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Type three secretion systems enable bacterial pathogens to inject effectors into the cytosol of eukaryotic hosts to reprogram cellular functions. It is technically challenging to label effectors and the secretion machinery without disrupting their structure/function. Herein, we present a new approach for labeling and visualization of previously intractable targets. Using genetic code expansion, we site-specifically labeled SsaP, the substrate specificity switch, and SifA, a here-to-fore unlabeled secreted effector. SsaP was secreted at later infection times; SsaP labeling demonstrated the stochasticity of injectisome and effector expression. SifA was labeled after secretion into host cells via fluorescent unnatural amino acids or non-fluorescent labels and a subsequent click reaction. We demonstrate the superiority of imaging after genetic code expansion compared to small molecule tags. It provides an alternative for labeling proteins that do not tolerate N- or C-terminal tags or fluorophores and thus is widely applicable to other secreted effectors and small proteins.

After Salmonella is phagocytosed, it resides in an acidic vacuole. Its cytoplasm acidifies to pH 5.6; acidification activates pathogenicity island 2 (SPI-2). SPI-2 encodes a type three secretion system whose effectors modify the vacuole, driving endosomal tubulation. Using super-resolution imaging in single bacterial cells, we show that low pH induces expression of the SPI-2 SsrA/B signaling system. Single particle tracking, atomic force microscopy, and single molecule unzipping assays identified pH-dependent stimulation of DNA binding by SsrB. A so-called phosphomimetic form (D56E) was unable to bind to DNA in live cells. Acid-dependent DNA binding was not intrinsic to regulators, as PhoP and OmpR binding was not pH-sensitive. The low level of SPI-2 injectisomes observed in single cells is not due to fluctuating SsrB levels. This work highlights the surprising role that acid pH plays in virulence and intracellular lifestyles of Salmonella; modifying acid survival pathways represents a target for inhibiting Salmonella., eLife digest Salmonellae are a group of bacteria that can cause vomiting and diarrhea if we consume contaminated food. Once in the bowel, the bacteria get inside our cells, where they stay in a compartment called the vacuole. This environment is very acidic, and the inside of the microbes also becomes more acidic in response. This change helps Salmonella to switch on genes that allow them to survive and infect humans, but it is still unclear how this mechanism takes place. To investigate this question, Liew, Foo et al. harnessed a recent technique called super-resolution imaging, which lets scientists see individual molecules in a cell. First, the technique was used to count a protein called SsrB as well as the enzyme that activates it, SsrA. The role of SsrB is to bind to DNA and turn on genes involved in making proteins that help Salmonella thrive. These studies revealed that the levels of SsrA/B proteins increased three-fold in an acidic environment. Then, Liew, Foo et al. followed SsrB inside cells, knowing that fast-moving particles are free in solution, while slow-moving particles are typically bound to DNA. In acidic conditions, the proportion of SsrB bound to DNA doubled. Finally, further experiments revealed that when the environment was acidic, SsrB became five times more likely to bind to DNA. Taken together, the results suggest that acidic conditions trigger a cascade of events which switch on genetic information that allows Salmonella to survive. If SsrB could be prevented from responding to acid stress, it could potentially stop Salmonella from surviving inside host cells. This knowledge should be applied to drive new treatment strategies for Salmonella and other microbes that infect human cells.