Control of M. tuberculosis ESAT-6 secretion and specific T cell recognition by PhoP

Abstract

Analysis of mycobacterial strains that have lost their ability to cause disease is a powerful approach to identify yet unknown virulence determinants and pathways involved in tuberculosis pathogenesis. Two of the most widely used attenuated strains in the history of tuberculosis research are Mycobacterium bovis BCG (BCG) and Mycobacterium tuberculosis H37Ra (H37Ra), which both lost their virulence during in vitro serial passage. Whereas the attenuation of BCG is due mainly to loss of the ESAT-6 secretion system, ESX-1, the reason why H37Ra is attenuated remained unknown. However, here we show that a point mutation (S219L) in the predicted DNA binding region of the regulator PhoP is involved in the attenuation of H37Ra via a mechanism that impacts on the secretion of the major T cell antigen ESAT-6. Only H37Ra "knock-ins" that carried an integrated cosmid with the wild-type phoP gene from M. tuberculosis H37Rv showed changes in colony morphology, increased virulence, ESAT-6 secretion, and induction of specific T cell responses, whereas other H37Ra constructs did not. This finding established a link between the PhoP regulator and ESAT-6 secretion that opens exciting new perspectives for elucidating virulence regulation in M. tuberculosis.

Figure 1. Macrophage Infection Studies

Bone marrow–derived murine macrophages (BMM) were infected with M. tuberculosis H37Ra, H37Ra::fadE5, H37Ra::phoP, and H37Rv at a MOI of ca. 1:1 and 10:1 bacteria per cell (A and B, respectively). The figure shows the means and standard deviations of CFU ratio values (CFU at days 1, 4, and 7 relative to CFU at 4 h) obtained in a representative experiment performed in quadruplicate. The significant difference levels in growth characteristics between H37Ra and other strains (H37Ra::phoP and H37Rv) were determined by analysis of variance (ANOVA) (*p < 0.025, **p < 0.01, ***p < 0.005).

Figure 2. Virulence in the Murine Model

Number of colony-forming units (CFU) in the lungs and spleens of severe combined immunodeficient (SCID) mice 1 d and 3 wk after intravenous (i.v.) infection with ca. 10⁶ CFU. The significance of differences between CFU values obtained in mice infected with H37Ra and other strains (H37Ra::phoP and H37Rv) was determined by ANOVA (*p < 0.025, ***p < 0.005).

Figure 3. T Cell Responses in Mice

ESAT-6- and CFP-10-specific T cell responses induced by various M. tuberculosis strains 2 wk after immunization. IFN-γ production by splenocytes in response to different antigens is expressed in ng/ml. (A) First panel of strains tested. (B) Second panel of strains tested for confirmation purposes.

Figure 4. Antigen-Specific IL-2 Production of T Cell Hybridomas

Analysis of IL-2 secretion by anti-ESAT-6 or anti-Ag85A T cell hybridomas in response to recognition of antigen presented by bone marrow–derived dendritic cells (BM-DC) incubated with homologous or control peptides or infected with H37Rv, H37Ra or recombinant H37Ra strains.

Figure 5. Secretion Analysis

In vitro expression and secretion of ESAT-6 from M. tuberculosis H37Rv, H37Ra, MT103 phoP ko (SO2), and recombinant H37Ra complemented with integrating cosmids carrying genes phoP or rpsL from H37Rv. Total protein concentrations were determined by using Bio-Rad protein assay, and 15-μg samples were subjected to SDS-PAGE. Detection was carried out by using monoclonal anti-ESAT-6 antibody Hyb 76–8a.

Figure 6. Quantitative RT-PCR

Expression levels of genes rv3614c, rv3865, rv3867, and phoP in duplicate cultures of M. tuberculosis H37Ra and H37Ra::phoP and H37Rv evaluated by quantitative RT-PCR (qRT-PCR). Genes rv3865 and rv3867 were included as controls as these genes are encoded in the region of difference 1 (RD1) and show similarity to rv3615/14c. The expression level of each gene in each strain is reported as the ratio between the expression level in M. tuberculosis H37Ra culture 1, used as reference. For each strain, values were normalized to the level of 16S rRNA. Note that in M. tuberculosis a single rrs gene is present. Primer and probe sequences of genes rv3614c, rv3865, rv3867, phoP, as well as of 16S rRNA are listed in Table S2.