were used for
construction of three libraries, which were sequenced on two separate
9.4.1 flow cells. The de novo assembly was performed using Flye
(v2.9-b1768) with the parameters --nano-hq --t 12. Since the genome
could not be fully resolved using existing assemblers, the terminal
inverted repeat edge from the unambiguous assembly repeat graph was
manually attached to both ends of the chromosome edge and the
orientations were verified by mapping of the reads on the new assembly
and manual inspection. This approach was later suggested by the Flye
assembler Misha. The Unicycler (v0.4.8) polishing module was used to
polish the nanopore assembly with Illumina data.
Genetic manipulation
To verify the BGC of alligamycin and its individual biosynthetic genes,
the classical CRISPR-Cas9 method, and advanced CRISPR-cBEST base
editing toolkit were used to construct gene-inactive mutants^[201]36.
The function-specific plasmids (Supplementary Table [202]4) were
constructed according to the respective protocol followed by
introducing into wild-type S. iranensis HM 35 by conjugation with donor
strain ET12567/pUZ8002 on SFM solid medium (soya flour 20.0 g; mannitol
20.0 g; and bacteria agar 20.0 g, in 1.0 L distilled water, pH = 7.2)
according to modified protocol. The exconjugants after resistance
screening (50 μg mL^−1 apramycin and 25 μg mL^−1 nalidixic acid) were
further verified by DNA extraction, PCR reaction, and Sanger
sequencing.
DNA polymerases (Q5® High-Fidelity 2X Master Mix with Standard Buffer
and OneTaq® 2X Master Mix with Standard Buffer) and restriction enzymes
(NcoI, EcoRI) were purchased from New England Biolabs. PCR
amplification and restriction enzyme digestions were carried out on
Bio-Rad’s thermal cyclers according to the manufacturer’s instructions.
Plasmid DNA extraction was performed using NucleoSpin Plasmid EasyPure
Kit (Macherey-Nagel, Germany). DNA purification was conducted on 1%
Tris-acetate-EDTA (TAE) agarose gel followed by using NucleoSpin Gel
and PCR Clean-up Kits (Macherey-Nagel, Germany). One Shot™ Mach1™ T1
Phage-Resistant Chemically Competent E. coli from Invitrogen™ was used
for transformation. All oligonucleotides were ordered from Integrated
DNA Technologies (Supplementary Data [203]6) and Sanger sequencing was
offered by Eurofins Genomics (Luxembourg).
UHPLC-HRESIMS sample preparation and analysis
An agar plug (6 mm diameter) of the bacterial culture was transferred
to a vial (Eppendorf) and extracted with 1 mL of isopropanol: ethyl
acetate 1:3 (v/v) with 1% formic acid under ultrasonication for 15 min.
The extracts were then transferred to new Eppendorf vials, evaporated
to dryness under N[2], and re-dissolved in methanol to meet the final
concentration of 10 mg/mL. After centrifugation at 10,000 rpm for
3 min, the supernatants were transferred to HPLC vials, diluted to
1 mg/mL with methanol and subjected to ultrahigh-performance liquid
chromatography-high resolution electrospray ionization mass
spectrometry (UHPLC-HRESIMS) analysis. Other samples during isolation
and purification process were all prepared with methanal and diluted to
1 mg/mL for UHPLC-HRESIMS analysis. HR-ESI-MS data were acquired on an
Agilent Infinity 1290 UHPLC system equipped with a diode array detector
and coupled to an Agilent 6545 QTOF MS equipped with Agilent Dual Jet
Stream ESI. Separation was achieved on a 250 × 2.1 mm i.d., 2.7 μm,
Poroshell 120 Phenyl Hexyl column (Agilent Technologies) held at 40 °C.
The sample, 1 μL, was eluted at a flow rate of 0.35 mL min^−1 using a
linear gradient from 10% acetonitrile/water buffered with 20 mM formic
acid to 100% acetonitrile in 15 min, held for 2 min and equilibrated
back to 10% acetonitrile/water in 0.1 min. Starting conditions were
held for 3 min before the following run. The MS settings were as
follows: drying gas temperature of 160 °C, a gas flow of 13 L min^−1,
the sheath gas temperature of 300 °C and flow of 16 L min^−1. Capillary
voltage was set to 4000 V and nozzle voltage to 500 V in positive mode.
All data were processed using Agilent MassHunter Qualitative Analysis
software (Agilent Technologies, USA). All solvents used for
chromatography and HR-MS were purchased from VWR Chemicals with LC-MS
grade, while for metabolites extraction, the solvents were of HPLC
grade.
Fermentation and isolation
The culture broth from large-scale fermentation (described above) was
filtered and loaded onto an Amberchrom 161c resin LC column
(200 × 20 cm, 6 L). Elution with a linear gradient of H[2]O-MeOH (from
30% to 100% v/v, flow rate 0.5 L min^−1, in 58 min) afforded seven
fractions (Fr.A-Fr.G). Fr.G was first fractionated by silica gel
chromatography with a CH[2]Cl[2]-CH[3]OH solvent system to yield 16
fractions, Fr.1-Fr.16. Fr.7 was further separated by a Sephadex LH-20
(MeOH) column, and twelve sub-fractions were obtained. The
sub-fractions were separated by semipreparative HPLC RP-C[18] using
MeCN-H[2]O as a solvent system to afford compounds 1 and 2.
Alligamycin A (1): white solid;
[MATH:
[α]
D25 :MATH]
15 (0.1 mg/mL, CH[3]OH); ^1H NMR (800 MHz, CDCl[3]): 7.91 (dd, 15.4 Hz,
11.8 Hz, 1H), 6.48 (s, 1H), 6.47 (d, 10.7 Hz, 1H), δ[H] 6.19 (d,
15.4 Hz, 1H), 5.15 (dq, 3.4 Hz, 2.1 Hz, 1H), 4.88 (d, 8.9 Hz, 1H), 4.15
(overlapping, 1H), 4.14 (overlapping, 1H), 4.12 (overlapping, 1H), 3.37
(overlapping, 1H), 3.37 (s, 3H), 3.23 (dt, 11,5 Hz, 9.6 Hz, 1H), 3.03
(m, 1H), 2.83 (dd, 8.8 Hz, 2.4 Hz, 1H), 2.81 (dd, 12.4 Hz, 2.9 Hz, 1H),
2.61 (dd, 16.3 Hz, 10.7 Hz, 1H), 2.50 (dd, 12.4 Hz, 10.1 Hz, 1H), 2.46
(t,7.4 Hz, 2H), 2.24 (dd, 16.3 Hz, 7.8 Hz, 1H), 2.15 (overlapping, 1H),
2.15 (s, 3H), 2.10 (overlapping, 2H), 2.10 (s, 3H), 1.97 (d, 14.1 Hz,
1H), 1.93 (overlapping, 1H), 1.93 (overlapping, 1H), 1.93 (overlapping,
1H), 1.81 (m, 1H), 1.66 (m, 1H), 1.58 (m, 1H), 1.58 (m, 2H), 1.51 (d,
4.7 Hz, 1H), 1.48 (overlapping, 1H), 1.46 (d, 4.10 Hz, 1H), 1.51
(overlapping, 1H), 1.45 (dd, 14.2 Hz, 3.5 Hz, 1H), 1.40 (overlapping,
1H), 1.38 (overlapping, 2H), 1.34 (overlapping, 1H), 1.33 (overlapping,
2H), 1.28 (overlapping, 1H), 1.26 (overlapping, 1H), 1.14 (m, 2H), 0.98
(d, 7.2 Hz, 3H), 0.95 (d, 6.8 Hz, 3H), 0.94 (d, 7.2 Hz, 3H), 0.93 (d,
6.8 Hz, 3H); ^13C NMR (200 MHz, CDCl[3]): δ[C] 209.6, 207.3, 201.3,
174.9, 168.6, 161.8, 156.4, 143.2, 137.8, 129.1, 128.5, 123.0, 101.0,
79.4, 76.5, 75.8, 72.0, 71.2, 65.0, 60.7, 59.6, 51.8, 48.4, 47.8, 47.2,
43.6, 41.5, 38.2, 37.0, 34.8, 33.9, 33.6, 32.0, 30.7, 30.0, 29.9, 29.2,
28.8, 27.4, 25.4, 25.4, 23.6, 19.5, 17.4, 14.3, 9.2, 8.4; UV/vis
(CH[3]CN/H[2]O) λ[max] 230, 270 nm; ECD λ[ext] (Δε) (CH[3]OH) 235
(–9.78), 267 (7.02), 301 (–3.31) nm; IR (ATR) v[max] 2932, 2748, 2704,
1810, 1713, 1686, 1619, 1457, 1382, 1173, 1134, 1085, 1057, 1013, 991,
947 cm^−1; ( + )-HR-ESI-MS m/z 881.4659 [M + Na]^+ (calcd for
C[47]H[70]O[14], 881.4658). Table of ^1H NMR and ^13C NMR data of 1 see
Supplementary Data [204]4. ECD spectrum of 1 see Supplementary
Fig. [205]12. IR spectrum of 1 see Supplementary Fig. [206]13.
Alligamycin B (2): white solid;
[MATH:
[α]
D25 :MATH]
18 (0.2 mg/mL, CH[3]OH); ^1H NMR (800 MHz, CDCl[3]): 7.94 (dd, 15.7 Hz,
11.7 Hz, 1H), 6.74 (d, 11.6 Hz, 1H), 6.52 (s, 1H), δ[H] 6.11 (d,
15.7 Hz, 1H), 5.14 (dq, 3.4 Hz, 2.1 Hz, 1H), 4.35 (d, 8.4 Hz, 1H), 4.14
(overlapping, 1H), 4.14 (overlapping, 1H), 4.13 (overlapping, 1H), 3.85
(s, 3H), 3.23 (dt, 11,5 Hz, 9.6 Hz, 1H), 3.45 (m, 1H), 3.36 (s, 3H),
2.97 (m, 1H), 2.83 (dd, 8.8 Hz, 2.4 Hz, 1H), 2.73 (dd, 13.2 Hz, 3.9 Hz,
1H), 2.61 (dd, 16.0 Hz, 10.5 Hz, 1H), 2.53 (dd, 13.2 Hz, 9.2 Hz, 1H),
2.46 (t,7.4 Hz, 2H), 2.24 (dd, 16.3 Hz, 7.8 Hz, 1H), 2.15 (overlapping,
1H), 2.13 (s, 3H), 2.10 (overlapping, 2H), 2.07 (s, 3H), 1.98
(overlapping, 1H), 1.97 (d, 14.1 Hz, 1H), 1.93 (overlapping, 1H), 1.93
(overlapping, 1H), 1.81 (m, 1H), 1.58 (m, 2H), 1.58 (m, 1H), 1.58 (m,
2H), 1.51 (overlapping, 1H), 1.48 (overlapping, 1H), 1.45 (dd, 14.2 Hz,
3.5 Hz, 1H), 1.66 (m, 1H), 1.40 (overlapping, 1H), 1.38 (overlapping,
2H), 1.34 (overlapping, 1H), 1.33 (overlapping, 2H), 1.26 (overlapping,
1H), 1.25 (overlapping, 1H), 1.15 (m, 2H), 0.96 (d, 7.2 Hz, 3H), 0.92
(d, 6.8 Hz, 3H, 0.87 (d, 7.2 Hz, 3H), 0.77 (d, 7.2 Hz, 3H); ^13C NMR
(200 MHz, CDCl[3]): δ[C] 209.6, 207.3, 201.6, 175.0, 168.3, 166.9,
156.4, 144.2, 141.0, 133.6, 125.9, 123.2, 101.0, 79.6, 75.8, 75.5,
72.0, 72.0, 64.9, 60.7, 58.7, 52.1, 51.8, 48.7, 47.9, 47.2, 43.6, 40.7,
33.6, 37.1, 37.0, 34.8, 34.0, 33.6, 30.7, 30.0, 30.0, 29.2, 28.8, 27.4,
25.4, 25.4, 23.6, 19.5, 17.3, 14.3, 11.6, 9.6; UV/vis (CH[3]CN/H[2]O)
λ[max] 230, 270 nm; IR (ATR) v[max] 2990, 2942, 2919, 2831, 2035, 1449,
1416, 1119, 1022 cm^−1; (-)-HR-ESI-MS m/z 889.4971 [M - H]^- (calcd for
C[48]H[74]O[15], 889.4955). Table of ^1H NMR and ^13C NMR data see
Supplementary Data [207]4.
NMR spectroscopy
NMR spectra were recorded on 800 MHz Bruker Avance III spectrometer
equipped with a TCI CryoProbe using standard pulse sequences.
Measurements were carried out using ^1H and ^13C NMR, ^1H−^13C
heteronuclear single quantum coherence (HSQC), ^1H−^13C heteronuclear
multiple bond correlation (HMBC), ^1H-^1H correlation spectroscopy
(COSY), ^1H-^1H nuclear overhauser effect spectroscopy (NOESY).
Chemical shifts (δ) were reported in parts per million (ppm) and the
^1H and ^13C NMR chemical shifts were referenced to the residual
solvent peaks at δ[H] 7.26 and δ[C] 77.16 ppm for CDCl[3]. Data are
described as follows: chemical shift, multiplicity (br = broad, s =
singlet, d = doublet, t = triplet, dd = doublet of doublet,
m = multiplet and ov = overlapped) and coupling constants (in Hertz).
All NMR data were processed using MestReNova 14.0.
Crystal data for alligamycin A
The suitable crystal was selected and mounted in a nylon loop directly
from the ethanol suspension and frozen in liquid nitrogen on a
Synchrotron diffractometer. X-ray data collection of 1 was performed on
an Agilent Supernova Diffractometer using CuKα radiation, and the
crystal was kept at 100 K during data collection. Using Olex2^[208]72,
the structure was solved with the XT^[209]73 structure solution program
using Intrinsic Phasing and refined with the SHELXL^[210]74 refinement
package using Least Squares minimization. Crystal data for
C[47]H[70]O[14] (M = 859.03 g/mol): monoclinic, space group P2[1] (no.
4), a = 10.118(7) Å, b = 16.799(7) Å, c = 14.081(5) Å,
β = 103.385(12)°, V = 2328(2) Å^3, Z = 2, T = 100 K, μ (synchrotron) =
0.089 mm^−1, Dcalc = 1.225 g/cm^3, 17,409 reflections measured
(3.836° ≤ 2θ ≤ 45.2°), 5762 unique (R[int] = 0.0646, R[sigma] = 0.0641)
which were used in all calculations. The final R[1] was 0.0646
(I > 2σ(I)) and wR[2] was 0.1523 (all data).
Sample preparation for proteomics analysis
Approximately 1.0 × 10^7 conidia mL^−1 of A. niger ATCC 1015 strain
were used to inoculate in 500 mL flasks containing 100 mL of liquid
cultures (PDB) (three biological replicates) and were incubated in a
reciprocal shaker at 28 °C in 180 rpm for 16 h. Afterwards, samples in
the treatment group were treated with 0.2 μg (final concentration of
2 ng/mL) of alligamycin A for 1 h and 4 hs. The mycelia were then
harvested by filtering, washed thoroughly with sterile water and
quickly frozen in liquid nitrogen. Pellets were lysed in 100 µL lysis
buffer (6 M guanidium hydrochloride, 10 mM TCEP, 40 mM CAA, 50 mM
HEPES, pH 8.5) by boiling the samples at 95 °C for 5 min, followed by
sonicating on high for 5 × 60 s on/30 s off using the Bioruptor Pico
sonication water bath (Diagenode) and lastly disrupting twice with the
TissueLyser (QIAGEN) going from 3 to 30 Hz in 1 min. Lysed samples were
centrifuged at 18,000 g for 10 min, and supernatants were transferred
to clean LoBind Eppendorf tubes. Protein concentration was determined
by BCA rapid gold (Thermo) and 10 µg of protein was taken forward for
digestion. Samples were diluted 1:3 with digestion buffer (10%
acetonitrile in 50 mM HEPES pH 8.5) and incubated with 1:100 enzyme to
protein ratio of LysC (MS Grade, Wako) at 37 °C for 4 h. Samples were
further diluted to a final 1:10 with more digestion buffer and digested
with 1:100 trypsin for 18 h at 37 °C. After digestion, samples were
acidified with TFA and desalted using the SOLAµ^TM SPE plate (HRP,
Thermo)^[211]75. Between each application, the solvents were spun
through by centrifugation at 350 g. For each sample, the filters were
activated with 200 µL of 100% methanol, then 200 µL of 80%
acetonitrile, 0.1% formic acid. The filters were subsequently
equilibrated 2× with 200 µL of 1% TFA, 3% acetonitrile, after which the
sample was loaded. After washing the tips twice with 200 µL of 0.1%
formic acid, the peptides were eluted into clean 0.5 mL Eppendorf tubes
using 40% acetonitrile, 0.1% formic acid. The eluted peptides were
concentrated in an Eppendorf Speedvac. Samples were reconstituted in
12 µL A* buffer with iRT peptides (Biognosys).
MS analysis for proteomics analysis
Peptides were loaded onto a 2 cm C[18] trap column (ThermoFisher
164946), connected in-line to a 15 cm C[18] reverse-phase analytical
column (Thermo EasySpray ES904) using 100% Buffer A (0.1% formic acid
in water) at 750 bar, using the Thermo EasyLC 1200 HPLC system, and the
column oven operating at 30 °C. Peptides were eluted over a 70 min
gradient ranging from 10% to 60% of Buffer B (80% acetonitrile, 0.1%
formic acid) at 250 µL/min, and the Orbitrap Exploris instrument
(ThermoFisher Scientific) was run in DIA mode with FAIMS ProTM
Interface (ThermoFisher Scientific) with CV of −45 V. Full MS spectra
were collected at a resolution of 120,000, with an AGC target of 300%
or maximum injection time set to ‘auto’ and a scan range of
400–1000 m/z. The MS2 spectra were obtained in DIA mode in the orbitrap
operating at a resolution of 60.000, with an AGC target 1000% or
maximum injection time set to ‘auto’, a normalized HCD collision energy
of 32. The isolation window was set to 6 m/z with a 1 m/z overlap and
window placement on. Each DIA experiment covered a range of 200 m/z
resulting in three DIA experiments (400–600 m/z, 600–800 m/z and
800–1000 m/z). Between the DIA experiments, a full MS scan is
performed. MS performance was verified for consistency by running
complex cell lysate quality control standards, and chromatography was
monitored to check for reproducibility.
Data process for proteomics analysis
The raw files were analyzed using Spectronaut^TM (version 17.4) spectra
were matched against the reviewed A. niger ATCC 1015 in NCBI database.
Trypsin was defined as the database with maximum two missed cleavages.
The minimum peptide length was set to seven amino acids. Dynamic
modifications were set as Oxidation (M) and Acetyl on protein
N-termini. Cysteine carbamidomethyl was set as a static modification.
All results were filtered to a 1% FDR, and protein quantitation was
done on the MS1 level. Only proteotypic peptides were used for
quantification and protein groups were inferred by IDPicker.
Antifungal susceptibility testing
In vitro antifungal susceptibility of alligamycin A against 34 fungal
species, represented by 38 clinical isolates, was tested in comparison
to approved antifungal drugs amphotericin B (AMB; European
Pharmacopoeia, Strasbourg, France); itraconazole (ITZ), and
voriconazole (VCZ; Pfizer Inc., Peapack, NJ, USA) using broth
microdilution technique following the European Committee on
Antimicrobial Susceptibility Testing (EUCAST) standard methodology for
yeasts or filamentous fungi respectively. In contrast to the EUCAST
protocol, microdilution plates were prepared by twofold serial
dilutions of the antifungal agents. Filamentous fungi were grown on
malt extract agar (MEA) for 2–7 days at 35 °C and yeasts were
cultivated on yeast extract peptone dextrose agar (YPD) for 24 h. Spore
or yeast cell suspensions were counted with a hemocytometer. Minimum
inhibitory concentrations (MIC) endpoints of filamentous fungi were
defined as 100% reduction in growth and were determined visually using
a mirror after 48 h of incubation at 35 °C. Microdilution plates of
yeasts were read with a microdilution plate reader (Infinite® M Nano
plus, Tecan) and MIC endpoints were defined as the lowest drug
concentration giving inhibition of growth of ≥50% of that of the
drug-free control. Since the mode of action of alligamycin A is
unknown, the minimum effective concentration (MEC) was determined as
well by reading the microplates with the aid of an inverted microscope
(Eclipse Ts2, Nikon). A. fumigatus ATCC 204305 and Candida parapsilosis
ATCC 22019 were used as reference strains^[212]76,[213]77. Utilizing
clinical isolates does not require ethical approval according to the
law of the Free State of Thuringia (ThürKHG §27a).
Cytotoxic activities of alligamycins
HL-60 cells (ATCC) were provided by the Olsen lab at the Department of
Drug Design and Pharmacology, University of Copenhagen. The cell line
was tested negative for mycoplasma contamination. Cytotoxicity against
the human cell line HL-60 was evaluated using Alamar Blue (Thermos
Scientific, Kansas, USA). The assay^[214]78 was performed in 96 well
plates (Costar 3595, Corning, New York, USA), with an assay volume of
200 µL. The software Prism 5.03 was used for data analysis (GraphPad
Software, USA)^[215]79.
Reporting summary
Further information on research design is available in the [216]Nature
Portfolio Reporting Summary linked to this article.
Supplementary information
[217]Supplementary information^ (7.4MB, pdf)
[218]41467_2024_53695_MOESM2_ESM.pdf^ (259.7KB, pdf)
Description of Additional Supplementary Files
[219]Supplementary data 1^ (3.8MB, xlsx)
[220]Supplementary data 2^ (69.9KB, xlsx)
[221]Supplementary data 3^ (12.6KB, xlsx)
[222]Supplementary data 4^ (14.5KB, xlsx)
[223]Supplementary data 5^ (13.5KB, xlsx)
[224]Supplementary data 6^ (11.4KB, xlsx)
[225]Supplementary data 7^ (167.4KB, pdf)
[226]Supplementary Data 8^ (208.3KB, pdf)
[227]Reporting summary^ (192.6KB, pdf)
[228]Peer Review file^ (1.1MB, pdf)
Source data
[229]Source data^ (1.3MB, xlsx)
Acknowledgements