A sulfated metabolite produced by stf3 negatively regulates the virulence of Mycobacterium tuberculosis

Abstract

Sulfated molecules have been shown to modulate isotypic interactions between cells of metazoans and heterotypic interactions between bacterial pathogens or symbionts and their eukaryotic host cells. Mycobacterium tuberculosis, the causative agent of tuberculosis, produces sulfated molecules that have eluded functional characterization for decades. We demonstrate here that a previously uncharacterized sulfated molecule, termed S881, is localized to the outer envelope of M. tuberculosis and negatively regulates the virulence of the organism in two mouse infection models. Furthermore, we show that the biosynthesis of S881 relies on the universal sulfate donor 3'-phosphoadenosine-5'-phosphosulfate and a previously uncharacterized sulfotransferase, stf3. These findings extend the known functions of sulfated molecules as general modulators of cell-cell interactions to include those between a bacterium and a human host.

Fig. 1.

Sulfate assimilation pathways and design of a global sulfation knockout. (A) Overview of the sulfate assimilation pathway of M. tuberculosis. CysD and CysN comprise ATP sulfurylase, which activates sulfate by forming APS from ATP and inorganic sulfate. APS has two fates: It can be phosphorylated by CysC (APS kinase) to form PAPS or reduced by CysH (APS reductase) to form sulfite. The Stf sulfotransferase family members use PAPS as the sulfate donor to generate molecules such as SL-1 and S881. (B) Sulfate assimilation genes are modular. Arrows depict the genomic arrangement of sulfate assimilation genes from representative bacterial species (cys gene letter used). The asterisk denotes the second M. avium cysN/cycC locus. The constitution of the M. tuberculosis cys operon after deletion of cysC is also shown (bottom).

Fig. 2.

ΔcysC is a global sulfation knockout of M. tuberculosis. (A) Inactivation of cysC does not inhibit the reductive branch of the sulfate assimilation pathway. Growth of WT (□), ΔcysC (○), and ΔcysH (▵) in vitro with sulfate as the sole sulfur source. ΔcysC is able to reduce sulfur and, therefore, grows identically to WT. ΔcysH has a known defect in sulfate reduction and does not grow under these conditions. (B) ΔcysC does not synthesize sulfolipid-1 (SL-1). Expanded view of the SL-1 region of mass spectra from crude M. tuberculosis extracts. Heterogeneity in the lipid lengths of SL-1 accounts for the observed series of isotope ion packets separated by m/z 14. SL-1 cannot be detected in extracts from ΔcysC. (C) ΔcysC has a specific defect in SL-1 biosynthesis. TLC analysis of crude extracts from ¹⁴C-propionic acid-labeled M. tuberculosis cultures. The arrowhead marks the position of SL-1, which is not observed in ΔcysC. The biosynthesis of other propionic acid-derived metabolites is unchanged in ΔcysC. (D) ΔcysC is deficient in sulfation of SL-1. Expanded view of the trehalose-2-sulfate region from mass spectra of crude extracts from WT and ΔcysC. The asterisk shows the position of the trehalose-2-sulfate peak in the WT extract. Trehalose-2-sulfate could not be detected in ΔcysC extracts.

Fig. 3.

S881 is a sulfated molecule localized to the outer envelope of M. tuberculosis and its biosynthesis depends on PAPS and stf3. (A) S881 is not synthesized by M. tuberculosis lacking PAPS (ΔcysC) or stf3. Mass spectra of crude extracts from the indicated M. tuberculosis strains in the m/z range of S881. The peak corresponding to S881, denoted by an asterisk, is absent from ΔcysC and Δstf3. Complementation of both strains restores the biosynthesis of S881 (ΔcysC-ctrl and Δstf3-ctrl). (B) Δstf3 has a specific defect in S881 biosynthesis. Expanded view of the SL-1 region of mass spectra from crude M. tuberculosis extracts is shown. Δstf3 does not have a sulfation defect, because SL-1 is synthesized at WT levels. (C) S881 contains a single sulfur atom. Mass spectra of extracts from M. tuberculosis grown with either Na₂³²SO₄ or Na₂³⁴SO₄ as the sole sulfur source. The asterisk indicates the primary isotope of S881 in each sample. (D) S881 is secreted to the outer envelope of M. tuberculosis. Mass spectra of fractions containing either cell or cell envelope-associated lipids from M. tuberculosis. (D Lower) Several peaks corresponding to metabolites present in the WT cell-associated lipid fraction are shown. (D Upper) A zoom-in of the S881 region. In the WT cell-associated fraction (WT-cell), S881 and other uncharacterized metabolites are present. In the WT cell envelope fraction (WT-env), only S881 is observed.

Fig. 4.

stf3 negatively regulates the virulence of M. tuberculosis. (A) Deletion of stf3 results in shorted TTD in a mouse model of tuberculosis. TTD analysis of BALB/c mice infected i.v. with WT (□), Δstf3 (○), ΔcysH (▵), or a control mutant (◇). Mice infected with Δstf3 succumb to infection faster than those infected with WT or the control mutant. Mice infected with the auxotroph do not die within the time frame of this experiment. ∗∗, P ≤ 0.01 for comparison of WT and Δstf3. (B) Expression of Stf3 is selected against in vivo. TTD analysis of BALB/c mice infected intravenously with WT (□), Δstf3 (○), or Δstf3 complemented by plasmid expression of Stf3 (▿, see Materials and Methods). Mice infected with Δstf3 succumb to infection faster than those infected with WT. (B Inset) Colony-forming units (cfu) recovered from the lungs of Δstf3-ctrl-infected mice at 12 weeks after infection. Samples were plated on hygromycin (hyg) or kanamycin (kan) and hygromycin. (C) The shortened TTD phenotype of Δstf3 is observed and can be complemented in the low-dose aerosol model of infection. TTD analysis of C57BL/6 mice infected by the aerosol route with 100–200 WT (□), Δstf3 (○), or stable complemented Δstf3 bacilli (▿, see Materials and Methods). In the low-dose aerosol model of tuberculosis, mice infected with Δstf3 succumb to infection faster than those infected with WT. Stable complementation of Δstf3 attenuates the mutant to WT virulence. (D) Δstf3 maintains a higher bacterial load in the lungs of infected mice. Three mice were killed at the indicated time points after low-dose aerosol infection with the indicated M. tuberculosis strain. Serial dilutions of lung homogenates were plated to determine cfu. ∗, P ≤ 0.05 for comparison of WT and Δstf3. (E) Infection of mice with Δstf3 leads to increased lung inflammation. Representative histological sections of lungs from C57BL/6 mice infected with the indicated M. tuberculosis strain. Lungs were harvested from mice in C at 10 weeks after infection. (Scale bars: 300 μm.)