Electrochemical Platforms for Solid-Phase Isothermal Amplification and Detection of Bacterial Genome

In this work, two sensing approaches for the specific quantification of the Salmonella genome are comparatively evaluated. These devices successfully integrate solid-phase isothermal HDA or RPA amplification in electrochemical platforms and support efficient detection of small amounts of the bacterial genome without thermal cycling, while using simple equipment. The detectability of the RPA-based sensing platform (105 genomes) was surpassed by that of the HDA-based one (10 genomes), though it could be enhanced by primer design optimization. This, together with a low operation temperature compatible with conventional surface chemistry and faster amplification, makes the RPA-based electrochemical platform a promising device for point-of-need analysis.

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.

Salmonella enterica’s “Choice”: Itaconic Acid Degradation or Bacteriocin Immunity Genes

Itaconic acid is an immunoregulatory metabolite produced by macrophages in response to pathogen invasion. It also exhibits antibacterial activity because it is an uncompetitive inhibitor of isocitrate lyase, whose activity is required for the glyoxylate shunt to be operational. Some bacteria, such as Yersinia pestis, encode enzymes that can degrade itaconic acid and therefore eliminate this metabolic inhibitor. Studies, primarily with Salmonella enterica subspecies enterica serovar Typhimurium, have demonstrated the presence of similar genes in this pathogen and the importance of these genes for the persistence of the pathogen in murine hosts. This minireview demonstrates that, based on Blast searches of 1063 complete Salmonella genome sequences, not all Salmonella serovars possess these genes. It is also shown that the growth of Salmonella isolates that do not possess these genes is sensitive to the acid under glucose-limiting conditions. Interestingly, most of the serovars without the three genes, including serovar Typhi, harbor DNA at the corresponding genomic location that encodes two open reading frames that are similar to bacteriocin immunity genes. It is hypothesized that these genes could be important for Salmonella that finds itself in strong competition with other Enterobacteriacea in the intestinal tract—for example, during inflammation.