The Lipopolysaccharide The Lipopolysaccharide of of Bordetella Bordetella

bronchiseptica bronchiseptica Acts as a Acts as a Protective Shield against Protective Shield against Antimicrobial PeptidesAntimicrobial Peptides

Andreas Banemann, Heike Andreas Banemann, Heike Deppisch, and Roy GrossDeppisch, and Roy Gross

Presented by Alaric SmithPresented by Alaric Smith

IntroductionIntroduction

►Bordetella Bordetella species species pertussis pertussis and and bronchiseptica bronchiseptica are closely relatedare closely related

► Infect mammalian mammalian upper Infect mammalian mammalian upper respiratory tractrespiratory tract

►Produce important virulence factors Produce important virulence factors (adhesins, adenylate cyclase toxin, etc.(adhesins, adenylate cyclase toxin, etc.

►All virulence genes are regulated by All virulence genes are regulated by BvgAS systemBvgAS system

►BvgAS BvgAS locus is highly unstablelocus is highly unstable

►Bacteria with mutations (“phase Bacteria with mutations (“phase variants”) in this locus produce no variants”) in this locus produce no virulence factorsvirulence factors

►Avirulent phase variants cannot Avirulent phase variants cannot colonize respiratory epitheliumcolonize respiratory epithelium

Differences between the two Differences between the two speciesspecies

►B. pertussis B. pertussis is an obligate human is an obligate human pathogen; causes whooping coughpathogen; causes whooping cough

►B. bronchiseptica B. bronchiseptica infects a wide range infects a wide range of mammalsof mammals

►B. pertussis B. pertussis unique virulence factors unique virulence factors (tracheal colonization factor, pertussis (tracheal colonization factor, pertussis toxintoxin

►LPS structure varies between the two LPS structure varies between the two speciesspecies

►B. pertussis B. pertussis requires Bvg-activated requires Bvg-activated factors for cell invasionfactors for cell invasion

►B. bronchiseptica B. bronchiseptica is more adept at is more adept at intercellular survivalintercellular survival

Characterization of virulence Characterization of virulence strategiesstrategies

►Analysis of susceptibility to Analysis of susceptibility to antimicrobial peptidesantimicrobial peptides

►These cationic peptides (such as These cationic peptides (such as defensins) protect against bacterial defensins) protect against bacterial colonizationcolonization

►Are produced in a wide range of phylaAre produced in a wide range of phyla►Arthropod and amphibian CPs were Arthropod and amphibian CPs were

used to distinguish between the two used to distinguish between the two species’ responsesspecies’ responses

Comparison of susceptibilityComparison of susceptibility

►Wild-type Wild-type B. bronchiseptica B. bronchiseptica is more is more resistant to CPs than resistant to CPs than B. pertussisB. pertussis

►Potency of the peptides studied was Potency of the peptides studied was ranked as follows: cecropin P > ranked as follows: cecropin P > cecropin B > magainine-II-amide > cecropin B > magainine-II-amide > protamine > melittin in protamine > melittin in B. B. BronchisepticaBronchiseptica

► In In B. pertussisB. pertussis protamine was more protamine was more effective than magainine-II-amide effective than magainine-II-amide

►HNP-1, a beta-defensin, did not affect HNP-1, a beta-defensin, did not affect B. bronchiseptica B. bronchiseptica virulence, but virulence, but significantly impaired significantly impaired B. pertussisB. pertussis

►Previous studies have shown Previous studies have shown B. B. bronchiseptica bronchiseptica to have high resistance to have high resistance to CPs compared to bacteria of other to CPs compared to bacteria of other generagenera

►Genetic activation of Genetic activation of bvgbvg locus in locus in B. B. bronchiseptica bronchiseptica increases susceptibility increases susceptibility to CPs (except HNP1)to CPs (except HNP1)

► Inactivation of the same locus in Inactivation of the same locus in B. B. pertussis pertussis produces milder effectsproduces milder effects

Are transposon-induced mutants Are transposon-induced mutants more susceptible to CPs?more susceptible to CPs?

►Transposon-induced mutants of Transposon-induced mutants of B. B. bronchiseptica bronchiseptica were created by delivery were created by delivery of Tn5, then antibiotically selected.of Tn5, then antibiotically selected.

►Mutants indistinguishable from WT in Mutants indistinguishable from WT in growth were compared to WT w/r/t growth were compared to WT w/r/t protamine and subsequently other CP protamine and subsequently other CP susceptibilitysusceptibility

►Mutants showed significantly increased Mutants showed significantly increased sensitivities to all CTs except cecropin Psensitivities to all CTs except cecropin P

Which Which B. bronchiseptica B. bronchiseptica genes genes are involved in SP resistance?are involved in SP resistance?

► Inverse PCR was used to amplify Inverse PCR was used to amplify transposon-inactivated genes in mutantstransposon-inactivated genes in mutants

► Inactivated genes were of the Inactivated genes were of the wlbwlb family, family, known to be involved in LPS synthesis known to be involved in LPS synthesis specifically (2,3-diNAcManA and FucNAcMe) specifically (2,3-diNAcManA and FucNAcMe) in in B. pertussis B. pertussis

► Inactivated gene in PS2 strain was Inactivated gene in PS2 strain was uncharacterizableuncharacterizable

► Sequencing showed high homology in Sequencing showed high homology in wlb wlb between the two between the two BordetellaBordetella species species

Confirmation of abnormal Confirmation of abnormal LPSLPS

► In WT In WT B. bronchiseptica, B. bronchiseptica, when run on a when run on a polyacrylamide gel and stained for LPS, polyacrylamide gel and stained for LPS, two bands are seen (Core LPS, and LPS two bands are seen (Core LPS, and LPS with O-specific side chains)with O-specific side chains)

►B. pertussis B. pertussis LPS lacks these side chainsLPS lacks these side chains►When separated on gels, When separated on gels, wlb-wlb-mutant mutant B. B.

bronchiseptica bronchiseptica show patterns similar to show patterns similar to WTWT B. pertussis B. pertussis

►PS2 strain showed normal LPS profilePS2 strain showed normal LPS profile

ConclusionConclusion

►Factors involved in transmembrane Factors involved in transmembrane peptide transport affect peptide peptide transport affect peptide resistance in resistance in BordetellaBordetella

►Highly charged LPS side-chains protect Highly charged LPS side-chains protect B. bronchiseptica B. bronchiseptica from CPs.from CPs.