Differential Growth of Francisella tularensis, Which Alters Expression of Virulence Factors, Dominant Antigens, and Surface-Carbohydrate Synthases, Governs the Apparent Virulence of Ft SchuS4 to Immunized Animals

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.

Development, Characterization, and Standardization of a Nose-Only Inhalation Exposure System for Exposure of Rabbits to Small-Particle Aerosols Containing Francisella tularensis

“…As part of these efforts, we report here that while the choice of culture medium for the aerosolized challenge agent did not affect the survival of unvaccinated rabbits, it did affect the survival of vaccinated rabbits. The latter observations mirror those reported for the intranasal challenge of vaccinated inbred mice (27). The implications of these findings and the impact on conducting pivotal vaccine efficacy studies for tularemia and other high-priority, aerosolized agents are discussed.…”

Development, characterization and standardization of a nose-only inhalation exposure system for exposure of rabbits to small particle aerosols containingFrancisella tularensis

“…As part of these efforts, we report here that while the choice of culture media for the aerosolized challenge agent did not affect survival of unvaccinated rabbits, it did affect survival of vaccinated rabbits. These latter observations mirror those reported for intranasal challenge of vaccinated inbred mice (26). The implications of these findings and the impact on conducting pivotral vaccine efficacy studies for tularemia and other high-priority, aerosolized agents will be discussed.…”

“…We recently reported that LVS-vaccinated, inbred mice survived better if challenged with MHB-grown SCHU S4 than BHI-grown SCHU S4 (26). Using the parameters defined above, we sought to evaluate whether the same pattern of vaccine-mediated protection would be seen in aerosol-challenged outbred rabbits.…”

“…BHI-grown Ft has been shown to more closely resemble Ft isolates from infected macrophages than MHB-grown Ft (37). We recently reported that LVS-vaccinated mice survived low-dose challenge with MHB-grown SCHU S4 better than after challenge with comparable doses of BHI-grown SCHU S4 (26). Here, we did not see 100% survival of LVS-vaccinated rabbits when challenged with ~500 cfu (~25 LD 50 ) MHB-grown SCHU S4, and even those that did survive developed a fever and lost weight post-challenge.…”

Stringent Response Governs the Virulence and Oxidative Stress Resistance of Francisella tularensis

“…The expression of several transcriptional regulators, including pmrA, is negatively regulated by RelA/SpoT, and this regulation is independent of the oxidative stress response. An upregulation of pmrA1 has also been reported in bacteria grown in Brain Heart Infusion broth, which mimic intramacrophage growth conditions (27). The upregulated expression of pmrA, specifically in the Δ relA Δ spoT mutant is intriguing and need an additional investigation.…”