MOLECULAR BIOLOGY OF PATHOGENS

Chlamydia trachomatis Serovar L2 Can Utilize Exogenous Lipoic Acid through the Action of the Lipoic Acid Ligase LplA1

Aishwarya V. Ramaswamy, Anthony T. Maurelli
DOI: 10.1128/JB.00717-10

ABSTRACT

Lipoic acid is an essential protein bound cofactor that is vital for the functioning of several important enzymes involved in central metabolism. Genomes of all sequenced chlamydiae show the presence of two genes encoding lipoic acid ligases and one gene encoding a lipoate synthase. However, the roles of these proteins in lipoic acid utilization or biosynthesis have not yet been characterized. The two distinct lipoic acid ligases in Chlamydia trachomatis serovar L2, LplA1Ct and LplA2Ct (encoded by the open reading frames ctl0537 and ctl0761) display moderate identity with Escherichia coli LplA (30 and 27%, respectively) but possess amino acid sequence motifs that are well conserved among all lipoyl protein ligases. The putative lipoic acid synthase LipACt, encoded by ctl0815, is ca. 43% identical to the E. coli LipA homolog. We demonstrate here the presence of lipoylated proteins in C. trachomatis serovar L2 and show that the lipoic acid ligase LplA1Ct is capable of utilizing exogenous lipoic acid for the lipoylation Therefore, host-derived lipoic acid may be important for intracellular growth and development. Based on genetic complementation in a surrogate host, our study also suggests that the C. trachomatis serovar L2 LipA homolog may not be functional in vivo.

Members of the family Chlamydiaceae are Gram-negative obligate intracellular pathogens that cause significant ocular, respiratory, and sexually transmitted disease (STD) in humans and animals. In humans, chlamydiae cause a wide range of diseases, including psittacosis, pneumonia, trachoma, and STDs. Chlamydia trachomatis-mediated genital infections are among the most prevalent of all STDs and result in a wide range of sequelae, including pelvic inflammatory disease, infertility, and ectopic pregnancy, thus posing an enormous public health problem throughout the world (4). C. trachomatis also causes trachoma, one of the leading causes of preventable blindness in the developing world (9).

Chlamydiae exhibit a biphasic developmental cycle that involves two morphologically distinct forms (1). The elementary body (EB) is the metabolically inactive form that initiates infection by attaching to and invading host epithelial cells. After invasion, the EBs differentiate into the metabolically active but noninfectious reticulate body (RB) within a vacuole known as the inclusion. After multiple rounds of binary fission ranging from 24 to 72 h, the RBs redifferentiate back into EBs. Eventually, cell lysis results in the release of EBs that can infect surrounding cells or be transmitted to a new host.

As obligate intracellular bacteria, chlamydiae depend on the host for much of their nutrient requirements and in general have undergone significant genome reduction (29). Consistent with this lifestyle, bioinformatic analyses of chlamydiae genomes show that while they possess pathways for the biosynthesis of some nutrients, other pathways are incomplete or entirely absent. Chlamydiae are known to be dependent on the host for a variety of metabolites, including purines, pyrimidines, cofactors, and amino acids (27) and have evolved several mechanisms, such as the use of specialized transporters, trafficking of host organelles, and modulation of signaling pathways to successfully tap into the nutrient-rich host environment (36). Characterizing the metabolic capabilities of intracellular pathogens is important because it enhances our understanding of the pathogen and its interactions with the host and enables the design of better drugs and vaccines.

Lipoic acid is a sulfur-containing cofactor that is universally required for aerobic metabolism (39). It is required for the activity of enzymes involved in oxidative and single carbon metabolism such as pyruvate dehydrogenase (PDH), 2-oxoglutarate dehydrogenase (2-OGDH), and branched-chain keto-acid dehydrogenase (BCKDH). These enzyme complexes typically consist of three subunits (E1, E2, and E3), and lipoic acid is attached to a conserved lysine residue in the E2 subunit via an amide bond (34). Another protein that undergoes lipoylation is the GcsH protein of the glycine cleavage system (GCS), which converts glycine to carbon dioxide, ammonia, and 5,10-methylenetetrahydrofolate (11). Protein-bound lipoic acid then serves as a carrier of reaction intermediates between the different subunits of the multienzyme complexes.

Details regarding the biosynthesis of lipoic acid and its utilization were largely obtained from studies conducted on Escherichia coli (11). Posttranslation modification of all apo-enzymes can occur by two distinct pathways in bacteria and plants. In the first, exogenous or salvaged lipoic acid is attached to unlipoylated E2 subunits by the action of a lipoic acid ligase (LplA) in an ATP-dependent manner (30) (Fig. 1). The pathway involving de novo biosynthesis of the cofactor is primarily mediated through the action of two enzymes, LipA and LipB. LipB is an octanoyl-ACP transferase that transfers an octanoyl group from acyl carrier protein (ACP) to the lipoyl-accepting domain of the apoenzyme, which subsequently becomes the substrate for sulfur insertion by LipA to form lipoylated holoenzyme (28, 45) (Fig. 1). Lipoic acid synthesis can also occur through the action of LplA which can transfer octanoic acid in an ATP-dependent manner to the apo E2 domain, which then becomes the substrate for the action of LipA (21) (Fig. 1).

FIG. 1.
FIG. 1.

Lipoylation pathways demonstrated in E. coli. Lipoic acid ligase (LplA) activates exogenous lipoic acid in an ATP-dependent manner and catalyzes its transfer to the apo E2-subunit of enzyme complexes that require lipoic acid to form the holo enzyme. De novo biosynthesis of lipoic acid involves the action of two enzymes, LipB and LipA. LipB is an octanoyl-ACP (acyl carrier protein) transferase that transfers the octanoyl moiety from octanoyl-ACP to the apo-E2 subunit. LipA uses the octanoylated-E2 domain as the substrate for the synthesis of lipoylated E2. Another pathway for the biosynthesis of lipoic acid involves the transfer of free octanoic acid by LplA to the apo-E2 domain, which then serves as a substrate for LipA to form lipoylated-E2. Enzymes predicted by genome annotation that are present (enclosed in solid line box) or absent (enclosed in dashed line box) in C. trachomatis are also indicated.

Although lipoic acid is a vital cofactor, details regarding its utilization by intracellular pathogens have started to emerge only recently (2, 10, 14, 24, 41). The obligate intracellular bacterium, C. trachomatis serovar L2, encodes for four proteins that are predicted to undergo lipoylation. Its genome also encodes for two putative lipoic acid ligases designated LplA1Ct and LplA2Ct and a LipACt homolog but no putative LipB homolog, suggesting that it possesses an incomplete pathway for de novo lipoic acid biosynthesis. Transcriptome studies of C. trachomatis serovar D have shown that both putative ligases genes and the lipoic acid synthase genes are expressed during growth and development (5, 6).

In this study we show the presence of lipoylated proteins in C. trachomatis serovar L2 and characterize the activity of the chlamydial lipoic acid ligase, LplA1Ct. Based on the present data, we propose that chlamydiae are capable of utilizing exogenous lipoic acid. However, LipACt was found to be nonfunctional in vivo in a heterologous system and did not allow us to determine its role in de novo biosynthesis of lipoic acid in the bacterium.

MATERIALS AND METHODS

Bacterial strains and growth conditions.The strains used in the present study are listed in Table 1. Strains were grown in Luria-Bertani (LB) medium or M9 minimal salts supplemented with 0.5% mannitol at 37°C. M9 minimal medium was supplemented with 0.5% mannitol as the carbon source, because gene expression under the ara promoter in pBAD18 used as the vector for complementation assays is repressed in the presence of glucose. Growth medium was supplemented with antibiotics for selection at the following concentrations: ampicillin (100 μg/ml), kanamycin (50 μg/ml), and chloramphenicol (25 μg/ml). When required, media were also supplemented with arabinose (0.2%), IPTG (isopropyl-β-d-thiogalactopyranoside; 1 mM), sodium acetate (10 mM), sodium succinate (10 mM), or lipoic acid (10 μM). Lipoic acid was obtained from Sigma. The lipoylated tripeptide DKLA was purchased from AnaSpec.

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TABLE 1.

Bacterial strains and plasmids used in this study

Quantitative determination of plating efficiency of the ligases was done as follows. Strains from exponential-phase cultures were washed three times in 1× phosphate-buffered saline (PBS; 1.05 mM KH2 PO4, 154 mM NaCl, 5.6 mM Na2HPO4), serially diluted, and plated on M9 mannitol medium without exogenous lipoic acid or M9 mannitol with arabinose and lipoic acid (10 μM). Efficiency of plating was calculated as the percentage ratio between the number of colonies obtained on plates without lipoic acid and those growing on plates with lipoic acid.

For growth curves, single colonies from appropriate LB plates were inoculated into M9 mannitol minimal with 0.2% arabinose, 10 mM sodium acetate, and 10 mM sodium succinate and then cultured overnight at 37°C. About 1 ml of the overnight culture was then washed three times in PBS and inoculated into fresh M9 mannitol medium containing 0.2% arabinose with or without 10 μM lipoic acid. Growth curves were performed by using a Bioscreen growth curve analyzer. The standard deviation represents the growth measurements of five replicates.

Strain and plasmid construction.ATM967 (E. coli ΔlplA ΔlipB::Kanr) and ATM1102 (E. coli ΔlplA ΔlipA::Kanr) were constructed by allelic exchange as described previously (12). Briefly, the kanamycin resistance cassette region (kan) from pKD4 (Table 1) with 5′ and 3′ overhangs identical to the 5′ and 3′ regions of E. coli lplA, lipA, and lipB (Table 1) was amplified by PCR. All primers are listed in Table 2. E. coli BW25113 containing pKD46 was used as the recipient for linear transformation for mutant construction. Kanamycin-resistant (Kanr) recombinants were purified and screened via PCR to ensure proper insertion of the cassette. P1L4 lysates grown on the ΔlplA::kan strain and ΔlipB::kan strain were used to transduce the standard prototroph, MG1655 with selection for Kanr. To construct the double mutant, the ΔlplA::kan allele was transduced into MG1655 and the transductant was transformed with pCP20 which expresses the Flp recombinase and mediates recombination between FRT sequences to excise the kan cassette. The Kans strain thus obtained was transduced with the ΔlipB::kan allele and selected on M9 minimal medium supplemented with mannitol, sodium acetate, sodium succinate, and kanamycin. The ΔlplA ΔlipB::kan double mutant was then stocked as ATM967. A P1L4 lysate grown on the BW25113 ΔlipA strain was used to transduce the MG1655 Kans ΔlplA strain to generate the ΔlplA ΔlipA::kan double mutant, which was stocked as ATM1102 (ΔlplA ΔlipA::kan).

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TABLE 2.

Primers used in this study

All plasmids used in the present study are described in Table 1. Plasmids pLplAEc, pLipAEc, and pLipBEc contain an optimized E. coli ribosome binding site (RBS) and complete open reading frames of E. coli lplA, lipB, and lipA, respectively, under the control of the ara promoter in pBAD18. Similarly, pLplA1Ct, pLplA2Ct, and pLipACt contain the same optimized RBS and complete open reading frames of ctl0537, ctl0761, and ctl0821, respectively, from C. trachomatis serovar L2 under the control of the ara promoter in pBAD18. All constructs were verified by sequencing at the Biomedical Instrumentation Center of the Uniformed Services University.

Protein expression and purification.LplA1Ct and LplA2Ct were amplified by PCR from C. trachomatis serovar L2 genomic DNA using Vent (New England Biolabs) and ligated into the XhoI site of the expression vector pGEX-6-P1 (GE Life Sciences), verified by sequencing, and transformed into ATM967 (ΔlplA ΔlipB::kan). ATM967 containing pGEX-6-P1::LplA1Ct or pGEX-6-P1::LplA2Ct was induced with 1 mM IPTG at room temperature overnight. Glutathione S-transferase (GST)-tagged proteins were then purified from bacterial lysates by using GST beads (GE Life Sciences) according to the manufacturer's instructions for batch purification. The N-terminal GST tag was cleaved from the recombinant protein by using PreScission protease (GE Life Sciences) and dialyzed against buffer containing 50 mM sodium phosphate (pH 7.6), 2 mM EDTA, 100 mM NaCl, 10 mM MgCl2, and 10% glycerol. The BCKDH-E2 subunit encoded by ctl0657 was amplified from C. trachomatis serovar L2 by PCR and also cloned into pGEX-6-P1 by using the XhoI restriction site. Recombinant BCKDH-E2 subunit was purified as described above, and the cleaved protein was dialyzed into 1× PBS plus 10% glycerol. LplAEc was purified as a His-tagged protein according to a previously described report (22). Briefly, E. coli BL21DE3 expressing LplAEc with an N-terminal His tag was grown at 37°C until reaching an optical density at 600 nm (OD600) of ∼0.5 and induced with 1 mM IPTG for 3 h at the same temperature. LplAEc was then purified under native conditions using HisPur cobalt resin (Pierce) according to the manufacturer's recommendations. Purified LplAEc was then dialyzed into buffer containing 20 mM sodium phosphate (pH 7.6), 2 mM EDTA, and 5% glycerol. All purified enzymes were stored at −80°C.

The purification of apo-PDH from ATM967 (ΔlplA ΔlipB::kan) was adapted from that of an earlier study (23). Briefly, ATM967 was grown in 100 ml of M9 minimal medium supplemented with mannitol, acetate, and succinate at 37°C. The cells were pelleted and resuspended in 12 ml of 0.1 M potassium phosphate (pH 7), lysed by sonication, and centrifuged for 30 min at 39,000 × g at 4°C. The supernatant was then centrifuged at 236,000 × g in a Beckman 45 Ti rotor for 8 h. The resulting pellet was resuspended in 5 ml of 50 mM potassium phosphate buffer and centrifuged for an additional 8 h at 236,000 × g. The supernatant was discarded, and the pellet was resuspended in 1 ml of potassium phosphate buffer containing 10% glycerol and stored at −80°C. Concentrations of all recombinant proteins were measured by using a BCA protein assay kit (Pierce).

Western blot analysis.For Western blot analyses, samples were resolved by SDS-PAGE and blotted. Lipoylated proteins were detected using α-lipoic acid antibody (1:10,000; Calbiochem), followed by the addition of donkey anti-rabbit IgG antibody conjugated to horseradish peroxidase (1:1,000; GE Life Sciences). Western blots were developed using the Pico Super Signal system (Pierce) and visualized by using a Fuji Intelligent Dark Box and Image Reader LAS-3000 software. For equal loading of samples, the protein concentration (A280) was determined by using a NanoDrop ND-1000 spectrophotometer.

Lipoic acid ligase assays.For lipoic acid ligase activity, purified LplA1Ct (1 μM) or LplAEc (0.3 μM) was incubated in 10 mM sodium phosphate (pH 7) containing 5 mM ATP, 0.3 mM dithiothreitol, and 5 mM MgCl2. E. coli apo-PDH/2-OGDH (1 μg) and chlamydial BCKDH-E2 (2 μM) were used as substrates in the reaction, and lipoic acid was used at a concentration of 1 mM. The reaction was incubated at 37°C for 1 h and analyzed by SDS-PAGE, followed by Western blotting with anti-lipoic acid antibody as described above.

RESULTS

Sequence alignments of LipACt, LplA1Ct, and LplA2Ct.Genes encoding for putative enzymes involved in de novo lipoic acid biosynthesis (lipA) and lipoic acid salvage (lplA) were identified by BLASTp using the E. coli homologs against the C. trachomatis serovar L2 genome (42). The LipA homolog (LipACt) shares 43 and 42% identity with lipoyl synthases from E. coli and Bacillus subtilis, respectively. LipACt possesses both sets of conserved cysteine residues involved in coordinating iron-sulfur (Fe-S) clusters, the first of which is common to all radical SAM superfamily enzymes and the second of which is unique to lipoic acid synthases (40) (Fig. 2A).

FIG. 2.
FIG. 2.

CLUSTAL W sequence alignments of chlamydial proteins predicted to be involved in lipoate utilization and synthesis with representative proteins. (A) The putative chlamydial LipA (LipACt) was aligned with lipoic acid synthases from Escherichia coli (E.c), Bacillus subtilis (B.s), Saccharomyces cerevisiae, (S.c), plant Arabidopsis thaliana (A.t), and Homo sapiens (H.s). Residues common to SAM radical super family of enzymes (I) and residues specific for lipoic acid synthases (II) are highlighted in gray. (B) Alignment of the putative lipoic acid ligases from C. trachomatis serovar L2 (LplA1Ct and LplA2Ct) with ligases from E. coli (E.c.) and Thermoplasma acidophilum (T.a.). Conserved regions are highlighted in gray, and residues involved in lipoyl-AMP binding are indicated by an inverted triangle (▾).

C. trachomatis serovar L2 also possesses two lipoic acid ligases, LplA1Ct and LplA2Ct (encoded by ctl0537 and ctl0761), that are only 30 and 27% identical to E. coli LplA (Fig. 2B) as determined by BLASTp analyses. These enzymes possess the typical BPL_LplA_LipB domain, which is characteristic to this family of enzymes (35). Although LplA1Ct showed homology to ligases from only prokaryotic organisms, LplA2Ct appeared to be more closely related to plant lipoic acid ligases (8). However, the two ligases did not show any similarity to the E. coli LipB transferase. Orthologs of both ligases have been identified in all of the sequenced chlamydiae, suggesting that they are a well-conserved family of genes (Table 3).

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TABLE 3.

Amino acid sequence identities of chlamydial lipoic acid ligases and lipoic acid synthase with the respective E. coli homologs

Evidence of lipoylation in C. trachomatis serovar L2 EBs and RBs.Chlamydiae possess at least three proteins that are predicted to undergo posttranslational modification with lipoic acid, including the E2 subunits of PDH, 2-OGDH, and BCKDH (encoded by ctl0499, ctl0311, and ctl0657). All of the enzyme subunits are predicted to possess a single lipoyl domain (13). In order to examine whether C. trachomatis serovar L2 possesses lipoylated proteins, renografin purified RBs and EBs were analyzed with a polyclonal antibody specific for lipoylated proteins (37). Western blot analysis showed that the anti-lipoic acid antibody recognized lipoylated proteins in both RB and in EB fractions (Fig. 3). The sizes of three of these lipoylated proteins are close to the predicted sizes of the chlamydial E2 subunits of PDH (∼46 kDa), 2-OGDH (∼40 kDa), and BCKDH (∼42 kDa). The lipoylated protein (∼14 kDa) present only in RBs is consistent with the predicted size of the GcsH subunit encoded by ctl0534.

FIG. 3.
FIG. 3.

Detection of lipoylated proteins in C. trachomatis serovar L2 RBs and EBs. Equal concentrations of Renografin-purified EBs and RBs were resolved by SDS-PAGE blotted and analyzed by Western blotting with anti-lipoic acid antibody. The sizes of E2 subunits of pyruvate dehydrogenase (PDH-E2), 2-oxoglutarate dehydrogenase (2-OGDH-E2), branched-chain keto-acid dehydrogenase (BCKDH-E2) and that of the glycine cleavage system protein H (GcsH) are indicated on the blot.

Functionality of lipACt, lplA1Ct, and lplA2Ct.Currently, the necessary tools to generate targeted knockout mutants in chlamydiae is lacking; thus, E. coli is used as the surrogate host for functional characterization of relevant genes. Toward this end, we first constructed a ΔlplA ΔlipA mutant of E. coli that was deficient in lipoic acid synthesis (ΔlipA) and utilization of exogenous lipoic acid (ΔlplA) but possesses a functional lipB. The resulting strain, ATM1102 (ΔlplA ΔlipA::kan) has an absolute requirement for acetate and succinate which provide metabolic bypasses to enzymes that require lipoic acid and served as the background for determining the functionality of the chlamydial lipoic acid synthase (lipACt). Because ATM1102 (ΔlplA ΔlipA::kan) possess a functional lipB gene, complementation with E. coli lipA (lipAEc) or functional chlamydial lipoyl synthase (lipACt) should restore de novo lipoic acid biosynthesis, thereby allowing bacteria to grow on M9 mannitol minimal medium in the presence of arabinose. ATM1102 was transformed with pLipAEc or pLipACt expressing lipAEc and lipACt, respectively, under the control of the arabinose-inducible promoter in pBAD18. Transformants were selected on M9 mannitol medium supplemented with acetate, succinate, and ampicillin. Select transformants were subsequently examined for growth in the presence of arabinose but in the absence of exogenous lipoic acid. Our complementation studies showed that upon induction with arabinose, lipAEc was able to complement the mutant strain (100% by efficiency of plating), whereas no complementation was observed with lipACt (<10−4% by efficiency of plating) under similar induction conditions. Similar results were obtained when complementation analysis was repeated with lipACt cloned into a high-copy vector (data not shown).

Next, we sought to assess the functionality of the two chlamydial ligase genes, lplA1Ct and lplA2Ct. For this, a second double mutant of E. coli (ΔlplA ΔlipB) was generated, and it was designated ATM967. This strain lacks the ability to synthesize lipoic acid (ΔlipB), as well as utilize exogenous lipoic acid (ΔlplA), and provides a better background for assessing ligase activity. To assay for putative ligase activity, ATM967 (ΔlplA ΔlipB::kan) was transformed with pLplAEc, pLplA1Ct, and pLplA2Ct expressing lplAEc, lplA1Ct, and lplA2Ct, respectively, under the control of arabinose-inducible promoter in pBAD. Transformants were screened for their ability to grow on M9 minimal medium in the presence of mannitol, 10 μg of lipoic acid/ml, and 0.2% arabinose. LplAEc was able to restore the ability to utilize exogenous lipoic acid (100% by efficiency of plating), while LplA1Ct complemented at an efficiency of 94% determined by the same method. However, no complementation was observed with LplA2Ct (<10−5%).

In order to further examine the functionality of the ligases and to see whether there was a delayed growth of the LplA2Ct transformed strain, the growth rates of ATM967 (ΔlplA ΔlipB::kan) expressing LplAEc, LplA1Ct, and LplA2Ct were measured in minimal medium with or without lipoic acid. As seen in Fig. 4A, in the absence of lipoic acid, the pLplA1Ct and the pLplAEc transformed strains showed growth kinetics that were identical to each other and grew faster than MG1655 transformed with pBAD18, presumably due to overexpression of the ligases. The MG1655 transformed with pBAD18 exhibited a longer lag most likely due to the presence of only a single copy of the chromosomal ligase. However, the pLplAEc- and pLplA1Ct-expressing ATM967 and the MG1655 control strains all had the same slope in exponential phase. A similar trend was seen for these strains even in the presence of lipoic acid (Fig. 4B), with growth kinetics of the LplAEc- and LplA1Ct-expressing strains being slightly faster than that of the MG1655 control. The growth rate of the pLplA2Ct transformed strain was similar in the presence or absence of lipoic acid, reaching an OD600 of only ∼0.3 even after 46 h of growth. The double mutant, ATM967, transformed with pBAD18 showed no growth throughout the experiment with or without lipoic acid.

FIG. 4.
FIG. 4.

Growth of lipoic acid ligase mutant and transformed strains in the presence or absence of lipoic acid. Strains (see inset) were grown in M9 mannitol medium containing 0.2% arabinose at 37°C in the absence (A) or presence of 10 μM lipoic acid (B). The absorbance (OD600) was measured in a Bioscreen growth curve analyzer and plotted against time (hours). Error bars represent the standard deviation from five replicates.

In order to confirm protein lipoylation in vivo, lysates of ATM967 (ΔlplA ΔlipB::kan) expressing lplAEc, lplA1Ct, and lplA2Ctwere blotted with the anti-lipoic acid antibody. Wild-type E. coli is known to possess three lipoylated proteins: PDH-E2 (65 kDa), 2-OGDH-E2 (45 kDa), and GcsH protein (∼15 kDa). As shown in Fig. 5, two lipoylated proteins corresponding to PDH-E2 and 2-OGDH-E2 were detected in the E. coli MG1655 lysates and in strains expressing lplAEc and lplA1Ct. The glycine cleavage protein GcsH was not observed in these lysates because the cultures were not induced by glycine. Interestingly, in lysates expressing lplAEc and lplA1Ct, lipoylation was also seen in the absence of exogenous lipoic acid, suggesting that the LplA1Ct may also be capable of utilizing endogenous octanoic acid as a substrate. The octanoylated apo-domain may then be modified into lipoylated holo-domain by the functional LipA in ATM967 (ΔlplA ΔlipB::kan) (Fig. 1). This observation is consistent with our growth curve studies and previous reports that have shown that LplAEc can also utilize free octanoic acid as a substrate (21, 28, 30). Therefore, LplA1Ct appears to function in a manner similar to that of LplAEc.

FIG. 5.
FIG. 5.

Detection of lipoylated proteins in lipoic acid ligase mutant and transformed strains. Lysates of transformed strains (LplAEc, LplA1Ct, and LplA2Ct), MG1655 (positive control), and ATM967 (negative control) grown in minimal medium with or without lipoic acid were analyzed for the presence of lipoylated proteins. Equal concentrations of the lysates were separated by SDS-PAGE, blotted, and probed with anti-lipoic acid antibody. Lipoylated proteins corresponding to PDH-E2 and 2-OGDH-E2 of E. coli were observed in the strains expressing LplAEc and LplA1Ct in the presence or absence of lipoic acid but not in the LplA2Ct-expressing strain.

We also examined the ability of LplA2Ct to utilize exogenous octanoic acid. ATM967 (ΔlplA ΔlipB::kan) was transformed with pLipBEc, pLplAEc, or pLplA2Ct and examined for its ability to grow on M9 mannitol medium supplemented with 50 μM octanoic acid. Although pLipBEc (98% efficiency of plating) and pLplAEc (80% efficiency of plating) were able to complement the double mutant upon induction with arabinose, no complementation was observed with pLplA2Ct (<10−5% efficiency of plating). In vivo lipoylation was not seen in cells expressing LplA2Ct in the presence or absence of lipoic acid (Fig. 4) and confirms the negative complementation data.

In vitro lipoylation activity of LplA1Ct.For in vitro assays, we used lysates of ATM967 (ΔlplA ΔlipB::kan) enriched for apo-PDH and 2-OGDH and recombinant chlamydial BCKDH-E2 as lipoate acceptors. The latter was overexpressed in ATM967 as a GST-tagged protein so that it could purified in the apo-form. Consistent with our in vivo complementation data, recombinant LplA1Ct was able to lipoylate apo-PDH and 2-OGDH-E2 subunits purified from E. coli ATM967 (Fig. 6A, left) as detected by Western blotting with anti-lipoic acid antibody. LplA1Ct was also able to lipoylate the recombinant chlamydial BCKDH-E2 subunit (Fig. 6B, left). Purified LplAEc served as a positive control for both reactions (Fig. 6A and B, right). Recombinant LplA2Ct was unable to lipoylate either substrate, suggesting that this enzyme was inactive under the conditions in which it was tested (data not shown).

FIG. 6.
FIG. 6.

In vitro lipoic acid ligase activity. Purified LplAEc (positive control) and LplA1Ct were assayed for activity in the presence or absence of various components as indicated. Purified LplA1Ct was assayed for activity using a mixture of E. coli apo-PDH/2-OGDH (panel A, left) and recombinant chlamydial BCKDH-E2 (panel B, left) as substrates at 37°C for 1 h. Reactions were analyzed by SDS-PAGE and blotted, and lipoylated proteins were detected using anti-lipoic acid antibody. Purified LplAEc served as a positive control and was also tested with apo-PDH/2-OGDH (panel A, right) and chlamydial BCKDH-E2 (panel B, right).

Previous reports have described the ability of lipoic acid ligases from Listeria monocytogenes to enable the utilization of lipoylated tripeptides as a source of lipoic acid (24). In order to determine whether the chlamydial ligases could function in a similar manner, we used a lipoylated tripeptide consisting of aspartate-lysine [lipoic acid]-alanine (DKLipA) as a source for lipoic acid in the in vitro assay. However, LplA1Ct and LplA2Ct were not able to utilize DKLipA to lipoylate the apo-enzyme substrates (data not shown).

DISCUSSION

Lipoic acid is an essential cofactor that is required for the posttranslational modification of several enzyme complexes involved in central metabolic processes. Organisms can obtain lipoic acid either by de novo biosynthesis or by salvage of lipoic acid from the environment. For example, E. coli, which has served as the model for unraveling the various steps involved in lipoic acid biosynthesis, is capable of synthesizing lipoic acid and utilizing exogenously available lipoic acid. Organisms such as Rickettsia spp. do not possess an LplA homolog but are predicted to synthesize lipoic acid through the action of LipA/B homologs (3). Others, such as Listeria spp., are lipoic acid auxotrophs (24). Genes involved in the synthesis or utilization of salvaged lipoic acid appear to play an important role in the growth and replication of some intracellular bacterial pathogens such as Burkholderia pseudomallei (33) and Listeria monocytogenes (32). Parasites such as Toxoplasma and Plasmodium have an absolute requirement for lipoic acid from host cells even though they possess a functional pathway for the de novo biosynthesis of the cofactor, which is produced and sequestered in the apicoplast (10, 14, 15). In Mycobacterium tuberculosis, the lipB gene is considerably upregulated in multiple drug-resistant strains, and attempts to construct a lipB mutant proved unsuccessful, suggesting that lipB is indispensable for growth (25). These examples underscore the importance of lipoic acid in the growth and replication of bacterial pathogens.

C. trachomatis serovar L2 possesses a putative lipoic acid synthase and two predicted lipoic acid ligase homologs, and this raised some intriguing questions about the biosynthesis and utilization of lipoic acid. Bacteria typically possess between three and four lipoylated proteins. Western blot analyses of C. trachomatis serovar L2 EB and RB lysates show the presence of lipoylated chlamydia proteins that correspond to predicted sizes of E2 subunits of PDH, 2-OGDH, and BCKDH. However, we were unable to confirm their identities due to the lack of specific antibodies. RB lysates showed the presence of an additional small lipoylated protein (<15 kDa) that may correspond to the GcsH subunit (ctl0534) of the glycine cleavage system. Interestingly, analysis of genome sequences by BLAST shows that the other subunits that form the glycine cleavage complex are absent in chlamydiae. In Saccharomyces cerevisiae, lipoylated GcsH is required for the lipoylation of PDH-E2 and 2-OGDH-E2 (38). Orphan GcsH proteins have also been reported in other systems (41). The presence of a putative lipoylated GcsH subunit in chlamydiae suggests that it may have a different role in metabolism. Alternately, this band may represent a breakdown product of one of the larger lipoylated subunits.

The presence of two lipoic acid ligases is not uncommon in bacteria and has been previously reported in lipoic acid auxotrophs such as L. monocytogenes wherein the ligases are paralogous, and each ligase is proposed to utilize different sources of lipoic acid (24). In organisms such as Toxoplasma and Plasmodium, the two ligases appear to be quite divergent at the sequence level and exhibit specificity for PDH-E2 or 2-OGDH-E2 subunits, respectively (2). Of the two putative ligases in C. trachomatis serovar L2, LplA1Ct was found to be functional in vitro and in vivo, whereas LplA2Ct appeared to be inactive in both the surrogate E. coli host and in in vitro lipoylation assays. The detection of lipoylated proteins in the LplA1Ct-expressing strain even in the absence of octanoic acid suggests that it can utilize free octanoic acid-like LplAEc (21).

Transcriptome studies of C. trachomatis serovar D, which has a similar intracellular progression as C. trachomatis L2, have shown that lplA1Ct gene expression is detected about 8 h postinfection, coincident with the differentiation of EBs into metabolically active RBs. However, the expression of lplA2Ct is detected only 16 h postinfection (6). Overall, relative expression of lplA1Ct is slightly higher than that of lplA2Ct throughout the developmental cycle (6), suggesting that it may play a more active role. As described earlier, LplA1Ct is more similar to other bacterial ligases, whereas LplA2Ct is more related to plant lipoic acid ligases and could be regarded as a remnant of this ancestral relationship between chlamydiae and cyanobacterial endosymbionts of chloroplasts (8). Inactivity of LplA2Ct in our system could be the result of incorrect assay conditions or the lack of a lipoic acid donor or substrate specific to LplA2Ct. Inherent difficulties associated with genetic manipulation of chlamydiae precluded gene inactivation experiments that could have elucidated the function of LplA2Ct.

The de novo pathway leading to the formation of lipoic acid involves the action of two enzymes, LipA and LipB. LipA belongs to the radical SAM family of enzymes that also includes enzymes such as biotin synthase (31). LipA binds to iron-sulfur clusters and mediates reductive cleavage of S-adenosylmethionine to generate 5′dA· radical, which ultimately results in the insertion of two sulfur atoms (presumably from the iron-sulfur cluster) into the octanoylated apodomain to form lipoic acid (11). Although LipACt shows a fairly high degree of homology to the E. coli LipA and has all of the canonical residues that are typical for this enzyme family, it failed to restore growth of an E. coli mutant lacking lipA. Homologs of LipA from other organisms have been shown to complement a similar E. coli lipA mutant, suggesting that heterologous enzymes are capable of functioning in vivo and complementing an E. coli mutant (26, 44). Bacillus subtilis possesses two ligases and a lipoic acid synthase but no putative lipB, an arrangement similar to that encountered in chlamydiae (26). The B. subtilis lipA is essential for growth of the bacteria in minimal medium without exogenous lipoic acid, suggesting that it is capable of de novo lipoic acid biosynthesis, even in the absence of a putative LipB transferase (26). The existence of a similar pathway in chlamydiae cannot be ruled out since we have shown that LplA1Ct expressed in ATM967 (ΔlplA ΔlipB::kan) can lipoylate target proteins even in the absence of exogenous lipoic acid. Due to the limitations imposed by the lack of genetic tools in chlamydiae, we used a lipoic acid synthase-deficient E. coli mutant as a surrogate host to examine the functionality of LipACt. Our complementation assay in this system suggests that LipACt is not functional in vivo. However, we cannot rule out the possibility that LipACt may not be functionally similar to E. coli LipA or that expression in the surrogate host could have resulted in an unstable protein or incomplete assembly of iron-sulfur clusters required for activity. In fact, several attempts to overexpress and purify LipACt in the soluble form proved unsuccessful and did not allow us to conclusively demonstrate the presence of the de novo pathway that perhaps utilizes LipACt and LplA1Ct.

Taken together, our data suggest that chlamydiae are capable of utilizing exogenous lipoic acid during growth and differentiation. Given the relatively low concentration of free lipoic acid in the intracellular environment (39), questions still remain as to the exact mechanism by which chlamydiae are able to gain access to and sequester the cofactor. Chlamydiae are known to alter mitochondrial function (19) and interact with the host mitochondria through transient or indirect interactions (36) and may therefore have access to the limited concentrations of free lipoic acid. Lipoic acid is a fairly small molecule (206 Da) and could thus freely diffuse into the inclusions (20). Another source for lipoic acid, as hypothesized for Toxoplasma (10), could be lipoylated peptides from breakdown of host enzymes, which could be taken up by the bacterium through an oligopeptide transporter and subsequently cleaved by nonspecific amidases to release free lipoic acid for further utilization.

Although antibiotic therapies are currently available for the treatment of ocular and genital tract infections caused by chlamydiae, reports of treatment failure and demonstration of in vitro resistance to antibiotics suggest that development of resistance to commonly used antibiotics against chlamydiae is possible (7, 43). Given the importance of lipoic acid and its role in the growth and virulence of other intracellular pathogens, genes involved in lipoic acid biosynthesis and utilization make attractive candidates for the development of novel therapeutics (25). In the present study, we have demonstrated the activity of the chlamydial lipoic acid ligase, LplA1Ct, which relies on exogenous lipoic acid obtained from the host. Therefore, LplA1Ct presents a potential target that can be exploited for the development of novel anti-infectives against chlamydiae.

ACKNOWLEDGMENTS

We thank J. Cronan (University of Illinois, Urbana) for providing pYFJ16 and R. Binet and J. Zheng (Food and Drug Administration, College Park, MD) for the use of the Bioscreen Growth curve analyzer. We also thank D. Fisher for critical reading of the manuscript and members of the Maurelli lab for many helpful discussions.

This study was funded by National Institutes of Health grant U19AI084044.

FOOTNOTES

    • Received 20 June 2010.
    • Accepted 14 September 2010.

  • ↵▿ Published ahead of print on 24 September 2010.

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