“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