Download The Lipopolysaccharide of Bordetella bronchiseptica Ac

The Lipopolysaccharide of
Bordetella bronchiseptica
Acts as a Protective Shield
against Antimicrobial
Peptides
Andreas Banemann, Heike
Deppisch, and Roy Gross
Presented by Alaric Smith
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
► BvgAS
locus is highly unstable
► Bacteria
with mutations (“phase variants”)
in this locus produce no virulence factors
► Avirulent
phase variants cannot colonize
respiratory epithelium
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
► LPS
structure varies between the two
species
► B.
pertussis requires Bvg-activated factors
► B.
bronchiseptica is more adept at
for cell invasion
intercellular survival
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
Comparison of susceptibility
B. bronchiseptica is more resistant
to CPs than B. pertussis
► Wild-type
► 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
a beta-defensin, did not affect B.
bronchiseptica virulence, but significantly
impaired B. pertussis
► HNP-1,
studies have shown B.
bronchiseptica to have high resistance to
CPs compared to bacteria of other genera
► Previous
activation of bvg locus in B.
bronchiseptica increases susceptibility to
CPs (except HNP1)
► Genetic
of the same locus in B.
pertussis produces milder effects
► Inactivation
Are transposon-induced mutants
more susceptible to CPs?
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
► Transposon-induced
Which B. bronchiseptica genes are
involved in SP resistance?
► Inverse
PCR was used to amplify transposoninactivated genes in mutants
► Inactivated genes were of the wlb family, known
to be involved in LPS synthesis specifically (2,3diNAcManA and FucNAcMe) in B. pertussis
► Inactivated gene in PS2 strain was
uncharacterizable
► Sequencing showed high homology in wlb
between the two Bordetella species
Confirmation of abnormal LPS
WT B. bronchiseptica, when run on a
polyacrylamide gel and stained for LPS, two
bands are seen (Core LPS, and LPS with Ospecific side chains)
► B. pertussis LPS lacks these side chains
► When separated on gels, wlb-mutant B.
bronchiseptica show patterns similar to WT
► In
B. pertussis
► PS2
strain showed normal LPS profile
Conclusion
► Factors
involved in transmembrane peptide
transport affect peptide resistance in
Bordetella
charged LPS side-chains protect B.
bronchiseptica from CPs.
► Highly