Coculture of a Pathogenic Actinomycete and Animal Cells to Produce Nocarjamide, a Cyclic Nonapeptide with Wnt Signal-Activating Effect

Article abstract of DOI:10.1021/acs.orglett.8b02522

A coculture method with a pathogenic actinomycete of the genus Nocardia and an animal cell line was designed to reconstruct and emulate the initial infection state, and a new cyclic nonapeptide, named nocarjamide (1), was obtained by coculture of Nocardia tenerifensis IFM 10554^T and the mouse macrophage-like cell line J774.1 in a modified Czapek-Dox medium. Nocarjamide (1) exhibited Wnt signal-activating effects.

Full text of DOI:10.1021/acs.orglett.8b02522

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Coculture of a Pathogenic Actinomycete and Animal Cells To
Produce Nocarjamide, a Cyclic Nonapeptide with Wnt Signal-
Activating Effect
Yasumasa Hara,† Midori A. Arai,† Kazufumi Toume,‡ Hyuma Masu,§ Tomoyuki Sato,†
Katsuko Komatsu,‡ Takashi Yaguchi,∥ and Masami Ishibashi*,†
†Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan
‡Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
§Center for Analytical Instrumentation, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan
∥Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan
S
* Supporting Information
ABSTRACT: A coculture method with a pathogenic actinomycete of the genus Nocardia and an animal cell line was designed
to reconstruct and emulate the initial infection state, and a new cyclic nonapeptide, named nocarjamide (1), was obtained by
coculture of Nocardia tenerifensis IFM 10554T and the mouse macrophage-like cell line J774.1 in a modified Czapek−Dox
medium. Nocarjamide (1) exhibited Wnt signal-activating effects.
ctinomycetes of the genus Nocardia are Gram-positive
search for novel, biologically active metabolites. Coculture in a
Abacteria that are widely present in natural environments liquid medium has been performed using various combinations
of microorganisms such as bacteria−fungi, bacteria−bacteria,
archaea−fungi, and fungi−fungi.4 Recently, we described a new
method for producing secondary metabolites by “coculture”
using pathogenic bacteria in combination with animal cells to
reconstruct and emulate the initial infection state.5 An
actinomycete, Nocardia tenerifensis IFM 10554T, was used as
the pathogenic bacteria, while a mouse macrophage-like cell
line, J774.1, was used as the animal cell. In the present study,
nocarjamide (1), a new cyclic nonapeptide with a Wnt signal-
activating effect, was obtained in a modified Czapek−Dox
(mCD)6 medium by performing this new coculture method
using N. tenerifensis IFM 10554T and J774.1 cells.
The bacterial strain for the coculture study was initially
selected from among 76 strains, all belonging to the genus
Nocardia, that were obtained from the Medical Mycology
Research Center at Chiba University. A phylogenetic tree was
prepared using members of the group that harbored the
such as soil and water. Many of these species possess
pathogenicity and infect the human lungs, skin, brain, and
other organs, causing nocardiosis. The study of the genus
Nocardia began with the isolation of the first Nocardia
actinomycete, Nocardia farcinica, by French veterinarian
Edmond Nocard in 1888.1 Although there are some reports
on secondary metabolites of Nocardia spp., such as nacardicin
A,2 nargenicins,3 and others, research on members of the genus
Nocardia has not progressed as rapidly as that on members of
the genus Streptomyces.
Microbial coculture is a method of culturing two or more
kinds of microorganisms in the same environment, thereby
potentially activating the biosynthetic genes responsible for the
production of secondary metabolites.4 This microbial coculture
is inspired by naturally occurring microbial communities,
where microbial interactions through secondary metabolites
are related to chemical defense and various other phenomena.
Coculture can be conducted in a solid or liquid medium and
has been widely used since the 1990s for research on the
interaction between microorganisms in nature and in the
Received: August 7, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b02522
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nocobactin-related biosynthetic gene cluster7 as an index,
yielding classification of the strains into six clades. Based on
these clades and the number of biosynthetic gene clusters, six
strains (N. altamirensis IFM 10819T, N. mexicana IFM 10801T,
N. tenerifensis IFM 10554T, N. terpenica IFM 0706T, N.
otitidiscaviarum IFM 0239T, and N. vulneris NBRC 108936T)
were selected for further study. A mouse macrophage-like cell
line, J774.1, was selected for use as the animal cell line for
coculturing in an effort to reconstruct the initial infection state.
Based on LC−UV analysis of the culture broth extract
obtained under various conditions, we focused on the extract
of Nocardia tenerifensis IFM 10554T cultured in the presence of
J774.1; this coculture extract yielded several specific peaks
(Figure 1). Thus, this bacterial strain was selected for further
study.
and J774.1, while compound 1 was not detected under single
culture conditions (i.e., with N. tenerifensis IFM 10554T or
J774.1 only) (Figure 1).
Nocarjamide (1) was revealed to have the molecular formula
C60H93N9O11 by high-resolution ITTOFMS (obsd m/z
1138.6897 [M + Na]+, calcd for C60H93N9O11Na, 1138.6892).
1
The H NMR spectrum of nocarjamide (1) measured in
DMSO-d6 (Figure S5, Supporting Information (SI)) showed
six NH [δH 8.80 (1H), 8.55 (1H), 8.42 (1H), 7.82 (1H), 7.58
(1H), 7.32 (1H)] and three NCH3 [δH 3.09 (3H), 2.69 (3H),
2.57 (3H)]. The cross-peaks observed in the 2D TOCSY
(Figure S8−S10, SI) and HMBC (Figure S12, SI) data of 1
suggested the presence of one alanine (Ala), one leucine
(Leu), one phenylalanine (Phe), one threonine (Thr), two
valines (Val1 and Val2), one N-methyl leucine (MeLeu), one
N-methyl phenylalanine (MePhe), one N-methylvaline
(MeVal), and one 3-methylbutanoic acid (MBA) residue
(Figure S22, Table S1, SI).
As shown in Figure 3, the HMBC spectrum of 1 in DMSO-
d6 showed correlations from MeVal-NCH3 (δH 2.57) to Ala-
Figure 1. Comparison of extracts of culture broth: (a) single culture
(J774.1, mCD), (b) single culture (N. tenerifensis, mCD), (c)
coculture (N. tenerifensis and J774.1, mCD).
A large-scale (0.8 L) coculture of N. tenerifensis IFM 10554T
and J774.1 in an mCD medium at a cell number ratio of 10:1,
respectively, was performed at 28 °C in 175 cm2 cell culture
flasks under static conditions for 2 weeks in atmospheric air.
After centrifugation of the culture broth, the supernatant and a
methanol extract of the mycelia and animal cells were
combined and subjected to partitioning between ethyl acetate
(EtOAc) and water. The EtOAc fraction was subjected to
fractionation by ODS column chromatography (the MeOH−
H2O system), followed by reversed-phase HPLC separation
(85% MeOH) to yield a new compound 1, named nocarjamide
(Figure 2). LC−MS analysis revealed that compound 1 was
produced only under coculture of N. tenerifensis IFM 10554T
Figure 3. HMBC correlations of 1.
CO (δC 172.1), Ala-NH (δH 7.32), and Val1-CO (δC 170.2)
from Val1-NH (δH 8.80) to Phe-CO (δC 172.0), from Phe-NH
(δH 7.58) to Leu-CO (δC 171.5), from Leu-NH (δH 7.82) to
MePhe-CO (δC 167.8), from MePhe-NCH3 (δH 2.69) to
MeLeu-CO (δC 172.3), from MeLeu-NCH3 (δH 3.09) to Val2-
CO (δC 175.3), and from Val2-NH (δH 8.55) to Thr-CO (δC
168.8), suggesting that all nine constituent amino acids were
connected by eight amide bonds to yield a sequence of Thr-
Val2-MeLeu-MePhe-Leu-Phe-Val1-Ala-MeVal. HMBC correla-
tions observed from Thr-NH (δH 8.42) to MBA-CO (δC
173.8) suggested that an N-terminal Thr was connected to
MBA by an amide bond, whereas HMBC correlations
observed from Thr-3 (δH 4.70) to MeVal-CO (δC 168.8)
implied that a C-terminal MeVal was connected to the Thr by
an ester bond (Figure 3).
The NOESY spectrum of 1 in DMSO-d6 (Figure S13, SI)
showed correlations between MeVal-2 (δH 3.96) and Ala-2 (δH
4.91); Ala-NH (δH 7.32) and Val1-2 (δH 5.09); Ala-NH (δH
7.32) and Val1-4, 5 (δH 0.89); Val1-NH (δH 8.80) and Phe-2
(δH 5.06); Val1-NH (δH 8.80) and Phe-3 (δH 2.59, 3.03); Phe-
NH (δH 7.58) and Leu-2 (δH 4.22); Phe-NH (δH 7.58) and
Figure 2. Structure of 1.
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Leu-3 (δH 1.34); Leu-NH (δH 7.82) and MePhe-2 (δH 4.36);
Leu-NH (δH 7.82) and MePhe-NCH3 (δH 2.69); MePhe-2 (δH
4.36) and MeLeu-2 (δH 4.98); MePhe-5 (δH 7.17) and
MeLeu-3 (δH −0.62); MePhe-5 (δH 7.17) and MeLeu-5 (δH
0.48); MeLeu-NCH3 (δH 3.09) and Val2-2 (δH 4.62); Val2-NH
(δH 8.55) and Thr-2 (δH 4.58); Val2-NH (δH 8.55) and Thr-3
(δH 4.70); Thr-NH (δH 8.42) and MBA-2 (δH 1.95, 2.05),
suggesting the planar structure of nocarjamide (1) (Figure 4).
This planar structure was also consistent with 2D NMR
analysis in CDCl3 (Figures S15−S21, Table S2, SI).
Figure 5. MS/MS and MS3 fragment analysis of 1.
Figure 4. NOESY correlations of 1.
Furthermore, when MS/MS analysis was carried out using a
positive ion peak m/z 1138 [M + Na]+ as a precursor ion, all of
the product ions were observed as sodium adducts. The
product ions of 1 observed at m/z 1025, 954, 855, and 708
indicated the sequential losses of the MeVal, Ala, Val1, and Phe
from the molecule of 1, respectively. The product ions at m/z
908, 809, and 662 were assigned to be the result of sequential
losses of MeVal-Ala, Val, and Phe coupled with the elimination
of CO and H2O from 1, respectively. The product ions at m/z
577 and 416 were assigned to be the dehydrated fragments of
MBA-Thr-Val2-MeLeu-MePhe and MBA-Thr-Val2-MeLeu,
respectively. Also, when MS3 analysis was carried out using a
product ion at m/z 954 in MS/MS as the precursor ion, the
product ion at m/z 307 indicated the fragment of MBA-Thr-
Val2. Additionally, the above-mentioned characteristic dehy-
drated product ions at m/z 908, 809, 662, 577, and 416
suggested the presence of the Thr residue in the molecule
(Figure 5 and Table S3, SI). These results were also consistent
with the planar structure of nocarjamide (1) as suggested by
NMR analysis.
The absolute configuration of amino acids of compound 1
was determined by the advanced Marfey’s method.9 Based on
the results of LC−MS, compound 1 was revealed to consist of
L-Ala, L-Leu, D-Phe, L-Thr, L-MeLeu, L-MePhe, and L-MeVal
(Figure S1, SI). It also was found that the two valines (Val1
and Val2) consist of one D-Val and one L-Val, although which
one is D or L remains undefined. The absolute configurations of
two valines were firmly established by an X-ray crystallographic
analysis of 1 (Figure 6), implying that Val1 and Val2 are D and
L, respectively. X-ray analysis of 1 revealed that two conformers
Figure 6. X-ray structure of nocarjamide (1).
are present in a 1:1 ratio in the crystal state of 1 (Figures S24−
S27, SI).8 As a result, the whole structure of nocarjamide was
concluded to be 1, as shown in Figure 2. In the H and 13C
1
NMR in CDCl3 solution of 1, the presence of two conformers
was also observed (Figures S15 and S16, Table S2, SI). The
ratio of conformers (1:1) was consistent between crystal and
solution (CDCl3) states, while the structure of two conformers
of the solution (CDCl3) state remained undefined.
The production conditions of compound 1 were next
examined using six kinds of media (Δsucrose, ΔNaNO3,
ΔKH2PO4, ΔKCl, ΔFeSO4, and ΔMgSO4, Figure S2, SI) to
reveal that the presence of NaNO3 and FeSO4 is important for
production of compound 1.
Compound 1 exhibited low cytotoxicity against several cell
lines (Figures S3 and S4, SI), while 1 exhibited a Wnt signal-
activation effect by using the TOPFlash luciferase assay
system;10 TCF/β-catenin transcriptional activity (TOPFlash
activity) was 1.8- and 2.2-fold higher in the presence of 20 and
40 μM compound 1, respectively (Figure 7). The FOPFlash
acitivity,10 using a construct with a mutated site to exclude
false-positive signals, was also examined to demonstrate that
TOPFlash activity was 1.5-fold higher than FOPFlash activity
at both 20 and 40 μM compound 1. Furthermore, we carried
out Western blot analysis using compound 1 in HEK293 cells.
β-Catenin is a key molecule in the Wnt pathway,11 and an
increase of β-catenin leads to upregulation of the Wnt signal
activity. c-Myc is a target protein of the Wnt pathway, and
upregulation of the Wnt pathway leads to an increase of the c-
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DOI: 10.1021/acs.orglett.8b02522
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: mish@chiba-u.jp.
ORCID
Yasumasa Hara: 0000-0003-0451-8731
Midori A. Arai: 0000-0003-0254-9550
Kazufumi Toume: 0000-0002-3315-145X
Masami Ishibashi: 0000-0002-2839-1045
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Figure 7. Effect of nocarjamide (1) on TCF/β-catenin transcriptional
This study was supported by JSPS KAKENHI Grant No.
17H03992 and the Strategic Priority Research Promotion
Program of Chiba University, “Phytochemical Plant Molecular
Sciences”.
activity. Data are presented as mean
SD of n = 3 independent
experiments, and the data in 0.1% DMSO aqueous solution are taken
as 100%. TOP: TOPFlash luciferase activity. FOP: FOPFlash
luciferase activity. Viability: cell viability.
REFERENCES
myc protein. As shown in Figure 8, compound 1 increased
protein levels of not only β-catenin but also c-myc. This result
■
(1) Nocard, E. Ann. Inst. Pasteur. 1888, 2, 293−302.
(2) Aoki, H.; Sakai, H.; Kohsaka, M.; Konomi, T.; Hosoda, J. J.
Antibiot. 1976, 29, 492−500.
(3) Celmer, W. D.; Cullen, W. P.; Moppett, C. E.; Jefferson, M. T.;
Huang, L. H.; Shibakawa, R.; Tone, J. U.S. Patent 4148883, 1977.
(4) Bertrand, S.; Bohni, N.; Schnee, S.; Schumpp, O.; Gindro, K.;
Wolfender, J. L. Biotechnol. Adv. 2014, 32, 1180−1204.
(5) Hara, Y.; Arai, M. A.; Sakai, K.; Ishikawa, N.; Gonoi, T.; Yaguchi,
T.; Ishibashi, M. J. Nat. Med. 2018, 72, 280−289.
(6) Sakai, K.; Komaki, H.; Gonoi, T. PLoS One 2015, 10, e0143264.
(7) Hoshino, Y.; Chiba, K.; Ishino, K.; Fukai, T.; Igarashi, Y.;
Yazawa, K.; Mikami, Y.; Ishikawa, J. J. Bacteriol. 2011, 193, 441−448.
(8) Stewart, W. E.; Siddall, T. H., III Chem. Rev. 1970, 70, 517−551.
(9) Harada, K.; Fujii, K.; Hayashi, K.; Suzuki, M. Tetrahedron Lett.
1996, 37, 3001−3004.
Figure 8. Effects of nocarjamide (1) on the expression of β-catenin
and c-myc in HEK293 cells.
clearly reveals that compound 1 activated Wnt signal activity
and was consistent with the result of the TOPFlash luciferase
activity test. The Wnt signal is known to play an important role
in various processes of vital phenomena such as the formation
of various tissues and the differentiation/proliferation of
eukaryotic cells.12 Regarding human pathology, suppression
of Wnt signaling has been implicated in osteoporosis−
pseudoglioma syndrome,13 Parkinson’s disease,14 and other
diseases. It is therefore important to identify compounds that
exhibit Wnt signal-modulating activity such as 1.
(10) Li, X.; Ohtsuki, T.; Koyano, T.; Kowithayakorn, T.; Ishibashi,
M. Chem. - Asian J. 2009, 4, 540−547.
(11) Fuentes, R. G.; Arai, M. A.; Ishibashi, M. Nat. Prod. Rep. 2015,
32, 1622−1628.
(12) Anastas, J. N.; Moon, R. T. Nat. Rev. Cancer 2013, 13, 11−26.
(13) Hoeppner, L. H.; Secreto, F. J.; Westendorf, J. J. Expert Opin.
Ther. Targets 2009, 13, 485−496.
(14) Berwick, D. C.; Harvey, K. Biochem. Soc. Trans. 2012, 40,
1123−1128.
In the present study, we demonstrated the effectiveness of a
method employing coculture of a pathogenic actinomycete of
the genus Nocardia with animal cells. This technique
succeeded in generating nocarjamide (1), a novel bioactive
natural product.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b02522.
Experimental procedures, spectral data, and X-ray crystal
analysis data of 1 (PDF)
Accession Codes
CCDC 1851304 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk, or by contacting The Cam-
bridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
D
DOI: 10.1021/acs.orglett.8b02522
Org. Lett. XXXX, XXX, XXX−XXX