Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system

Abstract

Although many bacterial pathogens use specialized secretion systems for virulence, no such systems have been described for Mycobacterium tuberculosis, a major pathogen of humans that proliferates in host macrophages. In a screen to identify genes required for virulence of M. tuberculosis, we have discovered three components and two substrates of the first Sec-independent secretion pathway described in M. tuberculosis, which we designate the Snm pathway. Here we demonstrate that the proteins Snm1, -2, and -4 are required for the secretion of ESAT-6 and CFP-10, small proteins previously identified as major T cell antigens. Snm2, a member of the AAA ATPase family, interacts with substrates and with Snm1, another AAA ATPase. We show that M. tuberculosis mutants lacking either the Snm system or these substrates exhibit defects in bacterial growth during the acute phase of a mouse infection and are attenuated for virulence. Strikingly, snm mutants fail to replicate in cultured macrophages and to inhibit macrophage inflammatory responses, two well established activities of wild-type M. tuberculosis bacilli. Thus, the Snm secretion pathway works to subvert normal macrophage responses and is a major determinant of M. tuberculosis virulence.

Fig. 1.

M. tuberculosis snm mutants fail to secrete ESAT-6 and CFP-10. (A) Schematic representation of the transposon insertion sites (black arrows) from three snm mutants within the M. tuberculosis genome. (B) Western blot detection of ESAT-6, CFP-10, and GroEL from cell supernatant (S) and cell pellet lysate (P) fractions of wild-type and mutant cells. Loading was normalized by OD₂₈₀, and efficiency of transfer was confirmed by Ponceau S staining of the membrane. The decreased level of ESAT-6 and CFP-10 detected in the pellet of the snm1 mutant was not observed in subsequent experiments. (C) Silver stain of wild-type and mutant (snm4) cell culture supernatants separated by SDS/PAGE. The protein band absent in the snm4 mutant is marked with an asterisk.

Fig. 2.

Components and substrates of the Snm secretion pathway interact. Diploid yeast strains harboring the indicated bait and prey yeast–two-hybrid constructs were replica plated to galactose + 5-bromo-4-chloro-3-indolyl β-d-galactoside indicator plates and allowed to develop overnight at 30°C. ESAT-6, CFP-10, and Snm2 were expressed as full-length fusion proteins and the Snm1 constructs expressed the N-terminal nontransmembrane domain portion of the protein (amino acids 252–747). Strains containing plasmids expressing only the C-terminal (Snm2-C, amino acids 248–591) or N-terminal (Snm2-N, amino acids 1–241) portions of Snm2 were also tested. Expression of all fusion proteins was confirmed by Western blotting by using antibodies that recognize LexA (baits) or the hemagglutinin epitope tag (preys).

Fig. 5.

Hypthetical model of Snm-mediated secretion of ESAT-6 and CFP-10; see text for details.

Fig. 3.

Snm-mediated secretion is required for M. tuberculosis virulence. (A) C57BL/6 mice were injected with 1 × 10⁶ cfu of each strain, and bacilli were harvested from lungs at 1, 5, 10, 15, and 21 d after infection. Error bars represent the SEM from two combined experiments by using five mice per timepoint per experiment. (B) cfu data from A normalized to initial inoculum (see Materials and Methods). (C) cfu isolated from spleens of mice infected in A.(D) cfu data from C normalized to initial inoculum. (E) Survival of BALB/c mice (n = 15 per group) infected with 10⁶ cfu of indicated strains.

Fig. 4.

Snm pathway mutants induce enhanced macrophage inflammatory responses. Bone marrow-derived macrophages were infected, and culture supernatants were collected and the concentration of IL-12 p40 (A) and TNF-α (B) was measured by ELISA. Total RNA was harvested from macrophage monolayers, and IL-12 p40 (C) and TNF-α (D) mRNA levels were measured by quantitative PCR and normalized to actin mRNA levels. Each sample was assayed in triplicate; error bars represent standard deviation from at least two experiments. (E) Nitrite concentration from culture supernatants was measured by using the Griess reaction. (F) M. tuberculosis-infected macrophages were infected at a multiplicity of infection of 1 and harvested immediately after the 2-h phagocytosis period (“0h”) and at the indicated time points, and bacterial cfu were determined by plating.