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

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


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

TcR

pMR6

exsFA

5′ flanking

sequence

exsFA

3′ flanking

sequence

KmR

chromosome

chromosome

exsFA

KmR


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)

  • Mechanical imaging of untreated spores

DexsFA

wt


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

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. 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?

free spores

7h

8h


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 colonies

on plate, heat to 80 °C

overlay agar

with 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

PCR from

chromosome

PCR from

pKL147

exsFA

3′ end

gfp

pRG25

chromosome

exsFA

chromosome

exsFA

gfp


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. 5

exsFA-gfp fusion

exsFA-gfp fusion

iunH-gfp fusion

6 hrs

GFP

spores

GFP

more

spores


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


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