Cloning, sequence analysis, and three-dimensional structure prediction of Universal stress protein A from Salmonella Typhimurium Isolated from the Poultry

doi:10.21203/rs.3.rs-1736627/v1

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Cloning, sequence analysis, and three-dimensional structure prediction of Universal stress protein A from Salmonella Typhimurium Isolated from the Poultry

https://doi.org/10.21203/rs.3.rs-1736627/v1

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Background

Salmonella Typhimurium is a zoonotic pathogen responsible for food and waterborne gastroenteritis in humans. Salmonella Typhimurium expresses Universal stress proteins (USPs) when subjected to different biotic and abiotic stresses. This class of proteins is becoming widely recognizedfor its role in bacterial stress resistance and protracted growth arrest. USPs possesses versatile structures arising from the fusion of the USP domain with various other catalytic motifs; they are possibly involved in a variety of reactions and cellular activities under stressful conditions.

Objective

The aim of this study is to obtain the universal stress protein A (uspA) gene of Salmonella Typhimurium and clone it into the pET28C plasmid for nucleotide sequencing. And also 3D structure prediction of USPA protein and to evaluate its phylogenetic gene tree model.

Methods

In this study, we constructed a recombinant clone of the uspA (pET28c_uspA) gene from Salmonella Typhimurium, and various methods, including sequencing, did conformation to the clone. The sequence was subsequently submitted to the NCBI GenBank database. Besides, the Three-dimensional structure for USPA protein was predicted by comparative modeling. A phylogenetic gene tree was also constructed to trace the evolutionary history of the uspA gene of Salmonella Typhimurium.

Results

The uspA gene of 435 bp in length was successfully cloned. Comparing the uspA gene sequence with that reported in GenBank showed that identities were 100%. Furthermore, Sequence analysis also revealed the uspA gene is 435 bp long, encodes a protein of 144 amino acids with a calculated molecular mass of 16 kDa, and belongs to the USP family. Predicted 3D structure and sequence alignments of USPA showed that all residues’ sites are highly similar among the genome. The stereo-chemical property of the 3D structure of USPA was found to be of good quality.

Conclusion

A confirmed uspA gene from Salmonella Typhimurium was reported in this study. This study laid a solid foundation for recombination and research of genes. Moreover, the results of this study would help to detect the functional domains of the protein to elucidate their structural/functional characteristics.

SalmonellaTyphimurium

Universal stress proteins

USPA

pathogenicity

Gastroenteritis

stress response

Salmonella enterica subsp. enterica is a substantial public health threat estimated to be responsible for over 300,000 deaths annually, most of which occur in developing countries (Majowicz et al., 2010, Buckle et al., 2012). The annual global burden of non-typhoidal salmonellosis was predicted to be around 93.8 million cases (with 155,000 deaths), with 80.3 million cases (80%) being foodborne(Majowicz et al., 2010). Salmonella Typhimurium,an important Zoonotic pathogen, is responsible for foodborne infections. The most common way to encounter Salmonella Typhimurium is to consume contaminated food or water (Ohl and Miller, 2001). In industrialized countries, the serovar SalmonellaEnteritidis (SE) and serovar SalmonellaTyphimurium (ST) are the two most commonly identified causes of infection (Hendriksen et al., 2011). Although the gut's acidity serves as an efficient barricade against this infection, but a substantial enough inoculum or contaminated food with buffering capacity may culminate in a productive infection with overt illness(Giannella et al., 1972).

During pathogenesis, Salmonella is exposed to a variety of stressful situations in the environment and within the host's body, including nutritional deficiency, temperature and pH variations, and oxidative and osmotic stressors(Chung et al., 2006, Fang et al., 2016). To develop an infection within the host, the bacteria must be able to overcome the host's immune reaction as well as the unfavorable circumstances.During the colonization of the host, the bacteria express genes required for each step of the infectious cycle which helps to resist host responses. In stress conditions such as heat shock, nutrient starvation, oxidants, uncouples DNA-damaging agents, or other stressors that may arrest the cell growth. Salmonella Typhimurium employs different mechanisms to evade the host immune response and survive in the hostile and stressful niche in the environment and within the host body. One of the weapons used by it is the universal stress proteins. Universal stress proteins (USPs) constitute a natural biological defense mechanism under various stress conditions. Universal stress proteins (USPs) are the class of proteins that play a vital role in the survival of living organisms, including bacteria, archaea, fungi, plants, and metazoans, in the face of various environmental challenges. As per the previous research, USPA protein is involved in a wide spectrum of cellular responses to biotic and abiotic stressors, as their name implies. Their physiological roles include ion scavenging, hypoxia responses, cell motility, and cell growth and development regulation. These proteins' defensive actions are critical in the pathogenicity of bacteria(Liu et al., 2007).USPs possess a wide variety of structural variations due to the vast range of additional functional motifs fused with the USP domain, which is consistent with their roles in stress resistance. Soon after the discovery of the amino acid sequence and protein structure of the first USP in E. coli, a substantial number of USP homologs from bacterial sources were discovered, resulting in the formation of enormous USP families(Kvint et al., 2003). USPA, USPC, USPD, USPE, USPF, and USPG are among the six universal stress proteins found in E. coli USPs. Their fused catalytic motifs may influence individual USPs' particular properties under stress circumstances. As a result, combining the USP domain with other specialized catalytic motifs is anticipated to result in various functions that safeguard organisms from multiple environmental challenges. The majority of these USPs are found in the cytoplasm(Kvint et al., 2003). According to certain reports USPA may possess the DNA binding capabilities(Mushegian and Koonin, 1996). Some studies show that E. coli mutants uspA and other usp mutants have been sensitive to UV-induced DNA damage(Kvint et al., 2003). Finally, based on the sequence similarity between UspA and the DNA binding helix of these proteins, it was hypothesised that USPA had a structural resemblance to human serum response factor (MefA2) and transcriptional regulators of the MADS-box protein family (Mushegian and Koonin, 1996). The likeness of USPA to the DNA binding domain of these proteins has been dismissed following the identification of the three-dimensional structure of the USPA orthologue of Haemophilus influenza (Sousa and McKay, 2001). It is also reported that the E. coli USPA is phosphorylated on serine and threonine sites in addition to being increased in response to growth arrest(Freestone et al., 1997). Despite advances in research, the biochemical activities of the USPA family of proteins and the methods by which they work remain unknown. This protein family obviously merits additional investigation.

In this study, the identification, cloning, sequence analysis, and structural modeling of a deduced amino acid sequence of Universal stress protein A from Salmonella Typhimurium isolated from poultry was done. A molecular phylogenetic analysis method based on DNA sequence was used for the preliminary identification. In order to address the origin and evolution of USPA, a comprehensive BLAST and phylogeny analysis revealed that Salmonella shared high similarities with other known pathogenic bacteria. Finally, homology modeling and sequence comparison were used to predict 3D structurefollowed by validation ofnew USPAmodel. The finding of this work contributes towards the necessity to discover the novel sources proficient for the structure/function characteristics of USPA.

Bacterial strains: In this study, we used Salmonella Typhimurium strain 3232 (isolated from poultry) in lab stock. Other than the Salmonella strain, we also used Escherichia coli strain DH5α and plasmid pET28c in thisstudy(Kumawat, 2016). The Salmonella Typhimurium strain was retrieved by using HAE agar. The plates were incubated at 37°C for 18 hrs. A single colony appearing on HAE agar was selected, and inoculated LB-broth was then incubated at 37 °C for 18hrs, and the grown culture was then used for genomic DNA isolation.

DNA isolation: The Genomic DNA from Salmonella Typhimurium was isolated using GenJET genomic DNA purification Kit (cat noK0721, thermo Scientific) as per the protocol with some minor changes. 3 ml of overnight grown culture was pelleted at 10000xg for 5 min and re-suspended in 180 µl of genomic digestion solution. Then 20 µl of proteinase K was added, mixed thoroughly, and incubated at 56 °C for 30 min. Then the mixture was treated with 20µl of RNase A solution and 200µl of genomic lysis solution. The mixture was vortexed to get ahomogeneous mixture. Then the lysate was washed by wash buffer I and wash buffer II, and finally, genomic DNA was eluted using the elution buffer. The genomic DNA concentration and quality were checked using UV absorbance and 1% agarose gel electrophoresis. This ST genomic DNA was used as the template for amplifying uspA by PCR.

PCR amplification of uspA gene from Salmonella Typhimurium

The primers used for the gene cloning were forward-ATATATGCTAGCATGGCTTATAAACACATTCT and reverse-ATATATGGATCCTTACTCTTCTTCGTCGC.The template DNA (50 ng) described above was mixed with the Thermo Scientific Dream Taq Green PCR 2x master mixture(50 µl), primers (0.5 µl) (10 pmol each), and nuclear-free water to make up the final volume of 100 µl and then amplified in Thermal cycler Veriti™. The PCR conditions used for amplification were 1 cycle at 95 ^°C for 5 minutes followed by 35 cycles at 95 ^°C for 5 minutes, 95^°C for the 30s, 48^°C for 30s, and then the final extension condition was 72^°C for 10 minutes. Amplified PCR products were analyzed with1 % agarose gel; separated DNA was stained with ethidium bromide (1 µg/µl) and then photographed using GeL Doc Azure Biosystem.

Cloning and Sequencing of uspA gene

The PCR amplified uspA gene product was first gel purified using GeneJET Gel Extraction Kit (cat. No. K0691, Thermo scientific) as per standard protocol with some modificationsand was then subjectedtodouble digestion by using restriction endonuclease enzymes NheI (cat. No. #ER0971, Thermo-scientific) for 3 hrs and BamHI(cat. No. #ER0051, Thermo scientific) for 2 h at 37 °C and finally enzymes were inactivated by setting the temperature at 80 °C for 10 min. Besides this, pET28c was also double digested by using the same restriction endonuclease enzymes NheI and BamHI to generate complementary overhangs. The double RE-digested pET28c vector was then dephosphorylated at 37 °C for 30 min by using the enzyme FastAP Thermosensitive Alkaline Phosphatase (cat.NO. EF0654, Thermo scientific), then followed by inactivation of enzymes at 80 °C for 10 min. The digested and dephosphorylated pET28c was also gel purified. Thendouble RE-digested uspA gene was ligated into the double digested pET28c vector by using enzyme T4 DNA Ligase (cat.No. EL0014 Thermo scientific), in the ratio of 3:1as per the manufacturer’s protocol. The ligated mixture of pET28c_uspA was then transformed into chemically competent E. coli DH5α cells and allowed to grow on LB-agar containing kanamycin (30mg/ml) plate for 18 h. On the following day, the colonies grown on the plate were screened for the presence of recombinant plasmid in four ways viz: colony PCR, plasmid PCR, re-digestion for release of the insert by NheI and BamHI, and finally by sequencing, using plasmid as a template and T7 promoter and T7 terminator as primers. The result of sequencing were assembled and analyzed.

Moreover, the nucleotide sequence of uspA was subjected to nucleotide BLAST to find the sequence similarities with other sequences which are available in the NCBI database. The final sequence alignment was done with the available uspA gene of Salmonella Typhimurium strain LT2. The sequence was submitted to NCBI Genebank.

Three-dimensional structure prediction and evaluation of USPA protein

The amino acid sequence of Universal stress protein A (USPA) Salmonella Typhimuriumwas retrieved from the UniProtKB database (Accession no: Q8ZLD7) (www.uniprot.org). The length of the target protein was 144 amino acid residues. To obtain the most similar template(s) for the target protein, database searching was performed against the PDB database using the BLASTp program. Based on higher sequence identity (68.57%),a template (PDB id:1JMV)was selected. Subsequently, multiple sequence alignment between the target and template sequences using the ClustalOmega program (https://www.ebi.ac.uk/Tools/msa/clustalo/). The target-template aligned sequences were submitted in PIR file format to the HHpred server (http://toolkit.tuebingen.mpg.de/hhpred). The HHpred is a fast and robust server for protein homology detection and structure prediction. The 3D structural models of USPA were predicted by the MODELLER10.2 software, which works in the back-end of the HHpred server. The target target-template aligned sequences were also submitted to the Swiss-model server for 3D structure prediction of the USPA protein. To evaluate and validate the stereochemical properties of 3D structures of the USPA protein predicted by both the servers, the predicted model (PDB file) was submitted to the Structural Analysis and Validation Server (https://saves.mbi.ucla.edu/).The SAVES is a metaserver for analyzing and validating protein structures in which five modules (Prochek, What_CheckErrat, Verify_3D, and Prove) are incorporated. The predicted 3D structure of USPA was visualized by PyMOL software(Yuan et al., 2017).

Phylogenetic Analysis of uspA gene

The gene sequences of other organisms related to the uspA gene of Salmonella Typhimurium, were obtained from the KEGG database (https://www.genome.jp/kegg/). Total 20 genes were found to be related with uspA gene. The multiple sequence alignment was performed using the Clustal omega server(Sievers and Higgins, 2021). The output file obtained in Clustal file was subsequently converted into the PHYLIP file format using ‘Clustal to Phylip Sequence Converter’. Finally, the phylogenetic tree was constructed using the PhyMLv3.0 server (Guindon et al., 2010). PhyML is a phylogeny tool based on the maximum-likelihood approach, a character-based method of phylogenetic analysis. Inferred tree was visualized by the PRESTO (a Phylogenetic tReEviSualisaTiOn), a tree visualization tool provided by ATGC-Bioinformatics platform (https://gite.lirmm.fr/atgc/Presto).

The USPs were first reported in bacterial systems as a 13.5 kDa cytoplasmic protein triggered by various stressors, including nutritional deprivation, toxic chemicals such as oxidants, heavy metals, acids, and antibiotics, and play an essential role in cell survival under stressful situations(Nyström and Neidhardt, 1992). Subsequently, many USPs in prokaryotes were identified and categorized (Tkaczuk et al., 2013). Proteins of the USPA superfamily play a crucial role in the body's defense mechanism against a variety of stressors. USPA is generated abundantly during growth arrest in E. coli in response to general stress conditions. Universal stress proteins have been reported to play a role in bacteria's persistence during stressful environmental conditions. Previous research has revealed that strains of E. coli(Nyström and Neidhardt, 1993, Nyström and Neidhardt, 1994, Nachin et al., 2005) and Salmonella Typhimurium (Liu et al., 2007) with a deletion of the uspA gene have a reduced ability to survive for lengthy periods of time during stasis caused by various stressors such as nutrient deprivation, temperature fluctuations, and oxidative stress. Although the biochemical mechanisms underlying the USPA proteins' ability to protect against stress are still mostly unclear. Here we demonstrated that the Salmonella Typhimurium strain possesses a functional uspA homolog and fallowing are the results of our study on USPA of Salmonella Typhimurium.

Identification of the uspA gene and sequencing

The uspA gene from the Salmonella Typhimurium strain was amplified by PCR using self-designed primers (Fig. 1). The amplicons were successfully cloned into the pET28c expression vector. The clones were then transformed into E. coli DH5α cells, and after growing on LB-agar with kanamysin (30mg/ml), the clones were screened by colony PCR, plasmid PCR, re-digestion by restriction enzymes to release insert and finally sequenced (Fig. 2). The nucleotide sequence was submitted to NCBI GenBank with the accession number OM105654.

The comparison of the sequence of uspA gene with that reported in GenBank showed that identities were 100% (Table 2). Furthermore, sequence analysis also revealed the uspA gene is 435 bp long, encodes a protein of 144 amino acids with a calculated molecular mass of 16 kDa, and belongs to the USP family. Also uspA of Salmonella Typhimurium was 100% identical to the uspA gene of Salmonella LT2 strain available in the database(Fig. 5).

Homology modeling and validation

The availability of the 3D structure of the target is most crucial to proceed for the structure-based drug design. Since the 3D structure of Universal stress protein A (USPA) of Salmonella Typhimurium, is not available in the Protein Data Bank (PDB), therefore its 3D structure was predicted by the homology modelling method using the Swiss model and HHpred server,respectively(Kumawat et al., 2020). To identify the best template(s) for the target protein, database searching was performed using the BLASTp program. A template (PDB ID: 1JMV_A) having 68.57% sequence identity was selected for predicting the 3D model of the USPA. The sequence identities and GMQE value are important parameters that are considered during the template(s) selection. The templates which are having high sequence identity (> 30%) and GMQE score closer to 1.0 are often selected for the reliable model generation of the target protein. The multiple sequence alignment was performed for the target-template sequences using the Clustal Omega program and aligned target-template sequences were submitted to the Swiss model server(Waterhouse et al., 2018)and HHpred server (Guindon et al., 2010, Gabler et al., 2020, Eswar et al., 2006) respectively. Then 3D model of the USPA was created (Fig. 3A). In order to evaluate the stereo-chemical properties as well as to find any anomaly in the predicted model, it was uploaded to the SAVES v6.0 server. The quality of models predicted by Swiss-model as well as HHpred server were compared, and it was found that better quality model was predicted by the Swiss-model server (Table 1).

Table 1

Comparison of quality of model predicted by Swiss-Model and HHpred Server
S.No.	Module	Swiss Model Server	HHpred Server
1.	Errat (Overall quality factor)	100	81.82
2.	Verrify 3D(% of the residues have averaged 3D-1D score > = 0.2)	81.02 (Passed)	70.71 (Failed)
3.	Procheck (Ramachandran plot)	i. Core region: 92.7% ii. Allowed region: 7.3% iii. Gen. allowed: 00 iv. Disallowed region: 00 v. Bad contacts: 00	i. Core region: 93.7% ii. Allowed region: 4.7% iii. Gen. allowed: 00 iv. Disallowed region: 1.6 v. Bad contacts: 01

Table 2

Result of nucleotide blast showing comparison of uspA gene of *S.enterica* serovar Typhimurium strain 3232 with other *Salmonella* serovar
S.No.	Description	Max Score	Total Score	Ident	Accession
1	Salmonella sp. A39 chromosome, complete genome	804	804	100%	CP084194.1
2.	Salmonella enterica subsp. enterica serovar Newlands strain ZC-S1 3rd chromosome, complete genome	804	804	100%	CP082916.1
3.	Salmonella enterica strain SP chromosome, complete genome	804	804	100%	CP077668.1
4.	Salmonella enterica subsp. enterica serovar Typhimurium strain CVM N18S2188 chromosome, complete genome	804	804	100%	CP082523.1
5.	Salmonella enterica subsp. enterica serovar Typhimurium strain CVM N18S2170 chromosome, complete genome	804	804	100%	CP082526.1
6.	Salmonella enterica subsp. enterica serovar Hadar strain CVM N18S2154 chromosome, complete genome	804	804	100%	CP082531.1
7.	Salmonella enterica subsp. enterica serovar Typhimurium strain CVM N18S1677 chromosome, complete genome	804	804	100%	CP082543.1
8.	Salmonella enterica subsp. enterica serovar Hadar strain CVM N18S1943 chromosome, complete genome	804	804	100%	CP082540.1
9.	Salmonella enterica subsp. enterica serovar Typhimurium strain CVM N18S1634 chromosome, complete genome	804	804	100%	CP082553.1
10.	Salmonella enterica subsp. enterica serovar Typhimurium strain CVM N18S1595 chromosome, complete genome	804	804	100%	CP082558.1
11.	Salmonella enterica subsp. enterica serovar Typhimurium strain CVM N18S0981 chromosome, complete genome	804	804	100%	CP082577.1
12.	Salmonella enterica subsp. enterica serovar Enteritidis strain SE211 chromosome, complete genome	804	804	100%	CP084532.1
13.	Salmonella enterica subsp. enterica serovar Typhimurium strain CVM N18S0666 chromosome, complete genome	804	804	100%	CP082596.1
14.	Salmonella enterica subsp. enterica serovar Typhimurium strain CVM N18S0645 chromosome, complete genome	804	804	100%	CP082674.1
15.	Salmonella enterica subsp. enterica serovar Typhimurium strain CVM N18S0597 chromosome, complete genome	804	804	100%	CP082681.1

Procheck results of swiss-model show that on the Ramachandran plot (RC), 92.7% of amino acid residues fall in core (most favored) regions, 7.3%in allowed, and no residue falls in the disallowed region. The wrong contacts in the 3D model were also found to be zero. These evaluation parameters suggest that the Swiss-model server predicted a good quality model of USPA(Fig. 3B). Moreover, the protein model in which > 90% residues fall in core regions on RC plot can be utilized for further studies such as molecular dynamics simulation or structure-based drug designing (Yadav et al., 2013).

Phylogenetic analysis of uspA gene

All the related uspA gene sequences were aligned by the multiple sequence alignment method using the Clustal omega server. The aligned sequences were given as input files in PHYLIP file format in the PhyMLv3.0 server, keeping default parameters. A phylogenetic tree was constructed using the SMS (Smart Model Selection) algorithm(Lefort et al., 2017)incorporated in the PhyML program. For tree construction, standard bootstrap value was set to as 100, which indicates how many times out of 100 the same branch was observed when repeating the phylogenetic reconstruction on a re-sampled set of input data. The phylogenetic tree was constructed and subsequently the tree layout was visualized in linear and radial view respectively (Fig. 4A, B).

The phylogenetic tree was constructed using a 0.5–2.5% scale bar. The number on the scale in Fig. 4A suggests the percentage of genetic variation in the corresponding taxon. The evolutionary history of the uspA gene was traced back to the phylogram depicted in Fig. 4A. The tree represents that the uspA gene of Salmonella Typhimurium is closely related to the uspA gene of bacteria Shigella sonnei, E. coli K-12 and Proteus mirabilis. All these share a common lineage with Serratia marcescens. Furthermore, these genes were also sharing a significant evolutionary relationship with other bacteria such as Salmonella enterica, Pseudomonas simiae, and Bacillus subtilis subsp. subtilis 168.

Interestingly, uspA gene from another plant, such as Brassica rapa, also shows some evolutionary relationship with bacteria. It indicates that uspA genes might have been evolved from bacteria to some other plants, which are commonly expressed in stress conditions. The uspA genes from Oryza sativa and Vitis vinifera are distantly related to other organisms, and have evolved from a separate lineage.

A confirmed uspA gene from Salmonella Typhimurium was reported in this study. This study laid a solid foundation for recombination and research of genes. Here,uspA of Salmonella Typhimurium was 100% identical to the uspA gene of Salmonella LT2 strain available in the database. Besides, 3D structure modeling of USPA protein was found to be of high quality with zero bad contacts, and the Ramachandran plot showed 92.7% amino acid residues in favoured regions and 7.3% in allowed regions, and none of the residue fall in disallowed regions which indicated that our 2D model is of high quality. 3D structure prediction also showed that USPA of salmonella Typhimuriumis a homodimer consisting of two polypeptide chains. A study of its evolution history through the construction of a phylogenetic tree depicted its close relationship with other uspA genes found in Shigella sonnei, E. coli K-12and Proteus mirabilis with common linage with Serratia marcescens. It also showed an evolutionary relationship with other organisms, as discussed above.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgments

The authors are thankful to the ICMR-NIREH &SHUATS for providing the necessary funds and facilities for conducting this study.

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No competing interests reported.

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Cloning, sequence analysis, and three-dimensional structure prediction of Universal stress protein A from Salmonella Typhimurium Isolated from the Poultry

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