Single cell, super-resolution imaging reveals an acid pH-dependent conformational switch in SsrB regulates SPI-2

Abstract

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.

EnvZ/OmpR Two-Component Signaling: An Archetype System That Can Function Noncanonically

“…These measurements were made possible by our ability to separate phosphorylated and unphosphorylated forms of the protein by HPLC (108), an approach that is now widely used with other RRs. High affinity binding by OmpR/OmpR~P is also observed in in vitro transcription studies (90,109), as well as by atomic force microscopy (3) and superresolution imaging (91). OmpR makes more phosphate backbone contacts and fewer specific base contacts (110) compared to RRs that recognize precise motifs such as PhoP, PhoB and others.…”

“…Footprinting analysis identified several OmpR binding sites upstream of ssrA and both upstream and within the ssrB coding sequence (112,113). The expression of the kinase SsrA is uncoupled from the expression of the response regulator SsrB (90,186). This is unusual compared to most two-component regulatory systems in which the ratio of kinase to response regulator is tightly controlled.…”

“…We hypothesize that the presence of the cognate HK has higher affinity/specificity for its cognate RR, preventing a lower affinity interaction with CpxR. Now that new tools and super-resolution imaging in bacteria is possible (39,90,91), it will be worthwhile to determine the extent to which OmpR is bound to EnvZ using single particle tracking photoactivation localization microscopy, (spt-PALM) and compare with OmpR binding to CpxA in an envZ null strain.…”

Salmonella biofilms program innate immunity for persistence in Caenorhabditis elegans

“…SsrB, as part of the SsrA/B TCRS, mediates transcriptional activation of SPI-2 effectors to enable survival of Salmonella inside an acidic vacuole in macrophages (4,10,12). At more neutral pH, in the intestinal lumen, the SsrA kinase, which normally phosphorylates SsrB, is down-regulated (4,13). SsrB then acts noncanonically to drive the biofilm pathway (reviewed in ref.…”

“…In the phosphorylated form, SsrB∼P activates the transcription of virulence genes that promote the intravacuolar lifestyle (4,10,11). At neutral intracellular pH 6.8 (12), the levels of SsrA kinase are low (13), and unphosphorylated SsrB drives the formation of biofilms by up-regulating the expression of the master regulator gene csgD (5 and reviewed in ref. 14).…”