Studies of the lipopolysaccharide from the intracellular pathogens Francisella tularensis and Francisella novicida




Cowley, Siobhán Clare

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Francisella tularensis and Francisella novicida are closely related facultative intracellular pathogens capable of survival and growth within macrophages. In this work we present evidence to show that F. tularensis uses phase variation to alter lipopolysaccharide (LPS) antigenicity, macrophage nitric oxide (NO) production, and microbial intramacrophage growth. The LPS and lipid A of F. tularensis LVS fail to stimulate production of significant levels of nitric oxide by rat macrophage monolayers. However, spontaneous variants of F. tularensis expressing an antigenically distinct LPS induce rat macrophages to produce increased levels of NO, thereby suppressing intracellular growth. This new form of LPS produced by F. tularensis is also the predominant form of LPS found normally in F. novicida. Rat macrophages infected with F. novicida produce high levels of NO and exhibit suppression of intracellular growth. LPS and lipid A isolated from F. novicida and variants of F. tularensis stimulate increased levels of NO production. In addition, a reverse phase shift can occur which returns the LPS of the F. tularensis variants to the original antigenic form, resulting in reduced macrophage NO production and restoration of intracellular growth. These results suggest that F. tularensis can modulate macrophage NO production through phase variation of its LPS. It was of interest to initiate a study that would ultimately characterize the molecular mechanism of LPS phase variation in Francisella tularensis . To this end, we used shuttle mutagenesis to create a mutant library of F. novicida. We mutagenized a size- restricted plasmid library of F. novicida with the erythromycin- resistant transposon TnMax2. Putative F. novicida LPS mutants created by shuttle mutagenesis were screened visually for aberrant colony phenotypes on agar plates. Of 10464 mutants screened, 5 unique F. novicida LPS mutants were isolated which exhibit three distinct LPS phenotypes as determined by Western immunoblot. A single mutant from each of the three phenotypic groups was further characterized with respect to DNA sequence analysis, intramacrophage growth, and sensitivity to detergent and serum complement. Furthermore, these three loci were shown to hybridize with a corresponding locus in F. tularensis LVS. However, there was no difference in the restriction pattern of the hybridizing bands between LVS and its LPS phase variants, thus indicating that no major genetic rearrangements or insertion/deletion of a large mobile genetic element occurs in these genes during the phase variation process of F. tularensis. The F. novicida valAB locus has previously been cloned, sequenced, and shown to be functionally homologous to the E. coli genes msbA/lpxK. In order to investigate the hypothesis that valAB is involved in transport of LPS to the cell surface, an E. coli strain harboring an NTG-mutagenized temperature sensitive (t.s.) allele of valAB, a nonfunctional copy of msbA/lpxK, and an IPTG-inducible copy of the gene encoding the Chlamydia trachomatis genus-specific LPS epitope (gseA) was constructed. In this study, DNA sequencing was used to locate the temperature sensitive mutations in the valAB locus. Two C to T transitions were found in the valA coding region which result in a S to F change at amino acid 543 and a T to I change at amino acid 458. The ability of E. coli cells harboring this t.s. copy of valAB to transport the Chlamydia LPS epitope across the inner membrane at the permissive and non-permissive temperatures was determined using sucrose density gradient centrifugation and ELISA. It was determined that there was increased association of the LPS epitope with the inner membrane at the non-permissive temperature, thus suggesting that ValA is required for transport of an LPS precursor across the inner membrane.



Endotoxins, Francisella tularensis, Pathogenic bacteria, Virulence (Microbiology)