A novel homoarginine-containing cyclic peptide pioamide with selective antipseudomonal activity isolated from the nematode symbiont Photorhabdus khanii.

Gram-negative bacteria harbor an outer membrane that physically protects them from the penetration of antibiotics into the cells. This barrier makes it challenging to develop antibiotics that effectively kill Gram-negative pathogens. The entomopathogenic bacterium Photorhabdus species produces various bioactive molecules and is receiving attention as an attractive source of novel antibiotics. We identified a novel antipseudomonal antibiotic, pioamide, from the culture supernatant of Photorhabdus khanii HGB1456, a strain that produces darobactin, which selectively kills Gram-negative bacteria. Pioamide, a pentapeptide antibiotic with a molecular weight of 704, exhibits selective activity against Pseudomonas aeruginosa but does not exhibit any activity against other bacteria or human cell lines. Whole-genome sequencing of spontaneous pioamide-resistant mutants of P. aeruginosa revealed mutations in pmrB, which encodes a two-component regulatory system response regulator that modifies the lipopolysaccharide composition in Gram-negative bacteria, conferring pioamide resistance to P. aeruginosa. Furthermore, the susceptibility of both P. aeruginosa PAO1 and the mutant strain PΔ6-Pore, which overexpresses porins and lacks six efflux pumps, to pioamide was identical, indicating that porins and the efflux pump exert no significant effect on the activity of pioamide. These results suggest that pioamide either targets the cell surface of P. aeruginosa or is incorporated via a species-specific uptake mechanism. Our findings highlight the potential of Photorhabdus strains as an attractive source for the discovery of antibiotics active against Gram-negative pathogens.

Importance: The rise of multidrug-resistant Gram-negative bacteria is a growing threat to global public health. Narrow-spectrum antibiotics minimize disruption of the host microbiota and reduce the risk of resistance development in off-target bacteria. In the field of antibacterial discovery, developing compounds effective against Pseudomonas aeruginosa remains particularly challenging. Although Photorhabdus species are known to produce various antibiotics, their potential remains largely underexplored. In this study, we applied differential screening to a highly concentrated culture extract of Photorhabdus khanii HGB1456 and discovered pioamide, a novel cyclic peptide with unusual selective activity against P. aeruginosa. Mutations in pmrB confer pioamide resistance to P. aeruginosa. However, the mechanism of action is distinct from that of colistin, which also involves resistance conferred by pmrB mutations. These findings underscore the untapped potential of Photorhabdus species as an attractive source of species-specific antibiotics and highlight the utility of differential screening for discovering compounds with targeted antibacterial activity.