Browsing by Author "Mohapatra, Nrusingh"

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    Further Characterization of the Capsule-Like Complex (CLC) Produced by Francisella tularensis Subspecies tularensis: Protective Efficacy and Similarity to Outer Membrane Vesicles
    Champion, Anna E.; Bandara, Aloka B.; Mohapatra, Nrusingh; Fulton, Kelly M.; Twine, Susan M.; Inzana, Thomas J. (Frontiers, 2018-06-15)
    Francisella tularensis is the etiologic agent of tularemia, and subspecies tularensis (type A) is the most virulent subspecies. The live vaccine strain (LVS) of subspecies holarctica produces a capsule-like complex (CLC) that consists of a large variety of glycoproteins. Expression of the CLC is greatly enhanced when the bacteria are subcultured in and grown on chemically defined medium. Deletion of two genes responsible for CLC glycosylation in LVS results in an attenuated mutant that is protective against respiratory tularemia in a mouse model. We sought to further characterize the CLC composition and to determine if a type A CLC glycosylationmutant would be attenuated inmice. The CLCs isolated from LVS extracted with 0.5% phenol or 1M urea were similar, as determined by gel electrophoresis and Western blotting, but the CLC extracted with urea was more water-soluble. The CLC extracted with either 0.5% phenol or 1M urea from type A strains was also similar to the CLC of LVS in antigenic properties, electrophoretic profile, and by transmission electron microscopy (TEM). The solubility of the CLC could be further enhanced by fractionation with Triton X-114 followed by N-Lauroylsarcosine detergents; the largest (>250 kDa) molecular size component appeared to be an aggregate of smaller components. Outer membrane vesicles/tubules (OMV/T) isolated by differential centrifugation and micro-filtration appeared similar to the CLC by TEM, and many of the proteins present in the OMV/T were also identified in soluble and insoluble fractions of the CLC. Further investigation is warranted to assess the relationship between OMV/T and the CLC. The CLC conjugated to keyhole limpet hemocyanin or flagellin was highly protective against high-dose LVS intradermal challenge and partially protective against intranasal challenge. A protective response was associated with a significant rise in cytokines IL-12, IL-10, and IFN-γ. However, a type A CLC glycosylation mutant remained virulent in BALB/cmice, and immunization with the CLC did not protectmice against high dose respiratory challenge with type A strain SCHU S4.
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    Glycosylation of a Capsule-Like Complex (CLC) by Francisella novicida Is Required for Virulence and Partial Protective Immunity in Mice
    Catanzaro, Kelly C. Freudenberger; Champion, Anna E.; Mohapatra, Nrusingh; Cecere, Thomas E.; Inzana, Thomas J. (Frontiers, 2017-05-30)
    Francisella tularensis is a Gram-negative bacterium and the etiologic agent of tularemia. F. tularensis may appear encapsulated when examined by transmission electron microscopy (TEM), which is due to production of an extracellular capsule-like complex (CLC) when the bacterium is grown under specific environmental conditions. Deletion of two glycosylation genes in the live vaccine strain (LVS) results in loss of apparent CLC and attenuation of LVS in mice. In contrast, F. novicida, which is also highly virulent for mice, is reported to be non-encapsulated. However, the F. novicida genome contains a putative polysaccharide locus with homology to the CLC glycosylation locus in F. tularensis. Following daily subculture of F. novicida in Chamberlain's defined medium, an electron dense material surrounding F. novicida, similar to the F. tularensis CLC, was evident. Extraction with urea effectively removed the CLC, and compositional analysis indicated the extract contained galactose, glucose, mannose, and multiple proteins, similar to those found in the F. tularensis CLC. The same glycosylation genes deleted in LVS were targeted for deletion in F. novicida by allelic exchange using the same mutagenesis vector used for mutagenesis of LVS. In contrast, this mutation also resulted in the loss of five additional genes immediately upstream of the targeted mutation (all within the glycosylation locus), resulting in strain F. novicida Delta 1212-1218. The subcultured mutant F. novicida Delta 1212-1218 was CLC-deficient and the CLC contained significantly less carbohydrate than the subcultured parent strain. The mutant was severely attenuated in BALB/c mice inoculated intranasally, as determined by the lower number of F. novicida Delta 1212-1218 recovered in tissues compared to the parent, and by clearance of the mutant by 10-14 days post-challenge. Mice immunized intranasally with F. novicida Delta 1212-1218 were partially protected against challenge with the parent, produced significantly reduced levels of inflammatory cytokines, and their spleens contained only areas of lymphoid hyperplasia, whereas controlmice challenged with the parent exhibited hypercytokinemia and splenic necrosis. Therefore, F. novicida is capable of producing a CLC similar to that of F. tularensis, and glycosylation of the CLC contributed to F. novicida virulence and immunoprotection.