PHYSIOLOGICAL-BASIS OF THE LOW-CALCIUM RESPONSE IN YERSINIA-PESTIS

It is established that duplication in vitro of that amount of Ca2+ (2.5 mM) and Mg2+ (1.5 mM) present in blood permits vegetative growth of Yersinia pestis with repression of virulence factors encoded by the Lcr plasmid (Lcr(+)); similar simulation of intracellular fluid (no Ca2+ and 20 mM Mg2+) promotes bacteriostasis with induction of these virulence determinants. However, proliferation of yersiniae in mice occurs primarily within necrotic focal lesions (supplied by Ca2+-deficient host cell cytoplasm) within visceral organs rather than in Ca2+-sufficient blood, The present study addressed this enigma by defining conditions necessary for achieving vegetative growth of Lcr(+) yersiniae at 37 degrees C in simulated intracellular fluid. Maximum optical densities were increased by substitution of K+ for Na+ and elimination of Cl-; the combination of Na+ pins L-glutamate was selectively toxic to Lcr(+) cells. This phenomenon was attributed in part to the absence of aspartase in Y. pestis (a lesion known to facilitate massive accumulation of L-aspartate via transamination of the oxalacetate pool by L-glutamate). Replacement of L-glutamate by exogenous L-aspartate or alpha-ketoglutarate reversed this toxicity by favoring retention of oxalacetate. Proliferation of Lcr(+) cells in a medium containing K+ and L-aspartate but lacking added Ca2+ and Na+ was markedly enhanced by increasing the concentration of fermentable carbohydrate. Accordingly, in the worst-case scenario (i.e., added Na+, Cl-, and L-glutamate), Lcr(+) yersiniae underwent restriction of growth after one doubling, and in the best-case scenario (i.e., added K+ and L-aspartate), the organisms completed more than five doublings, thereby achieving full-scale growth. Both of these Ca2+-deficient media promoted maximum induction of Mg2+-induced V antigen, a virulence factor encoded by the Lcr plasmid.