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J Bacteriol. May 2000; 182(10): 2732–2740.
PMCID: PMC101980

Identification of Genes Encoding Exported Mycobacterium tuberculosis Proteins Using a Tn552′phoA In Vitro Transposition System

Abstract

Secreted and cell envelope-associated proteins are important to both Mycobacterium tuberculosis pathogenesis and the generation of protective immunity to M. tuberculosis. We used an in vitro Tn552′phoA transposition system to identify exported proteins of M. tuberculosis. The system is simple and efficient, and the transposon inserts randomly into target DNA. M. tuberculosis genomic libraries were targeted with Tn552′phoA transposons, and these libraries were screened in M. smegmatis for active PhoA translational fusions. Thirty-two different M. tuberculosis open reading frames were identified; eight contain standard signal peptides, six contain lipoprotein signal peptides, and seventeen contain one or more transmembrane domains. Four of these proteins had not yet been assigned as exported proteins in the M. tuberculosis databases. This collection of exported proteins includes factors that are known to participate in the immune response of M. tuberculosis and proteins with homologies, suggesting a role in pathogenesis. Nine of the proteins appear to be unique to mycobacteria and represent promising candidates for factors that participate in protective immunity and virulence. This technology of creating comprehensive fusion libraries should be applicable to other organisms.

Mycobacterium tuberculosis continues to pose a serious health threat to people throughout the world. With an estimated 2.9 million people having died from tuberculosis in 1997, M. tuberculosis remains the infectious agent responsible for the most deaths worldwide (64). In order to develop new drugs, treatments, and vaccines for tuberculosis, a better understanding of M. tuberculosis pathogenesis and M. tuberculosis antigens that elicit a protective immune response is required.

Secreted and cell envelope-associated proteins are likely to play a critical role in M. tuberculosis disease. Research on several bacterial pathogens has revealed that the majority of virulence factors are secreted (22), and it was recently shown that the secreted ERP protein contributes to the virulence of M. tuberculosis (6). A key component of M. tuberculosis infection is the ability of the bacillus to survive within phagocytic cells. Since secreted and surface proteins are ideally positioned to interact with the host, they could facilitate this survival by influencing phagosome maturation, by enabling access to the cytoplasm, or by countering the antimicrobial attacks of the phagocyte. Secreted proteins of M. tuberculosis also play an important role in the generation of a protective immune response. The most striking demonstration of this property comes from experiments in which mice or guinea pigs were immunized with extracellular proteins and significant protective immunity ensued (2, 32, 33, 45, 51). It has been reported that M. tuberculosis secretes an extensive number of proteins, and there are even more proteins that must be cell surface associated (3, 50, 56). Many of these proteins have not yet been identified and even fewer have been tested for a role in virulence and protective immunity.

PhoA (Escherichia coli alkaline phosphatase) protein fusions have been used in many different organisms to identify exported proteins, including ones that are important to bacterial virulence (7, 17, 26, 36, 49). Importantly, PhoA has been shown to function as a reporter for secreted proteins in M. smegmatis (60). Moreover, when a multicopy plasmid library of phoA fusions to M. tuberculosis genomic DNA was screened in M. smegmatis, four active PhoA fusions were identified, one of which involved the ERP protein (see above) (40). Typically, phoA fusions are made either by using in vivo transposition with transposons such as TnphoA (a derivative of Tn5) or by cloning genomic DNA upstream of a truncated ′phoA gene (43). The ′phoA gene in both of these cases lacks signals for expression and export. Since PhoA is active only when it is located outside of the cytosol, enzymatically active PhoA fusions identify proteins that have export signals.

A simple and efficient in vitro transposition system for Tn552, a transposon first identified in Staphylococcus aureus (52), has recently been developed (25, 39). An extensive analysis of transposon insertion sites produced by this in vitro system demonstrated that the transposon inserts randomly within different target DNAs, including mycobacterial DNA (25). Thus, it appears to be ideal for constructing large complex libraries of gene fusions.

We set out to use this system to create a random library of phoA fusions in cosmids containing M. tuberculosis genomic DNA to identify genes encoding exported proteins. We constructed Tn552′phoA transposons and inserted them into cosmids containing M. tuberculosis DNA by using in vitro transposition reactions. The resulting population of transposon-containing cosmids was integrated into the M. smegmatis genome in single copy, and colonies were screened for active phoA fusions.

To our knowledge, this is the first report of an in vitro transposition system for producing phoA fusions. It provides a simple alternative to constructing libraries by the standard methods. Initial screening of our M. tuberculosis DNA-′phoA fusion libraries identified 31 secreted and membrane proteins. An additional feature of this system is that cosmids containing a transposon insertion can later be used as a substrate for allele exchange in M. tuberculosis. This should enable a relatively rapid transition from the identification of an exported protein to the construction of the corresponding M. tuberculosis mutant and evaluation of its role in pathogenesis and protective immunity.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

E. coli strains DH10B (ElectroMAX DH10B), DH5α, and STABL-2 were obtained from Life Technologies, Inc. In vitro transposition reactions were electroporated into DH10B cells. DH5α was used for all transformations involving plasmid constructions and plasmid rescue. STABL-2 cells were used to recover packaged cosmids libraries. E. coli strains were grown in Luria-Bertani (LB) media. Antibiotics were used at the following concentrations: 10 μg/ml, tetracycline; 40 μg/ml, kanamycin; 50 μg/ml, carbenicillin; 100 μg/ml, ampicillin; or 150 μg/ml, hygromycin. LB plates with 40 μg of 5-bromo-4-chloro-3-indolylphosphate (BCIP) (Sigma, Inc.) were used for screening phoA fusions in E. coli.

Transposon containing cosmid DNA was introduced into M. smegmatis mc2155 (55) by electroporation as previously described (48). Following electroporation, cells were expressed for 3 h at 37°C and then plated on LB agar containing 0.2% glucose, 0.05% Tween 80, 60 μg of BCIP per ml, and either 20 μg of kanamycin or 50 μg of hygromycin per ml.

Molecular biology procedures.

Standard molecular biology techniques for cloning and Southern analysis were done as described by Sambrook et al. (53). Restriction enzymes and Vent polymerase were obtained from New England Biolabs, Inc. KspI was obtained from Boehringer Mannheim, Inc.

Plasmid constructions.

Plasmids and cosmids used in this study are listed in Table Table1.1. Primer IRR1 (5′-GGATCCCAACTTCTCATTTATAAGGTTAA-3′) introduced a T-for-A exchange at nucleotide 41 of inverted repeat right (IRR) to create an open reading frame (ORF). Together with the IRL1 primer (5′-GATATCTTGACGTTCTCATTTACTAG), inverse PCR using Vent polymerase was done using pAL101 as a template. The resulting DNA product was circularized with ligase and now contained a 120-bp minitransposon with a BamHI and an EcoRV site between the two transposon end sequences. This transposon was cloned as a SpeI fragment into a pLitmus29 derivative that lacked the BamHI and EcoRV sites to generate pYUB910.

TABLE 1
Plasmids used in this study

Each of the transposons used in this report (Tn552′phoA · kan, Tn552′phoA · oriE1 · kan, and Tn552′phoA · oriE1 · hyg) are on plasmids (pTG481, pTG509, and pYUB1051, respectively) that contain the oriE1 and no other antibiotic markers.

M. tuberculosis cosmid libraries.

The integrating cosmid library of M. tuberculosis H37Rv has been previously described (4). Using the same strategy, an integrating cosmid library of M. tuberculosis CDC1551 was constructed in pYUB1052. Chromosomal CDC1551 DNA was generously provided by C. H. King (Emory University, Atlanta, Ga.) and was subjected to partial digestion with Sau3AI (average size, 30 kb). The chromosomal DNA was ligated to BclI- and XbaI-cut pYUB1052 and packaged using the Gigapack III Gold Packaging Extract (Stratagene, Inc.).

In vitro transposition reactions.

Transposition reactions targeting pBR322 and pYUB1000 were carried out as previously described (25) and employed His-tagged Tn552 transposase (39). Transposition reactions with pYUB412::H37Rv and pYUB1052::CDC1551 library DNAs were done with the following modifications. Cosmid library DNA was isolated from pooled cells scraped off plates using the Qiagen Maxi kit (Qiagen, Inc.). The library DNA was subjected to a hexadecyltrimethylammonium bromide (CTAB) extraction. The NaCl concentration of the DNA solution was adjusted to 0.7 M, and a 1/10 volume of CTAB-NaCl solution (10% CTAB in 0.7 M NaCl) was added. The DNA was then extracted twice with chloroform-isoamyl alcohol and precipitated with isopropanol. The DNA was washed with 70% ethanol and resuspended in Tris-EDTA buffer. Twenty-microliter reaction mixtures containing 400 ng of library DNA, 0.01 pmol of transposon (1/10 of the amount used in Griffin et al. [25]), and 30 ng of TnpA were incubated at 37°C for 60 min and then diluted 10 times, and 1 μl was electroplated into E. coli DH10B cells. Transposon-containing cosmids were selected as ampicillin-kanamycin- or ampicillin-hygromycin-resistant E. coli transformants.

To generate the pool of E. coli transformants that contained 500,000 transposition events, 42 electroporation reactions were done (a single electroporation yielded 1.2 × 104 E. coli colonies containing cosmids with transposon insertions). These colonies were scraped off selective plates and pooled, and transposon-containing cosmid DNA was isolated.

Recovery of transposon insertions.

Genomic DNA was isolated from M. smegmatis as previously described (19). Tn552′phoA · kan transposon insertions were recovered by digesting genomic DNA with KspI, a restriction enzyme which cuts frequently in M. tuberculosis DNA but does not cut the transposon, and cloning it into pKSII vector DNA. For recovery of Tn552′phoA · oriE1 · kan insertions, genomic DNA was digested with KspI, diluted, and self-ligated. For recovery of Tn552′phoA · oriE1 · hyg, insertions the genomic DNA was cut with Psp0M1 and ligated. Recovered plasmids that were ampicillin resistant (due to the presence of the bla gene of pYUB412) represent transposon insertions into the vector backbone and were not analyzed further.

DNA sequence analysis.

The DNA sequence was determined by using the Applied Biosystems Big Dye Terminator Cycle Sequencing kit (Perkin-Elmer) and an Applied Biosystems 377 automated DNA sequencer. Sequences across the junctions of the transposon and its target site in genomic DNA were obtained with the phoA2 sequencing primer (5′-CGTCCAGGACGCTACTTGTG-3′), the aph1 primer (5′-AGGCCTGGTATGAGTCAGCAAC-3′), and the hygro1 primer (5′-CGACGGATCATCTTGACGTTCT). These primers are directed outward from inside the transposon.

Protein sequence analysis.

The following sites were used: the NCBI Advanced Blast (http://www.ncbi.nlm.nih.gov/blast/), the Sanger Centre M. tuberculosis Sequencing Project (http://www.sanger.ac.uk/Projects/M_tuberculosis/), Tuberculist (http://www.pasteur.fr./Bio/Tuberculist/), PSORT (http://psort.nibb.ac.jp :8800/), TMHMM (http://www.cbs.dtu.dk/services/TMHMM-1.0/), and TopPred2 (http://www.biokemi.su.se/~server/toppred2Server.cgi).

Alkaline phosphatase assays on intact cells.

M. smegmatis strains containing phoA fusions were grown to saturation and then diluted to a final optical density at 600 nm (OD600) of 0.005. These cultures were then grown for 40 h at 37°C. The OD600 of each strain was measured at the start of the experiment. A 0.5-ml portion of cell culture was pelleted and resuspended in equal volume of 1 M Tris (pH 8.0). Then, 0.1 ml of cells was added to 1.0 ml of 2 mM p-nitrophenyl phosphate plus disodium salt (Sigma, Inc.) in 1 M Tris (pH 8.0). The reaction mixture was incubated at 37°C in the dark until a yellow reaction product was visible. Next, 0.1 ml of 1 M K2HPO4 was added to terminate the reaction. The bacteria were pelleted, and the OD420 of 1.0 ml of the supernatant was measured. Alkaline phosphatase units were determined by the following formula: 1,000 × OD420/(minutes of reaction)(OD600)(0.1-ml volume of cells). The negative control strain used in these assays was mc22757=mc2155 attB::Phsp60′phoA. This strain was constructed by integration of pYUB1057 into mc2155.

RESULTS

Tn552′phoA transposons.

Several Tn552′phoA transposons were constructed, each containing a truncated phoA gene that lacks signals for expression and secretion. The elements also contain a selectable marker that functions in both mycobacteria and E. coli. To allow expression of ′phoA, an uninterrupted reading frame must run through the upstream Tn552 end and into ′phoA. Examination of both ends of Tn552 showed that all inward reading frames contained at least one stop codon. We chose one reading frame that contained only a single stop codon and introduced a mutation at a nonconserved position to generate an ORF (Fig. (Fig.1A,1A, see Materials and Methods). The mutation was an A-to-T substitution at bp 41 of the IRR. The other end, the inverted repeat left (IRL), was not changed. The ′phoA gene fragment was cloned next to the modified IRR, and the Tn903 kan gene, encoding kanamycin resistance in both E. coli and mycobacteria, was inserted downstream of the ′phoA gene. The resulting transposon is Tn552′phoA · kan (Fig. (Fig.1B).1B).

FIG. 1
Tn552′phoA transposons. (A) A representative Tn552′phoA element is shown. The 48-bp Tn552 ends are represented by arrowheads. The nucleotide sequence of the modified end with the mutation to create the ORF is shown with its amino acid ...

In order to facilitate recovery of transposon insertions, we introduced the pUC19 origin of replication (oriE1) between the ′phoA and kan genes to make Tn552′phoA · oriE1 · kan (Fig. (Fig.1B).1B). A third element, Tn552′phoA · oriE1 · hyg, was constructed by replacing the kan gene of Tn552′phoA · oriE1 · kan with the hyg gene, which confers hygromycin resistance in E. coli and mycobacteria (Fig. (Fig.11B).

In all cases, substrates for the transposition reaction were made by SpeI digestion of the transposon-containing plasmids. This releases the transposon as a linear fragment with exposed CAOH 3′ ends (essential for strand transfer to the target DNA). The in vitro transposition reactions simply involve the addition of purified transposase to the transposon and target DNAs (Fig. (Fig.1C).1C). The transposon insertions are then recovered by electroporation into E. coli cells, and selecting for both the cosmid marker (ampicillin resistance) and the transposon marker (kanamycin or hygromycin resistance).

Characterization of Tn552′phoA insertions into pBR322.

The Tn552′phoA · kan element was tested using pBR322 as a target DNA. PhoA fusion proteins that are exported beyond the cytoplasm are enzymatically active and capable of hydrolyzing BCIP (the chromogenic substrate of PhoA) to produce blue colonies. Insertions into both the bla and tet genes gave rise to active phoA fusions as expected (data not shown). Out of 413 tetracycline-resistant transformants 121 were ampicillin sensitive, which is indicative of transposon insertions into bla. Of these 121 transformants, 20 were blue (16.5%), which is the expected proportion of insertions to be in the correct orientation and reading frame.

Active bla::Tn552′phoA insertions were isolated in two independent experiments. We chose 15 from one experiment and 13 from the other for DNA sequence analysis. All fusions were inserted after the first nucleotide of a codon as was expected for the insertions to be in the correct reading frame (Fig. (Fig.1C).1C). The insertions were distributed throughout the coding sequence in a total of 23 sites (data not shown).

Isolation of Tn552′phoA insertions in cosmid pYUB1000.

To test the feasibility of the system in mycobacteria, we picked as a target, pYUB1000, a cosmid with 44 kb of M. tuberculosis genomic DNA. We chose this cosmid because it contains the fbpB gene which encodes antigen 85B, one of the best-characterized secreted proteins of M. tuberculosis. Antigen 85B has an N-terminal standard signal peptide, which promotes its translocation across the cytoplasmic membrane and makes it a good model protein for this system.

Two separate transposition experiments into pYUB1000 were carried out. In the first, the Tn552′phoA · kan transposon was used, and a pool of 2,500 independent transposon insertions was generated. In the second experiment, the Tn552′phoA · oriE1 · kan transposon was employed, and a pool of 10,500 transposon insertions was produced. Cosmid DNA was prepared separately from each of these pools and was electroporated into M. smegmatis. pYUB1000 contains the L5 mycobacteriophage attachment site (attP) and integrase (int) gene, which enable stable integration of the cosmid into the M. smegmatis chromosome at the attB site (38). This allowed us to screen for active PhoA fusions that are expressed from single rather than multiple copies of a gene. The M. smegmatis transformants were screened on LB plates containing kanamycin and BCIP, and approximately 1 of every 225 transformants was blue. Twenty blue colonies were picked, genomic DNA was isolated, and the sequence flanking the transposon insertion site was determined (see Materials and Methods).

Characterization of active phoA fusions in pYUB1000.

The majority of these 20 blue colonies had transposons inserted in frame in the fbpB gene (encoding antigen 85B), demonstrating that this system can serve to identify secreted proteins of M. tuberculosis. Amongst the 16 fbpB::Tn552′phoA fusions analyzed, 10 different sites of insertion were identified (Table (Table2).2). Since the library of independent transposition events was first amplified in E. coli before transformation into M. smegmatis for screening, it is likely that multiple insertions at the same site reflect siblings.

TABLE 2
Active phoA fusions identified in pYUB1000

Four of the twenty blue colonies contained transposons that were not inserted in fbpB (Table (Table2).2). In two cases, the transposon was inserted in frame in an ORF that was annotated by the Sanger/Welcome Trust genome sequencing project as lppC (rv1911c) (18). The N terminus of LppC has a consensus lipoprotein signal peptide, for translocation of this protein across the cytoplasmic membrane, and a properly positioned cysteine residue, for lipid modification to tether the protein to the cell envelope.

The remaining two strains had rv1887::Tn552′phoA insertions. rv1887 encodes a type II transmembrane protein with a single hydrophobic membrane-spanning domain. The amino terminus of these fusions includes the predicted transmembrane domain of Rv1887, and the observed activity indicates that ′PhoA is located in the extracytoplasmic domain of the protein.

To confirm that the isolated fusions were responsible for the observed PhoA activity, Southern analysis using the kan gene as a probe was carried out on the 20 strains described above (data not shown). The majority (>80%) of the strains contained only a single transposon, and strains with a single transposon insertion in the three genes, fbpB, lppC, and rv1887 were all identified. Three strains had two transposons present. The presence of two transposons in a single strain complicates the identification of the responsible phoA fusion. Therefore, we undertook Southern analysis on all subsequent strains to verify the presence of a single transposon insertion.

Construction of Tn552′phoA · oriE1 · kan and Tn552′phoA · oriE1 · hyg insertions in genomic libraries of M. tuberculosis.

Following our successful tests of this transposon system, we chose to target an integrating cosmid library of M. tuberculosis H37Rv with Tn552′phoA · oriE1 · kan. In order to reduce the number of cosmids which contained multiple transposon insertions, we used 1/10 of the amount of transposon used in the previous reactions (see Materials and Methods). Cosmid DNA was isolated from a pool of E. coli cells containing 500,000 transposition events. This library of phoA fusions throughout the M. tuberculosis genome was then electroporated into M. smegmatis.

We initially screened 27,000 M. smegmatis transformants and observed 285 blue colonies. These fell into two classes: in about half of the colonies the color appeared early (within 4 days of plating the transformation), but the remainder took up to 14 days to produce a visible color. Representative blue colonies from each class were picked and analyzed in the manner described above.

Sequencing of transposon insertions in “early” blue colonies revealed that an unexpectedly high proportion were in the cosmid vector backbone. Of the first 15 “early” blue colonies, 8 had the transposon inserted in frame with a fortuitous ORF in the vector that could encode a transmembrane domain and consequently produce PhoA activity. Since insertions at this site had not been identified among 60 randomly selected Tn552′phoA · oriE1 · kan insertions in individual cosmids, this was not a “hot” spot for Tn552 insertion (R. Lukose and W. R. Jacobs, unpublished data). Rather, the repeated identification of active fusions in this site most likely reflects that vector sequences are present at a much higher copy number (~150-fold) than any specific M. tuberculosis gene in the cosmid library. Therefore, one is more likely to identify insertions into any vector sequences capable of giving rise to active phoA fusions than into a M. tuberculosis locus. Recovery of insertions into the vector bla gene, which is potentially a major source of active phoA fusions, is prevented by selecting ampicillin-resistant transposon insertions. We subsequently modified the cosmid vector, deleting the complicating region, to make the vector pYUB1052. A new integrating library of M. tuberculosis CDC1551 DNA was constructed (see Materials and Methods), and this library was targeted with Tn552′phoA · oriE1 · hyg. Our preliminary analysis of this library suggests that active phoA fusions are now restricted to insertions in M. tuberculosis DNA.

Analysis of active phoA fusions identified in the M. tuberculosis cosmid libraries.

We have characterized 41 M. tuberculosis DNA-′phoA fusions: 18 from the “early” and 23 from the “late” blue classes. These insertions identify 31 different orf's which can produce active PhoA fusions (Table (Table3).3). With only one exception, analysis of the protein sequence confirms their identification as exported proteins; they contain either a standard signal peptide (8 examples), lipoprotein signal peptide (6 examples), or transmembrane domain (16 examples). We have placed each of these ORFs into one of three groups. These groups are defined by the available information concerning a given protein's function in M. tuberculosis. Group I is comprised of proteins which have either been functionally characterized in M. tuberculosis or have informative homology that enables a reasonable prediction of function. Group II contains proteins which exhibit homology to conserved hypothetical proteins of nonmycobacterial organisms but for which the function is unknown (“FUN”). Group III contains FUN proteins that are apparently unique to mycobacteria. With the exception of homologues in other Mycobacterium species, such as M. bovis and M. leprae, BLAST analysis at the National Center for Biotechnology Information (NCBI) does not identify any proteins that are homologous to the group III proteins in GenBank or peptide sequence databases. Individual gene products are described in Table Table33 and in the Discussion.

TABLE 3
Active phoA fusions identified in M. tuberculosis cosmid libraries

There was one PhoA fusion that we identified which did not involve an ORF with a recognizable signal peptide or transmembrane domain. It has been annotated as InfB (Rv2839c) (18) and, as stated in the Discussion, we feel it is unlikely that it is truly exported in M. tuberculosis.

Quantitative alkaline phosphatase assays.

In order to compare the PhoA fusion strains, assays were carried out on liquid cultures of whole and/or intact cells to allow quantitation of the alkaline phosphatase activity associated with the cell envelope. We found that the overall number of alkaline phosphatase units was low. This most likely reflects difficulties with the substrate entering the highly impermeable cell wall of mycobacteria. However, attempts to permeabilize the cells with chloroform-sodium dodecyl sulfate treatment, as is generally done with E. coli strains, appeared to cause some cell lysis, and such optimization was not pursued. The background level of PhoA activity was determined with a negative control strain (mc22757) which contains a single copy of the truncated ′phoA (lacking a signal peptide for secretion) driven by the hsp60 promoter. Strains that had been picked as “early” blue fusions exhibited levels of PhoA activity that range from 44 times to 275 times the background level (Fig. (Fig.2A).2A). The “late” blue strains exhibited levels of PhoA activity that ranged from 3 times to 42 times the background level (Fig. (Fig.2B).2B).

FIG. 2
Quantitative alkaline phosphatase activity of M. tuberculosis DNA-phoA fusions. Alkaline phosphatase activity was measured as described in Materials and Methods. The values presented are the average of at least three independent experiments, with error ...

DISCUSSION

Tn552′phoA in vitro transposition system.

The in vitro transposition system, described here, is a powerful method for creating comprehensive libraries of phoA fusions. From a single reaction using cosmid target DNA, we could recover a pool of more than 106 cosmids with independent insertions. Such large numbers of randomly positioned transposon insertions would be difficult to achieve with the standard methods of creating phoA fusion libraries (in vivo transposition with TnphoA or cloning into ′phoA-containing vectors). An equally attractive property of this system is that the Tn552 transposon inserts randomly into target DNA. Transposon insertion sites reveal no obvious sequence or GC content preferences, and target sequences ranging from exclusively AT to exclusively GC have been observed (25). Analysis of the transposon insertion sites identified during the course of this work revealed the average GC content to be 65.9%, essentially the same as the overall composition of the M. tuberculosis genome (65.6% GC). This contrasts with the Ty1 in vitro transposition system for which transposition on Leishmania major DNA (61% GC) resulted in transposon insertion sites with an average GC content of 43% (24).

Library design and screening.

We chose to create our library of phoA fusions in an integrating cosmid library of M. tuberculosis. This allows us to screen for active PhoA fusions expressed from their native promoters in single copy. This approach should avoid problems associated with multicopy ′phoA vectors: false positives can result from very high expression of intracellular PhoA fusions, and overexpressed proteins (including fusion proteins) can be toxic (11). In fact, we found that M. smegmatis expressing a multicopy lppC::Tn552′phoA fusion exhibits poor growth and appears to delete the vector construct. Thus, LppC is likely to be an example of an exported protein that may only be identified by screening a library of single-copy phoA fusions.

Since we targeted cosmid libraries, our active phoA fusions do not simultaneously create M. tuberculosis mutants. However, they do create gene disruptions that can be introduced into the M. tuberculosis genome by allelic exchange (35). Additionally, our system has an advantage of being able to identify essential secreted proteins.

Ideally, screening the phoA libraries should be done in the host from which the library was created; however, the presence of endogenous phosphatase activity in M. tuberculosis makes this difficult. As a substitute, we screened the library in M. smegmatis. This fast-growing and nonpathogenic mycobacterium has been shown to properly express, secrete, and process several M. tuberculosis exported proteins, some of which have no homologues in M. smegmatis (23, 30, 63). Moreover, expression of an integrating M. tuberculosis library in M. smegmatis does not confer virulence (4). Consequently, it is safe to carry out our screen in a Biosafety Level 2 facility. A limitation to our strategy is that M. smegmatis may not express or export all M. tuberculosis exported proteins. In addition, M. tuberculosis genes that are expressed only upon interaction of the bacillus with its eukaryotic host could be missed.

Identification of exported M. tuberculosis proteins.

Insertions of Tn552′phoA successfully identified secreted proteins and cell envelope-associated proteins (most notably membrane proteins) of M. tuberculosis. Initial screening of our Tn552′phoA fusion libraries identified eight proteins with standard signal peptides, six proteins with lipoprotein signal peptides, and seventeen proteins with one or more transmembrane domains. To date, this represents the largest collection of exported proteins identified in a genetic screen in mycobacteria. Only three of these proteins had been identified in prior genetic screens (16, 21, 40).

A significant goal of this project is to demonstrate export of proteins predicted to be exported by the analysis of the complete M. tuberculosis genome sequence and to identify any additional proteins that were not predicted. In fact, 4 of the 31 exported proteins (CtpC, Rv1887, Rv1006, and Rv0455c), even though they contain recognizable export signals, are currently not predicted to be exported by the Sanger Center/Welcome Trust genome sequencing project (18) or the Tuberculist genome analysis worldwide web server.

Several proteins that do not contain recognizable signal sequences appear to be secreted by M. tuberculosis (8, 29, 66). Due to the lack of knowledge concerning the secretion of such proteins, it is currently impossible to predict these atypical secreted proteins by analyzing the genome sequence, and we hope that our Tn552′phoA system will help identify such proteins. One PhoA fusion we analyzed involved an ORF (Rv2839c) that had neither a signal peptide nor a membrane-spanning domain. However, we think it unlikely that the Rv2839c-′PhoA fusion is truly exported. The strong homology of Rv2839c to the translation initiation factor 2 of other bacteria is hard to reconcile with extracytoplasmic localization. Furthermore, this strain exhibited a low level of PhoA activity (Fig. (Fig.2B)2B) despite the likelihood that this PhoA fusion is highly expressed (9). This example serves as a reminder that false positives can be identified by this strategy, even though the phoA fusions are present in a single copy.

We identified six proteins which were previously reported to be secreted or cell wall-associated proteins of M. tuberculosis (antigen 85B, antigen 85A, PhoS1, PepA, 19-kDa lipoprotein, and MPT63) (8). Four of these proteins are on the list of 10 “secreted proteins” of Wiker et al. (62) and three of these proteins have been reported to be the most abundant proteins in culture filtrates of M. tuberculosis (32). These six proteins were identified among both the “early” and “late” blue fusions, indicating that both classes of PhoA fusions are worth further screening. The low level of activity associated with late blue fusions may reflect a reduced level of expression, stability, or secretion of a given protein. Furthermore, as we have seen with the MmpL4 fusions, longer periplasmic fusions tend to have reduced levels of PhoA activity (42).

The proteins we identified were split into three groups based on the information available regarding function (Table (Table3).3). Group I includes proteins for which there is sufficient information to enable a prediction of function. A number of the proteins in this group may play crucial roles in M. tuberculosis pathogenesis.

The CtpC and PhoS1 transporters may be essential to nutrient acquisition to allow the bacilli to survive in the phagosome (12, 15). The MmpL3, MmpL4, and MmpL5 proteins belong to the MmpL family of transporter proteins in M. tuberculosis (18). These proteins exhibit similarity to the resistance-nodulation-division family of transporters (47; I. T. Paulsen, M. K. Sliwinski, and M. H. J. Saier, http://www.biology.ucsd.edu/~ipaulsen/transport/) and could play important roles in pathogenesis. In fact, signature-tagged transposon mutagenesis experiments have recently shown that MmpL2, MmpL4, and MmpL7 are required for growth of M. tuberculosis in lungs of mice (13, 20). It is worth noting that we identified four different active MmpL4-′PhoA fusions, which helps to define two large periplasmic domains in this polytopic protein involved in pathogenesis.

FbpA (antigen 85A) and FbpB (antigen 85B) are homologous proteins which have mycolyltransferase and fibronectin-binding activity (1, 5). The latter activity has led to the proposal that these proteins may participate in M. tuberculosis infection.

Two of the group I proteins are homologous to factors that participate in the virulence of other bacterial pathogens. PepA is homologous to the DegP periplasmic protease (58). In Salmonella typhimurium, Yersinia enterocolitica, and Brucella abortus degP mutants exhibit decreased survival in macrophages and are attenuated (reviewed in reference 46). Since PepA has been shown to be expressed by M. avium subsp. paratuberculosis in infected sheep (14), PepA is likely to be expressed by M. tuberculosis growing in macrophages and to play an important role in facilitating survival. Rv1566c is homologous to the p60 invasion-associated protein of Listeria monocytogenes (37). In L. monocytogenes p60 is a murein hydrolase (65) which also participates in host cell invasion (31). Rv1566c may participate in binding or uptake of M. tuberculosis by host cells. In order to directly test the role of PepA and Rv1566c in the pathogenesis of M. tuberculosis, we have constructed the corresponding M. tuberculosis mutants, and we are testing these mutant strains for virulence defects in the mouse model of tuberculosis (M. Braunstein, P. M. Morin, and W. R. Jacobs, unpublished data).

The remaining two groups of proteins are comprised of FUN proteins, the majority of which have never been studied. Group II includes six FUN proteins which exhibit homology to protein families and/or conserved hypothetical proteins. Perhaps the most interesting proteins are those from group III. The nine FUN proteins in group III seem to be unique to mycobacteria since there are no homologous proteins identified by BLAST analysis at the NCBI. This apparent restriction of group III proteins to mycobacteria suggests that they may contribute to the unique physiology and life style of mycobacteria. For the majority of proteins in this group it remains to be determined whether the proteins are further restricted to slow-growing and pathogenic mycobacteria. Nonetheless, these proteins represent good candidates for proteins that participate in virulence or can serve as diagnostics for M. tuberculosis infection. Of course, additional experiments are required to test these hypotheses.

Since most of the proteins listed in Table Table33 were identified on the basis of a single active phoA fusion, we have clearly not exhausted the potential of the fusion library. However, some of the “early” blue fusions have been identified multiple times, which may indicate that a limited number of exported proteins are expressed at the level required to produce this degree of activity. We are continuing to screen for additional exported proteins. We anticipate that we will be able to identify many of the secreted proteins (~100) present in culture filtrates of M. tuberculosis (3, 50, 56). In conclusion, we have identified a large number of exported proteins of M. tuberculosis and in doing so have greatly increased our knowledge of the proteins localized to the surface and secreted by the bacillus. These proteins include several which have previously been shown to elicit immune responses (antigen 85B, antigen 85A, PhoS1, PepA, 19-kDa lipoprotein, MPT63, and LppX) (10, 14, 28, 34, 41, 44, 54, 61), proteins with homology suggestive of a role in virulence, and many FUN proteins. By using the recovered transposon insertions as allele exchange substrates in M. tuberculosis, we will create M. tuberculosis mutants of exported proteins and test the role of individual exported proteins in virulence and protective immunity.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grant AI26170 to W.R.J. and GM28470 to N.D.F.G. M.B. is a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation.

We gratefully thank R. Lukose and T. R. Weisbrod for DNA sequencing, S. Cole and C. H. King for cosmids and genomic DNA, M. Larsen for critical reading of the manuscript, and M. Goldberg and S. Bardarov for helpful discussions.

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