Rv1818c-encoded PE_PGRS protein of Mycobacterium tuberculosis is surface exposed and influences bacterial cell structure

Authors

  • Giovanni Delogu,

    Corresponding author
    1. Department of Biomedical Sciences, University of Sassari, Viale San Pietro 43/b, 07100 Sassari, Italy.
    Search for more papers by this author
    • Present address: Istituto di Microbiologia, Università Cattolica del Sacro Cuore, Largo F. Vito, 00168 Roma, Italy.

  • Cinzia Pusceddu,

    1. Department of Biomedical Sciences, University of Sassari, Viale San Pietro 43/b, 07100 Sassari, Italy.
    Search for more papers by this author
  • Alessandra Bua,

    1. Department of Biomedical Sciences, University of Sassari, Viale San Pietro 43/b, 07100 Sassari, Italy.
    Search for more papers by this author
  • Giovanni Fadda,

    1. Institute of Microbiology, Catholic University of the Sacred Heart, Rome, Italy.
    Search for more papers by this author
  • Michael J. Brennan,

    1. Laboratory of Mycobacterial Diseases and Cellular Immunology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA.
    Search for more papers by this author
  • Stefania Zanetti

    1. Department of Biomedical Sciences, University of Sassari, Viale San Pietro 43/b, 07100 Sassari, Italy.
    Search for more papers by this author

E-mail gdelogu@rm.unicatt.it; Tel. (+39) 06 3015 4964; Fax (+39) 06 305 1152.

Summary

Identification of the novel PE multigene family was an unexpected finding of the genomic sequencing of Mycobacterium tuberculosis. Presently, the biological role of the PE and PE_PGRS proteins encoded by this unique family of mycobacterial genes remains unknown. In this report, a representative PE_PGRS gene (Rv1818c/PE_PGRS33) was selected to investigate the role of these proteins. Cell fractionation studies and fluorescence analysis of recombinant strains of Mycobacterium smegmatis and M. tuberculosis expressing green fluorescent protein (GFP)-tagged proteins indicated that the Rv1818c gene product localized in the mycobacterial cell wall, mostly at the bacterial cell poles, where it is exposed to the extracellular milieu. Further analysis of this PE_PGRS protein showed that the PE domain is necessary for subcellular localization. In addition, the PGRS domain, but not PE, affects bacterial shape and colony morphology when Rv1818c is overexpressed in M. smegmatis and M. tuberculosis. Taken together, the results indicate that PE_PGRS and PE proteins can be associated with the mycobacterial cell wall and influence cellular structure as well as the formation of mycobacterial colonies. Regulated expression of PE genes could have implications for the survival and pathogenesis of mycobacteria within the human host and in other environmental niches.

Introduction

The complete sequence of the Mycobacterium tuberculosis genome (Cole et al., 1998) has provided exciting new information that is helping to unravel the complex biology of this ancient human pathogen (Domenech et al., 2001). The finding that the genome contains numerous genes belonging to two highly homologous gene families (PE and PPE) represented one of the most interesting outcomes of this genomic endeavour (Cole, 1999). Almost 100 genes belonging to the PE protein family were detected scattered throughout the genome of M. tuberculosis strain H37Rv and CDC1551 (Fleischmann et al., 2002). Of these genes, 38 code for highly homologous proteins with an average length of 110 amino acids in M. tuberculosis H37Rv (PE genes). The remaining PE_PGRS genes encode more complex proteins in which the PE domain is fused at the C-terminus with sequences varying in size and containing a domain rich in the amino acids alanine and glycine (PGRS domain) (Brennan and Delogu, 2002). Overall, the striking redundancy of these proteins in M. tuberculosis and the occurrence of related proteins in other mycobacterial species suggest that they may play an important role in the biology of M. tuberculosis and other pathogenic mycobacteria. However, the function of this large family of proteins remains unclear.

A few studies have provided some insights into the role of the PE_PGRS proteins. Ramakrishnan et al. (2000) showed that Mycobacterium marinum expresses a homologue of M. tuberculosis PE_PGRS protein Rv1651c in granulomatous tissues, suggesting that PE_PGRS proteins are involved in the chronic stages of mycobacterial infections. Expression of PE_PGRS proteins during infection was demonstrated in the mouse model of pulmonary tuberculosis (Delogu and Brennan, 2001) and in clinical studies involving patients infected with M. tuberculosis (Espitia et al., 1999). Results obtained using a BCG strain, in which the Rv1818c gene was inactivated by transposon mutagenesis, provided evidence that the gene is expressed in axenic culture and that at least some PE_PGRS proteins may be exposed on the surface of mycobacteria (Brennan et al., 2001). Banu et al. (2002) demonstrated that a number of PE_PGRS genes are expressed in culture and provided further evidence that these proteins are localized on the surface of mycobacteria. Recently, in a work based on statistical prediction, Lamichhane et al. (2003) suggested that the PE_PGRS gene family is enriched in genes with a high probability of being essential in M. tuberculosis.

Although these data strongly support a role for the PE_PGRS proteins in the biology and possibly pathogenesis of M. tuberculosis, understanding the function of PE_PGRS and PE proteins will require extensive investigations. However, experimental analysis of proteins belonging to the PE family presents a technical challenge. The redundancy of genes and their expression products makes it difficult to develop specific molecular and immunological tools to study individual PE and PE_PGRS proteins. A case in point is the cross-reactivity of antibodies raised against specific PE_PGRS proteins (Brennan et al., 2001; Banu et al., 2002). In this study, we have implemented a molecular approach to investigate PE_PGRS proteins. Using a prototype PE_PGRS gene, Rv1818c, a set of in frame derivatives was overexpressed in M. tuberculosis and M. smegmatis to localize the protein within the bacterial cell and to identify functional domains.

Results

Changes in colony morphology are associated with the overexpression of a PE_PGRS protein

The Rv1818c gene was cloned in frame with the hsp60 constitutive promoter in plasmid pMV206. In order to define the functions of the PE and PGRS domains, we also cloned a 5′ PE domain found in the Rv1818c gene sequence (Fig. 1). Expression of Rv1818c in M. smegmatis and M. tuberculosis strains was confirmed by immunoblot analysis using an anti-Rv1818c antibody (data not shown) (Brennan et al., 2001). Colonies of M. smegmatis and M. tuberculosis transformed with pMV1818cPE_PGRS appeared to be slightly smaller in size than controls, while colonies of pMV1818cPE appeared to be significantly larger than controls (Fig. 2A). Major changes in colony morphology were also observed by microscopy when the two mycobacterial species were transformed with the plasmids encoding Rv1818c, or its PE domain, and cultured on solid media (Supplementary material, Fig.S1).

Figure 1.

Schematic representation of the mycobacterial constructs used in this study. aThe estimated molecular weight (MW) for each protein is indicated in kDa.

Figure 2.

Effect on colony morphology resulting from overexpression of the 1818cPE_PGRS, 1818cPE. Diagram showing the colony diameter in M. smegmatis and M. tuberculosis grown on solid media for 7 and 30 days respectively. At least 12 colonies per group were measured in a single representative experiment.

The recombinant strains, for both M. tuberculosis and M. smegmatis, showed the same growth rate in liquid cultures as determined by colony-forming unit (cfu) counting (data not shown). A construct composed of the mtb39A gene, encoding a protein belonging to the PPE family (Dillon et al., 1999), fused to the PE domain of Rv1818c showed no differences in colony morphology compared with controls (data not shown). This indicates that the effect on colony morphology is associated with the PGRS domain of the Rv1818c protein (data not shown). These data clearly indicate that expression of high levels of Rv1818c has a significant effect on colony morphology without affecting bacteria multiplication. Interestingly, although overexpression of the PE domain favours the spreading of the colony on solid media without affecting colony morphology, changes in colony phenotype are associated with the overexpression of the PGRS domain of Rv1818c.

Overexpression of Rv1818c protein affects mycobacterial cell structure

Most of the phenotypic changes in colony morphology in mycobacteria, as well as in other bacteria, correspond to biochemical or physiological modifications that effect the cell wall or plasmatic membrane (Glickman et al., 2000; Pym et al., 2002). We therefore investigated whether the overexpression of Rv1818c and its PE domain had any significant effect on cellular structure. Scanning electron microscopy (SEM) of M. tuberculosis transformants overexpressing 1818cPE_PGRS revealed a striking difference in cell size, with the pMV1818cPE_PGRS transformants ranging from 6 to 10 µm in length (Fig. 3). In comparison, control and 1818cPE transformants showed the typical length for the mycobacterial cell of 1.5–2 µm (Supplementary material, Fig.S2). The differences in cell structure were also confirmed in experiments in which the transformants were grown in liquid media and analysed by SEM (Supplementary material, Fig.S2). Taken together, these results indicate that overexpression of the whole Rv1818c gene has profound effects on bacterial and colony architecture. The lack of any significant effect of PE expression on mycobacterial cell structure suggests that the differences observed are correlated with the presence of the glycine/alanine-rich PGRS domain.

Figure 3.

Scanning electron microscopic analyses of M. tuberculosis pMV1818cPE_PGRS recombinant strain. Recombinant strain overexpressing Rv1818c gene was grown in solid media for up to 26 days, then fixed and processed for SEM analysis. Magnification is 10 000× for and (B) and 5000× for (C) and (D). Bar is shown at bottom left (2 µm).

Localization of Rv1818c in the mycobacterial cell

Ultrastructural modifications observed in Rv1818c-overexpressing bacteria suggest that the Rv1818c protein localizes in the mycobacterial cell wall. In order to evaluate this hypothesis, we tagged the Rv1818c and its PE domain with the enhanced green fluorescent protein (EGFP) as indicated in Fig. 1. To investigate the localization of the PE_PGRS protein in the mycobacterial cells, the M. smegmatis and M. tuberculosis recombinant GFP strains were analysed by fluorescent microscopy. As indicated in Fig. 4A, most of the M. tuberculosis pMV1818cPE_PGRS–GFP transformants showed an intense fluorescence localized to one or both poles of the cell. In some cases, it was also possible to detect cells in which this bean-shaped intense fluorescence was localized to the centre of the cells (Fig. 4B). The M. tuberculosis pMV1818cPE–GFP transformant also demonstrated the deposition of protein at polar regions of the bacteria, but the fluorescence was less intense (Fig. 4C). M. tuberculosis transformants expressing only the GFP protein showed a uniform and intense fluorescence, without any sign of compartmentalization within mycobacterial cells (Fig. 4D). Similar results were obtained with the M. smegmatis GFP-tagged recombinant strains (data not shown), suggesting that the proteins under study follow a similar cellular localization in this fast-growing mycobacteria that does not naturally express Rv1818c.

Figure 4.

Fluorescence analysis of the M. tuberculosis transformants grown in liquid media and then analysed by fluorescence microscopy at 1000×. (B–D) were further enlarged using a digitalized form as indicated in Experimental procedures.
A and B. pMV1818cPE_PGRS–GFP.
C. pMV1818cPE–GFP.
D. pMVGFP.

To investigate the subcellular localization of the protein under study, the recombinant M. smegmatis strains expressing the tagged proteins were subjected to cell fractionation studies and analysed by immunoblot using anti-GFP antibodies. Analysis of the whole-cell lysates of these M. smegmatis strains indicated that the GFP and GFP fusion proteins migrated according to the expected mass for each protein (Fig. 5A). The majority of the 1818cPE_PGRS–GFP, 1818cPE–GFP fusion proteins were detected in the insoluble fraction of the cell preparation, and very little protein was detected in the soluble fraction (Fig. 5B and C). Conversely, most of the GFP protein expressed by the pMVGFP recombinant strain was detected in the soluble fraction of the cell lysates (Fig. 5C). These results suggest that Rv1818c is associated with the mycobacteria cell wall fraction and that the PE domain is sufficient to target the PE_PGRS protein to this cellular compartment.

Figure 5.

Immunoblot analysis carried out on the M. smegmatis recombinant strains using an anti-GFP antibody. Samples prepared as indicated in Experimental procedures were analysed using 12% SDS–PAGE.
A. Whole-cell lysates (WCL).
B. Insoluble fractions (pellet).
C. Soluble fraction (SUP). 1, pMV18; 2, pMVGFP; 3, pMV1818cPE–GFP; 4, pMV1818cPE_PGRS–GFP. Arrows indicate the overexpressed proteins.

To determine whether the cell wall-associated Rv 1818c protein is exposed on the mycobacterial surface, we performed a trypsin sensitivity assay (Uzzau et al., 1999) on selected recombinant M. smegmatis strains. As shown in Fig. 6A, the Rv1818c protein overexpressed by M. smegmatis (strain pMV1818cPE_PGRS) was rapidly digested by the trypsin treatment, with the reaction reaching completion after 5 min. A similar pattern was obtained when the M. smegmatis pMV1818cPE_PGRS–GFP and pMV1818cPE–GFP recombinant strains were subjected to the same assay and analysed in immunoblot using the anti-GFP antibody (data not shown). Conversely, the recombinant M. smegmatis strain expressing only GFP (pMVGFP) was protected from trypsin digestion (Fig. 6B). These results confirm that Rv1818c protein is exposed at the mycobacterial cell surface. Taken together, these results suggest that the Rv1818c PE_PGRS gene product localizes in the mycobacterial cell wall, mostly at the bacterial cell poles, where it is exposed to the extracellular milieu.

Figure 6.

Trypsin sensitivity assay performed on selected M. smegmatis recombinant strains. Live bacteria were incubated with (+) or without (–) trypsin at different time points, and the whole-cell lysates obtained from the M. smegmatis pMV1818cPE_PGRS (A) and pMVGFP (B) were run in 12% SDS–PAGE. Immunoblots were performed using anti-1818cPE_PGRS (A) and anti-GFP (B) antibodies.

Discussion

The investigation of the role of the PE family of proteins in the biology of mycobacteria is a significant challenge because of the large number of highly homologous genes dispersed throughout the genome. Traditional strategies used to assess protein functions such as the generation of deletion mutants and the development of specific antibodies are more difficult to implement in this large closely related family of proteins. To overcome this problem, one approach has been to overexpress genes of interest in order to assess the impact of protein expression on cell structure and function. Such an approach has also been used to investigate the role of proteins in the genome of species closely related to mycobacteria such as Corynebacterium glutamicum (Puech et al., 2000). In the case of mycobacteria, protein overexpression has been achieved by cloning the gene under the control of a strong and constitutive promoter, such as hsp60, in a multicopy plasmid (Stover et al., 1991). Using this strategy, expression constructs have been used to define the relative contribution of gene products involved in isoniazid and ethionamide detoxification (Larsen et al., 2002) and in lipoarabinomannan biosynthesis (Schaeffer et al., 1999). We have developed a system for the overexpression of a prototype PE_PGRS gene (Rv1818c) to examine the role of a PE_PGRS protein in the biology of the mycobacterial cell.

Rv1818c was selected as a prototype because, in previous studies, it has been shown that it is expressed by M. tuberculosis during infection, and it is representative of a typical protein encoded by members of the PE_PGRS family (Brennan et al., 2001; Delogu and Brennan, 2001; Banu et al., 2002). Most PE_PGRS proteins contain two domains, PE and PGRS, that appear, in some cases, to be joined by a putative transmembrane region and, in some of them, an extra domain is present at the C-terminus that can vary in size and sequence within the family (Brennan and Delogu, 2002). The PE domain is about 100 amino acids in length and is highly conserved within the PE family of proteins (Cole et al., 1998). It remains to be determined whether all or some of the ≈ 38 genes belonging to the PE family are expressed, how they are regulated and what is their function. In order to understand the role of the PE domain and to examine the role of the PGRS domain, we also cloned and expressed the gene fragment of Rv1818c encoding the first 140 amino acids of Rv1818c, which contains the PE domain and the putative transmembrane region (Brennan and Delogu, 2002).

Expression of the Rv1818c gene resulted in a significant modification of the colony morphology phenotype in both M. smegmatis and M. tuberculosis. In M. smegmatis, these changes were dramatic, probably because M. smegmatis does not naturally expresses any PE genes, as indicated by a search of the genome. Colonies of M. smegmatis and M. tuberculosis containing pMV1818cPE_PGRS showed a smooth morphology and a cellular organization different from that of bacteria containing the vector only. Analysis of the M. smegmatis and M. tuberculosis strains transformed with the plasmid pMV1818cPE indicates that overexpression of the PE domain of Rv1818c has a minor effect on colony morphology. In both mycobacterial species, the pMV1818cPE recombinant strains form colonies with a larger diameter than controls. As the recombinant strains pMV18, pMV1818cPE and pMV1818cPE_PGRS all show the same growth curve in liquid media (data not shown), the larger colonies are not the result of an increased growth rate resulting from the PE overexpression.

Colony morphology in mycobacteria is commonly affected by bacterial cell surface components involved in cell wall structure or biosynthesis. For instance, in Mycobacterium avium, it has been demonstrated that diminished expression of glycopeptidolipids (GPL), a structural component of the mycobacterial cell wall, results in a switch from a smooth to a rough colony. Morphological phenotypes have also been associated with differences in the virulence of mycobacterial strains (Belisle and Brennan, 1989; 1994; Reddy et al., 1996). In M. tuberculosis, changes in colony morphology have been observed in mutants lacking genes specifically involved in the biosynthesis of mycolic acids (Glickman et al., 2000). Also, it has been demonstrated recently that the complementation of a BCG strain with a fragment of the RD1 locus containing Esat-6, as well as other antigens including a PPE protein, results in a strain more virulent than the parent and affects colony morphology (Pym et al., 2002). Our data show that the overexpression in M. smegmatis and M. tuberculosis of PE_PGRS Rv1818c, which is found in the cell wall of mycobacteria, impacts on colony morphology. To date, we have no evidence to indicate whether overexpression of the recombinant proteins influences virulence.

Scanning electron microscopic analyses of colonies of M. tuberculosis pMV1818cPE_PGRS indicated that overexpression of Rv1818c results in bacterial cells significantly elongated compared with control cells, while analyses of the M. tuberculosis pMV1818cPE cells did not show significant cellular differences compared with controls. These results suggest that the presence of the glycine/alanine-rich PGRS domain of Rv1818c is associated with these changes in cellular architecture. It could be hypothesized that the PE_PGRS proteins may play a structural role, analogous to the glycine-rich proteins found in plant cell walls or in spider silk elastic structures (Ye et al., 1991; Hayashi and Lewis, 2000; Brennan and Delogu, 2002). Accumulation of PE_PGRS protein after overexpression of Rv1818c may interfere with cell wall organization and result in the observable phenotypical changes.

Cell fractionation studies provide further evidence that, although the GFP protein itself is detected in the cellular soluble fraction, and therefore mostly in the cytoplasm, the PE and PE_PGRS GFP-tagged proteins are detected mostly in the insoluble fraction and therefore are likely to be membrane- or cell wall-associated components. Trypsin sensitivity assays (Uzzau et al., 1999) performed on live recombinant mycobacterial strains demonstrated that the gene product encoded by the Rv1818c gene is exposed on the surface of mycobacteria. These results provide further evidence that the PE_PGRS proteins are present on the surface of mycobacteria and may be available for interaction with host components (Espitia et al., 1999; Brennan et al., 2001; Banu et al., 2002).

Fusion with GFP has been used extensively to study protein localization in bacteria (Lin et al., 1997; Kim and Wang, 1998; Chen et al., 1999; Ghigo et al., 1999). Fluorescence micrographs of the M. tuberculosis pMV1818cPE_PGRS–GFP recombinant strain indicates that the fusion protein 1818cPE_PGRS–GFP localizes mostly at the cell poles or in compartments at the centre of the cells forming bean-shaped aggregates. Similar to what has been observed in other bacterial species, cells expressing simply GFP showed a uniform distribution of the fluorescent protein, indicating that the observed localization of 1818cPE_PGRS–GFP reflects a property of the Rv 1818c protein and is not influenced by GFP (Dhandayuthapani et al., 1995; Kim and Wang, 1998). Fluorescence analysis of the M. tuberculosis 1818cPE–GFP strain indicates that the PE protein, which in this case also contains the putative transmembrane region but no glycine–alanine repeats, is compartmentalized much like the PE_PGRS protein, although there is no indication of centralized accumulation. Therefore, the PE domain may contain the information sufficient to transport PE_PGRS proteins to their correct cellular location. Protein localization at cell poles has been observed for the chromosome partition protein Spo0J of Bacillus subtilis (Lin et al., 1997), for the proteins FtsL and FtsQ involved in the cell division process (Chen et al., 1999; Ghigo et al., 1999) and for the SopB protein involved in the partition of single-copy plasmid F (Kim and Wang, 1998). These proteins localize and accumulate regularly at the poles or other sites at specific steps of the cell cycle. Of interest also is the fact that the growth zones in mycobacterial morphogenesis follow a similar pattern (Daniel and Errington, 2003). It is interesting that the Rv 1818c protein localizes in the mycobacterial cell wall following a similar pattern, and it would be of great interest to investigate whether the PE_PGRS proteins are directly involved in such basic processes in mycobacterial biology.

Our results, together with recent data predicting that the PE_PGRS gene family is enriched in essential genes in M. tuberculosis (Lamichhane et al., 2003), provide new insights in understanding the biological role of this unique protein family, and may even open a new avenue for antimycobacterial drug and vaccine development. Further studies are required in order to understand better the significance of cellular elongation and subcellular localization. For instance, fusion of the PE proteins with shorter peptides such as the HA or 3Xflag epitopes (Uzzau et al., 2001) may provide a better system to define the localization of the protein within the mycobacterial cell using immunochemical methods. Moreover, the characterization of the expression level for some of these genes through the use of gene reporter technologies (Tyagi et al., 2000) may offer important information on how these genes are regulated and provide better genetic systems that could reduce any bias resulting from overexpression of the protein.

The elucidation of the role and function of the PE/PE_PGRS proteins in M. tuberculosis will be an important step towards a better understanding of the biology of one of the most successful human pathogens. In this study, we provide experimental data that localize PE_PGRS proteins to the mycobacterial cell wall and show that expression of PE_PGRS protein can affect cellular structure. The results suggest that the sequences within the PE domain may be important in translocating the PE-PGRS protein to the cell wall where these proteins may function as structural proteins, in antigenic variation or in bacteria–host interactions as has been suggested previously (Banu et al., 2002; Brennan and Delogu, 2002).

Experimental procedures

Construction of vectors and recombinants

The Rv1818c gene of M. tuberculosis H37Rv was amplified and cloned in the pMV206 vector as indicated previously to obtain the pMV1-23 (pMV1818cPE–PGRS) plasmid (Brennan et al., 2001). Briefly, the 1500 bp fragment containing the Rv1818c gene was inserted in frame with the 390 bp fragment containing the hsp60 promoter region in the pMV206 mycobacteria shuttle plasmid. The amino-terminal fragment containing the PE region of the protein was amplified using the forward primer 5′-ACGTCCATGGGCTCATTTGTGGTC ACGATCCCGGAG-3′ containing an NcoI site, and the reverse primer 5′-ACGGATCCCTAGTTGCCGATCAAGATTC CGCCGTC-3′ containing a BamHI site and a stop codon, to amplify a fragment of 423 bp, coding for the first 140 amino acids of the Rv1818c gene. The gene fragment was amplified from M. tuberculosis H37Rv DNA using the Vent polymerase (New England Biolabs) and cloned in the pCRBlunt vector (Invitrogen). The NcoI–BamHI fragment was then inserted in the pMV18 vector (Brennan et al., 2001) to make the pMV1818cPE plasmid. The two fragments of Rv1818c were also amplified using a reverse primer lacking a stop codon and with an XbaI adaptor using similar procedures. The gene coding for GFP was amplified from the plasmid pEGFP-C1 (Clontech) using the forward primer 5′-ACGCTAGCATGGT GAGCAAGGGCGAGGAGCTG-3′ and the reverse primer 5′-ACGGATCCTTATCTAGATCCGGTGGATCC-3′ containing an NheI and a BamHI site, respectively, and cloned in pCRBlunt using similar methods to those described above. The GFP gene was fused to the two Rv1818c gene fragments using the XbaI–NheI compatible ends to make plasmids pMV1818cPE–GFP and pMV1818cPE_PGRS–GFP expressing the PE domain or the intact Rv1818c fused to the GFP protein. The mtb39A gene, encoding for a PPE protein (Dillon et al., 1999), which has been amplified and cloned previously (Delogu et al., 2002), was fused to the fragment coding for the PE domain exploiting the XbaI–NheI compatibility to make the plasmid pMV1818cPE–PPE.

Microorganisms, media and transformation

Mycobacterium tuberculosis H37Rv and M. smegmatis mc2155 were grown in liquid media 7H9 supplemented with albumin–dextrose–catalase enrichment (Microbiol) and Tween 80 (0.05%) until an OD600 of 0.6–0.9 was reached. Cell preparation and electroporation were carried out using standard protocols (Bardarov et al., 1997). Transformants were selected in 7H11 solid media supplemented with oleic acid–albumin–dextrose–catalase (Microbiol) and containing 50 µg ml−1 hygromycin (Sigma-Aldrich).

Fluorescent analysis

Mycobacterium smegmatis and M. tuberculosis transformants were grown in liquid media in 24-well plates containing glass coverslips previously treated with poly-lysine. After incubation, supernatants were gently removed, and cells were fixed with a 4% paraformaldehyde solution. Centrifugation was carried out to improve attachment of the bacteria to glass coverslips. Samples were washed with phosphate-buffered saline (PBS), then with PBS containing 0.5% bovine serum albumin (BSA), followed by PBS, and then mounted on slides using FluoromountG (Electron Microscopy Sciences). Slides were analysed on an Olympus BX51 microscope, and pictures were captured using the Optronics Magnafire camera (model S99802) with Olympus dp-soft software (version 3.2).

Electron microscopy

Mycobacterium smegmatis transformants were grown in solid media for 7 days, while M. tuberculosis transformants were grown in solid media for 30 days. Fragments of solid media containing the selected colonies were cut and fixed with a glutaraldehyde solution (2.5%) in PBS for 2–24 h. For liquid cultures, bacteria adhering to coverslips were fixed with the glutaraldehyde solution. Samples were washed three times with cacodylate buffer and then fixed with osmium tetroxide solution (1%) in PBS for 1.5 h. Samples were washed three times with cacodylate buffer, three times with distilled water and then dehydrated. Samples were then covered with gold and analysed in a scanning electron microscope Zeiss SM 962.

Cell fractionation and immunoblot analysis

Mycobacterial strains were grown in Sauton's media containing hygromycin at 50 µg ml−1 up to the late log phase. Cells were centrifuged and washed once with cold PBS. To prepare the whole-cell lysate (WCL), a fraction of the pellet was fully resuspended in deionized water, vortexed, and then an equal volume of 2× sample buffer was added and samples were boiled for 10 min. An equal amount of cell pellet was resuspended by vortexing in PBS–Tween 20 containing the protease inhibitor P-8849 (Sigma-Aldrich) and subjected to five cycles of sonication. Centrifugation was carried out at 12 000 g to pellet the insoluble fraction, and supernatants were transferred to fresh tubes. The pellet was treated as indicated above for the WCL.

Samples were subjected to SDS–PAGE, transferred on nitrocellulose membranes (Invitrogen) and detected using anti-GFP antibody (Zymed Laboratories) or anti-Rv1818c antibody (Brennan et al., 2001) as primary antibodies and horseradish peroxidase-conjugated or alkaline phosphatase-conjugated anti-mouse IgG (Sigma-Aldrich).

Trypsin sensitivity assay

Limited protease digestion was performed as described previously (Fukuda et al., 1995; Uzzau et al., 1999). Briefly, 20 ml bacterial cultures were harvested at the late log phase, centrifuged and washed twice with cold TBS buffer (Tris-HCl, pH 7.2, NaCl 150 mM, KCl 3 mM), then resuspended in 1.5 ml of TBS. A sample of 200 µl of this suspension was incubated with trypsin (Difco) to a final concentration of 100 µg ml−1 on ice for 5–120 min. The trypsin digestion was terminated by adding soybean trypsin inhibitor (Sigma-Aldrich) to a final concentration of 200 µg ml−1. Samples were then centrifuged, and the pellet was washed three times in TBS, dissolved in SDS sample buffer and analysed in immunoblot as described above.

Acknowledgements

We would like to thank Salvatore Marceddu and Domenico Delogu for technical assistance with the electron microscopy studies, and Sergio Uzzau for advice with the trypsin sensitivity assay and for reviewing the manuscript. This work was supported by grant MM06248818-004 from the Italian MIUR and by a grant from the US National Vaccine Program Office (M.J.B.).

Supplementary material

The following material is available from http://www.blackwellpublishing.com/products/journals/suppmat/mmi/mmi4007/mmi4007sm.htm

Fig.S1.  Pictures showing colonies of M. smegmatis (A–C) and M. tuberculosis (D–F) transformants grown on solid media for 7 and 30 days, respectively, and transformed with the plasmid pMV18 (A and D), pMV1818cPE_PGRS (B and E) and pMV1818cPE (C and F).

Fig.S2.  Scanning electron microscopic analyses of M. tuberculosis transformants.

Ancillary