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Title: Glycosylation of a Capsule-Like Complex (CLC) by Francisella novicida Is Required for Virulence and Partial Protective Immunity in Mice

Abstract

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. But, 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 ofmore » the targeted mutation (all within the glycosylation locus), resulting in strain F. novicida Δ11212–1218. The subcultured mutant F. novicida Δ11212–1218 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 Δ11212–1218 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 Δ11212–1218 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 control mice challenged with the parent exhibited hypercytokinemia and splenic necrosis. Thus, 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.« less

Authors:
 [1];  [1];  [1];  [1];  [2]
  1. Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States). Center for Molecular Medicine and Infectous Diseases, Dept. of Biomedical Sciences and Pathobiology
  2. Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States). Center for Molecular Medicine and Infectous Diseases, Dept. of Biomedical Sciences and Pathobiology; Virginia Technical Carilion School of Medicine, Roanoke, VA (United States). Dept. of Biomedical Sciences
Publication Date:
Research Org.:
Univ. of Georgia, Athens, GA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1389272
Grant/Contract Number:
FG02-93ER20097
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Frontiers in Microbiology
Additional Journal Information:
Journal Volume: 8; Journal ID: ISSN 1664-302X
Publisher:
Frontiers Research Foundation
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; 59 BASIC BIOLOGICAL SCIENCES; Francisella novicida; capsule-like complex; virulence in mice; tularemia; glycosylation; protective immunity

Citation Formats

Freudenberger Catanzaro, Kelly C., Champion, Anna E., Mohapatra, Nrusingh, Cecere, Thomas, and Inzana, Thomas J.. Glycosylation of a Capsule-Like Complex (CLC) by Francisella novicida Is Required for Virulence and Partial Protective Immunity in Mice. United States: N. p., 2017. Web. doi:10.3389/fmicb.2017.00935.
Freudenberger Catanzaro, Kelly C., Champion, Anna E., Mohapatra, Nrusingh, Cecere, Thomas, & Inzana, Thomas J.. Glycosylation of a Capsule-Like Complex (CLC) by Francisella novicida Is Required for Virulence and Partial Protective Immunity in Mice. United States. doi:10.3389/fmicb.2017.00935.
Freudenberger Catanzaro, Kelly C., Champion, Anna E., Mohapatra, Nrusingh, Cecere, Thomas, and Inzana, Thomas J.. 2017. "Glycosylation of a Capsule-Like Complex (CLC) by Francisella novicida Is Required for Virulence and Partial Protective Immunity in Mice". United States. doi:10.3389/fmicb.2017.00935. https://www.osti.gov/servlets/purl/1389272.
@article{osti_1389272,
title = {Glycosylation of a Capsule-Like Complex (CLC) by Francisella novicida Is Required for Virulence and Partial Protective Immunity in Mice},
author = {Freudenberger Catanzaro, Kelly C. and Champion, Anna E. and Mohapatra, Nrusingh and Cecere, Thomas and Inzana, Thomas J.},
abstractNote = {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. But, 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 Δ11212–1218. The subcultured mutant F. novicida Δ11212–1218 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 Δ11212–1218 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 Δ11212–1218 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 control mice challenged with the parent exhibited hypercytokinemia and splenic necrosis. Thus, 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.},
doi = {10.3389/fmicb.2017.00935},
journal = {Frontiers in Microbiology},
number = ,
volume = 8,
place = {United States},
year = 2017,
month = 5
}

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