Mycobacterium tuberculosis Small RNA MTS1338 Confers Pathogenic Properties to Non-Pathogenic Mycobacterium smegmatis

Abstract

Small non-coding RNAs play a key role in bacterial adaptation to various stresses. Mycobacterium tuberculosis small RNA MTS1338 is upregulated during mycobacteria infection of macrophages, suggesting its involvement in the interaction of the pathogen with the host. In this study, we explored the functional effects of MTS1338 by expressing it in non-pathogenic Mycobacterium smegmatis that lacks the MTS1338 gene. The results indicated that MTS1338 slowed the growth of the recombinant mycobacteria in culture and increased their survival in RAW 264.7 macrophages, where the MTS1338-expressing strain significantly (p < 0.05) reduced the number of mature phagolysosomes and changed the production of cytokines IL-1β, IL-6, IL-10, IL-12, TGF-β, and TNF-α compared to those of the control strain. Proteomic and secretomic profiling of recombinant and control strains revealed differential expression of proteins involved in the synthesis of main cell wall components and in the regulation of iron metabolism (ESX-3 secretion system) and response to hypoxia (furA, whiB4, phoP). These effects of MTS1338 expression are characteristic for M. tuberculosis during infection, suggesting that in pathogenic mycobacteria MTS1338 plays the role of a virulence factor supporting the residence of M. tuberculosis in the host.

Keywords: MTS1338; Mycobacterium smegmatis; Mycobacterium tuberculosis; macrophages; proteomics; small non-coding RNA; virulence factor.

Figure 1

MTS1338 regulated the growth and survival of mycobacteria. (A) Growth of control (pMV261-transformed) and MTS1338-expressing strains in Nutrient Broth medium supplemented with 0.05% Tween-80. (B) Survival of control and MTS1338 strains in RAW264.7 macrophages infected at multiplicity of infection (MOI) 10:1. Colony forming units (CFUs) were counted at 3 and 24 h post infection (hpi), and the results presented relative to time “0” taken as 100%. (C) MTS1338 expression in RAW264.7 macrophages infected by MTS1338-expressing MSmeg was analyzed by qRT-PCR at the indicated time post-infection. The data are presented as the mean ± SD of three independent experiments: * p < 0.05 and ** p < 0.01. ND, non detected.

Figure 2

MTS1338-expressing MSmeg inhibited phagosome maturation in RAW264.7 macrophages. (A) Representative fluorescence images of RAW 264.7 cells infected with MSmeg-GFP and MSmeg-GFP-MTS1338 for 6 h. Green, MSmeg; red, LAMP-1 or LysoTracker; blue, nuclei. Colocalization of bacteria with LAMP-1 or LysoTracker is indicated in orange. (B) Quantitative analysis of mycobacteria colocalization with LAMP-1 and LysoTracker. The data are presented as the mean ± SD of about 600 mycobacterial cells analyzed per each sample: * p < 0.05. Scale bars, 10 μm.

Figure 3

(A,B) Expression of cytokines in macrophages infected with MTS1338 and control strains. RAW264.7 macrophages were infected with control and MTS1338-expressing mycobacteria and analyzed for mRNA expression at 4 and 24 hpi. (C) Secretion of IL-6 by infected RAW264.7 cells at 24 hpi. The data are presented as the mean ± SD of three independent experiments: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 (by Student’s t-test). ND, not detected.

Figure 4

Confirmation of differential expression by qRT-PCR. mRNA expression of MSMEG_0965 (A), MSMEG_5872 (B), MSMEG_3886 (C), MSMEG_2433 (D), MSMEG_0615 (E) was determined in exponential (EXP) and stationary (STAT) cultures and normalized to that of 16S rRNA. * p < 0.05, ** p < 0.01.