Rv2224c is a cell envelope-associated predicted hydrolase. (A) Rv2224c is predicted to be a lipoprotein with an N-terminal type II signal peptide. The N region has lysine and/or arginine, and the H region has hydrophobic residues. Cysteine at amino acid residue 31 within the conserved “lipobox” sequence is the putative lipoprotein attachment site, where diglyceride units are added by thioester linkage, followed by cleavage at a site immediately preceding the lipidated cysteine, which becomes the N terminus of the mature lipoprotein (6). Rv2224c contains the α/β hydrolase fold sequence and a catalytic triad typically present in serine proteases, esterases, and lipases, consisting of the catalytic nucleophile serine active site (S₂₂₈), which associates with the proton carrier histidine (H₄₉₀), and a charge relaying aspartic acid (D₄₆₃). (B) Cell fractionation of M. bovis bacillus Calmette–Guérin-expressing myc-tagged Rv2224c and Western blots probed to detect the myc epitope, GroEL1, or Ag85 proteins. Lane 1, whole-cell extract; lane 2, cell wall; lane 3, cytoplasm; lane 4, cell membrane.

Rv2224c is required for optimal Mtb growth in vivo. (A) Mixed infection (1:1, wild type:Rv2224c::tn), by using 5 × 10⁵ cfus per strain, of C57BL/6 mice (four per time point) by i.v. infection. cfus were determined at 1, 7, 28, and 56 days after infection as described in Materials and Methods. Data are represented as fold increase in mean cfus relative to mean cfus on day 1. (B) Mixed infections to assess in vivo competition between wild type versus mutant Rv2224c::tn (wild type:Rv2224c::tn, gray bars), wild type versus mutant Rv2224c::tn complemented with intact Rv2224c (wild type:Rv2224c::tn + 2224c, black bars), or wild type versus mutant Rv2224c::tn-expressing Rv2224c containing the S228A mutation (wild type:Rv2224c::tn + S228A, hatched bars). cfus of each strain were determined at 1 and 28 days after infection from lungs. Five mice were used per strain for each time point. Data are represented as log ratios of mean cfus of mutant per mean cfus of wild type.

Disruption of Rv2224c prolongs survival and reduces lung immunopathology of infected mice. (A and B) Single infections of C57BL/6 mice with wild type or Rv2224c::tn and Mtb cfus in lungs (A) and spleens (B) over time. Four mice were used per strain for each time point. cfu data are represented as fold increase in cfus relative to cfus on day 1. (C) Survival of C57BL/6 mice infected with wild type or Rv2224c::tn. Twelve mice were infected in each group. Median survival time was 195 days (wild type) and 336 days (Rv2224c::tn) (P < 0.0001 by log-rank test). (D) (Top) Gross pathology of lungs at 20 weeks after infection. (Middle and Bottom) H&E-stained lung tissue sections from wild-type- or mutant-infected mice at 20 weeks. (Scale bars, Middle, 1 mm; Bottom, 50 μm.)

Rv2224c modulates innate immune responses. (A and B) Survival of RAG^−/− mice infected by i.v. route at 2 × 10⁶ cfus per strain (median survival: wild type, 37 days; Rv2224c::tn, 64 days; P < 0.0001 by log-rank test) (A) and aerosol route (median survival: wild type, 50 days; Rv2224c::tn, 84 days; P < 0.0001 by log-rank test) (B). Twelve mice per strain were infected in both A and B. (C) H&E-stained lung tissue sections from wild-type- or mutant-infected mice at 35 days after infection. (D) Auramine/rhodamine staining of lung tissue sections from aerosol-infected RAG^−/− mice on day 35 (Top) and time of death (Middle and Bottom). (E) cfus from lungs of aerosol-infected RAG^−/− mice. Mean time of death for each strain is indicated. Four mice were used per group per time point. Error bars represent standards deviation from the mean.

Rv2224c is required for survival in macrophages. (A) Macrophages were infected with wild type or Rv2224c::tn at an moi of 1 in triplicate and lysed at 1, 3, 5, and 7 days after infection, and the intracellular bacteria were plated for cfus. Data represent four independent experiments and are shown as fold increases in cfus, normalized to cfu on day 1 for each strain. (B) Secretion of cytokines and chemokines by macrophages 48 h after infection with wild type or Rv2224c::tn. Data are representative of three independent experiments. Error bars represent standard deviation from the mean. (C) Survival index of wild-type Mtb or Rv2224c::tn in the presence of lysozyme (ratio of cfu of each strain in the presence/absence of lysozyme). Data are representative of four independent experiments, each in triplicate, per concentration of lysozyme.

Rv2224c promotes processing of GroEL2. (A) Western blot showing GroEL2 in wild-type whole-cell pellets (P) and as a smaller form in the culture supernatant (S). In Rv2224c::tn pellets, the larger form of GroEL2 is present in both P and S, with defective processing of GroEL2 to the smaller form. GroEL1 is exclusively present in P and absent from S. (B) Localization of GroEL2 in cell fractions of M. bovis bacillus Calmette–Guérin. GroEL2 is present in cell wall, cell membrane, and cytoplasmic fractions. GroEL1 is largely cytoplasmic. Lane 1, whole-cell extract; lane 2, cell wall; lane 3, cytoplasm; lane 4, cell membrane. (C) Complementation of GroEL2 processing defect in Rv2224c::tn by expression in trans of wild-type Rv2224c, mutated versions (S228A and C31A), or both Rv2223c and Rv2224c (operon).