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.

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.

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. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate