Molecular Prediction and Correlation of the Structure and Function of Universal Stress Protein A (UspA) from Salmonella Typhimurium.

Salmonella Typhimurium (ST) is a zoonotic pathogen that can cause gastroenteritis in humans when they consume contaminated food or water. When exposed to various stressors, both from living organisms (biotic) and the environment (abiotic), Salmonella Typhimurium produces Universal Stress Proteins (USPs). These proteins are gaining recognition for their crucial role in bacterial stress resistance and the ability to enter a prolonged state of growth arrest. Additionally, USPs exhibit diverse structures due to the fusion of the USP domain with different catalytic motifs, enabling them to participate in various reactions and cellular activities during stressful conditions. In this particular study, researchers cloned and analyzed the uspA gene obtained from poultry-derived strains of Salmonella Typhimurium. The gene comprises 435 base pairs, encoding a USP family protein consisting of 144 amino acids. Phylogenetic analysis demonstrated a close relationship between the uspA genes of Salmonella Typhimurium and those found in other bacterial species. We used molecular dynamics simulations and 3D structure prediction to ensure that the USPA protein was stable. Furthermore, we also carried out motif search and network analysis of protein-protein interactions. The findings from this study offer valuable insights for the development of inhibitors targeted against Salmonella Typhimurium.

Purification and characterization of Cyclophilin: a protein associated with protein folding in Salmonella Typhimurium.

Salmonella Typhimurium (ST) is a Gram-negative zoonotic pathogenic bacterium that causes infectious disease in humans as well as in animals. It causes foodborne diarrheal or gastrointestinal illness and fever called salmonellosis, which is a leading cause of millions of deaths worldwide. Salmonellaenterica serovar Typhimurium (S. Typhimurium) during its pathogenesis take away the actin cytoskeleton of their host cells and this is the crucial step of its infection cycle. Cyclophilin A, a type of peptidyl-prolyl isomerase that's encoded by the ppiA gene in ST, plays pleiotropic roles in maintaining bacterial physiology. In this investigation, the proteomic characterization of the peptidyl-prolyl cis-trans isomerase- A (Cyclophilin A) from Salmonella Typhimurium is reported. Cyclophilin A (CypA) protein from Salmonella Typhimurium proved to be highly conserved and homologous protein sequence compared to other organisms. This protein was expressed in Escherichia coli followed by its purification in a recombinant form protein exhibited a characteristic PPIases activity (Vmax = 0.8752 ± 0.13892 µmoles/min, Km = 0.9315 ± 0.5670 µM) in comparison to control. The mass spectrometry analysis of Cyp A protein-peptide showed a highest sequence similarity with the cyclophilin protein of Salmonella. PPIases proteins (enzyme) data suggest that Ppi-A has roles in the protein folding that may be contributing to the virulence of Salmonella by isomerization of protein outline. These results suggest an active and vital role of this protein in protein folding along with regulation in Salmonella Typhimurium.

Salmonella Typhimurium peptidyl-prolyl cis-trans isomerase C (PPIase C) plays a substantial role in protein folding to maintain the protein structure.

Salmonella is a well-known food-borne pathogen causing disease in humans and animals worldwide. Peptidyl-prolyl isomerases (PPIases) catalyse the cis-trans isomerisation of prolyl bound, which is a slow and rate-limiting step of protein folding. Here, we present the biochemical and molecular characterisation of a novel multi-domain parvulin-type, PPIases-C from the pathogenic bacteria Salmonella Typhimurium, annotated as rPpiC. The recombinant plasmid PpiC_pET28c was used for protein induction using 1.5 mM concentration of isopropyl-ß-D-thiogalactopyranoside at 30 °C. Subsequently, the protein was identified by using the LC-MS technique showing high match score and sequence coverage with available PPIases-C proteins database. Using the succinyl-ala-phe-pro-phe-p nitroanilide as a substrate, Vmax of the enzyme was found to be 0.8187 ± 0.1352 µmoles/min and Km = 1.6014 ± 0.8449 µM, respectively. With this, we conclude that rPpiC protein is an active form of protein from Salmonella Typhimurium and plays an important role in protein folding.

Molecular, Structural, and Functional Diversity of Universal Stress Proteins (USPs) in Bacteria, Plants, and Their Biotechnological Applications.

Universal stress proteins (USPs) are widely distributed and play crucial roles in cellular responses to biotic and abiotic stresses. These roles include regulating cell growth and development, cell motility, hypoxia responses, and ion sequestration. With the increasing frequency and intensity of extreme weather events due to climate change, pathogens have developed different strategies to withstand environmental stresses, in which USPs play a significant role in their survival and virulence. In this study, we analyzed the importance of USPs in various organisms, such as archaea, plants, and fungi, as a parameter that influences their survival. We discussed the different types Of USPs and their role, aiming to carry out fundamental research in this field to identify significant constraints for better understanding of USP functions at molecular level. Additionally, we discussed concepts and research techniques that could help overcome these hurdles and facilitate new molecular approaches to better understand and target USPs as important stress adaptation and survival regulators. Although the precise characteristics of USPs are still unclear, numerous innovative uses have already been developed, tested, and implemented. Complementary approaches to basic research and applications, as well as new technology and analytical techniques, may offer insights into the cryptic but crucial activities of USPs in various living systems.

Identification of Salmonella Typhimurium Peptidyl-prolyl cis-trans Isomerase B (PPIase B) and Assessment of their Role in the Protein Folding.

BACKGROUND: Peptidyl-prolyl cis-trans isomerase (PPIases) enzyme plays a vital role in protein folding. It catalyses the cis-trans isomerisation of peptide bonds, an essential step for newly synthesized protein to acquire its correct functional conformation in both prokaryotes and eukaryotes. OBJECTIVE: The present study showed the biochemical and molecular characterisation of cyclophilins (PpiB), a type of peptidyl-prolyl isomerases proteins from the pathogenic bacteria Salmonella Typhimurium. METHODS: Salmonella Typhimurium is one of the leading serovars responsible for human and animal salmonellosis globally, with the majority of human cases originating through the food chain. Here successful expression and purification of PpiB protein have been demonstrated and LC-MS based analyses showed high protein score and similarity with other PPi protein. Further the enzymatic activity of the purified recombinant PpiB was determined using Succinyl-Ala-Phe-Pro- Phe-p nitroanilide as substrate and enzyme-catalysed reaction. RESULT: Km and Vmax were calculated and found to be Vm = 1.023 ± .06400 min/µg, Km = 0.6219 ± 0.1701 µM, respectively. We have reported for the first time the presence of Salmonella PPIase-B (PpiB) protein isoforms in salmonella genome having PPi activity. CONCLUSION: Taken together, our data clearly showed that Salmonella Cyclophilin B (PpiB) protein is active and involved in diverse biological processes and highly similar to the different domain of Cyclophilin proteins.

Comparative evaluation of structure and characteristic of peptidyl-prolyl cis-trans isomerase proteins and their function in Salmonella Typhimurium stress responses and virulence.

Peptidyl-prolyl cis-trans isomerases (PPIase) exhibit chaperone activity and assist in protein folding by increasing the rate of cis-trans transition on proline-peptide bonds. The current study aimed to identify and characterize three genes, ppiA, ppiB, and ppiC, which encode proteins of the PPIase family in the bacterium Salmonella enterica serovar Typhimurium. Salmonella Typhimurium is a facultative intracellular zoonotic pathogen that causes food- and water-borne gastroenteritis in humans (leading to bacteremia in immune-compromised subjects). Recombinant clones for the three genes were constructed and sequenced and the sequences submitted to NCBI GenBank. Three-dimensional structures for the corresponding proteins were predicted by comparative modeling. A maximum-likelihood phylogenetic gene tree constructed for the three genes showed a low evolutionary mean diversity, indicating strong evolutionary conservation. Further, single-gene deletion mutant strains, generated for the respective genes, were observed to be more susceptible to the stationary phase of growth and heat stress conditions and showed reduced survival within macrophage cells line. The present study thus indicates that ppiA, ppiB, and ppiC genes are conserved among Salmonella genome, are critical for the growth of Salmonella Typhimurium in the examined stress conditions, and may play a role in its responses and virulence.