Background

Flagella are helical, corkscrew-like appendages that, depending on their clockwise or counterclockwise rotation, push or pull bacterial cells. They act like tiny propellers allowing bacteria to move through liquids or across hydrated surfaces. For the assembly of the bacterial flagellum, a flagellar type 3 secretion (T3S) system initially exports early component substrates that build the hook-basal body (HBB) structure, which is the main component making up the flagellar motor. Upon HBB completion, the flagellar T3S system undergoes a secretion substrate specificity switch, resulting in an export selectivity for late substrate proteins, which include flagellin that assembles into the long external filament that acts as the propeller of the flagellum.

The export of T3S substrates is traditionally evaluated using trichloroacetic acid (TCA) precipitation of the supernatant of cultured cells, followed by western blot analysis of the secreted substrates. This technique is very useful at assessing protein translocation but is limited to the study of substrates that are translocated outside the cells. We have developed a reporter assay using β-lactamase (Bla), lacking its Sec secretion signal, for the secretion of flagellar proteins into the periplasm through the flagellar T3S system. Bla is an enzyme that cleaves and inactivates β-lactam antibiotics, such as ampicillin (Ap), through hydrolysis of the peptide bond of the characteristic four-membered β-lactam ring. The inactivation of the antibiotic provides Ap-resistance (Ap^R) to the bacterium; for that, Bla needs to be transported into the periplasm where it folds into an active conformation. Such translocation into the periplasm occurs through the Sec-secretion pathway. Secreted proteins through the Sec-dependent pathway are readily recognized by an N-terminal signal sequence, which is cleaved during the process of secretion into the periplasm to yield a mature protein. Flagellar proteins are exported through the T3S pathway. Fusing Bla without its N-terminal signal sequence to the C-terminal of flagellar proteins results in the transport of the Fla-Bla fusions into the periplasm, where Bla is active and confers Ap^R (Lee and Hughes, 2006; Hirano et al., 2009; Erhardt and Hughes, 2010; Singer et al., 2014; Hendriksen et al., 2021; Qu et al., 2022). In cells expressing intact flagellar structures, Fla-Bla fusions are secreted into the periplasm transiently after completion of the flagellar core T3S system until outer membrane penetration. Once the flagellar structure penetrates the outer membrane, the Fla-Bla fusions are secreted into the extracellular medium. Mutants defective in rod assembly continuously secrete Fla-Bla fusion into the periplasm, which results in higher levels of Ap^R. By using assays to assess minimum inhibitory concentrations of ampicillin, we can estimate how much of the Fla-Bla fusion is exported and provide a quick estimate of secreted flagellar substrate levels. The use of Bla as a reporter for the flagellar T3S substrates also provides a positive selection for secretion. Using this technique, we were able, for example, to localize important sites of recognition in an early substrate’s secretion signal by the flagellar T3S apparatus located at the cytoplasmic base of flagellum (Qu et al., 2022). This system is not specific to Salmonella; Bla fusions to T3S proteins have been used in other Gram-negative pathogens, such as enteropathogenic and enterohemorrhagic Escherichia coli and Yersinia enterocolitica (Charpentier and Oswald, 2004; Diepold et al. 2015). In these systems, Bla fusions were used to measure the translocation of effector proteins in living host cells and in the extracellular medium, using a fluorescent β-lactamase substrate. Thus, fusion to Bla can be used to measure translocation of proteins either into the periplasm or the extracellular medium. We use secretion of Bla fusions in the periplasm because of the powerful selection of Ap^R. Here, we describe in detail the protocol used to assay the minimum inhibitory concentration to ampicillin for the assessment of the translocation efficiency of protein fusions in the periplasm and for positive selections using this system.