fumarase deficiency causes protein and metabolite ... chemical biology article fumarase deﬁciency...

Article

Fumarase Deficiency Causes Protein andMetabolite
Succination and Intoxicates Mycobacteriumtuberculosis
Graphical Abstract

Highlights

d Fumarase is essential for growth of Mtb

d Fumarase deficiency inMtb is bactericidal in vitro and during

mouse infection

d Fumarate accumulation leads to metabolite and protein

succination

d Fumarase-deficient Mtb is hypersusceptible to oxidative

stress

Ruecker et al., 2017, Cell Chemical Biology 24, 1–10March 16, 2017 ª 2017 Elsevier Ltd.http://dx.doi.org/10.1016/j.chembiol.2017.01.005

Authors

Nadine Ruecker, Robert Jansen,

Carolina Trujillo, ..., Henrik Molina,

Kyu Y. Rhee, Sabine Ehrt

[email protected]

In Brief

Ruecker et al. report that Mycobacterium

tuberculosis is uniquely vulnerable to

fumarase deficiency. Fumarase depletion

perturbed essential metabolic pathways

and led to intracellular fumarate

accumulation. Fumarate caused

metabolite and protein succination, a

covalent chemical modification of

cysteine thiol residues. This affected two

ofMtb’s major antioxidants, catalase and

mycothiol, causing hypersusceptibility to

oxidative stress.

mailto:[email protected].�edu

http://dx.doi.org/10.1016/j.chembiol.2017.01.005

Please cite this article in press as: Ruecker et al., Fumarase Deficiency Causes Protein and Metabolite Succination and Intoxicates Mycobacteriumtuberculosis, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.01.005

Cell Chemical Biology

Article

Fumarase Deficiency Causes Protein and MetaboliteSuccination and IntoxicatesMycobacterium tuberculosisNadine Ruecker,1 Robert Jansen,2 Carolina Trujillo,1 Susan Puckett,1,5 Pradeepa Jayachandran,1,6 Gerardo G. Piroli,3

Norma Frizzell,3 Henrik Molina,4 Kyu Y. Rhee,2 and Sabine Ehrt1,7,*1Department of Microbiology and Immunology2Department of MedicineWeill Cornell Medical College, New York, NY 10065, USA3Department of Pharmacology, Physiology & Neuroscience, School of Medicine, University of South Carolina, Columbia, SC 29209, USA4Proteomics Resource Center, Rockefeller University, New York, NY 10065, USA5Present address: Department of Medical Microbiology, University Medical Center Utrecht, 3584 CX Utrecht, the Netherlands6Present address: Department of Biological Sciences and RNA Institute, University at Albany, Albany, NY 12222, USA7Lead Contact

*Correspondence: [email protected]


SUMMARY

Enzymes of central carbon metabolism are essen-tial mediators of Mycobacterium tuberculosis(Mtb) physiology and pathogenicity, but are oftenperceived to lack sufficient species selectivity tobe pursued as potential drug targets. Fumarase(Fum) is an enzyme of the canonical tricarboxylicacid cycle and is dispensable in many organisms.Transposon mutagenesis studies in Mtb, however,indicate that Fum is required for optimal growth.Here, we report the generation and characteriza-tion of a genetically engineered Mtb strain in whichFum expression is conditionally regulated. This re-vealed that Fum deficiency is bactericidal in vitroand during both the acute and chronic phases ofmouse infection. This essentiality is linked tomarked accumulations of fumarate resulting in pro-tein and metabolite succination, a covalent modifi-cation of cysteine thiol residues. These resultsidentify Mtb Fum as a potentially species-specificdrug target whose inactivation may kill Mtbthrough a covalently irreversible form of metabolictoxicity.

INTRODUCTION

Mycobacterium tuberculosis (Mtb) remains a global health threat

causing 1.5 million fatal infections annually (http://www.who.int/

tb/en). Tuberculosis (TB) treatment requires a regimen of four

drugs for a period of at least 6 months and demands

careful implementation and high patient compliance. Healthcare

mismanagement and poor patient adherence favor the occur-

rence of multidrug-resistant strains, leading to an even higher

demand for new chemotherapeutics with novel mechanisms of

action.

Cell

Mtb’s central carbon metabolism (CCM) provides not only

energy for growth, but also pathogenicity. Several studies

have highlighted the importance of CCM enzymes for Mtb’s

ability to grow and persist in mice (Marrero et al., 2010, 2013;

McKinney et al., 2000; Munoz-Elıas and McKinney, 2005;

Pandey and Sassetti, 2008; Puckett et al., 2014; Trujillo et al.,

2014; Venugopal et al., 2011). Knowledge about Mtb’s

metabolism, therefore, benefits the understanding of TB patho-

genesis and can identify potential new targets and chemothera-

peutic interventions.

Fumarase catalyzes the reversible hydration of fumarate to

malate, a reaction of the tricarboxylic acid (TCA) cycle. The

TCA cycle oxidizes acetyl coenzyme A to carbon dioxide and

generates reducing equivalents for ATP production. It also pro-

vides precursors for amino acid and nucleotide biosynthesis.

However, fumarate is not only generated in the TCA cycle, but

is also a product of argininosuccinate lyase (ArgH) in the urea cy-

cle and of adenylosuccinate lyase (PurB) in purine biosynthesis

(Figure 1A).

Two classes of fumarases can be differentiated (Woods et al.,

1988). Class I fumarases occur only in prokaryotes, are thermo-

labile, iron dependent, and form homodimers. Class II fumarases

can be found in both prokaryotes and in eukaryotic mitochon-

dria, they are thermostable, iron independent, and form homote-

tramers. Several prokaryotes possess multiple fumarases

including E. coli, which encodes fumarases of both classes.

Expression of each depends on growth rate, oxygen availability,

and carbon source (Park and Gunsalus, 1995; Tseng et al.,

2001). Mtb encodes a single class II-type fumarase gene, fum

(rv1098c). Fum shares 45% sequence identity with the human

enzyme, including a conserved active site serine residue (Cole

et al., 1998; Mechaly et al., 2012). Somewhat unexpectedly,

transposon mutagenesis studies predicted Fum to be essential

for optimal growth of Mtb in vitro (Griffin et al., 2011; Zhang

et al., 2012). This was surprising because Mtb encodes a func-

tional glyoxylate shunt that generates succinate and malate

and should, therefore, bypass the Fum reaction (Eoh and

Rhee, 2013) (Figure 1A). Mtb has also been reported to be

capable of co-catabolizing different carbon substrates, making

Chemical Biology 24, 1–10, March 16, 2017 ª 2017 Elsevier Ltd. 1

mailto:[email protected]


http://www.who.int/tb/en

http://www.who.int/tb/en

Figure 1. Fum Depletion Kills Mtb

(A) Schematic depicting the TCA cycle, the urea

cycle, and reactions leading to fumarate produc-

tion. ArgH, argininosuccinate lyase; PurB, ad-

enylosuccinate lyase.

(B and C) Growth (B) and survival (C) of WT and

Fum-DUC in 7H9 complete medium with (+) and

without (�) atc. Colony-forming units (CFU) were

determined by culturing serial dilutions on agar

plates at different time points post inoculation. See

also Figure S2.

(D) Fum amounts in Fum-DUC were determined by

immunoblot. The proteasome subunit B (PrcB)

serves as loading control.

(E) Quantitative Fum immunoblotting in Fum-DUC

extracts from cultures on day 7 after atc addition.

Serial dilutions of WT extract were used for relative

quantification. PrcB served as loading control.

(F) Impact of Fum depletion on starved Mtb. WT

and Fum-DUC were cultured in PBS with 0.05%

tyloxapol for 10 days, before atc was added (ar-

row). CFU were determined by culturing serial di-

lutions on agar plates at different time points post

inoculation.

(G) Fum depletion in Fum-DUC during PBS star-

vation was analyzed by immunoblot. PrcB serves

as loading control. All data are representative of at

least two independent experiments. See also Fig-

ures S1 and S2.


Fum dispensable in the presence of a glycolytic carbon source

and a fatty acid source (Beste et al., 2011; de Carvalho et al.,

2010). Fumarase null mutants in other bacteria, and even eukary-

otes, have been reported to be viable (Carls and Hanson, 1971;

Mercado-Lubo et al., 2009; Song et al., 2013; Xu et al., 2013).

Interestingly, a small-molecule inhibitor of Fum that potently

and selectively inhibitsMtb Fum, via binding to an allosteric reg-

ulatory site, was recently reported (Kasbekar et al., 2016). How-

ever, this compound only slowed growth and failed to kill Mtb,

leaving the phenotypic consequences of Fum inactivation in

Mtb unexplored.

We sought to address this ambiguity and generated a condi-

tional knockdown strain of Mtb Fum. We found that Fum deple-

tion not only inhibited growth, but was also bactericidal in

replicating conditions in vitro and during acute and chronic

mouse infection. Surprisingly, and in contrast to previous studies

2 Cell Chemical Biology 24, 1–10, March 16, 2017

of other CCM enzymes in Mtb, we

were unable to metabolically complement

Fum deficiency, indicating a dominant-

negative toxicity. Metabolic profiling of

a Fum-depleted strain revealed severe

accumulation of fumarate. Immunoblot

analysis, followed by protein mass

spectrometry and metabolomic profiling,

further revealed that fumarate covalently

reacted with thiol residues resulting in

succination of numerous proteins andme-

tabolites, including mycothiol. The domi-

nant-negative consequences of Fum defi-

ciency and heightened susceptibility to

peroxide stress differentiate Mtb from

many other bacteria and eukaryotes, and identify Fum as poten-

tially attractive drug target.

RESULTS

Fum Depletion in Mtb Cannot Be Metabolically RescuedIn Vitro and Is Detrimental during Acute and ChronicMouse InfectionTo study the function of Fum inMtb, we generated a conditional

knockdown mutant (Fum-DUC) in strain H37Rv, using the previ-

ously described dual control (DUC) genetic switch in which

expression of Fum is controlled by both inducible transcriptional

silencing and proteolytic degradation (Kim et al., 2013) (Fig-

ure S1). Depletion of Fum following addition of anhydrotetracy-

cline (atc) inhibited Mtb growth and led to a rapid decline in

viability in nutrient-rich 7H9 medium (Figures 1B and 1C) and

Figure 2. MtbRequires Fum to Establish and

Maintain Infection in Mice

(A and B) Bacterial burden in lungs (A) and spleens

(B) from mice infected with WT Mtb and the Fum-

DUC strain. Mice were fed doxy-containing chow

as indicated. Lung and spleen homogenates were

cultured on agar plates to determine bacterial

burden at the indicated time points post infection.

Data are means ± SD of four mice per time point.

Data are representative of two independent infec-

tion experiments. See also Figure S3.


when cultured on either glucose or acetate as single-carbon

sources (Figures S2A and S2C). Fum protein levels declined

upon atc treatment (Figures 1D and S2B), while in wild-type

(WT) Mtb atc did not affect viability or Fum protein levels

(Figure S2B).

To evaluate to what degree Fum has to be depleted to result

in growth inhibition, we quantified the relative Fum protein

amount in Fum-DUC on day 7 after atc addition (Figure 1E).

Fum amount was �3% of that in WT Mtb, suggesting that

approximately 97% depletion of Fum is required to achieve

growth inhibition.

A previous study of Fum-deficient cancer cells reported

malate deficiency and arginine auxotrophy (Adam et al., 2013)

arising from catabolism of arginine by ArgH (Figure 1A). This

enzyme is encoded by argH in Mtb, which is predicted to be

essential for in vitro growth (Griffin et al., 2011; Sassetti et al.,

2003). However, supplementation with either arginine or malate

at concentrations that maximally supported growth of the parent

strain (Figure S2D) failed to complement Fum deficiency

(Figure S2C).

In contrast, loss ofMtb viability was negligible when Fum-DUC

was starved in carbon-free Sauton’s medium, suggesting a po-

tential link between Fum essentiality and metabolic activity (Fig-

ure S2C). Accordingly, addition of atc to Fum-DUC after 10 days

of starvation in PBS resulted in substantial depletion of Fum, but

only moderate (1 log) reduction in viability after 28 days (Figures

1F and 1G), supporting the relative dispensability of Fum in non-

replicating Mtb.

To determine the importance of Fum during infection, we

infected mice via aerosol with either WT or Fum-DUC Mtb

and fed doxycycline (doxy)-containing food starting at day

10 post infection to deplete Fum. This led to rapid clearance

of Fum-DUC from the lungs and lack of dissemination to the

spleen (Figures 2A and 2B). Fum-DUC infected mice fed

with doxy chow starting at day 35, during the chronic phase

of infection, exhibited a similar decline in bacterial numbers

in lungs and spleens, with no recoverable bacteria at day

112. This decline in bacterial burden was accompanied

by a matching reduction in lung pathology with hardly any

detectable lesions on days 56 and 112 in lungs from Fum-

DUC-infected mice that had been treated with doxy (Fig-

ure S3). These results thus demonstrate that Fum is required

for both establishment and maintenance of Mtb infection

in mice.

Fum Depletion Results in Fumarate Accumulation andToxicityConsistent with its annotated function, metabolic profiling of

Fum-depleted Mtb by liquid chromatography-mass spectrom-

etry (LC-MS) revealed a 100-fold increase in the fumarate pool

size (Figures 3 and S4). However, in contrast to the conse-

quences of Fum depletion in eukaryotic cells (Adam et al.,

2013), neither malate nor arginine pools were depleted, consis-

tent with their inability to rescue viability (Figure S2C). We further

observed an impact on fumarate-producing reactions in the urea

cycle and in purine biosynthesis. Argininosuccinate, the sub-

strate of ArgH, encoded by rv1659, accumulated 100-fold, while

levels of the dephosphorylated form of succinyl-aminoimidazole

carboxamide ribotide (SAICAR), SAICAr, a product of PurB,

encoded by rv0777, accumulated 50-fold, consistent with

fumarate-mediated feedback inhibition as observed in fuma-

rase-deficient cancer cells (Adam et al., 2013; Jurecka et al.,

2014); however, we did not detect an increase in dephosphory-

lated adenylosuccinate or succinyladenosine, as observed in

human cells with PurB deficiency (Jurecka et al., 2014).

In addition to the dramatic accumulation of cytosolic fumarate

pools in Fum-DUC, we observed a similar 100-fold increase in

extracellular fumarate levels (Figure 4A). A fumarate transporter

has not been identified in Mtb, but fumarate is likely actively

secreted, because diffusion of fumarate across lipid membranes

is very inefficient (Janausch et al., 2001). To inhibit secretion of

intracellular fumarate via concentration gradient-dependent

mechanisms (Engel et al., 1994; Eoh and Rhee, 2013), we

cultured Fum-DUC in the presence of extracellular fumarate

and depleted Fum. This accelerated death of Fum-DUC, but

not WT, in a concentration-dependent manner (Figures 4B and

4C). Addition of dimethylfumarate (DMF), a more reactive diester

of fumarate with increased membrane permeability (Schmidt

and Dringen, 2010), killed WT Mtb, and Fum-depleted Mtb was

hypersusceptible to DMF treatment (Figures 4C and 4D). These

results thus implicate intracellular fumarate accumulation as a

primary mediator of toxicity in Fum-deficient Mtb.

Fumarate Accumulation Leads to Succination ofMycothiol in Mtb

Fumarate is an unsaturated dicarboxylic acid and can react

with free cysteine thiol groups via Michael addition, generating

S-(2-succino) compounds (Alderson et al., 2006) (Figure 5A).

Thismodification is defined as succination and forms a thioether,

Cell Chemical Biology 24, 1–10, March 16, 2017 3

Figure 3. Metabolic Consequences of Fum Deficiency

Intrabacterial pool sizes of selected metabolites in the indicated Mtb strains after 24 hr cultivation on filters on top of 7H9 medium with 0.2% glucose with or

without atc. Pool sizes are expressed as area under the curve normalized to protein content. Data are mean values ± SD of three biological replicates and are

representative of two independent experiments. Statistically significant differences between atc-treated and untreated cultures that were observed in two

biologically independent experiments are reported. *p% 0.05, **p% 0.005 by Student’s t test. The differences between pool sizes in atc-treated and untreated

WT were not reproducibly statistically significant. Enzymatic reactions are depicted as arrows; the dashed arrow indicates that the respective enzyme has not

been identified in Mtb; black dotted arrows indicate spontaneous dephosphorylation reactions. AICAR, 1-(5’-phosphoribosyl)-5-amino-4-imidazolecarbox-

amide; AMP, adenosine 5’-monophosphate; ArgH, argininosuccinate lyase; PurB, adenylosuccinate lyase; SAICAR, succinylaminoimidazole carboxamide

ribose-50-phosphate; SAICAr, succinylaminoimidazole carboxamide ribose. See also Figure S4.


in contrast to succinylation, which generates a thioester when

succinyl-coenzyme A reacts with lysine residues (Zhang et al.,

2011). In eukaryotes, this reactivity results in succination

of glutathione (GSH) (Sullivan et al., 2013). Accordingly, we

observed succination of mycothiol (MSH), the mycobacterial

counterpart (Figures 5B and 5C). In addition to succinated

MSH, we also detected free succinocysteine, the reaction prod-

uct of cysteine and fumarate (Figures 5D and 5E).

Fumarate Accumulation Leads to Succination ofProteinsSuccination results in a similar modification of protein cysteine

residues in eukaryotes (Alderson et al., 2006; Merkley et al.,

2014). We therefore analyzed whole-cell extracts from WT and

Fum-DUC Mtb using an antibody against S-(2-succino)cysteine

(2SC) (Nagai et al., 2007) and observed numerous 2SC-positive

signals specifically in lysates of Fum-DUC treated with atc (Fig-

ure 6A). Expression of fumarase with an active site mutation


(S318A) neither restored viability nor prevented succination,

confirming that succination was attributable to loss of Fum activ-

ity (Figure S5A). In addition, succination occurred specifically in

cells accumulating fumarate and was not just a marker of cell

death (Figure S5B).

Multiple proteins appeared succinated in Fum-DUC, with the

most intense signal at a molecular range of 75–100 kDa, which

was also present in WT treated with DMF (Figure S5C). To iden-

tify succinated proteins, we analyzed trypsin-digested gel pieces

of the corresponding molecular weight range by liquid chroma-

tography-tandem mass spectrometry (LC-MS/MS) querying

the data for the expected mass shifts corresponding to succina-

tion (H4C4O4: 116.010959 Da) and dimethylsuccination (H8C6O4:

144.042259 Da). We identified three proteins containing succi-

nated cysteine residues in both Fum-DUC-treated samples,

as well as WT Mtb-treated with DMF (Table 1). These corre-

sponded to aminopeptidase N (Rv2467), catalase-peroxidase

KatG (Rv1908c), and the 1,4-alpha-glucan branching enzyme

Figure 4. Intracellular Fumarate Accumula-

tion Results in Toxicity

(A) Secretion of fumarate and succinate. Pool sizes

of secreted metabolites are expressed as area

under the curve (AUC) and normalized to the pro-

tein content of the bacteria on the filter. Values are

the averages ± SD of biological triplicates. *p %

0.05, **p % 0.005 by Student’s t test. Data are

representative of two independent experiments.

(B) Addition of fumarate to the extracellular medium

accelerates death of Fum-depleted Mtb. Fum-

DUC was cultivated in medium containing

increasing amounts of fumarate with or without atc.

CFU were monitored at the indicated time points.

Data are representative of three independent

experiments.

(C) Effect of extracellular fumarate and dime-

thylfumarate (DMF) on WT Mtb. CFU values are

averages ± SD of biological triplicates and are

representative of two independent experiments.

(D) Effect of extracellular dimethylfumarate (DMF)

on Fum-DUC. Fum-DUC was treated with atc for

48 hr to deplete Fum or left untreated and then

exposed DMF. CFU values are averages ± SD of

biological triplicates. Data are representative of

three independent experiments.


GlgB (Rv1326c) (Figure 6B). In all cases, only one specific

cysteine residue was found modified.

Fum Depletion Leads to Increased Susceptibility toOxidative StressGiven the foregoing discovery of fumarate accumulation, and

linked formation of succinated catalase and succinated MSH,

two of Mtb’s antioxidants, in Fum-depleted Mtb, we sought to

determine if these modifications affected susceptibility to oxida-

tive stress. We treated Fum-DUC with a low concentration of

H2O2 in the presence and absence of atc. Only Fum-depleted

Mtb was susceptible to killing by this low concentration of

H2O2 (Figure 6C) indicating that Fum deficiency heightened

Mtb’s susceptibility to peroxide stress.

DISCUSSION

This work reveals Mtb’s vulnerability to fumarase deficiency.

Fumarase depletion in culture resulted in death of Mtb and we

determined that an approximate 97% reduction in fumarase

amount impaired growth and prevented regrowth on agar plates.

The vulnerability of antibiotic targets can vary widely, and not all

effective drug targets are highly vulnerable to inhibition (Wei

et al., 2011). While we cannot predict how efficiently Fum must

be depleted to abolish replication and kill Mtb in vivo, the rapid

loss of viability in response to Fum depletion in mouse lungs

and spleens suggests that it may be an effective target.

Loss of Mtb’s fumarase resulted in a marked intracellular

accumulation of fumarate, the chemical reactivity of which led

to the covalent modification of proteins and metabolites. The ci-

dality resulting from fumarase depletion might be caused by the

inactivation of specific client proteins. Fumarate-mediated pro-

tein and metabolite succination targeted twomajor antioxidants,

catalase andMSH, and crippledMtb’s ability to withstand oxida-

tive stress. Inactivation of fumarase in primary mouse kidney

cells similarly resulted in formation of succinated GSH and was

associated with persistent oxidative stress and cellular senes-

cence (Zheng et al., 2015).

Interestingly, Fum depletion killed, but never sterilized, in vitro

cultures of replicatingMtb. The small subpopulation of surviving

bacteria likely reflects a conditional essentiality linked to Mtb’s

metabolic or replicative activity. This is supported by the modest

impact of Fum depletion on starved Mtb. Notwithstanding, Fum

depletion sensitized Mtb to oxidative stress, which may have

contributed to the severe impact of Fum depletion during acute

and chronic mouse infection, resulting in sterilization. Continued

slow replication and metabolic activity might also contribute to

the vulnerability of Mtb to Fum depletion during chronic mouse

infection (Gill et al., 2009).

The quantitative analysis of only small fractions of the

mycobacterial proteome and metabolome revealed that

fumarate accumulation resulted in succination of both

cysteine thiol-containing metabolites and proteins. Cysteine

has many important cellular functions: it acts as a nucleophile

for catalysis in the active sites of proteins, it contributes to

inter- and intramolecular protein stability through disulfide

bounds, and it is the sulfur donor for the generation of impor-

tant cofactors including thiamine, coenzyme A, pantetheine,

and MSH in Mtb. In the absence of clear measures of stoichi-

ometry, it is unclear whether and how 2SC modification of

proteins may impact their activity, but the data demonstrate

that succination only occurred in Fum-deficient Mtb. The thiol


Figure 5. Fumarate Accumulation Leads to

Metabolite Succination

(A) The electrophile fumarate can react with

cysteine thiol residues in a Michael addition

generating S-(2-succino) compounds.

(B) Intracellular pool sizes of a metabolite with a

mass to charge ratio (m/z) of 603.158 in extracts

fromMtb Fum-DUC treated with atc for 24 hr. *p%

0.05, by Student’s t test.

(C) Comparison ofMS-MS fragmentation spectra of

603.158 with fragmentation spectra of MSH after

incubation with fumarate.

(D) Intracellular pool sizes of a metabolite with

an m/z of 238.038. *p % 0.05, by Student’s t test.

(E) Comparison of MS/MS fragmentation spectra

of 238.038 with fragmentation spectra of succino-

cysteine.


of MSH is the major redox buffer in Mtb (found in millimolar

quantities in the cytoplasm of Mtb; Buchmeier et al., 2003),

and is rendered potentially idle by succination. Loss of

MSH biosynthesis, although dispensable for viability of Mtb,

increased susceptibility to hydrogen peroxide and low pH

(Buchmeier et al., 2003, 2006). This is consistent with our

observation of increased susceptibility of Fum-depleted Mtb

to peroxide stress.

Enzymes function within the context of extensively inter-

connected pathways and inhibition of a single enzyme

can impact multiple pathways (Eoh and Rhee, 2013, 2014).

This is also the case with Fum. Moreover, the dominant-

negative toxicity of Fum deficiency may kill Mtb via effects

on multiple pathways, albeit restricted to replicating Mtb.

Mtb’s vulnerability to Fum depletion is unique and not

observed in other organisms, revealing Fum as a poten-

tially attractive, species-specific target for new and urgently

needed TB drugs.

SIGNIFICANCE

The development of novel chemotherapeutics against

tuberculosis requires a better understanding of the essential


processes in Mycobacterium tubercu-

losis (Mtb). Fumarase functions in the

tricarboxylic acid (TCA) cycle, which

plays a central role inMtb’s metabolism

and pathogenesis. Here, we demon-

strate that fumarase is essential for

growth of Mtb, in contrast to its

dispensability inmany other organisms.

Fumarase depletion perturbed essen-

tial metabolic pathways and led to

intracellular fumarate accumulation,

which in turn caused metabolite and

protein succination, a covalent chemi-

cal modification of cysteine thiol resi-

dues. This affected two of Mtb’s major

antioxidants, catalase and mycothiol,

causing hypersusceptibility to oxidative

stress. Importantly, Fumarase deple-

tion killed Mtb not only in vitro, but also during the acute

and chronic phases of infection in a mouse model of tuber-

culosis. These studies revealMtb’s vulnerability to fumarase

depletion and identify fumarase as a potentially attractive,

species-selective drug target in Mtb.

EXPERIMENTAL PROCEDURES

Culture Conditions

Mtb H37Rv WT and mutant strains were grown aerated in standing flasks at

37�C with 5% CO2 in Middlebrook 7H9 liquid medium containing 0.2%

glucose, 0.5% glycerol, 5% BSA, 0.085% sodium chloride, and 0.05% Tween

80. For agar plates Middlebrook 7H10 agar with 10% commercial OADC sup-

plement (final concentration 0.5% BSA, 0.2% glucose, 0.085% NaCl, 0.006%

oleic acid, and 0.0003% catalase) and 0.5% glycerol was used.

Carbon-defined minimal medium consisted of modified Sauton’s medium

(Allen, 1998): asparagine was replaced by 0.5 g/L ammonium sulfate and

Tween 80 was replaced by 0.05% tyloxapol. Acetate and or glucose were

added to yield a concentration of 0.2% (w/v) each. Arginine and malate were

used as indicated. To ensure adaptation of the bacteria to the medium, the

bacteria were cultured in the respective medium for 7 days before atc

was added.

H37Rv DFba was cultured in the presence of fatty acid-free BSA and

with 0.4% glycerol, 0.4% glucose, and 0.05% tyloxapol as described

previously (Puckett et al., 2014). Antibiotics were used in the following

Figure 6. Fumarate Accumulation Leads to Protein Succination

(A) Detection of 2SC proteins in lysates of Fum-DUC grown with or without atc. For each condition 5 mg protein was loaded. Succinated GAPDH (Alderson et al.,

2006) was loaded as a positive control for succination (labeled C). PrcB serves as loading control.

(B) Tandem MS spectra of the triply charged tryptic cysteine-containing peptide: LQV TYE GCE PHT VAD AYR, from 1,4-alpha-glucan branching enzyme GlgB

(Rv1326c). Fragment ion (y and b ions)-annotated spectra are shown (top to bottom) of the iodoacetamide-modified cysteine peptide (m/z of 703.6605) matched

in the ‘‘WT + atc’’ sample, the succinated peptide (m/z of 723.3422) matched in the ‘‘Fum-DUC + atc,’’ sample and the dimethylsuccinated peptide (m/z of

732.6677) matched in the ‘‘WT + DMF’’ sample, respectively. All peptides were matched with mass accuracy better than 2 ppm. See also Figure S5.

(C) Fum deficiency increases susceptibility to hydrogen peroxide (H2O2). Fum-DUC was exposed to 5 mMH2O2 in the presence or absence of atc for 30 hr. CFU

values are averages ± SD of biological triplicates. Data are representative of three independent experiments.


concentrations: hygromycin (50 mg/mL), zeocin (25 mg/mL), and kanamycin

(25 mg/mL). atc was added at 500 ng/mL to deplete Fum.

For metabolite analysis,Mtb strains at OD580 �1 were seeded onto 0.22 mM

nitrocellulose filters on top of 7H10 agar plates containing 0.5% BSA, 0.085%

NaCl, 0.2% glucose, and 0.5% glycerol. Every 3 days, filters were moved to

fresh plates, to gain biomass. After 7 days filters were transferred to a swim-

ming pool setup (Eoh and Rhee, 2013; Maksymiuk et al., 2015), using liquid

7H9 medium with or without 500 ng/mL atc.

To assess the susceptibility to peroxide, Fum-DUC was treated either with

5 mM hydrogen peroxide or atc or both. After 4 hr, 10 hr, and 24 hr, 5 mM

hydrogen peroxide was replenished.

Metabolite Extraction, Detection, Fragmentation, and Analysis

To extract metabolites bacteria were quenched in cold LC-MS-grade ace-

tonitrile:methanol:water (40:40:20), scraped off of filters, and mechanically

lysed using a bead beater prior to metabolite analysis. Lysates were ster-

ilized using a 0.22 mm filter. Secreted fumarate and succinate were quan-

tified in the medium underneath the filter. Prior to LC-MS analysis, extracts

were diluted 1:1 with mobile phase (acetonitrile with 0.5% formic acid) and

centrifuged (10 min 10,000 3 g). LC-MS analysis was performed using

an Agilent 1,200 Series Liquid Chromatography system with a Cogent

Diamond Hydride Type C column (Microsolv Technologies) coupled to

an Agilent Accurate-Mass TOF 6230, as described previously (Eoh and

Rhee, 2013).

Standards for metabolite identification were obtained from Sigma, except

S-(2-succinyl)-L-cysteine (www.polypeptide.com), SAICAr (MyBioSource),

and succinyladenosine (MyBioSource). Succinated MSH was prepared by

incubating MSH and 500 mM fumarate for 24 hr at 37�C. Targeted data anal-

ysis was performed using Profinder B.06.00 software (Agilent). Metabolite

concentrations were normalized to biomass based on measurement of

residual peptide content in individual samples using the BCA assay (Thermo

Scientific). Fragmentation spectra were obtained using the same conditions


http://www.polypeptide.com

Table 1. Identification of Succinated Proteins in Mtb

Protein Peptide WT + atc Fum-DUC + atc WT + DMF

Aminopeptidase N, PepN HVQTLGALLEGEPAAC[Succ]a

GLAGLAVDTDLR

0 4 2

HVQTLGALLEGEPAAC[diMet-Succ]b

GLAGLAVDTDLR

0 0 41

HVQTLGALLEGEPAAC[IAA]c

GLAGLAVDTDLR

100 96 58

1,4-a-Glucan branching enzyme, GlgB LQVTYEGC[Succ] EPHTVADAYR 0 16 0

LQVTYEGC[diMet-Succ] EPHTVADAYR 0 0 89

LQVTYEGC[IAA] EPHTVADAYR 100 84 11

Catalase-peroxidase, KatG PEQHPPITETTTGAASNGC[Succ]

PVVGHMK

0 49 1

PEQHPPITETTTGAASNGC[diMet-Succ]

PVVGHMK

0 0 90

PEQHPPITETTTGAASNGC[IAA]

PVVGHMK

100 51 8

Listed are peptides that were found modified, either succinated (+116) or dimethyl-succinated (+144). Unmodified cysteines were carbamidomethy-

lated (+57). Values represent the percent distribution of the indicated modifications in each sample.a2-Succinyl (116.010959 H4C4O4).b2-Dimethylsuccinyl (144.042259 H8C6O4).cCarbamidomethyl (57.021464 H3C2NO).


as described under LC-MS analysis, but using an Agilent Accurate-Mass

Q-TOF 6520 operated at a narrow isolation width and a collision energy of 20.

Proteomics Profiling and Identification of Succinated Peptides

Lysates from Mtb strains grown in culture were separated by SDS-PAGE. A

section of the gel (�75–100 kDa), corresponding to the strongest succination

signal, was excised and trypsin digested as described previously (Shevchenko

et al., 1996). Extracted peptides were analyzed by LC-MS/MS (Ultimate 3000

nano-HPLC system coupled to a Q Exactive Plus mass spectrometer [Thermo

Scientific]). Peptides were separated on a C18 column (12 cm/75 mm, 3 mm

beads, Nikkyo Technology) at 200 nL/min with a gradient increasing from

1% buffer B/95% buffer A to 45% buffer B/55% buffer A in 137 min

(buffer A, 0.1% formic acid; buffer B, 0.1% formic acid in acetonitrile) and

analyzed in a data-dependent (DDA)manner. Six cysteine-containing peptides

found to be modified by 2-succinate and/or 2-dimethylsuccinate: HVQ TLG

ALL EGE PAA CGL AGL AVD TDL R [3+], LQV TYE GCE PHT VAD AYR [3+],

LWV NPD CGL K [2+], PEQ HPP ITE TTT GAA SNG CPV VGH MK [4+], IAV

QPG GCA GLR [2+], and acetyl-STA VNS CTE APA SR [2+] were targeted in

a follow-up parallel reaction monitoring (PRM) (Peterson et al., 2012) experi-

ment. For the six sequences, cysteines were considered as being modified

by iodoacetamide, 2-succinate, as well as 2-dimethylsuccinate. PRM tandem

MS spectra were recorded at 17,500 resolution with m/z 100 as lowest mass.

Normalized collision energy was set at 27, with an AGC target and maximum

injection time being 2 3 105, and 60 ms, respectively.

Data analysis: DDA data were extracted and queried against UniProt

Mycobacterium_H37Rv database concatenated (December 2015) with com-

mon contaminants (Bunkenborg et al., 2010) using Proteome Discoverer 1.4

(Thermo Scientific) and Mascot 2.5.1 (Matrix Science). Acetyl (Protein

N-term), Oxidation (M), succinyl (C), 2-dimethylsuccinyl (C), and carbamido-

methyl (C) were allowed as variable modifications and 10 ppm and 20 mDa

were used as mass accuracy for precursors and fragment ions, respectively.

Matched peptides were filtered using 1% false discovery rate calculated by

Percolator (Spivak et al., 2009) and in addition requiring that a peptide was

matched as rank 1 and that precursor mass accuracy was better than 5

ppm. The area of the three most abundant peptides per protein (Silva et al.,

2006) was used to estimate the abundance of matched proteins.

PRM data were analyzed using Skyline v. 3.5 (MacLean et al., 2010). MS2

data were used to validate the targeted peptides, while MS1 data were used

to quantitate the peptides. Multiple charge states for each peptide were

used for quantitation.


Immunoblot Analysis

Immunoblot analyses were performed on protein extracts from liquid cultures.

Rabbit polyclonal antiserum against recombinant fumarase was generated by

GenScript. Immunoblot analysis was done as described previously (Puckett

et al., 2014) using rabbit polyclonal antiserum against Fum, Fba (Puckett

et al., 2014), the proteasome subunit B (PrcB) (a gift fromG. Lin and C. Nathan),

and 2SC (Nagai et al., 2007). For anti-2SC immunoblotting, lysates were

cleared by incubation with 93 volumes of ice-cold acetone. After 30 min incu-

bation, proteins were pelleted. Pellets were resuspended in radioimmunopre-

cipitation assay buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.1%

Triton X-100, 0.1%SDS, and 0.5% sodium deoxycholate [pH 7.4], 2 mMdieth-

ylenetriaminepentaacetic acid, and protease inhibitor). Protein concentration

was determined with BCA Assay Kit (Thermo Scientific).

Mouse Infections

Aerosol infections of 7-week-old female C57BL/6 mice (Jackson Laboratory)

were performed using a Glas-Col inhalation exposure system with early log

phase Mtb cultures prepared as single-cell suspensions in PBS to deliver

100–200 bacilli per mouse. At the indicated time points, doxy food (2,000

ppm, Research Diets) was administered in lieu of regular mouse chow for

the rest of the experiment. To quantify bacteria, serial dilutions of lung and

spleen homogenates were cultured on Middlebrook 7H10 plates to determine

the number of colony-forming units. The left lobe of the mouse lungs was fixed

in 10% formalin in PBS and used for staining with H&E. Procedures involving

mice were performed according to NIH guidelines for housing and care of lab-

oratory animals, and were reviewed and approved by the Institutional Animal

Care and Use Committee of Weill Cornell Medical College.

SUPPLEMENTAL INFORMATION

Supplemental Information includes five figures and can be found with this

article online at http://dx.doi.org/10.1016/j.chembiol.2017.01.005.

AUTHOR CONTRIBUTIONS

N.R., R.J., C.T., S.P., P.J., G.G.P., and H.M. performed the experiments. N.R.,

R.J., C.T., S.P., P.J., G.G.P., N.F., H.M., K.Y.R., and S.E. contributed to exper-

imental design and data analysis. N.R. and S.E. wrote the manuscript, which

was edited by all authors.



ACKNOWLEDGMENTS

We thank Hyungjin Eoh for assistance with preliminary experiments; Dirk

Schnappinger for discussions and advice; Gang Lin and Carl Nathan for

PrcB-specific antiserum; and Weizhen Xu for help with data analysis. This

work was supported by a grant from the NIH (AI063446 to S.E.) and grants

from the Bill and Melinda Gates Foundation (grants 42848 and OPP1024065

to Dirk Schnappinger).

Received: September 11, 2016

Revised: December 7, 2016

Accepted: January 19, 2017

Published: February 16, 2017

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Cell Chemical Biology, Volume 24

Supplemental Information

Fumarase Deficiency Causes Protein and Metabolite

Succination and Intoxicates

Mycobacterium tuberculosis

Nadine Ruecker, Robert Jansen, Carolina Trujillo, Susan Puckett, PradeepaJayachandran, Gerardo G. Piroli, Norma Frizzell, Henrik Molina, Kyu Y.Rhee, and Sabine Ehrt

Supplemental Figures

Figure S1, related to Figure 1. Confirmation of the generation of Fum-DUC. (A) Southern blot design showing the genomic locus of fumC and the same locus after successful replacement of fumC with a hygromycin resistance cassette via homologous recombination. The strain carries an additional copy of fumC with a C-terminal DAS-tag under the control of reverse Tet repressor (T38S38) and expresses SspB under the control of wt TetR (TSC10). (B) Southern blot showing successful replacement of the native fumC gene. Chromosomal DNAs were restricted with PmlI and SacI and hybridized with the probe indicated in A.

WT Fum locus

Fum-DUC Fum locus attL5 and tweety integration sites

glpx fumC Rv1097c Rv1096 phoH2

SacI PmlI probe

4733bp

glpx fumC Rv1097c Rv1096 phoH2

SacI PmlI probe

1209bp

SacI

HygR

fumC-DAS T38S38 sspB ZeoR KanR TSC10

A B

Fum-DUC WT

- 5 kb - 4 kb

- 3 kb

- 2 kb

- 1 kb

Figure S2, related to Figure 1. Fum depletion kills Mtb and cannot be metabolically rescued. (A) Growth and survival of WT and Fum-DUC in 7H9 complete medium with (+) and without (-) atc. Colony forming units (CFU) were determined by culturing serial dilutions on agar plates at different time points post inoculation. (B) Fum amounts in WT and Fum-DUC were determined by immunoblot. The proteasome subunit B (PrcB) serves as loading control. (C) Survival of Fum-depleted Mtb in carbon defined Sauton’s media. Fum-DUC was cultured in Sauton’s media with the indicated carbon sources for 7 days and then atc was added (day 0). CFU were determined by culturing serial dilutions on agar plates at different time points post atc addition. Data are means of triplicate cultures ± SD. (D) Growth of Mtb strains with various concentrations of arginine and malate. WT and Fum-DUC were grown in Sauton’s carbon defined medium with 0.2% glucose, 0.05% tyloxapol and the indicated arginine and malate concentrations in 96-well plates for 26 days. The mean OD580 values of three replicates are depicted as heat map.

Figure S3, related to Figure 2. Mtb requires Fum to establish and maintain infection in mice. Hematoxylin and eosin stained lung sections were isolated on day 56 and day 112 from mice infected with WT or Fum-DUC. Scale bar, 1 mm. Cellular infiltration was evident on day 56 and increased in magnitude in mouse lungs infected with WT Mtb and in lungs infected with Fum-DUC but not treated with doxy. In contrast, mice infected with Fum-DUC and treated with doxy starting day 10 or day 35 did not show similar cellular infiltrates. These data are consistent with the growth of WT Mtb and Fum-DUC in doxy-free mice and killing of Fum-DUC in doxy-treated mice.

Figure S4, related to Figure 3. (A) Viability of bacteria after growth on filters was determined by CFU. Depicted are mean values of biological triplicates ± SD. (B) Fum levels in bacteria grown on filters were determined by immunoblot. PrcB serves as a loading control.

Figure S5, related to Figure 6. Loss of Fum activity results in succination and DMF treatment causes dimethyl-succination in WT Mtb. Fum-DUC was complemented with an integrative plasmid expressing wild type Fum (Comp-Fum) or Fum with an active site mutation (Mechaly et al., 2012) (Comp-Fum-S318A). Both complementation plasmids contained only the fumC gene transcribed from the hsp60 promoter. Expression of Fum and succinated proteins was detected by immunoblot from bacteria grown in liquid culture. For each strain and condition 12 µg of protein was loaded. (B) Protein succination is not a general marker of death. Mtb lacking fructose-1,6-bisphophate aldolase (∆Fba) dies in glycerol containing media but not in glucose containing media (Puckett et al., 2014). Death of ∆Fba is not accompanied by appearance of succinated proteins. For each strain and condition 10 µg of protein was loaded. (C) Immunoblot with the anti-2SC antibody of atc-treated Fum-DUC and WT lysates (48hr treatment with atc) and lysate of WT Mtb treated with DMF for 24 h. For each strain and condition 60 µg of protein was loaded. The area around 75-100 kDa was excised for each sample and analyzed by mass spectrometry.

Supplemental References Mechaly, A.E., Haouz, A., Miras, I., Barilone, N., Weber, P., Shepard, W., Alzari, P.M., and Bellinzoni, M. (2012). Conformational changes upon ligand binding in the essential class II fumarase Rv1098c from Mycobacterium tuberculosis. FEBS Lett 586, 1606–1611.

Puckett, S., Trujillo, C., Eoh, H., Marrero, J., Spencer, J., Jackson, M., Schnappinger, D., Rhee, K., and Ehrt, S. (2014). Inactivation of Fructose-1,6-Bisphosphate Aldolase Prevents Optimal Co-catabolism of Glycolytic and Gluconeogenic Carbon Substrates in Mycobacterium tuberculosis. PLoS Pathog 10, e1004144.

fumarase deficiency causes protein and metabolite ... chemical biology article fumarase deﬁciency...

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