Small molecule perimeter defense in entomopathogenic bacteria

Article abstract of DOI:10.1073/pnas.1201160109

Two Gram-negative insect pathogens, Xenorhabdus nematophila and Photorhabdus luminescens, produce rhabduscin, an amidoglycosyl- and vinyl-isonitrile-functionalized tyrosine derivative. Heterologous expression of the rhabduscin pathway in Escherichia coli, precursor-directed biosynthesis of rhabduscin analogs, biochemical assays, and visualization using both stimulated Raman scattering and confocal fluorescence microscopy established rhabduscin's role as a potent nanomolar-level inhibitor of phenoloxidase, a key component of the insect's innate immune system, as well as rhabduscin's localization at the bacterial cell surface. Stimulated Raman scattering microscopy visualized rhabduscin at the periphery of wild-type X. nematophila cells and E. coli cells heterologously expressing the rhabduscin pathway. Precursor-directed biosynthesis created rhabduscin mimics in X. nematophila pathway mutants that could be accessed at the bacterial cell surface by an extracellular bioorthogonal probe, as judged by confocal fluorescence microscopy. Biochemical assays using both wild-type and mutant X. nematophila cells showed that rhabduscin was necessary and sufficient for potent inhibition (low nM) of phenoloxidases, the enzymes responsible for producing melanin (the hard black polymer insects generate to seal off microbial pathogens). These observations suggest a model in which rhabduscin's physical association at the bacterial cell surface provides a highly effective inhibitor concentration directly at the site of phenoloxidase contact. This class of molecules is not limited to insect pathogens, as the human pathogen Vibrio cholerae also encodes rhabduscin's aglycone, and bacterial cell-coated immunosuppressants could be a general strategy to combat host defenses.

Full text of DOI:10.1073/pnas.1201160109

Small molecule perimeter defense in
entomopathogenic bacteria
Jason M. Crawforda,1,2, Cyril Portmanna,1, Xu Zhangb, Maarten B. J. Roeffaersb,3, and Jon Clardya,b,4
aDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; and bDepartment of Chemistry and
Chemical Biology, Harvard University, Cambridge, MA 02138
Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved May 3, 2012 (received for review January 20, 2012)
Two Gram-negative insect pathogens, Xenorhabdus nematophila
and Photorhabdus luminescens, produce rhabduscin, an amido-
glycosyl- and vinyl-isonitrile-functionalized tyrosine derivative.
Heterologous expression of the rhabduscin pathway in Escherichia
coli, precursor-directed biosynthesis of rhabduscin analogs, bio-
chemical assays, and visualization using both stimulated Raman
scattering and confocal fluorescence microscopy established rhab-
duscin’s role as a potent nanomolar-level inhibitor of phenoloxi-
dase, a key component of the insect’s innate immune system, as
well as rhabduscin’s localization at the bacterial cell surface. Stimu-
lated Raman scattering microscopy visualized rhabduscin at the
periphery of wild-type X. nematophila cells and E. coli cells hetero-
logously expressing the rhabduscin pathway. Precursor-directed
biosynthesis created rhabduscin mimics in X. nematophila pathway
mutants that could be accessed at the bacterial cell surface by an
extracellular bioorthogonal probe, as judged by confocal fluores-
cence microscopy. Biochemical assays using both wild-type and mu-
tant X. nematophila cells showed that rhabduscin was necessary
and sufficient for potent inhibition (low nM) of phenoloxidases,
the enzymes responsible for producing melanin (the hard black
polymer insects generate to seal off microbial pathogens). These
observations suggest a model in which rhabduscin’s physical
association at the bacterial cell surface provides a highly effective
inhibitor concentration directly at the site of phenoloxidase con-
tact. This class of molecules is not limited to insect pathogens,
as the human pathogen Vibrio cholerae also encodes rhabduscin’s
aglycone, and bacterial cell-coated immunosuppressants could be a
general strategy to combat host defenses.
(5) by homology to the known isonitrile-forming biosynthetic
genes isnA and isnB (6, 7) along with a glycosyltransferase, and
this analysis was validated by heterologous production of rhabdus-
cin in genetically transformed Escherichia coli (4). The P. lumines-
cens TT01 genome (8) also contained the rhabduscin biosynthetic
genes, and rhabduscin production was detected in this genus as
well. A structurally related molecule, byelyankacin (structure 2),
which was first identified in an Enterobacter species (9) and then
later in metagenomic libraries (10), exhibits potent human mela-
nogenesis–inhibitory activity by inhibiting tyrosinase—a phenolox-
idase-type enzyme. Taken together, these observations led to the
proposal that rhabduscin’s natural function is inhibiting the mel-
anin-producing insect phenoloxidase (PO), a critical enzymatic
innate immune response to invading pathogens (4).
Innate immunity is a largely conserved feature in animals, and
the insect’s immune system parallels vertebrate innate immunity
in many regards (11). The insect has pattern-recognition recep-
tors (PRRs) that, upon microbial invasion, recognize pathogen-
associated molecular patterns (PAMPs) and mount both humoral
and cellular immune responses with high spatial and temporal
control (12). In the pro-PO system, PAMP recognition stimulates
a serine protease cascade leading to pro-PO cleavage and cata-
lytically competent PO (13, 14). PO is the terminal component
in invertebrate innate immunity that accepts phenolic substrates
initially to generate short-lived cytotoxic quinone products, which
are in turn converted to the long-lived and rigid polymer called
melanin. The tightly controlled activation of the melanization
complex, which binds to bacterial cells and polysaccharides, leads
to localization of cytotoxic products around and melanin deposi-
tion directly on the intruding microorganism, encapsulating the
pathogen. PO also enhances pathogen phagocytosis by hemocytes
and contributes in wound healing by sclerotization, providing a
formidable defense against microbial invaders (13, 14).
oxidase inhibitors ∣ innate immunity ∣ natural products ∣ virulence factors
enorhabdus and Photorhabdus species are enteric mutualis-
X
tic gammaproteobacteria of their respective Steinernema and
While P. luminescens was previously shown to produce multi-
potent bacterial stilbenes that exhibit micromolar PO inhibition
against the model Manduca sexta insect larvae and contribute to
virulence (15–17), rhabduscin’s natural role has not been experi-
mentally assessed. To assign a specific function to rhabduscin
in immunosuppression, we began a detailed investigation of rhab-
duscin’s cellular localization and biological activity. Here, we
Heterorhabditis nematode hosts (1–3). The free-living infective
juvenile-stage nematode hunts insect larvae in the soil, invades
the insect’s circulatory system, and regurgitates its mutualistic
bacteria to initiate insect pathogenesis (entomopathogenesis).
The bacteria produce an assortment of small molecules and pro-
teins to regulate the multipartite symbiosis by overcoming the
insect’s innate immune system, killing the insect host and micro-
bial competitors, and supporting proper nematode development.
A bacteria–nematode pair can efficiently parasitize a range of in-
sect larvae, making the duo an effective biocontrol agent in agri-
cultural applications. The bacteria’s coordination of two vastly
different objectives in two different animal hosts provides an ex-
cellent model system for investigating mutualistic and pathogenic
bacteria–animal interactions (1–3).
Author contributions: J.M.C., C.P., and J.C. designed research; J.M.C., C.P., X.Z., and M.B.J.R.
performed research; J.M.C., C.P., X.Z., and M.B.J.R. contributed new reagents/analytic
tools; J.M.C., C.P., X.Z., M.B.J.R., and J.C. analyzed data; and J.M.C., C.P., and J.C. wrote
the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
We previously found that the bacterial uptake of L-proline, an
Freely available online through the PNAS open access option.
1J.M.C. and C.P. contributed equally to this work.
abundant amino acid in insect hemolymph, enhanced the bacter-
ial proton motive force and antibiotic production in the primary-
phase phenotypic variants of X. nematophila ATCC19061 and
P. luminescens TT01 (4). Rhabduscin (structure 1), a tyrosine-
derived amidoglycosyl- and vinyl-isonitrile product (Fig. 1), was
up-regulated in X. nematophila by about an order of magnitude in
2Present address: Department of Chemistry, Chemical Biology Institute, Yale University,
New Haven, CT 06520.
3Present address: Department of Microbial and Molecular Systems, Katholieke Universiteit
Leuven, B-3001 Heverlee, Belgium.
4To whom correspondence should be addressed. E-mail: jon_clardy@hms.harvard.edu.
L
-proline–supplemented cultures. A candidate rhabduscin gene
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1201160109/-/DCSupplemental.
cluster was identified in the X. nematophila ATCC19061 genome
www.pnas.org/cgi/doi/10.1073/pnas.1201160109
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Fig. 1. Rhabduscin (1) and byelyankacin (2) biosynthesis.
demonstrate that rhabduscin harbors a potent nanomolar-level
immunosuppressive activity against PO, which is dramatically
more potent than the previously described stilbenes. Strikingly,
rhabduscin binds at the periphery of bacterial cells, as visualized
by stimulated Raman scattering and confocal fluorescence micro-
scopic techniques, where its location and potent PO-inhibitory
activity contribute substantially to virulence.
Results and Discussion
Rhabduscin Biosynthesis and its Cellular Localization. The organiza-
tion of the biosynthetic genes for rhabduscin (1) in P. luminescens
differs dramatically from that found in X. nematophila, and hetero-
logous expression in E. coli was used to clarify the differences.
In X. nematophila, we recently demonstrated that a small three-
gene cluster (XNC1_1221-XNC1_1223), which includes isnA, isnB,
and a glycosyltransferase gene, conferred rhabduscin production in
the heterologous host E. coli (4). Genome synteny analysis, using
the MicroScope bioinformatics platform (18), showed a similar
set of genes in P. luminescens, but a large gap, more than 1,000
genes, separated isnA and isnB (plu2816 and plu2817) from two
glycosyltransferase homologs (plu1760 and plu1762) (Fig. 2A). It
seemed likely that both glycosyltransferase genes contributed to
rhabduscin biosynthesis in the P. luminescens biosynthetic pathway
either by transferring different activated sugars onto the vinyl-iso-
cyanide aglycone 3 or by expressing redundant enzymatic func-
tions. We constructed a series of heterologous expression
vectors, which included isnA, isnB, GT1760 or GT1762, isnA–isnB,
and isnA–isnB-GT1760 or -GT1762, to monitor metabolite pro-
duction in E. coli. As expected, both isnA and isnB were required
for aglycone 3 production (Fig. 2B). E. coli cells producing isnA,
isnB, and either GT1760 or GT1762 redundantly produced
rhabduscin and a second minor glycoside. High-resolution mass
spectrometry (HR–MS) indicated that the minor compound
has a molecular formula of C15H17NO5Na [calculated, 314.1004
ðM þ NaÞþ; observed, 314.1012]. One-dimensional (1H) NMR
chemical shift and coupling constant analyses in addition to
two-dimensional (gCOSY, gHSQC, gHMBC, and NOESY) corre-
lations indicated that the minor glycoside was byelyankacin (9) (2)
(SI Appendix, Table S1). The isonitrile functional group is sensitive
to hydrolysis, especially in acidic conditions, and the resulting
hydrolyzed formamide derivatives (1H O and 3H O) could also
Fig. 2. (A) Rhabduscin biosynthetic gene cluster in Photorhabdus luminescens
and heterologous expression of the different combinations of P. luminescens
genes. (B) LC/MS ultraviolet traces and products from the heterologous expres-
sion of P. luminescens genes in E. coli. (C) Rhabduscin biosynthetic gene cluster
in Xenorhabdus nematophila and genetic mutants. (D) LC/MS ultraviolet traces
and products from X. nematophila wild-type, ΔGT, and ΔisnAB.
in signal localization, suggesting that the sugar substituent con-
tributes to rhabduscin placement (SI Appendix, Fig. S1).
To extend these results into a wild-type host, we first con-
structed scarless-deletion mutants of the vinyl-isocyanide bio-
synthetic genes, isnA–isnB, and the glycosyltransferase gene in
X. nematophila (Fig. 2C). X. nematophila was selected because we
2
2
had previously found that L-proline up-regulates production of
be detected.
rhabduscin in this host by about an order of magnitude in
rich laboratory medium (4), and also because we wanted to in-
vestigate rhabduscin’s contribution to the cellular inhibition of
phenoloxidase. P. luminescens TT01 was less suitable because it
robustly produces additional phenoloxidase inhibitors belonging
The isonitrile functional group, which occurs rarely in naturally
produced molecules, has a characteristic Raman resonance
(observed 2;121 cm−1 for rhabduscin) that should be distinct
for rhabduscin in E. coli. Robust production of rhabduscin in the
heterologous host combined with its unique Raman shift enabled
observation of its cellular localization by stimulated Raman scat-
tering (SRS) microscopy (19, 20). Within the limits of spatial re-
solution for SRS, it was observed that rhabduscin’s 2;121 cm−1
Raman signal was localized to the periphery of the overproducing
E. coli cells, while the background signal in control cells lacking
the gene cluster was low (Fig. 3 A and B). E. coli cells overprodu-
cing rhabduscin’s aglycone qualitatively showed less congruency
to the bacterial stilbene class (15), and L-proline supplementation
does not stimulate rhabduscin production (4). Deletion of the
X. nematophila glycosyltransferase alone led to accumulation of
the aglycone intermediate 3, and deletion of isnA–isnB comple-
tely destroyed isonitrile biosynthesis (Fig. 2D). Similar to the
E. coli results described above, rhabduscin’s distinct Raman sig-
nal localized to the periphery of wild-type X. nematophila cells,
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Fig. 3. SRS microscopy analyzing the spatial distribution of rhabduscin
based on the vibrational resonance of the isonitrile functional group at
2;121 cm−1. (Scale bar, 10 μm.) In this technique, two laser beams (pump and
Stokes) with the frequency difference matching the vibrational frequency of
the isonitrile group are utilized to drive the transition coherently. The spec-
trum is identical to spontaneous Raman spectrum but with much higher ac-
quisition speed, allowing fast imaging (19). Because most naturally produced
molecules do not have Raman signatures in this spectral region, the signal
is distinctive for rhabduscin. The wild-type X. nematophila signal level of
rhabduscin is lower than the E. coli overproducer, indicating lower expression
level, but the difference in signal between wild-type and control cells is big
enough to measure different cell phenotypes.
whereas the control ΔisnAB strain lacking rhabduscin only exhib-
ited weak background signal (Fig. 3 C and D).
Fig. 4. Precursor-directed biosynthesis of glycoside derivatives. (A) (E)-3-
(4-hydroxyphenyl)acrylonitrile (6) was obtained via a Heck reaction between
4-iodophenol (4) and acrylonitrile (5) (29), which resulted in a 15/85 cis/trans
mixture. The P. luminescens glycosyltransferases (GTs) both glycosylated the
aglycone mimic to produce glycosides 7 and 8. (B) (E)-4-(2-azidovinyl)phenol
(12) was obtained in three steps starting from 4-ethynylphenyl acetate (9); 10
was prepared by hydroboration of 9 (30); 11 was obtained via a copper-
catalyzed cross-coupling between sodium azide and boronic acid 10 by
adapting a previously described procedure (31); 11 was deprotected with po-
tassium carbonate to afford phenolic compound 12, which was accepted by
the GTs to afford glycoside 13. SI Appendix provides further details and spec-
tral data.
Synthesis of Substrate Mimics and Precursor-Directed Biosynthesis.
With the heterologous glycosyltransferase expression constructs
in hand, we began to investigate glycosyltransferase substrate
promiscuity for the amino acid-derived part of the molecule. We
challenged both P. luminescens glycosyltransferases individually
expressed in E. coli with a panel of synthetic and commercial agly-
cone substrate mimics. We first synthesized the sterically conser-
vative nitrile mimic 6, which simply inverts the N─C triple bond
(Fig. 4A). We then synthesized the azide derivative 12 (Fig. 4B),
which could serve as a glycosyltransferase substrate mimic and
allow orthogonal probes to be introduced for further cellular
localization studies. The less conservative commercial mimics in-
cluded para-coumaric acid, 4-hydroxy-benzylideneacetone, (4-hy-
droxybenzylidene)malononitrile, trans-4-hydroxy-β-nitrostyrene,
and umbelliferone. Using a precursor-directed biosynthetic strat-
egy, the synthetic nitrile 6 and the deprotected azide 12 mimics
were fed to BL21(DE3) Star pLysS E. coli cells overexpressing
GT1760 or GT1762. The products were extracted with ethyl acet-
ate and analyzed by C18 reversed-phase LC/MS. The nitrile mimic
was nicely converted to its corresponding rhabduscin (7, 46–71%
conversion) or byelyankacin (8, 13–42%) glycoside derivatives
(Fig. 4 and SI Appendix, Table S2), and the azide mimic was con-
verted to its rhabduscin glycoside derivative (13, 61–93%). Of the
commercial substrates, only benzylideneacetone and umbellifer-
one incorporated at minor levels (≤3%), indicating limited sub-
strate tolerance in glycoside formation.
Confocal Fluorescence Microscopic Analyses of Metabolite Localiza-
tion. Because azide-aglycone mimic 12 was accepted by the gly-
cosyltransferase in vivo to generate the azide-glycoside 13
(Fig. 4B), their azide chemical handles could be used to introduce
bioorthogonal probes in vivo via click chemistry (21). To evaluate
whether the small molecule inhibitors are accessible at the bac-
terial cell surface, rather than embedded in the cell membrane,
we used exogenous addition of green fluorescent protein (GFP)
as an easily detectable surrogate of insect phenoloxidase. In
addition, to avoid competition with the natural isonitrile small
molecules, we selected the X. nematophila strain disabled in iso-
nitrile biosynthesis (the ΔisnAB strain). The glycosyltransferase
was left intact in this mutant so that the synthetic mimics could
be used for precursor-directed glycoside biosynthesis, bypassing
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vinyl-isonitrile biosynthesis. Stationary-phase X. nematophila
mutant cultures supplemented with the synthetic azide 12 or the
biosynthetic azide-glycoside 13 were first reacted with a biotin–
alkyne conjugate, washed, and then followed by the binding of a
streptavidin–GFP conjugate. Similar to the SRS results, the fluor-
escence localized to the periphery of the cells within the limits of
resolution only in the presence of the inhibitor mimics (Fig. 5),
supporting that the small molecule inhibitors would be accessible
to inhibit the extracellular phenoloxidase response of an insect’s
innate immune system. To determine if this peripheral localiza-
tion resulted from covalent linkage to the cell’s peripheral
lipopolysaccharide (LPS), the LPS was purified (Gentaur LPS ex-
traction kit) and separated over a polyacrylamide gel. The fluor-
escent signal partitioned to an initial phenol/chloroform wash
fraction, and no fluorescence was detected in the denaturing LPS
gel, suggesting that localization is physical, not covalent. Further
supporting a physical interaction, the isonitrile-containing mole-
cules are present in both cell preparations and in the cleared
spent medium as a diffusion product.
from Photorhabdus species, which have been linked to phenolox-
idase inhibition in the model tobacco hornworm larvae, Manduca
sexta (15). Strikingly, conservative replacement of the isonitrile
with the nitrile in the synthetic aglycone mimic 6 largely destroys
tyrosinase inhibition, demonstrating the isonitrile’s crucial role
for inhibitory activity. The azide mimic 12 also exhibited weak
activity. Phenoloxidases are copper-containing enzymes (23),
and it is possible that the isonitrile’s ability to bind copper,
and other metal ions, plays a role in enzyme inhibition.
When a bacterium invades an insect host, the insect deposits
melanin onto the periphery of the invading cell, which exhibits
a bactericidal activity and traps the bacteria in nodules (24).
To determine whether the cells themselves can inhibit tyrosinase,
we subjected washed X. nematophila cells to tyrosinase-inhibitory
assays. X. nematophila wild-type (initial OD600 ¼ 0.71, 64% inhi-
bition) and the glycosyltransferase deletion mutant (initial
OD600 ¼ 0.86, 65% inhibition) both inhibited tyrosinase activity
at similar levels, whereas the isonitrile isnAB biosynthetic gene
deletion (initial OD600 ¼ 0.77, 0% inhibition) destroyed the cell’s
inhibitory activity. The inhibitory activity remained persistently
associated with the cells under physiologically relevant condi-
tions, as iterative phosphate-buffered saline (PBS) washes did
not flush the activity away (SI Appendix, Fig. S5). Taken together,
these results identify rhabduscin as a potent immunosuppressant
that localizes to the periphery of the bacterial cells producing it
and enables them to inhibit phenoloxidase, thereby suppressing
one of the insect’s primary innate immune defense strategies.
Metalloenzyme Inhibition: Tyrosinase and Phenoloxidase. The periph-
eral localization and hemolymph- or L-proline-induced up-regu-
lation of rhabduscin suggested that it had a functional role in
bacterial pathogenesis. In an earlier screen for skin-whitening
agents, the α- L-rhamnopyranoside byelyankacin (2) was reported
to be a potent tyrosinase inhibitor, the key enzyme in human mel-
anin formation (9). Establishing whether rhabduscin plays a simi-
lar role first required establishing the absolute stereochemistry of
the attached sugar, because only the relative configuration for
rhabduscin was reported previously (4). Mosher ester analysis
(22) indicates that rhabduscin (1) contains a β-L-acetamidopyr-
anoside (SI Appendix, Figs. S2 and S3 and Table S3).
Given the reported activity and insect phenoloxidase’s critical
role in innate immune defense, we tested the compounds for in-
hibition against a model tyrosinase from mushroom and the phe-
noloxidase from waxmoth larvae (Galleria mellonella), a model
insect host. The aglycone intermediate 3, byelyankacin, and rhab-
duscin all showed low nanomolar-level inhibition (IC50) against
both mushroom tyrosinase and insect phenoloxidase, a level
roughly three orders of magnitude more potent than the commer-
cial whitening agent kojic acid (Table 1 and SI Appendix, Fig. S4).
The isonitrile-containing natural products are also roughly three
orders of magnitude more potent than the bacterial stilbenes
Rhabduscin is a Virulence Factor. Having obtained evidence for
rhabduscin’s nanomolar-level activity against phenoloxidase and
its localization at or near the periphery of the bacterial cell, we
wanted to determine the compound’s importance in pathogenesis
in an animal model. X. nematophila wild-type, ΔGT, ΔisnAB, and
a PBS control were individually injected into the hemolymph of
ten Galleria mellonella larvae. Wild-type bacteria killed all of the
larvae within 50 h, and PBS killed none of the larvae throughout
the 96 h experiment (SI Appendix, Fig. S6). The ΔGT mutant was
less virulent than wild-type, but this bacterium grew more slowly
than wild-type, presumably because of the toxicity of accumulated
aglycone. The ΔisnAB bacteria only killed half of the larvae dur-
ing the experiment, and the dead larvae were generally darker
compared to the wild-type–infected dead larvae because of heigh-
Fig. 5. Confocal fluorescence microscopy. (A) X. nemato-
phila ΔisnAB fed with the two azide mimics 12 and 13 com-
pared to PBS control. Brightness and contrast were adjusted
identically for compared image sets. The azide mimics were
first reacted with a biotin-alkyne conjugate followed by the
binding of a streptavidin-GFP conjugate. (B) Enlargement
of X. nematophila ΔisnAB fed with 13. The enlarged region
is indicated in A by the white rectangle.
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prepared and analyzed using similar conditions as previously described (4)
(SI Appendix, Table S4). Aliquots of stationary-phase cultures were visualized
directly on poly-L-lysine-coated slides by SRS microscopy without manipula-
tion (SI Appendix).
Table 1. Inhibition (IC50) of tyrosinase from mushroom and
hemolymph-activated phenoloxidase from Galleria mellonella
(waxmoth larvae)
Tyrosinase
Phenoloxidase
Compound
inhibition [nM]
inhibition [nM]
Heterologous Expression of P. luminescens and V. cholerae Genes. All genes ex-
cept VC1949 were cloned into pETDuet-1 (Novagen) and expressed in BL21
(DE3) Star pLysS E. coli using standard molecular biological procedures (SI
Appendix, Table S4). E. coli cells harboring the P. luminescens rhabduscin
pathway or harboring the pETDuet-1 control vector were used for SRS micro-
scopic experiments (SI Appendix), and aliquots of stationary-phase cells were
visualized directly. A codon-optimized VC1949 in pJExpress401 was obtained
from DNA2.0.
Rhabduscin (1)
Byelyankacin (2)
Aglycone-NC 3
Aglycone-CN 6
Aglycone-N3 12
Kojic acid
15.1 1.0
37.1 2.5
7.9 0.8
>400;000*
>400;000*
22,173 2,924
64.1 2.0
184.9 14.5
61.7 6.2
ND
ND
ND
*Low inhibitory activity precluded an accurate IC50 measurement.
Precursor-Directed Biosynthesis of Glycoside Derivatives. BL21(DE3) Star pLysS
E. coli containing the P. luminescens glycosyltransferase genes (either
pEGT1760 or pEGT1762) were grown overnight at 37 °C and 250 rpm as LB
suspension cultures with 0.1 mg∕mL ampicillin. Fifty-μL aliquots of overnight
stationary-phase culture were used to inoculate 5-mL portions of M9 minimal
medium supplemented with 0.5% (m∕vol) casamino acids and 0.1 mg∕mL
tened melanization. These semiquantitative results demonstrate
that rhabduscin contributes to an effective pathogenesis.
Isonitrile Biosynthesis in Human and Other Pathogens. The rhabdus-
cin biosynthetic pathway is not limited to the insect pathogens
X. nematophila and P. luminescens. For example, sequence ana-
lysis indicates that the related insect pathogen Xenorhabdus
bovienii contains a homologous gene cluster (XBJ1_1355-XBJ1_
1353). Like P. luminescens, two gene sets separated by over 1,000
genes reside in Photorhabdus asymbiotica, an emerging human
pathogen also capable of infecting insects. P. asymbiotica appears
to contain three adjacent copies of the glycosyltransferase
(PAU_02755-PAU_02757) in addition to the isnA and isnB genes
(PAU_01721-PAU_01720). The conservation across multiple
Xenorhabdus and Photorhabdus species and the redundant glyco-
syltransferase copies in P. luminescens and P. asymbiotica empha-
size the pathway’s important role.
ampicillin. The cultures were grown at 37 °C and 250 rpm to an OD600
¼
0.5–0.6. Glycosyltransferase gene expression was induced with IPTG (1 mM
final) and supplemented with small molecule precursors (100 μM) in DMSO.
The induced cultures were incubated at 25 °C and 250 rpm to a final time of
24 h. The whole cultures were rigorously extracted with 5 mL of ethyl acetate,
separated by centrifugation; the top 4 mL of ethyl acetate was transferred to
a glass vial and dried, and the resulting residue was dissolved in 500 μL
methanol for reversed-phase LC/MS analysis.
Chemoenzymatic Synthesis of Rhabduscin-Azide (13). Two 4-L Erlenmeyer
flasks, each containing 1 L of M9 minimal medium supplemented with casa-
mino acids (0.5%) and ampicillin (0.1 g∕L), were inoculated with 5 mL of an
overnight BL21(DE3) Star pLysS E. coli culture harboring pEGT1762 (LB,
0.1 mg∕mL of ampicillin). The cultures were grown at 37 °C and 250 rpm to
an OD600 ¼ 0.5–0.6. Expression of the cluster was induced by the addition of
IPTG (1 mM final), and, at the same time, synthetic (12) (32 mg, 0.20 mmol)
dissolved in DMSO (5 mL) was added to each culture. The cultures were
grown at 25 °C and 250 rpm for a total time of 24 h. The combined whole
cultures were extracted with ethyl acetate (three times, 2 L total). The com-
bined organic fractions were dried (Na2SO4), filtered, and concentrated un-
der reduced pressure to afford an orange solid (220 mg). The residue was
purified by preparative HPLC (Luna C18, 5 μm, 250 • 21.2 mm, Phenomenex)
with an acetonitrile∶water gradient at 10 mL∕ min: 0–15 min, 30% to 45%
acetonitrile; 15–16 min, 45% to 100% acetonitrile; and 16–21 min, 100%
acetonitrile. The combined fractions (retention time ¼ 13 min) were concen-
trated under reduced pressure to afford (13) as a pale yellow compound
A single isnA–isnB fusion protein (VC1949) was reported in
the related gammaproteobacterium Vibrio cholerae (10), the cau-
sative agent of cholera, but to date, no small molecule has been
reported for this Vibrio pathway. We heterologously expressed a
codon-optimized version of this isnA–isnB fusion protein in
E. coli, but a biosynthetic small molecule product was not de-
tected. Closer inspection of the genes surrounding VC1949 in the
V. cholerae O1 biovar El Tor N16961 genome revealed an addi-
tional isnB homolog (VC1944) (SI Appendix, Fig. S7A). Coex-
pression of VC1949 with VC1944 or VC1944-VC1945 in E. coli
led to aglycone 3 production (SI Appendix, Fig. S7B). Both the
(9.0 mg, 0.026 mmol, 6.5%). 1H-NMR: (600 MHz, CD3OD)
δ 7.28 (d,
vinyl-isonitrile 3 and its hydrolysis product 3H O were observed.
2
J ¼ 8.8 Hz, 2H), 7.00 (d, J ¼ 8.8 Hz, 2H), 6.78 (d, J ¼ 13.8 Hz, 1H), 6.21 (d,
J ¼ 13.8 Hz, 1H), 4.85 (m, 1H), 4.29 (dd, J ¼ 4.7, 1.4 Hz, 1H), 3.92 (qd,
J ¼ 1.4, 6.4 Hz, 1H), 3.78 (dd, J ¼ 10.0, 4.7 Hz, 1H), 3.67 (dd, J ¼ 10.0,
7.6 Hz, 1H), 2.05 (s, 3H), 1.17 (d, J ¼ 6.4 Hz, 3H). 13C NMR: (151 MHz,
CD3OD) δ 174.8, 158.4, 131.0, 128.0, 126.9, 120.1, 117.9, 102.7, 73.6, 72.3,
71.2, 54.9, 22.5, 17.0. HR-MS: (ESI) calculated for C16H20N4O5Na 371.1331
ðM þ NaÞþ, found 371.1335.
While it is not clear from these experiments whether 3 is further
modified, based on the studies above, it is reasonable to speculate
that this related pathway in V. cholerae contributes to pathogen-
esis by inhibiting metalloenzymes in the human innate immune
system.
Conclusions
Gram-negative bacterial pathogens often modify their outermost
LPS layer to modulate the effects of their target host’s immune
system (25, 26). Additionally, the LPS layer’s hydrophobicity
minimizes hydrophobic small molecules, including many antibio-
tics, from reaching the inside of the cell. Rhabduscin, byelyanka-
cin, and their common aglycone represent another protective
strategy: one in which they serve as substrate mimic inhibitors for
phenoloxidase, a copper-dependent metalloenzyme that plays a
central role in invertebrate immunity by depositing melanin onto
the cell surface of invading microorganisms. Decorating the bac-
teria’s outermost layer with these inhibitors provides a spatially
appropriate and high local concentration to defend effectively
against this terminal phenoloxidase response and thereby support
the bacteria’s pathogenic lifestyle.
Mushroom Tyrosinase-Inhibition Assay. Mushroom tyrosinase was purchased
from Sigma–Aldrich (T3824). The assay was adapted from the Pomerantz
method (27), using 96-well plates (costar 3370) with the absorbance being
monitored in a Molecular Devices FlexStation 3. Fifty μL of a 2-mM DOPA so-
lution in 0.1-M phosphate buffer (pH 6.8) was added to 50 μL of the inhibitor
(varying concentrations) dissolved in 10% DMSO in water. The resulting solu-
tion was incubated for 10 min at room temperature before the addition of
50 μL of a 135-U∕mL mushroom tyrosinase solution in phosphate buffer. The
96-well plate was agitated for 3 s before starting to measure absorbance at
475 nm every 20 s. Kojic acid was used as a positive control and a 10% DMSO
water solution was used as a blank. The initial rate was obtained by linear
regression of the first 3 min of dopachrome formation. The fractional activity
was obtained by dividing the inhibited rate by the uninhibited rate obtained
from the blank reaction. The inhibition curves and IC50 values were calculated
using nonlinear regression sigmoid GraphPad with Prism 5 for Windows.
Materials and Methods
Construction and Metabolite Analysis of Deletion Mutants. Scarless-deletion
mutants of genes isnA–isnB and glycosyltransferase Xn_1223 in X. nemato-
phila, and subsequent product analysis of organic extracts by LC/MS, were
Mushroom Tyrosinase Inhibition Using X. nematophila Cells. The cell-inhibition
assay was conducted similarly to the small molecule-inhibition assay de-
scribed above, but a suspension of cells was used as the inhibitor. X. nema-
tophila wild-type, ΔisnAB, and ΔGT mutants were grown overnight in
Crawford et al.
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suspension (5 mL of LB) from a single colony at 30 °C and 250 rpm. Five-mL
cultures (2-g tryptone, 5-g yeast extract, and 10-g NaCl per L supplemented
with 0.1 M final of L-proline) were inoculated with 100 μL of the overnight
amide-propargyl biotin (3 μM final, Invitrogen B10185) using Click-iT Cell Buf-
fer Kit (Invitrogen C10269) following the supplier’s procedure. The only
modification to the supplier’s procedure was that the cells were resuspended
in half the volume of buffer before adding the second half of the buffer
mixed with the active compounds. After 30 min of reaction time, the cells
were centrifuged at 1;500 × g for 4 min and washed three times with
500 μL of PBS before being resuspended in 500 μL PBS. A solution of 100 μL
PBS and 10 μL streptavidin-Renilla reniformis GFP (Avidity, 320 mg∕mL) was
added to a 100-μL aliquot, and the mixture was reacted for 15 min at room
temperature. The cells were then centrifuged at 1;500 × g for 4 min and
washed two times with PBS before being resuspended in 100 μL PBS for mi-
croscopy.
culture and grown at 30 °C and 250 rpm for 48 h. The cells were centrifuged
and washed once with PBS before being diluted to an OD600 of approxi-
mately 0.75 for use in the assay described above. To assess inhibitor persis-
tence on the cells, a subsequent experiment was conducted with iterative
washing steps. Here, X. nematophila was grown and analyzed exactly the
same, except cellular inhibition was measured after each successive wash cy-
cle. A 15-mL bacterial suspension was washed 14 times with PBS by centrifu-
gation (800 × g, 5 min) and cell resuspension. At each washing round, an
aliquot of 500 μL of cell suspension was removed for analysis, and the volume
of PBS added for the next washing round was reduced by 500 μL to minimize
cell dilution.
Waxmoth Larvae Survival After X. nematophila Infection. Waxmoth larvae
(Galleria mellonella) were obtained from Berkshire Biological. Virulence as-
says were adapted from Eleftherianos et al. (28). X. nematophila wild-type,
ΔisnAB, and ΔGT were grown overnight in liquid culture (5 mL, LB) at 30 °C
and 250 rpm. One hundred μL of the overnight cultures were centrifuged
(6 min at 3;300 × g), the spent medium was discarded, and the cells were
resuspended in 1 mL of PBS. The quantity of cells was estimated with
OD600 (estimation: 109 cells∕mL for OD600 ¼ 1). The suspension was diluted
to obtain an approximate concentration of 100, 1,000, and 10,000 cells per
10 μL. Prior to injection, the rear ends of the larvae were washed with 70%
ethanol. The larvae were then injected into the hemocoel with 10 μL of cell
suspension using 31-gauge disposable insulin syringes (BD). PBS-injected in-
sects served as a control set. Larvae were kept at room temperature with
food, and mortality (no reaction when poked) was monitored for 100 h.
Ten larvae were used for each condition.
Hemolymph Phenoloxidase–Inhibition Assay. The assay was a modification of
the procedure described by Eleftherianos et al. (28). Waxmoth larvae were
chilled on ice for 15 min before sterilizing their surface with 70% ethanol.
The larvae were then decapitated and bled into a Falcon tube on ice. The
collected hemolymph was diluted in
a 3∶1 (vol∕vol) ratio with PBS
(50 mM, pH ¼ 6.5) and centrifuged at 14;700 × g and 4 °C for 10 min to ob-
tain the plasma. Activation of the phenoloxidase was performed directly in
the 96-well assay plate. Ten μL of diluted hemolymph plasma were added to
2 μL of E. coli LPS (5 mg∕mL; Sigma-Aldrich) for activation and 115 μL of PBS
(50 mM, pH ¼ 6.5), and the solution was kept at room temperature for 1 h.
Then, 50 μL of the inhibitor dissolved in 10% DMSO in PBS followed by 25 μL
of 20-mM 4-methyl catechol were added. The 96-well plate was agitated for
3 s before starting to measure absorbance at 490 nm every min for 1 h. IC50
values were obtained as described for the tyrosinase-inhibitory assays.
ACKNOWLEDGMENTS. We thank Xiaoliang Sunney Xie (Harvard University,
Cambridge, MA) for facilitating SRS microscopic measurements in his lab.
We thank Shugeng Cao (Harvard Medical School, Boston, MA) for helpful
discussions and reviewing a preliminary version of the manuscript. We also
thank Jennifer C. Waters and Wendy C. Salmon at the Harvard Medical School
Nikon Imaging Center (Boston, MA) for assistance with confocal fluorescence
microscopy. This work was supported by National Institutes of Health Grant
R01 GM086258 (to J.C.); National Institutes of Health Pathway to Indepen-
dence Award (Grant 1K99 GM097096-01, to J.M.C.); a Damon Runyon Cancer
Research Foundation postdoctoral fellowship (DRG-2002-09, to J.M.C.); and
the Swiss National Science Foundation (PBELP2-130880, to C.P.).
Preparation of Cells for Confocal Fluorescence Microscopy. X. nematophila
ΔisnAB was grown overnight in suspension (5 mL of LB) from a single colony
at 30 °C and 250 rpm. Five-mL cultures (2-g tryptone, 5-g yeast extract, and
10-g NaCl per L, and 0.1 M of L-proline) were inoculated with 150 μL of the
overnight culture and were grown at 30 °C and 250 rpm for 6 h before adding
25 μL of DMSO stock solution of 12, 13, or blank (100 μM final). The cultures
were grown for 72 h at 30 °C and 250 rpm, where 25 μL of DMSO or azide 12
or 13 stock solutions were added after 29 and 56 h. Aliquots of 500 μL were
centrifuged at 4;500 × g for 5 min and the cells were then washed with
500 μL PBS and centrifuged. The cells were then reacted with PEG4 carbox-
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