Abstract
The gram-negative bacterium Francisella tularensis (Ft) is both a potential biological weapon and a naturally occurring microbe that survives in arthropods, fresh water amoeba, and mammals with distinct phenotypes in various environments. Previously, we used a number of measurements to characterize Ft grown in Brain-Heart Infusion (BHI) broth as (1) more similar to infection-derived bacteria, and (2) slightly more virulent in naïve animals, compared to Ft grown in Mueller Hinton Broth (MHB). In these studies we observed that the free amino acids in MHB repress expression of select Ft virulence factors by an unknown mechanism. Here, we tested the hypotheses that Ft grown in BHI (BHI-Ft) accurately displays a full protein composition more similar to that reported for infection-derived Ft and that this similarity would make BHI-Ft more susceptible to pre-existing, vaccine-induced immunity than MHB-Ft. We performed comprehensive proteomic analysis of Ft grown in MHB, BHI, and BHI supplemented with casamino acids (BCA) and compared our findings to published “omics” data derived from Ft grown in vivo. Based on the abundance of ~1,000 proteins, the fingerprint of BHI-Ft is one of nutrient-deprived bacteria that—through induction of a stringent-starvation-like response—have induced the FevR regulon for expression of the bacterium's virulence factors, immuno-dominant antigens, and surface-carbohydrate synthases. To test the notion that increased abundance of dominant antigens expressed by BHI-Ft would render these bacteria more susceptible to pre-existing, vaccine-induced immunity, we employed a battery of LVS-vaccination and S4-challenge protocols using MHB- and BHI-grown Ft S4. Contrary to our hypothesis, these experiments reveal that LVS-immunization provides a barrier to infection that is significantly more effective against an MHB-S4 challenge than a BHI-S4 challenge. The differences in apparent virulence to immunized mice are profoundly greater than those observed with primary infection of naïve mice. Our findings suggest that tularemia vaccination studies should be critically evaluated in regard to the growth conditions of the challenge agent.
Highlights
Francisella tularensis (Ft) is an extremely infectious gramnegative bacterium that is readily aerosolized
Late-log phase Ft grown in MHB, BHI, or BHI supplemented with casamino acids (BCA) were harvested by centrifugation (8,000 × g, 15 min, 20◦C)
In an effort to identify in vitro cultivation conditions for Ft that yielded “host-adapted”-like bacteria, we used a variety of biochemical and immunological approaches to compare Ft grown in MHB and BHI to Ft grown within macrophages (M s)
Summary
Francisella tularensis (Ft) is an extremely infectious gramnegative bacterium that is readily aerosolized. Antibiotic-resistant strains of this bacterium were developed by at least one nation’s biological weapons program (Oyston et al, 2004) The risk of such an agent being maliciously employed, in concert with the current lack of a licensed tularemia vaccine, has prompted intense interest in Ft. The risk of such an agent being maliciously employed, in concert with the current lack of a licensed tularemia vaccine, has prompted intense interest in Ft This pathogen is naturally-occurring in diverse environments including warmand cold-blooded hosts (Oyston et al, 2004) and has both intracellular (replicative) and extracellular (transmissive) phases (Abd et al, 2003; Forestal et al, 2007; Yu et al, 2007; BarHaim et al, 2008; Thelaus et al, 2009). Mounting evidence indicates that as Ft progresses through the intracellular cycle (phagocytosis, phagosomal escape, logarithmic cytoplasmic replication, induction of autophagic vacuoles, and cellular egress) the bacterium adapts by modulating expression of many Ft genes (de Bruin et al, 2007; Chong et al, 2008; Wehrly et al, 2009)
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