In the course of a proteomic analysis designed to discover
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“In the course of a proteomic analysis designed to discover spore coat proteins, we identified several previously described exosporium proteins.”. Rationale. Anthrax: infection by B. anthracis spores Understanding of disease Prevention of or response to deliberate release as a bioweapon.

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In the course of a proteomic analysis designed to discover

“In the course of a proteomic analysis designed to discover

spore coat proteins, we identified several previously

described exosporium proteins.”


Rationale

Rationale

  • Anthrax: infection by B. anthracis spores

    • Understanding of disease

    • Prevention of or response to deliberate release as a bioweapon


Exosporium background

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

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

Hypothesis

  • No overall hypothesis

  • Objective: characterize the role of ExsFA in exosporium


Mutant construction

Mutant construction

  • B. anthracis “Ames strain,” virulent

    • exsFA mutant is RG124

  • B. anthracis “Sterne strain,” attenuated

    • exsFA mutant is Ames-JAB-5

TcR

pMR6

exsFA

5′ flanking

sequence

exsFA

3′ flanking

sequence

KmR

chromosome

chromosome

exsFA

KmR


Results electron microscopy

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.

Sylvestre et al.

DexsFA

wt


Results atomic force microscopy afm

Results: Atomic force microscopy (AFM)

  • Mechanical imaging of untreated spores

DexsFA

wt


Results atomic force microscopy afm fig 1

Results: Atomic force microscopy (AFM) – Fig. 1

  • Loss of ridges on mutant spore coat

wt Sterne

mutant


Results immunofluorescence microscopy ifm fig 2

Results: Immunofluorescence microscopy (IFM) – Fig. 2

  • BclA normally located around forespore by 7h

1 cell

bright field + Hoechst dye: binds DNA, blue fluorescence

forespore

mother cell chromosome

mouse anti-BclA mAb

fluorescent goat anti-mouse Ab


Results immunofluorescence microscopy ifm fig 21

Results: Immunofluorescence microscopy (IFM) – Fig. 2

free spores


Results immunofluorescence microscopy ifm fig 22

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?

free spores

7h

8h


Results germination fig 3

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 31

Results: Germination – Fig. 3

  • Reduced germination by loss of OD in Sterne strain with RPMI-BHI medium


Results germination fig 32

Results: Germination – Fig. 3

  • Late events monitored by tetrazolium overlay

  • No defect in mutants

sporulate colonies

on plate, heat to 80 °C

overlay agar

with TTC


Results virulence fig 4

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

gfp Fusion construction

PCR from

chromosome

PCR from

pKL147

exsFA

3′ end

gfp

pRG25

chromosome

exsFA

chromosome

exsFA

gfp


Results localization of exsfa and exsfb fig 5

Results: Localization of ExsFA and ExsFB – Fig. 5

WT

exsFA-gfp fusion

DNA

stain

vegetative

DNA

stain

3 hrs

DNA

stain

6 hrs

GFP

DNA

stain

spores

GFP


Results localization of exsfa and exsfb fig 51

Results: Localization of ExsFA and ExsFB – Fig. 5

exsFA-gfp fusion

exsFA-gfp fusion

iunH-gfp fusion

6 hrs

GFP

spores

GFP

more

spores


Results localization of exsfa and exsfb fig 52

Results: Localization of ExsFA and ExsFB – Fig. 5

  • IFM with anti-GFP antibody


Results localization of exsfa and exsfb fig 53

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

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 paper1

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


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