The Lipopolysaccharide of Bordetella bronchiseptica Acts as a Protective Shield against Antimicrobial Peptides

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The Lipopolysaccharide of Bordetella
bronchiseptica Acts as a Protective Shield
against Antimicrobial Peptides

• Andreas Banemann, Heike Deppisch, and Roy Gross
• Presented by Alaric Smith

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Introduction

• Bordetella species pertussis and bronchiseptica
  are closely related
• Infect mammalian mammalian upper respiratory
  tract
• Produce important virulence factors (adhesins,
  adenylate cyclase toxin, etc.
• All virulence genes are regulated by BvgAS system

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• BvgAS locus is highly unstable
• Bacteria with mutations (phase variants) in
  this locus produce no virulence factors
• Avirulent phase variants cannot colonize
  respiratory epithelium

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Differences between the two species

• B. pertussis is an obligate human pathogen
  causes whooping cough
• B. bronchiseptica infects a wide range of mammals
• B. pertussis unique virulence factors (tracheal
  colonization factor, pertussis toxin

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• LPS structure varies between the two species
• B. pertussis requires Bvg-activated factors for
  cell invasion
• B. bronchiseptica is more adept at intercellular
  survival

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Characterization of virulence strategies

• Analysis of susceptibility to antimicrobial
  peptides
• These cationic peptides (such as defensins)
  protect against bacterial colonization
• Are produced in a wide range of phyla
• Arthropod and amphibian CPs were used to
  distinguish between the two species responses

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Comparison of susceptibility

• Wild-type B. bronchiseptica is more resistant to
  CPs than B. pertussis
• Potency of the peptides studied was ranked as
  follows cecropin P gt cecropin B gt
  magainine-II-amide gt protamine gt melittin in B.
  Bronchiseptica
• In B. pertussis protamine was more effective than
  magainine-II-amide

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• HNP-1, a beta-defensin, did not affect B.
  bronchiseptica virulence, but significantly
  impaired B. pertussis
• Previous studies have shown B. bronchiseptica to
  have high resistance to CPs compared to bacteria
  of other genera

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• Genetic activation of bvg locus in B.
  bronchiseptica increases susceptibility to CPs
  (except HNP1)
• Inactivation of the same locus in B. pertussis
  produces milder effects

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Are transposon-induced mutants more susceptible
to CPs?

• Transposon-induced mutants of B. bronchiseptica
  were created by delivery of Tn5, then
  antibiotically selected.
• Mutants indistinguishable from WT in growth were
  compared to WT w/r/t protamine and subsequently
  other CP susceptibility
• Mutants showed significantly increased
  sensitivities to all CTs except cecropin P

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Which B. bronchiseptica genes are involved in SP
resistance?

• Inverse PCR was used to amplify
  transposon-inactivated genes in mutants
• Inactivated genes were of the wlb family, known
  to be involved in LPS synthesis specifically
  (2,3-diNAcManA and FucNAcMe) in B. pertussis
• Inactivated gene in PS2 strain was
  uncharacterizable
• Sequencing showed high homology in wlb between
  the two Bordetella species

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Confirmation of abnormal LPS

• In WT B. bronchiseptica, when run on a
  polyacrylamide gel and stained for LPS, two bands
  are seen (Core LPS, and LPS with O-specific side
  chains)
• B. pertussis LPS lacks these side chains
• When separated on gels, wlb-mutant B.
  bronchiseptica show patterns similar to WT B.
  pertussis
• PS2 strain showed normal LPS profile

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Conclusion

• Factors involved in transmembrane peptide
  transport affect peptide resistance in Bordetella
• Highly charged LPS side-chains protect B.
  bronchiseptica from CPs.