M. tuberculosis PrrA binds the dosR promoter and regulates mycobacterial adaptation to hypoxia.

The PrrAB two-component system (TCS) is essential for Mycobacterium tuberculosis viability. Previously, it was demonstrated that PrrA binds DNA in the absence of PrrB-mediated transphosphorylation and that non-cognate serine/threonine-kinases phosphorylate PrrA threonine-6 (T6). Therefore, we investigated the differential binding affinity and regulatory properties of the M. tuberculosis-derived wild-type PrrA, PrrA phosphomimetic (D58E, T6E), and PrrA phosphoablative (D58A, T6A) proteins with the prrAMtb, dosRMtb, and cydAMtb genes. While we hypothesized greater DNA binding affinity and more pronounced regulation by PrrA phosphomimetic variants, recombinant, wild-type PrrAMtb bound DNA with greatest affinity. Collectively, wild-type PrrAMtb recombinant protein displayed the highest binding affinity to the dosRMtb promoter (KD 3.46 ± 2.09 nM), followed by the prrAMtb promoter (KD 9.00 ± 2.66 nM). To establish PrrAMtb regulatory activity, we constructed M. smegmatis ΔprrABMsmeg::prrAMtb strains with each of the PrrAMtb variants and extrachromosomal prrAMtb, dosRMtb, and cydAMtb promoter-mCherry reporter fusions. Our findings showed that PrrAMtb is autoregulatory and induces dosRMtb expression only during in vitro, hypoxic growth. Combined expression of prrABMtb in M. smegmatis ΔprrAB significantly induced cydAMtb promoter-mCherry expression. Our studies advanced the understanding of PrrA function and PrrAB phosphorylation-mediated regulatory mechanisms and control of mycobacterial dosR and cydA hypoxic and low-oxygen responsive genes.

High-Throughput Screening Identifies Synthetic Peptides with Antibacterial Activity against Mycobacterium abscessus and Serum Stability.

The rise in antibiotic resistance in bacteria has spawned new technological approaches for identifying novel antimicrobials with narrow specificity. Current antibiotic treatment regimens and antituberculosis drugs are not effective in treating Mycobacterium abscessus. Meanwhile, antimicrobial peptides are gaining prominence as alternative antimicrobials due to their specificity toward anionic bacterial membranes, rapid action, and limited development of resistance. To rapidly identify antimicrobial peptide candidates, our group has developed a high-density peptide microarray consisting of 125,000 random synthetic peptides screened for interaction with the mycobacterial cell surface of M. abscessus morphotypes. From the array screening, peptides positive for interaction were synthesized and their antimicrobial activity was validated. Overall, six peptides inhibited the M. abscessus smooth morphotype (IC50 = 1.7 µM for all peptides) and had reduced activity against the M. abscessus rough morphotype (IC50 range: 13-82 µM). Peptides ASU2056 and ASU2060 had minimum inhibitory concentration values of 32 and 8 µM, respectively, against the M. abscessus smooth morphotype. Additionally, ASU2060 (8 µM) was active against Escherichia coli, including multidrug-resistant E. coli clinical isolates, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus. ASU2056 and ASU2060 exhibited no significant hemolytic activity at biologically relevant concentrations, further supporting these peptides as promising therapeutic candidates. Moreover, ASU2060 retained antibacterial activity after preincubation in human serum for 24 h. With antimicrobial resistance on the rise, methods such as those presented here will streamline the peptide discovery process for targeted antimicrobial peptides.