In-silico characterization of GABAT protein found in gut-brain axis associated bacteria of healthy individuals and multiple sclerosis patients

Background Multiple sclerosis (MS) is a neurodegenerative disease characterized by inflammation and demyelination of neurons. There is evidence to suggest that level of a neurotransmitter gamma-aminobutyric acid (GABA), due to the degradation by γ-aminobutyric acid transaminase (GABAT), is reduced in certain areas of the brain in MS patients. MS is always accompanied by gut bacteria dysbiosis. In healthy individuals, Faecalibacterium sp. while in MS patients A. calcoaceticus, Clostridium sp. and S. typhimurium are found abundantly. Although all these microbes produce GABAT but only in MS patients this enzyme significantly degrades GABA. Objective Present study is an attempt to characterize the GABAT protein sequences of these bacteria. Methodology Sequences of GABAT protein were retrieved from Uniprot database. Sequences were analyzed by Protparam, Gneg-mPLoc, SOSUI, PFP-FunDSeqE, Pepwheel program, PROTEUS and Alphafold and SAVES servers, MEME suite and HDOCK server. Results In healthy individuals gastrointestinal tract (GIT) bacteria, GABAT protein was present in inner-membrane with α helix content (61 and 62%) and β sheet content (5%), 4-helical cytokines functional domains. It has greater number of B-cell epitopes and more complex 3D configuration as compared to MS patients GIT bacterial enzymes. Conclusion Present study might enable us to modify the GABAT encoding gene and enzyme through site-directed mutagenesis in pathogenic bacteria thus reducing their potential of causing MS.

Due to the association of gut bacteria with innate pathways of immune system and various physiological mechanisms of CNS, their dysbiosis might lead to neurodegenerative pathologies (Ma et al., 2019).One of these pathological conditions is multiple sclerosis (MS) (Cheng et al., 2019).MS is a chronic autoimmune and neurodegenerative disease characterized by inflammation and demyelination of neurons particularly in the CNS (Farshbafnadi et al., 2021).There is evidence to suggest that GABA level is reduced in several areas of the brain in individuals with MS.GABA is a neurotransmitter that plays an important role in regulating neuronal excitability in the CNS.Demyelination of neurons results in memory impairment, sensory and motor deficits such as muscular spasms or stiffness and numbness in legs.It also leads to cognitive impairment including difficulty with memory, attention, and problem-solving.MS is a consequence of interplay between environment and organism's genetic makeup (Schapiro, 2014).The MS causing factors include more than 200 mutations, sanitation, antibiotics use, lifestyle, vitamin D deficiency, Epstein-Barr virus and childhood and adulthood obesity (Belbasis et al., 2020).Additionally, disturbance in gut inhabiting bacteria, postbiotics, permeability of intestine and nervous system activities have also been observed as contributing factors of MS (Buscarinu et al., 2017;Camara-Lemarroy et al., 2020;Escribano et al., 2017;Ghezzi et al., 2021;Kriesel et al., 2019;Laman et al., 2020;Mirza and Mao-Draayer, 2017).
GABA is an inhibitory neurotransmitter produced in GABAergic neurons from L-glutamate in the presence of L-glutamic acid decarboxylase (Chang et al., 2003;Petroff, 2002).It is an important neurotransmitter that contributes to processes such as cortical adaptation, synaptic plasticity, and neural reorganization.(Wu and Sun, 2015).The degradation of GABA begins with the action of γ-aminobutyric acid transaminase (GABAT), which converts it into succinic semialdehyde.This is then further metabolized into succinate within the mitochondria, which enters the Krebs cycle.Apart from its role as a neurotransmitter, GABA also functions as an anti-inflammatory molecule.Under normal circumstances, the amount of GABA being degraded is replenished through the conversion of L-glutamate.(Ramachandran V. S., 2002).Studies have proved that gut dwelling bacteria play a significant role in MS.Mechanisms through which these bacteria contribute to MS may include, weakening of immune system and induction of autoimmune myelin sheath deterioration, production of GABA degrading enzyme GABAT, GABA fermentation and initiating inflammation by inhibiting anti-inflammatory IL10 expressing human CD4 + CD25 + T cells (Cekanaviciute et al., 2017;Ochoa-Repáraz et al., 2018;Shahi et al., 2017;Strandwitz et al., 2019).A high abundance of gut bacteria producing GABAT) could result in a reduced amount of GABA in the neurons of the prefrontal cortex, left sensorymotor cortex, and right hippocampus, potentially contributing to the development of MS.In such instances, there may also be a decrease in the levels of glutamate, which is another important neurotransmitter in the nervous system (Nantes et al., 2017).Degradation of GABA by GABAT cannot be fully compensated, which can result in a significant reduction in GABA levels (Fig. 1A).One of the major consequences of this GABA reduction is physical disability in MS patients.(Cawley et al., 2015).
Taking in account the involvement of GABAT enzyme in the reduction of GABA levels as well as the abundance of Acinetobacter calcoaceticus, Clostridium species, and Salmonella typhimurium in the gut of MS patients, we may suggest that GABAT enzymes from these bacterial species are involved in GABA degradation and the onset of MS.Although the gene encoding for GABAT is also present in gut bacteria of healthy individuals, including various strains of Faecalibacterium species, however, these microbes are not typically associated with MS.Aim of this study is to identify the variations in GABAT proteins that may contribute to its virulence in Acinetobacter calcoaceticus, Clostridium species, and Salmonella typhimurium, while being non-virulent in Faecalibacterium species.

Methodology
The methodology followed in present study is summarized in Fig. 1B.

MEGA 11 software
To study the evolutionary relationship among the bacteria documented in present study with reference to GABAT protein, multiple sequence alignment was performed using CLUSTAL Omega Multiple Sequence Alignment tool (https://www.ebi.ac.uk/tools/msa/clustalo, accessed on 9 January 2024).Neighbor joining tree was constructed using the aligned sequences after gaps removal, using MEGA 11 software (https://www.megasoftware.net,accessed on 9 January 2024).The bootstrapping procedure with 100 bootstrap value was selected.

Protparam tool
Retrieved sequences were subjected to Protparam tool (https://web.expasy.org/protparam/,accessed on 19 January 2023) to analyze the physicochemical properties of GABAT protein in present study bacteria (Gasteiger et al., 2005).Properties computed include number of amino acids, molecular weight, isoelectric point (pI), grand average of hydropathy (GRAVY), half-life, instability index and aliphatic index.

Pepwheel program
To compute the hydrophilic, aliphatic and positively charged residues of GABAT proteins, Pepwheel program (https://bioinformatics. nl/cgi-bin/emboss/pepwheel, accessed on 20 January 2023) was consulted (Ramachandran G. N. and Sasisekharan, 1968).Results were obtained in the form of a helical wheel showing aliphatic, hydrophilic and positively charged residues as squares, diamonds and octagons, respectively.

MEME suite 5.5.0
Conserved motifs in GABAT protein sequences were determined using MEME suite 5.5.0 (https://meme.sdsc.edu/meme/meme.html,28 January 2023) (Bailey et al., 2009).For motifs prediction, by default values were used for all the parameters except the number of motifs.Ten motifs were predicted for each protein sequence.

HDOCK server
In order to check the best possible inhibitors of GABAT proteins from MS associated bacteria, binding affinities of proteins with Food and Drug Administration (FDA) approved GABAT inhibitors, HDOCK server (hdo ck.phys.hust.edu.cn,accessed on 28 January 2023) was used (Yan et al., 2020).Two anti-GABAT drugs, isoniazid and benzodiazepine were used for this analysis.

Phylogeny of present study bacteria
Phylogenetic tree is represented in (Fig. 2).According to this tree, Faecalibacterium sp.An58 and Faecalibacterium sp.An121 shared the same clade with bootstrap value of 100.Acinetobacter calcoaceticus III and V were more closely related to each other as compared to other.Acinetobacter calcoaceticus II, IV and IV originated from same branch point with bootstrap value of 100 so were more related.Acinetobacter calcoaceticus I and Clostridium sp.II were related with bootstrap value of 88.Salmonella typhimurium was found to be distantly related with other bacteria.

Prediction of physicochemical properties
Physicochemical properties of GABAT protein computed for present study bacteria are given in Table 1.The pI was observed in the range of 5.48-6.43.Highest value was observed in Clostridium sp.I and lowest in Faecalibacterium sp.An121.GRAVY was found to be ranging between − 0.235 and 0.070 with the highest and lowest values recorded in Acinetobacter calcoaceticus II and Acinetobacter calcoaceticus I, respectively.Half-life was found same for all the bacterial GABAT proteins.Instability index was observed above 40 in Faecalibacterium sp. while below 40 in the bacteria associated with MS patients GIT.Highest value of aliphatic index was found in Acinetobacter calcoaceticus I and lowest in Clostridium sp.II.In all other bacteria, aliphatic index was intermediate between these values.

Prediction of hydrophilic, aliphatic and positively charged residues
Aliphatic, hydrophobic and positively charged residues content were found in the range of 32-44, 40-63 and 15-26, respectively (Supplementary data Fig. 1, Table 2).Number of aliphatic and hydrophilic residues were comparable between the bacteria from healthy individuals and MS patients gut.However, content of positively charged amino acids was lesser in strains of Faecalibacterium sp.than other bacteria documented in study.

Hydropathy plots prediction
The values of average of hydropathicity analyzed using SOSUI tool were same as predicted using Protparam.GABAT proteins from all the bacteria of present study were found to lack signal peptides (Supplementary data Fig. 2).The hydropathy values were recorded in the range of − 2 to +2.

Sub-cellular localization
Sub-cellular localization analyses revealed that GABAT protein is located in inner-membrane of cell in strains of Faecalibacterium species as compared to GIT inhabiting bacteria of MS patients in which protein was found in cytoplasm (Table 2).

Functional domains prediction
Functional domains predicted using PFP-FunDSeqE revealed the presence of 4-helical cytokines in strains of Faecalibacterium sp. while (TIM)-barrel was found in all the MS patients GIT inhabiting bacteria (Table 2).

Two dimensional structure prediction
The 2D structure was predicted in terms of α-helix, β-sheet and coil contents (Supplementary data Fig. 3, Table 3).The α-helix was found to be the highest in strains of Faecalibacterium sp.i.e. 61 to 62 % as compared to the MS associated bacteria in which the values were observed in the range of 37 and 44.Beta sheet content was lowest in Faecalibacterium sp.strains (5 %) as compared to the value of 16-22 in other bacterial GABAT proteins.Coil content was comparable among the bacteria from healthy individuals and MS patients and was in the range of 33-45.

Three dimensional structure prediction
The 3D structures were computed using Alphafold which showed that GABAT protein among the GIT bacteria of MS patients were significantly different in 3D form as compared to strains of Faecalibacterium sp (Fig. 3, Supplementary data Table 2).Although the structure also vary among strains of Acinetobacter calcoaceticus, Clostridium and Salmonella species but that variation was very small.Number of amino acid residues in allowed region were in the range of 88.4 to 95.1 showing stability of predicted protein models.

B-cell epitopes prediction
B-cell epitopes were found highest in two strains of Faecalibacterium sp.i.e. 18 and 19.On the other hand, in bacteria associated with MS, the epitopes were found in very small number i.e.Acinetobacter calcoaceticus I (9), Acinetobacter calcoaceticus II (6), Acinetobacter calcoaceticus III (4), Acinetobacter calcoaceticus IV and VI, Clostridium sp.I and II (5), Acinetobacter calcoaceticus V and Salmonella typhimurium (7) (Supplementary data Fig. 4, Supplementary data Table 3).

Conserved domains
Total ten conserved motifs were predicted in GABAT proteins of present study bacteria (Fig. 4, Supplementary data Table 4).Among these bacteria, Faecalibacterium sp.An58 and An121 shared eight motifs

Docking results
Docking analysis of GABAT proteins from MS associated bacteria with isoniazid and benzodiazepine revealed high binding affinities of protein for isoniazid with docking scores (-213.15 to − 267.42) in all the bacteria as compared to benzodiazepine with docking scores (-78.12 to − 87.88) (Fig. 5, Supplementary data Fig. 5, Table 4).

Discussion
The interference of gut associated bacteria in MS patients metabolism has been considerably explored and reported (Bhargava and Mowry, 2014;Cantarel et al., 2015;Cox et al., 2021;Durack and Lynch, 2019;Parker et al., 2020).Several of these studies also reported the role of GABAT in metabolic reduction of GABA in MS patients (Kowalczyk and Kulig, 2014;Nantes et al., 2017).To our knowledge, there has been no prior investigation focusing specifically on the characterization of GABAT in gut-inhabiting bacteria, particularly in the context of MS.Hence, this study represents first attempt to examine the various attributes of GABAT in these bacteria.
A study involving a control group of thirty-three healthy individuals and experimental group of twenty-two Chinese MS patients revealed the reduction in abundance of Faecalibacterium in MS effected individuals (Ling et al., 2020).A systematic review targeted the alpha and beta diversity among the MS patients by interpreting the literature published in ScienceDirect, Scopus, PubMed, Cochrane, CINAHL and Proquest databases and confirmed the reduction in proportion of Faecalibacterium (Ordoñez-Rodriguez et al., 2023).A study investigated the microbial composition of experimental group comprising of MS patients and control group comprising of healthy individuals.Both these groups contained seventy-one subjects.Acinetobacter was not only abundantly  The pI is a measure of the pH at which a protein has a net charge of zero.It is a characteristic property of proteins and depends on the amino acid sequence of the protein.Activity of GABAT is influenced by the pH of its environment.Previous studies have shown that an alkaline pH is the optimal condition for this enzyme.(Bloch-Tardy et al., 1974).The pI values of GABAT in gut-associated bacteria of MS patients were found more alkaline (i.e., above pH 7) as compared to those in healthy individuals.Specifically, in Acinetobacter calcoaceticus I, Clostridium sp.I and II (which are associated with MS patients), the pI values of GABAT were higher as compared to the values of GABAT in other MS-associated and non-MS associated bacteria.This suggests that this protein would act as a more hydrophilic molecule at alkaline pH and the GABAT protein in the gut-associated bacteria of MS patients may behave differently at this pH, which may have implications for GABA degradation in MS (van Oss, 1997).
The half-life of all bacterial proteins was found to be longer, indicating their potential use as probiotics.This suggests that non-MS associated bacteria with less active GABAT enzymes have the potential to be used as probiotics to improve the gut microbiota of MS patients.Aliphatic index has direct relation with thermostability of globular proteins (Morya et al., 2012).Higher values were observed in majority of the diseased patients bacterial GABAT revealing their greater thermostability.
Instability index is a measure of in-vitro stability.The value below 40 shows greater protein stability (Gamage et al., 2019).In present study, GABAT from MS gut bacteria with instability index of 28.62 to 38.23 might be more stable in laboratory and can be easily manipulated through mutagenesis.
Analyses of conserved motifs revealed that the GABAT protein in healthy individuals' gut bacteria is not related to the same protein in bacteria inhabiting MS patients, as they do not share any of the motifs.However, both strains of Faecalibacterium species are related to each other, and Acinetobacter calcoaceticus II, III, IV, V, and VI are also related to some extent.Salmonella typhimurium and Clostridium species are also not related to any of the bacteria documented in the present study.The number of B-cell epitopes in a protein reflects its potential to stimulate the immune system.Current study found that the GABAT protein in non-MS associated bacteria had a greater number of epitopes as compared to   MS-associated bacteria, indicating a greater potential for immune stimulation in the former group.This significant difference in epitope numbers may also explain the production of anti-inflammatory molecules in healthy individuals and the production of pro-inflammatory molecules in MS patients (Latvala, 2014).Additional experiments can be designed to examine the variations in immune-modulatory potential of GABAT proteins between individuals with and without the disease.The differences observed in the functional domains, 2D and 3D configurations, conserved motifs, and B-cell epitopes of GABAT proteins between MS-associated and non-MS associated bacteria may be the underlying cause of its substantial involvement in MS pathology.These variations are justifying the strong link between dysbiosis and MS.Considering this, the MS can be treated significantly via replacing the gut bacteria of patients with the Faecalibacterium species.

Conclusion
Findings of this study could aid in elucidating the mechanisms behind MS pathogenesis caused by microbial dysbiosis.Currently, no diagnostic or prognostic biomarkers have been identified for MS.However, GABAT-producing gut bacteria targeted in present study i. e. Acinetobacter calcoaceticus, Clostridium sp. and Salmonella typhimurium could serve as potential prognostic biomarkers for MS in the future.Additionally, this study supports the idea of fecal microbiota transplantation (FMT) as a means of restoring gut microbiota and potentially reducing the abundance of GABA-degrading bacteria.Therefore, controlling GABA neurotransmission could be an effective strategy for protecting neurons in MS patients.In addition to this, GABAT protein of the MS patients gut associated bacteria can be mutated to enhance the number of immunogenic B-cell epitopes thus increasing its ability to stimulate the immune system.
Statements and Declarations: Funding: N/A.Author contributions: Nadia Hussain wrote the manuscript, Fatima Muccee perceived the idea, retrieved data from database, designed methodology and performed analysis.
Submission declaration and verification: The work is not published previously and it is not under consideration for publication elsewhere.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 1 .
Fig. 1.Graphical representation of present study (Ramachandran V. S., 2002).(A)Mechanism of action of GABAT in microbiota-gut-brain axis of healthy individuals and multiple sclerosis patients (B) Layout of methodology involved in present study

Fig. 2 .
Fig. 2. Phylogenetic tree constructed using MEGA 11 software to analyze the evolutionary relationship among the present study bacteria with reference to GABAT protein.

Fig. 4 .
Fig. 4. E-value and location of conserved protein motifs of GABAT proteins of GIT bacteria of healthy individuals and multiple sclerosis patients predicted using MEME suite.

Table 1
Prediction of physicochemical properties of GABAT proteins documented in present study using Protparam tool.

Table 2
Prediction of sub-cellular localization, functional domains, aliphatic, hydrophilic and positively charged residues of GABAT protein in present study bacteria using Gneg-mPLoc, PFP-FunDSeqE and Pepwheel program, respectively.

Table 3
Prediction of 2D configuration of GABAT proteins in present study bacteria using PROTEUS Structure Prediction Server.

Table 4
Scores reflecting binding affinities of GABAT proteins found in GIT bacteria of MS patients, with FDA approved drugs isoniazid and benzodiazepine, predicted using HDOCK server.