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Lipid Mass Spectrometry: Core Technologic Research and Development
IV.)
A.)
PAGE
MASS SPECTROMETRY OF COMPLEX LIPIDS FROM PATHOGENIC
MICROORGANISMS ………………………………………………………………………..
3
Complex Lipids of Kinetoplastid Parasites …………………………………………………..
3
A.1.) Leishmania major sphingolipids and ethanolamine lipids …………………………………..
A.1.a.) Sphingolipids are essential for differentiation but not growth in Leishmania ………………..
A.1.b.) Leishmania salvage and remodelling of host sphingolipids in amastigote survival and
acidocalcisome biogenesis. ……………………………………………………………………..
A.1.c.) Characterization of inositol phosphorylceramides from Leishmania major by tandem
mass spectrometry with electrospray ionization. ……………………………………………….
A.1.d.) Redirection of sphingolipid metabolism toward de novo synthesis of ethanolamine in
Leishmania. ……………………………………………………………………………………..
A.1.e.) 55. Degradation of host sphingomyelin us essential for Leishmania virulence……………….
A.1.f.) 72. Sphingosine kinase A is a pleiotropic and essential enzyme for Leishmania survival and
virulence…………………………………………………………………………………………
A.1.g.) 70. Structural characterization of Leishmania infantum diradyl-glycerophosphoethanolamine
lipids with cyclopropane fatty acid substituents by electrospray ionization multiple-stage
linear ion-trap tandem mass spectrometry with high m/z resolution………………………….
A.1.h.) 57. Deletion of UDP-glucose pyrophosphorylase reveals a UDP-glucose independent UDPgalactose salvage pathway in Leishmania major……………………………………………….
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A.2.) Trypanosoma brucei and Trypanosoma cruzi sphingolipids ……………………………….
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A.2.a.) 51.Developmentally regulated sphingolipid synthesis in African trypanosomes………………… 13
A.2.b.) 58.Cell-free synthesis and functional characterization of sphingolipid synthases from
parasitic Trypanosomatid protozoa………………………………………………………………. 15
B.)
Complex Lipids of Pathogenic Bacteria ……………………………………….......................
B.1.) Gram-Negative Bacteria: Salmonella species ……………………….…………………………
B.1.a.) Characterization of acylphosphatidylglycerols from Salmonella typhimurium by tandem
mass spectrometry with electrospray ionization. ……………………………………………….
B.1.b.) The PmrA-regulated pmrC gene mediates phosphoethanolamine modification of Lipid A
and polymyxin resistance in Salmonella enterica. ……………………………………………..
B.1.c.) PhoP-regulated Salmonella resistance to the antimicrobial peptides magainin 2 and
polymyxin B. …………………………………………………………………………………….
B.1.d.) Identification of the lipopolysaccharide modifications controlled by the Salmonella
PmrA/PmrB system mediating resistance to Fe(III) and Al(III). ……………………………..
B.1.e.) 78.The PmrAB system-inducing conditions control both Lipid A remodeling and O-antigen
length distribution, influencing the Salmonella typhimurium-host interactions………………
B.2.) Gram-Negative Bacteria: Escherichia coli: Characterization of cardiolipin from Escherichia
coli by ESI with multiple stage quadrupole ion-trap mass spectrometric analysis of
[M-2H+Na]- ionS…………………………………………………………………………………
B.3.) Gram-Positive Bacteria: Streptococcus pyogenes and Bacillus anthracis ……………………
B.3.a.) Anionic lipids enriched at the ExPortal of Streptococcus pyogenes. …………………………..
B.3.b.) 87.The Bacillus anthracis protein MprF is required for synthesis of lysylphosphatidyl1
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C.)
glycerols and for resistance to cationic antimicrobial peptides……………………………........
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Complex Lipids of Mycobacterium tuberculosis related species…………………………….
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C.1.) Identification and macrophage-activating activity of glycolipids released from
intracellular Mycobacterium bovis BCG. …………………………………..............................
C.2.) Mycobacterium tuberculosis Rv2252 encodes a diacylglycerol kinase involved in the
biosynthesis of phosphatidylinositol mannosides (PIMs). ……………………………………
C.3.) Structural characterization of phosphatidyl-myo-inositol mannosides from Mycobacterium
bovis Bacillus Calmette Guérin by multiple-stage quadrupole ion-trap mass spectrometry
with electrospray ionization. I. PIMs and lyso-PIMs. …………………………………………
C.4.) Structural characterization of phosphatidyl-myo-inositol mannosides from Mycobacterium
bovis Bacillus Calmette Gúerin by multiple-stage quadrupole ion-trap mass spectrometry
with electrospray ionization. II. Monoacyl- and diacyl-PIMs. ………………………………..
C.5.) 85.Mycobacterium abscessus glycopeptidolipids mask underlying cell wall phosphatidyl-myoinositol mannosides blocking induction of human macrophage TNF- α by preventing
interaction with TLR2……………………………………………………………………………
C.6.) 84.Caseation of human tuberculosis granulomas correlates with elevated host lipid
metabolism………………………………………………………………………………………..
C.7.) 82.aprABC: a Mycobacterium tuberculosis complex-specific locus that modulates
pH-driven adaptation to the macrophage phagosome…………………………………………..
C.8.) 61. Characterization of sulfolipids of Mycobacterium tuberculosis H37Rv by multiple-stage
linear ion-trap high-resolution mass spectrometry with electrospray ionization reveals that
the family of Sulfolipid II predominates…………………………………………………………
C.9.) 60.Characterization of mycolic acids from the pathogen Rhodococcus equi by tandem
mass spectrometry with electrospray ionization…………………………………………………
C.10.) 62.Structural definition of trehalose 6-monomycolates and trehalose 6,6'-dimycolates from the
pathogen Rhodococcus equi by multiple-stage linear ion-trap mass spectrometry with
electrospray ionization……………………………………………………………………………
C.11.) 64. Structural determination of glycopeptidolipids of Mycobacterium smegmatis by highresolution multiple-stage linear ion-trap mass spectrometry with electrospray ionization…….
C.12.) 77.Diversion of phagosome trafficking by pathogenic Rhodococcus equi depends on mycolic
acid chain length………………………………………………………………………………….
C.13.) 74.MmpL11 protein transports mycolic acid-containing lipids to the mycobacterial cell
wall and contributes to biofilm formation in Mycobacterium smegmatis………………………
C.14.) 69.Characterization of mycobacterial triacylglycerols and monomeromycolyl diacylglycerols
from Mycobacterium smegmatis biofilm by electrospray ionization multiple-stage and
high-resolution mass spectrometry………………………………………………………………
D.)
Pathogenic Microorganism Lipids Literature Cited …………………………........................
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IV.)
Core Technologic Research and Development: Mass Spectrometry of Complex Lipids from
Pathogenic Microorganisms
A.)
Complex Lipids of Kinetoplastid Parasites
A.1.) Leishmania major sphingolipids and ethanolamine lipids.
A.1.a.) Sphingolipids are essential for differentiation but not growth in Leishmania. Sphingolipids (SLs) play
critical roles in eukaryotic cells in the formation of lipid rafts, membrane trafficking, and signal transduction.
We created a SL-null mutant in the protozoan parasite Leishmania major through targeted deletion of the key de
novo biosynthetic enzyme serine palmitoyltransferase subunit 2 (SPT2) (1). Although SLs are typically
essential, spt2- Leishmania were viable, yet were completely deficient in de novo sphingolipid synthesis, and
lacked inositol phosphorylceramides and other SLs. Remarkably, spt2- parasites maintained 'lipid rafts' as
Figure 1. Negative ion ESI/MS spectra of total lipids purified from log phase WT (A) and spt2- (B) cells.
The assigned identities of peaks are indicated. Abbreviations: p18:0/18:2-PE, 1-O-octdec-1’-enyl-2-octadecadienoyl-snglycero-3-phosphoethanolamine (plasmalogen); p18:0/18:1-PE, 1-O-octadec-1’-enyl 2-octadecenoyl-sn-glycerophosphoethanolamine; d16:1/18:0-PI-Cer, phosphorylinositol N-stearoyl-hexadecesphing-4-enine (IPC-d16:1); d18:1/18:0-PI-Cer,
phosphorylinositol N-stearoylsphingosine (IPC-d18:1); 18:0/18:1-PI, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositol;
16:/18:1-PI, 1-palmitoyl-2-oleyoyl-sn-glycero-3-phosphoinositol; a18:0/18:1-PI, 1-O-octadecanyl-2-octadecenoyl-sn-glycero-3-phosphoinositol (plasmanyl inositol).
defined by Triton X-100 detergent resistant membrane formation. Upon entry to stationary phase spt2- failed to
differentiate to infective metacyclic parasites and died instead. Death occurred not by apoptosis or changes in
metacyclic gene expression, but from catastrophic accumulation of small vesicles characteristic of the
multivesicular body/multivesicular tubule network. Stage specificity may reflect changes in membrane structure
3
as well as elevated demands in vesicular trafficking required for parasite remodeling during differentiation. SLdeficient Leishmania provide a useful biological setting for tests of essential SL enzymes in other organisms
where SL perturbation is lethal (1).
A.1.b.) Leishmania salvage and remodelling of host sphingolipids in amastigote survival and acidocalcisome
biogenesis. Sphingolipids (SLs) play essential roles in most eukaryotes, but in the trypanosomatid protozoan
Leishmania major their functions differ significantly. It was previously demonstrated that although null mutants
defective in de novo sphingoid base synthesis (spt2–) lack SLs, they grow well and retain lipid rafts while
Figure 2. Amastigotes contain abundant parasite-specific IPCs. Lipids from log phase WT (A) or
spt2- (B) promastigotes, from foot pads of uninfected mice (C), or amastigotes purified from mice infected with WT (D) or
spt2- (E) parasites, were extracted and analyzed by negative-ion ESI/MS using a precursor ion scan of m/z 241 (specific for
IPCs and PIs). Before lipid extraction, a PI standard (16:0/16:0-PI at m/z 809.8) was added as internal standard.
replicating as promastigotes in vitro, but they experience catastrophic defects in membrane trafficking on entry
into stationary phase and fail to differentiate to the infective metacyclic form. Here this mutant is shown to
retain the ability to enter macrophages silently and inhibit activation, although, as expected, most parasites were
destroyed (2). In contrast, in mouse infections, after a delay rapidly progressive lesions appear, and purified
amastigotes are fully virulent to macrophages and mice. Mass spectrometry of spt2– amastigote lipids revealed
the presence of high levels of parasite-specific inositol phosphorylceramides (IPCs) not synthesized by the
mammalian host. Inhibitor studies reveal that salvage occurs at the level of complex SLs, suggesting that
4
parasites carry out ‘headgroup’ remodeling. Additionally, a new defect of the spt2– promastigotes is observed
that involves ‘empty’ acidocalcisomes (ACs), which may point to the origin of this organelle from the
lysosome-related organelle/multivesicular body biogenesis pathway. Nonetheless, ACs in spt2– amastigotes
appear quantitatively and morphologically normal. Thus, salvage of SLs and other molecules by intracellular
amastigotes is important in AC biogenesis and parasite survival in the host (2).
A.1.c.) Characterization of inositol phosphorylceramides from Leishmania major by tandem mass
spectrometry with electrospray ionization. We have developed tandem mass spectrometric approaches,
including multiple stage ion-trap and source collisionally activated dissociation (CAD) tandem mass
spectrometry with electrospray ionization (ESI) to characterize inositol phosphorylceramide (IPC) species as [M
- H]- and [M - 2H + Li]- ions in negative-ion mode and as [M + H]+, [M + Li]+, and [M - H + 2Li]+ ions in
positive-ion mode (3). Following CAD in an ion-trap or a triple-stage quadrupole instrument, IPC [M - H]- ions
+
Figure 3. Fragmentation of Li adducts of inositol-phosphorylceramides upon ESI/MS/MS.
yield fragment ions reflecting only the inositol and the fatty acyl substituent. In contrast, spectra from MS3 of
[M - H - Inositol]- ions contain abundant ions that identify the fatty acid and long-chain base (LCB) moieties.
Product-ion spectra from MS2 and MS3 of [M - 2H + Alk]-, [M + H]+, [M + Alk]+, and [M - H + 2Alk]+ ions also
contain fragment ions that permit unambiguous assignment of the fatty acyl substituent and the LCB, but the
abundance of those precursor ions is about ten-fold lower than that of [M - H]- ions, which limits sensitivity. In
addition to the major fragmentation pathways involving elimination of the inositol or inositol monophosphate
moiety, several structurally informative ions result from rearrangement processes, and the fragmentation
processes are similar to those we previously reported for ceramides. Although the MSn (n = 2, 3) tandem mass
spectrometry permits detailed structural assignment of Leishmania major IPC species, including isomer
discrimination, constant neutral loss scans provide a simple method for detecting IPC species in biological
mixtures (3).
A.1.d.) Redirection of sphingolipid metabolism toward de novo synthesis of ethanolamine in Leishmania. In
most eukaryotes, sphingolipids (SLs) are critical membrane components and signaling molecules, but
mutants of the trypanosomatid protozoan Leishmania lacking serine palmitoyltransferase (spt2-) and SLs grow
well but are defective in stationary phase differentiation and virulence. Similar phenotypes are observed in
sphingolipid (SL) mutant lacking the degradatory enzyme sphingosine 1-phosphate lyase (spl-). This epistatic
interaction implicates a metabolite downstream of SLs, we have shown that, unlike other organisms, the
Leishmania SL pathway has evolved to be the major route for ethanolamine (EtN) synthesis, as EtN
5
Figure 4. Synthesis of Sphingolipids and Phosphatidylethanolamine in Eukaryotes. Open block arrows
represent pathways not present in Leishmania. Filled block arrows represent dominant metabolic routes in Leishmania
promastigotes. ADS1 = 1-alkyl dihydroxyacetone phosphate synthase; SDC = serine decarboxylase*; PSS =
phosphatidylserine synthase*; BE = base exchange enzyme (phosphatidylserine synthase 2 or PSS2); PSD =
phosphatidylserine decarboxylase; Smy = sphingomyelin’ GSL = glycosphingolipid; DAG = diacylglycerol; DHAP =
dihydroxyacetone phosphate; * denotes absent in Leishmania.
supplementation completely reversed the viability and differentiation defects of both mutants (4). Thus,
Leishmania has undergone two major metabolic shifts: first in de-emphasizing the metabolic roles of SLs
themselves in growth, signaling, and maintenance of membrane microdomains, which may arise from the
unique combination of abundant parasite lipids; Second, freed of typical SL functional constraints and a lack of
alternative routes to produce EtN, Leishmania redirected SL metabolism toward bulk EtN synthesis. These
results thus reveal a striking example of remodeling of the SL metabolic pathway in Leishmania (4).
A.1.e.) Degradation of host sphingomyelin is essential for Leishmania virulence. In eukaryotes, sphingolipids
(SLs) are important membrane components and powerful signaling molecules. In Leishmania, the major group
of SLs is inositol phosphorylceramide (IPC), which is common in yeast and Trypanosomatids but absent in
mammals. In contrast, sphingomyelin is not synthesized by Leishmania but is abundant in mammals. In the
promastigote stage in vitro, Leishmania use SL metabolism as a major pathway to produce ethanolamine (EtN),
a metabolite essential for survival and differentiation from non-virulent procyclics to highly virulent
metacyclics. To further probe SL metabolism, we identified a gene encoding a putative neutral
sphingomyelinase (SMase) and/or IPC hydrolase (IPCase), designated ISCL (Inositol phosphoSphingolipid
phospholipase C-Like) (5). Despite the lack of sphingomyelin synthesis, L. major promastigotes exhibited a
potent SMase activity which was abolished upon deletion of ISCL, and increased following over-expression by
episomal complementation. ISCL-dependent activity with sphingomyelin was about 20 fold greater than that
6
Fig. 5. Restoration of stationary phase morphology and acute virulence in iscl2 mutants by mammalian
neutral SMases and fungal ISC1ps. (A)–(B) Morphology and virulence of iscl2 parasites with human neutral SMase 1 (iscl2 SSU::hNSM1),
murine neutral SMase 2 (iscl2 SSU::mNSM2), or pIR1SAT vector only (iscl2 SSU::pIR) were examined as described. (C)–(D) Similar experiments were
performed using iscl2 parasites transfected with pIR1SAT-ScISC1 (iscl2/+ScISC1), pIR1SAT-CnISC1 (iscl2/+CnISC1), and pIR1SAT vector only (iscl2/+pIR).
In (A) and (C), promastigotes were grown to stationary phase and the percentage of round cells (day 1 through day 5 in stationary phase) was determined. In
(B) and (D), infectivity of stationary phase promastigotes was examined by footpad infection assay using BALB/c mice (26107 cells/mouse in B and 16106
cells in D) and the progression of lesions was monitored weekly.
SPHINGOMYELINASE
SERINE
PALMITOYL
TRANSFERASE
SPHINGOSINE-1PO4 LYASE
INOSITOL
PHOSPHORYL
CERAMIDE
SYNTHASE
INOSITOL PHOSPHO-SPHINGO-LIPID
PHOSPHOLIPASE C1
INOSITOL
PHOSPHOSPHINGOLIPID
PHOSPHOLIPASE C1
Fig. 6. Metabolism of Sphingolipids in L. major.
Fig. 7. Degradation of sphingomyelin (A) and IPC (B).
SPT: serine palmitoyltransferase; SPL: sphingosine- 1-phosphate lyase;
ISC1p: inositol phospho-sphingolipid phospholipase C 1 protein or IPCase;
IPCS: IPC synthase. Note that phosphoethanolamine can be produced via
an IPC-independent pathway or an indirect pathway that requires the
synthesis and degradation of IPC.
In mammals, sphingomyelin degradation by SMase is a major route to
produce ceramide, an important sig-naling molecule. Leishmania do not
synthesize sphin-gomyelin but contain high abundance of IPC. Fungi also
synthesize IPC & hydrolyze IPC with inositol phosphosphingolipid
phospholipase C (ISC1p, B), a homolog of mammalian neutral SMase
seen with IPC. Null mutants of ISCL (iscl2) showed modest accumulation of IPC, but grew and differentiated
normally in vitro. Interestingly, iscl2 mutants did not induce lesion pathology in the susceptible BALB/c mice,
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yet persisted indefinitely at low levels at the site of infection. Notably, the acute virulence of iscl2 was
completely restored by the expression of ISCL or heterologous mammalian or fungal SMases, but not by fungal
proteins exhibiting only IPCase activity. Together, these findings strongly suggest that degradation of hostderived sphingomyelin plays a pivotal role in the proliferation of Leishmania in mammalian hosts and the
manifestation of acute disease pathology (5). (PLoS Pathog. 2009; 5(12): 1000692).
A.1.f.) Sphingosine kinase A is a pleiotropic and essential enzyme for Leishmania survival and virulence.
Sphingosine kinase is a key enzyme in sphingolipid metabolism, catalyzing the conversion of sphingosine or
dihydrosphingosine into sphingosine-1-phosphate or dihydrosphingosine-1-phosphate respectively. In
mammals, sphingosine-1-phosphate is a powerful signaling molecule regulating cell growth, differentiation,
apoptosis and immunity. Functions of sphingosine kinase or sphingosine-1-phosphate in pathogenic protozoans
Fig. 8. Loss of SKa leads to catastrophic defects in culture due to sphingoid base accumulation. (A and
B) Ska− mutants exhibit severe growth defects in culture. Promastigotes were inoculated at 1.0 × 105 cells ml−1 and
incubated at 27°C. Culture densities (A) and percentages of dead cells (B) were monitored daily. (C and D) Rescue of ska−
mutants by MYR + EtN. Ska− promastigotes were inoculated at 1.0 × 105 cells ml−1 in the presence of EtN (200 µM), MYR
(1 µM) or EtN + MYR (200 and 1 µM respectively) and incubated at 27°C. Culture densities (C) and percentages of dead
cells (D) were monitored daily.
are virtually unknown. While most organisms possess two closely related sphingosine kinases, only one
sphingosine kinase homologue (SKa) can be identified in Leishmania, which are vector-borne protozoan
parasites responsible for leishmaniasis. Leishmania SKa is a large, cytoplasmic enzyme capable of
phosphorylating both sphingosine and dihydrosphingosine. Remarkably, deletion of SKa leads to catastrophic
defects in both the insect stage and mammalian stage of Leishmania parasites (6). Genetic and biochemical
8
analyses demonstrate that proper expression of SKa is essential for Leishmania parasites to remove toxic
metabolites, to survive stressful conditions, and to cause disease in mice. Therefore, SKa is a pleiotropic
enzyme with vital roles throughout the life cycle of Leishmania. The essentiality of SKa and its apparent
divergence from mammalian counterparts suggests that this enzyme can be selectively targeted to reduce
Leishmania infection (6). (Molecular Microbiology 2013; 90: 489–501.)
Fig. 9. Sphingolipid Metabolism in Leishmania. Left Panel: Sphingolipid biosynthesis and involvement in
ethanolamine generation in Leishmania. Right Panel: Negative-ion ESI/MS of total lipids from log phase
promastigotes of WT (a), ska- (b), & ska-/+ ska (c). [M-H] ions for ceramide, PLE, IPC, and PI are labeled in red.
A.1.g.) Structural characterization of Leishmania infantum diradyl-glycerophosphoethanolamine lipids with
cyclopropane fatty acid substituents by electrospray ionization multiple-stage linear ion-trap tandem mass
spectrometry with high m/z resolution. Leishmania infantum glycerophosphoethanolamine (GPE) lipids
include an unusual subfamily with fatty acid substituents that contain a cyclopropane moiety. Such
cyclopropane fatty acid (CFA)-containing GPE lipids include plasmenylethanolamine (plasmalogen) species.
We developed an ESI-LIT-MSn approach using high m/z resolution to characterize the structures of such lipids
by CAD of [M-H]- and [M-H+2Li]+ ions (7). MSn data from each of those ions provided complementary
information that permitted complete structural characterization, including identification of the fatty acid or fatty
aldehyde residues and their positions on the glycerol backbone, as well as the location(s) of double bonds and of
cyclopropane moieties in the fatty chains. The resultant structural data supported previously reported CFA
biosynthetic pathways. A GPE lipid subclass was also identified that had not previously been reported in L.
infantum in which the head-group amine is substituted with two methyl groups. This ESI-LIT-MSn approach
permitted unambiguous identification of diradyl-GPE lipids in complex biological mixtures that included
numerous isomers that would be difficult to identify with conventional analytical methods (7). (J. Mass
Spectrom. 2014; 49: 201-9.)
9
Fig. 10. BIOSYNTHESIS OF PHOSPHOLIPID-BOUND CYCLOPROPANE FATTY
ACIDS IN LEISHMANIA INFANTUM.
Table 1. High resolution and ESI-LIT-MSn analyses of L. infantum diradylglycerophosphoethanolamine species.
10
n
+
Fig. 11. ESI-LIT-MS of L. infantum diradyl-GPE lipid [M–H+2Li] ions.
2
+
3
4
4
(a) MS of [M–H+2Li] (m/z 728); (b) MS of m/z 605 (728→605); (c) MS of m/z 293 (728→605→293); (d) MS of m/z
4
2
+
295 (728→605→295); (e) MS of m/z 309 (728→605→309); (f) MS of [M-H+2Li] of 9,10-methyleneoctadecanoic acid
standard (m/z 309), which is similar to Panel e, providing the structural assignment. Insets of Panels a, c, d and f are
proposed fragmentation pathways that specify position(s) of double bond(s) and/or of cyclopropane moieties.
A.1.h.) Deletion of UDP-glucose pyrophosphorylase reveals a UDP-glucose independent UDP-galactose
salvage pathway in Leishmania major. The nucleotide sugar UDP-galactose (UDP-Gal) is essential for the
biosynthesis of several abundant glycocon-jugates forming the surface glycocalyx of the protozoan parasite
Leishmania major (8). Current data suggest that UDP-Gal could arise de novo by epimerization of UDP-glucose
(UDP-Glc) or by a salvage pathway involving phosphorylation of Gal and the action of UDP-glucose:α-Dgalactose-1-phosphate uridylyltransferase as described by Leloir. Since both pathways require UDP-Glc,
inactivation of the UDP-glucose pyrophosphorylase (UGP) catalyzing activation of glucose-1 phosphate to
UDP-Glc was expected to deprive parasites of UDP-Gal required for Leishmania glycocalyx formation.
Targeted deletion of the gene encoding UGP, however, only partially affected the synthesis of the Gal-rich
phosphoglycans. Moreover, no alteration in the abundant Gal-containing glycoinositolphospholipids was found
in the deletion mutant. Consistent with these findings, the virulence of the UGP-deficient mutant was only
modestly affected. These data suggest that Leishmania elaborates a UDP-Glc independent salvage pathway for
UDP-Gal biosynthesis (8). (Glycobiology 2010; 20: 872–882).
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Fig. 12. Biosynthesis of UDP-α-D-galactose in various organisms.
UDP-α-D-galactose (UDP-Gal) is synthesized de novo by
epimerization of UDP-α-D-glucose (UDP-Glc) by the UDP-glucose 4-epimerase (UDP-Glc 4-epimerase, EC:5.1.3.2). In addition, α-D-galactose-1-phosphate (α-DGal-1-P) produced from α-D-galactose (α-D-Gal) by the galactokinase (GK, EC:2.7.1.6) is activated by the UDP-glucose:α-D-galactose-1-phosphate
uridylyltransferase (Gal-1-P uridylyltransferase, EC:2.7.7.12). These reactions depend on UDP-Glc production from α-D-glucose-1-phosphate (α-D-Glc-1-P) by the
UTP:α-D-glucose-1-phosphate uridylyltransferase also named UDP-glucose pyrophosphorylase (UGP, EC:2.7.7.9). The phosphoglucomutase (PGM, EC:5.4.2.2)
mediating theinterconversion of α-D-Glc-1-P and α-D-glucose-6-P (α-D-Glc-6-P) connects the galactose metabolism to gluconeogenesis and glycolysis. α-D-Glc6-P may also originate from phosphorylation of free glucose (α-D-Glc) by the glucokinase (EC:2.7.1.1) or hexokinase (HK, EC:2.7.1.2). The conversion of α-D-Gal1-P into UDP-Gal described in mammals by Isselbacher is thought to be due to a weak UTP:α-D-galactose-1-phosphate uridylyltransferase activity (EC:2.7.7.10)
of UGP. In plants, a third pathway for UDP-Gal biosynthesis is mediated by an unspecific UDP-sugar pyrophosphorylase (USP, EC:2.7.7.64). The pathways
proposed for Leishmania parasites are based on analysis of the genome and the existence of a UDP-glucose independent pathway for UDP-Gal biosynthesis
demonstrated in this work. Activation of α-D-Glc-1-P and α-D-Gal-1-P by USP would explain the production of UDP-Glc and UDP-Gal in the L. major ugp− mutant.
12
(See Figure on Preceding Page.)
Fig. 13. GIPLs structures are unaffected in the ugp− mutant.
(A) Schematic of Leishmania GIPLs. Light shaded circles, Galp; light circles with f, Galf; dark circles, Man; half-shaded
squares, GlcN; hexagons, myoinositol; & P, phosphate. (B) Negative-ion MALDI spectra of GIPLs from ugp− mutant (top
panel) & wild-type (lower panel) parasites. Identities of major ions are indicated by schematics in A & can be inferred from the
GIPL3
structure,
which
is
Galα1-6Galα1-3Galfβ1-3Manα1-3-Manα1-4GlcNα1-6myo-inositol-1-HPO4-3(sn-1-alkyl-2acylglycerol). Nos. of C atoms & C═C double bonds in acyl & alkyl chains are indicated above each peak.
A.2.) Trypanosoma brucei and Trypanosoma cruzi sphingolipids.
A.2.a.) Developmentally regulated sphingolipid synthesis in African trypanosomes. Sphingolipids are essential
components of eukaryotic membranes, and many unicellular eukaryotes, including kinetoplastid protozoa, are
Fig. 14. T. brucei sphingolipid synthase locus. A. Organization
of ~9 kb TbSLS locus on Chromosome 9 is presented schematically from
5′end of upstream intergenic region to 3′end of downstream intergenic
region. Black boxes, TbSLS1–4 genomic open reading frames (orfs) from
first in-frame Met codons. Grey boxes, extent of highly similar 5′UTRs.
Arrowhead over TbSLS4 indicates site of in situ in-frame introduction of
HA epitope tag. Thin bar over TbSLS1 orf repre-sents relative position of
757 nt dsRNA target sequence. Thick bar under TbSLS1 orf represents
relative position of the 283 nt target sequence of northern probe. Pair-wise
nucleotide sequence identities of RNAi target regions range from 87.3%
(TbSLS1 vs. TbSLS2) to 98.9% (TbSLS3 vs. TbSLS4). Numbering of
TbSLS genes is consecutive. B. Deduced N-terminal amino acid
sequences of TbSLS orfs. Numbering is relative to 1st in-frame Met
residue downstream of experimentally determined trans-splice acceptor
site in corresponding mRNAs (arrowhead). Sequences are aligned to 1st
Met codon (Met-18) of genomic TbSLS1 orf. Identity is indicated by dots,
and positions of 5′stop codons in TbSLS2 & TbSLS3 genomic orfs are
indicated by asterisks (codon -15). Initiator Met codons for each orf as
originally annotated in the T. brucei genomic database are underlined.
a
Table 2. Composition of sphingolipids from T. brucei and transgenic L. major .
thought to synthesize exclusively inositol phosphorylceramide (IPC). Here sphingolipids from Trypanosoma
brucei were characterized, and a trypanosome sphingolipid synthase gene family (TbSLS1–4) that is
orthologous to Leishmania IPC synthase (9). Procyclic trypanosomes contain IPC, but also sphingomyelin,
while surprisingly bloodstream-stage parasites contain sphingomyelin and ethanolamine phosphorylceramide
(EPC), but no detectable IPC. In vivo fluorescent ceramide labeling confirmed stage-specific biosynthesis of
both sphingomyelin and IPC. Expression of TbSLS4 in Leishmania resulted in production of sphingomyelin
13
Fig. 15. Total lipid profiles of bloodstream-form and procyclic trypanosomes.
Negative-ion (A and C) and positive-ion
(B and D) ESI/MS scans of total lipids purified from bloodstream (BSF; A and B) and procyclic (PCF, C and D) trypanosomes are presented. Internal standards
included in the extraction steps were phosphatidylethanolamine [PE Int. Std. (12:0/12:0), m/z 578.6] and phosphatidylcholine [PC Int. Std. (12:0/12:0), protonated
m/z 622.6, sodiated m/z 644.6]. Identities and structure of major sphingolipid species, as well as select prominent glycerolphospholipids were confirmed by
multiple-stage mass spectrometry (MSn, n = 2, 3, 4) and are indicated as appropriate. Abbreviations: PE, phosphatidylethanolamine; PC, phosphatidylcholine;
PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; EPC, ethanolamine phosphorylceramide; IPC, inositol phosphorylceramide; p, plasmalogen;
+
+
a, plasmanyl glycerophospholipid. Note that in the positive-ion mode a variable mixture of H and Na I adduct ons are detected for each choline-containing lipid.
and EPC suggesting that the TbSLS gene family has bi-functional synthase activity. RNAi silencing of
TbSLS1–4 in bloodstream trypanosomes led to rapid growth arrest and eventual cell death. Ceramide levels
Fig. 16. Total lipid profiles of TbSLS-silenced bloodstream-form trypanosomes. Clonal bloodstream cells containing
the TbSLS RNAi construct were cultured for 24 h without (A) or with (B) tetracycline to induce specific dsRNA synthesis. Total lipids were
extracted and analysed in negative-ion mode.
14
were increased more than threefold by silencing, suggesting a toxic downstream effect mediated by this potent
Fig. 17. TbSLS topology model.
A. Diagram of TbSLS. Methods for predicting topology are described in Experimental procedures. Conserved domains
(D1–D4) of SMS sequences as defined by Huitema et al. (2004) are indicated by red bars. Selected conserved residues
within these domains are indicated in one-letter code, or F for bulky hydrophobic. Key residues of the putative catalytic
triad (TbSMS1, H210, H253, D257) in TM4 and TM6 are indicated in silhouette.
B. Alignment of the D2 domain sequences (upper case) from TbSLS1, human SMS2 (Q8NHU3) and L. major IPCS
(LmjF35.4990). Vertical lines indicate identity and conserved residues from the consensus motif are indicated below.
Transmembrane domain residues (silhouette) are: TbSLS1, 144–164; human SMS2, 154–174; LmjIPCS, 158–180.
intracellular messenger. Topology predictions support a revised six-transmembrane domain model for the
kinetoplastid sphingolipid synthases consistent with the proposed mammalian sphingomyelin synthase
structure. This work reveals novel diversity and regulation in sphingolipid metabolism in this important group
of human parasites (9). (Molecular Microbiology 2008; 70: 281–296.)
A.2.b.) Cell-free synthesis and functional characterization of sphingolipid synthases from parasitic
Trypanosomatid protozoa. The Trypanosoma brucei genome has four highly similar genes encoding
sphingolipid synthases (TbSLS1–4). TbSLSs are polytopic membrane proteins that are essential for viability of
the pathogenic bloodstream stage of this human protozoan parasite and, consequently, can be considered as
potential drug targets. TbSLS4 was shown previously to be a bifunctional sphingomyelin/ethanolamine
phosphorylceramide synthase, whereas functions of the others were not characterized (10). Using a recently
described liposome-supplemented cell-free synthesis system, which eliminates complications from background
cellular activities, we now unambiguously define the enzymatic specificity of the entire gene family. TbSLS1
produces inositol phosphorylceramide, TbSLS2 produces ethanolamine phosphorylceramide, and TbSLS3 is
bifunctional, like TbSLS4. These findings indicate that TbSLS1 is uniquely responsible for synthesis of inositol
phosphorylceramide in insect stage parasites, in agreement with published expression array data. This approach
15
also revealed that the Trypanosoma cruzi ortholog
(TcSLS1) is a dedicated inositol phosphorylceramide
synthase. The cell-free synthesis system allowed rapid
optimization of the reaction conditions for these enzymes
and site-specific mutagenesis to alter end product
specificity. A single residue at position 252 (TbSLS1,
Ser252; TbSLS3, Phe252) strongly influences enzymatic
specificity. We also have used this system to demonstrate
that aureobasidin A, a potent inhibitor of fungal inositol
phosphorylceramide synthases, does not significantly
affect any of the TbSLS activities, consistent with the
phylogenetic distance of these two clades of sphingolipid
synthases. These results represent the first application of
cell-free synthesis for the rapid preparation and functional
annotation of integral membrane proteins and thus
Fig. 18. Sphingolipid synthesis pathway.
3-KDS, 3-keto-dihydrosphingosine; PL, phosphol-ipid; DAG,
diacylglycerol. Homologs of all indicated enzymes (italics) are
found in the T. brucei and L. major genomes. The proposed site
of action of aureobasidin A (AbA?) is indicated.
Fig. 19. ESI-MS lipid analysis of the TbSLS1HA transgenic bloodstream cell line.
A & C. Negative-ion ESI-MS scans of
total lipids from control & TbSLS1HA cells, respectively. Internal PE standard, endogenous EPC & IPC peaks are indicated. A small peak for d18:1/16:0 IPC is
detected in TbSLS1HA cells, but not in control parental cells. B & D. The corresponding tandem quadrupole mass spectra from precursor of m/z 241 (dehydrated
inositol phosphate anion) scans are presented. Ions in these scans represent species initially containing inositol phosphate, i.e., IPC & PI. The most prominent ion
m/z 778.2 seen (Panel D) (TbSLS1HA cell line) is d18:1/16:0-IPC. Low levels of this species are also seen in control cell scans (Panel D). Additional IPC ions in
Panel D: m/z 766.7, d17:0/16:0-IPC; m/z 780.8, d18:0/16:0-IPC; m/z 796.9, t18:0/16:0-IPC; & m/z 806.9, d20:1/16:0-IIPC.PC.
16
Fig. 20. Site-specific mutagenesis of TbSLSs.
A, alignment of Trypanosomatid SLS Loop V residues. Top, diagram of the modeled topology of Trypanosomatid SLSs
(adapted from Refs. 28 and 11). Transmembrane domains and solvent exposed loops are numbered in Arabic and
Roman numerals, respectively. Key catalytic residues are silhouetted. Residue 252 targeted for mutagenesis is indicated
by an open circle. Bottom, alignment of the Loop V region from trypanosomatid SLSs as indicated. Key catalytic residues
are underlined, and residue 252 is silhouetted. Proven enzymatic activities are in parentheses. Numbering is according to
the T. brucei orthologs. B, TbSLS1 and TbSLS3 mutants were produced in the cell-free system, and normalized amounts
of translation product were assayed for enzymatic activity as in Fig. 4. Top, SDS-PAGE and Coomassie Blue staining of
purified proteoliposomes. Equivalent synthesis of matched wild type and mutant TbSLSs is evident. The vertical white
stripe indicates intervening lanes that were digitally excised from the gel. Equivalent amounts of each cell-free translation
product were used in the SLS assays. Bottom, NBD-SL product analysis was performed in parallel on a single plate. A
phosphorimage is presented with mobility standards indicated. WT, wild type.
illustrate its utility in studying otherwise intractable enzyme systems (10). (J. Biol. Chem. 2010; 285: 20580–
20587).
B.)
Complex Lipids of Pathogenic Bacteria
B.1.) Gram-Negative Bacteria: Salmonella species
B.1.a.) Characterization of acylphosphatidylglycerols from Salmonella typhimurium by tandem mass
spectrometry with electrospray ionization. Acylphosphatidylglycerol (Acyl-PG), a polar lipid that contains
three fatty acyl groups, has been isolated from Salmonella bacteria and characterized by tandem quadrupole
and quadrupole ion-trap mass spectrometry with electrospray ionization. For acyl-PG species with various acyl
groups (A-B/C-PG, where A, B, and C differ from each other) (RxCO2-) that arise from sn-2 (R2CO2-) are more
abundant than those that arise from sn-3' (R3’CO2-). In addition, R3’CO2- is much more abundant than R1CO2-)
17
(11). This permits assignment of the positives of the fatty acyl substituents. In addition, the fragment ion
reflecting loss of the sn-2 fatty acid substituent as a ketene is more abundant than the ion reflecting loss of that
substituent as a free acid (i.e., [M - H - R'2CH=CO]- > [M - H – R2CO2H]-). In contrast, the sn-1 or sn-3' fatty
acid substituents are eliminated more readily is a free acid than as a ketene (i.e., [M - H – R1CO2H]- > [M - H R'1CH=CO]-; [M - H - R3’CO2H]- > [M - H -R'3’CH=CO]-). The identity of the sn-3' acyl moiety is also reflected
by an acyl-glycerophosphate
anion in the product-ion
spectrum from a triple-stage
quadrupole (TSQ) instrument
but not in that from an iontrap
mass
spectrometer
(ITMS). However, the MS2spectrum obtained with an
ITMS is featured by the ion
series that abundances of
[M-H-R'2CH=CO–R3CO2H-74]->
[M-H-R'2CH=CO–R1CO2H-74]-;
[M-H-R'1(or3’)CH=CO–R3’(or 1)CO2H-74]-.
Structural identification of
Figure 21. Structure of acylphosphatidylglycerols in Salmonella.
acyl-PG species with 2 identical fatty acyl substituents at
sn-1, sn-2, or sn-3' or of members of mixtures that contain isomers can be achieved with this approach (11).
B.1.b.) The PmrA-regulated pmrC gene mediates phosphoethanolamine modification of lipid A and
polymyxin resistance in Salmonella enterica. The PmrA/PmrB regulatory system of Salmonella enterica
controls the modification of Lipid A with aminoarabinose and phosphoethanolamine. The aminoarabinose
modification is required for resistance to the antibiotic polymyxin B, as mutations of the PmrA-activated pbg
18
(See Figure on Preceding Page) Figure 22. Lipid A species profiles from wild-type (14028s) (A), pbgP
(EG9241) (B), ΔpmrC1.1 (EG14590) (C), ΔpmrC1.1/ppmrC (EG14595) (D), and ΔpmrC1.1/vector (EG14656) (E) strains
grown to logarithmic phase in N-minimal medium, pH 5.8, with 10 µM MgCl2, and analyzed by negative-ion-mode MALDITOF mass spectrometry. These profiles show that the pmrC mutant lacks lipid A species modified with
phosphoethanolamine.
operon or ugd gene result in strains that lack aminoarabinose in their lipid A molecules and are more susceptible
to polymyxin B. Additional PmrA-regulated genes appear to participate in polymyxin B resistance, as pbgP and
ugd mutants are not as sensitive to polymyxin B as a pmrA mutant. Moreover, the role that the
phosphoethanolamine modification of lipid A plays in the resistance to polymyxin B has remained unknown.
We addressed both of these questions by establishing that the PmrA-activated pmrC gene encodes an inner
membrane protein that is required for the incorporation of phosphoethanolamine into lipid A and for polymyxin
B resistance (12). The PmrC protein consists of an N-terminal region with five transmembrane domains
followed by a large periplasmic region harboring the putative enzymatic domain. A pbgP pmrC double mutant
resembled a pmrA mutant both in its lipid A profile and in its susceptibility to polymyxin B, indicating that the
PmrA-dependent modification of lipid A with aminoarabinose and phosphoethanolamine is responsible for
PmrA-regulated polymyxin B resistance (12).
B.1.c.) PhoP-regulated Salmonella resistance to the antimicrobial peptides magainin 2 and polymyxin B. In
Salmonella enterica, the PhoP-PhoQ two-component system governs resistance to structurally different
antimicrobial peptides including the alpha-helical magainin 2, the beta-sheet defensins and the cyclic
Figure 23. PhoP-regulated lipid A modifications associated with resistance to antimicrobial peptides.
The formation of monophosphorylated lipid A is mediated by the UgtL protein, which is required for resistance to both magainin 2 and
polymyxin B. Although UgtL is shown as acting at the 1 position of lipid A, it is presently unclear whether it affects the 1 and/or 4’
positions. The PagP-mediated transfer of a palmitate from phospholipids to lipid A is required for resistance to magainin 2 but not to
polymyxin B. PmrA-controlled loci encode enzymes involved in the biosynthesis and incorporation into lipid A of 4-aminoarabinose,
which is necessary for resistance to polymyxin B but not magainin 2. Although the 4-aminoarabinose modification is shown as acting at
the 4’ position of lipid A, it is presently unclear whether it affects the 1 and/or 4’ positions.
19
lipopeptide polymyxin B. To identify the PhoP-regulated determinants mediating peptide resistance, we
prepared a plasmid library from a phoP mutant, introduced it into a phoP mutant and selected for magaininresistant clones (13). One of the clones harboured the PhoP-activated ugtL gene, deletion of which rendered
Salmonella susceptible to magainin 2 and polymyxin B, but not defensin HNP-1. We established that ugtL
encodes an inner membrane protein that promotes the formation of monophosphorylated lipid A in the
lipopolysaccharide. Inactivation of both ugtL and the regulatory gene pmrA, which controls lipid A
modifications required for resistance to polymxyin B (but not to magainin 2) and is post-transcriptionally
activated by the PhoP-PhoQ system, resulted in a strain that was as susceptible to polymyxin B as a phoP
mutant. The most frequently recovered clone harboured the yqjA gene, which we show is PhoP regulated and
required for resistance to magainin 2 but not to polymyxin B or defensin HNP-1. Thus, different Lipid A PhoPmediated modifications are required for resistance to different antimicrobial peptides (13).
B.1.d.) Identification of the lipopolysaccharide modifications controlled by the Salmonella PmrA/PmrB
system mediating resistance to Fe(III) and Al(III). Iron is an essential metal but can be toxic in excess. While
several homeostatic mechanisms prevent oxygen-dependent killing promoted by Fe(II), little is known about
how cells cope with Fe(III), which kills by oxygen-independent means. Several Gram-negative bacterial species
Figure 24. PmrG is a Phosphatase in Salmonella that Targets the Hep(II) Phosphate of
Lipopolsysaccharide (LPS) and Localizes to the Periplasm. Schematic representation of LPS phosphates
targeted by Fe(III) resistance determinants. PmrC protein mediates modification of lipid A phosphates with
phosphoethanolamine whereas Ugd and PbgPE proteins modify lipid A phosphates with 4-aminoarabinose. PmrG protein
removes phosphate from Hep(II) in the LPA inner core region normally introduced by RfaY protein.
harbour a regulatory system - termed PmrA/PmrB - that is activated by and required for resistance to Fe(III).
We now report the identification of the PmrA-regulated determinants mediating resistance to Fe(III) and Al(III)
in Salmonella enterica serovar Typhimurium (14). We establish that these determinants remodel two regions of
20
the lipopolysaccharide, decreasing the negative charge of this major constituent of the outer membrane (8).
Remodeling entails the covalent modification of the two phosphates in the lipid A region with
phosphoethanolamine and 4-aminoarabinose, which has been previously implicated in resistance to polymyxin
B, as well as dephosphorylation of the Hep(II) phosphate in the core region by the PmrG protein. A mutant
lacking the PmrA-regulated Fe(III) resistance genes bound more Fe(III) than the wild-type strain and was
defective for survival in soil, suggesting that these PmrA-regulated lipopolysaccharide modifications aid
Salmonella's survival and spread in non-host environments (14).
B.1.e.) The PmrAB system-inducing conditions control both Lipid A remodeling and O-antigen length
distribution, influencing the Salmonella typhimurium-host interactions. The Salmonella enterica serovar
Typhimurium lipopolysaccharide consisting of covalently linked lipid A, non-repeating core oligosaccharide,
and the O-antigen polysaccharide is the most exposed component of the cell envelope. Previous studies have
demonstrated that all of these regions act against the host immunity barrier. Here, the role and interaction of
Fig. 25. Analysis of the lipid A
profiles from wzzst mutant.
Lipid A from wild-type (14028s)
(A), pmrA (EG13307) (B), and
wzzst
(EG14929)
(C)
S.
Typhimurium strains grown to
logarithmic phase in N-minimal
2+
medium with low Mg
were
analyzed by negative ion mode
MALDI-TOF-mass spectrometry.
These profiles show that the
wzzst mutant lacks diphosphorylated Lipid A bearing L-Ara4N
(m/z
1928),
L-Ara4N
and
palmitic acid (m/z 2166), and
pEtN and palmitic acid (m/z
2158) species but that this
mutant harbors a new lipid A
species of m/z 2281. D, S.
Typhimurium
Lipid
A
modifications associated with
PhoPQ and PmrAB activation
through pagP, pmrC, and wzzst
gene regulation, respectively.
The PagP protein catalyzes the
palmitic acid transfer to Lipid A,
whereas PmrC is required for
modification of Lipid A with
pEtN, resulting in a lipid A
species of m/z 2158 (left). In the
absence of wzzst, a new pEtN
group (ca.123 average mass
units) could be added at the 1or
4’-position
of
the
diphosphorylated m/z 2158 Lipid
A, leading to formation of the
species represented by the ion
of m/z 2281 (right).
21
Fig. 26. Formation of the Lipid A species of m/z 2281 requires pmrC and pagP. Shown are negative ion mode MALDITOF mass spectra of Lipid A from pmrC (EG13633) (A), wzzst pmrC (MDs1015) (B), pagP (EG13678) (C), and wzzst pagP
2+
(MDs1016) (D) mutants grown in low Mg conditions to induce the PmrAB system in a PhoPQ-dependent manner.
PmrAB-dependent gene products required for the lipopolysaccharide component synthesis or modification
22
mainly during the Salmonella infection were defined (15). The PmrAB two-component system activation
promotes a remodeling of Lipid A and the core region by addition of 4-aminoarabinose and/or
Fig. 27. Role of pbgE2 and pbgE3 genes in the O-antigen length distribution. A, graphic representation of the
pbgPE operon harboring 7 genes required for L-Ara4N incorporation into Lipid A. B, production of VL-, L-, and S-type O-antigen in response
8
to PmrAB system-inducing conditions. The SDS-PAGE analysis of LPS was performed using samples isolated from 10 cells of wild-type
2+
(14028s), wzzfepE (MDs1443), wzzst (EG14929), pbgE2 (MDs1102), and pbgE3 (MDs1103) strains grown in low Mg (left and middle
+
+
2+
panels) or from pbgE2 and pbgE3 mutants carrying ppbgE2 (pbgE2 ) and ppbgE3 (pbgE3 ) plasmids, respectively, grown in low Mg and 0.5
mM IPTG (right panel). The VL-, L-, and S-type O-antigen and the number of O-subunits attached to the lipid A-core are indicated on the left.
C, alignment of the PX2PX4SPKX1X10GGMXGAG domain conserved in PbgE2 (filled line box) and PbgE3 (dotted line box) proteins with the
reported Wzzst functional sequence domain of S. Typhimurium.
phosphoethanolamine. These PmrA-dependent activities are produced by activation of ugd, pbgPE, pmrC, cpta,
and pmrG transcription. In addition, under PmrA regulator activation, expression of wzzfepE and wzzst genes is
induced, and their products are required to determine the O-antigen chain length. Here Wzzst protein is
23
demonstrated to be necessary to maintain the balance of 4-aminoarabinose and phosphoethanolamine lipid A
modifications. Moreover, the interaction of the PmrA-dependent pbgE2 and pbgE3 gene products is shown to
be important for the formation of the short O-antigen region. These results establish that PmrAB is the global
regulatory system that controls lipopolysaccharide modification, leading to a coordinate regulation of 4aminoarabinose incorporation and O-antigen chain length to respond against the host defense mechanisms (15).
(J. Biol. Chem. 2012: 287: 38778–38789).
B.2.) Gram-Negative Bacteria: Escherichia coli: Characterization of cardiolipin from Escherichia coli by
electrospray ionization with multiple stage quadrupole ion-trap mass spectrometric analysis of [M-2H+Na]ions. We have developed a multiple-stage ion-trap (IT) mass spectrometric approach with electrospray
-
Figure 28. Fragmentation of [M - 2H + Na] ions of cardiolipin from Escherichia coli upon
ESI/MS/MS.
ionization (ESI) for structural characterization of cardiolipin (CL) species by collisionally activated
dissociation (CAD) of their [M - 2H + Na]- ions (16). CL is a 1,3-bisphosphatidyl-sn-glycerol that consists of
four fatty acyl chains and three glycerol moieties designated A, B, and central glycerol, respectively. Upon
CAD, CL [M - 2H + Na]- ions yield two prominent fragment ions that arise from the differential losses of the
diacylglycerol (DAG) moieties containing A or B glycerol, respectively. Ions that arise from loss of the DAG
moiety containing glycerol B are more abundant than those arising from DAG that contains glycerol A. This
permits assignment of the two phosphatidyl moieties attached to the 1'- or 3'-position of the central glycerol.
The structures of those two ions, including the identities of the fatty acyl substituents and their positions on the
glycerol A and B backbones, are determined by MS3 experiments. This approach permits assignment of the
structures of CL species, including isomers, in a mixture isolated from Escherichia coli (16).
B.3.) Gram-Positive Bacteria: Streptococcus pyogenes
B.3.a.) Anionic lipids enriched at the ExPortal of Streptococcus pyogenes. The ExPortal of Streptococcus
pyogenes is a membrane microdomain dedicated to the secretion and folding of proteins. We investigated the
lipid composition of the ExPortal by examining the distribution of anionic membrane phospholipids (17).
24
Staining with 10-N-nonyl-acridine orange revealed a single microdomain enriched with an anionic phospholipid
whose staining characteristics and behavior in a cardiolipin-deficient mutant were characteristic of
phosphatidylglycerol. Furthermore, the location of the microdomain corresponded to the site of active protein
secretion at the ExPortal. These results indicate that the ExPortal is an asymmetric lipid microdomain,
whose enriched content of anionic phospholipids may play an important role in ExPortal organization and
protein trafficking (17).
Figure 29. Lipid profile of streptococcal membranes. The electrospray ionization/mass spectrometry spectra of
-
-
the lipid extracts arising from the [M-H] ions of cardiolipin from Cls cells (panel A) and from the WT (panel B). Panels C
and D show the [M-H] ions of phosphatidylglycerol species from the extracts shown in panels A and B, respectively. Ions
from the (12:0)4-cardiolipin internal standard seen at m/z 1239.8 (panels A and B) are [M-H] ions, while the ions seen at
22m/z 619.7 (panels C and D) are [M-2H] ions. Both the [M-H] (panel A) and [M-2H] (panel C) ions of cardiolipin are
absent in the lipid extract from Cls cells but are abundant in the lipid extract from WT cells (panels B and D). In contrast,
phosphatidylglycerol is abundant from Cls cells (pane C) and is of relatively low abundance in the WT (panel D).
B.3.b.) The Bacillus anthracis protein MprF is required for synthesis of lysylphosphatidylglycerols and for
resistance to cationic antimicrobial peptides. During inhalational anthrax, Bacillus anthracis survives and
replicates in alveolar macrophages, followed by rapid invasion into the host’s bloodstream, where it multiplies
to cause heavy bacteremia. B. anthracis must therefore defend itself from host immune functions encountered
during both the intracellular and the extracellular stages of anthrax infection. In both of these niches, cationic
antimicrobial peptides are an essential component of the host’s innate immune response that targets B.
anthracis. However, the genetic determinants of B. anthracis contributing to resistance to these peptides are
largely unknown. Here we generated Tn917 transposon mutants in the ΔANR strain (pXO1- pXO2-) of B.
25
anthracis and screened them for altered protamine susceptibility. A protamine-sensitive mutant identified
Fig. 30. B. anthracis mprF mutant is
hypersusceptible to a linear
cationic peptide protamine.
Wild-type Sterne 34F2, wild-type/
pCN55 (empty vector) (SH0001),
ΔmprF::Kmr
mutant
(SH0002),
ΔmprF::Kmr/pmprF (SH0003), and
mprF::Tn917ermr (SH0004) strains
(ca. 5 x 104 cells) were spotted onto
LB agar plates supplemented with
protamine
at
the
indicated
concentrations. The images scanned
were obtained after 48 hours of
incubation at 37°C.
Fig. 31. B. anthracis mprF mutant is unable to synthesize
lysinylated phospholipids.
2D-TLC profiles of 32P-labeled phospholipids (A) and L[14C]lysine-labeled lipids (B) from wild-type Sterne 34F2,
ΔmprF::Kmr, or ΔmprF::Kmr/pmprF strains. The total
radiolabeled lipid samples were prepared from each strain
and separated by 2D-TLC and visualized using a
phosphorimager. Wild-type lipid samples subjected to
preparatory 2D-TLC and iodine-stained displayed profiles
similar to that of the wild-type sample in panel A. The circled
area (indicated approximately with a dotted line) on the
preparatory 2D-TLC with wild-type lipid samples was
extracted as a mixture for mass spectrometric analyses. CL,
cardiolipin;
PE,
phosphatidylethanolamine;
PG,
phosphatidylglycerol.
carried the transposon inserted in the BA1486 gene encoding a putative membrane protein homologous to MprF
known in several gram-positive pathogens (18). A mutant strain with the BAS1375 gene (the orthologue of
BA1486) deleted in the Sterne 34F2 strain (pXO1+ pXO2-) of B. anthracis exhibited hypersusceptibility not
only to protamine but also to α-helical cathelicidin LL-37 and β-sheet defensin human neutrophil peptide 1
compared to the wild-type Sterne strain. Analysis of membrane lipids using isotopic labeling demonstrated that
the BAS1375 deletion mutant is unable to synthesize lysinylated phosphatidylglycerols, and this defect is
rescued by genetic complementation. Further, the structures of these lysylphosphatidylglycerols were
determined using various mass spectrometric analyses. These results demonstrate that in B. anthracis a
functional MprF is required for the biosynthesis of lysylphosphatidylglycerols, which is critical for resistance to
cationic antimicrobial peptides (18). (J. Bacteriology 2009; 191: 1311–1319.)
26
Figure 32. Mass spectrometric analyses of lysylphospholipids of B. anthracis. Lysylphospholipid samples
n
were prepared and analyzed by ESI-LIT-MS . (A) ESI/MS spectrum obtained in the positive-ion mode; (B)
2
2
+
3
LIT/MS spectrum of neutral loss scan of 300; (C) LIT/MS spectrum of the [M+H] ion at m/z 851; (D) LIT/MS
spectrum of the same ion at m/z 551 (851à551).
27
C.)
Complex Lipids of Mycobacterium tuberculosis and related species
C.1.) Identification and macrophage-activating activity of glycolipids released from intracellular
Mycobacterium bovis BCG. Intracellular mycobacteria release cell wall glycolipids into the endosomal network
of infected macrophages. We have characterized the glycolipids ofMycobacterium bovis BCG (BCG) that are
released into murine bone marrow-derived macrophages (BMMØ) (19). Intracellularly released mycobacterial
lipids were harvested from BMMØ that had been infected with 14C-labelled BCG. Released BCG lipids were
Figure 33. Mass Spectrometry of culture-derived BCG lipids that co-migrate with intracellularly
released lipids. A and B. ESI mass spectra of the [M-H]- ions of 1,2-diacyl-sn-glycero-3-phosphatidylinositol
dimannoside (PIM 2) species differing in the degree of additional acylation. The major species of PIM2a (A) at m/z 1413
(C 16:0-C19:0-PIM 2) contains and additional C16:0, whereas the major species of PIM 2b (B) at m/z 1693 (C16:0, C19:0C19:0/C16:0-PIM 2) contains an additional C16:0 and C19:0.
resolved by thin-layer chromatography, and they migrated similarly to phosphatidylinositol dimannosides
(PIM2), mono- and diphosphatidylglycerol, phosphatidylethanolamine, trehalose mono- and dimycolates and
the phenolic glycolipid, mycoside B. Culture-derived BCG lipids that co-migrated with the intracellularly
released lipids were purified and identified by electrospray ionization mass spectrometry. When delivered on
polystyrene microspheres, fluorescently tagged BCG lipids were also released into the BMMØ, in a manner
similar to release from viable or heat-killed BCG bacilli. To determine whether the released lipids elicited
macrophage responses, BCG lipid-coated microspheres were delivered to interferon gamma-primed
macrophages (BMMØ or thioglycollate-elicited peritoneal macrophages), and reactive nitrogen intermediates
as well as tumour necrosis factor-alpha and monocyte chemoattractant protein-1 production were induced.
When fractionated BCG lipids were delivered on the microspheres, PIM2 species reproduced the macrophageactivating activity of total BCG lipids. These results demonstrate that intracellular mycobacteria release a
28
heterogeneous mix of lipids, some of which elicit the production of proinflammatory cytokines from
macrophages that could potentially contribute to the granulomatous response in tuberculous diseases (19).
C.2.) Mycobacterium tuberculosis Rv2252 encodes a diacylglycerol kinase involved in the biosynthesis of
phosphatidylinositol mannosides (PIMs). Phosphorylated lipids play important roles in biological systems, not
only as structural moieties but also as modulators of cellular function. Phospholipids of pathogenic bacteria play
roles both as membrane components and as factors that modulate the infectious process. Mycobacterium
Figure 34. Mycobacterium Tuberculosis Expresses a Unique Diacylglycerol Kinase Specific for the
Diglyceride Lipid Core of Mycobacterial Phosphatidylinositol-Mannosides.
tuberculosis has an extremely diverse repertoire of biologically active phosphorylated lipids that, in the absence
of a specialized protein translocation system, appear to constitute the main means of communication with the
host. Many of these lipids are derived from phosphatidylinositol (PI) that is differentially processed to give rise
to phosphatidylinositol mannosides (PIMs) or lipoarabinomannan. In preliminary studies on the lipid processing
enzymes associated with the bacterial cell wall, a kinase activity was noted that gave rise to a novel lipid species
released by the bacterium. It was determined that this kinase activity was encoded by the ORF Rv2252 (20).
Rv2252 demonstrates the capacity to phosphorylate various amphipathic lipids of host and bacterial origin, in
particular a M. tuberculosis-derived diacylglycerol. Targeted deletion of the rv2252 gene resulted in disruption
of the production of certain higher order PIM species, suggesting a role for Rv2252 in the biosynthetic pathway
of PI, a PIM precursor (20).
C.3.) Structural characterization of phosphatidyl-myo-inositol mannosides from Mycobacterium bovis
Bacillus Calmette Guérin by multiple-stage quadrupole ion-trap mass spectrometry with electrospray
ionization. I. PIMs and lyso-PIMs. We have developed multiple-stage ion-trap mass spectrometric approaches
to characterize structures of phosphatidylinositol and phosphatidyl-myoinositol mannosides (PIMs) in a
complex mixture from Mycobacterium bovis Bacillus Calmette Guérin (21). Positions of the fatty acyl
substituents of PIMs at the glycerol backbone can be assigned are reflected by the fact that ions arising from
losses of the sn-2 fatty acid substituent as a free acid or as a ketene, respectively (i.e., [M - H – R2CO2H]- and
[M - H – R2CHCO]-), are more abundant than ions arising from analogous losses at sn-1 (i.e., [M - H –
R1CO2H]- and [M - H – R1CHCO]-) in MS2 product-ion spectra of [M - H]- ions produced by electrospray
29
ionization (ESI). [M - H – R2CO2H]- and [M - H – R1CO2H]- ions decompose to yield a pair of unique ions
Figure 35. Structure of phosphatidyl-myo-inositol mannosides from Mycobacterium bovis.
corresponding to losses of 74 and 56 Da (i.e., [M - H – RxCO2H - 56]- and [M - H – RxCO2H - 74]-, x = 1 or 2),
respectively. This probably reflects elimination of various glycerol-derived moieties. The relative abundance of
members of this ion-pair in the MS3 spectrum of [M - H – R2CO2H]- is quite difference from that in the MS3
spectrum of the [M - H – R1CO2H]-, and this permits assignment of the fatty acid substituents and their positions
on the glycerol backbone. Product-ion spectra of [M - H]- ions from 2-lyso-PIM and 1-lyso-PIM are
distinguishable and both spectra contain a unique ion that arises from primary loss of the fatty acid substituent,
followed by loss of a bicyclic glycerophosphate ester moiety of 136 Da. Combined information from MS2 and
MS3 product-ion spectra permit detailed assignments of PIM structures, including isomer discrimination (21).
C.4.) Structural characterization of M. bovis Bacillus Calmette Gúerin phosphatidyl-myo-inositol
mannosides by quadrupole ion-trap MSn with electrospray ionization. II. Monoacyl- and diacyl-PIMs.
Multiple-stage
ion-trap MS approaches for characterizing monoacyl-PIM (triacylated PIM) and
diacyl-PIM (tetracylated
PIM)
have been developed (22). Identification of the
fatty acid substituents and their
positions on the
glycerol backbone
can be achieved as
described above.
The identity of the
glycoside moiety
Figure 36. Fragmentation of of phosphatidyl-myo-inositol mannosides from M. bovis
upon ESI/MS/MS.
and its acylation
state is reflected
by a prominent acylglycoside ion arising from cleavage of the diacylglycerol moiety ([M - H - diacylglycerol]-)
30
in the MS2-spectra of monoacyl-PIM and diacyl-PIM. Fatty acid substituents on the 2-O-mannoside (i.e.,
R3CO2H) can be distinguished from those on the inositol (i.e., R4CO2H) because the MS3-spectrum of [M - H diacylglycerol]- contains a prominent ion arising from further loss of the fatty acid substituent on the 2-Omannoside (i.e., [M - H - diacylglycerol – R3CO2H]-), while the ion arising from loss of the fatty acid
substituent on the inositol moiety (i.e., the [M-H-diacylglycerol–R4CO2H]- ion) is of low abundance. The fatty
acyl substituent on the inositol moiety can be identified by the MS4 product-ion spectrum of [M-H\diacylglycerol– R3CO2H]-, which contains a prominent ion corresponding to loss of R4CO2H. An [M - H acylmannose]- ion is also observed in the MS2-spectra and also reveals the identity of the fatty acid substituent
on the 2-O-mannoside moiety. Combined information from MS2, MS3, and MS4 multiple-stage product-ion
spectra permit identification of monoacyl-PIMs and diacyl-PIMs in a mixture from M. bovis Bacillus Calmette
Guérin (22).
C.5.) Mycobacterium abscessus glycopeptidolipids mask underlying cell wall phosphatidyl-myo-inositol
mannosides blocking induction of human macrophage TNF-α by preventing interaction with TLR2.
C
MACROPHAGE
TNFα PRDUCTION
MACROPHAGE
TNFα PRDUCTION
ROUGH
ROUGH
ROUGH
Figure 37. TNF-α response M. abscesssus variants and involvement of TLR2.
ROUGH
390R
SMOOTH
390S
ROUGH
390V
GLYCOPEPTIDOLIPIDS
SMOOTH ROUGH ROUGH
A–B, M. abscessus 390R variants. A, Human MDM
were challenged with M. abscessus 390R, 390V, or 390S. Culture supernatants were collected at various intervals after addition of bacteria and assayed by
ELISA for TNF-α. p < 0.01 for 390R and 390V vs 390S. B, Human MDM were preincubated with Ab to TLR2 or isotype control Ab and then challenged
with M. abscessus 390R. Culture supernatants were collected at 2 h after addition of bacteria and assayed by ELISA for TNF-α. p < 0.01 comparing 390R
plus anti-TLR2 to 390R and 390R plus isotype control Ab. C. Equal weights of total lipid extracts from CF-R and CF-S were analyzed by TLC and visualized
with 1-naphthol, which detects GPLs. Lanes 1, 2, and 3, M. abscessus 390R, 390S, and 390V, respectively.
31
MONOACYL-PIM2
m/z 1399
ROUGH
390R
PI m/z 851
DIACYL-PIM2
m/z 1664
PI m/z 851
MONOACYL-PIM2
m/z 1399
SMOOTH
390S
DIACYL-PIM2
m/z 1664
Fig. 38. Mass spectrometric comparison of M. abscessus PIMs. Crude lipid extracts of M. abscessus (A, 390R;
and B, 390S) were analyzed by tandem quadrupole ion MS. Major ions of phosphatidylinositol as well as the monoacyl and
diacyl forms of PIM2, PIM3, and PIM6 were present in similar proportions in all variants as indicated.
Mycobacterium abscessus causes disease in patients wit h structural abnormalities of the lung, and it is an
emerging pathogen in patients with cystic fibrosis. Colonization of the airways by nontuberculous mycobacteria
is a harbinger of invasive lung disease. Colonization is facilitated by biofilm formation, with M. abscessus
glycopeptidolipids playing an important role. M. abscessus can transition between a noninvasive, biofilmforming, smooth colony phenotype that expresses glycopeptidolipid, and an invasive rough colony phenotype
that expresses minimal amounts of glycopeptidolipid and is unable to form biofilms (23). The ability of this
pathogen to transition between these phenotypes may have particular relevance to lung infection in cystic
fibrosis patients since the altered pulmonary physiology of these patients makes them particularly susceptible to
colonization by biofilm-forming bacteria. Here rough variants of M. abscessus were found to stimulate the
human macrophage innate immune response through TLR2, while smooth variants do not. Temperaturedependent loss or physical removal of glycopeptidolipid from the cell wall of one of the smooth variants leads
to TLR2 stimulation. This response is stimulated in part through phosphatidyl-myoinositol mannosides that are
present in the cell wall of both rough and smooth variants. Mannose-binding lectins bind to rough variants, but
32
lectin binding to an isogenic smooth variant is markedly reduced. This suggests that glycopeptidolipid in the
outermost portion of the M. abscessus cell wall masks underlying cell wall lipids involved in stimulating the
innate immune response, thereby facilitating colonization (23). Conversely spontaneous “unmasking” of cell
wall lipids may promote airway inflammation. (J. Immunology 2009) 183: 1997–2007).
C.6.) Caseation of human tuberculosis granulomas correlates with elevated host lipid metabolism. The
progression of human tuberculosis (TB) to active disease and transmission involves the development of a
caseous granuloma that cavitates and releases infectious Mycobacterium tuberculosis bacilli. Here a genomewide microarray analysis was used to determine that genes for lipid sequestration and metabolism are highly
expressed in caseous TB granulomas (24). Immunohistological analysis of these granulomas confirmed the
disproportionate abundance of proteins involved in lipid metabolism in cells surrounding the caseum; namely,
adipophilin, acyl-CoA synthetase long-chain family member 1 and saposin C. Biochemical analysis of the lipid
species within the caseum identified cholesterol, cholesteryl esters, triacylglycerols and lactosylceramide, which
implicated low-density lipoprotein-derived lipids as the most likely source. M. tuberculosis infection in vitro
induced lipid droplet formation in murine and human macrophages. Furthermore, the M. tuberculosis cell wall
lipid trehalose dimycolate induced a strong granulomatous response in mice, which was accompanied by foam
cell formation. These results provide molecular and biochemical evidence that development of the human TB
granuloma to caseation correlates with pathogen-mediated dysregulation of host lipid metabolism. (24). (EMBO
Mol Med. 2010; 2: 258-74).
D. ESI-MS/MS
TRIACYLGLYCEROLS
A. EI-MS CHOLESTEROL
[M+NH4]
+
CHOLESTEROL
B. GC-MS TIC
+
E. ESI-MS/MS LACTOSYLCERAMIDES [M+Na]
C. ESI-MS/MS CHOLESTEROL ESTERS
PAR 369
SCAN
CNL 162 SCAN
33
(See Figure on Preceding Page)
Figure 39. Tuberculous granuloma lipid analysis by mass spectrometry.
The identities of cholesterol (A: electron impact gas chromatography/mass spectrometry (EI-MS), B: total ion current (TIC)), cholesteryl
esters (C), triacylglycerols (D), and lactosylceramide (E) were confirmed by mass spectrometry.
Figure 40. A model illustrating the linkages between Mtb-infection, foam cell formation and accumulation of
caseum in the human TB granuloma. A. Intracellular Mtb bacilli synthesize and release cell wall components
inside their host cells. These lipids have previously been found to accumulate in the internal vesicles in multi-vesicular
bodies, which are exocytosed from the cell in vesicular form. B. Because of the release of these vesicles, both infected
and uninfected macrophages are exposed to cell wall mycolates and induced to form foamy macrophages. The
foamy macrophages have been found to support the maintenance and growth of persistent bacteria. C. These cells are
proposed to die via an inflammatory, necrotic process and release their lipid droplets into the extracellular milieu
within the granuloma. As a result of the fibrotic capsule, the human granuloma is an enclosed, isolated structure with
minimal vasculature. The enclosed nature of the human granuloma leads to accumulation of necrotic debris as caseum. In
this model, this process is an integral part of the pathology that leads to active disease and transmission.
C.7.) aprABC: a Mycobacterium tuberculosis complex-specific locus that modulates pH-driven adaptation to
the macrophage phagosome. Following phagocytosis by macrophages, Mycobacterium tuberculosis (Mtb)
senses the intracellular environment and remodels its gene expression for growth in the phagosome. Here an
acid and phagosome regulated (aprABC) locus is identified that is unique to the Mtb complex and whose gene
expression is induced during growth in acidic environments in vitro and in macrophages. Using the aprA
promoter, a strain was generated that exhibits high levels of inducible fluorescence in response to growth in
acidic medium in vitro and in macrophages (25). aprABC expression was found to be dependent on the two34
component regulator phoPR, linking phoPR signaling to pH sensing. Deletion of the aprABC locus was found
to cause defects in gene expression that impact aggregation, intracellular growth, and the relative levels of
storage and cell wall lipids. A model is proposed in which phoPR senses the acidic pH of the phagosome and
induces aprABC expression to fine-tune processes unique for intracellular adaptation of Mtb complex bacteria
(25). (Molecular Microbiology 2011; 80: 678–694).
Figure 41. The aprABC locus is specific to the Mtb complex and exhibits sustained induction during growth
in acidic medium in vitro and in MØ. A. Schematic of genes in the region surrounding the aprABC locus. 1 =
Rv2392, 2 = Rv2393, 3 = Rv2394, 4 = Rv2395, 5 = Rv2397. Genes 1 and 5 are involved in sulphur metabolism and are
conserved across Mycobacterium species. The phylogenetic tree is an approximate representation of the relationships between
the species. The gene colors signify the conservation of genes in a particular species. For example, genes coloured red are
specific to the Mtb complex species, while genes coloured blue are conserved in the Mtb complex as well as M. marinum and M.
ulcerans. B and C. Semi-quantitative real-time PCR shows in vitro induction of the aprABC locus at pH 6.0 (B)
and pH 5.5 (C) when compared with expression at pH 7.0. D. The aprABC locus is induced in resting mouse
MØ when compared with the 2 h MØ medium control. Expression of aprABC locus genes is slightly repressed during
the 2 h incubation in MØ medium contributing to the higher magnitude of aprABC locus expression in the MØ as compared with
in vitro.
35
Figure 42. The deletion of the aprABC locus alters the accumulation of mycobacterial lipids. Identification
+
of lipid species by MALDI-TOF MS. Spectra of [M+Na] ions of three identified species: Band 1: Triacylgly-cerols, Band 2:
Phthiocerol A dimycocerate, Band 3: Phthiodiolone dimycocerate.
C.8.) Characterization of sulfolipids of Mycobacterium tuberculosis H37Rv by multiple-stage linear ion-trap
high-resolution mass spectrometry with electrospray ionization reveals that the family of Sulfolipid II
predominates. Mycobacterium tuberculosis, the causative agent of tuberculosis, contains a wide array of
complex lipids and lipoglycans in its cell wall. Among them, the sulfated glycolipid (designated sulfolipid) is
thought to mediate specific host−pathogen interactions during infection. Sulfolipids (SLs), including sulfolipid I
36
(SL-I) and sulfolipid II (SL-II), are 2,3,6,6-tetraacyltrehalose-2-sulfates. SL-I was identified as a family of
homologous 2-palmitoyl(stearoyl)-3-phthioceranoyl-6,6‘-bis-(hydroxyphthioceranoy1)trehalose 2’-sulfates and
was believed to be the principal sulfolipid of M. tuberculosis strain H37Rv (26). After mycobacterial cultured
and extraction of sulfolipids using various conditions, including those originally described, high resolution
multiple-stage linear ion-trap mass spectrometry with electrospray ionization was used to characterize the
structure of the principal SL. In contrast to previous reports, SL-II rather than SL-I was found to be the principal
M. tuberculosis strain H37Rv sulfolipid class. SL-II comprises a family of homologous 2-stearoyl(palmitoyl)3,6,6’-tris(hydroxyphthioceranoy1)-trehalose 2’-sulfates. We identified numerous isomers resulting with
various combinations of hydroxyphthioceranoyl substituents at positions 6 and 6’ of the trehalose backbone for
each of the SLII species in the family. These findings re-define the structure of this important lipid family that
was misassigned using conventional analytical techniques 40 years ago (26). (Biochemistry 2011; 50:
9135−9147.)
Figure 43. High-resolution ESI-MS spectrum of the [M-H]− ions of sulfolipids of H37Rv grown in
Middlebrook 7H9 broth. The spectrum was obtained on an FTICR instrument. High-resolution (R =
500000) readily distinguished SL-I (monoisotopic ions labeled with ∗) class from SL-II (monoisotopic
ions labeled with ▼), the principal sulfolipid class from M. tuberculosis strain H37Rv (inset). Elemental
compositions deduced from accurate m/z measurements also define SL-I & SL-II.
37
Figure 44. Fragmentation Processes Proposed for 2,3,6,6’-Tetraacyl-α ,α ’-D-trehalose-2’-Sulfate (Sulfolipid
II). Pathways for the most prominent [M−H]− ion (m/z 2459, monoisotopic), which predominantly represents [18:0,hC40,hC40,hC40]-SL,
although other isomers are also present. The structures of the indicated fragment ions are supported by their elemental compositions deduced
n
from accurate mass measurements via high-resolution MS mass spectrometry.
n
Figure 45. Fragment Ions from MS of the major hydroxyphthioceranoic acid substituents in SL-II and proposed
pathways to structurally informative product ions. Structures of indicated fragment ions are supported by high-resolution LIT MSn.
38
C.9.) Characterization of mycolic acids from the pathogen Rhodococcus equi by tandem mass spectrometry
with electrospray ionization. An ESI-LITMSn approach was developed for
structural characterization of mycolic
acids, the long-chain α-alkyl-β-hydroxy
fatty acids unique to mycobacteria and
related taxa (27). On collisionally
activated dissociation in a linear ion trap
or tandem quadrupole mass spectrometer,
the [M-H]- ions of mycolic acid generated
by electrospray ionization undergo
dissociation to eliminate the meroaldehyde
residue, leading to formation of
carboxylate anions containing α-alkyl
FIGURE 46. FRAGMENTATION PROCESSES PROPOSED FOR MYCOLIC A CID [M-H] IONS
chains. The structural information from
these fragment ions affords structural
assignment of the mycolic acids,
including the lengths of the meromycolate
chain and the α-branch. This study
revealed that the mycolic acids isolated
from pathogenic Rhodococcus equi 103
contained a series of homologous ions
having C30 to C50 chain with 0–2 double
bonds. The α-branch ranged from C10 to
C18 with 0 to 1 double bond, in which
16:0 and 14:0 are the most prominent,
whereas the meromycolate chain ranged
from C14 to C34 with 0 to 2 double
bonds. The major molecular species
consisted of more than 3 isomers that
differ by the lengths of the a-branch or
meromycolate chain, and up to 10 isobaric
isomers were identified for some minor
ions.
Tandem
quadrupole
mass
spectrometry with precursor ion and
neutral loss scans was used to profile
specific classes of mycolic acid molecules
in biological mixtures. Tandem spectral
scans to identify precursor ions for m/z
255 (16:0-carboxylate anion) and m/z 227
(14:0-carboxylate anion) may provide a
simple specific means to classify
Rhodococcus species, whereas tandem
spectral scans for neutral loss of Fig. 47. ESI/MS spectra of [M-H]- ions (A) and corresponding [M-D]- ions
after H/D exchange of mycolic acids from wild-type R. equi strain
meroaldehyde residues provides a simple (B)
103. The 1-Da mass shift in panel B reflects H/D exchange.
approach to determine mycolic acid
molecules with specific meromycolate chain in mixtures (27). (Anal. Biochem. 2011; 409: 112–122).
-
39
2
-
Fig. 48. ESI-LIT-MS of R. equi mycolic acid [M-H] ions m/z 535 (A), m/z 575 (B), m/z 659 (C), and m/z 673 (D).
Table 3. Mycolic acid composition of Rhodococcus equi strain 103..
40
C.10.) Structural definition of trehalose 6-monomycolates and trehalose 6,6'-dimycolates from the pathogen
Rhodococcus equi by multiple-stage linear ion-trap mass spectrometry with electrospray ionization. The cell
wall of the pathogenic bacterium Rhodococcus equi (R. equi) contains abundant trehalose monomycolate
(TMM) and trehalose dimycolate (TDM) species, which are glycolipids that contain mycolic acid residues. An
electrospray ionization (ESI)-linear ion trap (LIT)-multiple-stage mass spectrometric (MSn) approach was
developed for TMM and TDM structural characterization by collisionally activated dissociation (CAD) of [M
Alk]+ (Alk = Na, Li) and of [M+X]– (X = CH3CO2, HCO2) (28). MSn (n = 2, 3, 4) of TMM or TDM [M+Alk]+
or [M+X]– adduct ions yielded abundant structurally informative product ions that permit assignment of the
length of the meromycolate chain and of the α-branch on the mycolyl residues, and this approach was used to
determine structures of TMM and TDM species isolated from pathogenic R. equi strain 103. Results indicate
that the major TMM and TDM molecules possess 6 and/or 6'-mycolyl groups that consist of mainly C14 and
C16 α-branches with meromycolate branches ranging from C18 to C28. This is similar to the composition of
free mycolic acid species found in the cell envelope. Up to 60 isomers with various lengths of the α-branch and
n
Figure 49. Multiple-stage mass spectrometric (MS ) structural characterization of 6-mycolyl-α,α'-D+
trehalose (TMM)) by CAD of [M+Na] ions. The indicated m/z values are ions observed for 18:0/16:0-TMM,
+
which is one of three isomers with an [M+Na] m/z 871.
of the meromycolate backbone were observed for some TDM species in the mixture. This ESI-LIT-MSn
approach provides a direct method for identification of various TMM and TDM isomers in complex biological
mixtures that has not been previously achieved with other analytical methods (28). (J. Am. Soc. Mass Spectrom.
2011; 22: 2160-2170.)
41
Figure 50. ESI-LIT-MSn structural characterization of 6,6'-dimycolyl-α,α'-D-trehalose (TDM)
by CAD of [M+Na]+.
C.11.) Structural determination of glycopeptidolipids of Mycobacterium smegmatis by high-resolution
multiple-stage linear ion-trap mass spectrometry with electrospray ionization. Glycopeptidolipids (GPLs) are
abundant in the cell walls of mycobacterial species of and consist of a tripeptide-amino alcohol core of D-PheD-allo-Thr-D-Ala-L-alaninol in an amide linkage to 3-hydroxy or 3-methoxy C26–34 fatty acyl chain at the Nterminal of D-Phe (29). Attached to the D-allo-Thr and the terminal L-alaninol are 6-deoxytalose (6-dTal) and
O-methyl rhamnose residues, respectively. GPLs are important cell-surface antigens implicated in the
pathogenicity of opportunistic mycobacteria of the Mycobacterium avium complex. An ESI-LIT-MSn approach
using high m/z resolution was developed for structural characterization of complex GPLs isolated from
Mycobacterium smegmatis, which is a rapidly growing but non-pathogenic mycobacterial species. Upon
resonance excitation in an ion trap, MSn of GPL [M+Na]+ ions yielded product ion spectra with b and y ion
series that specify the peptide sequence. Other product ions specified the locations of the 6-dTal and O-methyl
rhamnose residues linked to the D-allo-Thr and terminal L-alaninol of the peptide core, respectively. Product
ions also identified modifications of the glycosides, including acetylation and methylation states and the
presence of a succinyl substituent. GPL families with 3-hydroxy fatty acyl or 3-methoxy fatty acyl substituents
were readily distinguished. GPL MS profiles reflect the conditions under which the cells were grown, and
several isomers were identified at many m/z values (29). This ESI-LIT-MSn approach yields detailed structural
characterization of GPL in complex biological mixtures, including discrimination of isomeric species that
would be difficult to identify with other analytical methods. (J. Mass Spectrom. 2012; 47: 1269–1281.)
42
C.12.) Diversion of phagosome trafficking by pathogenic Rhodococcus equi depends on mycolic acid chain
length. Rhodococcus equi is a close relative of Mycobacterium spp. and a facultative intracellular pathogen
which arrests phagosome maturation in macrophages before the late endocytic stage. Here, screening a
transposon mutant library of R. equi for mutants with decreased capability to prevent phagolysosome formation
yielded a mutant in the gene for β-ketoacyl-(acyl carrier protein)-synthase A (KasA), a key enzyme of the longchain mycolic acid synthesizing FAS-II system (30). The longest kasA mutant mycolic acid chains were 10
carbon units shorter than those of wild-type bacteria. When non-pathogenic E. coli were coated with purified
wild-type trehalose dimycolates, phagolysosome formation was reduced substantially, but this was not the case
with shorter kasA mutant-derived trehalose dimycolate. The mutant was moderately attenuated in macrophages
and in a mouse infection model, but was fully cytotoxic. Although loss of KasA is lethal in mycobacteria, the R.
equi kasA mutant multiplication in broth was normal, which indicates that long-chain mycolic acid compounds
are not necessarily required for cellular integrity and viability of the bacteria that typically produce them. This
study demonstrates a central role of mycolic acid chain length in diversion of phagosome trafficking by R. equi
(30). (Cellular Microbiology 2013; 15: 458–473).
Figure 51. Organization of Kas genes and processing functions of mycolic acid precursors. A.
Structure of a typical mycolic acid from Rhodococcus equi. B. The mycobacterial FAS-I enzyme
system generates C14–C24 fatty acids which are either used directly or are transferred to the
KasA-dependent FAS-II system for extension by two carbon units per enzymatic cycle up to 42 carbon
units. Further extension of long KasA-dependent mero-segments to very long ones (up to C60) is further
dependent on KasB. KasB also participates in cyclopropanation in mycobacteria, a modification absent
from R. equi mycolic acids. R. equi FAS-I produces C10–C16 fatty acids which are elongated up to 34
carbon units by FAS-II. The resulting FAS-II-derived fatty acids are incorporated into mycolic acids as
their mero-segments and the FAS-I-derived C12–C18 as a-alkyl chains. ‘D’ denotes the missing
enzymatic activity in the R. equi kasA mutant. Scheme based on Slayden and Barry (2002), Gao et al.
(2003), Brown et al. (2005), Takayama et al. (2005) and Hsu et al. (2011b). C. Organization of the kasA
gene regions on the M. tuberculosis and R. equi chromosomes is identical but R. equi lacks a
kasB gene (dotted line). The insertion position of the transposon in mutant FA11 is denoted by a ‘T’ in
an open triangle.
43
Figure 52. Mass spectrometry of mycolic acids from wild-type and kasA mutant cells. Lipid
extracts of R. equi were analysed by ESI-LIT-MS, and the m/z region of mycolic acids [M+H]+ ions
extracted from (A) strain 103+, from (B) mutant 103+/kasA, and from (C) mutant 103+/kasA
complemented with wild-type kasA are shown as well as the mass spectra of TDM from strain 103+
(E), from mutant 103+/kasA (F) and from strain 103+ grown in the presence of 0.2 mg ml-1
thiolactomycin (G). Elemental formulae are indicated for some of the compounds. (D and H)
Structures of the longest TDM variant from 103+ ([M+Na]+ ion = C98H186O15Na+, 1626.3 Da) and
from 103+/kasA ([M+Na]+ ion = C 82H158O15Na+, 1406.1 Da) respectively.
44
Figure 53. Sensitivity of the R. equi and M. smegmatis to FAS inhibitors. Bacteria were grown in
rich media (LB for Mycobacterium smegmatis, BHI for R. equi) in the presence of the indicated concentrations
(mg/mL) of (A) thiolactomycin, (B) isoniazid or (C) triclosan. DMSO carrier controls contained the DMSO
amounts corresponding to the 10 mg/mL drug samples. At the indicated times, samples were removed and
absorbance at 600 nm was recorded. Data are presented as means and standard deviations from 3–6
independent experiments.
45
C.13.) MmpL11 protein transports mycolic acid-containing lipids to the mycobacterial cell wall and
contributes to biofilm formation in Mycobacterium smegmatis. A growing body of evidence indicates that
MmpL (mycobacterial membrane protein large) transporters are dedicated to cell wall biosynthesis and
transport mycobacterial lipids. How MmpL transporters function and the identities of their substrates have not
been fully elucidated. We have characterized the Mycobacterium smegmatis MmpL11 (31). We showed
previously that M. smegmatis lacking MmpL11 has reduced membrane permeability that results in resistance to
host antimicrobial peptides. Further characterization of the M. smegmatis mmpL11 mutant coupled with mass
spectrometric identification of the MmpL11substrates revealed that mmpL11 mutants accumulate mycolic acid
precursors and fail to transport monomeromycolyl diacylglycerol and mycolate ester wax to the bacterial
surface. M. smegmatis mmpL11 mutant was distinct from that by wild type M. smegmatis. Analysis of cell wall
lipids revealed that the mmpL11 mutant failed to export the mycolic acid-containing lipids monomeromycolyl
diacylglycerol and mycolate ester wax to the bacterial surface. In addition, analysis of total lipids indicated that
the mycolic acid-containing precursor molecule mycolyl phospholipid accumulated in the mmpL11 mutant
compared with wild-type mycobacteria. MmpL11 is encoded at a chromosomal locus that is conserved across
pathogenic and nonpathogenic mycobacteria. This suggests that MmpL11 contributes to mycobacterial cell wall
biosynthesis, and MmpL11 is known to play a conserved role in mycobacterial cell wall biogenesis that is
important for M. tuberculosis virulence. Phenotypes of the M. smegmatis mmpL11 mutant are complemented
by the expression of M. smegmatis or M. tuberculosis MmpL11, suggesting that MmpL11 plays a conserved
role in mycobacterial cell wall biogenesis (31). (J. Biol. Chem. 2013; 288: 24213–24222).
MYCOLATE ESTER
+
WAX [M+Li]
MYCOLATE ESTER
+
WAX [M+Na]
MONOMEROMYCOLYY
L DIACYLGLYCEROL
+
[M+NH4]
Fig. 55. Model depicting MmpL3 and MmpL11 function in
mycobacterial cell wall biogenesis. MmpL3 & MmpL11 are
conserved mycobacterial cell wall lipid transporters. Mycolic acid
biosynthesis yields the intermediate MycPL whose mycolate is
incorporated into TMM (transported by MmpL3) & into MMDAG &
mycolate ester wax (WE) (transported by MmpL11).
Fig. 54. ESI high-resolution MS of Mono-Meromycolyl
+
Diacylglycerol (MMDAG) and mycolate ester wax. [M+Li] (A) and
+
[M+Na] ions (B) of apolar lipid B (mycolate ester wax), and ESI/MS of
+
[M+NH4] of apolar lipid C (C) (MMDAG). Apolar lipid B contains
n
+
pentatriacontatrienyl mycolate esters shown by MS of [M+Li ];
+
reported [M+Na] ions were misassigned as mycolyl-DAG.
C.14.) Characterization of mycobacterial triacylglycerols and monomeromycolyl diacylglycerols from
Mycobacterium smegmatis biofilm by electrospray ionization multiple-stage and high-resolution mass
spectrometry. Storage of triacylglycerols (TAGs) is essential for non-replicating persistence of mycobacteria
46
Table 4. High resolution mass measurements and LIT MSn analysis of TAG species from M.
smegmatis biofilm.
and permits survival and subsequent regrowth when they resume replication after resolution of state stress
conditions, but the detailed structures of mycobacterial TAG has remained largely unexplored. We developed
an ESI-LIT-MSn approach using high resolution m/z measurements for structural characterization of
mycobacterial TAG species, including a novel monomeromycolyl diacylglycerol (MMDAG) lipid subclass
47
isolated from biofilms of Mycobacterium smegmatis, a rapidly growing but non-pathogenic mycobacterium
used as a model for Mycobacterium tuberculosis (32). Our results demonstrate that the major isomers of TAG
and MMDAG species from M. smegmatis consists of a common structure in which Δ918:1- and 16:0-fatty acyl
substituents are exclusively located at the sn-1 and sn-2 positions on the glycerol backbone, respectively.
Several isomers were observed for most TAG and MMDAG species, and hundreds of structures are present in
this mycobacterial neutral lipid family. Importantly, our studies revealed the structures of MMDAG, a novel
subclass of TAG that has not been previously been characterized by direct mass spectrometric approaches (32).
(Anal. Bioanal. Chem. 2013; 405: 7415–7426).
Table 5. High-resolution m/z measurements and LIT MSn of [M+Li]+ ions of M. smegmatis biofilm
mmDAG species.
48
D.)
1.)
2.)
3.)
4.)
5.)
6.)
7.)
8.)
9.)
10.)
11.)
12.)
13.)
14.)
15.)
16.)
17.)
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50
32.)
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PMID: 23852148
51