Response to reactive nitrogen intermediates in Mycobacterium tuberculosis: induction of the 16-kilodalton alpha-crystallin homolog by exposure to nitric oxide donors

Abstract

In contrast to the apparent paucity of Mycobacterium tuberculosis response to reactive oxygen intermediates, this organism has evolved a specific response to nitric oxide challenge. Exposure of M. tuberculosis to NO donors induces the synthesis of a set of polypeptides that have been collectively termed Nox. In this work, the most prominent Nox polypeptide, Nox16, was identified by immunoblotting and by N-terminal sequencing as the alpha-crystallin-related, 16-kDa small heat shock protein, sHsp16. A panel of chemically diverse donors of nitric oxide, with the exception of nitroprusside, induced sHsp16 (Nox16). Nitroprusside, a coordination complex of Fe2+ with a nitrosonium (NO+) ion, induced a 19-kDa polypeptide (Nox19) homologous to the nonheme bacterial ferritins. We conclude that the NO response in M. tuberculosis is dominated by increased synthesis of the alpha-crystallin homolog sHsp16, previously implicated in stationary-phase processes and found in this study to be a major M. tuberculosis protein induced upon exposure to reactive nitrogen intermediates.

FIG. 1

Characterization of the M. tuberculosis response to a panel of nitric oxide donors. Autoradiograms of 2-D gels show newly synthesized polypeptides radiolabeled with [³⁵S]Met and [³⁵S]Cys in 1-ml aliquots of M. tuberculosis H37Rv cultured in Youmans medium for 8 days at 37°C and exposed to NO donors (Alexis Biochem., and Research Biochem.) as indicated. Positioning of autoradiograms is according to pH gradient, which ranged from 7.47 to 5.25 from left to right. Equal amounts of protein in cell homogenates were loaded. Metabolic labeling with [³⁵S]methionine and [³⁵S]cysteine (NEN protein labeling mix) (25), 2-D gel electrophoresis, and electroblotting were performed as previously described (6, 25). (A) Untreated control. (B) SNAP (500 μM). (C) DETA/NO (500 μM). (D) PAPA/NONOate (500 μM). (E) GSNO (500 μM). (F) Sodium nitroprusside dihydrate (NP [1 mM]). The high-virulence M. tuberculosis strain I2646 (from D. B. Young) was used. Similar results were obtained with H37Rv (data not shown).

FIG. 2

Identification of Nox16 as the 16-kDa α-crystallin homolog of M. tuberculosis. (A) SDS-PAGE analysis of metabolically labeled polypeptides in 1-ml aliquots from a 6-day M. tuberculosis I2646 culture grown in Youmans medium. Equal amounts of protein were loaded. Induction of Nox16 is detectable in M. tuberculosis treated with SNAP, DETA/NO, PAPA/NONOate, and GSNO (concentrations as in Fig. 1), but is not observed in the aliquots treated with SIN-1 or nitroprusside. Nox16 is also absent in the unstimulated control. (B) Western blot analysis of panel A with monoclonal antibody TB68, which is specific for sHsp16. (C) N-terminal sequence analysis of Nox16 and its alignment with the corresponding sequence of MMP (31) and that of the 14- or 16-kDa antigen (48) which is identical to that of the 16-kDa α-crystallin homolog in M. tuberculosis. Menad., menadione.

FIG. 3

N-terminal sequence determination of the nitroprusside-inducible Nox19. M. tuberculosis H37Rv culture (100 ml of Youmans medium) was divided into two aliquots, and 500 μl of a 100 mM nitroprusside solution in H₂O was added to a final concentration of 1 mM. The culture was incubated with continuous stirring overnight at 37°C. The bacterial pellets were homogenized with glass beads in a Mini bead beater (Biospec Products, Bartlesville, Okla.) for 3 min at maximum speed. The nitroprusside-treated homogenate was mixed with a metabolically labeled homogenate (1-ml aliquot of the nitroprusside-treated culture radiolabeled with ³⁵S) and clarified by a 5-min spin in an Eppendorf microcentrifuge. Next, the supernatant was centrifuged at 230,000 × g for 2 h at 4°C. The pellet was resuspended in 200 μl of H₂O and spun at 3,000 × g for 1 h at 4°C, followed by 1 h at 230,000 × g, and the final supernatant was subjected to further analysis. (A) SDS-PAGE analysis of protein extracts from M. tuberculosis H37Rv. Lanes: 1 and 2, Ponceau red-stained proteins from a 230,000 × g pellet isolated from unstimulated and stimulated (1 mM nitroprusside) 50-ml culture aliquots of M. tuberculosis; 3, autoradiogram corresponding to lane 2; 4 and 5, a pair of radiolabeled extracts from nitroprusside-stimulated and unstimulated 1-ml culture aliquots. The major radioactive signal in lane 3 comigrates with the major protein band in lane 2 and with the inducible 19-kDa band in lane 4, indicating that Nox19 is enriched in the sample analyzed in lane 2. (B) 2-D gel electrophoresis of an aliquot from the 230,000 × g pellet from the nitroprusside-stimulated culture shown in panel A, lane 2. The major spot on the autoradiogram had an apparent molecular mass of 19 kDa and overlapped with the major protein spot on the blot visualized by Ponceau red. This protein spot was used for N-terminal sequence analysis. (C) N-terminal sequence of Nox19, aligned with the polypeptide 24 previously reported to copurify with the 30S ribosomal subunit from M. bovis BCG (40).

FIG. 4

M. tuberculosis Nox19 (Ftn [BfrB]) is a homolog of bacterial nonheme ferritins. Shown is a multiple sequence alignment of M. tuberculosis (Mt) Ftn (BfrB) (Nox19 [the sequence is based on the N-terminal amino acid sequence in Fig. 3 and the corresponding M. tuberculosis H37Rv genomic translated sequence, where it was termed BfrB subsequent to the completion of this work]) with the previously characterized eucaryotic-type nonheme ferritins from H. pylori (Hp Pfr), C. jejuni (Cj Cft), E. coli ferritin (Ec Ftn1 [also known as RsgA]), and additional homologs from the genomic E. coli (Ec Ftn2) and Haemophilus influenzae (Hi Ftn1 and Ftn2) databases. An asterisk indicates that the open reading frame corresponding to H. influenzae Ftn1 in the database has been truncated to match the start codon corresponding to other bacterial nonheme ferritins. Identical residues are boxed; additional similarities and conservative substitutions among bacterial nonheme ferritins are noticeable, but are not indicated.