In vitro antileishmanial activity of parasporal-associated protein preparation from Lysinibacillus xylanilyticus SAN 1.5 involving mitochondrial membrane potential disruption in Leishmania amazonensis promastigotes and genomic characterization

Leishmaniasis is among the top ten neglected tropical diseases, requiring new and more efficient treatment options. In this study, lyophilized parasporal-associated protein preparations from nine spore-forming bacterial isolates were tested for activity against Leishmania amazonensis promastigotes. Protein preparation from bacterial isolate SAN 1.5 showed the best activity in the initial analysis, therefore it was selected for further evaluation. SAN 1.5 produced ellipsoidal spores and small round parasporal inclusions. DNA amplification and sequencing of the 16 S region showed more than 99% identity with the genus Lysinibacillus . Whole genome sequencing followed by phylogenetic analysis showed 96.28% identity to Lysinibacillus xylanilyticus . No genes encoding the commonly recognized parasporal crystal toxins, such as Cry or Cyt proteins, were identified in the SAN 1.5 genome. Nevertheless, S-layer-like proteins were identified in the annotated SAN 1.5 genome. This finding is of interest because S-layer proteins have previously been reported to form parasporal inclusions in certain Bacillus strains. However, the expression of the candidate S-layer protein and its contribution to the observed parasporal structures and antileishmanial activity remain to be experimentally confirmed. In the quantitative bioassay experiments against the promastigotes using different concentrations of the protein preparation, maximum inhibitory effects observed were 46% and 67% after 24 and 48 h, respectively, with a half maximal inhibitory concentration (IC 50 ) value of 18.04 µg/mL (95% CI 9.94–32.72) after 48 h. The protein preparation showed no impact on the morphology or plasma membrane integrity of promastigotes. Nevertheless, 24 h after the treatment, protein preparation from SAN 1.5 (1/2 ×; 1 × and 2 × IC 50 ) was able to reduce the mitochondrial membrane potential by about 50%, suggesting mitochondrial involvement in the observed antileishmanial effect. These findings highlight bacterial parasporal-associated protein preparations as promising leads for alternative antileishmanial strategies, with their distinct mitochondrial-associated activity warranting further investigation, particularly in view of the limitations and adverse effects associated with currently available therapies.