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T3SS-1-dependent secretion of fusion protein SipA-CyaA’. Bacterial strains expressing SipA-CyaA’ were grown in vitro under conditions that induce the expression of SPI-1 genes (i.e., LB medium containing 300 mM NaCl) or SPI-2 genes (i.e., N-minimal medium adjusted to pH 5.8). Proteins from culture supernatants and bacterial lysates were subjected to SDS-PAGE in 12% polyacrylamide gels and transferred to PVDF membranes. SipA-CyaA’ fusion protein and DnaK were detected by Western blot using a commercial mouse anti-CyaA’ monoclonal antibody or a commercial mouse anti-DnaK monoclonal antibody. In both cases, an anti-mouse IgG conjugated with horseradish peroxidase was used as secondary antibody.
Source publication
The type III secretion systems (T3SS) encoded in pathogenicity islands SPI-1 and SPI-2 are key virulence factors of Salmonella. These systems translocate proteins known as effectors into eukaryotic cells during infection. To characterize the functionality of T3SS effectors, gene fusions to the CyaA’ reporter of Bordetella pertussis are often used....
Citations
... Fernández et al. [19] report the construction of plasmids that are useful to generate chromosomal CyaA' translational fusions by homologous recombination using the Red recombination system. These fusions can be used to measure the level of expression of a gene and the secretion of a fusion to the culture supernatant by Western blot using anti-CyaA' antibodies. ...
Type III secretion systems (T3SSs) are molecular devices that are essential for the communication of many Gram-negative bacteria with their eukaryotic hosts [...]
Salmonella enterica (S. enterica) is a causative agent of numerous foodborne outbreaks, as current industrial measures may be <90% effective. Therefore, bacteriophages have been suggested as an antimicrobial treatment against S. enterica, but it is currently unclear if there is an optimal bacteriophage multiplicity of infection (MOI) against S. enterica. Two bacteriophage cocktails at MOIs 1, 10, 100, 1000 and 10,000 were co-inoculated against four S. enterica strains (S. Enteritidis, S. Newport, S. Muenchen and S. Typhimurium), and populations were estimated on days 0–3. The transcriptional profiles of 20 genes previously indicated to be differentially expressed after bacteriophage treatment were studied by extracting RNA from all four S. enterica strains after bacteriophage SE14, SF5 and SF6 treatment on days 0, 1 and 3, and RT-qPCR was conducted to determine the expression of the 20 selected genes. The results showed that an MOI of 1000 was the most optimal in reducing S. Enteritidis populations to undetectable levels from day 0 to 3. The cas1 (SOS response) and mod (DNA modification and recombination) genes were highly upregulated between 2.5- and 5-fold on day 0 for S. Enteritidis S5-483 and S. Typhimurium S5-536 at MOIs of 1000 and 10,000. On day 3, hsdS (DNA modification and recombination) was upregulated by ~1-fold for S. enteritidis S5-483 after an MOI of 1000. Understanding an optimal bacteriophage MOI can be beneficial to implementing effective and optimal bacteriophage treatments in the industry. Knowledge of S. enterica’s transcriptional response after bacteriophage treatment provides further insight into how S. enterica can survive bacteriophage infection.
