Phylogenetic and Proteomic Analysis of Pyruvate-Ferredoxin Oxidoreductase; a Redox Enzyme Involved in the Pharmacological Activation of Nitro-Based Prodrugs in Protozoan and Bacterial Species
Authors: Duwor Seth , Mäser Pascal , Brites Daniela 
Published: November 2023

Introduction: The distribution of typical bacterial redox enzymes such as pyruvate-ferredoxin oxidoreductase (PFOR) in protozoa remains interestingly puzzling. Previous studies have demonstrated diverse cellular localizations of PFOR in amitochondriate anaerobic protozoa such as Trichomonas vaginalis. PFOR is of particular pharmacological importance because it catalyzes the reductive bio-activation of nitro-based prodrugs to cytotoxic radical metabolites. Metronidazole was developed primarily as an antiprotozoal agent against infections caused by T. vaginalis. However, its antimicrobial spectrum was subsequently expanded to cover anaerobic bacterial infections. It has been shown that mutations in the genes encoding PFOR result in inherent resistance of T. vaginalis and anaerobic bacteria to metronidazole.

Aims: To analyze the phylogenetic distribution of PFOR in selected protozoa and bacteria using proteins encoded by housekeeping genes as controls. Using comparative bioinformatics to test the hypothesis that PFOR was most likely acquired through horizontal gene transfer from bacteria, proteome-wide analysis and gene expression analysis to identify other genes that were putatively acquired through horizontal gene transfer from bacteria to protozoa.

Methods: Sequence similarity queries were performed using the proteins of interest against the NCBI non-redundant protein sequence database with BLASTP version 2.12.0+ from the NCBI website. Multiple sequence alignments were performed with MEGA version X software using the Muscle algorithm. These were then exported in Mega format to construct phylogenetic trees using the neighbor-joining algorithm and the Poisson substitution model. Complete proteomes of representative protozoan and bacterial species were downloaded in fasta format from the ensemblgenomes database. HMM profile libraries were used to screen the proteomes with hmmscan of the HMMer package. Whole proteome BLAST searches were performed between protozoan species of interest against closely related protozoa. The results were compared to the proteome BLAST against the intestinal bacteria Desulfovibrio vulgaris. Gene enrichment analysis was performed between the exclusively present proteins in the protozoa and D. vulgaris. Only results with p-values &lt;0.05 were considered for analysis.

Results and conclusions: A plausible explanation for the restricted occurrence of PFOR in protozoa is based on the hypothesis that bacteria serve as potential sources of genes that enhance optimal adaptation of protozoa in hostile environments. The expanded cladograms of Entamoeba and Cryptosporidium with their closely related genera substantiated this hypothesis. The exclusively expressed proteins obtained from E. histolytica and the putative bacterial gene donor, D. vulgaris, showed an over-representation of eleven genes involved in small molecule metabolism, generation of precursor metabolites, and carbohydrate metabolism. If these results can be reproduced in other PFOR-possessing protozoa, it would provide a more validated evidence to support the horizontal transfer of pfor from bacteria. Subsequent syntenic analyses of the significantly enriched genes would be required to provide further information regarding the positional relatedness of these genes at the chromosomal level. Since metronidazole is an established and well tolerated drug for treating infections caused by PFOR-possessing pathogens, it can be considered as a potential drug candidate for the treatment of infections caused by Cryptosporidium, Spironucleus and Blastocystis.