Identification of Poly(ADP-ribose) Polymerase 9 (PARP9) as a Potent Suppressor for Mycobacterium tuberculosis Infection

Abstract

ADP-ribosylation is a reversible and dynamic post-translational modification mediated by ADP-ribosyltransferases (ARTs). Poly(ADP-ribose) polymerases (PARPs) are an important family of human ARTs. ADP-ribosylation and PARPs have crucial functions in host-pathogen interaction, especially in viral infections. However, the functions and potential molecular mechanisms of ADP-ribosylation and PARPs in Mycobacterium infection remain unknown. In this study, bioinformatics analysis revealed significantly changed expression levels of several PARPs in tuberculosis patients compared to healthy individuals. Moreover, the expression levels of these PARPs returned to normal following tuberculosis treatment. Then, the changes in the expression levels of PARPs during Mycobacterium infection were validated in Tohoku Hospital Pediatrics-1 (THP1)-induced differentiated macrophages infected with Mycobacterium model strains bacillus Calmette-Guérin (BCG) and in human lung adenocarcinoma A549 cells infected with Mycobacterium smegmatis (Ms), respectively. The mRNA levels of PARP9, PARP10, PARP12, and PARP14 were most significantly increased during infection, with corresponding increases in protein levels, indicating the possible biological functions of these PARPs during Mycobacterium infection. In addition, the biological function of host PARP9 in Mycobacterium infection was further studied. PARP9 deficiency significantly increased the infection efficiency and intracellular proliferation ability of Ms, which was reversed by the reconstruction of PARP9. Collectively, this study updates the understanding of changes in PARP expression during Mycobacterium infection and provides evidence supporting PARP9 as a potent suppressor for Mycobacterium infection.

Supplementary information: The online version contains supplementary material available at 10.1007/s43657-023-00112-2.

Keywords: Mycobacterium; Mycobacterium smegmatis; Poly(ADP-ribose) polymerase 9; Poly(ADP-ribose) polymerases; Tuberculosis.

Fig. 1

Transcription levels of PARPs in TB patients. a Transcription levels of PARPs in TB patients (red) compared with healthy donors (HD, blue). b Transcription levels of PARPs before treatment (red) or six months after treatment (blue) in TB patients. c Transcription levels of PARPs in whole blood compared 242 TB patients (red) with 498 normal individuals (blue). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 2

Transcription levels of PARPs in lungs of mice before (blue) or 21 days after Mtb infection (red). **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 3

Expression changes of pro-inflammatory cytokines and PARPs infected with BCG or Ms at the mRNA level. a mRNA expression of proinflammatory cytokines (TNF-α, IL-6, and IFN-γ) and PARPs which significantly changed in macrophages after BCG infected at 0 (grey), 12 (green), 24 (blue), and 48 (orange) hours. b mRNA expression of proinflammatory cytokines (IL-6 and TNF-α) and four PARPs (PARP9, PARP10, PARP12, and PARP14) showing significant changes in A549 cell line after Ms infection. *p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 4

Protein expression changes of PARP9, PARP10, PARP12, and PARP14 in two cell lines after Mtb model strain infection. a Protein expression of PARP9, PARP10, PARP12, and PARP14 in macrophages after BCG infection at 0, 12, 24, and 48 h. b Protein expression of PARP9, PARP10, PARP12, and PARP14 in A549 cells after Ms infection. *p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 5

Analysis of Ms infection efficiency and intracellular proliferation ability in PARP9-deficient A549 cells. a Two sgRNAs targeting the third exon of the PARP9 gene. b Electrophoresis results of genomic PCR products of wild-type (WT) and PARP9-deficient (KO1 and KO2) A549 cells. c Validation of PARP9 expression in the PARP9-dificient A549 cells. d Bar chart showing CFU counts in WT and PARP9-deficient A549 cells at different time points (0, 6, 12, and 48 h) post-infection and analysis of infection efficiency. e Relative CFU fold changes (normalized to 0 h) of WT and PARP9-deficient A549 cells at different time points post-infection and analysis of intracellular proliferation ability. ns, p > 0.05, **p < 0.01; ***p < 0.001; ***p < 0.0001

Fig. 6

Analysis of Ms infection efficiency and intracellular proliferation ability in PARP9-reconstructed A549 cells. a Reconstitution of PARP9 in the PARP9-deficient A549 cell line (KO2) and validation of PARP9 expression. b Bar chart showing CFU counts in WT, PARP9-deficient (KO2), and PARP9-reconstructed (KR1 and KR2) A549 cells at different time points (0, 6, 12, and 48 h) post-infection and analysis of infection efficiency. c Relative CFU fold changes (normalized to 0 h) of WT, PARP9-deficient (KO2), and PARP9-reconstructed (KR1 and KR2) A549 cells at different time points (0, 6, 12, and 48 h) post-infection and analysis of intracellular proliferation ability. ns, p > 0.05, *p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001