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Physiological basis of the low calcium response in Yersinia pestis
1Department of Microbiology, Michigan State University, East Lansing 48824.
Abstract:
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 Ca(2+)-deficient host cell cytoplasm) within visceral organs rather than in Ca(2+)-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+ plus 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 Ca(2+)-deficient media promoted maximum induction of Mg(2+)-induced V antigen, a virulence factor encoded by the Lcr plasmid.
Insights
Yersinia pestis grows vegetatively in simulated intracellular fluid by optimizing ion concentrations and nutrient availability. This research clarifies conditions for Y. pestis proliferation, crucial for understanding plague pathogenesis.
Area of Science:
- Microbiology
- Pathogenesis
- Bacterial Physiology
Background:
- Yersinia pestis exhibits differential growth and virulence factor expression based on environmental ion concentrations (Ca2+, Mg2+).
- In vivo proliferation occurs in Ca2+-deficient intracellular lesions, contrasting with in vitro observations in Ca2+-rich blood.
- This discrepancy highlights a need to understand Y. pestis growth dynamics in simulated intracellular environments.
Purpose of the Study:
- To define conditions for Yersinia pestis vegetative growth at 37°C in simulated intracellular fluid.
- To investigate the role of specific ions (Na+, K+, Cl-) and metabolites (L-glutamate, L-aspartate) in Y. pestis proliferation and virulence gene expression.
- To reconcile in vitro findings with in vivo observations of Y. pestis growth in host lesions.
Main Methods:
- Culturing Yersinia pestis in various simulated intracellular fluid media with controlled ion concentrations and nutrient compositions.
- Measuring bacterial growth using optical densities.
- Assessing virulence factor (V antigen) induction under different growth conditions.
Main Results:
- Substitution of K+ for Na+ and elimination of Cl- enhanced Y. pestis growth in simulated intracellular fluid.
- L-glutamate was toxic to Y. pestis in the presence of Na+, attributed to aspartase deficiency and L-aspartate accumulation.
- Replacement of L-glutamate with L-aspartate or alpha-ketoglutarate reversed toxicity and favored growth.
- Increased fermentable carbohydrate concentration further enhanced proliferation in optimal media.
- Both Ca2+-deficient media promoted Mg2+-induced V antigen expression, a key virulence factor.
Conclusions:
- Specific ionic compositions (K+-rich, Na+- and Cl--poor) and nutrient availability (L-aspartate, fermentable carbohydrates) are critical for Yersinia pestis vegetative growth in simulated intracellular environments.
- Understanding these conditions is essential for elucidating Y. pestis pathogenesis within host tissues.
- The study successfully reconciled in vitro growth characteristics with in vivo proliferation patterns observed in necrotic lesions.