Abstract
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. CyaA’ is a calmodulin-dependent adenylate cyclase that is only active within eukaryotic cells. Thus, the translocation of an effector fused to CyaA’ can be evaluated by measuring cAMP levels in infected cells. Here, we report the construction of plasmids pCyaA’-Kan and pCyaA’-Cam, which contain the ORF encoding CyaA’ adjacent to a cassette that confers resistance to kanamycin or chloramphenicol, respectively, flanked by Flp recombinase target (FRT) sites. A PCR product from pCyaA’-Kan or pCyaA’-Cam containing these genetic elements can be introduced into the bacterial chromosome to generate gene fusions by homologous recombination using the Red recombination system from bacteriophage λ. Subsequently, the resistance cassette can be removed by recombination between the FRT sites using the Flp recombinase. As a proof of concept, the plasmids pCyaA’-Kan and pCyaA’-Cam were used to generate unmarked chromosomal fusions of 10 T3SS effectors to CyaA’ in S. Typhimurium. Each fusion protein was detected by Western blot using an anti-CyaA’ monoclonal antibody when the corresponding mutant strain was grown under conditions that induce the expression of the native gene. In addition, T3SS-1-dependent secretion of fusion protein SipA-CyaA’ during in vitro growth was verified by Western blot analysis of culture supernatants. Finally, efficient translocation of SipA-CyaA’ into HeLa cells was evidenced by increased intracellular cAMP levels at different times of infection. Therefore, the plasmids pCyaA’-Kan and pCyaA’-Cam can be used to generate unmarked chromosomal cyaA’ translational fusion to study regulated expression, secretion and translocation of Salmonella T3SS effectors into eukaryotic cells.
Highlights
Salmonella enterica comprises over 2500 serotypes that are able to infect a wide range of animal hosts causing a variety of diseases ranging from gastroenteritis to systemic infections [1,2]
S. enterica harbors two independent T3SS encoded in pathogenicity islands SPI-1 and SPI-2 (T3SS-1 and T3SS-2, respectively)
At least 7 effectors are known to be secreted through T3SSI-1, 22 through T3SS-2, and 9 through both systems
Summary
Salmonella enterica comprises over 2500 serotypes that are able to infect a wide range of animal hosts causing a variety of diseases ranging from gastroenteritis to systemic infections [1,2]. During the course of infection, S. enterica injects effector proteins into the cytoplasm of host cells using type III secretion systems (T3SS). This process is relevant for Salmonella virulence, as most T3SS effectors subvert host cellular functions through their. Microorganisms 2021, 9, 475 enzymatic activities and physical interactions, promoting bacterial survival and colonization (reviewed in [2]). Effectors include a signal sequence at the N-terminal region (first 20–30 residues) required for secretion through T3SS. These sequences lack a discernible consensus, which hinders the identification of possible effector proteins [4]
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