Bacterial protein-O-mannosylating enzyme is crucial for virulence of Mycobacterium tuberculosis

Abstract

A posttranslational protein O-mannosylation process resembling that found in fungi and animals has been reported in the major human pathogen Mycobacterium tuberculosis (Mtb) and related actinobacteria. However, the role and incidence of this process, which is essential in eukaryotes, have never been explored in Mtb. We thus analyzed the impact of interrupting O-mannosylation in the nonpathogenic saprophyte Mycobacterium smegmatis and in the human pathogen Mtb by inactivating the respective putative protein mannosyl transferase genes Msmeg_5447 and Rv1002c. Loss of protein O-mannosylation in both mutant strains was unambiguously demonstrated by efficient mass spectrometry-based glycoproteomics analysis. Unexpectedly, although the M. smegmatis phenotype was unaffected by the lack of manno-proteins, the Mtb mutant had severely impacted growth in vitro and in cellulo associated with a strong attenuation of its pathogenicity in immunocompromised mice. These data are unique in providing evidence of the biological significance of protein O-mannosylation in mycobacteria and demonstrate the crucial contribution of this protein posttranslational modification to Mtb virulence in the host.

Fig. 1.

The Msmeg_5447 gene encoding the putative ortholog of the Mtb Rv1002c mannosyl transferase gene is dispensable for M. smegmatis growth in vitro. Effects of Msmeg_5447 inactivation on the growth (A) and the sensitivity of the M. smegmatis ΔM5447 mutant to the antituberculosis drug Ciprofloxacin (B), and detergent (SDS)-induced cell wall stress (C). Growth and survival were followed by monitoring the OD at 600 nm (A) or the level of MTT reduction by the metabolically active cells at 570 nm (B and C). (D and E) Transmission electron micrographs of negatively stained M. smegmatis wild-type (Wt) and ΔM5447 mutant cells.

Fig. 2.

Inactivation of the Msmeg_5447 gene interrupts the O-mannosylation of the M. smegmatis FasC protein. (A) Identification of the Msmeg_5447 protein target by silver nitrate-stained SDS/PAGE of the culture filtrate protein extract from M. smegmatis WT, ΔM5447 mutant, and complemented mutant (ΔM5447:M5447) revealing the electrophoretic mobility alteration of a major protein gel band (arrows). (B) Amino acid sequence of the recombinant His-Tagged rFasC (SignalP 3.0 predicted signal peptide is in italic; NetOGlyc 3.1 predicted S and T glycosylation sites are boldface; Histidine tag is into brackets; yellow highlights the amino acid sequence covered by mass spectrometry analysis). (C) Carbohydrate content analysis and (D) Coomassie blue-stained SDS/PAGE of the rFasC, the α-exo-mannosidase–treated rFasC, and the α-exo-mannosidase sham control (Standard: manno-heptose). (E) Identification of the triglycosylated rFasC N-terminal peptide P_30–55 by CID MS/MS [(P_30–55Hex₃+3H)³⁺ at m/z 1680.23]. Underlined masses correspond to fragment ions resulting from one to three neutral losses of hexose from the parent ion. (F) FasC N-terminal peptide P_30–55 sequence reporting the major informative peptide cleavages (b, c, and y peptide fragment ions) observed in the positive CID and ETD MS/MS spectra (Fig. S2). (G) Reconstructed ion chromatograms (mass tolerance: 10 ppm, Sum of [M+3H]³⁺ [M+2H]²⁺) of the native FasC P_30-55 glycoforms detected in the culture filtrates of the WT and the mannosyl transferase mutants. (H) Cumulated normalized abundances of the native FasC P_30–55 glycoforms observed in the mannosyl transferase mutants and their respective complemented strains.

Fig. 3.

Inactivation of the M. tuberculosis Rv1002c gene impairs Apa protein O-mannosylation and impacts on Mtb growth. (A) Impact of the Rv1002c inactivation on the Mtb ΔRv1002c mutant growth in Middlebrook 7H9 broth with Albumin-Dextrose-Catalase or dextrose alone (Inset). (B) Growth impairment of the mutant on solid Middlebrook 7H11 agar medium. Serial dilution of cultures containing 10⁸ bacteria per milliliter were plated and colony sizes were compared after 4 wk of growth at 37 °C. (C, Right) Relative abundances of the Apa peptides detected in the culture filtrates of wild-type (Wt), ΔRv1002c (Mt), and complemented ΔRv1002c:Rv1002c (Cp) mutant (sum of the abundances are set at 100% for each strain). (Left) Weighted deviation from the mean relative abundance of each peptide in the three strains. (D) LC-MS reconstructed ion chromatograms of the P_278–321, P_285–321, and P_287–321 Apa C-terminal peptides and their monoglycosylated forms (*) detected in the different Mtb strains; (reconstructed ion chromatograms parameters as in Fig. 2).

Fig. 4.

Rv1002c-dependent protein O-mannosylation is required for Mtb intracellular persistence and proliferation and for full virulence in SCID mice. (A–C) Persistence and (B–D) intracellular proliferation of GFP-tagged Mtb wild-type (Wt), ΔRv1002c mutant (Mt), and complemented mutant (Cp) in the mouse alveolar macrophage cell line (A and B) and primary human blood monocyte-derived macrophages (C and D). [Data (Table S5) were analyzed by one-way Anova tests: *P < 0.05, **P < 0.01]. Survival of immunocompromised SCID mice infected intranasally with 10³ Mtb WT, ΔRv1002c mutant, or complemented mutant cells (E), or with 10³, 10⁴, or 10⁵ ΔRv1002c mutant cells or 10³ Mtb WT and complemented mutant cells (F). Numbers correspond to the median survival time.