Universal stress protein Rv2624c alters abundance of arginine and enhances intracellular survival by ATP binding in mycobacteria

Abstract

The universal stress protein family is a family of stress-induced proteins. Universal stress proteins affect latency and antibiotic resistance in mycobacteria. Here, we showed that Mycobacterium smegmatis overexpressing M. tuberculosis universal stress protein Rv2624c exhibits increased survival in human monocyte THP-1 cells. Transcriptome analysis suggested that Rv2624c affects histidine metabolism, and arginine and proline metabolism. LC-MS/MS analysis showed that Rv2624c affects the abundance of arginine, a modulator of both mycobacteria and infected THP-1 cells. Biochemical analysis showed that Rv2624c is a nucleotide-binding universal stress protein, and an Rv2624c mutant incapable of binding ATP abrogated the growth advantage in THP-1 cells. Rv2624c may therefore modulate metabolic pathways in an ATP-dependent manner, changing the abundance of arginine and thus increasing survival in THP-1 cells.

Figure 1. Overexpression of rv2624c in M. smegmatis increases survival in the Wayne hypoxia model.

(A) Genetic organization of the rv2624c gene locus. (B) Growth curve of pMV261/mc²155, pMV261-Rv2623/mc²155 and pMV261-Rv2624c/mc²155 cells in 7H9 medium. *p < 0.05, **p < 0.01. (C) Overexpression of Rv2624c increased survival under hypoxia. Growth curves of pMV261/mc²155 and pMV261-Rv2624c/mc²155 under hypoxia. The results shown are representative of three independent experiments. *p < 0.05, **p < 0.01. (D) Overexpression of rv2624c in M. smegmatis increases survival in human monocyte THP-1 cells. *p < 0.05, ***p < 0.001.

Figure 2. Overview of the differential expression profiles of pMV261/mc²155 and pMV261-Rv2624c/mc²155 cells.

(A) M. smegmatis metabolic pathways in which DEGs are enriched. KEGG-User Data Mapping assigned DEGs to “histidine metabolism” and “arginine and proline metabolism”. (B) Mass spectrometry of arginine (m/z 175, 116, 70). (C) Ion intensities of fragment ions of arginine. *p < 0.05.

Figure 3. Rv2624c sequence analysis.

Alignment of the universal stress protein domain of Rv2624c with other universal stress protein family proteins. The compared universal stress protein sequences are from DNAMAN V6 software. The predicted ATP-binding sites are indicated below the sequence.

Figure 4. Rv2624c is a nucleotide-binding universal stress protein.

(A) Gel filtration analysis of native His₆-Rv2624c. The upper panel indicates the retention time of the corresponding standard proteins. The lower panel indicates the retention time of His₆-Rv2624c. (B) The ATP-binding capacity of mutant Rv2624c was compared with that of wild-type Rv2624c. *p < 0.05.

Figure 5. ATP binding by Rv2624c is required for its ability to increase survival in infected THP-1 cells.

(A) The ATP-binding deficiencies of Rv2624c mutants did not influence growth on 7H10 medium. The mycobacterial strains indicated were serially diluted and were spotted onto solid 7H10 medium with 10% OADC. Pictures were taken after 3 d of incubation at 37 °C. (B) ATP-binding ability affects the survival of mycobacterial strains in THP-1 cells. Rv2624c mutant D17E with abrogated ATP-binding ability attenuated survival in THP-1 cells. The viability of Rv2624c mutant G109A, which had reduced ATP-binding ability, was comparable with that of wild-type Rv2624c. All results presented are representative of three independent experiments. p < 0.001.