Campylobacterjejuni, a Gram-negative enteric pathogen, is the leading cause of bacterial
gastroenteritis in the developed world. A C. jejuni strain 8 1-176 transposon library was used to
screen for mutants over-producing a calcofluor white (CFW)-reactive polymer implicated in
biofilm formation. This identified two lipooligosaccharide (LOS) core mutants: one defective
for a two-domain glycosyltransferase (lgtF), and the other defective in a heptosyltransferase
(waaF). To determine if other LOS core mutants displayed a similar phenotype, and to explore
other biological outcomes of step-wise LOS truncations on C. jejuni stress resistance and
pathogenesis, mutant strains defective for GaiT and CstII were also constructed. Silver stain and
mass spectrometry analyses confirmed the sequential truncation of sialic acid (ΔcstII), galactose
(ΔgalT), two glucoses (ΔlgtF), and heptose II (ΔwaaF). While the ΔlgtF and ΔwaaF mutants
exhibited enhanced biofilm formation and ΔlgtF displayed increased sensitivity to complement
killing, no effect for these phenotypes and only modest alterations in CFW reactivity were seen
with partial outer core truncations. Deletion of LgtF had no effect on mouse colonization in vivo,
or on invasion and intracellular survival in epithelial cells in vitro. In contrast, the ΔwaaF
mutant exhibited a significant defect in intracellular survival in vitro. Interestingly, the mutants
exhibited stepwise increases in susceptibility to the antimicrobial peptide LL-37, with /waaF
and ΔlgtF being more susceptible and ΔgalT and ΔstII being more resistant than wild type. In
contrast, all of the mutants were highly susceptible to polymyxin B. This is the first report of C.
jejuni susceptibility to LL-37 and of LOS affecting polymyxin B resistance. Each of these
appears to be independent of overt effects on outer membrane protein expression, membrane
stability, or surface hydrophobicity. Together, our data indicate that the length and specific
moieties of the LOS play important roles in C. jejuni biology, and suggest a dynamic interplay of
the LOS with other stress resistance factors.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:BVAU./5032 |
| Date | 11 1900 |
| Creators | Naito, Mizue |
| Publisher | University of British Columbia |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | 1899388 bytes, application/pdf |
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