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

### **Dmitry A. Maslov \*, Kirill V. Shur, Aleksey A. Vatlin and Valery N. Danilenko**

Laboratory of Bacterial Genetics, Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow 119333, Russia; shurkirill@gmail.com (K.V.S.); vatlin\_alexey123@mail.ru (A.A.V.); valerid@vigg.ru (V.N.D.)

**\*** Correspondence: Maslov\_da@vigg.ru

Received: 4 February 2020; Accepted: 25 February 2020; Published: 28 February 2020

**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.

**Keywords:** *Mycobacterium smegmatis*; imidazo[1,2-*b*][1,2,4,5]tetrazine; MmpS5-MmpL5; efflux; drug discovery; drug resistance; tuberculosis
