# Enzymatic hydrolysis of trehalose dimycolate releases free mycolic acids during mycobacterial growth in biofilms

Anil K. Ojha, Xavier Trivelli, Yann Guerardel, Laurent Kremer, Graham F. Hatfull

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## To cite this version:

Anil K. Ojha, Xavier Trivelli, Yann Guerardel, Laurent Kremer, Graham F. Hatfull. Enzymatic hydrolysis of trehalose dimycolate releases free mycolic acids during mycobacterial growth in biofilms. Journal of Biological Chemistry, 2010, 285 (23), pp.17380-17389. 10.1074/jbc.M110.112813. hal-00641552

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# Enzymatic Hydrolysis of Trehalose Dimycolate Releases Free Mycolic Acids during Mycobacterial Growth in Biofilms*§

Received for publication, February 9, 2010, and in revised form, April 2, 2010. Published, JBC Papers in Press, April 7, 2010, DOI 10.1074/jbc.M110.112813

Anil K. Ojha†<fnref n="1" />, Xavier Trivelli§, Yann Guerardel§, Laurent Kremer¶<fnref n="2" />, and Graham F. Hatfull||

From the ‡Department of Infectious Diseases and Microbiology and the ||Department of Biological Sciences and Pittsburgh Bacteriophage Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, ¶Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Universités de Montpellier II et I, CNRS, UMR 5235, case 107, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France, and §UGSF UMR 8576 CNRS, Université de Lille-1, 59650 Vill neuve d'Ascq, France

Mycobacterial species, like other microbes, spontaneously form multicellular drug-tolerant biofilms when grown *in vitro* in detergent-free liquid media. The structure of *Mycobacterium tuberculosis* biofilms is formed through genetically programmed pathways and is built upon a large abundance of novel extracellular free mycolic acids (FM), although the mechanism of FM synthesis remained unclear. Here we show that the FM in *Mycobacterium smegmatis* biofilms is produced through the enzymatic release from constitutively present mycolyl derivatives. One of the precursors for FM is newly synthesized trehalose dimycolate (TDM), which is cleaved by a novel TDM-specific serine esterase, Msmeg_1529. Disruption of Msmeg_1529 leads to undetectable hydrolytic activity, reduced levels of FM in the mutant, and retarded biofilm growth. Furthermore, enzymatic hydrolysis of TDM remains conserved in *M. tuberculosis*, suggesting the presence of a TDM-specific esterase in this pathogen. Overall, this study provides the first evidence for an enzymatic release of free mycolic acids from cell envelope mycolates during mycobacterial growth.

Many microbial species grow as organized communities on surfaces and interfaces, forming a range of multicellular structures from colonies on submerged biotic and abiotic surfaces to pellicles at the air-medium interfaces (1–6). Individual cells within these biofilms are typically embedded in a complex architecture of extracellular matrices primarily composed of secreted polymeric substances including polysaccharides, lipids, proteins, and DNA (1, 3, 7–9). Biofilm development involves coordinated communication between cells and programs of gene expression needed for individual cells to respond to varying microenvironments within the biofilm structure (10–12).

The architectural complexities of microbial biofilms, built on the scaffold of a extracellular matrix, lead to a widespread heterogeneity in the interior microenvironments to which the resident cells presumably adapt by adjusting their physiological

and metabolic activities (13, 14). Therefore, heterogeneous microenvironments of biofilms foster a range of phenotypic diversity in the population and may even trigger cellular differentiation in the community (15, 16). Taken together, mature biofilms provide suitable microenvironments for the development of a subpopulation of stress-tolerant cells, which can persist against a wide range of environmental challenges including antibiotics (17, 18). The extraordinary drug tolerance of microbes in biofilms is particularly relevant in clinical settings where pathogens responsible for chronic infectious diseases such as cystic fibrosis, otitis media, infective endocarditis, and urinary tract infections colonize the host surfaces to form persistent and drug tolerant communities (19–22).

*Mycobacterium tuberculosis*, the causative agent of human tuberculosis, kills about 2 million people annually, and treatment of its infection requires 6–9 months of multidrug chemotherapy (23). These extended periods of chemotherapy are required to minimize relapse and reoccurrence of disease, presumably by eradicating the subpopulation of cells that escape the early phase of treatment (24). Although the basis for the tolerance of these persisters is not clear, the duration of treatment as well as the rate of relapse correlates closely with the bacterial burden in lung lesions such that the large number of bacilli associated with caseating granulomas and cavities may be especially difficult to eliminate by chemotherapy (25, 26). The microbiological nature of *M. tuberculosis* in these high burden infections remains unclear, although we note that *in vitro* *M. tuberculosis* bacilli form parallel rope-like cords in detergent-free growth medium as well as pellicles at the air-medium interface (27). These *M. tuberculosis* cords are rich in an extracellular glycolipid, trehalose dimycolate (TDM),<fnref n="3" /> that has been shown to induce granulomatous responses in animal models (28–30), and mutants defective in cording show loss of lethal chronic persistence in mice (31).

Recent studies show that development of multicellular structures of mycobacteria has distinct physiological and genetic requirements and promote drug tolerance in the population (32–37); these structures can, therefore, be broadly categorized as biofilms. Mutants of *Mycobacterium smegmatis* and *Mycobacterium marinum* with defective biosynthesis of either cell

<footnote>* This work was supported, in whole or in part, by National Institutes of Health Grants AI079288 (to A. K. O.) and AI064494 (to G. F. H.). This work was also supported by an award from the American Lung Association (to A. K. O.).</footnote>

<footnote>§ The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1–S3 and Figs. S1–S3.</footnote>

<footnote n="1">¹ To whom correspondence should be addressed: 130 DeSoto St., Pittsburgh, PA 15261. E-mail: ano7@pitt.edu.</footnote>

<footnote n="2">² Supported by a grant from the CNRS.</footnote>

<footnote n="3">³ The abbreviations used are: TDM, trehalose dimycolate; FM, free mycolic acid; TG, triacylglyceride; mAGP, mycolylarabinogalactan-peptidoglycan; TMM, trehalose monomycolate; ORF, open reading frame; Mtb, *M. tuberculosis*; BSA, bovine serum albumin.</footnote>
wall or cell surface lipids like glycopeptidolipids, mycolyldiacyl-
glycerol, and lipooligosaccharides fail to form biofilms (33, 38,
39). However, the composition of mycobacterial biofilms is sig-
nificantly different from that of other bacteria, containing an
extracellular matrix rich in lipids rather than polysaccharides
(8, 34, 37). Previously, we observed that mycolic acid synthesis
during biofilm maturation of *M. smegmatis* was regulated by
*groEL1* through its direct interaction with KasA (a β-keto-acyl
carrier protein synthase), a component of the multienzyme
complex type II fatty acid synthase involved in mycolic acid
synthesis (34). We further found that *M. tuberculosis* biofilms
also have substantial amounts of extracellular free mycolic
acids (FM) (37). The abundance of FM in biofilms was novel in
a way that mycolic acids have generally been understood to be
present as esters of sugars, although the processes whereby FM
is synthesized and released during biofilm formation remain
unclear.

Here we show that FM-rich *M. smegmatis* biofilms contain enzymatic activity that hydrolyzes trehalose dimycolate to release FM. We also identify a gene, Msmeg_1529, that encodes a cutinase-like serine carboxyesterase responsible for TDM hydrolysis. Disruption of Msmeg_1529 leads to loss of hydrolytic activity in the mutant. We further show that disrupted TDM synthesis through mutation in mycolyltransferases or TDM hydrolysis through mutation in Msmeg_1529 impairs biofilm development of *M. smegmatis*.

EXPERIMENTAL PROCEDURES

*Bacterial Strains, Plasmids, and Growth Conditions*—All *M. smegmatis* strains were maintained in 7H9ADCTween liquid medium or on 7H10ADC agar. Mutants were grown in 150 µg/ml hygromycin and the complemented strains in 150 µg/ml hygromycin and 40 µg/ml kanamycin. For experiments, the strains were grown either planktonically or as biofilms in biofilm media as described previously (34). An attenuated strain of *M. tuberculosis* *mc2*7000 (37) was used as a parent wild type in this study. The strain was maintained in 7H9OADC with 100 µg/ml pantothenic acid in liquid culture and on 7H11OADC + pantothenic acid on plates. For experiments, the strain was cultured in Sauton's medium with pantothenic acid before processing.

*Genetic Manipulations of M. smegmatis*—All the genetic
manipulations in *M. smegmatis* were carried using a recently
developed recombineering technique in mycobacteria (53).
The allelic exchange substrate for every deletion was prepared
by amplifying the left and right arms of the gene using primers
listed in supplemental Table S1. The kan', hyg' recombinant
colonies were screened for correct gene replacement by PCR.
For complementation, the mutants were rescued of the recom-
bineering plasmid, pJV53, and then transformed with comple-
menting plasmids. The transformants were selected on
7H10ADC/hyg/kan. The complementing plasmids, pMs6398
and pMs1529, were constructed by cloning the PCR- amplified
full-length ORF downstream of Phsp60 of pJL37 (54).
The primers used for amplification of Msmeg_6398 and
Msmeg_1529 ORF are listed in supplemental Table S2.

*Lipid Purification and Analysis*—The total organic extracta-
ble apolar and polar lipids of *mc2*155, cultured either as biofilms

or as a planktonic suspension, were extracted in petroleum
ether as described previously (37). For analysis of radiolabeled
lipids, the cultures were exposed to [¹⁴C]glucose for 6 h before
extraction in petroleum ether. The extracted lipids were devel-
oped on one- or two-dimensional TLC in the solvent systems as
indicated for every experiment. The radiolabeled lipids were
analyzed by autoradiography. The FM were developed in chlo-
roform:methanol (96:4) and visualized by spraying the TLC
plate with 5% molybdophosphoric acid in ethanol and charring
the plate at 120 °C for 10 min. The TDM was developed in
chloroform:methanol:water (90:10:1) and visualized by char-
ring the plate with 10% H₂SO₄ in ethanol at 120 °C for 10 min.
Methyl esters of FM and total cellular mycolic acids were pre-
pared as described previously and resolved in hexane:diethyl
ether (95:5) before visualizing the plate with 5% molybdophos-
phoric acids.

*Structural Analysis of TDM and FM*—The lipid spot migrat-
ing similar to *M. tuberculosis* FM in solvent C (37) was
extracted from the silica plate in chloroform:methanol (2:1)
and analyzed by NMR and mass spectrometry. Similarly, lipid
comigrating with a standard *Mtb* TDM (Sigma) was extracted
from silica plate in chloroform:methanol (2:1) and analyzed by
mass spectrometry. Mass spectrometry analyses of purified
mycolates and TDM were done on a Voyager Elite reflectron
matrix-assisted laser desorption ionization time-of-flight mass
spectrometer (PerSeptive Biosystems, Framingham, MA)
equipped with a 337-nm UV laser. Samples were solubilized in
1 ml of chloroform/methanol (2:1) and mixed on target with 1
ml of a 2,5-dihydroxybenzoic acid matrix solution (10 mg/ml
dissolved in chloroform/methanol 2:1). For NMR analyses the
same molecules were dissolved in deuterated chloroform con-
taining 0.01% of tetramethylsilane and transferred into Shigemi
tubes matched for D₂O. Then 0.1 ml of deuterium oxide was
added to avoid solvent evaporation during long acquisition.
One-dimensional proton NMR spectra were recorded at 299 K
on an 800 MHz Avance II Bruker spectrometers equipped with
a ¹H/¹³C/¹⁵N/²H probe. After structural confirmation of TDM
and FM, the purified lipids were used as reference for all subse-
quent TLCs.

*In Vitro Assay for the Synthesis of FM*—For all the *in vitro*
assays described, radiolabeled substrate, either total cellular lip-
ids, TDM, or cell wall, equivalent to 100,000 cpm were homo-
geneously resuspended in the assay buffer (50 mM Tris-HCl, pH
7.5, 100 mM NaCl, 25 mM MgCl₂, 1 mM phenylmethylsulfonyl
fluoride, and 0.2% Triton N101) by mild sonication. The homo-
genate was then mixed to 100 µg of proteins from *M. smegmatis*
lysates prepared from either planktonic or biofilm cells lysed by
sonication in the assay buffer. The mixture was incubated at
37 °C for 2 h. The lipids in the reaction mixture were extracted
in an equal volume of petroleum ether, dried, and analyzed on
TLC developed in chloroform:methanol 96:4. For testing the
heat sensitivity of the active component, the biofilm lysate was
incubated at 95 °C for 15 min before the assay. For assays with
either purified TDM or cell wall, the radiolabeled homogenate
in the assay was substituted with equivalent counts of TDM or
cell wall, isolated using the previously described methods. For
assay with purified proteins, 5 µg of Msmeg_1529 or D29 LysB
were used.

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## Lipids of Mycobacterial Biofilms

<figure><img src="image_4.png" /><figcaption>FIGURE 1. Regulated synthesis of FM in *M. smegmatis* biofilms. A, two-dimensional radio-thin layer chromatography of ¹⁴C apolar and polar lipids equivalent to 50,000 cpm extracted from planktonic and biofilm cultures of *M. smegmatis* is shown. The TLCs were developed in solvent (Sol.) systems A, C, and D as described previously (37). The lipids with significantly different level of ¹⁴C incorporation in the two cultures are annotated as Spot 1, Spot 2, and TG. The direction of migration in each dimension is marked at the *left bottom*. B, radio-TLC of ¹⁴C apolar lipids equivalent to 50,000 cpm extracted from 2-, 3-, and 4-day (d) biofilms of *M. smegmatis*, *mc*²¹⁵⁵ wild-type (wt), ΔgroEL1 (Δg), and ΔgroEL1 complemented with *pMsgroEL1 (comp)* is shown. The arrow denotes FM. The quantity of FM is normalized to the 2-day biofilms of wild-type *M. smegmatis*. C, shown is exposure of 2-, 3-, and 4-day biofilms to 400 µg/ml rifampicin. Percentage survival was calculated using untreated culture at the respective stage as a reference.</figcaption></figure>

**Cloning and Characterization of Serine Esterases from** *M. smegmatis*—A search of serine esterase from *M. smegmatis* genome webpage of the J. Craig Venter Institute (Comprehensive Microbial Resource website of the J. Craig Venter Institute) revealed six ORFs. All six ORFs along with Msmeg_6398 were amplified using the primers listed in **supplemental Table S3**. The amplicons were cloned in the NdeI and XhoI sites of pET21b, and the recombinant plasmid expressing the ORFs was sequenced to confirm the clone identity. Each recombinant clone was then transformed into *Escherichia coli* strain BL-21 DE3. The transformants were grown in LB-ampicillin until A₀.₆ before being induced with 1 mM isopropyl 1-thio-β-D-galactopyranoside for 3 h. The induced cells were harvested, washed, and sonicated in assay buffer. 100 µg of total culture lysate protein was mixed with 100,000 cpm of purified [¹⁴C]TDM, and the esterase activity in the lysates were assayed as described above.

*Drug Tolerance Assay of M. smegmatis Biofilms*—A drug tolerance assay of *M. smegmatis* *mc*²¹⁵⁵ in biofilms cultures was

done as described previously (37). Cells were grown in biofilms for 5 days before exposing to 400 µg/ml rifampicin for 7 days. Before harvesting cells, the biofilms plates were incubated with 0.2% Tween 80 to solubilize the biofilms. The solubilized biofilms were then rocked in the presence of detergent and glass beads for 24 h at 4 °C. The homogeneous suspension of cells was then diluted and plated on 7H10 agar to determine their colony-forming units.

## RESULTS

*Regulated Synthesis of FM in M. smegmatis Biofilms*—We previously described the presence of FM in the biofilms of *M. tuberculosis* (37), although the mechanisms underlying FM synthesis remained unclear. To further study the mechanism of FM synthesis, we sought to exploit the fast growing and non-pathogenic species *M. smegmatis*, which was preliminarily observed to make a lipid in biofilms that co-migrated with *Mtb* FM (37). Before undertaking a detailed study on FM synthesis in this species, we extended those preliminary studies and performed a complete lipid analysis using the same technique that we described for *Mtb* (37). We examined total polar and apolar lipid synthesis in planktonic and biofilm cultures of *M. smegmatis* by ¹⁴C-labeling and two-dimensional thin layer chromatography (Fig. 1A).

Apart from the large abundance of a lipid (marked as *spot 1*) corresponding to the position of *Mtb* FM (37), we also noted two other major changes in biofilm cells; that is, increased levels of triacylglyceride (TG) and decreased levels of an unknown lipid (*spot 2*). There are other minor changes in the total lipids of the two cultures that we have not pursued further.

Spot 1 was purified and structurally characterized by ¹H NMR and mass spectrometry to be a mixture of free alpha and epoxy mycolic acids (supplemental Fig. S1). Further detailed analysis of this lipid by TLC is described in **supplemental Fig. S1**. The identity of spot 2 is described below.

Because the nonessential *M. smegmatis* chaperone GroEL1 associates directly with the type II fatty acid synthase enzyme KasA during biofilm formation and is, therefore, directly implicated in *M. smegmatis* mycolic acid synthesis (34), we asked whether the defect in the later stages of biofilm development of ΔgroEL-1 mutant correlate with decreased accumulation of FM. We determined the stage-specific synthesis of FM by
radiolabeling the newly synthesized lipid for 6 h with [¹⁴C]acetate. We observed an ~2.5-fold increase in radiolabeled mycolic acids from day 2 to day 4 of biofilm growth of wild type, and the induction was largely abrogated in the ΔgroEL1 mutant and could be restored in the complemented strain (Fig. 1B).

Because a matured structure is important for drug tolerance behavior of *M. tuberculosis* biofilms (37), we predicted that the drug tolerance in the wild-type *M. smegmatis* biofilms would also correlate with the induced synthesis of FM (Fig. 1B) and would progressively increase from the 2- to 4-day stage. Upon exposure to rifampicin there was a very sharp increase in the number of drug tolerant bacilli in 4-day biofilms compared with 2- and 3-day biofilms (Fig. 1C). Overall, these data together established a tight correlation between FM synthesis, structural maturation, and drug tolerance properties of *M. smegmatis* biofilms.

*FM Is Made through Hydrolysis of Trehalose Dimycolate*—Because GroEL1 is known to associate with the fatty acid synthase II enzyme KasA (34), it seems unlikely that synthesis of FM in *M. smegmatis* biofilms involves an entirely new enzymatic pathway but, rather, derives from mycolic acid derivatives made by FAS-II. However, mycolic acids are typically esterified to other molecules with the major forms being mycolylarabinojalactan-peptidoglycan (mAGP), in which the mycolic acids are covalently linked to the cell wall components, and TDM, in which two mycolic acid chains are linked to the sugar trehalose (40). These two molecules are, therefore, the major candidates for substrates for the generation of FM, although cellular esterases that hydrolyze these have not been described. We note though that the mycobacteriophage lysin B enzyme has recently been shown to cleave mAGP (41), and there are several mycobacterial cutinase-like proteins for which lipolytic activity has been reported (42).

To test the hypothesis that biofilm-grown cells contain an enzymatic activity responsible for synthesizing FM from precursor mycolic acid-containing substrates, planktonically grown *M. smegmatis* cells were labeled with [¹⁴C]glucose, and a total lysate was prepared; we then incubated this with a total extract prepared from unlabeled biofilm-grown cells (Fig. 2A). As shown in Fig. 2A, the planktonically grown substrate did not contain FM, but it was generated upon incubation with the biofilm extract (Fig. 2A). The activity in the biofilm extract is likely to be enzyme-mediated as heat treatment abolished the activity. Having determined that the biofilm extract contains the enzymatic activity of interest, we examined whether it was capable of generating FM from purified mAGP. However, no activity was observed, indicating that mAGP is not the immediate precursor for FM synthesis (Fig. 2B). We then tested this with TDM as a substrate using ¹⁴C-radiolabeled TDM purified from planktonically grown *M. smegmatis* (Fig. 2C). Because TDM from *M. smegmatis* is relatively uncharacterized, we purified the apolar lipid co-migrating with *M. tuberculosis* TDM and confirmed its identity by NMR and mass spectrometry (supplemental Fig. S2). We observed that the biofilm lysate has potent activity that released FM from TDM. However, we also observed a detectable activity in the planktonically grown cells (Fig. 2C). Although the product from planktonic lysate in Fig. 2C (fifth lane) appears to migrate slightly faster than reference

<figure><img src="image_5.png" /><figcaption>**FIGURE 2.** FM is released from TDM in the presence of mycobacterial lysates. A, radio-TLC of the lipids in reaction mixtures of ¹⁴C-labeled *M. smegmatis* planktonic lysate with either lysates of *M. smegmatis* biofilms (*bf*), heat-inactivated biofilm lysates (*heat bf*), or with BSA is shown. B, radio-TLC of the lipids in the reaction mixture of purified ¹⁴C-labeled cell wall (*MsCW*) with buffer containing BSA, biofilm lysate (*bf*), heat-inactivated biofilms lysate (*bf heat*), and planktonic lysate (*plnk*) is shown. C, radio-TLC of the reaction mixture as described in panel B is shown except that ¹⁴C-MsTDM was used instead of *MsCW* as a substrate. D, radio-TLC shows lipids in the reaction mixture of purified ¹⁴C-MsTDM with *M. tuberculosis* biofilms lysate (*bf*) and buffer containing BSA. Purified FM was used as a control in panels A–D. TLCs in panel A–D were developed in chloroform:methanol 96:4.</figcaption></figure>

FM (first lane), the difference in mobility was not reproducible. An activity of TDM esterase was also observed in lysates prepared from *M. tuberculosis* biofilms (Fig. 2D), suggesting that both species contain enzymes capable of cleaving TDM to generate FM.

*Reduced Synthesis of TDM in Maturing Biofilms*—If TDM is a precursor for FM synthesis, then we predict that it is present at reduced levels as cells form mature biofilms. Indeed we observed that the synthesis of ¹⁴C-labeled TDM is reduced as cells mature from 2- to 4-day-grown *M. smegmatis* biofilms (Fig. 3A). We postulate that this likely arises from an increase in subsequent processing of synthesized TDM to yield FM. We note that a reduction also occurs in the ΔgroEL1 mutant, although this could arise from loss of synthesis (see Fig. 1B) as opposed to enhanced processing (Fig. 3A). Although the levels of newly synthesized TDM decreased during biofilm growth, the total amount of TDM in both the wild-type and mutant strains remained unchanged (Fig. 3B). It is likely that the site of TDM synthesis is spatially separated from its ultimate location on the outside of the cell envelope, which is then not accessible to the proposed biofilms esterase. We propose that when active synthesis of FM begins, the newly synthesized TDM that has yet

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FIGURE 3. **Decreased synthesis of TDM in maturing biofilms.** A, shown is radio-TLC of ¹⁴C apolar lipids corresponding to 50,000 cpm from 2-, 3-, and 4-day (d) biofilms of *M. smegmatis*, *mc*²¹⁵⁵ wild-type (*wt*), and *mc*²¹⁵⁵-ΔgroEL1 extracted in petroleum ether. The plot shows relative amount of spots corresponding to [¹⁴C]TDM using 2-day biofilms of wild type as reference. B, the total lipid content in each sample of panel A was visualized by charring the plate with 10%*H*₂*SO*₄ in ethanol. A cold purified TDM was used as a control (ctrl). The position of TDM is marked by arrow. The plot shows the relative amount of total TDM using 2-day biofilms of wild type as reference. C, TLC of Spot 2, originally identified with differential ¹⁴C incorporation in planktonic and biofilm cultures (Fig. 1 panel A), along with purified *M. tuberculosis* TDM (*Mtb TDM*) and *M. smegmatis* (*MsTDM*) is shown. D, shown is TLC of total apolar lipids equivalent to 50,000 cpm from ¹⁴C-labeled planktonic (*plnk*) and 4-day biofilm (*bf*) cultures seen after charring as described above. A cold purified MsTDM was used as a control. The TLCs were developed in chloroform:methanol:water (90:10:1).

to be delivered to its outer membrane location is captured and
converted to FM by the esterase before translocation.

Finally, we note that if newly synthesized TDM is the precursor for FM, then spot 2 in Fig. 1A, which is abundant in planktonically grown cells but barely detectable in biofilms (Fig. 1A), may correspond to TDM. To confirm this we purified spot 2 after two-dimensional TLC separation and showed that it has similar migration patterns as TDM isolated from both *M. smegmatis* and *M. tuberculosis* (Fig. 3C). Because spot 2 in Fig. 1A was identified by a radio-TLC, we predicted that the difference in TDM levels of planktonic and 4-day biofilm cultures, like the one observed for early and late stage biofilms (Figs. 3, A and B),

will remain limited only to the newly made TDM. We looked at
the total TDM in the apolar lipids of the two cultures by char-
ring the TLC plate, and indeed we observed that the levels of
total TDM in the cultures were very similar, although we note a
clear accumulation of total FM in the biofilm culture (Fig. 3D).
Taken together, these observations strongly support the
hypothesis that biofilm formation occurs through a transition
in growth phase in which FM is generated by esterase-depen-
dent cleavage of newly synthesized TDM.

*An Antigen 85 Homologue Is Required for Biofilm Formation—*
We reasoned that if TDM is a precursor for FM and that these
are required for mature biofilm formation, then mutants defec-
tive in TDM synthesis would also be altered in biofilm develop-
ment. TDM synthesis is dependent on mycolyltransferases,
members of the serine carboxyesterases superfamily but origi-
nally identified as fibronectin-binding proteins (fbp), and mem-
bers of the antigen-85 (Ag85) complex, which transfer mycolyl
chains from 6-hydroxy group of trehalose monomycolate
(TMM) to the 6-hydroxy group of a second TMM molecule to
form TDM (43). They can also transfer mycolyl chains from
TMM to arabinogalactan-peptidoglycan to form the mAGP
complex (43). *M. tuberculosis* encodes four homologues of
mycolyltransferases (*fbpA*:Rv3804c, *fbpB*:Rv1886c, *fbpC*:
Rv0129c and *fbpD*:Rv3803c), of which FbpD is likely devoid of
enzymatic activity (44). Although there is some functional
redundancy in the remaining three, loss of FbpA activity leads
to a strong depletion of TDM (45), suggesting that FbpA plays a
major role in TDM synthesis. A similar role for FbpA has been
suggested in *M. smegmatis* (46).

Apart from *fbpA* (Msmeg_6398), we noted four additional
mycolyltransferases in *M. smegmatis*, *fbpB* (Msmeg_2078),
*fbpC* (Msmeg_6396), and two genes closely related to *fbpD*
(Msmeg_3580, and Msmeg_6399), and we constructed deletion
mutants of each of these. Each of the mutants was examined for
biofilm formation, and we observed that the Δ*fbpA* mutant
showed a strong biofilm defect (Fig. 4A) that could be com-
pletely restored by complementation (Fig. 4A); the other four
mutants formed normal biofilms (supplemental Fig. S3). TDM
synthesis was depleted by about 43% in the Δ*fbpA* mutant, con-
sistent with previous findings (46) (Fig. 4B). As predicted, a
similar partial reduction in the synthesis of FM was also
observed in the Δ*fpbA* mutant (Fig. 4C), consistent with the
hypothesis that newly synthesized TDM is a substrate for FM
synthesis.

*Msmeg_1529 Encodes a TDM-specific Esterase*—The release of FM from TDM by biofilm lysates of *M. smegmatis* and *M. tuberculosis* suggests the presence of an esterase that hydrolyzes the linkage between the sugar and lipid moieties in TDM. This is unlikely to be catalyzed by the mycolyltransferases discussed above as these have strong specificity for TMM (43). However, such enzymes may share common mechanisms with the mycolyltransferases, which use a catalytic serine (within a conserved GXSXG motif) and have conserved glutamine and histidine residues in the active site (47). We note that the recently described cutinase-like phage-encoded lysin B is also a serine esterase, with conserved serine, aspartic acid, and histidine residues in the active site, and cleaves mycolylarabinogalactan to release FM (41). In a search for related enzymes with potential serine-esterase activity, we looked for ORFs annotated as serine esterase in the *M. smegmatis* genome web page (Comprehensive Microbial Resource at the J. Craig Venter Institute) and identified six ORFs (Fig. 5A). All six candidate ORFs were cloned and expressed in *E. coli*, and total cell lysates from induced cells were tested for the ability to generate FM from radiolabeled *M. smegmatis* TDM. Five of the ORFs as well as FbpA (Msmeg_6398) had no activity, but one (Msmeg_1529) was evidently active (Fig. 5B). The Msmeg_1529 protein was purified to greater than 90% homogeneity from *E. coli* (Fig. 5C), and its activity was verified on purified *M. smegmatis* TDM (Fig. 5D). Interestingly this enzyme is specific for TDM, and no activity

was observed on purified mAGP (Fig. 5E); it is, however, active
on *M. tuberculosis* TDM (Fig. 5F). This activity is distinct from
the phage lysin B protein, which acts preferentially on mAGP
even though it has some activity on TDM (Fig. 5, D and E). We
propose to call this enzyme a TDM hydrolase.

A Δ*Msmeg1529* *Mutant Is Altered in Biofilm Formation*—
Given the *in vitro* activity of Msmeg_1529 and the proposed
relationship between TDM and FM during biofilm formation,
we constructed a mutant with a deletion in this ORF. Expect-
edly, the lysate of Δ*Msmeg_1529* mutant had undetectable
hydrolytic activity against purified TDM, indicating that
Msmeg_1529 is the major enzyme in *M. smegmatis* responsible
for TDM hydrolysis (Fig. 6A). However, Δ*Msmeg_1529* mutant
is capable of forming biofilms but with a retarded rate as com-
pared with wild type (Fig. 6B). The difference was clearly
noticeable at 3- and 4-day stages (Fig. 6B), but upon prolonged
incubation the mutant biofilms were indistinguishable from the
parent strain both in appearance as well as in rifampicin toler-
ance (data not shown). The delayed development in the mutant
was complemented by expression of Msmeg_1529 by an episо-
mal plasmid, pMs_1529 (Fig. 6B). The compromised TDM
hydrolysis and partial defect in the biofilm growth of
Msmeg_1529 suggested that the mutant biofilms would also be
compromised in making FM. We indeed found nearly 50%
reduction in the synthesis of FM in the mutant biofilms at the
4-day stage, and the synthesis was restored to the normal level
in the complemented strain (Fig. 6C). Interestingly, we also
observed a dramatic reduction in the synthesis of TG in the
mutant, suggesting that the induction of FM and TG in
*M. smegmatis* biofilms could be linked (Fig. 6C). The FM and
TG in the mutant, however, were restored to wild-type levels
upon extended incubation (data not shown).

We further reasoned that if FM synthesis is only partially
reduced in Δ*Msmeg1529* mutant, then there would be alternate esterase-dependent pathway to produce FM in *M. smegmatis*. We tested the presence of alternate pathways in Δ*Msmeg1529* using the assay described in Fig. 2A, and we found that the biofilm lysate of the mutant was able to generate FM when mixed with ¹⁴C-label planktonic lysate (Fig. 6D). However, identity of alternate esterase and its substrate remains unknown, although mAGP of the cell wall as a substrate is ruled out from the experiment described in Fig. 2B.

DISCUSSION

Although growth of mycobacterial surface pellicles has been
reported for many years, it is only recently that these structures
have been revisited from a biofilm perspective and found to
involve a specific developmental genetic program and harbor
drug-tolerant cells in the extracellular matrix presumably made
of secreted FM (37). Although the role of mycobacterial bio-
films in their pathogenicity remains unclear, the presence of
drug tolerant persisters and altered lipid profiles suggests that
this is an important question to address.

Here we show that FM is newly synthesized during growth of
both *M. smegmatis* and *M. tuberculosis* biofilms. The data
reported here also show that newly synthesized but not the
previously made TDM is one of the precursors for FM and is
processed by a TDM-specific esterase. It is perhaps not surpris-

<figure><img src="image_11.png" /></figure>
<figure><img src="image_12.png" /><figcaption>A</figcaption></figure>

mc²¹⁵-wt

ΔfbpA

ΔfbpA : pMs6398

<figure><img src="image_13.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_14.png" /></figure>

FIGURE 4. Defective biofilms of *mc*²¹⁵:fbpA mutant. A, 5-day biofilms of *M. smegmatis*, *mc*²¹⁵ wild-type (*wt*), *mc*²¹⁵:ΔfbpA, and *mc*²¹⁵:ΔfbpA:pMs6398 (complemented) strains are shown. B, shown is radio-TLC, developed in chloroform:methanol:water (90:10:1), of apolar lipids from *M. smegmatis* wild type (*wt*) and ΔfbpA mutant showing incorporation of ¹⁴C into MsTDM. The plot shows the relative amount of MsTDM from the two strains. C, shown is radio-TLC of the apolar lipids from the wild-type and mutant strains developed in chloroform:methanol (96:4) showing ¹⁴C incorporation into FM. The plot shows the relative amount of FM in the two strains. 5-Day biofilms of the strains were labeled for 6 h with [¹⁴C]glycerol, and total apolar lipids equivalent to 50,000 cpm from each sample were loaded and compared with either purified MsTDM in *panel B* (marked with an arrow) or purified FM in *panel C*.

ing that mAGP is not a precursor for FM synthesis, as the only previously described enzyme that cleaves mAGP to release FM (Lysin B) is also involved in cell lysis during phage growth. However, it is known from previous studies that although TDM is the major component of cord factor and plays a role in *M. tuberculosis* pathogenicity (29, 31), mutants with reduced levels of TDM are still viable.

We propose that the distinction between newly synthesized TDM and pre-existing TDM in the synthesis of FM is determined by cellular localization of the enzyme and its cognate substrate. Synthesis of mycolic acids leading to formation of

TMM is thought to occur in the cytoplasm and that TMM is transported across the cytoplasmic membrane where it serves as a precursor for linkage of mycolic acids to arabinogalactan-peptidoglycan or for TDM synthesis (40); both of these reactions are catalyzed by the Ag85 complex (40). We propose that TDM is generated within the periplasmic space between the mycobacterial inner and outer membranes and that this is the site of its conversion into FM by esterases such as Msmeg_1529. The FM is then secreted to form the biofilm matrix. We further propose that the Msmeg_1529 enzyme is restricted to this location and, therefore, does not have access to pre-existing TDM

<figure><img src="image_15.png" /></figure>
<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

FIGURE 5. **Msmeg_1529** is a TDM-specific hydrolase. A, multiple sequence alignment of serine esterases from *M. smegmatis* by ClustalX is shown. The conserved catalytic triad of GXSXG, Asp, and His are annotated. B, shown is radio-TLC of apolar lipids extracted in petroleum ether from a reaction mixture containing purified ¹⁴C-MsTDM lysates of recombinant *E. coli* expressing putative *M. smegmatis* serine esterase identified in panel A. The recombinant strains expressed the ORFs (marked below each lane) under isopropyl 1-thio-β-D-galactopyranoside-inducible LacI-dependent expression system. Reactions with BSA and recombinant *E. coli* containing empty vector (pET21b) were used as negative control. C, SDS-PAGE shows TDM hydrolase (TDMH) (*Msmeg_1529*) purified from recombinant strain used in panel B. D, shown is radio-TLC of lipids extracted from the reaction mixture of pure ¹⁴C-MsTDM with either TDM hydrolase or LysB from mycobacteriophage D29. E, radio-TLC of lipids extracted from reaction mixture containing pure [¹⁴C]mAGP with either TDM hydrolase or LysB. F, shown is radio-TLC of lipids extracted from a reaction mixture containing pure ¹⁴C-MtTDM (TDM from *M. tuberculosis*) with TDM hydrolase. Buffer with BSA as a negative control and pure FM as a reference was used in panels D, E, and F.

that is an integrated component of the outer membrane. Two further lines of evidence support a specific role for Msmeg_1529. First, expression of this gene is induced about 4-fold in biofilm formation (48); second, Msmeg_1529 contains a strongly predicted signal peptide at its N terminus, consistent with its proposed site of action outside of the cytoplasmic membrane, where newly synthesized TDM is made.

There appears to be considerable redundancy in many of the enzymatic pathways involved in mycolic acid synthesis and processing. For example, there are at least four mycolyltransferases in both *M. tuberculosis* and in *M. smegmatis*, and although FbpA plays a major role in forming both mAGP and TDM, it is not an essential gene (49). Likewise, although only Msmeg_1529 from the six putative esterases we overexpressed has the ability to convert TDM to FM in vitro, the Δ*Msmeg_1529* mutant showed only a partial reduction in FM synthesis, suggesting that there are other pathways for FM synthesis. This is further supported by the presence of mycolyl esterase activity in the Δ*Msmeg_1529* mutant. Further identification

of novel pathways involved in FM synthesis will be critical for understanding the role of this lipid in biofilm formation.

The specific role of GroEL1 in biofilm formation remains unclear. Because GroEL1 interacts directly with KasA during biofilm formation (34), it acts at a relatively early stage before TMM transport across the membrane and before the processing of TDM into FM. An intriguing possibility is that GroEL1 helps to deliver newly synthesized TMM to a specific ABC transporter, which is turn is associated on the cell wall side of the membrane with Msmeg_1529 and perhaps other esterases. This newly synthesized TMM would then be targeted for conversion to TDM by antigen-85 followed by closely coupled processing into FM by Msmeg_1529. The recent report of phosphorylation of GroEL1 provides a possible mechanism for activation of its role during biofilm growth (50).

The pathways for mycolic acid synthesis and processing are closely related in *M. smegmatis* and *M. tuberculosis*. In this study we show that *M. tuberculosis* biofilm-grown cells also possess an activity that generates FM from purified radiola-

<figure><img src="image_19.png" /></figure>
## Lipids of Mycobacterial Biofilms

<figure><img src="image_20.png" /></figure>

FIGURE 6. Phenotype of Δ*Msmeg_1529* mutant. A, radio-TLC shows the petroleum ether-extracted ¹⁴C lipids from the reaction mixture containing [¹⁴C]TDM and lysates of either wild-type (wt) or Δ*Msmeg_1529*. BSA was used as a negative control. Purified FM (marked with an arrow) was used as a reference. B, 3- and 4-day biofilms of *mc²¹⁵⁵* (wild type), *mc²¹⁵⁵:ΔMsmeg_1529*, and *mc²¹⁵⁵:ΔMsmeg_1529* complemented with pMsmeg_1529 are shown. C, shown is radio-TLC of ¹⁴C-labeled apolar lipids equivalent to 50,000 cpm made at the 4-day stage of the three strains described in panel B. Lipids were labeled with [¹⁴C]acetate for 6 h before petroleum ether extraction. FM and TG are annotated. D, radio-TLC shows the petroleum ether-extracted ¹⁴C lipids from the reaction mixture containing ¹⁴C-labeled planktonic (plnk) lysate and lysate from either wild-type or Δ*Msmeg_1529-*, or Δ*Msmeg_1529-complemented strains. BSA in the buffer was used as a negative control, and purified FM from *M. smegmatis* (marked with an arrow) was used as a reference.

beled TDM from *M. smegmatis* (Fig. 2D). Seven cutinase-like proteins, with esterase activities in some of them, have been described in *M. tuberculosis* (42, 51), and we suggest that one or more of these behaves similarly to Msmeg_1529, although none has yet been shown to cleave either TDM or mAGP. Rv3452, designated Culp4, is the closest relative of Msmeg_1529, has a putative signal peptide, and has been reported to be cell wall-associated (52). We, therefore, suggest that Rv3452 is a likely candidate for TDM hydrolase in *M. tuberculosis*.

We demonstrated previously that *M. tuberculosis* biofilms contain a subset of cells that show high tolerance to antibiotics (37), and we have shown here (Fig. 1C) that the drug tolerance of *M. smegmatis* biofilm correlates with induced synthesis of FM. Thus, FM is very likely to play an important role in development of drug tolerant persisters. Interestingly, it has also been reported that a Δ*fbpA* mutant of *M. smegmatis* is hypersensitive to anti-mycobacterial drugs, consistent with a role of TDM in FM synthesis and consequent development of drug

tolerant persisters. Intriguingly, although both Δ*fbpA* and Δ*Msmeg_1529* mutants have comparable defects in FM synthesis, Δ*fbpA* is severely defective in biofilm growth. The severity of the Δ*fbpA* mutant could be due to possible defect in its cell wall biosynthesis as the gene has been previously implicated in the synthesis of mycolyl esters of cell wall (43, 45).

Although complete identification of pathways involved in FM synthesis remains to be identified, identification of a TDM hydrolase provides an important insight into the dynamic nature of mycobacterial cell wall during their growth in biofilms. Furthermore, given that *Mtb* TDM is a highly immunogenic lipid and can be hydrolyzed by Msmeg_1529, the esterase can also be exploited as a reagent to understand the pathogenesis of tuberculosis.

Acknowledgments—We acknowledge technical support provided by Amy Vogelsberger and Christina Ferreira.

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<figure><img src="image_22.png" /></figure>