Acyl Histidines: New N-Acyl Amides from Legionella pneumophila

Article abstract of DOI:10.1002/cbic.201600618

Legionella pneumophila, the causative agent of Legionnaires' disease, is a Gram-negative gammaproteobacterial pathogen that infects and intracellularly replicates in human macrophages and a variety of protozoa. L. pneumophila encodes an orphan biosynthetic gene cluster (BGC) that contains isocyanide-associated biosynthetic genes and is upregulated during infection. Because isocyanide-functionalized metabolites are known to harbor invertebrate innate immunosuppressive activities in bacterial pathogen–insect interactions, we used pathway-targeted molecular networking and tetrazine-based chemoseletive ligation chemistry to characterize the metabolites from the orphan pathway in L. pneumophila. We also assessed their intracellular growth contributions in an amoeba and in murine bone-marrow-derived macrophages. Unexpectedly, two distinct groups of aromatic amino acid-derived metabolites were identified from the pathway, including a known tyrosine-derived isocyanide and a family of new N-acyl-l-histidine metabolites.

Full text of DOI:10.1002/cbic.201600618

Accepted Article
Title: Acyl-histidines: New N-acyl amides from Legionella pneumophila
Authors: Thomas Tørring, Stephanie R Shames, Wooyoung Cho, Craig
R Roy, and Jason Crawford
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Acyl-histidines: New N-acyl amides from Legionella pneumophila
Thomas Tørring[a], Stephanie R. Shames[b], Wooyoung Cho [c,d], Craig R. Roy [b] and Jason M.
Crawford*[b,c,d]
Abstract: Legionella pneumophila, the causative agent of
Legionnaires’ disease, is a Gram-negative Gammaproteobacterial
pathogen that infects and intracellularly replicates in human
macrophages and a variety of protozoa. L. pneumophila encodes an
orphan biosynthetic gene cluster (BGC) that contains isocyanide-
associated biosynthetic genes and is upregulated during infection.
Because isocyanide-functionalized metabolites are known to harbor
invertebrate innate immunosuppressive activities in bacterial
pathogen-insect interactions, we used pathway-targeted molecular
networking and tetrazine-based chemoseletive ligation chemistry to
characterize the metabolites from the orphan pathway in L.
pneumophila. We also assessed their intracellular growth
contributions in an amoeba and in murine bone marrow derived
macrophages. Unexpectedly, two distinct groups of aromatic amino
acid-derived metabolites were identified from the pathway, including
a known tyrosine-derived isocyanide and a family of new N-acyl-L-
histidine metabolites.
admission to an intensive care unit, and with a total mortality
rate of approximately 9%.[2] Infection normally occurs through
inhalation of contaminated aerosols from hot tubs, hot water
tanks, or cooling towers.
In macrophages, L. pneumophila is taken up by phagocytosis,
but the bacterium employs a type 4 secretion system known as
Dot/Icm
(defect
in
organelle
trafficking/
intracellular
multiplication) to prevent the normal fusion with lysosomes and
create a specialized Legionella-containing vacuole (LCV). L.
pneumophila is predicted to employ a staggering number of
more than 300 effector proteins during infection.[3,4] The
remaining effector proteins are hypothesized to stem from the
broad range of hosts infected by L. pneumophila. This is also
supported by a high degree of functional redundancy where
single gene deletion mutants rarely result in significant
intracellular growth defects.[3,4] While the protein effectors have
been intensely studied over the past decade, few secondary
metabolites have been thoroughly investigated in Legionella
strains. A recent survey described several predicted biosynthetic
gene clusters (BGCs) encoding polyketide synthases (PKSs)
and nonribosomal peptide synthetases (NRPSs) spread among
various Legionella spp. As part of that study, legionellol, a
polyketide-derived surfactant that affected sliding mobility was
identified.[5] Other secondary metabolites from Legionella include
the siderophore legiobactin[6] and the quorum sensing auto-
inducer LAI-1.[7] We were intrigued by a BGC in L. pneumophila
containing the isocyanide-associated genes isnA and isnB
because previous work has demonstrated that isocyanide-
containing secondary metabolites dramatically affect innate
immunosuppression in an invertebrate animal infection model,
Galleria mellonella.[8] Given the fact that the isnA and isnB
genes are clustered in various human pathogens, we aimed to
elucidate the structures of the secondary metabolites encoded
by this BGC using heterologous expression, a chemoselective
tetrazine probe, gene deletion in wild-type L. pneumophila, and
intracellular growth in both amoebae and macrophages.
Unexpectedly, the BGC produced two distinct groups of
secondary metabolites derived from aromatic amino acid
substrates, one of which constitutes the first metabolite
members of histidine-derived N-acyl amides. N-acyl-amides are
well known in lipidomics and chemical signaling where the acyl-
groups are often appended via an acyl-transferase.[9]
Interestingly, the biosynthesis of N-acyl-histidines was not
derived from an acyl-transferase, but rather an ATP-grasp
enzyme.
Introduction
Most commonly referred to as an accidental human pathogen,
Legionella pneumophila thrives in both anthropogenic and
environmental water systems where it is found replicating as an
intracellular pathogen in protozoa such as amoebae.[1] L.
pneumophila produces a wide variety of often functionally
redundant virulence factors enabling it to infect a spectrum of
protozoa. This host diversity has facilitated its ability to infect
and intracellularly replicate in human macrophages.[1] Human
infection results in either a mild influenza-like disease called
Pontiac fever or the much more severe pneumomia termed
Legionellosis (Legionnaires’ disease). In the United States alone,
for example, about 5000 individuals are diagnosed with
Legionellosis every year, roughly half of which required
[a]
[b]
Dr. T. Tørring
Interdiscplinary Nanoscience Center, Aarhus University
8000 Aarhus C, Denmark
Dr. S. R. Shames, Prof. C. R. Roy, Prof. J. M. Crawford
Department of Microbial Pathogenesis, Yale School of Medicine,
New Haven, CT, USA
E-mail: jason.crawford@yale.edu
[c]
[d]
W. Cho, Prof. J.M. Crawford
Department of Chemistry, Yale University,
New Haven, CT, USA
W. Cho, Prof. J.M. Crawford
Chemical Biology Institute, Yale University,
West Haven, CT, USA
Supporting information for this article is given via a link at the end of
the document.
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efflux transporter (lpg0177), and an ATP-grasp protein (lpg0178).
Interestingly, the gene cluster is up-regulated at the
transcriptional level during intracellular bacterial growth in
human macrophages,[15] suggesting a potential role in infection.
lpg0173 lpg0174 lpg0175
lpg0176
lpg0177
lpg0178
lpg0179
lpg0173 - LysR regulator
lpg0175 - IsnB
lpg0174 - IsnA
lpg0176 - FAD-dependent monooxygenase
lpg0178 - ATP grasp
lpg0177 - major facilitor transporter
lpg0179 - methyltransferase
Pathway-targeted analysis pinpoints new acyl-histidines in
L. pneumophila.
We first cloned the operon lpg0174-lpg0178 (LegA) and the O-
methyltransferase lpg0179 (LegB) downstream of two
independent isopropyl-β-D-1-thiogalactopyranoside (IPTG)-
inducible T7 phage promoters in pETDuet (pLegAB, Supporting
Figure 1, Supporting Table 1). The construct was transformed
into E. coli BAP-1[16] for heterologous expression and metabolite
analysis. The cultures were grown in lysogeny broth (LB)
supplemented with ampicillin under aerobic conditions and at a
temperature of 30°C. n-Butanol extracts of 5 mL cultures of
induced E. coli BAP-1 carrying either pLegAB or its empty vector
control were analyzed by comparative metabolomics and a
Figure 1. Acyl-histidine and isocyanide biosynthetic gene cluster. Biosynthetic
genes are colored red. Regulatory and transport genes are colored blue.
Results and Discussion
L. pneumophila encodes an “orphan” isocyanide
biosynthetic gene cluster.
Homologs of isocyanide biosynthetic genes, isnA (lpg0174) and
isnB (lpg0175),[10-12] in L. pneumophila (subsp. pneumophila
strain Philadelphia 1)[13] are clustered on a predicted five-gene
operon (lpg0174-lpg0178, Figure 1). The operon is flanked by a
putative lysR-family transcriptional regulator (lpg0173), which is
molecular
networking
technique,[17]
“pathway-targeted”
molecular networking.[18,19] Molecular networking allows for
metabolite connections based on tandem MS fragmentation
relationships, and pathway-targeted molecular networking maps
only the ions dependent on the presence of the pathway. Our
pathway-targeted analysis of the E. coli strain in positive ion
detection mode resulted in one distinct molecular family that was
also detected in wild-type L. pneumophila (Figure 2, Supporting
Figure 2). Two abundant metabolite ions in this molecular family,
322.2130 and 310.2119, were targeted for isolation and
likely involved in its regulation, and
a
predicted O-
methyltranferase (lpg0179). L. pneumophila strain Corby
encodes an orthologous pathway, and a transposon mutant in
the O-methyltransferase (lpc0263) was reported to lead to a
defect in the strain’s natural lysozomal avoidance phenotype,
although the mutant phenotype was not verified by genetic
complementation.[14] In addition to the isocyanide biosynthetic
genes in the operon, it encodes homologs of a FAD-dependent
oxidoreductase (lpg0176), a major facilitator superfamily (MFS)
structural elucidation.
Their high-resolution masses were
consistent with the molecular formulas, C17H27N3O3 (calc, M+H+,
322.2125) and C16H27N3O3 (calc, M+H+, 310.2125), respectively.
The metabolites were extracted from a large-scale culture (6 L)
with n-butanol and were purified by a series of normal- and
reversed-phase chromatographic steps guided by mass
spectrometry, resulting in the isolation of new N-acyl-L-histidine
metabolites 1 (<1 mg) and 2 (2 mg). Their planar structures were
determined through one- (1H) and two-dimensional (gCOSY,
gHSQCAD, and gHMBCAD) NMR experiments (Supporting
Tables 2-3). The acyl groups of these major metabolites
featured a straight-chain decanoyl-moiety (2) and a rare β,γ-
cyclopropanated decanoyl-moiety (1), supporting a biosynthetic
preference for C10-chain-lengths.
a)
310.21
322.21
321.144
282.18
297.218
331.201
254.151
N
N
b)
NH
OH
NH
OH
O
H
O
H
N
H
N
H
O
N
(1) - 322 m/z - I
(2) - 310 m/z - I, F, SO
N
The absolute configurations of acyl-histidines 1 and 2 were
supported by chemical degradations, 1H-NMR resonance
signatures, and bacterial genetics. Acid hydrolysis of 1 and 2
and subsequent Marfey’s analysis[20] established S-
configurations in their histidine moieties (Supporting Figure 3).
This completed the structure of 2 as N-decanoyl-L-histidine. The
cyclopropane ring system in 1 can be assigned as cis based on
interpretation of the NMR data and the signature negative
chemical shift value of the methylene proton cis to the alkyl
substituents (Figure 3b).[21,22]
NH
NH
OH
O
O
OH
N
H
N
H
(4) - 254 m/z -OF
O
(3) - 282 m/z - F,S
Figure 2. Pathway-targeted molecular networking. a) Red color nodes mark
molecular ions observed in both E. coli expressing the pathway and L.
pneumophila. Blue color is associated with ions that could only be observed in
the E. coli strain. Nodes are interconnected by tandem-MS relationships.
Metabolomic analysis was conducted in positive ion mode. b) New N-acyl-L-
histidine metabolites (1-4) identified and characterized in this study. I, isolated;
F, confirmed by free fatty acid feeding studies; and S, confirmed by synthesis.
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NH3
NH3
a)
c)
O
O
P
O
P
O
O
O
100000
50000
0
322 m/z
O
O
O
O
O
O
O
O
O
O
cfa
H
H
19
19.5
20
300000
200000
100000
0
310 m/z
b)
N
NH
OH
H H
O
H
H
N
19
19.5
20
H
O
E. coli
E. coli cfa mutant
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
Figure 3. Biosynthesis and stereochemical assignment of cyclopropropanated N-acyl-L-histidine 1. a) Cfa-catalyzed SAM-dependent methylation of cis-
unsaturated fatty acid moiety in cyclopropanated phospholipid biosynthesis. b) 1H-NMR spectrum supporting a cis-configuration of the cyclopropane moiety. The
characteristic chemical shift value supporting a cis conformation is highlighted in the red box. c) LC-MS extracted ion count (EIC) chromatograms of E. coli wild-
type (red) and cfa mutant (blue) containing the dual acyl-histidine-isocyanide biosynthetic gene cluster.
We hypothesized that this acyl-group was derived from an
endogenous cyclopropanated decanoic acid. Cyclopropanation
of unsaturated fatty acids in E. coli is observed on existing
phospholipids. This reaction is carried out by a SAM-dependent
methyltransferase, cyclopropane fatty acid synthase (CFAS)
(Figure 3a). To determine whether the cyclopropane moiety in 1
originates from CFAS, we introduced the pathway (pLegAB) and
pTARA:500[23] (a plasmid with an arabinose-inducible expression
system for the T7 phage RNA polymerase) into E. coli BW25113
and its corresponding cfa mutant strain.[24] n-Butanol extracts
from both strains were analyzed using LC-MS. While the
production of 2 with a straight-chain fatty acid was only slightly
lower in the cfa mutant, the production of 1 was completely
abolished (Figure 3c). This experiment allowed us to assign the
cfa-dependent biosynthetic origin of the cyclopropanated acyl-
group in 1 and support a 3S, 4S stereochemical assignment.[25]
The gene, lpg1131 in L. pneumophila, encodes a CFAS
homolog (protein identity/similarity: 54%/71%), and we therefore
find it likely that 1 is produced in a similar manner in L.
entering stationary phase,[26] the cyclopropanated substrate is
likely recruited from phospholipid catabolism.
The chromatographic behaviors, tandem MS fragmentations,
predicted molecular formulas, and pathway-targeted molecular
network data for the remaining detectable ions 308.1962 (5),
282.1804 (3), and 254.1508 (4) suggested additional N-acyl-L-
histidine analogs, featuring decenoyl-, octanoyl-, and hexanoyl-
side chains. Synthetic standards of 2, 3, and 4 were prepared.
The synthetic standards shared identical retention times, high-
resolution masses, and fragmentation patterns with their
corresponding
assignments.
natural
products,
confirming
structural
pneumophila.
Considering
that
membrane-associated
phospholipids represent the known substrates for CFAS, the
cyclopropanated substrate is likely recruited from the
phospholipid catabolism which is a typical occurance in cells
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a)
b)
E. coli
L. pneumophila
4000000
3000000
2000000
1000000
0
2
2
400000
200000
0
1
3000000
2000000
1000000
0
1
80000
40000
0
7
7.5
8
8.5
9
9.5
10
7
7.5
8
8.5
9
9.5
10
Lpg174-178
lpg178 deleted
L. pneumophila wildtype
lpg178 mutant
Figure 4. Genetic dependency of lpg0178 on N-acyl-L-histidine biosynthesis. a) Extracted ion chromatograms of the 310 m/z and 322 m/z from n-butanol extracts
of E. coli grown in lysogeny broth containing the biosynthetic gene cluster with (red) and without (blue) the gene encoding the ATP-grasp enzyme lpg0178. b)
Extracted ion chromatograms of the 310 m/z and 322 m/z from n-butanol extracts of L. pneumophila grown in buffered charcoal yeast extract containing the
biosynthetic gene cluster with (green) and without (yellow) the gene encoding the ATP-grasp enzyme lpg0178. Tandem-MS spectra of the compounds can be
viewed in Supporting Figures 15 and 16. Co-injection experiments of 1 from E. coli and from L. pneumophila support an identical structure (Supporting Figure 6).
a)
biosynthesis (Figure 4a, Supporting Figure 5). Similarly, we
analyzed n-butanol extracts of wild-type L. pneumophila and a
N
N
C
N
N
N
N
N
series of transposon mutants in the pathway covering every
gene (lpg0173 through lpg0179). Both 1 and 2 could be
observed in wild-type L. pneumophila (Figure 4b) and a co-
injection of extracts supports an identical stereoconfiguration of
1 (Supporting Figure 6). The metabolites were detected in the L.
pneumophila transposon mutants (albeit at lower intensities)
except for mutations in the ATP-grasp gene, in which they were
completely abrogated (see Supporting Figure 7). Mutants in the
methyltransferase lpg0179 located in the gene cluster (not the
CFAS homolog) showed production of both 1 and 2 at intensities
comparable to wild-type, indicating that lpg0179 was not
involved in the cyclopropanation reaction (see Supporting Figure
8). These experiments validate the function of the ATP-grasp
protein and its involvement in N-acyl-L-histidine biosynthesis in L.
pneumophila. Based on protein homologies, the ATP-grasp
protein (PF13535) likely functions as an ATP-dependent ligase,
recruiting free medium-chain-length fatty acids from primary
metabolism and linking them to free L-histidine substrates. This
biosynthetic strategy is in stark contrast not only to the SAM-
derived biosynthesis of acyl-homoserine lactones,[27] but also to
the N-acyl transferase family (PF13444) responsible for
N
N
+
N
HO
N
6
N
HO
b)
0.2
Legionella isnA and isnB
0.2
0.1
pETDuet background
0.1
0
0
3
Vibrio isnA and isnB
0.2
Photorhadus isnA and isnB
2
1
0
0.1
0
6.5
7
7.5
6.5
7
7.5
synthesizing
N-acyl-tyrosines,
N-acyl-arginines,
and
commendamide.[28] In further support of this notion,
supplementation of the culture medium with free C6-, C8-, and
C10-chain length fatty acids led to dramatically enhanced
production of their corresponding N-acyl-L-histidines (Supporting
Figure 9). Unsaturated C10-fatty acids with an α/β-cis- (a known
Pseudomonas biofilm inhibitor[29]), an α/β-trans-, and a β/γ-cis-
double bond were also readily accepted leading to their N-
decenoyl-L-histidines. Lastly, it is worth noting that L-histidine
biosynthesis is up-regulated during intracellular bacterial growth
in human macrophages.[15]
Figure 5. Trapping genetically encoded isocyanide metabolites with
a
tetrazine probe. a) Chemoselective reaction between an isocyanide (known
from P. luminescence and V. cholerae) and a tetrazine probe. b) EICs of the
resulting trapped isocyanide (354.135 m/z) in E. coli with a pETDuet negative
control background, the dual isocyanide / N-acyl-L-histidine biosynthetic gene
cluster (lpg0174-lpg0178) from L. pneumophila, isnA-isnB from V. cholerae,
and isnA-isnB from P. luminescens.
To determine the genetic dependencies on metabolite
production, we constructed individual deletions of every
biosynthetic gene in the pathway (Δlpg0174, Δlpg0175,
Δlpg0176, Δlpg0178, and Δlpg0179) for heterologous production
in E. coli BAP-1 (Supporting Figure 4). The ATP-grasp gene
(lpg0178) was the only gene required for N-acyl-L-histidine 1-5
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A tetrazine probe identifies an isocyanide metabolite
Bacterial genetics enabled preliminary interrogation of
pathway function
The N-acyl-L-histidines described above do not support the
presence of isocyanide biosynthetic genes in the gene cluster.
To identify isocyanide-functionalized metabolites, we designed a
general isocyanide detection scheme using established
tetrazine-coupling chemistry. Tetrazines have been shown to
chemoselectively react with isocyanides in a [4+1] cycloaddition
reaction followed by release of N2 even in complex cellular
enviroments.[30] We found that addition of 3,6-di-2-pyridyl-
1,2,4,5-tetrazine and methanol to either M9 medium containing
casamino acids or bacterial cultures in the same medium (Vibrio
cholerae O1 biovar El Tor N16961 was used for method
Based on observation of the up-regulation of this gene cluster
during intracellular infection, we examined the impact of
isocyanide and acyl-histidine production on intracellular growth
of L. pneumophila in murine bone marrow-derived macrophages
(BMDMs) and the natural protozoan host Acanthamoeba
castellanii. NLRC4-/- BMDM, which are permissive to infection
with flagellated L. pneumophila,[32] and A. castellanii were
infected with wild-type (SRS43), lpg0174::Tn, or lpg0178::Tn
mutant L. pneumophila followed by enumeration of colony
forming units (CFUs) recovered over 72 hours. We observed a
significant defect in intracellular replication of the lpg0178::Tn
mutant compared to wild-type L. pneumophila in NLRC4-/-
BMDM (*P < 0.05, **P < 0.01, 13). The lpg0178::Tn mutant also
exhibited a subtle but significant growth attenuation in A.
castellanii (*P < 0.05, **P < 0.01, Supporting Figure 13). We
subsequently examined the impact of isocyanide production on
intracellular replication of L. pneumophila and found that the
lpg0174::Tn mutant could replicate to the same extent as the
wild-type strain in NLRC4-/- BMDM and A. castellanii (Supporting
Figure 13). This prompted us to construct clean deletions of
lpg0178 and of the entire lpg0173-lpg0179 gene locus (∆locus)
to verify the phenotype observed for the transposon mutants.
However, we were unable to distinguish between wild type and
the nonpolar lpg0178 or ∆locus mutants (data not shown),
suggesting that the prior transposon insertion led to deleterious
polar or non-specific effects on the pathogen. Our experiments
may call into question the reported transposon-mediated
inactivation of lpc0263 (ortholog to lpg0179) and its negative
effect on virulence in the related strain, L. pneumophila
Corby.[14]
development) could trap
a
supplemented isocyanide-
functionalized molecule (4-methoxyphenyl isocyanide) with
lower than 1 µM detection limits even on a simple single
quadrupole LC/MS (Supporting Figure 10). This adduct could
readily be extracted with n-butanol and identified by its signature
UV-visible spectrum and product mass by LC/MS, supporting a
general route for isocyanide-functionalized metabolite discovery.
With the LC/MS-based isocyanide-detection scheme, we then
screened E. coli heterologously expressing the known
isocyanide biosynthetic genes (isnA and isnB) from
Photorhabdus luminescens and Vibrio chlolerae.[8,31] We
compared these cultures to E. coli harboring the orphan L.
pneumophila pathway (lpg0173-lpg0179). In all cases, only a
single tetrazine-isocyanide adduct was detected in positive ion
mode, which matched the known isocyanide 6 (Figure 5). With
this knowledge, we extracted unreacted cultures with ethyl
acetate and we were able to identify free isocyanide 6 in the
cultures using negative ion mode mass spectrometry
(Supporting Figure 11). This molecule represents a nanomolar-
level inhibitor of phenoloxidase,
a critical component of
invertebrate innate immune systems.[8] While the isocyanide
could readily be identified in the heterologous production
cultures, 6 was not detected in wild-type L. pneumophila under
the conditions of our experiment. In wild-type L. pneumophila,
the isocyanide biosynthetic genes may be functionally “silent”
under our cultivation conditions or 6 is further processed to other
currently undetectable metabolites. While looking for other
potential isocyanide metabolites, we determined that the
tetrazine probe formed an adduct with palmitoleic acid
(Supporting Figure 12). This off-target reactivity on a common
precursor should be generally considered in the various
applications of tetrazine probes, particularly in “bioorthogonal”
applications. Collectively, our probe-based experiments confirm
that L. pneumophila has the genetic potential to produce
isocyanide products/intermediates and that the single orphan
gene cluster encodes not one, but two distinct types of aromatic
amino acid-derived natural products, N-acyl-L-histidines and the
tyrosine-derived isocyanide. The clustered organization of genes
suggests that there is a possible benefit to the coordinate
transcriptional regulation of these distinct families of natural
products in L. pneumophila.
We also investigated whether the BGC had any effect on in vitro
bacterial growth, survival in water, and biofilm formation relative
to wild-type. However, no significant changes were observed
under the conditions of our experiments. Finally, representative
acyl-histidine 2 was inactive against yeast (Saccharomyces
cerevisiae, no zone of inhibition around discs loaded with 100 µg
of 2) and bacteria (E. coli, Staphylococcus aureus, Enterococcus
faecalis, and Pseudomonas aeruginosa, no zone of inhibition
around discs loaded with 50 µg of 2). Well-known N-acyl amides,
such as anandamide, are known to play key roles in human
receptor-mediated cell signaling.[33] Recently Brady and
coworkers described an N-acyl amide – commendamide – from
a human commensal bacteria that activated a NF-κB dependent
gene reporter assay in HEK293 cells.[28] Consequently, we
screened 2 for NF-κB activation in HEK293 cells (up to 100
µg/ml of 2), but we did not observe an effect. The pathway’s
specific role(s) could be functionally redundant with one or more
of the many other Legionella virulence factors encoded in the
genome, and/or the pathway regulates a currently unexamined
cellular phenotype(s). These functional contributions remain
subjects of future investigations.
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pETDuet™-1 was also transformed into E. coli BAP-1 cells to
serve as control background for metabolomic analysis.
Conclusions
In conclusion, we identified a BGC in L. pneumophila that
produces two distinct groups of aromatic amino acid-derived
secondary metabolites, one of which, to the best of our
knowledge represents the first N-acyl amide metabolites
containing L-histidine. Moreover, we observed incorporation of a
rare cyclopropane-containing fatty acid that is likely recruited
through endogenous phospholipid catabolism. The enzyme
responsible for N-acylation, lpg0178 from the ATP-grasp family
(PF13535) represents an alternative to the N-acyl transferase
family (PF13444) responsible for synthesizing N-acyl-tyrosines,
N-acyl-arginines, and commendamide through preactivated
carboxylates[28] or the transacylation from glycerophospholipids
employed in the biosynthesis of the well-known human N-acyl
amide anandamide.[34] The biosynthetic genes for the new N-
acyl-histidine metabolites reported here are up-regulated during
intracellular L. pneumophila macrophage infection, lending
further support to the general notion that structurally diverse N-
acyl amides could regulate a variety of human cellular signaling
programs.
Construction of deletion mutants for heterologous
expression.
1) pELegAB::Δlpg0174: The genes lpg0175-lpg0178 were
amplified using the Phusion HF polymerase according to the
manufacturer’s protocol with primers Leg-Δ174-5 and Leg-A3
(Supporting Table 3). The product was digested with NcoI and
SacI and ligated into the same sites in pLegB.
2)
pELegAB::Δlpg0175: The upstream (lpg0174) and downstream
(lpg0176-0178) regions of lpg0175 were amplified by PCR
using primer pairs Leg-A5/Leg-Δ175-3 and Leg-Δ175-5/Leg-A3.
The two products were purified and used as templates in an
overlap extension PCR with primers Leg-A5 and Leg-A3. The
full-length product was digested with NcoI and SacI and ligated
into the corresponding sites in pLegB. 3) pELegAB::Δlpg0176:
The lpg0176 gene was deleted from the pLegAB plasmid using
a quick-change-like PCR protocol using the primers Leg-Δ176-5
and Leg-Δ176-3. After PCR amplification, the parental plasmid
was digested with DpnI. The product was gel purified and
transformed into chemically competent E. coli DH5α. The
deletion was validated by sequencing and the plasmid was
transformed into chemically competent E. coli BAP-1 for analysis.
4) pELegAB::Δlpg0178: Genes lpg0174-lpg0177 were amplified
using the Phusion HF polymerase and primers Leg-A5 and Leg-
Δ178-3. The product was digested with NcoI and SacI and
ligated into the same sites in pLegB. All deletion constructs were
validated by sequencing and were transformed into chemically
competent E. coli BAP-1 for analysis. Vector maps of these
constructs are shown in Supporting Figure 4.
Experimental Section
Bacterial strains and growth conditions.
Legionella pneumophila (subsp. pneumophila strain Philadelphia
1) was cultured on N-(2-Acetamido)-2-aminoethanesulfonic acid
(ACES)-buffered charcoal yeast extract (CYE) and grown at
37˚C as described.[35] All L. pneumophila strains were grown in
the presence of 100 µg mL-1 streptomycin (Sigma-Aldrich).
When necessary, media were supplemented with 5 µg mL-1
chloramphenicol (Sigma-Aldrich). Legionella pneumophila strain
SRS43 was generated by allelic-exchange mutagenesis to
incorporate wild-type thyA into the Lp02 thymidine auxotroph.[36]
Transposon mutants in lpg0173-lpg0179 were generated by
Construction of L. pneumophila deletion mutants
Clean deletions of lpg0178 and lpg0173-lpg0179 were
generated by allelic-exchange mutagenesis as previously
described.[38] For the lpg0178 deletion, 5’ and 3’ flanking regions
were amplified using primer pairs 0178KO-5F/0178KO-5R and
0178KO-3F/0178KO-3R, respectively. For the lpg0173-lpg0179
deletion, 5’ and 3’ flanking regions were amplified with locusKO-
5F/locusKO-5R and locusKO-3F/locusKO-3R, respectively. The
digested PCR products were ligated into pSR47s[39] to create
deletion constructs pSR47s::∆lpg0178 or pSR47s::∆locus, which
were maintained in E. coli DH5αλpir. Deletion constructs were
introduced into wild-type L. pneumophila by conjugation.
Chromosomal deletions were confirmed by PCR.
electroporation of pSRS_Cm1,
derivative of pSAM_Bt[37]
into SRS43 and selection of
transposon mutants on CYE supplemented with
chloramphenicol.
a chloramphenicol resistant
,
Construction of plasmids for heterologous expression.
Genomic DNA from L. pneumophila was used as a template for
PCR reactions, using the Phusion HF polymerase according to
the manufacturer’s protocol with primers Leg-A5 and Leg-A3 or
LegB5 and LegB3 (Supporting Table 1). The amplification
products were purified using a PCR clean-up kit (Macherey-
Nagel). The LegA PCR product was digested with NcoI and SacI
and ligated into the corresponding sites in pETDuet™-1
(Novagen) to generate pLegA. The LegB PCR product was
digested with NdeI and XhoI and ligated into the corresponding
sites in pETDuet™-1 to generate pLegB. To generate pLegAB,
the LegA PCR product was digested with NcoI and SacI and
ligated into the same sites in pLegB. Vector maps are shown in
Supporting Figure 1. All constructs were validated by restriction
analysis and sequencing and were transformed into chemically
competent E. coli BAP-1 cells for analysis. The parental plasmid
In vitro L. pneumophila assays
Replication of L. pneumophila wild-type and ∆locus in broth
culture was performed as previously described.[40] The OD600
was measured over 24 hours.
For bacterial survival in water, L. pneumophila wild-type and
∆locus were resuspended from a 2 day heavy patch in defined
water medium (50 mg/L NaCl, 50 mg/L KCl, 20 mg/L KH2PO4,
pH 6.9). Bacteria were seeded into a 6-well plate and CFUs
were enumerated every 7 days for one month as described.[41]
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The ability of L. pneumophila wild-type or ∆locus to form biofilms
in the presence of the Pseudomonas biofilm inhibitor cis-2-
decenoic acid (Sigma) was assayed. Briefly, bacteria were
harvested from 2 day heavy patches into AYE media and diluted
to OD600 = 2.0 Strains were aliquoted into sterile 96-well U-
bottom plates and incubated at 25˚C for 6 hours. Supernatant
and non-adherent cells were aspirated and AYE supplemented
with 0, 1, 10, 100, or 1000 nM cis-2-decenoic acid was added to
each well (n=8) and plates were incubated at 25˚C for 7 days.
Non-adherent cells and supernatants were aspirated and
biofilms were assayed as described.[42]
was used for subsequent tandem mass spectrometry (MS2)
using targeted auto-MS2 mode. MS2 data were acquired in
positive mode from 80-1500 m/z at a ms/ms scan rate of 1
spectra/sec with fixed collision energies of 10, 20 and 40. The
same UPLC method and column conditions were used as
described above. The MS2 data files were used to build mass
spectral networking files using the open source software
platform Cytoscape version 3.1.0 (http://www.cytoscape.org).
Clusters were built based on cosine cutoff of 0.7. Routine
screening was performed by monitoring the desired masses on
an Agilent 6120 Infinity series quadrupole mass spectrometer
coupled to an Agilent 1260 series HPLC system. The mass
spectrometer was operated with an atmospheric pressure
electrospray ionization (API-ES) source in positive ion mode.
LC-MS chromatographic analysis was performed over a Kinetex
C18 HPLC column (250 x 4.6 mm, 100Å, 5 µm particle size,
Phenomenex) with a H2O:MeCN gradient at 0.5 mL/min: 0-2 min,
5% MeCN; 2-30 min, 5 to 98% MeCN; 30-38 min, hold at 98%
MeCN.
Solvent extraction and identification of pathway-dependent
metabolites.
LB agar plates with ampicillin (100 µg/ml) were streaked with
glycerol stocks of E. coli BAP-1 with pETDuet™-1 or pLegAB.
After incubation overnight at 37°C, single colonies were used to
inoculate LB liquid cultures (5 ml with ampicillin) in triplicates (or
quadruplicates). After overnight growth (37°C, 250 rpm), these
samples were used as starter cultures to inoculate fresh LB
liquid cultures in triplicates (or quadruplicates). These cultures
were grown to approximately OD600 = 0.4, at which time they
were cooled on ice and induced with IPTG (100 µM). The
induced cultures were grown at 30°C and 250 rpm for 2 days.
Determination of cyclopropane dependency on cfa.
To determine whether the cyclopropane group in 1 is dependent
on cfa, we transformed the Keio parent E. coli strain
(BW25113)[43] and its cfa mutant (JW1653-1)[24] with pLegAB
The cultures were vigorously extracted with n-BuOH (6 ml added, and pTARA:500 (pTara:500 was a gift from Matthew Bennett,
4 removed), and the organic fraction was reduced in vacuo, re-
dissolved in MeOH, and analyzed using a LC-HR-ESI-QTOF-MS
(Agilent iFunnel 6550 system) and a single-quadrupole LC-MS
(Agilent 6120 system). This protocol was selected as a result of
preliminarily screening a number of cultivation conditions: IPTG
concentrations (0.1, 0.25, 0.5, and 1.0) or autoinduction growth
conditions, temperature (20, 25, 30, and 37°C), base media
(M9+casamino acids and LB), and time (1, 2, and 3 days). The
pathway-dependent metabolites were identified by high-
resolution mass spectrometry using an electrospray ionization
(ESI) source on an Agilent iFunnel 6550 quadrupole time-of-
flight mass spectrometer (Q-TOF-MS) coupled to an Agilent
Infinity 1290 HPLC using a Phenomenex Kinetex C18 column
(100 x 2.10 mm, 1.7 µ, 100Å). UPLC conditions consisted of a
H2O:acetonitrile (MeCN) gradient containing 0.1% formic acid:
0-2 min, 5% MeCN; 2-18 min, 5 to 98% MeCN; hold for 5 min,
98% MeCN. Column temperature was set at 25°C and solvent
flow was set at 0.3 mL/min. Mass spectra were acquired using
Dual Agilent Jet Stream (AJS) ESI in positive mode scanning
from 50-1700 m/z at 1.00 spectra/second. The capillary and
nozzle voltages were set at 3500V and 1000V, respectively. The
source parameters were set with a gas temperature at 225°C
and flow at 12 L/min, nebulizer at 50 psig, sheath gas
temperature at 275°C and flow at 12 L/min. MS data were
acquired with MassHunter Workstation Data Acquisition and
processed with MassHunter Qualitative Analysis (Agilent
Technologies). The centroid MS data was processed with
MassHunter Qualitative Analysis and statistically analyzed using
MassHunter Mass Profiler Professional (Agilent Technologies).
A list of unique ions associated with pLegAB was generated by
removing the MOFs found in at least one of the control
replicates (pETDuet) and only keeping those that were present
in all four experimental biological replicates. This inclusion list
Addgene plasmid #60717).[23] Wild type and mutant cultures
were analyzed as described under “Solvent extraction and
identification of pathway-dependent metabolites” with the
exception of a longer post-induction incubation period (one
week).
Testing antimicrobial activity.
The antibacterial effect of 2 and 3 was investigated using
synthetic materials against E. coli BAP1, Staphylococcus
aureus DSM
20231, Enterococcus
faecalis DSM
20478,
and Pseudomonas aeruginosa PAO1 DSM 19880. Glycerol
stocks of all four strains were streaked on LB agar plates and
grown overnight at 37°C. These cells were used to initiate starter
cultures in LB, which were grown overnight at 37°C and 200 rpm.
The starter cultures were used to inoculate fresh 5 ml LB liquid
cultures, and these were grown until approximately OD600 = 0.5.
Aliquots (200 µl) were then spread uniformly on Mueller Hinton
Agar plates. Discs were prepared as blank, 20 µg 3, 50 µg 3, 20
µg 2, and 50 µg 2. The disc assays were monitored after
overnight growth at 37°C.
N-acyl-histidine effects on yeast were investigated using
representative 2 (20, 50, and 100 µg loaded on paper discs)
against S. cerevisiae strain DSY-5 (MATalpha leu2 trp1 ura3-52
his3::GAL1-GAL4 pep4 prb1-1122). Aliquots (100 µl) from an
overnight starter culture in YP broth 1:1, 1:10, and 1:100 were
spread uniformly onto YP agar plate. After application of the
discs, the plates were incubated overnight at 28°C.
Testing NF-κB activation
The reporter cell line used to screen 2 for NF-κB activation
(HEK-BlueTM Null2-k; Invivogen, San Diego, California, USA) is a
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human embryonic kidney (HEK 293) cell line transfected with an
NF-κB/AP-1-SEAP (secreted embryonic alkaline phosphatase)
reporter gene. HEK-BlueTM Null2-k was maintained according to
the manufacturer’s instructions. Two-fold serial dilutions of the
positive control, human TNF-α (100 ng/mL in DMSO; Cell
Signaling Technology, Danvers, Massachusetts, USA) and of
synthetic 2 (100 µg/mL in DMSO) were done in triplicate in a 96-
well microplate. HEK-BlueTM Null2-k cells were grown to
approximately 80% confluence and the assay was carried out
according to the manufacturer’s protocol with HEK-BlueTM
Detection (Invivogen, San Diego, California, USA), a cell culture
medium that allows for colorimetric detection of SEAP. After 16
h of stimulation at 37°C 5% CO2, SEAP activity was measured
with Perkin Elmer EnVision 2100 Plate Reader at 630 nm.
each in triplicates) were induced with IPTG (100 µM) and grown
for two days at 30°C and 250 rpm. Then 3,6-di-2-pyridyl-1,2,4,5
tetrazine (1 mg/ml, 0.5 ml) in MeOH was added and left to react
for 1 h at 37°C. The culture was extracted with n-butanol (6 ml
added, 4 ml removed) and the organic phase was reduced in
vacuo, re-dissolved in MeOH (500 µl), and analyzed using the
method described above in “Solvent extraction and identification
of pathway-dependent metabolites.”
Isolation of (1) and (2) from E. coli.
6 x 1 L of LB liquid media with ampicillin (100 µg/ml) in 4 L
Erlenmeyer flasks were inoculated with an overnight culture
derived from a single colony. The cultures were grown at 37°C
and 250 rpm until OD600 = 0.4, at which time they were cooled
on ice and induced with IPTG (final conc. 0.1 mM). The cultures
were grown for an additional 48 h at 30°C and 250 rpm. The
cultures were combined and extracted twice with equal portions
of n-BuOH (12 L total). The combined organic extracts were
filtered, reduced in vacuo, re-dissolved in water (500 ml), and
fractionated over a C18 column (LiChroprep, RP18, 40-63 µm,
Merck). Seven fractions (500 ml each) were collected (H2O and
10%, 20%, 40%, 60%, 80%, and 100% MeOH, respectively).
LC-MS analysis identified the target masses in the 60% and
80% MeOH fractions. These fractions were combined and
evaporated in vacuo yielding 2 g of crude material. The material
was subjected to normal phase LC fractionation on a Biotage
system (DCM/MeOH, SNAP-KP-Sil 25g) using a gradient (75
ml/min, 5% MeOH, 30 sec; 5-45%, 4 min, 45-85%, 40 sec; 85%,
1min40sec). Fractions (20ml each) 2-13 contained m/z 310 and
m/z 322 and yielded 600 mg of crude material. The wide
retention window indicated acidic functional groups, and further
Feeding free fatty acids to heterologous host.
Starter cultures of E. coli BAP-1 pLegAB were prepared from
single colonies and grown overnight at 37°C and 250 rpm in LB
liquid medium supplemented with ampicillin (100 µg/ml). Fresh
cultures were inoculated from the starter cultures, and these
were grown until OD600 = 0.4. All cultures (5 ml each in
triplicates) were induced with IPTG (100 µM) and a range of free
fatty acids (decanoic acid, octanoic acid, hexanoic acid, cis-2-
decenoic acid, trans-2-decenoic acid, and cis-3-decenoic acid)
dissolved in dimethyl sulfoxide (DMSO; final supplemented fatty
acid concentration, 10 µM). A DMSO solvent control was
included as a negative control. The cultures were grown at 30°C
for two days before extracting with n-butanol. The extracts were
reduced in vacuo, re-dissolved in MeOH, and analyzed using a
Waters Acquity UPLC instrument coupled to a Bruker Q-TOF
maXis Impact mass spectrometer operated in positive
electrospray ionization (ESI+) mode. UPLC separation was
achieved on an Acquity UPLC HSS T3 column (2.1 mm x 100
mm, 1.8 µm). The column temperature was set at 50°C. Mobile
phase A: H2O with 0.1% formic acid; Mobile phase B:
MeCN/Methanol 50/50 with 0.1% formic acid. The mobile phase
was kept at 0% B for 2 min, followed by a linear gradient to 40%
B over 4 min, to 60% over 0.5 min, 88% over 4.5 min, and finally
increased to 100% over 0.5 min. The flow rate was 0.4 mL/min
and the total analysis time per sample was 21 min. The injection
volume was 10 µL, and the sample temperature was maintained
at 6°C. Calibration was performed at the beginning of each run
by natrium formate acetate (ESI+). A mass range of 50-1000
m/z and a sampling rate of 4 Hz were used. The capillary
voltage was 4000 V (ESI+). The nebulizing gas pressure was 4
bar and the drying gas flow and temperature were 11 L/min and
220°C, respectively.
purifications
were
performed
using
reversed-phase
chromatography. After preparative HPLC (H2O:MeCN, Agilent
Polaris C18-A 5µm 250 x 21.2mm) using a gradient (10 ml/min,
5% MeCN, 2 min; 5-98%, 2-30 min), fractions containing 310
and 322 ions (fractions 24-26, approx. 65% MeCN) were
reduced in vacuo yielding 43 mg. Semi-preparative RP-HPLC
(H2O:MeCN, Agilent Phenyl-hexyl 5µm 250 x 10 mm) using a
gradient (2.5 ml/min, 30% MeCN, 2 min; 30-45%, 2-30 min)
gave a mixture of the 310 and 322 ions that then could be
separated using RP-HPLC (Phenomenex Luna C8 5µ 250 x 10
mm) and an isocratic (30%) method, yielding 1 (<1 mg) and 2 (2
mg). 1D- and 2D- NMR spectra were recorded on an Agilent
600 MHz NMR equipped with
Datastation) in methanol-d
a
cold-probe (VnmrJ 3.2
.
Chemical shifts, coupling
4
constants, and annotation are listed in the supporting material.
Marfey’s analysis of (1) and (2).
Screening for isocyanides using a tetrazine probe.
The isolated metabolites 1 and 2 (approx. 100 µg each) were
dissolved in aqueous HCl (6 M) and heated to 115°C for 1 hour
and then reduced in vacuo. The residue was re-suspended in
water (0.5 mL) and lyophilized overnight to remove residual acid.
The residue was dissolved in sodium bicarbonate (1 M, 50 µl)
Starter cultures of E. coli BAP-1 with pETDuet, pLegAB,
pLegAB-Δlpg0176 or the Vibrio cholerae isocyanide biosynthetic
gene constructs, pEVC1944+pJVC1949[8], were prepared from
single colonies and grown overnight at 37°C and 250 rpm in LB
liquid medium supplemented with appropriate antibiotics,
ampicillin (100 µg/ml) or kanamycin (50 µg/ml). Fresh cultures
similarly supplemented with antibiotics were inoculated using the
starter cultures and grown until an OD600 = 0.4. All cultures (5 ml
and N -(2,4-dinitro-5-fluorophenyl)-L-alaninamide (10 mg/ml, 30
α
µl) in acetone was added. The mixture was heated to 80°C for 2-
3 min and then reduced in vacuo. The residue was dissolved in
H2O:MeCN (50:50), cleared by centrifugation, and analyzed by
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LC-MS. Comparative standards of L-histidine and D-histidine
were prepared and analyzed using the same protocols.
C8: 1H NMR (400 MHz, Methanol-d4) δ 8.68 (d, J = 1.4 Hz, 1H),
7.25 (d, J = 1.3 Hz, 1H), 4.68 (dd, J = 8.6, 5.2 Hz, 1H), 3.24 (d*,
J = 5.2, 1H), 3.04 (dd, J = 15.2, 8.7 Hz, 1H), 2.18 (td, J = 7.4, 3.9
Hz, 2H), 1.60 – 1.42 (m, 2H), 1.37 – 1.17 (m, 8H), 0.88 (t, J =
6.70, 3H). *The second part of an expected dd is obscured by
the water peak. 13C NMR (101 MHz, MeOD) δ 176.1, 174.2,
134.9, 132.0, 118.2, 53.2, 36.9, 32.9, 30.2, 28.7, 26.9, 23.7,
Synthesis of cis-3-decenoic acid from 3-decyn-1-ol.
3-decynoic acid: Following the protocol of Qiu et al.[44] a round-
bottomed flask (25 ml) was charged with Bobbitt’s salt (930 mg,
3.10 mmol, 2.4 eq) and 9:1 mixture of MeCN:H2O (5 ml). After
addition of 3-decyn-1-ol (200 mg, 1.29 mmol) the reaction was
left for 48 hours at room temperature. The reaction mixture was
concentrated in vacuo, diluted with diethylether and extracted
three times with aq. NaOH (1 M), then the aqueous phase was
acidified with conc. HCl and extracted with diethylether five
times. The combined organic phase was washed with brine,
dried over Na2SO4 and reduced to a slightly yellow solid in
+
14.4. HRMS (ESI): Calc. C12H24N3O3 : 282.1812; found:
282.1827 [M + H]+.
C6: 1H NMR (400 MHz, Methanol-d4) δ 8.52 (s, 1H), 7.19 (s,
1H), 4.58 (dd, J = 8.1, 5.2 Hz, 1H), 3.23 (dd, J = 15.2, 5.2 Hz,
1H), 3.02 (dd, J = 15.1, 8.1 Hz, 1H), 2.17 (td, J = 7.4, 1.8 Hz,
2H), 1.53 (p, J = 7.6 Hz, 2H), 1.25 (m, 4H), 0.87 (t, J = 7.1 Hz,
3H). 13C NMR (101 MHz, MeOD) δ 175.9, 175.5, 134.8, 132.5,
118.2, 54.1, 37.0, 32.5, 29.2, 26.5, 23.4, 14.3.
1
vacuo (165 mg, 76%). H NMR (400 MHz, Chloroform-d) δ 3.33
(t, J = 2.4 Hz, 2H), 2.20 (tt, J = 7.2, 2.5 Hz, 2H), 1.50 (p, J = 7.1
Hz, 2H), 1.44 – 1.22 (m, 8H), 0.89 (t, J = 6.9 Hz, 3H). 13C NMR
(101 MHz, CDCl3) δ 175.3, 84.8, 70.6, 31.5, 28.7, 28.7, 26.1,
+
HRMS (ESI): Calc.C12H20N3O3 : 254.1499; found: 254.1503 [M +
H]+
-
22.7, 18.9, 14.2. HRMS (ESI): Calc. C10H15O2 : 167.1078; found:
167.1091 [M - H]-
Legionella pneumophila intracellular replication
cis-3-decenoic acid: To a stirring mixture of Nickel(II) acetate
tetrahydrate (250 mg) in absolute ethanol (6 ml) saturated with
hydrogen was added the filtrate (1.25 ml) of a solution made
from NaBH4 (125 mg), aqueous NaOH (2 M, 150 µl) and
absolute ethanol (3 ml). To the resulting black suspension was
slowly added ethylenediamine (175 µl) and 3-decynoic acid (150
mg). The reaction was left for 1 hour and then diluted with brine
(20 ml). After acidifying the mixture with HCl (20%) the product
was extracted with diethylether (4 x 50ml). The combined
organic phase was washed with brine and dried over Na2S04
NLRC4-/- mice on a C57BL/6 background were generously
provided by Richard Flavell and have been described
previously.[46] C57BL/6 wild-type mice were purchased from
Jackson Laboratories. NLRC4-/- BMDMs were generated and
maintained as previously described[47]
.
Differentiated BMDMs were maintained in RPMI (Gibco)
supplemented with 10% HI FBS and 7.5% macrophage colony
stimulating factor (M-CSF) and seeded at 2 x 105/well in 24 well
tissue culture treated dishes. Growth curves were performed as
previously described and colony forming units (CFU) were
enumerated at 1h (t0), 24h, 48h , and 72h p.i.[48]
1
yielding cis-3-decenoic acid (124 mg, 82%). H NMR (400 MHz,
Chloroform-d) δ 5.73 – 5.42 (m, 2H), 3.14 (d, J = 6.7 Hz, 2H),
2.04 (q, J = 7.0 Hz, 2H), 1.45 – 1.17 (m, 8H), 0.88 (t, J = 6.7 Hz,
3H). 13C NMR (101 MHz, CDCl3) δ 178.3, 134.4, 120.0, 32.8,
Acanthamoeba castellanii Neff (ATCC #30010) were maintained
in PYG media and cultured at 25˚C as previously described.[49]
For infections, cells were seeded into 24 well tissue culture
dishes at 2.5 x 105/well in PYG medium and allowed to adhere
for 2h prior to infection. Cells were washed in warm Ac buffer
and infected with the indicated strains at a multiplicity of infection
(MOI) of 0.8 in pre-warmed Ac buffer (4 mM MgSO4, 0.4 M
CaCl2, 0.1% sodium citrate dehydrate, 0.05 mM Fe(NH4)2(SO4)2
• 6H2O, 2.5 mM NaH2PO3, 2.5 mM K2HPO3, pH 6.5). At 1h p.i.,
cells were washed gently 3x with sterile phosphate buffered
saline (PBS; Gibco) and Ac buffer was replaced. Bacteria were
recovered by collecting supernatants and A. castellanii were
lysed by passage through a 27 gauge needle three times.
Lysates were plated on CYE and CFU were enumerated at 1h
(t0), 24h, 48h, and 72h p.i. Fold replication was calculated by
normalizing CFU counts at t0 to 1. Statistical analyses were
performed at each time point using Students t-test and
GraphPad software.
-
31.8, 29.4, 29.1, 27.6, 22.8, 14.2. HRMS (ESI): Calc. C10H17O2 :
169.1234; found: 169.1240 [M - H]-
Synthesis of N-acyl-L-histidines
The N-acyl-L-histidines were prepared using the protocol of Coin
et al.[45] and a Fmoc-His(Trt)-Wang resin with the following
exceptions: only one coupling reaction was performed with the
corresponding fatty acid (decanoic-, octanoic-, or hexanoic acid),
no final Fmoc protection for obvious reasons, and final
deprotection and cleavage was performed in TFA:H2O (95:5).
The crude cleavage mixture was reduced in vacuo, redissolved
in aq. AcOH (20%), washed once with DCM and purified by
sequential HPLC-purification using
a
semi-prep LC-MS
(Phenomenex, Luna, 5 µ, C18(2), 100 Å, 250 x 10 mm).
C10: 1H NMR (400 MHz, Methanol-d4) δ 8.41 (d, J = 1.4 Hz, 1H),
7.14 (d, J = 1.3 Hz, 1H), 4.53 (dd, J = 7.7, 5.2 Hz, 1H), 3.19 (dd,
J = 15.2, 5.0 Hz, 1H), 3.00 (dd, J = 15.1, 7.7 Hz, 1H), 2.18 (td, J
= 7.3, 1.9 Hz, 2H), 1.57 – 1.49 (m, 2H), 1.27 (s, 12H), 0.88 (t, J
= 7.01, 3H). 13C NMR (101 MHz, MeOD) δ 175.8, 173.5, 134.9,
132.8, 118.2, 54.6, 37.1, 33.1, 30.6, 30.5, 30.5, 30.3, 29.6, 26.9,
Acknowledgments
+
23.7, 14.4. HRMS (ESI): Calc. C14H28N3O3 : 310.2125; found:
Our work on the discovery of new metabolites from bacterial
symbionts was supported by the Searle Scholars Program (grant
310.2143 [M + H]+.
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[21]
[22]
[23]
D. T. Longone, A. H. Miller, Chem. Commun. 1967.
13-SSP-210 to JMC), the Damon Runyon Cancer Research
Foundation (grant DRR-39-16 to JMC), and the National
Institutes of Health (grant 1DP2-CA186575 to JMC). Our work
on the functional roles of Legionella effectors was supported by
the National Institutes of Health (grants AI041699 and AI048770
P. H. Buist, D. B. Maclean, Can. J. Chem. 2011, 59, 828–838.
D. L. Shis, M. R. Bennett, Proc. Natl. Acad. Sci. U.S.A. 2013,
110, 5028–5033.
T. Baba, T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba,
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10.1002/cbic.201600618
ChemBioChem
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We identified a biosynthetic gene
Thomas Tørring, Stephanie R.
Shames, Wooyoung Cho, Craig R. Roy
and Jason M. Crawford*
cluster from the human pathogen
Legionella pneumophila that
produces two distinct groups of
secondary metabolites. One of these
groups represents a new histidine-
derived addition to the biologically
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Acyl-histidines: New N-acyl amides
from Legionella pneumophila
N
important class of N-acyl amides.
NH
OH
O
H
H
N
H
O
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