Growth of calcium-blind mutants of Yersinia pestis at 37 °C in permissive Ca2+-deficient environments

Abstract

Cells of wild-type Yersinia pestis exhibit a low-calcium response (LCR) defined as bacteriostasis with expression of a pCD-encoded type III secretion system (T3SS) during cultivation at 37 °C without added Ca2+ versus vegetative growth with downregulation of the T3SS with Ca2+ (≥2.5 mM). Bacteriostasis is known to reflect cumulative toxicity of Na+, l-glutamic acid and culture pH; control of these variables enables full-scale growth (‘rescue’) in the absence of Ca2+. Several T3SS regulatory proteins modulate the LCR, because their absence promotes a Ca2+-blind phenotype in which growth at 37 °C ceases and the T3SS is constitutive even with added Ca2+. This study analysed the connection between the LCR and Ca2+ by determining the response of selected Ca2+-blind mutants grown in Ca2+-deficient rescue media containing Na+ plus l-glutamate (pH 5.5), where the T3SS is not expressed, l-glutamate alone (pH 6.5), where l-aspartate is fully catabolized, and Na+ alone (pH 9.0), where the electrogenic sodium pump NADH : ubiquinone oxidoreductase becomes activated. All three conditions supported essentially full-scale Ca2+-independent growth at 37 °C of wild-type Y. pestis as well as lcrG and yopN mutants (possessing a complete but dysregulated T3SS), indicating that bacteriostasis reflects a Na+-dependent lesion in bioenergetics. In contrast, mutants lacking the negative regulator YopD or the YopD chaperone (LcrH) failed to grow in any rescue medium and are therefore truly temperature-sensitive. The Ca2+-blind yopD phenotype was fully suppressed in a Ca2+-independent background lacking the injectisome-associated inner-membrane component YscV but not peripheral YscK, suggesting that the core translocon energizes YopD.

YopD Self-assembly and Binding to LcrV Facilitate Type III Secretion Activity by Yersinia pseudotuberculosis

“…It might be concerned with defective proton motive force generation, reduced ATP pools, and the inability to export toxic levels of Na ϩ from the cytoplasm (64,67,68). At 37°C Yersinia appears to be sensitive to Na ϩ but only during active T3S; the presence of Ca 2ϩ in the growth medium inhibits Ysc-Yop T3S, concomitantly relieving Na ϩ sensitivity and reinitiating growth (69). It follows that the Na ϩ and temperature sensitivity displayed by a yopD null mutant constitutively producing an active T3SS cannot be suppressed by the addition of Ca 2ϩ (a so-called Ca 2ϩ blind phenotype) (69).…”

“…At 37°C Yersinia appears to be sensitive to Na ϩ but only during active T3S; the presence of Ca 2ϩ in the growth medium inhibits Ysc-Yop T3S, concomitantly relieving Na ϩ sensitivity and reinitiating growth (69). It follows that the Na ϩ and temperature sensitivity displayed by a yopD null mutant constitutively producing an active T3SS cannot be suppressed by the addition of Ca 2ϩ (a so-called Ca 2ϩ blind phenotype) (69). This led to a proposal that another component of the Ysc-Yop T3SS may facilitate the elevation of toxic Na ϩ levels inside the bacteria, thus inhibiting a fundamental cellular process needed for vegetative growth (64,69).…”

“…It follows that the Na ϩ and temperature sensitivity displayed by a yopD null mutant constitutively producing an active T3SS cannot be suppressed by the addition of Ca 2ϩ (a so-called Ca 2ϩ blind phenotype) (69). This led to a proposal that another component of the Ysc-Yop T3SS may facilitate the elevation of toxic Na ϩ levels inside the bacteria, thus inhibiting a fundamental cellular process needed for vegetative growth (64,69). Indeed, one possible candidate could be YscV (also termed LcrD), a core inner membrane component of the Ysc-Yop T3SS, because its depletion from a yopD null mutant subsequently restored Ca 2ϩ -dependent growth to these bacteria at elevated temperature (69).…”

“…This led to a proposal that another component of the Ysc-Yop T3SS may facilitate the elevation of toxic Na ϩ levels inside the bacteria, thus inhibiting a fundamental cellular process needed for vegetative growth (64,69). Indeed, one possible candidate could be YscV (also termed LcrD), a core inner membrane component of the Ysc-Yop T3SS, because its depletion from a yopD null mutant subsequently restored Ca 2ϩ -dependent growth to these bacteria at elevated temperature (69). It is possible that our YopD mutants with intermediary temperature sensitivity, unlike either parental Yersinia or the full-length yopD null mutant, may provide opportunities to better understand this intriguing YopD-YscV regulatory connection.…”

Identification of host factors required for Yersinia pestis macrophage intracellular survival and their impact on vacuole maturation, acidification and trafficking.

“…Furthermore, consecutive passages at 37°C in the absence of Ca 2+ results in loss of the pCD1 plasmid, which encodes the T3SS, and attenuation in mice [336,337]. To date, there is little known about the biological relevance for the LCR [1,[338][339][340][341]. Szatmari and colleagues recently demonstrated that Rab11b interacts with Hook, a negative regulator of endosome maturation, to facilitate crosstalk between recycling endosomes and induction of autophagy [254].…”