Salmonella Typhimurium'S Effector Ssei'S Effects

Salmonella Typhimurium is the model organism for severe food-borne illnesses in humans and other food producing animals. While causing acute self-limiting gastroenteritis, it causes a more severe illness in immunocompromised patients. Salmonella Typhimurium toxicity is due to the outer membrane containing lipopolysaccharides(LPS) that protect the bacteria from the environment. The LPS is made of O-antigen, polysaccharide core, and lipid A. The O-antigen can acetylate by changing conformations which causes difficulty in host antibody recognition. Lipid A is made of phosphorylated glucosamines connected to fatty acids. The phosphate groups cause and determine the level of toxicity or virulence factor.

The virulence factors depend on two type III secretion systems (T3SS), SPI-1 and SPI-2 of Salmonella. The mechanism of Salmonella-specific translocator (Ssel) was unknown until Brink et al., studied the mode of action on SPI-2 effector Ssel involving the intracellular proliferation of the pathogen. Recombinant wildtype Ssel deamidated a glutamine residue in a G protein, which impaired the GTP hydrolysis causing a change in the phenotype. Consequently, the G protein became permanently active. G protein activation leads to signaling cascades of cAMP level decline and an increase phosphoinositol-3-kinase. Furthermore, causing an increase in phosphorylation of Pl3K downstream effectors Akt and mTOR. Catalyzed deamidation of the G protein inhibits dendritic cells. S. enterica servor Typhimurium NTC12023 is the parent strain of the mutant Ssel strain. There is a high amino acid sequence similarity in Ssel and the deamidase domain of Pasturella multiocida toxin (PMT). Evidently, Crystallographic studies show similarity in amino acid sequences of the Ssel protein and the Pasturella multocisa toxin (PMT) deamidase domain. Therefore, a receptor binding and translocation domain of PMT assisted in cellular uptake of Ssel.

To detect deamination of G proteins by bacterial effectors, Brink and colleagues found that Ssel coexpressed with the α subunit of Gi2 in E. Coli. As determined by immunoblot analysis, a monoclonal antibody(GαQE) recognizes the Gα after deamidation of a glutamine residue. GαQE lacked expression Gαiz solely, proving that Ssel involves deamidation. Moreover, Mass spectrometric analysis identified 3-part peptide of Gαiz with a glutamine residue that hydrolyzes GTP. Tandem MS analysis shows that when Gln-205 loses amide group, there is a glutamic acid in that position. There was no deamidation when Gαiz coexpressed with mutant Ssel. However, Recombinantly expressed wild type Ssel caused deamidation of purified Gαiz. (Brink, 2018) Furthermore, The C terminus deamidation domain of Ssel was combined with the N terminal end of PMT. PMT-Ssel was tested for cellular activity.

First, the concentrations of PMT-Sselc in Human embryonic kidney cells 293 (HEK-293) was increased, results show deamination of Gα proteins based on the immunoblot analysis. However, cell treatment with C178A mutant of PMT-Sselc showed no deamidation. Secondly, HEK-293 cells took up PMT and PMT-Ssel. Results showed one deamidation signal migrating led by PMT-Ssel and PMT showed two deamination signals with the same molecular mass as Gα proteins. (Brink, 2018) This proves that Ssel acts as a deamidase in G proteins because there was only one signal with Ssel incorporated with PMT. The main hypothesis researched to prove that Salmonella SPI-2 mode of action was the Ssel effector deamidating proteins. One of the main findings of the paper is the mode of action of Ssel. Brink et al., used earlier studies stating that activation of AKT leads to pro survival and anti-apoptotic outcomes on the cell types of mammals. Cell death determined the release of lactL DH from RAW254.7 macrophages. Studies show that cells infected by mutant Salmonella increases LDH release than the wildtype Salmonella. In brief, Ssel plays a role after the pathogen invades and changes the immune responses. Statistical data shows the effects of Ssel during infection. During fluorescence microscopy of fixed cells, RAW264.7 were infected with the wt S. Typhimurium at multiplicity of infection(MOI), the average number of bacteria affecting cells of 1 for 5 hours. RAW264.7 cells were infected with a MOI of 30 every 30 minutes. During time-resolved immunoblot analysis, p-Akt measured with the wildtype and the mutant Salmonella from 3 independent experiments. The first hour shows the mutant has higher chemiluminescence (area units) that the wildtype but, in the second and fourth hours, there is a dramatic increase of chemiluminescence of the wildtype and a decrease of the mutant over time. Wildtype Salmonella shows strong phosphorylation of Akt. The PI3K inhibitor LY29 added leads to dependent inhibition of Akt phosphorylation.

These statistical analyses used are proper, it justifies the information written and help to explain the cellular effect of Ssel during infection. This shows that deamidation occurred after the bacteria entered the cells. The FRET approach determined levels of cAMP during deamidation of G protein. The fluorescent resonance energy transfer (FRET) approach determined how cAMP levels responded to Ssel. Bone marrow derived mature dendritic cells were infected in a 3D model. In order to investigate whether G subunits impaired chemotaxis by studying Gnai deleted mice and purified G proteins in vitro with deamidation assays. Future directions should look for more G proteins that are activated by SPI-2 effector Ssel. More research will offer new perspectives to effects of SPI-2 and signal factors through chemotaxis. Also, studying the biological effects of effectors in other pathogens with similar amino acid sequences. This was seen PMT and Ssel, a way for the eukaryotic cells to uptake Ssel. More research will address the effector and chemotaxis with other signal factors.

Brink et al., findings show a strength in their research. Brink et al., elucidated the mode of action of the effector SPI-2 which explains microbial pathogenesis. They started their research finding earlier studies and continuing the research with the unknown mechanism of Ssel. For example, comparing amino acid sequences of the PMT toxin and Ssel, finding similar modes of actions.

In conclusion, toxins secreted by bacteria, that are not taken up by eukaryotic cells, can find bacteria with similar characteristics in toxins even if the primary sequence is only 20%.