MmpS5-MmpL5 Transporters Provide Mycobacterium smegmatis Resistance to imidazo[1,2-b][1,2,4,5]tetrazines

Abstract

The emergence and spread of drug-resistant Mycobacterium tuberculosis strains (including MDR, XDR, and TDR) force scientists worldwide to search for new anti-tuberculosis drugs. We have previously reported a number of imidazo[1,2-b] [1,2,4,5]tetrazines-putative inhibitors of mycobacterial eukaryotic-type serine-threonine protein-kinases, active against M. tuberculosis. Whole genomic sequences of spontaneous drug-resistant M. smegmatis mutants revealed four genes possibly involved in imidazo[1,2-b][1,2,4,5]tetrazines resistance; however, the exact mechanism of resistance remain unknown. We used different approaches (construction of targeted mutants, overexpression of the wild-type (w.t.) and mutant genes, and gene-expression studies) to assess the role of the previously identified mutations. We show that mutations in MSMEG_1380 gene lead to overexpression of the mmpS5-mmpL5 operon in M. smegmatis, thus providing resistance to imidazo[1,2-b][1,2,4,5]tetrazines by increased efflux through the MmpS5-MmpL5 system, similarly to the mechanisms of resistance described for M. tuberculosis and M. abscessus. Mycobacterial MmpS5-MmpL5 transporters should be considered as an MDR-efflux system and they should be taken into account at early stages of anti-tuberculosis drug development.

Identification of Mutations Conferring Tryptanthrin Resistance to Mycobacterium smegmatis

“…The MmpS5-MmpL5 efflux system was previously shown to provide resistance to multiple drugs in different mycobacterial species [ 25 , 26 , 27 ], including resistance to bedaquiline in clinical M. tuberculosis isolates [ 28 ]. We determined the minimal inhibitory concentrations (MICs) of 1a and 1b on three M. smegmatis strains differing in mmpS5-mmL5 operon expression levels: mc2 155 , Δmmp5 and atr9c .…”

“…deletion in the mmpS5-mmpL5 operon, and hypersensitive to drugs, subjected to MmpS5-MmpL5 efflux, such as imidazo[1,2- b ][1,2,4,5]tetrazines [ 29 ]. M. smegmatis atr9c, on the contrary, carries a mutation in MSMEG_1380 , leading to overexpression of mmpS5-mmpL5 genes, and is resistant to drugs, subjected to MmpS5-MmpL5 efflux [ 27 ]. Thus, comparing MIC values on these three strains can be used for prescreening antimycobacterial drugs candidates for potential MmpS5-MmpL5 efflux mediated drug resistance.…”

“…MmpS5-MmpL5 is an important multidrug-efflux-pump system, which is able to provide mycobacteria resistance to multiple drugs such as bedaquiline, clofazimine [ 39 ], azoles [ 25 ], thiacetazone derivatives [ 26 ], and imidazo[1,2- b ][1,2,4,5]tetrazines [ 27 ]. We used a test-system involving two M. smegmatis recombinant strains to show that mmpS5-mmpL5 operon overexpression provides resistance to tryptanthrin ( 1a ), while deletion of mmpS5-mmpL5 operon leads to hypersensitivity to 8-fluorotryptanthrin ( 1b ).…”

Resistance against Membrane-Inserting MmpL3 Inhibitor through Upregulation of MmpL5 in Mycobacterium tuberculosis

“…In conclusion, we report the identification of a pump-based resistance mechanism to the spiroketal indolyl Mannich base lead SIMBL MmpL5-mediated resistance has been reported for multiple antimycobacterials (10,(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28).…”

“…MmpR5 was reported to repress expression of its neighboring, divergently transcribed siderophore transporter and multisubstrate efflux pump gene mmpL5 ( m ycobacterial m embrane p rotein l arge 5) and is hence named MmpR5 ( m ycobacterial m embrane p rotein r epressor 5) ( 10 – 16 ). Notably, numerous MmpR5 mutations have been associated with mycobacterial resistance to a range of chemically and mechanistically diverse drugs, including azoles, bedaquiline, clofazimine, the ionophores nigericin and A23187 (calcimycin), thiacetazone, and imidazo[1,2- b ][1,2,4,5]tetrazine derivatives ( 10 , 16 – 28 ). In fact, the SIMBL resistance mutation in the DNA-binding domain of MmpR5 detected in M. tuberculosis M1 (A202G/S68G) is known to confer resistance to bedaquiline and clofazimine ( 18 , 19 ).…”