A Phagosomally Expressed Gene, rv0428c, of Mycobacterium tuberculosis Demonstrates Acetyl Transferase Activity and Plays a Protective Role Under Stress Conditions

Abstract

Mycobacterium tuberculosis genome is composed of several hypothetical gene products that need to be characterized for understanding the physiology of bacteria. Rv0428c was one of the 11 proteins exclusively identified within the phagosomal compartment of macrophages infected with mycobacteria and marked as hypothetical. The expression of rv0428c gene was upregulated under acidic and nutritive stress conditions in M. tuberculosis H37Ra, which was supported by potential sigma factor binding sites in the region upstream to the rv0428c gene. The bioinformatics analysis predicted it to be a GCN5- acetyl transferase, belonging to the Histone acetyl transferase (HAT) family. The docking analysis predicted formation of hydrogen bonds and hydrophobic interactions between donor acetyl-co-A and histone H3 tail region. rv0428c gene was cloned and expressed in E. coli. The protein was purified to homogeneity and was fairly stable over a wide range of pH 5.0-9.0 and temperature up to 40 °C. The HAT activity of purified Rv0428c was confirmed by in vitro acetylation assay using recombinant H3 histone expressed in bacteria as substrate, which increased in time dependent manner. The results suggested that it is the second confirmed acetyl transferase in M. tuberculosis H37Rv. Furthermore, rv0428c was over expressed in surrogate host M. smegmatis, which led to enhanced growth rate and altered colony morphology. The expression of rv0428c in M. smegmatis promoted the survival of bacteria under acidic and nutritive stress conditions. In conclusion, Rv0428c, a phagosomal acetyl transferase of M. tuberculosis, might be involved in survival under stress conditions.

Keywords: Chloramphenicol; Histone acetyl transferase (HAT); Mycobacterium tuberculosis; Stress.

Fig. 1

Expression analysis of rv0428c under different in-vitro stress conditions by real- time PCR. A RT-PCR analysis, Amplification of Sig A gene as internal control and rv0428c gene from prepared cDNAs under different stress conditions. L1 control, L2 acidic, L3 nutritive, L4 Oxidative, L5 iron B qPCR analysis of rv0428c expression under various in-vitro stress conditions C Diagrammatic representation of putative binding sites in the promoter region of rv0428c gene, sigF binding site is highlighted in red color in a box, sigE binding site is highlighted in blue color. Given values are expressed in mean ± SD performed in three independent experiments. Statistical analysis was assessed using student’s t-test (*p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001)

Fig. 2

A Map showing the genomic organization of rv0428c in M. tuberculosis H37Rv and its counterparts mb0436c and ml1930 in M. bovis and M. leprae B Multiple sequence alignment of Rv0428c protein sequence with its orthologs was obtained with the Clustal Ω program and conserved regions were assigned to the aligned file by Espript 3.0. Red Shading indicates residues identical in all the protein sequences C Molecular 3D model of Rv0428c: The helices were represented in red, β-sheets in cyan and random coils in green. Image was generated through Discovery studio 4 D The superposition of the structure of bacterial GCN5-related N-acetyltransferase from Kribella flavida and the 3-D structure model of the Rv0428c protein. PDB_ID: 4IUS is represented in maroon and Rv0428c in cyan

Fig. 3

Docking analysis of Rv0428c protein A Docking of acetyl-co-A in the binding pocket of Rv0428c protein. B Cavity residues of Rv0428c protein involved in interaction with acetyl-co-A C Cavity residues of Rv0428c protein involved in interaction with histone H3 tail region. D Zoomed in view of interaction between histone H3 tail and Rv0428c protein, The green coloured helical structure represents Rv0428c protein and red colour corresponds to histone H3 tail

Fig. 4

A Expression analysis of recombinant Rv0428c protein on 10% SDS PAGE L1 Protein marker, L2 Pellet of uninduced rRv0428c culture, L3 Pellet of culture induced with 60 µm IPTG at 18 °C and L4 Purified rRv0428c protein B Far–UV CD spectra of recombinant purified rRv0428c protein measured in 10 mM phosphate buffer (pH 8.0) at 25 °C at wavelength 190–260 nm C Far UV CD spectra recorded at temperatures ranging from 30–70 °C demonstrated gradual decrease in the negative ellipticity with subsequent increase of temperature D Far UV CD spectra of rRV0428c protein after incubating the protein for 1 h with different buffers of pH- 5.0–9.0 demonstrated that Rv0428c protein was stable over a wide range of pH E Fluorescence spectra of purified rRv0428c protein at different temperatures (20 to 90 °C) recorded from 310 to 400 nm wavelength demonstrated gradual decrease in the intrinsic fluorescence with the subsequent increase in temperature

Fig. 5

In vitro acetylation assay for rRv0428c protein, Time dependent acetylation of H3 by rRv0428c, L1 Nucleocytoplasmic fraction from eukaryotic AGS cell line, L2 RecH3 + rRv0428c (1 h), L3 RecH3 + rRv0428c (2 h), L4 RecH3 + rRv0428c (3 h), L5 Rec-H3 + AGS NC fraction (1 h), L6 Rec-H3 + AGS NC fraction (2 h), L7 Rec-H3 + AGS NC fraction (3 h), L8 Core histones from AGS cell line

Fig. 6

A Effect of rv0428c on colony morphology: Enlarged view of single colony of Msmeg-pVV16 and Msmeg-pVV16-rv0428c B Growth pattern analysis of Msmeg-pVV16 and Msmeg-pVV16-rv0428c at different time intervals- 24 h, 48 h, 72 h and 96 h Given values are expressed in mean ± SD performed in three independent experiments. Statistical analysis was assessed using student’s t-test (*p ≤ 0.05 and **p ≤ 0.01)

Fig. 7

A Survival of Msmeg-pVV16-rv0428c in comparison to Msmeg-pVV16 at pH 6.0 and 5.0 B Survival of Msmeg-pVV16-rv0428c and Msmeg-pVV16 under nutritive deprived conditions. Data are representative of three independent biological replicates and shown as mean ± SD. Statistical analysis was assessed using student’s t-test (*p ≤ 0.05)

Fig. 8

Plate assay for drug susceptibility testing of chloramphenicol (0.5–10 μg/ml) using resazurin A Effect of Rv0428c on drug susceptibility of Msmeg-pVV16-rv0428c and Msmeg-pVV16 cultures: Mid log-phase recombinant M. smegmatis cultures diluted in 7H9 medium without tween-80 were treated with indicated concentrations of chloramphenicol, with M indicating negative control containing media alone. Resazurin dye in 20% tween 80 was added after 4 h of incubation at 37 °C and the colour change was monitored after 24 h. Pink colour indicates live bacteria, while blue indicates dead bacteria B Survival of Msmeg-pVV16-rv0428c and Msmeg-pVV16 was monitored by counting the CFU/mL after treatment with chloramphenicol. Results were expressed in % survival (CFU counts without drug treatment was considered to be the 100% survival). Data are representative of three independent biological replicates and shown as mean ± SD. Statistical analysis was assessed using student’s t-test (*p ≤ 0.05 and **p ≤ 0.01)