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The Mycobacterium tuberculosis ESAT-6 Homologue in Listeria monocytogenes Is Dispensable for Growth In Vitro and In Vivo

Departments of Pediatrics,¹ Immunology, University of Washington School of Medicine, Seattle, Washington 98195²

Received 25 February 2005/ Returned for modification 19 April 2005/ Accepted 9 May 2005

	ABSTRACT

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ESAT-6 is a virulence determinant in Mycobacterium tuberculosis and a member of a conserved group of proteins in a variety of other bacteria. A targeted deletion of the homologous gene in Listeria was generated, and in contrast to that observed for mycobacteria, this locus was not required for Listeria virulence.

	TEXT

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Infection with Mycobacterium tuberculosis is a major global public health problem. Over one-third of the world's population is infected, and an estimated 2 million deaths occur each year as a result. Comparative genome analysis between the nonpathogenic mycobacterium bacille Calmette-Guerin and the pathogenic mycobacterium M. tuberculosis or Mycobacterium bovis has revealed a 9.5-kb region (RD1) to be important for virulence (1, 2, 9). Spontaneous deletion of this region is thought to be the primary mutation that occurred in M. bovis during serial passage by Calmette and Guerin between 1908 and 1921, resulting in the current widely used vaccine strain BCG. This hypothesis is supported by the severe in vivo attenuation of M. tuberculosis after targeted deletion of this region and restoration of virulence by complementation of this region in BCG (7, 8, 13).

In M. tuberculosis, 11 open reading frames (ORFs) are present in the 9.5-kb RD1 region. Three of these ORFs encode critical components of a secretion system that exports two additional protein products encoded within RD1, ESAT-6 and CFP-10 (14). Both ESAT-6 and CFP-10 contain immunodominant epitopes in the T-cell response to M. tuberculosis infection (12). Although the mechanism of action of ESAT-6 is unknown, this specific locus is essential for mycobacterial virulence because targeted deletion of this gene in either M. tuberculosis or M. bovis results in profound attenuation following in vivo infection (14, 15).

Homologues of the M. tuberculosis ESAT-6 protein have been identified in a variety of other bacterial species. While initial studies identified homologues in high-G+C bacterial species such as actinobacteria and other mycobacteria (6), further analyses have identified more distant homologues in various species of gram-positive bacteria in the low-G+C group, including Bacillus spp., Clostridium spp., Staphylococcus spp., and Listeria spp. (10). Although the level of sequence similarity of these proteins compared with ESAT-6 is relatively low (30% similarity and 15% identity), conservation of the internal tryptophan-X-glycine (W-X-G) and 100-residue length supports the inclusion of these proteins in an ESAT-6 superfamily of "WXG100" proteins. Recently, the homologue of M. tuberculosis ESAT6 in Staphylococcus aureus has been identified and found to play an essential role in S. aureus virulence following intravenous infection (3).

Listeria monocytogenes is a gram-positive bacterium that causes human clinical disease ranging from self-resolving gastroenteritis to more serious illnesses, including abortion, sepsis, and central nervous system infection. Neonates and other immunocompromised hosts are especially susceptible to more serious infection. The pathogenesis and virulence determinants of L. monocytogenes have been well characterized with in vitro and in vivo infection models (4). During infection, L. monocytogenes coordinates the expression of an array of bacterial gene products to gain access and reside within the intracytoplasmic compartment of infected cells, thereby evading humoral immunity, and as a result protective immunity to this infection is mediated predominantly by cellular mediators (11). L. monocytogenes infection is currently widely used as a model to study and identify host mediators of innate and adaptive immunity to intracellular bacterial pathogens.

The L. monocytogenes ESAT-6 homologue is a 99-residue protein and is encoded by the lm00056 locus (Lmesat6) identified by genome sequencing (National Center for Biotechnology Information accession no. AL591973). At the protein level, it is 14% identical and 30% similar to M. tuberculosis ESAT-6 and contains the conserved W-X-G motif (Fig. 1A). During infection, transcription of this locus can be detected in the spleens of infected mice by reverse transcription-PCR (Fig. 1B). The importance of this protein in the virulence of other bacterial and mycobacterial pathogens and the conserved nature of homologous proteins in diverse bacterial species suggested they may play a conserved role in bacterial replication and/or pathogenesis. Thus, we sought to examine the role of this locus in L. monocytogenes infections by comparing the in vitro and in vivo virulence properties of an Lmesat6 deletion mutant with wild-type (WT) L. monocytogenes. A targeted internal 170-bp deletion within the Lmesat6 open reading frame was generated by homologous recombination after cloning upstream and downstream fragments into the temperature-sensitive construct pKSV7 (Fig. 1C and D) and electroporation into WT L. monocytogenes strain 10403s using previously described methods (5).

View larger version (43K): [in this window] [in a new window]   FIG. 1. (A) Alignment of M. tuberculosis ESAT-6TB and the homologous protein in L. monocytogenes, ESAT-6LM. Identical residues are in bold, and similar residues are shaded. (B) Reverse transcription-PCR demonstrating expression of the L. monocytogenes ESAT-6 locus (lmo0056; National Center for Biotechnology Information accession no. AL591973) by bacteria recovered ex vivo from the spleens of mice infected for 72 h with 105 CFU of L. monocytogenes strain 10403s. RT, reverse transcriptase. (C) L. monocytogenes genomic map containing the L. monocytogenes ESAT-6 locus (lmo0056) and the construct used for deletion of bases 57 to 227 in the 294-base coding region. Five hundred base pairs of the genomic upstream sequence flanking the deletion sites was cloned into the HindIII/KpnI sites of the temperature-sensitive plasmid pKSV7 using the following primers: forward primer 5'-aagcttccagtgaactgccgc-3' and reverse primer 5'-ggatccgtaagttttcgcgcgatc-3'. The following primers were used for the downstream flanking region: forward primer 5'-ggatccttgagaagacagcaaacg-3' and reverse primer 5'-ggtaccactttctgcaactccgcg-3'. Underlined regions indicate introduced restriction sites. (D) PCR of genomic DNA isolated from L. monocytogenes strains 10403s (WT) and 10403s esat6 demonstrating the 170-bp deletion.

We further evaluated the importance of Lmesat6 during in vivo infection. Following intravenous infection with either WT L. monocytogenes or L. monocytogenes esat6, bacterial replication uniformly occurred in the spleens and liver at day 3 compared with the initial inocula, and bacterial clearance uniformly occurred by day 7 (Fig. 2). However, at each of these time points, no significant differences in bacterial burden were detected in either organ between mice infected with L. monocytogenes esat6 and WT L. monocytogenes. In the time course of primary L. monocytogenes infection, innate immune cells such as macrophages, neutrophils, and natural killer cells are responsible for control of bacterial replication in the first 5 days following infection, while immunity thereafter is mediated by antigen-specific CD8 and CD4 T cells. Thus, the normal bacterial clearance from day 3 to day 7 following infection by L. monocytogenes esat6-infected mice suggests that the L. monocytogenes homologue of ESAT6 is not required for generation of L. monocytogenes antigen-specific CD8 and CD4 T cells. This was tested by examining the percentage and total numbers of L. monocytogenes-specific CD8 and CD4 T cells after infection with either WT L. monocytogenes or L. monocytogenes esat6. At the peak of the L. monocytogenes-specific T-cell response (day 7), splenocytes from infected mice were stimulated with either the major histocompatibility complex (MHC) class I antigenic peptide LLO 91-99 (CD8 T cells) or the class II peptide LLO 189-201 (CD4 T cells) and specific cells were quantified by cell surface and intracellular cytokine staining as previously described (16) (Fig. 3). While infection with both strains elicited a robust CD8 and CD4 T-cell response, there was no difference in either the percentage or the total number of L. monocytogenes-specific CD8 or CD4 T cells after infection with L. monocytogenes esat6 or WT L. monocytogenes.

View larger version (21K): [in this window] [in a new window]   FIG. 2. Numbers of L. monocytogenes bacteria recovered from the spleens (top panel) and livers (bottom panel) of mice (C57BL/6 [H-2b] x B10.D2 [H-2d]) inoculated intravenously with 2.0 x 104 CFU of either 10403s (squares) or 10403s esat6 (triangles) on day 3 (closed symbols) and day 7 (open symbols) postinfection. Each datum point indicates the 1og10 CFU from an individual mouse. These data are pooled from two independent experiments that yielded similar results. Bar, geometric mean. Liver day 7 number of CFU of 10403s compared with the number of 10403s esat6 CFU (P = 0.59) (Mann-Whitney test; Graph Pad, San Diego, CA).

View larger version (23K): [in this window] [in a new window]   FIG. 3. (A) Percentage of gamma interferon (IFN-)-positive splenic CD8 and CD4 T cells on day 7 after infection with 10403s (solid bars) or 10403s esat6 (shaded bars) after in vitro stimulation with MHC class I-restricted L. monocytogenes peptide LLO 91-99 or class II-restricted L. monocytogenes peptide LLO 189-201 with corresponding controls. (B) Calculated total numbers of gamma interferon-producing CD8 and CD4 T cells per spleen in mice infected with 10403s (solid bars) or 10403s esat6 (shaded bars) after stimulation with the indicated MHC class I- or class II-restricted L. monocytogenes-specific peptides. These data represent six mice per group combined from two independent experiments. Bar, standard error. Percent CD8 10403s cells compared with 10403s esat6 cells, P = 0.48; percent CD4 10403s cells compared with 10403s esat6 cells, P = 0.59; total number of CD8 10403s cells compared with 10403s esat6 cells, P = 0.24; total number of CD4 10403s cells versus 10403s esat6 cells, P = 1.0 (Mann-Whitney test; Graph Pad, San Diego, CA).

	ACKNOWLEDGMENTS

 
We acknowledge Ester Li, Carleen Collins, Nancy Freitag, Adeline Hajjar, Tobias Kollmann, David Sherman, and Kevin Urdahl for helpful discussion and critical review of the manuscript.

This work was supported by NIH grant HD18184 (to C.B.W.). S.S.W. is an NICHD Fellow of the Pediatric Scientist Development Program (NICHD grant award K12-HD00850).

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Pediatrics, University of Washington School of Medicine, 1959 NE Pacific Street, Box 357650, Seattle, WA 98195. Phone: (206) 221-2819. Fax: (206) 543-1013. E-mail: singsing{at}u.washington.edu.

Editor: J. L. Flynn

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