scholarly journals Probing Differences in Gene Essentiality Between the Human and Animal Adapted Lineages of the Mycobacterium tuberculosis Complex Using TnSeq

2021 ◽  
Vol 8 ◽  
Author(s):  
Amanda J. Gibson ◽  
Ian J. Passmore ◽  
Valwynne Faulkner ◽  
Dong Xia ◽  
Irene Nobeli ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nucleic Acid ◽  
Genetic Basis ◽  
Functional Categories ◽  
Tuberculosis Complex ◽  
Gene Essentiality ◽  
Peptidoglycan Synthesis ◽  
Human Counterpart ◽  
Species Specific

Members of the Mycobacterium tuberculosis complex (MTBC) show distinct host adaptations, preferences and phenotypes despite being >99% identical at the nucleic acid level. Previous studies have explored gene expression changes between the members, however few studies have probed differences in gene essentiality. To better understand the functional impacts of the nucleic acid differences between Mycobacterium bovis and Mycobacterium tuberculosis, we used the Mycomar T7 phagemid delivery system to generate whole genome transposon libraries in laboratory strains of both species and compared the essentiality status of genes during growth under identical in vitro conditions. Libraries contained insertions in 54% of possible TA sites in M. bovis and 40% of those present in M. tuberculosis, achieving similar saturation levels to those previously reported for the MTBC. The distributions of essentiality across the functional categories were similar in both species. 527 genes were found to be essential in M. bovis whereas 477 genes were essential in M. tuberculosis and 370 essential genes were common in both species. CRISPRi was successfully utilised in both species to determine the impacts of silencing genes including wag31, a gene involved in peptidoglycan synthesis and Rv2182c/Mb2204c, a gene involved in glycerophospholipid metabolism. We observed species specific differences in the response to gene silencing, with the inhibition of expression of Mb2204c in M. bovis showing significantly less growth impact than silencing its orthologue (Rv2182c) in M. tuberculosis. Given that glycerophospholipid metabolism is a validated pathway for antimicrobials, our observations suggest that target vulnerability in the animal adapted lineages cannot be assumed to be the same as the human counterpart. This is of relevance for zoonotic tuberculosis as it implies that the development of antimicrobials targeting the human adapted lineage might not necessarily be effective against the animal adapted lineage. The generation of a transposon library and the first reported utilisation of CRISPRi in M. bovis will enable the use of these tools to further probe the genetic basis of survival under disease relevant conditions.

scholarly journals Probing differences in gene essentiality between the human and animal adapted lineages of the Mycobacterium tuberculosis complex using TnSeq

2021 ◽  
Author(s):  
Amanda J Gibson ◽  
Ian J Passmore ◽  
Valwynne Faulkner ◽  
Dong Xia ◽  
Irene Nobeli ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nucleic Acid ◽  
Mycobacterium Tuberculosis Complex ◽  
Functional Categories ◽  
Tuberculosis Complex ◽  
Gene Essentiality ◽  
Peptidoglycan Synthesis ◽  
Human Counterpart ◽  
Species Specific

Members of the Mycobacterium tuberculosis complex (MTBC) show distinct host adaptations, preferences and phenotypes despite being >99% identical at the nucleic acid level. Previous studies have explored gene expression changes between the members, however few studies have probed differences in gene essentiality. To better understand the functional impacts of the nucleic acid differences between Mycobacterium bovis and Mycobacterium tuberculosis we used the Mycomar T7 phagemid delivery system to generate whole genome transposon libraries in laboratory strains of both species and compared the essentiality status of genes during growth under identical in vitro conditions. Libraries contained insertions in 54% of possible TA sites in M. bovis and 40% of those present in M. tuberculosis, achieving similar saturation levels to those previously reported for the MTBC. The distributions of essentiality across the functional categories were similar in both species. 527 genes were found to be essential in M. bovis whereas 477 genes were essential in M. tuberculosis and 370 essential genes were common in both species. CRISPRi was successfully utilised in both species to determine the impacts of silencing genes including wag31, a gene involved in peptidoglycan synthesis and Rv2182c/Mb2204c, a gene involved in glycerophospholipid metabolism. We observed species specific differences in the response to gene silencing, with the inhibition of expression of Mb2204c in M. bovis showing significantly less growth impact than silencing its ortholog (Rv2182c) in M. tuberculosis. Given that glycerophospholipid metabolism is a validated pathway for antimicrobials, our observations suggest that target vulnerability in the animal adapted lineages cannot be assumed to be the same as the human counterpart. This is of relevance for zoonotic tuberculosis as it implies that the development of antimicrobials targeting the human adapted lineage might not necessarily be effective against the animal adapted lineage. The generation of a transposon library and the first reported utilisation of CRISPRi in M. bovis will enable the use of these tools to further probe the genetic basis of survival under disease relevant conditions.

scholarly journals Detection of rifampin- and ciprofloxacin-resistant Mycobacterium tuberculosis by using species-specific assays for precursor rRNA.

1996 ◽  
Vol 40 (8) ◽  
pp. 1790-1795 ◽  
Author(s):  
G A Cangelosi ◽  
W H Brabant ◽  
T B Britschgi ◽  
C K Wallis
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nucleic Acid ◽  
Lower Limit ◽  
Cultured Cells ◽  
Rrna Synthesis ◽  
Bacterial Cells ◽  
Mycobacterium Species ◽  
Antibiotic Resistant ◽  
Species Specific

rRNA precursor (pre-rRNA) molecules carry terminal stems which are removed during rRNA synthesis to form the mature rRNA subunits. Their abundance in bacterial cells can be markedly affected by antibiotics which directly or indirectly inhibit RNA synthesis. We evaluated the feasibility of rapidly detecting antibiotic-resistant Mycobacterium tuberculosis strains by measuring the effects of brief in vitro antibiotic exposure on mycobacterial pre-rRNA. By hybridizing extracted M. tuberculosis nucleic acid with radiolabeled nucleic acid probes specific for pre-16S rRNA stem sequences, we detected clear responses to rifampin and ciprofloxacin within 24 and 48 h, respectively, of exposure of cultured cells to these drugs. Detectable pre-rRNA was depleted in susceptible cells but remained abundant in resistant cells. In contrast, no measurable responses to isoniazid or ethambutol were observed. Probes for pre-rRNA were specific for the M. tuberculosis complex when tested against a panel of eight Mycobacterium species and 48 other bacteria. After 24 h of incubation with rifampin, resistant M. tuberculosis strains were detectable in a reverse transcriptase PCR assay for pre-rRNA with a calculated lower limit of sensitivity of approximately 10(2) cells. Susceptible cells were negative in this assay at over 500 times the calculated lower limit of sensitivity. This general approach may prove useful for rapidly testing the susceptibility of slowly growing Mycobacterium species to the rifamycin and fluoroquinolone drugs and, with possible modifications, to other drugs as well.

scholarly journals In Vitro Activities of Linezolid against Clinical Isolates of Mycobacterium tuberculosis Complex Isolated in Taiwan over 10 Years

2008 ◽  
Vol 52 (6) ◽  
pp. 2226-2227 ◽  
Author(s):  
Tsi-Shu Huang ◽  
Yung-Ching Liu ◽  
Cheng-Len Sy ◽  
Yao-Shen Chen ◽  
Hui-Zin Tu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Tuberculosis Complex ◽  
Baseline Period ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Tuberculosis Complex

ABSTRACT Significant increases in the MIC90s of linezolid in multidrug-resistant Mycobacterium tuberculosis isolates were seen between the baseline period of 2001 to 2003 (0.5 μg/ml) and 2004 (2 μg/ml). The MICs were 4 μg/ml in three strains. Both fluoroquinolones (except levofloxacin) and kanamycin were found to have statistically significant degrees of concordance with linezolid.

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