Lysosome repositioning as an autophagy escape mechanism by Mycobacterium tuberculosis Beijing strain

Induction of host cell autophagy by starvation was shown to enhance lysosomal delivery to mycobacterial phagosomes, resulting in the restriction of Mycobacterium tuberculosis reference strain H37Rv. Our previous study showed that strains belonging to M. tuberculosis Beijing genotype resisted starvation-induced autophagic elimination but the factors involved remained unclear. Here, we conducted RNA-Seq of macrophages infected with the autophagy-resistant Beijing strain (BJN) compared to macrophages infected with H37Rv upon autophagy induction by starvation. Results identified several genes uniquely upregulated in BJN-infected macrophages but not in H37Rv-infected cells, including those encoding Kxd1 and Plekhm2, which function in lysosome positioning towards the cell periphery. Unlike H37Rv, BJN suppressed enhanced lysosome positioning towards the perinuclear region and lysosomal delivery to its phagosome upon autophagy induction by starvation, while depletion of Kxd1 and Plekhm2 reverted such effects, resulting in restriction of BJN intracellular survival upon autophagy induction by starvation. Taken together, these data indicated that Kxd1 and Plekhm2 are important for the BJN strain to suppress lysosome positioning towards the perinuclear region and lysosomal delivery into its phagosome during autophagy induction by starvation to evade starvation-induced autophagic restriction.

Ekspresi Protein Resuscitation promoting factor-D Mycobacterium tuberculosis pada sel CHO-K1 secara in-vitro

Abstrak Protein Resuscitation promoting factor–D (Rpf-D) merupakan protein transmembran dari Mycobacterium tuberculosis dan dieskpresikan pada fase reaktivasi infeksi laten menjadi aktif. Protein Rpf-D tersusun atas 154 asam amino berukuran 16 kDa yang dikode oleh 456 basa dari famili gen-Rpf. Pada studi sebelumnya, kami telah berhasil mengklona gen rpfD dari Mycobacterium tuberculosis strain Beijing kedalam plasmid pcDNA3.1, dikenal sebagai pcDNA3.1-rpfD. Dalam studi ini, kami mentransfeksi sel mamalia CHO-K1 dengan plasmid rekombinan pcDNA3.1-rpfD dan dilanjutkan dengan pewarnaan imunologis menggunakan serum mencit yang telah diimunisasi dengan pcDNA3.1-rpfD sebagai vaksin DNA. Hasil pewarnaan menunjukkan bahwa protein RpfD mampu diekspresikan pada sel mamalia. Selain itu, protein rekombinan yang diekspresikan ini terbukti bersifat imunogenik dan mampu menginduksi respon imun humoral pada hewan uji mencit Balb/C berupa IgG1, IgG2a, IgG2b dan IgG3. Hasil penelitian ini menunjukan potensi pemanfaatan protein rekombinan RpfD untuk penelitian lanjutan pengembangan vaksin ataupun uji diagnosis tuberkulosis. Kata kunci: Resuscitation promoting factor-D, Mycobacterium tuberculosis, sel CHO-K1, transfeksi, immunostaining Abstract Resuscitation promoting factor-D (Rpf-D) protein is known as a component of Mycobacterium tuberculosis cell wall which highly expressed in reactivation state of latent tuberculosis to turn in to an active infection. Rpf-D protein is a small protein consist of 154 amino acids encoded by 465 nucleotides in Rpf-gene family and having 16 kDa in size. In our previous study, we had successfully cloned Rpf-D gene of Mycobacterium tuberculosis Beijing strain into pcDNA3.1 plasmid expression system, which named as pcDNA3.1-rpfD later on. In this study, we transfect pcDNA3.1-rpfD into CHO-K1 mammalian cell line followed by immunostaining using mice sera immunized with pcDNA3.1-rpfD as DNA vaccine. Our result shows that our recombinant pcDNA3.1-rpfD construct can express recombinant RpfD protein in mammalian cells. On the other hand, this expressed recombinant protein has been proven to be immunogenic and able to induce a humoral immune response in Balb/C mice especially IgG1, IgG2a, IgG2b, and IgG3. Our study has shown the potency of recombinant RpfD protein which can be further developed as vaccine or diagnostic approach for tuberculosis. Keywords: Resuscitation promoting factor-D, Mycobacterium tuberculosis, CHO-K1 cells, transfection, immunostaining

The role of Mycobacterium tuberculosis complex species on apoptosis and necroptosis state of macrophages derived from active pulmonary tuberculosis patients

Objective: The role of Mycobacterium tuberculosis complex (MTBC) species in tuberculosis (TB) infection in human is still questioned. The aim of this study was to determine whether M. tuberculosis and M. bovis is associated with apoptosis and necroptosis by measuring the expression of specific signaling pathways components (Fas-associated protein with death domain (FADD) and receptor interacting protein 3 (RIP3)), and the level of apoptosis. Results: We recruited 30 patients with pulmonary TB; 24 patients were infected with M. tuberculosis Beijing strain and six patients with M. bovis BCG strain. M. tuberculosis-infected patients were more likely to have severe lung damage compared to those infected with M. bovis (odds ratio [OR]: 7.60; 95% confidence interval [CI]: 1.07-54.09). M. tuberculosis infection was associated with lower expression of FADD and lower apoptosis level of macrophages compared to M. bovis. No significant different of RIP3 between MTBC species groups. In conclusion, M. tuberculosis Beijing strain was associated with severe pulmonary damage, inhibited FADD expression and reduced apoptosis level of macrophages derived from pulmonary TB patients. This suggests that the M. tuberculosis Beijing strain is potentially to be used as determinant of disease progressivity and tissue damage in TB cases.

The role of Mycobacterium tuberculosis complex species on apoptosis and necroptosis state of macrophages derived from active pulmonary tuberculosis patients

Objective: The role of Mycobacterium tuberculosis complex (MTBC) species in tuberculosis (TB) infection in human is still questioned. The aim of this study was to determine whether M. tuberculosis and M. bovis is associated with apoptosis and necroptosis by measuring the expression of specific signaling pathways components (Fas-associated protein with death domain (FADD) and receptor interacting protein 3 (RIP3)), and the level of apoptosis.Results: We recruited 30 patients of pulmonary TB; 24 patients were infected with M. tuberculosis Beijing strain and six patients with M. bovis BCG strain. M. tuberculosis-infected patients were more likely to have severe lung damage compared to those infected with M. bovis (odds ratio [OR]: 7.60; 95% confidence interval [CI]: 1.07-54.09). M. tuberculosis infection was associated with lower expression of FADD and lower apoptosis level of macrophages compared to M. bovis. No significant different of RIP3 between MTBC species groups. In conclusion, M. tuberculosis Beijing strain was associated with severe pulmonary damage, inhibited FADD expression and reduced apoptosis level of macrophages derived from TB. This suggests that the M. tuberculosis Beijing strain is potentially to be used as determinant of disease progressivity and tissue damage in TB cases.

The role of Mycobacterium tuberculosis complex strain on apoptosis and necroptosis state of macrophages derived from active pulmonary tuberculosis patients

Objective The role of Mycobacterium tuberculosis complex (MTBC) strain in tuberculosis (TB) infection in human is still questioned. The aim of this study was to determine whether MTBC strain is associated with apoptosis and necroptosis by measuring the expression of specific signaling pathways components (Fas-associated protein with death domain (FADD) and receptor interacting protein 3 (RIP3)), as well as the level of apoptosis. Results We recruited 24 TB patients infected with M. tuberculosis Beijing strain and six patients with M. bovis BCG strain. Data indicated that those who infected with M. tuberculosis were more frequent to have severe lung damage than M. bovis (odds ratio [OR]: 7.60; 95% confidence interval [CI]: 1.07-54.09). M. tuberculosis infection was also associated with lower expression of FADD and lower apoptosis level of macrophage cells compared to M. bovis . No significant different of RIP3 between strain groups. In conclusion, M. tuberculosis Beijing strain is associated with severe pulmonary damage, inhibits FADD expression and reduces apoptosis level in macrophages derived from TB. This suggests that MTBC strain potentially be used as determinant of progressivity of disease and tissue damage in TB cases.

The role of Mycobacterium tuberculosis complex strain on apoptosis and necroptosis state of macrophages derived from active pulmonary tuberculosis patients

Objective The role of Mycobacterium tuberculosis complex (MTBC) strain in tuberculosis (TB) infection in human is still questioned. The aim of this study was to determine whether MTBC strain is associated with apoptosis and necroptosis by measuring the expression of specific signaling pathways components (Fas-associated protein with death domain (FADD) and receptor interacting protein 3 (RIP3)), as well as the level of apoptosis.Results We recruited 24 TB patients infected with M. tuberculosis Beijing strain and six patients with M. bovis BCG strain. Data indicated that those who infected with M. tuberculosis were more frequent to have severe lung damage than M. bovis (odds ratio [OR]: 7.60; 95% confidence interval [CI]: 1.07-54.09). M. tuberculosis infection was also associated with lower expression of FADD and lower apoptosis level of macrophage cells compared to M. bovis . No significant different of RIP3 between strain groups. In conclusion, M. tuberculosis Beijing strain is associated with severe pulmonary damage, inhibits FADD expression and reduces apoptosis level in macrophages derived from TB. This suggests that MTBC strain potentially be used as determinant of progressivity of disease and tissue damage in TB cases.

ACID-FAST BACILLI CONVERSION OF BEIJING AND NON-BEIJING STRAIN OF PULMONARY TUBERCULOSIS IN SOUTH SULAWESI

Beijing strains are a major part of the Mycobacterium tuberculosis Asian phylogenetic lineage. Beijing strains represent about 50% of all TB strains in East Asia and at least 13% of strains worldwide. Beijing strain of Mycobacterium tuberculosis is presumed as the factor of the increase in bacteria virulence and drug resistance, and the contributor in treatment failure. The aim of this study was to analyze the association between acid-fast bacilli conversion with strain genotipe Beijing and non-Beijing of pulmonary tuberculosis in South Sulawesi. The design of research was observational analytic with prospective approach. The sampling technique used consecutive sampling. Data were taken from active pulmonary tuberculosis patients’ medical record in Balai Besar Kesehatan Paru Masyarakat Makassar (Pulmonary Health Center of Makassar) and Community Health Center in Gowa Regency, South Sulawesi from March to June 2018. Collected sputum samples were screened for AFB and identified as Beijing strain and non Beijing strains using Multiplex PCR in Tropical Disease Institute of Universitas Airlangga. The results is showed that the characteristics of the respondents consisted of 12 respondents (33.3%) aged 56-65 years, 25 respondents (69.4%) men and 28 respondents (77.8%) had low category gradation of AFB smear. Univariate analysis showed 6 respondents (16.7%) with Beijing strains, 30 respondents (83.3%) with non-Beijing strains, 32 respondents (88.9%) conversion sputum AFB and 4 respondents (11.1%) non conversion sputum AFB. Bivariate analysis with Chi-Square statistical test shows that p value 0.022 < 0,05, that means there was association of Beijing strains with BTA conversion. Microscopic examination of BTA can be used to monitor and evaluate the treatment of new pulmonary TB patients undergoing treatment and the Beijing Mycobacterium tuberculosis strain has a significant correlation with the treatment failure of anti-tuberculosis drugs in South Sulawesi.