ABC: ATP Binding Cassette; TMD: Transmembrane domains; NDB: Nucleotide-binding domains; ATP: Adenosine triphosphate; SBP: Substrate-binding protein; TMHMM: Transmembrane Hidden Markov Model; KEGG: Kyoto Encyclopedia of genes and Genomes; BLAST: Basic Local tool Alignment Search Tool; SMART: Simples Modular Architecture Research Tool; AcSpm: Acetyl-spermine; SPD: Spermidine; PUT: Putrescine; RMSD: rootmean-square engineering.

The bacterium of the genus Enterobacter cloacae are gram-negative strains, facultative anaerobes, belonging to Enterobacteriaceae. Saprophytic microorganisms are from the environment, but they are also found in the gastrointestinal microbiota of animals and humans. Outside the gastrointestinal tract, it is shown to be a pathogen responsible for several infections, namely, endocarditis, septic arthritis, osteomyelitis, and skin and soft tissue infections. The possible mechanisms of pathogenicity of E.cloacae are complex with the involvement of a series of virulence factors, whose role in the evolution of the disease is still unknown [1]. Antibiotics are one of the most important interventions to control diseases caused by bacteria, however, their misuse has promoted bacterial resistance, becoming a global health problem, as in the case of E.cloacae that affects patients hospitalized in intensive care [2, 3]. Studies carried out between the years 1998 to 2003 reported three outbreaks with up to 23 systemic infections and a mortality rate of 34%. In the last 15 years, 5% of cases of nosocomial sepsis, 5% of nosocomial pneumonia, and 10% of cases of post-surgical peritonitis caused by E.cloacae have been reported. In addition, several studies point to E.cloacae with resistance to several antibiotics, due to the production of beta-lactamase [1].

Obtaining nutrients from the environment by bacteria, including pathogenic ones, is mainly done through transport through the cell membrane, making use of specialized protein complexes. ATP-Binding Cassette type transporters form a superfamily of membrane proteins that carry out the transport of molecules and substrates across the membrane, through import and export mechanisms [4]. Exporter-type transporters are found from simple prokaryotes to complex eukaryotes, participating in the pathogenesis of bacteria, but are involved in other processes such as toxin export and drug resistance, DNA repair, export of peptides, proteins, polysaccharides, and lipids, in addition to participating in cell division [4-6]. Importer-type transporters are only present in prokaryotic organisms and are primarily responsible for the uptake and absorption of essential nutrient sources of phosphorus, nitrogen, sulfur, and other compounds. The relationship between the uptake system using ABC transporters and the physiological processes demonstrated in different bacteria is evident since the internalization of nutrients and different compounds favors the diversity of intracellular activities involved in bacterial virulence and pathogenesis [7,8]. The basic structure of ABC transporters consists of two transmembrane domains (TMDs), which form a pore across the cell membrane, selectively allowing substrate transport, two highly conserved nucleotide-binding domains (NDBs), which carry out the hydrolysis of the molecule adenosine triphosphate (ATP), providing energy for substrate translocation. Also, transporters of the import system have an extra protein component, located in the periplasm region, with a substratebinding protein (SBP) responsible for specifically capturing and delivering the substrate to the transmembrane domain for its internalization [4,7].

Among all the substrates necessary for the survival of pathogenic bacteria are the polyamines formed by two or more amine fractions along the aliphatic chain. These molecules are involved with the pathogenesis process of bacteria and their intracellular concentration and internalization of the environment [9]. The need to constantly obtain polyamines by the cell is due to the involvement of polyamines in processes such as biofilm formation, cell growth, resistance to oxidative stress, and nitrogen storage [10,11]. Previous studies have described several import ABC transporters for virulence in Escherichia coli and Pseudomonas aeruginosa such as PotABCD and PotFGHI transporters, responsible for transporting spermidine and putrescine, respectively, in E.coli; and SpuDEFGHI, in which SpuD performs putrescine uptake and SpuE promotes spermidine uptake in P.aeruginosa [12,13].

Periplasmic-binding proteins, PotD and PotF, present in the PotABCD and PotFGHI system, present in Enterobacter cloacae, are described as polyamine binders and, given the importance of these molecules for the growth and survival of pathogenic bacteria, the present work aimed to identify and to characterize the proteins that make up the PotABCD and PotFGHI complexes from in silico analyzes and molecular docking, seeking possible ligands that already exist in the literature capable of partially or completely inhibiting the uptake of polyamines by ABC transporters.

Search and identification of ABC transporters in Enterobacter cloacae

The identification of the proteins that constitute the ABC polyamine transporter of Enterobacter cloacae bacteria was performed using the online database Kyoto Encyclopedia of Genes and Genomes and the Basic Local tool Alignment Search Tool BLAST [14] allowing the identification of homologous proteins. The amino acid sequences of homologous proteins were aligned with those of Enterobacter cloacae using the Clustal Omega [15] program to identify conserved amino acids in different bacterial species.

Transmembrane domain, signal peptide region, and protein domain

The permease proteins of the transporters were identified from the analysis of transmembrane domains performed by the program TMHMM [16]. The software reviews transmembrane regions based on the method of hidden Markov models [17] and uses algorithms to identify existing patterns in a given sequence of amino acids, in this case, the presence of transmembrane helices [18,19]. The SBPs proteins were identified from the presence of signal peptide by the Signal P 6.0 program [20]. The signal peptide is characterized by short amino acid sequences, usually present in the N-terminal region of the protein, being responsible for directing the proteins to the plasmatic membrane [21,22]. Other domains such as highly conserved AAA+ in ATPase proteins [23] were identified from the Simples Modular Architecture Research Tool (SMART) program combined with the Uniprot database [24].

Homology modeling of ABC transporters protein and molecular docking

Homology modeling consists of determining the threedimensional structure of a protein from the alignment of amino acid sequences between the target protein and one or more template proteins of known structure [25,26]. The HHPred program [27], was used to carry out the molecular modeling by homology of the SBPs proteins of the ABC transportation of polyamines from E. cloacae and the generated models were validated by the Ramachandran diagram that tests the quality of the three-dimensional structures since it provides the conformation of the phi (φ) and psi (ψ) torsion angles of the amino acid sequence. These twist angles are defined for each of the amino acid residues [28]. The PyMol program [29] was used for the visualization of the models, prediction of the binding site, virtual sorting, and estimation of binding affinity with the ligands [30].

Using the Auto Dock Vina software [31], molecular docking was performed to evaluate the scoring function used in the protein-ligand fit, predicting the binding mode and affinity of a ligand concerning a protein [32]. Molecular docking is a method capable of identifying possible drug candidates, which uses predictions of binding and affinity between a protein and a ligand. The AutoDock Vina program is based on the Monte Carlo method which presents scoring functions to estimate the free energy in the receptor and an exploratory method to show the positional and conformational space of the ligand on the receptor [32,33]. From the literature data, molecules possible to interact with SBPs and promote transporter inhibition were selected for this analysis.

Enterobacter cloacae feature two complete polyamine uptake transporters

Two polyamine-importing ABC transporters were identified in Enterobacter cloacae bacteria encoded by the potABCD and potFGHI operons (Figure 1). TMHMM analysis identified six transmembrane regions in PotB, PotC, PotH, and PotI proteins, suggesting that these proteins are TMD of ABC transporters. The SignalP 6.0 program identified a probable signal peptide region in the N-terminal region of the PotD and PotF proteins already described as SBPs, and in the PotD protein, the signal peptide is predicted to be located between amino acids 1 to 19 and in the PotF protein between amino acids 1 to 26.

Analysis of the amino acid sequences of the PotA and PotG proteins resulted in the identification of the AAA domain present in the NDB protein of ABC transporters as the P-loop region that contains the alpha-beta-Rossman loop formed by ABC signature nucleotide binding motifs and the Walker A and Walker B regions [35,23] strongly suggesting that PotA and PotG proteins are NDB proteins.

The periplasmic protein-binding pocket conserves amino acids that interact with polyamines

The multiple alignments of SBP protein sequences of polyamine transporters from previously studied bacteria such as Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, Shigella flexneri, Xanthomonas citri and Pseudomonas aeruginosa with the PotD and PotF proteins of E. cloacae, allowed identifying conserved amino acids in the binding pocket of these proteins (Figure 2). The characteristics of the amino acids present in the PotD and PotF binding pocket determine the molecules that interact with these proteins, one of these characteristics being the hydrophobic amino acids. Therefore, the protein-ligand interaction is promoted by the collapse of the organizational structure of water, resulting in entropic gain associated with this disorganization of the system [36]. Such interaction occurs in a specific way, causing a conformational change in the protein, bringing the two globular domains closer together, and changing the protein from an open conformation to a closed one [8,37]. The amino acids identified in the PotD protein that constitute its binding pocket are: W11, T12, E13, Y14, W206, W232 e D234; and the amino acids that make up the PotF protein binding pocket are: W11, S12, D13, Y14, W218, D221, F250 e D252 (Figure 2A). Previous studies carried out in E.coli, P.aeruginosa, and X.citri bacteria, disclosed the presence of conserved amino acids that provide a recognition specificity in the question of the spermidine and putrescine transport, being these, in PotD appearance of a threonine (T12), glutamate (E13) and a tryptophan (W232) and in PotF the presence of serine (S12), aspartate (D13,221,252) and phenylalanine (F250) [13,8] all of them observed in proteins of this study.

For molecular modeling by homology, the following proteins were identified as template proteins for PotD: PotD from Escherichia coli (PDB ID: 1POT), showing 100% sequence identification, and PotD from Streptococcus pneumoniae (PDB ID: 4EQB) with 99.98% identify. PotF proteins from Escherichia coli (PDB ID: 7OYW) served as the template for PotF, presenting 100% identity; Pseudomonas aeruginosa SpuD (PDB ID: 3TTN) with 99.97% identify and Escherichia coli PotF (PDB ID: 1A99) with 89.80% identify. The architecture of the generated templates was observed by the PyMol program (Figure 3A), indicating similarities in the structure of the PotD and PotF proteins, since the conservation of the ancestral structure is crucial for the maintenance of the protein function [38]. This similarity can be observed from the mean square deviation (rootmean-square engineering - RMSD) that compares the folded protein structures, giving the atomic coordinates after an overlap between two structures. The lower the RMSD value, the more similar the detected structures [39]. The aligner between PotD and PotF proteins in PyMol showed an RMSD of 0.656, indicating high similarity between the structures.

Previous studies have shown structural conservation in substrate-binding proteins, and these proteins generally have two globular domains connected through hinge regions which connect the globular domains. Based on the connection regions, it is possible to classify these proteins as type I, II, or III, and type I SBPs have three pleated beta strands in their hinge region. Type II proteins have only two pleated beta strands and type III proteins have an alpha helix connecting the domains [37]. The analysis of the generated models showed that the PotD and PotF proteins present two pleated beta-sheets between the two domains, N and C-terminal, characteristic of type II periplasmic proteins (Figure 3A). The organizational form of folding of polypeptide chains presents the N-terminal and C-terminal region of the polypeptide chain forming domain 1 and the remaining middle region forming domain 2 [37].

Molecular docking identified possible inhibitors for polyamine uptake

Molecular docking indicated a possible interaction between PotD and spermidine, and between PotF and putrescine. The current literature on possible ligands capable of interacting and inhibiting the uptake of polyamines is scarce, but in one study, the cystamine molecule was identified as a competitive inhibitor by nature concerning putrescine, showing itself as a possible inhibitor of its transport [40]. A study carried out with simple conjugations of molecules with spermine, demonstrated that the Acetyl-spermine conjugate (AcSpm) presents a possible ability to inhibit the uptake of polyamines [41]. The best candidates for molecular docking inhibitors were determined from the Gibbs Free Energy of the interaction [42,43]. When carrying out the molecular docking of the PotD protein with the Acetyl-spermine ligand, nine different positions were generated, and three of these positions interacted with the PotD protein binding pocket, with the free energy of -3.3 kcal/mol, -2.8 kcal/mol, and -2.7 kcal/mol (Figure 4A), comparing these values with those obtained from spermidine docking, -3.4 kcal/mol, -3.3 kcal/mol, and -3.2 kcal/ mol (Figure 4B). The docking of the PotF protein also generated nine different positions, of which cystamine showed interaction with the binding pocket in three positions with the free energy of -3.6 kcal/mol, -3.5 kcal/mol, and -3.0 kcal/mol (Figure 4C), in which it was cataloged and compared with the Gibbs free energy values generated from the docking of putrescine, namely -3.9 kcal/mol, -3.6 kcal/mol, and -3.5 kcal/mol (Figure 4D).

Spermine-conjugated molecules can act as a potent inhibitor of polyamine transport in the breast cancer cell line as the Acetylspermine ligand selected in this study for docking with the PotD protein. Acetyl-spermine is a simplified synthetic analog that can bind with equal affinity to the ABC transporter because it is a spermine-conjugated molecule. In this way, it maintains the physicochemical characteristics of polyamines, including hydrophobicity, which enables protein-ligand interaction, inhibiting spermidine uptake [41].

On the other hand, the cystamine molecule is an organic disulfide obtained by the oxidative dimerization of cystamine that presents the connection of a sulfur pair in which the radical ends are interacting with amine groups. This physical-chemical characteristic, in which the sulfur atom is hydrophobic, favorably allows the protein-ligand interaction, since the pocket amino acids are also hydrophobic. Still, in terms of size, cystamine is an inhibitor similar to polyamines, facilitating its entry to interact with the pocket [44].

From the results obtained in this research, it is possible to suggest that two transporters import polyamines present in the Enterobacter cloacae bacterium, formed by NDB, TMD, and SBP proteins. The genes encoding these proteins are organized in the form of potABCD and potFGHI operons and are possible spermidine and putrescine transporters, respectively. The PotD and PotF proteins were highly conserved with most of the SBPs described in the literature, presenting two globular domains linked by beta-pleated strands. The binding pocket of both proteins conserved amino acids involved in the interaction with their respective polyamines, suggesting that each one transports a different polyamine, PotD being a possible spermidine scavenger and PotF possibly a putrescine scavenger. These findings were corroborated by molecular docking performed. Still, the cystamine and Acetyl-spermine molecules were identified as possible inhibitors of the PotF and PotD uptake action, respectively, because they present favorable free energy for effective interaction with the binding pockets. These initial data open possibilities for the identification of new molecules that may serve as inhibitors for polyamine uptake in resistant bacteria, thus, more studies will be carried out to identify new inhibitors of the action of ABC transporters in resistant bacteria and to understand their mechanisms of action.

We would like to thank Universidade São Francisco for the scientific initiation program and support in carrying out this project.

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