Molecular analysis of GerP and spore- associated proteins ...
“In the course of a proteomic analysis designed to discover spore coat proteins, we identified...
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Transcript of “In the course of a proteomic analysis designed to discover spore coat proteins, we identified...
“In the course of a proteomic analysis designed to discoverspore coat proteins, we identified several previouslydescribed exosporium proteins.”
Rationale
Anthrax: infection by B. anthracis spores• Understanding of disease• Prevention of or response to deliberate
release as a bioweapon
Exosporium background
Present in some Bacillus species Significant variation in structure Means of attachment to spore unknown Functions little understood
• Attachment to host cells• Resistance to oxidative burst• Reduces innate immune response• Mediates phagocytosis• Regulates stickiness• Affects germination• May contain enzymes
Exosporium proteins
20 proteins and glycoproteins Lipids, carbohydrates Orthologs of B. subtilis coat proteins
• CotE (attachment?)• CotO (assembly?)• CotY, ExsY
Unique B. anthracis proteins• BclA – major protein component• ExsFA – basal layer, BclA assembly and projections = BxpB• ExsFB – paralog of ExsFA• BclB – stability
ExsFA-BclA-ExsY complex
Hypothesis
No overall hypothesis Objective: characterize the role of ExsFA in exosporium
Mutant construction
B. anthracis “Ames strain,” virulent• exsFA mutant is RG124
B. anthracis “Sterne strain,” attenuated• exsFA mutant is Ames-JAB-5
exsFA
chromosome
KmR
chromosome
pMR6
KmR
exsFA5′ flankingsequence
exsFA3′ flankingsequence
TcR
Results: Electron microscopy
Growth and sporulation unaffected TEM: “nap” missing from spores of both strains
• Same finding as Steichen et al.• Sylvestre et al. reported fewer projections
Steichen et al.
wt exsFA
Sylvestre et al.
Results: Atomic force microscopy (AFM)
Mechanical imaging of untreated spores
wt exsFA
Results: Atomic force microscopy (AFM) – Fig. 1
Loss of ridges on mutant spore coat
wt Sterne mutant
Results: Immunofluorescence microscopy (IFM) – Fig. 2
BclA normally located around forespore by 7h
bright field + Hoechst dye: binds DNA, blue fluorescence
1 cell
mother cell chromosome
forespore
mouse anti-BclA mAbfluorescent goat anti-mouse Ab
Results: Immunofluorescence microscopy (IFM) – Fig. 2
free spores
Results: Immunofluorescence microscopy (IFM) – Fig. 2
Some BclA in mother cell at 7h BclA around forespore at 8h and in free spores but polar Associated with “cap” portion of exosporium?
7h
8h
free spores
Results: Germination – Fig. 3
Syto-9 dye taken up by germinating spore during rehydration (early) Increased green fluorescence = germination Mutant shows reduced germination, especially in Ames strain
Results: Germination – Fig. 3
Reduced germination by loss of OD in Sterne strain with RPMI-BHI medium
Results: Germination – Fig. 3
Late events monitored by tetrazolium overlay No defect in mutants
sporulate colonieson plate, heat to 80 °C
overlay agarwith TTC
Results: Virulence – Fig. 4
Infected guinea pigs by i.m. and inhalation routes No difference in virulence between wild-type and mutant
intramuscular inhalation
gfp Fusion construction
pRG25
exsFA
chromosome
gfp
PCR frompKL147
exsFA3′ end
PCR fromchromosome
exsFA
chromosome
gfp
Results: Localization of ExsFA and ExsFB – Fig. 5
DNAstain
WT exsFA-gfp fusion
vegetative
DNAstain
3 hrs
DNAstain6 hrs
DNAstainspores
GFP
GFP
spores
Results: Localization of ExsFA and ExsFB – Fig. 5
exsFA-gfp fusion
6 hrs GFP
GFP
exsFA-gfp fusion iunH-gfp fusion
morespores
Results: Localization of ExsFA and ExsFB – Fig. 5
IFM with anti-GFP antibody
Results: Localization of ExsFA and ExsFB – Fig. 5
IFM with anti-GFP antibody in cotE and bclA mutants
What is the importance of this paper?
ExsFA (perhaps C terminus) required for exosporium “nap” ExsFA plays a role in germination (contrary to others’ results) ExsFA is not involved in virulence ExsFA appears to be localized to the basal layer of the exosporium ExsFB and IunH appear to be localized to the interspace
What is the importance of this paper?
Nap is dispensable for virulence: targeting the exosporium is a bad idea Interesting but challenging to identify function of nap Unusual paralogs (3rd in B. cereus) – adaptive role? First step toward separating interspace and exosporium proteins/assembly