Distantly Related Homologue of UhpT in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa (PA) is an opportunistic Gram-negative bacteria that affects patients in intensive care units and chronic respiratory disease patients. Compared to other bacteria, it has a wide genome (around 6.3-Mb) that supports its metabolic versatility and antimicrobial resistance. Fosfomycin (FF) is primarily used as an oral treatment for urinary tract infections (UTIs). FF diffuses inside the cell via glycerol-3-phosphate transporter (GlpT) PA, as well as in other bacteria. In other bacteria, such as E. coli, glucose-6-phosphate transporter (UhpT) functions as FF transporter. Since mutant GlpT leads to FF resistant PA, it is assumed that GlpT is the only FF transporter. However, it is also assumed that PA uses glucose-6-phosphate and, thus, homologous proteins of UhpT may be present in its genome. Here, we present an attempt to find a distant related homologue of UhpT in PA. A Hidden Markov Model (HMM) was created to seek for Major facilitator family (MFS) domain in 21 PA genomes of 14 CF patients annotated with prokka and the statistical analysis was performed (MCC: 0.84, ACC: 0.99). Then, the HMM was applied to PA genomes. Besides the actual GlpT, annotated as glpt_1, one more GlpT protein was found in 21 out of 21 genomes, annotated as glpt_2. Since glpt_2 clusters closer to UhpT than GlpT, glpt_2 was selected to build a model. Computing a structural superimposition, the model and the template of UhpT have 0.6 Å of RMSD. The model of glpt_2 has some characteristics that are fundamental to UhpT functions. The binding site, consisting of 2 arginines (Arg46 and Arg275) and Lys45, is totally conserved, as well as the topology of the structure. Asp90 is also conserved in glpt_2 model. No studies aimed at searching for distant related homologous of UhpT. Since the high genetic exchange and high mutational rate in bacteria, it is likely that PA has a UhpT-like protein in the PA genome. The binding site is superimposable to UhpT protein as well as the overall topology. In fact, the 12 TMs are completely comparable, suggesting a well-defined folding of the protein across the bilayer lipid membrane. To enforce our hypothesis, in all 21 PA genomes, we also found a protein annotated as membrane sensor protein UhpC, important for expression and function of UhpT in E. coli. Since PA strains are wild-type, we can assume that most of the PA have proteins like this. The presence of a homologue of UhpT suggests that this protein is conserved in PA genome.