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New insights about the EptA protein and its correlation with the pmrC gene in polymyxin resistance in Pseudomonas aeruginosa

Highlights • Computational biology.• Bacterial resistance.• Pseudomonas aeruginosa.• Gram-negative bacteria.• Polymyxin.


Introduction
Polymyxins are classified into five groups, named: A, B, C, D and E (Neiva et al., 2013). Although, currently only polymyxins B and E are commercially available (Pogue et al., 2017), or used in clinical interventions (Lorenzo et al., 2011), due to high toxicity of the other isoforms (Falagas and Kasiakou, 2005). This group of antibiotics was discovered in 1947 (Li et al., 2005), and polymyxin E was the first to be used in the clinic, in 1959 (Yu et al., 2015). Polymyxins were identified in Bacillus polimyxa strains (Stansly and Schlosser, 1947;Girardello and Gales, 2012), subspecies: colistinus Koyamae (Lorenzo et al., 2011). Polymyxins B and E have potent antimicrobial activity against several species of Gram-negative bacteria (Mendes and Burdmann, 2009;Carvalho and Cogo, 2014). Some hypotheses could explain the mechanism of polymyxins resistance acquired by P. aeruginosa: (I) Adaptive mechanism: Gradual adaptation, due to the presence of this antimicrobial component associated with the culture medium used in experimental settings or in clinical diagnosis (Zavascki et al., 2010).
Another possibility is the interference of this drug during active transport through the membrane, specifically in the lipid A portion of bacterial lipopolysaccharides (LPS) (Mendes and Burdmann, 2009) resulting in loss of OMPs (Outer Membrane Proteins) or reduction of interactions between polymyxin and the envelope (Moore et al., 1984); (II) Genetic mutation mechanism: Associated with increased levels of H 1 protein (H 1 -T6SS), and cations replacement, minimizing the Mg 2+ concentration and increasing the Ca 2+ concentration in cell membrane, reducing possible electrostatic interactions with polymyxin (Zhang et al., 2011;Fair and Tor, 2014;Morita et al., 2014). In 2010, it was shown that H 1 -T6SS in P. aeruginosa has three effector proteins, called Tse 1-3 (type VI secretion exporters 1-3), where Tse 1 and Tse 3 can cleave peptidoglycan associated with the bacterial envelope (Hood et al., 2010), and this function of H 1 -T6SS is directly linked to antibiotic resistance in biofilms (Zhang et al., 2011).
Furthermore, absence of 2-hydroxy-laureate and presence of 4-aminoarabinose in bacteria membrane and an increased A palmitate concentration in lipid A (Bonomo and Szabo, 2006), besides mutations in the PhoPQ and PmrAB systems that induces modified LPS operon (arn), resulting in addition to Lara4N (4-amino-4-deoxy-L-arabinose) in the lipid A portion of the LPS, could potentialize the resistance to polymyxins in P. aeruginosa (Schurek, 2009). Different modifications in lipid A may occur, including changes in the number of acyl chains and phosphoethanolamine addition (Tran et al., 2005;Herrera et al., 2010). Other possible resistance factors to polymyxins could be the induction of the pmrCAB operon. These genes comprise a three-component system: response regulator (pmrA), histidine kinase sensor (pmrB), and the protein that adds phosphoethanolamine to lipid A (pmrC).
Thus, polymyxins resistance could be related to increased expression of pmrC gene. In P. aeruginosa, N. meningitidis, S. enterica and S. plymuthica the product of the pmrC gene it is it is known as EptAs, whereas in the bacterium Escherichia coli it is known as MCR 1 , and Klebsiella pneumonia it is known as MCR 1.9 . In this context, P. aeruginosa, a Gram-negative bacterium, in the last decade has received much attention due to its capacity to cause serious infections in Brazilian hospitals (Lopes, 2009;Silva Filho and Silva, 2013;Bomfim and Knob, 2013). For this reason, this work aims to study the physicochemical properties of EptA proteins expressed in P. aeruginosa genomes deposited at the National Center for Biotechnology Information -NCBI (https://www.ncbi.nlm.nih.gov/) database, to improve the understanding of the mechanisms of resistance to polymyxins developed by this bacterial species.

Obtaining and analysing gene sequences
In this experimental approach, the National Center for Biotechnology Information -NCBI (https://www.ncbi.nlm.nih.gov/) database was initially evaluated to identify clusters that encode isoforms of the enzyme Lipid A phosphoethanolamine transferase (EptA) in P. aeruginosa genome. For this purpose, the EptA sequence (product of the mcr 1 gene of E. coli, Access in GenBank: ASK04346.1) was used as bait to obtain target protein sequences from the Protein Blast tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Prediction of secondary structure and protein domain
Secondary structures of the EptA isoforms were predicted using the SopMa -Secondary Structure Prediction Method server (Geourjon and Deléage, 1995). For protein domain prediction all .fasta extension sequences were submitted to Conserved Domains Database (CDD) and Resources -NCBI -NIH server (Lu, 2020).

Phylogenetic analysis
The phylogenetic analysis was obtained from the alignment of EptA from P. aeruginosa sequences. For determination of the phylogenetic tree, the Neighbor Joining method (NJ) was used with pairwise deletion option and reliability index of 1000 replicas of bootstrap. The analysis was performed in the MEGA X Molecular Evolutionary Genetics Analysis -Phylogenetic (Kumar et al., 2018) software.

Results and discussions
P. aeruginosa is classified as a Gram-negative bacterium and are very versatile, capable of spreading on soils (Al-Saleh and Akbar, 2015), marshes (Teixeira et al., 2016), coastal marine habitats (Habbu et al., 2016), as well as plants and animals (Ellison et al., 2013). In this context, the easy environmental adaptation and dissemination have aroused the curiosity of many researchers around the world, resulting in several P. aeruginosa genomes being sequenced (genomes data were obtained from the National Center for Biotechnology Information -NCBI (https://www.ncbi.nlm.nih.gov/). The microorganism was found in many researches conducted in intensive care units (ICUs) (Basso et al., 2016). In Belém-PA, Brazil, infections caused by P. aeruginosa resulted in the second highest mortality rate in ICUs, only behind Mycobacterium tuberculosis species (Barros et al., 2016). Polymyxins B and E (colistin) are antibiotics that P. aeruginosa developed resistance against (Lorenzo et al., 2011;Barros et al., 2016;Lee et al., 2012), although the mechanism leading to it is not very clear (Stefani et al., 2017;Gutu, 2013).
On the other hand, in microorganisms as Acinetobacter baumannii, E. coli and Klebsiella pneumoniae this mechanism is well known, being a direct result of the mobile colistin resistance mcr 1 gene (Olaitan et al., 2014;Chang et al., 2012;Liu, 2016). It is a common sense that the emergence of polymyxin-resistant strains could be associated with the use of colistin in agriculture and animal farms (Zheng et al., 2018;Economou and Gousia, 2015;Nguyen et al., 2016;Zheng et al., 2018). The Protein Blast server (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was searched for sequences related to the mcr 1 gene of E. coli present in genomes belonging to Pseudomonas genus. 101 protein sequences were identified, of which 80 were associated with P. aeruginosa, 4 with P. citronellolis, 4 with P. nitroreducens, 2 with P. alcaligenes, 2 with P. jinjuensis, and 9 other species, with all sequences belonging to Pseudomonas genus. Only the isoforms catalogued for the P. aeruginosa species were analysed, being distributed currently, among 18 known genomes, according to the identification of the rates (Table 1).
To avoid redundant data, all 80 EptA sequences from P. aeruginosa species were subjected to identity matrix analysis using the MEGA X software (Teixeira et al., 2016) resulting in only 59 unique sequences. Then, these sequences, were evaluated regarding the protein domain that they belong. After this evaluation, only 31 EptA isoforms remained, all belonging to the DUF 1705 superfamily, similar to proteins encoded by E. coli (Stoesser et al., 2016), Salmonella enterica (Doumith, 2016), K. pneumonia (Di Pilato et al., 2016), Serratia plymuthica strain AS 13 e Neisseria meningitides (Zhang et al., 2019), all Gram-negative bacteria. Some members of this superfamily are putative bacterial membrane proteins.
The number of amino acids presents in EptA isoforms of P. aeruginosa ranged from 551 to 572 amino acids (aa), and it was possible to recognize three groups based on the sequence size: (I) eleven isoforms Table 1 List of Gram-negative bacteria, Pseudomonas aeruginosa genomes, deposited at the National Center for Biotechnology Information -NCBI (https://www.ncbi. nlm.nih.gov/). formed by 551 aa; (II) a single isoform formed by 567 aa; and (III) two isoforms formed by 572 aa in length. The results obtained in this work in relation to the size of the sequences in P. aeruginosa EptA enzymes showed that it is larger than other proteins from E. coli MCR 1 , K. pneumoniae MCR 1.9 and S. enterica MCR 1.6 (GenBank ids: ASK04346.1, ARA74236.1 and WP_077248208.1, respectively) which has 541 aa (Compain et al., 2014), N. meningitidis EptA (GenBank id: ELL31702.1) which has 544 aa, and S. plymuthica EptA (GenBank id: KYG15235.1) which has 545 aa.
These results could be due to sequencing processes, or even location of EptA gene in a region of the P. aeruginosa genome difficult to sequence. These enzymes are still in process of evolution since the reports of polymyxins resistance in P. aeruginosa are very recent (Olaitan et al., 2014) for this reason the EptA isoform could currently have a smaller length than other microorganisms. The most frequent amino acids in P. aeruginosa EptAs belongs to the group of the aliphatic amino acids, including: leucine (13.6% to 17.1%), alanine (7.6% to 11.4%), glycine (6.7% to 7.9%), valine (5.2% to 7.8%) and the polar uncharged serine (5.8% to 6.6%), respectively. Moreover, amino acids in lower frequency are: the aromatic amino acid tryptophan (1.3% to 1.6%), the aliphatic amino acid methionine (1.7% to 2.2%), the polar uncharged amino acid cysteine (1.9% to 2.2%), and the positively charged amino acid histidine (1.7% to 2.4%).
When the EptAs in P. aeruginosa were compared with other microorganisms, a pattern of higher and lower frequency of amino acids was found, suggesting that these isoforms were derived from a single gene. When compared to the proteins MCR 1 of E. coli, MCR 1.9 of K. pneumoniae, and EptAs of S. enterica, N. meningitidis and S. plymuthica the same pattern repetition in relation amino acids frequency was found, with high rates of leucines, valines, alanines and serines. Only in the EptA isoforms from P. aeruginosa the glycine residues are among the five most frequent amino acids, being replaced by threonine (uncharged amino acid). In relation to the less frequent amino acids: methionine, cysteine and histidine, there is also repetition in the EptAs of these microorganisms (E. coli, K. pneumoniae, N. meningitidis, S. enterica, and S. plymuthica).
Only phenylalanine the fifth most frequent amino acid in EptA isoforms of P. aeruginosa, is replaced by tyrosine in the EptA of S. plymuthica, while the methionine amino acid, second most frequent in P. aeruginosa EptA, is replaced by glycine. Few differences in frequency of each amino acid may be directly associated with the different lengths of polypeptide chains that form these proteins in these microorganisms. The molecular mass (average) predicted for EptA isoforms of P. aeruginosa, showed variations from 61.36 to 62.80 kDa (Table 2). In this analysis two generic groups were observed based on their molecular masses (I) formed by 10 isoforms (32.26%, molecular mass approximately 61.5 kDa); (II) formed by 21 isoforms (67.74%, molecular mass approximately 62.5 kDa). In relation to the protein molecular masses of E. coli MCR 1 (60,12 kDa), K. pneumoniae MCR 1.9 (60.09 kDa) and EptAs of S. enterica (60.10 kDa), N. meningitidis (61.36 kDa) and S. plymuthica (61.64 kDa). This result may be due to the same reasons discussed above about the lengths of these proteins sequences. Although all these EptAs proteins belong to the DUF 1705 superfamily (Lu, 2020), the isoforms expressed in P. aeruginosa, classified in group (I), are likely to be more active against polymyxins B and E, because their size is closer to the EptA of E. coli (Stoesser et al., 2016), S. enterica (Doumith, 2016), K. pneumonia (Di Pilato et al., 2016), S. plymuthica AS 13 strain.
The concentration of hydrogen ions in a solution where the ionization of the acid groups are equal to the ionization of the basic groups is known as the isoelectric point (Highberger, 1939). Data obtained on the Table 2 Identification of EptAs amino acid sequences encoded by the bacterium Pseudomonas aeruginosa based on access from the National Center for Biotechnology Information -NCBI (https://www.ncbi.nlm.nih.gov/). Domain analysis: Position-Specific Scoring Matrix (PSSM id), Domain (starting and ending amino acid), and Bit-score E-value were obtained after access to the Conserved Domain Database -CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). Physicochemical analysis: Molecular weight (rM), Theoretical (pI), Instability index (II), Aliphatic index (AI), and Grand average of hydropathicity (GRAVY) were obtained after access to the ProtParam tool (https://web.expasy.org/protparam/). All data revisited and checked on April 20th, 2021.

GenBank id
Size ( showed basic pI. This variation in isoelectric points presented by EptAs expressed in P. aeruginosa may be directly associated to its adaptive potential in different environments (Kumar et al., 2018;Teixeira et al., 2016;Al-Saleh and Akbar, 2015).
The instability index provides an estimate of how much a protein can remain stable during an experiment conducted in a test tube (Guruprasad et al., 1990). The instability index provides an inverse estimate of the metabolic stability of a protein (https://web.expasy.org/protp aram/). All EptAs in P. aeruginosa sequences analysed in this study predicted instability index values ranging from 33.76 to 41.87, so all these isoforms are relatively stable analysis in test tubes ( Table 2). The analysis of the instability index indicated values of 22.65 for E. coli MCR 1 and S. enterica EptA protein (Doumith, 2016;Di Pilato et al., 2016), 21.78 for K. pneumoniae MCR 1.9 (Zhang et al., 2019), 35.77 for N. meningitidis EptA and 41,11 for S. plymuthica EptA. Based on observed rates for P. aeruginosa EptA, the proteins from E. coli MCR 1 , K. pneumoniae MCR 1.9 , S. enterica and N. meningitidis EptAs are stable. The S. plymuthica species was the only one that presented unstable EptA. Similarly, in P. aeruginosa, two groups EptA were observed, the first group formed by 18 stables proteins (58.06%), and second by 13 unstable proteins (41.94%).
The protein aliphatic index is defined as the relative volume occupied by aliphatic side chains: alanine, valine, isoleucine and leucine (Ikai, 1980). It is generally considered a positive factor to increase protein thermostability, therefore, it demonstrates how much protein molecules are able to withstand heat denaturation. The results obtained on thermostability for the EptA isoforms of P. aeruginosa have shown values between 98.67 and 102.63 corroborating the previous data, indicating that many of these proteins are really stable (Ikai, 1980). When the aliphatic index of the other proteins was evaluated it presented values of 92.83 for E. coli MCR 1 and S. enterica EptA, 93,03 for S. plymuthica EptA, 93.36 for K pneumoniae MCR 1.9 , and 96,21 for N. meningitidis EptA. Based on rates observed for P. aeruginosa EptA, the majority of the proteins are stable at 25 • C temperature as presented in the species E. coli, K. pneumoniae, S. enterica, and N. meningitidis. It is possible that P. aeruginosa EptA can tolerate higher temperatures because these proteins are important both in environmental adaptation as well in vivo febrile hosts infection (Ikai, 1980;Zhang et al., 2012;Chan et al., 2016).
The GRAVY value (hydrophobicity index) for a peptide or protein is calculated based on the sum of all amino acids hydropathy values, divided by the total number of residues present in the sequence (Oliveira et al., 2020). High positive values means that the amino acids located in the protein region are more hydrophobic. These scales are commonly used to predict the transmembrane alpha helices of membrane proteins (Kyte and Doolittle, 1982). GRAVY analysis from P. aeruginosa EptAs profile revealed that 29 isoforms (93.55%) are hydrophobic and 2 (6.45%) are hydrophilic (Table 2). This result indicates that mostly of these enzymes may be in transmembrane regions in the P. aeruginosa bacteria, while the other group of these enzymes must have differentiated their metabolic function. In other bacterial species, the evaluation of GRAVY indicated a value of 0,067 for S. enterica EptA, 0, 018 for N. meningitidis EptA, 0.065 for E. coli MCR 1 , 0,067 for S. enterica EptA and 0.069 for K. pneumonia MCR 1.9 , all with hydrophobic profile.
Although, the hydrophilic P. aeruginosa EptAs role is not known, they are very similar to LptB protein, when the amino acid sequences are analysed, both are soluble proteins without transmembrane domain. According to Sperandeo et al., LptB was identified in an internal membrane complex in E. coli, forming a 140 kDa protein agglomerate, although no other proteins were detected (Sperandeo, 2007;Stenberg et al., 2005).
We suggest that hydrophobic and hydrophilic EptA in P. aeruginosa may interact in a similar way like LptA and LptB in E. coli and K. pneumoniae, together with a transmembrane partner unidentified yet, resulting in a membrane-associated complex required to transport deleterious molecules to this microorganism. On the other hand, the bacterial LPS modification, with the cations substitution present in the phosphate groups by L-Ara4N, neutralizing the lipids A charges, or PEtN modification, increases the net load from − 1.5 to − 1 (Nikaido, 2003). The L-Ara4N modification is the most effective of the two modifications due to the nature of the charge change. The net positive charge resulting from the modified LPS reduces its binding to polymyxins, leading to resistance (Olaitan et al., 2014). The composition of amino acid residues is directly associated with the structure and protein functions.
Although such differences can be observed in P. aeruginosa EptAs, this phenomenon could be explained due to variations observed in the polypeptides sequences, which could be associated with the thermostability shown by this isoforms (Ikai, 1980;Zhang et al., 2012), considering the combination of selective pressure factors that have acted on these proteins for decades (pressure, pH, temperature), besides the mutations that have accumulated. According to the analysis performed using Conserved Domains Database -CDD (Lu, 2020), all EptAs expressed by P. aeruginosa belong to the superfamily DUF 1705 majority members of this family are putative bacterial membrane proteins, and this domain is found immediately in the N-terminal region, likewise all those used in this work as standard, included, MCR 1 of E. coli, MCR 1.9 of K. pneumoniae and EptAs of S. enterica, N. meningitidis and S. plymuthica.
The alignment of the EptAs sequences was performed using the Molecular Evolutionary Genetics Analysis -MEGA X software. It revealed low amino acid variability along the polypeptide chains, however, these substitutions are equivalent, what means they belong to the same functional group (Data not shown). The largest consensus area is located between the amino acids 240 until the C-terminal region of these proteins. It is also possible to observe that the N-terminal region has a higher variation in these proteins. The dendogram was generated with support of the MEGA X software (Teixeira et al., 2016), with the aid of sequence alignment previously performed. It was shown that EptAs from P. aeruginosa formed two distinct clades as can be seen in Fig. 1: Clade I concentrates 93.55% of the identified isoforms. Interestingly, the MCR 1 proteins of E. coli, MCR 1.9 of K. pneumoniae and EptA of S. enterica were organized in the clade I, such result corroborates with the other results previously presented, strengthening other findings that predict the participation of these proteins in the mechanism of the resistance to polimixins by members from Enterobacteriaceae family (Nikaido, 2003;Alves and Behar, 2013;Andino and Hanning, 2015). In relation to, the clades II and III, both are formed by only one isoform (WP_034053930.1 and WP_031694049.1, respectively).
Although the S. plymuthica species are classified as an Enterobacteriaceae, in clinical settings, members belonging to the Serratia genus, generally cause infections in the respiratory system (Domingo et al., 1994), urinary tract (Jain et al., 2017) and can cause necrotic cellulitis (Mahlen, 2011), instead of infections in the gastrointestinal tract, as observed with other microorganisms in this family. In addition, small mammals, vegetables and water seem to be the natural environment of S. plymuthica species (Grimont and Grimont, 2006). These S. plymuthica characteristics may be the cause of their differentiated grouping in relation to other enterobacteria as E. coli, K. pneumoniae and S. enterica. In the same way as S. plymuthica, N. meningitidis, was classified in a group of its own, because this species presents different biology among the other species discussed in this study. N. meningitidis is a Gram-negative Diplococcus and humans are the only natural host (Souza and Gagliani, 2011), causing cerebrospinal meningitis and/or septicaemia (Coureuil et al., 2013).
Considering the cladogram, it is possible to identify the way of acquisition of antimicrobial resistance to polymyxins. P. aeruginosa appears to be the species that initially developed resistance to polymyxins, followed by enterobacteria K. pneumoniae, E. coli and S. enterica, and then N. meningitidis and S. plymuthica bacteria. Finally, P. aeruginosa through EtpA proteins began to resist the action of polymyxins.

Conclusion
In conclusion these results showed the high degree of similarity between EptA proteins of P. aeruginosa with MCR 1 of E. coli, MCR 1.9 of K. pneumoniae and EptA proteins of S. enterica, N. meningitidis and S. plymuthica, based on their physicochemical properties, we demonstrated 31 protein isoforms in P. aeruginosa (EptA) found in to 18 genomes from P. aeruginosa closely related to proteins known to provide resistance to polymyxin in other species of Gram-negative bacteria of clinical interest. EptAs from P. aeruginosa studied here, belonging to DUF 1705 family as indicated by values bit-score between 559.81 and 629.78. In addition, these EptAs showing lengths between 551 and 572 amino acids, molecular mass values between 61.36 -62. 80 kDa, isoelectric point between 6.10 to 7.17, instability index between 33.76 to 41.87, aliphatic index between 98.67 to 102.63 and the hydropathy index between -0.008 to 0.094. However, further studies are needed to identify the actual biochemical contribution of EptAs in P. aeruginosa species.