1. The Lipopolysaccharide of Bordetella bronchiseptica Acts as a Protective Shield against Antimicrobial Peptides Andreas Banemann, Heike Deppisch, and Roy Gross Presented by Alaric Smith

2. 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

3. ► BvgAS locus is highly unstable ► Bacteria with mutations (“phase variants”) in this locus produce no virulence factors ► Avirulent phase variants cannot colonize respiratory epithelium

4. 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

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

6. 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

8. 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 > cecropin B > magainine-II-amide > protamine > melittin in B. Bronchiseptica ► In B. pertussis protamine was more effective than magainine-II-amide

9. ► 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

11. ► 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

13. 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

15. 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

17. 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

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