Pathogens utilize a diverse set of signal transduction mechanisms to respond to host-derived stresses, with phosphotransfer-mediated two component systems (TCS) playing key roles in virulence factor regulation. Staphylococcus aureus encodes an alternative protease-mediated TCS prototype known as BlaRI involved in inducible β-lactam resistance. BlaR senses extracellular β-lactams, leading to activation of a cytoplasmic protease domain able to cleave DNA-bound BlaI dimers, de-repressing blaRI and blaZ (β-lactamase). The two known mycobacterial BlaRI-type systems in M. tuberculosis (Mtb) and M. abscessus ( Mab) are characterized by BlaR orthologs with conserved zinc metalloprotease domain but lacking an extracellular β-lactam binding domain. BlaIR Mtb and BlaIR Mab (renamed BlaIR to reflect inverted genomic organization) regulate β-lactamase expression ( Mtb only) and respiration (both Mtb and Mab). In this study, we have identified a second BlaRI-type system in Mab, MAB_4287-4288 (BlaIR2). Using RT-PCR and EMSA, we established that BlaIR2 Mab is encoded within a five gene operon, a unique characteristic in BlaRI-type systems, and is auto-regulatory. Identification of putative BlaI2 Mab binding motifs revealed a predicted regulon comprised of several genes involved in respiration, with some overlap with the BlaI1 Mab regulon. Finally, our data demonstrated that BlaIR2 Mab was also induced by the respiration inhibitor clofazimine (CFZ) similarly to BlaIR1 Mab, with evidence of possible crosstalk between BlaRI systems. Overall, this study established MAB_4287-4288 (BlaIR2 Mab) as a second BlaRI-type system in Mab, whose role may overlap or intersect that of BlaRI1 Mab. However, the activation mechanism and full role of BlaIR2 Mab in Mab stress responses and pathogenesis remains to be elucidated.

Mycobacterium abscessus (Mab) is highly drug resistant, and understanding regulation of antibiotic resistance is critical to future antibiotic development. Regulatory mechanisms controlling Mab’s β-lactamase (Bla Mab) that mediates β-lactam resistance remain unknown. S. aureus encodes a prototypical protease-mediated two-component system BlaRI regulating the β-lactamase BlaZ. BlaR binds extracellular β-lactams, activating an intracellular peptidase domain which cleaves BlaI to derepress blaZ. Mtb encodes homologs of BlaRI, that regulate the Mtb β-lactamase, blaC, but also additional genes related to respiration. We identified orthologs of blaRIMtb in Mab and hypothesized that they regulate blaMab. Surprisingly, neither deletion of blaRIMab nor overexpression of only blaIMab altered blaMab expression or β-lactam susceptibility. However, BlaI Mab did bind to conserved motifs upstream of several Mab genes involved in respiration, yielding a putative regulon that partially overlapped with BlaI Mtb. Prompted by evidence that respiration inhibitors including clofazimine (CFZ) induce the BlaI regulon in Mtb, we found that CFZ triggers induction of blaIRMab and its downstream regulon. Highlighting an important role for BlaRI Mab in adapting to disruptions in energy metabolism, constitutive repression of the BlaI Mab regulon rendered Mab highly susceptible to CFZ. In addition to our unexpected findings that BlaIR Mab does not regulate β-lactam resistance, this study highlights the novel role for mycobacterial BlaRI-type regulators in regulating electron transport and respiration.