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Infection and Immunity, April 2004, p. 2379-2382, Vol. 72, No. 4
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.4.2379-2382.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

The Hydrophobic Domain of the Mycobacterial Erp Protein Is Not Essential for the Virulence of Mycobacterium tuberculosis

Dana Kocíncová, Berit Sondén, Yann Bordat, Elisabeth Pivert, Leila de Mendonça-Lima,{dagger} Brigitte Gicquel, and Jean-Marc Reyrat*{ddagger}

Unité de Génétique Mycobactérienne, Institut Pasteur, Paris Cedex 15, F-75724, France

Received 26 September 2003/ Returned for modification 18 November 2003/ Accepted 1 December 2003


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ABSTRACT
 
Erp (exported repetitive protein) is a member of a mycobacterium-specific family of extracellular proteins. A hydrophobic region that is localized at the C-terminal domain and that represents a quarter of the protein is highly conserved across species. Here we show that this hydrophobic region is not essential for restoring the virulence and tissue damage of an erp::aph mutant strain of M. tuberculosis as assessed by bacterial counts and lung histology analysis in a mouse model of tuberculosis.


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TEXT
 
The genus Mycobacterium is very large and encompasses intra- and extracellular pathogens and environmental opportunistic pathogens as well as saprophytes (20). Mycobacterium tuberculosis, a facultative intracellular pathogen that persists within professional phagocytes, is responsible for more than 1.5 millions deaths per year (WHO information tuberculosis fact sheet, 2002; World Health Organization [http://www.who.int/mediacentre/factsheets/who104/en/index.html]). The situation is worsened by the AIDS epidemic and the deteriorating socioeconomic conditions of increasing numbers of people. Moreover, the recent emergence of multidrug-resistant strains, together with the relative inefficacy of the M. tuberculosis BCG vaccine, has aggravated the situation.

The development and the utilization of classical bacterial genetics in M. tuberculosis (19) have enabled the characterization of virulence factors (5, 8; for a review, see reference 11). To date, most of the characterized virulence factors have been products that are involved in the structure and function of the cell wall, a particularity of the Corynebacteria-Mycobacteria-Nocardia group of Actinomycetales (9, 20). The erp gene of M. tuberculosis has been characterized before completion of the genome sequence through the use of the phoA fusion method that helps to entrap genes coding for exported and secreted products (2, 15). Erp has been shown to be a crucial virulence factor. Indeed, disruption of the erp gene by insertion of an antibiotic element results in a marked decrease in virulence, with lower levels of survival and multiplication both in vitro in cell culture assays and in vivo in the mouse model of infection (3). The erp gene was later shown to be present not only in pathogenic mycobacteria but also in saprophytic and opportunistic pathogenic mycobacteria (17). However, no homologue has been found in other bacterial species, making Erp a mycobacterial signature.

The Erp protein has a composite structure made of three domains. Both the amino (1 to 80)- and carboxy (176 to 284)-terminal domains are conserved, while the central domain is subject to high-level interspecies variability. It was recently shown that the nature of the erp allele strongly affects the number and the size of the lung lesions of infected animals (18). None of the three domains exhibit homology to other characterized proteins. The central domain consists of tandem repeats of five amino acids based on a PGLTS motif (variable both in the number and quality of repeats between mycobacterial species). The central regions are identical within members of the tuberculosis (TB) complex and are not subject to allelic variation among M. tuberculosis isolates (3, 17). The two other domains (amino and carboxy terminal) are highly conserved between species. The amino-terminal domain contains a canonical signal sequence likely involved in its sec-dependent secretion, and the site of cleavage is predicted to be localized at amino acid 23 (2). The carboxy-terminal contains an hydrophobic region extended by a stretch rich in proline and alanine (17). Interestingly, this hydrophobic region is conserved in M. leprae, in which a dramatic proteome reduction has been observed (7). It has been hypothesized that this hydrophobic region might anchor the Erp protein at the surface of the bacillus and hence that it might play a role in the function of the protein (2, 3). However, the Erp protein has been shown to traffic into subcellular compartments of infected cells (3).

In this study, by expressing a truncated Erp protein in M. tuberculosis we have investigated the role of the most hydrophobic region of the carboxy-terminal domain in the virulence of the tubercle bacillus.

Construction of the C-terminal deletion mutant. In the course of a previous study, a spontaneous mutant lacking the most hydrophobic region of the carboxy-terminal domain was serendipitously obtained (Fig. 1A). Indeed, construct pML101, containing the M. tuberculosis erp::aph gene (lacking its C-terminal portion) in pNIP40b, was generated by amplifying M. tuberculosis genomic DNA with oligonucleotide primers erp-C1 and erp-C2 (18). Purified PCR product digested with XbaI was ligated into the XbaI site of dephosphorylated pNIP40b (16). Sequencing of pML101 revealed the deletion of an A residue at position 631, leading to a frameshift mutation and a premature stop codon. The corresponding protein lacks the last 74 amino acids, corresponding to a molecular mass of approximately 8 kDa. The M. tuberculosis erp::aph mutant strain (3) was transformed by either pML101 or pML10, a vector carrying the full-length erp gene (3), and selected on Middlebrook 7H11 (Difco) agar plates containing kanamycin (20 µg ml-1) and hygromycin (50 µg ml-1) at 37°C; these complemented strains are named M. tuberculosis erp::aph/TB{Delta}C and M. tuberculosis erp::aph/TB, respectively.



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  		FIG. 1. (A) Kyte-Doolittle plot of the of the erp gene product of M. tuberculosis. The deletion is indicated by an arrow. All data respecting protein features and sequences, together with the protein alignment, can be found at http://mic.sgmjournals.org/cgi/content/full/147/8/2315/DC1. (B) Denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblot, showing the TB-Erp and TB-Erp{Delta}C protein present in the supernatant fraction of the M. tuberculosis erp::aph/TB and M. tuberculosis erp::aph/TB{Delta}C strains. rErp is the purified recombinant TB-Erp (30 ng) and was used as an internal control.

These strains were grown in Middlebrook 7H9 medium (Difco) supplemented with 0.05% (vol/vol) Tween 80 and 10% ADC (Difco). Antibiotics were added as required. To quantify the expression of the complemented strains, 50 µg of total proteins from supernatant fraction was probed using an anti-TB Erp antiserum. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 15% polyacrylamide gels (14) and transferred to Hybond P membranes (Amersham Pharmacia). A rabbit TB-Erp antiserum (3) was used as first antibody at a 1:5,000 dilution, and bound antibodies were revealed as previously described (18). No cross-reacting band was observed in the M. tuberculosis erp::aph mutant, whereas a clear signal of a similar intensity was observed in the case of M. tuberculosis erp::aph/TB and M. tuberculosis erp::aph/TB{Delta}C, demonstrating that the ErpTB{Delta}C protein is expressed stably and at a similar level (Fig. 1B). The reduction in size (8 kDa) was in agreement with the calculated mass of ErpTB{Delta}C.

In vivo analysis. The erp::aph mutant of M. tuberculosis is very severely hampered with respect to multiplication in vivo (3). Indeed, infection with the erp mutant of M. tuberculosis leads to a less that 1% bacterial M. tuberculosis erp::aph/TB load in the lung after long periods of time (3, 18). The virulence of the complemented M. tuberculosis strains was assayed using the mouse model of tuberculosis.

Mice (n = 5) were infected intravenously with 105 CFU of either the erp::aph mutant or complemented strains in a total volume of 0.5 ml as previously described (12) and maintained in specific pathogen-free containment. Mice were sacrificed 1, 21, 42, or 80 days after infection. The course of the infection was monitored over time by homogenizing the lung and spleen and counting viable bacilli, as described elsewhere (6). On day 1, similar numbers of CFU from the spleen and for the lung were obtained for all strains, confirming that the inoculum sizes did not differ between the various complemented strains. As reported previously, the M. tuberculosis erp::aph mutant was severely attenuated, leading to a bacterial M. tuberculosis erp::aph/TB load in the spleen of about 4% and a load in the lung of 0.6% on day 42 (3). On days 21, 42, and 80, the bacterial loads observed in the spleen and in the lung for M. tuberculosis strains erp::aph/TB and M. tuberculosis erp::aph/TB{Delta}C did not differ significantly, demonstrating that these alleles had similar functional roles in this context (Fig. 2A).




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  		FIG. 2. (A) Total CFU counts of M. tuberculosis erp::aph, M. tuberculosis erp::aph/TB, and M. tuberculosis erp::aph/TB{Delta}C mutantstrains in the spleens and lungs of infected animals at various time points. Data are expressed as the log10 value of the mean number of bacteria ± standard deviation recovered from each mouse (n = 5). Two independent experiments were carried out. (B) Representative photomicrograph of lung tissue sections harvested on day 80 from mice infected with the M. tuberculosis erp::aph/TB (panel 1), M. tuberculosis erp::aph (panel 2), or M. tuberculosis erp::aph/TB{Delta}C (panel 3) mutant strain and from naïve mice (panel 4). Arrows, cellular recruitment and infiltration; stars, foamy macrophage; a, alveolus; v, blood vessel. All plates are shown at x100 magnification. The qualitative histological analysis was repeated twice independently.

Histological analysis. As CFU counts and histology analysis can sometimes produce discrepant results (13, 18), a histology analysis was undertaken to evaluate the damage caused by these three strains. The right superior, median, and inferior pulmonary lobes of the right lungs of three additional mice were embedded in paraffin, stained with hematoxylin and eosin, and examined under a light microscope as previously described (21). Sections were evaluated by three individuals, including a veterinary pathologist blinded to the time point and strain considered. On day 42 (data not shown), both infiltrations (which consist of lymphocytes combined with cells derived from the phagocyte mononuclear system that invade the lung tissue and that are not organized in a granuloma) and granulomas with foamy macrophages were observed in the lungs of mice infected with M. tuberculosis erp::aph/TB. In mice infected with the M. tuberculosis erp::aph mutant, little perivascular and peribronchial infiltration was observed. M. tuberculosis erp::aph/TB{Delta}C infection resulted in damage compared to the results seen with M. tuberculosis erp::aph/TB infection, with both infiltration and granulomas with foamy macrophages appearing. The situation was similar on day 80 (Fig. 2B). These data demonstrate that the C-terminal domain of the Erp protein is not necessary for either organ multiplication or persistence or for induction of lung damage.

Despite intensive efforts such as genome sequencing and genetic tool development, the mechanism of virulence of the tubercle bacilli remains poorly characterized (11, 20). Some virulence factors have been characterized through the use of signature tag mutagenesis (5, 8), and several studies concerning the virulence factors of M. tuberculosis pinpointed the cell wall as a major player in this process. The surface of mycobacteria is a very complex structure composed of proteins, sugars, and lipids, some of which have been shown to play a role in virulence, as shown by studies of the fbpA gene, which encodes a mycoloyl transferase (1), the pcaA gene, which encodes a protein involved in mycolic acid cyclopropane ring synthesis (10), and the phthiocerol dimycocerosate locus, which is involved in the synthesis of lipid of the cell envelope (6).

In similarity to the results seen with gram-negative bacteria, this outer membrane contains porins and delineates a pseudo periplasm (4). Erp, which is specific to mycobacteria, is one of the numerous actors for the cell wall and likely plays an important function in its structure. Both the amino- and the carboxy-terminal domains are conserved between species, suggesting an important role for these domains.

In this study, we have shown that the most hydrophobic part of the carboxy-terminal domain is not essential for animal infection. Interestingly, this carboxy-terminal domain has been conserved through evolution even in M. leprae, in which a dramatic proteome reduction has been observed (7). It is remarkable that a region that represents more than one-fourth of the total protein length and that has been conserved through speciation appears to be dispensable for the virulence property of the tubercle bacillus. One may speculate that this region interacts with some uncharacterized cell wall products required in some situations other than virulence, such as desiccation or long-time persistence. Another hypothesis is that this hydrophobic domain is required in other mycobacterial species and that it is a relic of evolution in M. tuberculosis. Our current view is that Erp is a protein present at the surface of the bacillus. The most important domains are clearly the amino-terminal domain, which is required for secretion, and the central region, which influences disease outcome (2, 18). We expect that it will be an interesting challenge to characterize the molecular function of this surface protein.


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ACKNOWLEDGMENTS
 
D.K. is funded by the Marie Curie Host Programme (HPMT-2000-00058). B.S. is funded by the E.U. (QLK2CT-2000-52077). L.M.-L. is funded by the Oswaldo Cruz Foundation (Fiocruz).

V. Pelicic is greatly acknowledged for critical improvement of the manuscript. J.-M.R. is chargé de recherche at Inserm.


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FOOTNOTES
 
* Corresponding author. Mailing address: Unité de Pathogénie des Infections Systémiques, UMR570, Faculté de Médecine Necker-Enfants Malades, 156 rue de Vaugirard, Paris Cedex 15, F-75730, France. Phone: (33 1) 40 61 53 79. Fax: (33 1) 40 61 56 77. E-mail: jmreyrat{at}necker.fr. Back

Editor: D. L. Burns

{dagger} Present address: Dept of Biochemistry and Molecular Biology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil. Back

{ddagger} Present address: Unité de Pathogénie des Infections Systémiques, UMR 570, Paris Cedex 15, F-75730 France. Back


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Infection and Immunity, April 2004, p. 2379-2382, Vol. 72, No. 4
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.4.2379-2382.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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