Myosin cross-reactive antigen of Streptococcus pyogenes M49 encodes a fatty acid double bond hydratase that plays a role in oleic acid detoxification and bacterial virulence

Article abstract of DOI:10.1074/jbc.M109.081851

The myosin cross-reactive antigen (MCRA) protein family is highly conserved among different bacterial species ranging from Gram-positive to Gram-negative bacteria. Besides their ubiquitous occurrence, knowledge about the biochemical and physiological function of MCRA proteins is scarce. Here, we show that MCRA protein from Streptococcus pyogenes M49 is a FAD enzyme, which acts as hydratase on (9Z)- and (12Z)-double bonds of C-16, C-18 non-esterified fatty acids. Products are 10-hydroxy and 10,13-dihydroxy fatty acids. Kinetic analysis suggests that FAD rather stabilizes the active conformation of the enzyme and is not directly involved in catalysis. Analysis of S. pyogenes M49 grown in the presence of either oleic or linoleic acid showed that 10-hydroxy and 10,13-dihydroxy derivatives were the only products. No further metabolism of these hydroxy fatty acids was detected. Deletion of the hydratase gene caused a 2-fold decrease in minimum inhibitory concentration against oleic acid but increased survival of the mutant strain in whole blood. Adherence and internalization properties to human keratinocytes were reduced in comparison with the wild type. Based on these results, we conclude that the previously identified MCRA protein can be classified as a FAD-containing double bond hydratase, within the carbon-oxygen lyase family, that plays a role in virulence of at least S. pyogenes M49.

Full text of DOI:10.1074/jbc.M109.081851

Lipids:
Myosin Cross-reactive Antigen of
Streptococcus pyogenes M49 Encodes a
Fatty Acid Double Bond Hydratase That
Plays a Role in Oleic Acid Detoxification
and Bacterial Virulence
Anton Volkov, Alena Liavonchanka, Olga
Kamneva, Tomas Fiedler, Cornelia Goebel,
Bernd Kreikemeyer and Ivo Feussner
J. Biol. Chem. 2010, 285:10353-10361.
doi: 10.1074/jbc.M109.081851 originally published online February 9, 2010
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 14, pp. 10353–10361, April 2, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Myosin Cross-reactive Antigen of Streptococcus pyogenes
M49 Encodes a Fatty Acid Double Bond Hydratase That Plays
□
S
a Role in Oleic Acid Detoxification and Bacterial Virulence
Received for publication, November 4, 2009, and in revised form, January 19, 2010 Published, JBC Papers in Press, February 9, 2010, DOI 10.1074/jbc.M109.081851
Anton Volkov‡1, Alena Liavonchanka‡2, Olga Kamneva§, Tomas Fiedler¶, Cornelia Goebel‡, Bernd Kreikemeyer¶3,
and Ivo Feussner‡4
From the ‡Department for Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences, Georg-August-University,
37077 Go¨ttingen, Germany, the ¶Institute of Medical Microbiology, Virology and Hygiene, University Hospital Rostock,
18057 Rostock, Germany, and the §Molecular Biology Department, College of Agriculture, University of Wyoming,
Laramie, Wyoming 82072
The myosin cross-reactive antigen (MCRA) protein family is
highly conserved among different bacterial species ranging from
Gram-positive to Gram-negative bacteria. Besides their ubiqui-
tous occurrence, knowledge about the biochemical and physio-
logical function of MCRA proteins is scarce. Here, we show that
MCRA protein from Streptococcus pyogenes M49 is a FAD
enzyme, which acts as hydratase on (9Z)- and (12Z)-double
bonds of C-16, C-18 non-esterified fatty acids. Products are
10-hydroxy and 10,13-dihydroxy fatty acids. Kinetic analysis
suggests that FAD rather stabilizes the active conformation of
the enzyme and is not directly involved in catalysis. Analysis of
S. pyogenes M49 grown in the presence of either oleic or linoleic
acid showed that 10-hydroxy and 10,13-dihydroxy derivatives
were the only products. No further metabolism of these hydroxy
fatty acids was detected. Deletion of the hydratase gene caused a
2-fold decrease in minimum inhibitory concentration against
oleic acid but increased survival of the mutant strain in whole
blood. Adherence and internalization properties to human ke-
ratinocytes were reduced in comparison with the wild type.
Based on these results, we conclude that the previously identi-
fied MCRA protein can be classified as a FAD-containing double
bond hydratase, within the carbon-oxygen lyase family, that
plays a role in virulence of at least S. pyogenes M49.
for antigens recognized by acute rheumatic fever sera. Its amino
acid sequence did not exhibit significant similarity to any strep-
tococcal protein with a known function but was conserved
among pathogenic groups A, C, and G of Streptococci (1).
A BLAST search with the MCRA protein sequence reveals
more than 148 conserved sequences across different Gram-
positive and Gram-negative bacteria (supplemental Fig. S1).
MCRA genes are not a part of any bacterial operon. Despite
such conservation level only for two members of the family so
far, biochemical features have been assigned: MCRA from Lac-
tobacillus reuteri PYR8 was suggested to be a (9Z,11E)-conju-
gated linoleic acid (CLA)-forming isomerase (2), and only
recently hydratase activity was shown for MCRA of Pseudomo-
nas sp. strain 3266 (3).
To analyze biochemical properties and physiological func-
tions of MCRA and their more ubiquitous activity as fatty acid
hydratase, we have chosen S. pyogenes M49 as a representative
of the group A streptococci (GAS). GAS species exclusively
colonize humans and cause a wide range of primary infections
of the skin, throat, and other mucosal surfaces, including phar-
yngitis and impetigo (4), and hence have a vast medical impor-
tance. We show that S. pyogenes MCRA is a FAD-containing
hydratase that adds water to (9Z)- and (12Z)-double bonds of
C-16 and C-18 fatty acids. Using a gene deletion strain, we show
that the hydroxylated fatty acids are not further metabolized.
Importantly, the mutant strain showed alteration in virulence
properties, such as blood survival and adherence and internal-
ization to human keratinocytes.
In 1994, the first member of the MCRA5 protein family was
identified in Streptococcus pyogenes as the result of a screening
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S9.
EXPERIMENTAL PROCEDURES
1 Supported by the Ph.D. program “Molecular Sciences and Biotechnology of
Crop Plants.”
Materials—Chemicals were obtained from Sigma and Carl
Roth & Co. (Karlsruhe, Germany). Agarose was from Biozym
Scientific GmbH (Hess. Oldendorf, Germany). All fatty acids
were purchased from Sigma or Cayman Chemical (Ann Arbor,
2 Recipient of a Georg-Christoph-Lichtenberg stipend of the Ph.D. program
“Molecular Biology.”
3 Work in this author’s laboratory was supported by a Bundesministerium fu¨r
Bildung und Forschung grant in the framework of the SysMO (Systems MI). Acetonitrile was from Fisher. Restriction enzymes were
Biology of Microorganisms) program, Grant FKZ 0313979B.
provided by MBI Fermentas (St. Leon-Roth, Germany).
Bacterial Strains and Culture Conditions—S. pyogenes (GAS)
serotype M49 strain 591 is a skin isolate originally obtained
4 To whom correspondence should be addressed: Georg-August-University,
Albrecht-von-Haller-Institute for Plant Sciences, Dept. for Plant Biochemis-
try, Justus-von-Liebig Weg 11, 377077 Goettingen, Germany. Tel.: 49-551-
395743; Fax: 49-551-395749; E-mail: ifeussn@uni-goettingen.de.
5 The abbreviations used are: MCRA, myosin cross-reactive antigen; 10,13-
DiHOA, 10,13-dihydroxyoctadecanoic acid; GAS, group A streptococci; GC,
gas chromatography; 10-HOA, 10-hydroxyoctadecanoic acid; 10-HOE,
(12Z)-10-hydroxy-12-octadecenoic acid; HPLC, high pressure liquid chro-
matography; MIC, minimal inhibitory concentration; MS, mass spectrome-
try; MS/MS, tandem MS; PAI, P. acnes isomerase; SPH, S. pyogenes
hydratase; THY, Todd-Hewitt broth supplemented with yeast extract; CLA,
(9Z,11E)-conjugated linoleic acid; MES, 4-morpholineethanesulfonic acid;
CFU, colony-forming unit(s).
APRIL 2, 2010•VOLUME 285•NUMBER 14
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Fatty Acid Hydratase from S. pyogenes
from R. Lutticken (Aachen, Germany). The Escherichia coli the PhusionTM hot start high fidelity DNA polymerase system
(Finnzymes) according to the manufacturer’s protocol.
Overproduction of the Recombinant Protein—E. coli BL21
Star strain was transformed with pET24a-SPH or pET28a-SPH,
and the bacteria was cultivated in 2ϫ YT medium (30 g of Tryp-
tone, 15 g of yeast extract, 5 g of NaCl per 1000 ml of H2O)
supplied with 50 ␮g/ml kanamycin at 37 °C until A600 ϳ0.6–
0.8. Then isopropyl 1-thio-␤-D-galactopyranoside was added to
0.1 mM, and the cells were shifted to 16 °C and harvested by
centrifugation (10 min at 9100 ϫ g) after an 18-h induction
time.
strains DH5␣, XL Blue and BL21 Star were purchased from
Invitrogen and served as a host for the plasmids. E. coli strains
DH5␣ and XL Blue BL21 were used for DNA cloning purposes,
and BL21 Star was used for the protein overproduction. The
GAS wild type strain and mutant derivates were cultured in
Todd-Hewitt broth or on Todd-Hewitt broth-agar (Oxoid-
Unipath, Wesel, Germany), both supplemented with 0.5% yeast
extract (THY). GAS mutants harboring recombinant pFW11
plasmid (5) were maintained in medium containing 100 ␮g/ml
spectinomycin except when being used for functional analyses.
GAS strains were grown as standing cultures at a temperature
of 37 °C under a 5% CO2-20% O2 atmosphere unless otherwise
indicated. All E. coli cultures were grown at 37 °C with agitation
at 180 rpm except when used for expression of recombinant
proteins.
Protein Purification—The non-tagged protein was purified
as described below, and all steps were done at 4 °C. Cells were
lysed by adding 0.1 mg/ml lysozyme and 1 ␮g/ml DNase I in
0.1 M Tris, pH 7.5, 0.05 M NaCl, 10 mM MgCl2 and centrifuged for
20 min at 70,000 ϫ g. The supernatant was loaded on pre-
equilibrated Source 30Q resin (25 ml, XK 16/20 column; GE
Nucleic Acid Techniques—Chromosomal and plasmid DNA
preparations, genetic manipulations, and other conventional
DNA techniques, including electroporation of GAS and E. coli
strains, were done as described previously (6).
¨
Healthcare) at a flow rate of 2 ml/min using an AKTA-Prime-
System (GE Healthcare). The column was washed with 3 col-
umn volumes of buffer A (0.1 M Tris, pH 7.5, 50 mM NaCl),
followed by washing with 3 column volumes of buffer B (0.1 M
MES, pH 6.0) at a flow rate of 3 ml/min, and the protein was
eluted by linear NaCl gradient from 50 mM to 0.6 M using buffer
C (0.1 M MES, pH 6.0, 1 M NaCl) at a flow rate of 2 ml/min,
within 20 min. The eluted protein peak was collected and con-
centrated by ultrafiltration on Vivaspin 20 (50,000 molecular
weight cut-off) (Sartorius Stedim Biotech GmbH, Goettingen,
Germany). For gel filtration, the concentrated protein extract
was loaded on a Superdex S200 26/60 pg column (GE Health-
care) equilibrated with buffer A. Elution was performed at a
flow rate of 1 ml/min. Fractions corresponding to the SPH
homodimers were combined and concentrated as above. In
case a buffer exchange was required, the enzyme was loaded on
a desalting 26/10 column (GE Healthcare) equilibrated with
an appropriate buffer. Protein concentration was estimated
Construction of Recombinant Vectors and S. pyogenes M49
Strains—To perform the deletion knock-out of sph, the
upstream and downstream fragments of the gene (GenBankTM
accession number ACI60731.1) were PCR-amplified using the
following primer pairs: upstream fragment (5Ј-TTTAGTGTC-
GACCGTCCAGGAGATC-3Ј (forward primer) and 5Ј-TTCA-
AGGATCCCTTGAATAGTCAAGCAAAG-3Ј (reverse primer)
containing SalI and BamHI recognition sites, respectively) and
the downstream fragment (5Ј-CATGGCATGCAGTAGTTA-
AAGCTGATAAG-3Ј (forward primer) and 5Ј-CTGCAGCC-
CGGGACCAAAAACCA-3Ј (reverse primer) containing SphI
and XmaI recognition sites, respectively). The PCRs were per-
formed with the PhusionTM hot start high fidelity DNA poly-
merase system (Finnzymes, Espoo, Finland) according to the
manufacturer’s protocol. For expression in E. coli, the sph open
reading frame was amplified with the primers 5Ј-AAAGCTA-
GCATGTATTATACTAGTGGTAATTACG-3Ј (forward) and
5Ј-AAAGCGGCCGCTTACATAAGATTAGCATCTTTG-
AGC-3Ј (reverse), containing NheI and NotI recognition sites,
respectively, and cloned into the pET24a and pET28a expres-
sion vectors (Novagen), respectively, yielding the plasmid
pET24a-SPH (non-tagged version) and pET28a-SPH (N-His-
tagged version).
Ϫ1
spectrophotometrically using calculated ⑀280 ϭ 104.335 M
cmϪ1. The N-His-tagged version was purified as follows. Cell
lysis and the supernatant preparation were performed as above.
The obtained supernatant was loaded on a HisTrap column
(GE Healthcare), the column was washed by 10 volumes of
buffer A, and the protein was eluted with buffer A containing
0.5 M imidazole.
Spectroscopic Assays and HPLC-MS/MS Cofactor Identi-
fication—For initial characterization, UV-visible spectra of
purified proteins in the range 300–800 nm were recorded with
the single-beam absorption spectrometer (Ultrospec 2100 pro,
GE Healthcare). Reference spectra of FAD and FMN were
recorded in the same way, using FAD and FMN concentrations
of 10–40 ␮M in buffer A. To identify a flavin cofactor, an aliquot
of 50 ␮M SPH in water was heated at 95 °C for 10 min, precipi-
tated protein was removed by centrifugation at 20,000 ϫ g for
10 min, and the supernatant was diluted 2-fold with acetonitrile
and used for MS analysis. 20 ␮M FAD was used as a reference in
the same solvent system. The analysis of constituents was per-
formed using an Agilent 1100 HPLC system (Agilent, Wald-
bronn, Germany) coupled to an Applied Biosystems 3200
hybrid triple quadrupole/linear ion trap mass spectrometer
Reverse Transcription-PCR—To analyze sph expression dur-
ing the exponential growth phase of wild type and the mutant,
total mRNA was isolated by the FastRNAꢀ Pro Blue Kit (MP
Biomedicals) according to the manufacturer’s protocol. RNA
concentration and the quality were measured spectrophoto-
metrically by Picodrop (Picodrop). Reverse transcription reac-
tion was performed by the SuperScriptꢀ III first strand synthe-
sis system (Invitrogen) according to the manufacturer’s
protocol; genomic DNA contamination was controlled by PCR
with the upstream region primers, which were used for gen-
eration of the deletion knock-out (see above); and gyrA was used
as the internal control (forward primer, 5Ј-CGACTTGTCTG-
AACGCCAAA-3Ј; reverse primer, 5Ј-TTATCACGTTCCAA-
ACCAGTCAA-3Ј). The PCR reactions were performed with (MDS Sciex, Toronto, Canada). Nanoelectrospray analysis was
10354 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 14•APRIL 2, 2010

Fatty Acid Hydratase from S. pyogenes
achieved using a chip ion source (TriVersa NanoMate, Advion A600 ϭ 2.5 were used. Substrates were either oleic acid or
BioSciences (Ithaca, NY)). Reversed-phase HPLC separation U-13C-labeled or non-labeled linoleic acid. Fatty acids were
was performed on an EC 250/2 Nucleodure 100-5 C18ec col- extracted by a chloroform/methanol (2:1) mixture, dissolved in
umn (250 ϫ 2.1 mm, 5-␮m particle size; Macherey and Nagel methanol, and converted to methyl esters with (trimethylsilyl)-
(Du¨ren, Germany)) with a methanol, 5 mM NH4OAc, pH 6.0 diazomethane, hydroxyl groups were modified with N,O-bis-
(20:80, v/v) solvent system and a flow rate of 200 ␮l/min. For (trimethylsilyl)-trifluoroacetamide. Esterified fatty acids were
stable nanoelectrospray analysis, 100 ␮l minϪ1 2-propanol/ace- transesterified by adding sodium methoxide in methanol/tolu-
tonitrile/water/formic acid (70:20:10:0.1, v/v/v/v) delivered by ene (2:1, v/v). GC-MS analysis was carried out using an Agilent
a 515 HPLC pump (Waters, Milford, MA) were added just after 5973 network mass-selective detector connected to an Agilent
the column via a mixing tee valve. By using another postcolumn 6890 gas chromatograph equipped with a capillary DB-23 col-
splitter, 637 nl minϪ1 of the eluent were directed to the nano- umn (30 m ϫ 0.25 mm; 0.25-␮m coating thickness; Agilent,
electrospray analysis chip. Ionization voltage was set to Ϫ1.7 Waldbronn, Germany). Helium was used as carrier gas (1
kV. FMN and FAD were ionized in a positive mode and deter- ml/min). The temperature gradient was 150 °C for 1 min, 150–
mined by product ion scanning. The collision energy, with 160 °C at 10 K/min, 160–200 °C at 7 K/min, 200–250 °C at 25
nitrogen in the collision cell, was 50 V. Declustering potential K/min, and 250 °C for 7 min. Electron energy of 70 eV, ion
was 80 V. Entrance potential was 11 V. The mass analyzers were source temperature of 230 °C, and temperature of 260 °C for
adjusted to a resolution of 0.7 atomic mass units full width at the transfer line were used (8).
half-height. The ion source temperature was 40 °C, and the cur-
tain gas was set at 10 (given in arbitrary units).
Blood Survival Assay—Overnight cultures of the wild type
and mutant strains were inoculated into fresh THY medium
Kinetic Parameters and Analysis of FAD Function—N-His- and grown to exponential growth phase. Bacteria was harvested
tagged SPH was purified as described above following buffer by centrifugation, set to an optical density at 600 nm of 0.25,
exchange in buffer A by the desalting column. The obtained and further diluted 1:10,000 in PBS. Twenty microliters of each
amount of enzyme was split into two equal parts and dialyzed suspension were incubated with 480 ␮l of serum or heparinized
against buffer A with and without a 5-fold excess of FAD at 4 °C blood for 3 h at 37 °C with agitation at 800 rpm. After this
overnight. The next day, the protein preparations were centri- incubation, the remaining CFU were determined by plating and
fuged and concentrated by ultrafiltration on Vivaspin 20 related to the serum inoculum, which was set to 100%. The CFU
(50,000 molecular weight cut-off), and the concentration was contained in this final suspension were determined by plating
measured by a Bradford protein assay (7). The enzyme was of serial dilutions on THY agar plates.
diluted to a concentration of 10 mg/ml, frozen in liquid nitro-
Eukaryotic Cell Adherence and Internalization Assays—As-
gen, and stored at Ϫ70 °C until used. Kinetic measurements sessment of eukaryotic cell adherence and internalization was
were performed in 200 ␮l of 50 mM MES, NaOH (pH 6.1), con- determined by an antibiotic protection assay. Briefly, for the
taining 50 mM NaCl, 2% ethanol and 10% glycerol with 32, 64, antibiotic protection assay, the early stationary phase bacteria
128, or 180 ␮M of oleic or linoleic acid. The reactions were were suspended in Dulbecco’s modified Eagle’s medium sup-
initiated by the addition of S. pyogenes M49 hydratase (SPH) to plemented with 10% fetal calf serum and were added to HaCaT
a final concentration of 500 nM and incubated at 37 °C for dif- cells (a human skin keratinocyte cell line) grown overnight to
ferent times (0.5–15 min). The reactions were stopped by the confluence, establishing a multiplicity of infection of 25. Previ-
addition of 1 ml of chloroform/methanol (2:1, v/v), and fatty ous studies indicated this multiplicity of infection to be in the
acids were extracted by 10 min of immediate intensive vortex- linear range of such experiments. After 2 h, the cells were
ing in the presence of heptadecanoic acid as an internal stand- washed with phosphate-buffered saline. One-half of the cells
ard. The organic phase was taken and dried under a stream of were lysed with distilled water, and the number of bacteria con-
liquid nitrogen. The pellets were dissolved in 200 ␮l of metha- tained in the lysate was assessed by viable counts. The other half
nol containing 0.1 M (trimethylsilyl)diazomethane and agitated of the eukaryotic cells were exposed to culture medium supple-
at room temperature for 10 min. Methyl esters were diluted in mented with penicillin and streptomycin for another 2 h. Then
acetonitrile and analyzed by the Agilent GC 6890 system cou- these cells were washed and lysed, and the bacterial numbers
pled with a flame ionization detector, where the column and the were counted as above.
parameters were identical to those described above. The Km
and Vmax values were estimated by nonlinear fitting tools of leic acid were determined by a series of double broth microdi-
OriginPro version 7.5 software. lutions according to the protocols of Andrews (9), followed by
Stopped-flow kinetic analysis was performed in degassed measurement of optical density after the overnight incubation.
MIC Determination—MICs for cerulenin and oleic and lino-
buffer A by mixing equal volumes of SPH and linoleic acid solu-
Protein Sequences and Phylogenetic Analysis—Members
tions to a final concentration of 5 ␮M enzyme and 180 ␮M and of the MCRA gene family were identified using a reciprocal
1.8 mM substrate, respectively. The formation of reduced FAD BLAST search. MCRA of S. pyogenes M49 591 (ZP_00366513.1)
species was monitored by using a stopped-flow spectropho- was used as an initial query sequence for a BLASTP (10) search
tometer (SX20, Applied Photophysics).
with conditional compositional score matrix adjustment against
GC-MS Analysis of Lipids—To determine the substrate spec- the non-redundant protein data base (11). Obtained sequences
ificity, 50 ␮g of a substrate were mixed with 1 ml of buffer A and were used for a BLASTP search of S. pyogenes M49 591 pro-
10 ␮g of purified SPH and incubated for 1–24 h at 37 °C. For teins, and only sequences that returned MCRA as a best hit
analysis of in vivo fatty acid metabolism, S. pyogenes cells at were used for further analysis. Sequences obtained after the
APRIL 2, 2010•VOLUME 285•NUMBER 14
JOURNAL OF BIOLOGICAL CHEMISTRY 10355

Fatty Acid Hydratase from S. pyogenes
previous step were aligned using MUSCLE (12), and sequences
Protein Purification—In order to characterize the MRCA
with less than 55% of alignment extension were excluded from protein from S. pyogenes M49, we purified the non-tagged ver-
the data set. A draft phylogeny for 243 protein sequences was sion after heterologous expression in E. coli Bl21 Star cells. The
reconstructed using the neighbor-joining method (13), as initial anion exchange chromatography yielded protein having
implemented in MEGA4 (14). 148 sequences covering widest a light yellow color (supplemental Fig. S3A, lines 1 and 2). With
range of taxa were selected for in depth phylogenetic analysis. the following size exclusion chromatography, the protein was
The JTT ϩ GAMMA (15, 16) model was determined as the found in two fractions. The main fraction represented the
model of the best fit according to AIC (17) by the PROTTEST homodimer of 134 kDa, and the second fraction represented
tool (18) with default parameters. Neighbor-joining phylogeny the monomer of 67 kDa. Both fractions contained active pro-
was reconstructed with the use of MEGA4 implementing the tein, but in the fractions harboring the homodimer, only traces
previously identified evolutionary model with 1000 bootstrap of other proteins were found and were used for further analysis
runs. The final tree was rooted using the ancestral node of all (supplemental Fig. S3A, line 3). However, during this step, yel-
ascomycete sequences as an outgroup. Sequences from strep- low color was lost. Therefore, N-His-tagged SPH was affinity-
tococcal, lactobacilli, and staphylococcal species were selected purified in one step (supplemental Fig. 3B). It eluted as intense
for detailed evolutionary analysis. Sequences identical at the yellow colored solution similar to the non-tagged enzyme after
amino acid level were excluded from the analysis. Protein the anion exchange chromatography. However, in both colored
sequences were aligned using MUSCLE, and gaps were inserted and non-colored enzyme preparations, hydratase activity was
into DNA sequences using the PAL2NAL tool (19). Best fit detectable.
evolutionary models for DNA were identified using the MOD-
Spectral Properties and Cofactor Identification—Concen-
ELTEST (20) program. The phylogenetic tree was recon- trated affinity-purified SPH protein had yellow color, and the
structed for DNA sequences using RAxML software (21) imple- UV-visible spectrum of SPH showed maxima around 370 and
menting the previously identified evolutionary model. The tree 450 nm characteristic for flavin cofactors as well as small addi-
was rooted in order to maximize a number of monophyletic tional peaks at 420 and 480 nm, respectively (supple-
groups. DNA sequence data were analyzed with the CODELM mental Fig. S4, solid line). To test for the presence of flavin
program of the PAML4 software package (22).
cofactor in SPH, the protein was heat-denatured, and the spec-
trum of the protein-free supernatant was recorded (sup-
plemental Fig. S4, thin solid line). In this case, the spectrum
RESULTS
SPH Protein Sequence Analysis—In order to identify all contained only two maxima at 447 and 373 nm, respectively,
known homologues of MCRA protein from S. pyogenes M49 and closely resembled the absorption patterns of free FAD and
591, we conducted a BLAST search using the GenBankTM data FMN, confirming that SPH contained a non-covalently bound
base. 243 gene sequences from organisms of different domains flavin cofactor. The cofactor was identified by mass spectrom-
of life were identified. The majority of these gene sequences are etry, and the spectra of the FAD standard and of the superna-
from Gram-positive and negative bacteria or from ascomyce- tant are shown in Fig. 1, A and B, respectively. The MS/MS
ϩ
tes. A phylogenetic analysis of 148 sequences covering the wid- spectrum of the parent ion M ϭ 786.1 unambiguously identi-
ϩ
est range of taxa revealed that the MCRA protein family con- fies the cofactor as FAD, with three major signals (M ϭ 136.1,
ϩ
ϩ
sists of three large clusters (supplemental Fig. S1). The first M ϭ 348.2, and M ϭ 439.2) found both in the FAD standard
cluster includes all of the analyzed ascomycete genes. The rest and in the sample (Fig. 1, C and D).
of the sequences form two additional clusters. Based on high
Products of SPH—In activity assays with purified SPH protein
sequence similarity (more than 30% identity for the most diver- and linoleic acid as a substrate, (12Z)-10-hydroxy-12-octadece-
gent sequences), one may assume similar biological function for noic acid (10-HOE) was detected by GC-MS as the main reac-
all of the family members. Disagreement of the gene tree topol- tion product (Fig. 2, A and D). The ion Mϩ ϭ 273 is the pre-
ogy with the accepted evolutionary relationship of considered dominant species in the spectrum and represents a C-10
organisms suggests evolution of the family through active hor- fragment in which the 10-hydroxy group is modified by
izontal gene transfer within at least the bacterial domain.
MS/MS. When an additional (12Z)-double bond is present in
Evolutionary analysis of MCRA genes from streptococcal, the substrate, it is also hydrated, yielding 10,13-dihydroxyocta-
lactobacilli, and staphylococcal species suggested that proteins decanoic acid (10,13-DiHOA) (Fig. 2, E and H). In order to
evolved under strong negative selection because of the low non- characterize the enzymatic preferences with respect to the dou-
synonymous/synonymous substitution rate over the branches ble bond position and configuration and the polarity of the fatty
of the gene tree (Kn/Ks rarely exceeds 1; supplemental Fig. acid headgroup, a range of different fatty acids and lipids were
S2, left). Alignment of the predicted FAD binding motif assayed (Table 1). SPH showed strict preference for the free
revealed a deviation from the canonical FAD binding motif carboxyl group and did not modify complex lipids. It also
GXGXXGX17–19E of the Rossmann fold for lactobacilli and required the (9Z)-double bond, whereas the (9E)-, (11E)-, and
staphylococcal proteins (supplemental Fig. S2, right). The third (11Z)-double bonds were not hydrated. Any combination of cis
glycine residue of the motif is substituted by serine in staphylo- and trans double bonds as well as double bonds in front of C-9
coccus (GXGXXSX15S and GXGXXSX21E) or by asparagine in abolished SPH activity. The chain length of the substrate was
lactobacillus (GXGXXN(A/S)X15(D/K/E) and GXGXXGX21E- limited to 16–18 carbons.
(G/S/A). The latter corresponds to members of the glutathione
reductase family (23).
The origin of oxygen in the hydroxylated fatty acids was ana-
lyzed with isotopically labeled water (Fig. 2, B, C, F, and G). In
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Fatty Acid Hydratase from S. pyogenes
suggest a stabilizing function of
FAD in SPH rather than its involve-
ment in catalysis.
Generation of ⌬sph Deletion
Strain of S. pyogenes M49—To gen-
erate a sph gene deletion mutation, a
replacement type targeting vector
pFW11 was used and ligated with
the upstream and downstream re-
gions of the sph gene. After restric-
tion and sequencing analysis, the
vector was transformed into S. pyo-
genes M49 cells, and clones were
selected in the presence of spectino-
mycin. Genomic DNA from surviv-
ing clones was isolated, and the
deletion of the sph gene was con-
firmed by PCR. A fragment of 1782
bp corresponding to the sph open
reading frame was amplified from
the wild type DNA but not from
the mutant DNA (supplemental
Fig. S7A). Reverse transcription-
PCR of total mRNA from wild type
FIGURE 1. Identification of FAD in SPH. A and B, mass spectra of FAD standard and supernatant of SPH sample
after protein precipitation, respectively. C and D, MS/MS spectra of molecular ion (Mϩ ϭ 786.1) corresponding
to A and B, respectively.
and the mutants confirmed sph
expression in the wild type and no
expression in the mutants during
the mass spectrum of 10-HOE obtained after incubation in the exponential phase of the growth (supplemental Fig. S7B).
2H2O, the mass shift of ϩ1 was evident for the C-10 fragment
Metabolism of Oleic and Linoleic Acid and Their Influence on
ϩ
described above (Fig. 2, A versus B, M ϭ 273 versus 274). the Growth of Wild Type and ⌬sph Mutant Strains—Oleic and
Furthermore, after the reaction in H218O, the mass of the C-10 linoleic acid are toxic to a variety of bacteria (24–26). There-
fragment increased by ϩ2 atomic mass units, indicating the fore, growth curves for wild type cells in the presence of differ-
addition of 18O from water (Fig. 2C, M ϭ 275). The structures ent concentrations of these fatty acids were recorded. We
ϩ
of 10,13-DiHOA confirmed these findings (Fig. 2, E–G).
found that a 10 ␮g/ml concentration of either fatty acid did not
Kinetic Parameters and Function of FAD—Next, properties inhibit growth. Next, growth kinetics of the wild type and
of the SPH holoenzyme (FAD-harboring form) and the apoen- mutant strains in THY medium without or with the addition of
zyme were compared. Supplemental Fig. 5 shows their UV- oleic and linoleic acid and 10% human serum alone were ana-
visible spectra together with that of a FAD standard. From the lyzed. As shown in supplemental Fig. S8, A–C, the mutant had
apoenzyme spectrum, we conclude that this form may contain a slight delay of 1 h in growth but very similar responses to all
only traces of the cofactor. For the holoenzyme the Km values additives compared with wild type. The addition of fatty acids
for 18:2 and 18:1 were 49 Ϯ 3 and 63 Ϯ 6 ␮M, respectively. kcat or serum enhanced growth speed. Because all mutant strains
values were 101 Ϯ 3 and 67 Ϯ 5 minϪ1, respectively. Linoleic behaved in a similar way, only one mutant strain was further
acid (18:2) was the preferred substrate under the given condi- analyzed.
tions: kcat/Km 2 ϫ 106 versus 1 ϫ 106
M
Ϫ1 minϪ1, respectively. In
Wild type bacteria converted 18:2 to 10-HOE and 10,13-Di-
contrast to the holoenzyme, the apoenzyme showed a 10–50- HOA and 18:1 to 10-hydroxyoctadecanoic acid (10-HOA),
times lower activity, making it impossible to reach substrate whereas the mutant strain did not metabolize the fatty acids to
saturating conditions and therefore to determine its kinetic any of these compounds (Figs. 3, A and B). Incubations of either
parameters.
wild type or mutant strain with U-13C-labeled 18:2 did not
To investigate direct involvement of FAD in catalysis, reveal any further transformation to less saturated/unsaturated
stopped-flow experiments with the holoenzyme in the presence fatty acids. As a control, we measured free fatty acid consump-
of linoleic acid were performed. No difference in the functional tion between control medium and medium containing the bac-
range of absorbance (300–600 nm) of FAD was detected within teria after the overnight incubation. As much as 43% of oleic
10–5000-ms time range at two different concentrations of lin- and 37% of linoleic acid were utilized by wild type bacteria, and
oleic acid (supplemental Fig. S6, A and B). Notably, absorbance 16 and 19% were utilized by the mutant, respectively, and there-
spectra did not change even after prolonged incubation for fore the overall contribution of the hydratase activity of SPH
1–10 min. Together with the fact that molecular oxygen is not may reach 50% of total free fatty acid utilization.
involved in this hydration reaction and thus the role of FAD in
Bacteria detoxify unsaturated fatty acids by hydrogenating
this process cannot be attributed to a redox reaction, these data them to less unsaturated or saturated fatty acids (25, 27), and
APRIL 2, 2010•VOLUME 285•NUMBER 14
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Fatty Acid Hydratase from S. pyogenes
To further analyze a possible
involvement of SPH in the bacterial
fatty acid metabolism, we deter-
mined MIC for cerulenin. Cerulenin
acts as an irreversible inhibitor of
␤-ketoacyl-acyl carrier protein syn-
thases, which are crucial for fatty
acid biosynthesis (28, 29). The
experiments were carried out in the
presence of oleic, linoleic, and pal-
mitic acid with or without human
serum albumin and with 10%
human serum alone. An increase of
the growth at similar levels was
recorded for both strains in the
presence of all additives (sup-
plemental Fig. S9). The addition of
either free fatty acids or fatty acids
supplemented with human serum
albumin (to increase availability of
fatty acids to the bacterial cells)
showed no phenotypic differences;
nor did the addition of 10% human
serum alone.
Analysis of ⌬sph Mutant Strain in
Human Blood and HaCaT Cells—
In order to characterize virulence-
associated roles of SPH, the wild
type and ⌬sph mutant strain were
compared for survival in human
blood and their capability to adhere
to and internalize into HaCaT cells.
The mutant strain survived 2 times
better in human blood than the wild
type (Fig. 5A). However, the mutant
strain was significantly attenuated
in its capacity to adhere to and inter-
nalize into HaCaT cells (Fig. 5, B
and C).
DISCUSSION
Because the first member of the
MCRA protein family was identified
in S. pyogenes (1), different func-
tions have been discussed for them,
ranging from their similarity to the
major histocompatibility complex
class II antigen to a catalytic activity
as CLA forming double bond
isomerase (2) or hydratase (3). A
FIGURE 2. Production of hydroxylated fatty acids by purified SPH. Linoleic acid was used as a substrate, and
reaction products were analyzed by GC-MS. Mass spectra of 10-HOE after reaction in H2O (A), in 2H2O (B), and in
H218O (C) show that one water molecule is added to the ⌬9 double bond. The mass of C-10 fragment (m/z ϭ
273) is changing due to the isotopic labeling either with 2H at position C-9 (ϩ1 atomic mass unit) or with 18O at
position C-10 (ϩ2 atomic mass units). The structure of 10-HOE and its fragmentation pattern are shown in D.
Mass spectra of 10,13-DiHOA obtained after reaction of SPH with linoleic acid in H2O (E), in 2H2O (F), and in
H218O (G) show that two water molecules are incorporated into the substrate linoleic acid across the ⌬9 and
⌬12 double bonds. The structure of 10,13-DiHOA and its fragmentation pattern are shown in H.
high degree of sequence similarity between the family members
and a low level of non-synonymous substitutions suggests a
negative selection and therefore a preservation of their yet
unclear function. Sequence analysis of the proteins of the gen-
era Streptococcus, Staphylococcus, and Lactobacillus revealed
hydratase activity may be involved in this process. To test this,
MICs of free oleic and linoleic acid for the wild type and the
mutant strain were determined. MICs for 18:1 and 18:2 were
256 and 16 ␮g/ml for the wild type and 128 and 16 ␮g/ml for the
mutant, respectively (Fig. 4). Hence, the mutant was 2 times
more sensitive toward oleic acid than the wild type. No differ- only a semiconserved FAD/dinucleotide binding motif similar
ence between the two strains in sensitivity to linoleic acid was to that of the glutathione reductase family (GXGXXGX17–19(E/
observed.
D)) (supplemental Fig. S2). GXGXXG is part of a loop connect-
10358 JOURNAL OF BIOLOGICAL CHEMISTRY
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Fatty Acid Hydratase from S. pyogenes
TABLE 1
Substrate specificity of SPH
The products were identified by GC/MS analysis after chloroform/methanol (2:1) extractions of the samples. The reactions were performed as described under “Experi-
mental Procedures.” Two repetitions for each experiment were conducted.
Substrate
Product(s)
Retention time
Fragmentation pattern
min
⌬9Z
⌬9Z
⌬9Z
⌬9E
14:1
16:1
18:1
18:1
18:2
No products detected
10-Hydroxyhexadecanoate
10-Hydroxyoctadecanoate
No products detected
6.88
8.1
357(M-1ϩ), 273, 187, 169
385(M-1ϩ), 273, 215, 169
⌬9Z,⌬12Z
(12Z)-10-Hydroxy-12-octadecenoate
10,13-Dihydroxyoctadecanoate
(12Z,15Z)-10-Hydroxy-12,15-octadecadienoate
No products detected
8.33
8.81
8.77
383(M-1ϩ), 273, 213, 173, 169
313, 273, 213, 173
⌬9Z,⌬12Z,⌬15Z
⌬6Z,⌬9Z,⌬12Z
⌬11Z,⌬14Z,⌬17Z
18:3
18:3
20:3
20:4
380(M-1ϩ), 273, 241, 169
No products detected
Z
⌬5Z,⌬8 ⌬11Z,⌬14Z
No products detected
18:2⌬9Z,⌬12Z methyl ester
No products detected
Linoleyl-CoA
No products detected
Dilinoleylphosphatidylcholine
Trilinoleylglycerol
No products detected
No products detected
FIGURE4. Minimalinhibitoryconcentrationofoleicacidforwildtypeand
⌬sph strains of S. pyogenes M49. Optical density was measured after over-
night growth in the presence of different concentrations of free oleic acid in
THY medium. Data represent mean and S.D. of three independent experi-
ments. Wt, wild type.
third glycine allows close packing of the helix with the ␤-sheet
(30, 31). According to our analysis, the most drastic changes in
the canonical motif were observed for MCRA proteins from
Staphylococcus and Lactobacillus species, where the third gly-
cine is substituted either with a serine or asparagine residue,
respectively (supplemental Fig. S2). The substitution of glycine
with these larger residues may perturb proper orientation of the
first ␣-helix with the ␤-sheet and therefore may significantly
affect FAD binding. In fact, the MCRA proteins from lactoba-
cilli have even lower affinity with FAD in comparison with SPH
(data not shown). Noticeably, MCRA proteins have different
numbers of amino acid residues between the third glycine and
the conserved glutamate residue compared with the canonical
motif, namely 15 or 21 residues instead of 17–19 for the gluta-
thione reductase family. The glutamate/aspartate residues are
involved in hydrogen bonding with the ribose 2Ј-hydroxyl of
the adenosine moiety (30), and therefore the different position-
ing may also affect the strength of FAD binding. In accordance
FIGURE 3. Total ion chromatograms of GC/MS runs for identification of
products formed from linoleic and oleic acid upon incubation with wild
type and ⌬sph strains of S. pyogenes M49. A, products were formed from
linoleic acid (18:2): 10-HOE and 10,13-DiHOA. None of these products were
formed after incubation with the mutant. B, 10-HOA was formed from oleic
acid (18:1) after incubation with the wild type (Wt), and 10-HOA was not
detected after incubation with the mutant. For the experiments, 2.5 OD of the
wild type and mutant cells were incubated with 50 ␮g of substrates in 200 ␮l
ofTHYmediumfor3hat37 °C, followedbyextractionwithchloroform/meth-
anol (2:1). Representative data for one of two repetitions for the respective
condition are presented.
ing the first ␤-strand and ␣-helix in the ␤␣␤␣␤-Rossmann fold with this observation, heterologously expressed SPH appeared
with the N-terminal end of the first ␣-helix pointing toward the to be a yellow protein after the first step of purification by anion
pyrophosphate moiety for charge compensation. The role of exchange chromatography but appeared colorless after the size
the glycine residues is well understood. The first glycine allows exclusion chromatography or dialysis. Purification of His-
for a tight turn of the main chain, which is important for posi- tagged protein allowed us to obtain colored enzyme solution of
tioning the second glycine. The second glycine permits close higher purity, which was used for flavin cofactor identification
contact of the main chain to the pyrophosphate of FAD, and the and analysis of kinetic parameters (supplemental Fig. S3). Most
APRIL 2, 2010•VOLUME 285•NUMBER 14
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Fatty Acid Hydratase from S. pyogenes
detected. Instead, we were able to show that the MCRA protein
from S. pyogenes, when it was heterologously expressed in
E. coli, acts as a hydratase. The enzyme is specific for (9Z)- and
(12Z)-double bonds of C-16 and C-18 fatty acids and catalyzes
the formation of 10-hydroxy and 10,13-dihydroxy derivatives
(Table 1 and Fig. 2), and no CLA-forming isomerase activity
was detected (32). Analysis of substrate specificity showed that
SPH strictly prefers free fatty acids over the esterified species
(Table 1). The ability to add a water molecule to a double bond
classifies SPH as a new member of the carbon-oxygen lyase family.
In contrast to the well studied enoyl-CoA hydratases involved in
fattyacid ␤-oxidation, whichaddawatermoleculetothepolarized
␣-double bond in CoA-thioester (33, 34), SPH hydrates a non-
activated carbon-carbon double bond. Our experiments show that
molecular oxygen is not involved in this hydration reaction; thus,
the role of FAD in this process cannot be attributed to a redox
reaction (Fig. 2). Additionally, FAD was not reduced during reac-
tion of the holoenzyme with linoleic acid at least under our condi-
tions(supplementalFig. S6). Kineticanalysisofholo-andapo-SPH
forms demonstrated that apo-SPH was at least 10–50 times less
active than the holoenzyme, whereas linoleic acid was the pre-
ferred substrate. Together, these data provide good evidence of a
structural role of FAD in SPH function.
There are several examples of flavoenzymes that require
FAD to maintain a proper architecture of the active site but
where the cofactor is not directly involved in catalysis (35–37).
The direct role of FAD in the hydrogen transfer during
(10E,12Z)-CLA formation by double bond isomerase from Pro-
pionibacterium acnes (PAI) was demonstrated previously (38,
39). SPH has similar substrate requirements as the PAI and
(9Z,11E)-CLA isomerase of the cell envelope preparation of
Butyrivibrio fibrisolvens (40). So far, PAI is the only CLA-form-
ing double bond isomerase for which structural information is
available. In contrast to SPH, PAI retained FAD during purifi-
cation and crystallization, and the crystal structures of the free
enzyme and its complex with (10E,12Z)-CLA provided insight
into the substrate binding and reaction mechanism (38). How-
ever, the sequence alignment of PAI and SPH did not reveal any
significant similarity between the two proteins except the short
stretch of the FAD binding motif. With respect to an involve-
ment of SPH in CLA formation, it is still possible that recombinant
MCRA enzymes from CLA-producing species like lactobacilli
require critical protein partners or a membrane environment in
order to retain a double bond isomerase activity.
FIGURE 5. Blood survival and adherence to and internalization into
HaCaT cells of wild type and ⌬sph strains of S. pyogenes M49. A, blood
survival in heparinized human whole blood. The number of surviving CFU in
whole human blood was represented as a percentage of CFU of the respec-
tive serum. Data represent means and S.D. of three independent experiments
with three different blood samples. B and C, adherence and internalization,
respectively, represented as a percentage from the CFU count of the original
inoculum. Data represent means and S.D. of three independent experiments.
The asterisks mark data with statistical significance (p Ͻ 0.05). Wt, wild type.
likely, the purification of His-tagged SPH avoids multiple wash-
To investigate the possible role of the hydroxy fatty acids
ing steps and therefore allows retention of a larger proportion formed by SPH in the transformation of unsaturated fatty acids
of the cofactor. HPLC-MS/MS analysis unambiguously identi- to saturated ones, we used feeding experiments with U-13C-
fied FAD as a non-covalent bound cofactor of SPH (Fig. 1).
labeled linoleic acid. Analysis of free and esterified fatty acid
The first attempt to identify an enzymatic activity for MCRA fraction by GC/MS from the wild type strain and the ⌬sph
protein was reported by Rosson et al. (2). Their study mutant showed conversion to the hydroxy derivatives by wild
focused on the isolation of a (9Z,11E)-CLA-forming double type cells but not by the mutant (Fig. 3). However, in neither the
bond isomerase from L. reuteri PYR8. In this case, the major- wild type strain nor in the mutant did we detect CLA formation.
ity of the activity was found in the membrane fraction. The This observation argues against a link between the SPH-depen-
isolated isomerase protein had more than 70% identity and dent hydration and CLA formation, at least under our experi-
85% similarity to the known MCRA from S. pyogenes, for which mental conditions. On the other hand, SPH seems to be
no enzymatic activity was previously reported. However, when involved in fatty acid metabolism by a yet unknown mechanism
the putative (9Z,11E)-CLA isomerase was heterologously because in the mutant strain, consumption of the free fatty
expressed in E. coli and B. subtilis, no isomerase activity was acids from the medium is reduced to about 50% of that of the
10360 JOURNAL OF BIOLOGICAL CHEMISTRY
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Fatty Acid Hydratase from S. pyogenes
bielski, A. (2001) Mol. Microbiol. 39, 392–406
wild type. However, it seems not to be involved in fatty acid
homeostasis because MICs of cerulenin for wild type and the
mutant in the presence of different fatty acids were similar
(supplemental Fig. S9).
7. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254
8. Liavonchanka, A., Rudolph, M. G., Tittmann, K., Hamberg, M., and Feuss-
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Unsaturated fatty acids are toxic for many bacteria (41) due
to deteriorating effect on bacterial cellular membrane (42).
They also inhibit enoyl-ACP reductase (FabI) and thus disrupt
bacterial fatty acid synthesis (43). We hypothesize that hydra-
tion of unsaturated fatty acids may represent an additional
detoxification mechanism in bacteria harboring MCRA en-
zymes. Such detoxification could be essential for bacterial col-
onization and survival in fatty acid-rich environments (e.g. skin
and inflamed tissues). In fact, the mutant appeared to be 2-fold
more sensitive to oleic acid than wild type, whereas no changes
in sensitivity to linoleic acid was observed (Fig. 4). The later may
be explained by the fact that linoleic acid is 10 times more toxic
for S. pyogenes M49.
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No growth inhibition was observed for the mutant in human
serum, which can be explained by a much lower concentration
(up to 5 orders of magnitude, 7.5 nM) of free oleic and linoleic
acid in the serum (44, 45) than the measured MIC (58 ␮M for
linoleic acid, 458 ␮M for oleic acid). This may explain as well
why the virulence properties are independent of the fatty acid
concentrations in the serum. Here, the mutant showed
increased survival in blood with reduced adherence and inter-
nalization properties to HaCaT cells (Fig. 5, A–C). Therefore,
the deletion of MCRA protein may lead to reduced opsoniza-
tion of the bacterial cell surface, resulting in lower efficiency
20. Posada, D., and Crandall, K. A. (1998) Bioinformatics 14, 817–818
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involvement of MCRA in pathogenicity, however, is connected
to its enzymatic activity remains an open question at this stage.
In summary, our study shows for the first time a detailed
characterization of biochemical and physiological properties
for a member of the MCRA protein family. The SPH protein of
S. pyogenes M49 was identified as fatty acid hydratase that har-
bors FAD as a cofactor that stabilizes its active conformation. It
forms 10-hydroxy and 10,13-dihydroxy derivatives from C-16
and C-18 non-esterified fatty acids. Moreover, the deletion of
SPH led to a 2-fold decrease in resistance to oleic acid,
increased survival of the mutant strain in the whole blood, and
reduced adhesion and internalization to human keratinocytes
in comparison with the wild type.
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Acknowledgments—We thank Prof. Kai Tittmann and Dr. Danilo
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discussions.
39. Liavonchanka, A., and Feussner, I. (2008) ChemBioChem 9, 1867–1872
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