Genome-wide identification of Mycobacterium tuberculosis exported proteins with roles in intracellular growth

Abstract

The exported proteins of Mycobacterium tuberculosis that are localized at the bacterial cell surface or secreted into the environment are ideally situated to interact with host factors and to function in virulence. In this study, we constructed a novel β-lactamase reporter transposon and used it directly in M. tuberculosis for genome-wide identification of exported proteins. From 177 β-lactam-resistant transposon mutants, we identified 111 different exported proteins. The majority of these proteins have no known function, and for nearly half of the proteins, our demonstration that they are exported when fused to a β-lactamase reporter is the first experimental proof of their extracytoplasmic localization. The transposon mutants in our banked library were of further value as a collection of mutants lacking individual exported proteins. By individually testing each of 111 mutants for growth in macrophages, six attenuated mutants with insertions in mce1A, mce1B, mce2F, rv0199, ctaC, and lppX were identified. Given that much of the M. tuberculosis genome encodes proteins of unknown function, our library of mapped transposon mutants is a valuable resource for efforts in functional genomics. This work also demonstrates the power of a β-lactamase reporter transposon that could be applied similarly to other bacterial pathogens.

FIG. 1.

Map of Tn′bla_TEM-1. Plasmid pJES124 contains the signal sequence-less ′bla_TEM-1 gene cloned downstream of the left inverted repeat (LIR) of a Himar1-based mariner transposon derived from pSH1. The codon sequence of the inverted repeat is shown in the same reading frame as the starting codons of ′BlaTEM-1. Tnpmar, mariner transposase; hygR, hygromycin resistance gene.

FIG. 2.

Evaluation of M. tuberculosis mce::Tn′bla_TEM-1 mutants in macrophages. (A) Representation of mce1, mce2, and mce4 operons in M. tuberculosis H37Rv. Black triangles indicate Tn′bla_TEM-1 insertion mutants with macrophage growth defects. White triangles mark Tn′bla_TEM-1 insertion mutants that were not attenuated. The mce1 operon has two possible start sites and spans from either fadD5 or yrbE1A to mas1D (12, 20). The mce2 and mce4 operons are transcribed in a single transcript comprised of the genes shown (11, 24). (B) Murine bone marrow-derived macrophages were infected with M. tuberculosis MBTB126, the mce1A::Tn′bla_TEM-1 mutant, or the mce1B::Tn′bla_TEM-1 mutant. At 4 h (day 0) and 3 and 5 days postinfection, macrophages were lysed and plated to enumerate intracellular bacteria. Error bars indicate standard deviations (SD) of the means. (C) MBTB126 carrying pMV261.kan, the mce2F::Tn′bla_TEM-1 mutant carrying pMV261.kan, and the mce2F::Tn′bla_TEM-1 mutant carrying a multicopy complementing vector expressing mce2F from a constitutive promoter (pJES153) were used to infect macrophages as described for panel B. *, P ≤ 0.05. Each graph shows representative data from one of at least three independent replicate experiments.

FIG. 3.

M. tuberculosis rv0199::Tn′bla_TEM-1 has a growth defect in macrophages. (A) Organization of genes surrounding Tn′bla_TEM-1 insertion (triangle) in rv0199. (B) Membrane topology of Rv0199 predicted by the TMHMM algorithm (49). The site of ′BlaTEM-1 fusion is indicated with a triangle. (C) Macrophages were infected with MBTB126 carrying the empty vector pMV261.kan, the rv0199::Tn′bla_TEM-1 mutant carrying pMV261.kan, or the rv0199::Tn′bla_TEM-1 mutant carrying a multicopy complementing vector expressing rv0199 from a constitutive promoter (pJES178). Error bars indicate SD of the means. *, P ≤ 0.05. The graph shows representative data from one of at least three independent replicate experiments.

FIG. 4.

M. tuberculosis ctaC::Tn′bla_TEM-1 has a growth defect in macrophages and a colony phenotype on agar plates. (A) Relative location of ctaC in the M. tuberculosis genome. The black triangle indicates the site of Tn′bla_TEM-1 insertion. (B) Membrane topology of CtaC predicted by the TMHMM algorithm (49). The site of ′BlaTEM-1 fusion is indicated with a triangle. (C) Macrophage infection as described in the legend for Fig. 3. Error bars indicate SD of the means. *, P ≤ 0.05. The graph shows representative data from one of at least three independent replicate experiments. (D) M. tuberculosis MBTB126 and the ctaC::Tn′bla_TEM-1 mutant were plated on 7H10 agar plates supplemented with Tween 80. Colonies were examined after 25 days at 37°C.

FIG. 5.

M. tuberculosis lppX::Tn′bla_TEM-1 has a growth defect in macrophages. (A) Representation of insertions in the PDIM locus of M. tuberculosis. The black arrowhead represents a Tn′bla_TEM-1 insertion that resulted in attenuation in macrophages. The white arrowhead indicates a Tn′bla_TEM-1 insertion that did not lead to attenuation. (B) Murine bone marrow-derived macrophages were infected with M. tuberculosis MBTB126 and the lppX::Tn′bla_TEM-1 mutant as described in the legend to Fig. 3. Error bars indicate SD of the means. *, P ≤ 0.05. Each graph shows representative data from one of at least three independent replicate experiments.