Ribosomal maturation factor (RimP) is essential for survival of nontuberculous mycobacteria Mycobacterium fortuitum under in vitro acidic stress conditions.

Mycobacterium fortuitum is an important human pathogenic NTM, which resists stress conditions inside macrophages by exploitation of specific genes. TnphoA-based transposon mutagenesis was employed to identify membrane genes responsible for survival of M. fortuitum under such stress conditions. A library of about 450 mutants was constructed after electroporation of vector pRT291 into wild-type M. fortuitum. On the basis of blue color development and alkaline phosphatase assay, 20 mutants were shortlisted to screen for growth and survival under acidic stress at pH 6.5, 5.5, 4.5, and 3.5. Mutant MT727 showed reduced growth and survival under acidic stress. The acid susceptible mutant MT727 was subjected to other in vitro stress conditions prevalent inside macrophages including oxidative, nutrient starvation and nitrosative stress. However, the mutant showed no appreciable difference in growth behavior under oxidative, nutrient starvation and nitrosative stress conditions in comparison to the wild type. Genomic and bioinformatics analysis of MT727 led to identification of putative ribosomal maturation factor RimP of M. fortuitum to be affected by mutagenesis, showing closest homology to M. abscessus RimP. In silico functional interaction of RimP protein using STRING database showed its interaction with proteins of ribosomal assembly and maturation. Results indicate role of rimP gene in survival of M. fortuitum under acidic stress conditions which may be further explored for use as a potential drug target against M. fortuitum and other mycobacterial infections.

Systematic study of non-natural short cationic lipopeptides as novel broad-spectrum antimicrobial agents.

We describe the design and synthesis of a new series of non-natural short cationic lipopeptides (MW = 700) as antimicrobial agents. All of the synthesized lipopeptides were tested against a range of microbes such as Gram-positive, Gram-negative bacteria, fungi including methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE). By systematic study of design template, we found that three ornithine residues conjugated with myristic acid are minimum requirement for a compound to be an antimicrobial agent. The most potent lipopeptide LP16 possesses broad-spectrum antimicrobial activity and has MICs in the range of 1.5-6.25 µg/mL against Escherichia coli, S. aureus, Pseudomonas aeruginosa, Bacillus subtilis, and MRSE. All lipopeptides showed high selectivity toward microbial strains as compared to human red blood cells (HC50 > 250 µg/mL). Moreover, most potent lipopeptides (LP16 and LP23) did not induce drug resistance in S. aureus even after 15 rounds of passaging. In addition, a representative lipopeptide (LP16) showed tryptic stability for 24 h. These results suggest the potential of short cationic lipopeptides to boost the discovery of future antimicrobial therapeutics.

Comparative mode of action of novel hybrid peptide CS-1a and its rearranged amphipathic analogue CS-2a.

Cell selective, naturally occurring, host defence cationic peptides present a good template for the design of novel peptides with the aim of achieving a short length with improved antimicrobial potency and selectivity. A novel, short peptide CS-1a (14 residues) was derived using a sequence hybridization approach on sarcotoxin I (39 residues) and cecropin B (35 residues). The sequence of CS-1a was rearranged to enhance amphipathicity with the help of a Schiffer-Edmundson diagram to obtain CS-2a. Both peptides showed good antibacterial activity in the concentration range 4-16 µg·mL(-1) against susceptible as well as drug-resistant bacterial strains, including the clinically relevant pathogens Acenatobacter sp. and methicillin-resistant Staphylococcus aureus. The major thrust of these peptides is their nonhaemolytic activity against human red blood cells up to a high concentration of 512 µg·mL(-1). Compared to CS-1a, amphipathic peptide CS-2a showed a more pronounced α-helical conformation, along with a better membrane insertion depth in bacterial mimic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) small unilamellar vesicles. With equivalent lipid-binding affinity, the two peptides assumed different pathways of membrane disruption, as demonstrated by calcein leakage and the results of transmission electron microscopy on model bacterial mimic large unilamellar vesicles. Extending the work from model membranes to intact Escherichia coli cells, differences in membrane perturbation were visible in microscopic images of peptide-treated E. coli. The present study describes two novel short peptides with potent activity, cell selectivity and divergent modes of action that will aid in the future design of peptides with better therapeutic potential.