Surface Layer Protein EA1 Is Not a Component of Bacillus anthracis Spores but Is a Persistent Contaminant in Spore Preparations

EA1 is an abundant, highly antigenic, surface layer proteinof Bacillus anthracis vegetative cells. Recent studies indicatethat EA1 is also a component of B. anthracis spores and a potentialmarker for spore detection. We show here that EA1 is not a sporecomponent but a persistent contaminant in spore preparations.

INTRODUCTION

Top

Introduction

Surface layers, or S-layers, are two-dimensional paracrystallinearrays that cover vegetative cells of many prokaryotic organisms(9). Typically, S-layers form by noncovalent, entropy-drivenself-assembly of identical protein or glycoprotein subunits.For some species, alternative S-layers and S-layer proteinsare present at the cell surface. In the case of Bacillus anthracis,the causative agent of anthrax, the S-layer is composed of eitherextracellular antigen 1 (EA1) or surface array protein (Sap),which is encoded by the chromosomal gene eag or sap, respectively(3). EA1 is the main S-layer protein produced in B. anthracisstrains carrying plasmid pXO1, which contains genes necessaryto produce anthrax toxins and other virulence-related proteins(4). In fact, EA1 is the most abundant protein and the majorcell-associated antigen in these strains (3).

Several recent studies indicate that EA1 is also a componentof the B. anthracis spore surface and could be used as a species-specificmolecular marker for detection of spores (2). Here we investigatethese possibilities and discuss the adverse consequences ofusing EA1 as a marker for B. anthracis spores.

Evidence indicating that EA1 is a spore component.

Top

Evidence indicating that ea1...

Three observations suggested that EA1 was present on sporesof B. anthracis. First, in a proteomic analysis of spore surfaceproteins of B. anthracis, Lai et al. reported the presence onhighly washed spores of a 91,362.5-Da (pI = 5.70) cell surfaceantigen containing S-layer homology domains (2). They also identifiedthis protein by matrix-assisted laser desorption ionization-timeof flight mass spectrometry as protein NP_654830 in the NationalCenter for Biotechnology Information database. Although notstated in this report, the sequence of this 862-amino-acid proteinis identical to that of EA1.

Second, from a small fraction of our B. anthracis spore preparations,we were able to extract a variable amount of an approximately100-kDa protein under conditions that solubilized proteins onthe spore surface. Most of our spore preparations were devoidof this protein. In all cases, spores were derived from theavirulent Sterne strain (pXO1⁺ pXO2^-; unable to produce vegetativecell capsule), which was grown in Difco sporulation medium at37°C with shaking for 36 to 48 h (5). Under these cultureconditions greater than 95% of the cells sporulate. The sporeswere washed extensively with water and pelleted through 50%Renografin to remove vegetative cell debris (12). This widelyused protocol is generally regarded as the most rigorous methodfor spore purification (1, 5). Spore extracts, which containedprimarily proteins of the outermost spore layer (i.e., the exosporium),were prepared by boiling 3 x 10⁸ spores from each preparationfor 8 min in 20 µl of sample buffer containing 125 mMTris-HCl (pH 6.8), 4% sodium dodecyl sulfate (SDS), 10% (vol/vol)2-mercaptoethanol, 1 mM dithiothreitol, 0.05% bromophenol blue,and 10% (vol/vol) glycerol (10). The samples were then spunat 10,600 x g for 3 min, and the proteins in the supernatantswere separated by SDS-polyacrylamide gel electrophoresis (PAGE)on a 4 to 15% polyacrylamide gradient gel (Ready Gels; Bio-RadLaboratories). The occasional presence of variable amounts ofthe $~$ 100-kDa protein band is depicted in Fig. 1A, which showsa Coomassie blue-stained gel of electrophoretically separatedproteins that had been extracted from three spore preparations.

View larger version (69K):
[in this window]
[in a new window]
FIG. 1. Variable amounts of the $~$ 100-kDa protein (EA1) were extracted from selected preparations of B. anthracis spores. A typical spore preparation (preparation 1) and two preparations with low (preparation 2) and unusually high (preparation 3) amounts of the $~$ 100-kDa protein (EA1) were analyzed. As a control, we also analyzed an extract of B. anthracis vegetative cells (Veg.). (A) Proteins were separated by SDS-PAGE and stained with Coomassie brilliant blue. The $~$ 100-kDa protein (EA1) (indicated by the filled arrowhead) was a major protein in spore preparation 3 and one of only two proteins observed in the vegetative cell extract (with 4 µg of protein loaded in the lane). (B) The electrophoretically separated spore and vegetative cell proteins described for panel A were analyzed by Western blotting. Identical results were obtained with either SA26 or M2-V129 as the primary antibody, and only the results with SA26 are shown.

To identify the $~$ 100-kDa protein, we excised the band from thegel and determined its amino-terminal amino acid sequence byautomated Edman degradation. The resulting sequence was AGKSFPDVPA,which corresponds uniquely to the amino terminus of EA1 afterremoval of a 29-amino-acid signal peptide (3, 7). In addition,we demonstrated that extracts of vegetative cells of B. anthracis(Sterne) contained a highly abundant protein that comigratedwith the $~$ 100-kDa protein variably extracted from spores (Fig.1A). This vegetative cell protein was previously identifiedas EA1 (3, 4).

Third, commercial mouse monoclonal antibodies (MAbs) advertisedas recognizing 92- to 94-kDa B. anthracis spore proteins wererecently made available. The MAbs are designated SA26 (or ab2281)and M2-V129 and are sold by Novus Biologicals, Inc. (suppliedby Abcam), and OEM Concepts, respectively. Reportedly, theseMAbs do not cross-react with B. anthracis vegetative cells orwith spores of B. globigii, B. subtilis, or B. cereus (the latterbeing the species most phylogenetically similar to B. anthracis[6]). The size of the spore antigen(s) recognized by MAbs SA26and M2-V129 and the highly antigenic character of EA1 promptedus to investigate the possibility that these MAbs were reactingwith EA1 present in spore extracts. Therefore, we analyzed byWestern blotting the separated proteins of the spore and vegetativecell extracts shown in Fig. 1A. After SDS-PAGE, the proteinswere transferred to duplicate nitrocellulose membranes and treatedas described in the manual for the Bio-Rad Immun-Blot assaykit. The membranes were probed separately with SA26 and M2-V129(each at 5 µg/ml), and bound MAb was detected with horseradishperoxidase-conjugated goat anti-mouse immunoglobulin G (IgG)at 2 µg/ml (Bio-Rad Laboratories). Identical results wereobtained with each MAb, which clearly show that both MAbs reactwith EA1 in extracts prepared from spores and vegetative cells(Fig. 1B). Cross-reactive bands that migrated faster than 100kDa in the gel presumably were proteolytic fragments of EA1.In addition, we demonstrated that SA26 and M2-V129 reacted withintact vegetative cells of the Sterne strain of B. anthracis(data not shown), contrary to the claims of the commercial suppliersof the MAbs.

Evidence that EA1 is not a spore component but a contaminant in spore preparations.

Top

Evidence that ea1 is...

To determine if EA1 was in fact a component of the spore surface,we used fluorescence microscopy to examine directly the bindingof (anti-EA1 MAb) SA26 to B. anthracis spores. Using all threespore preparations analyzed in Fig. 1, we dried 10⁶ spores (in10 µl) of each onto separate poly-L-lysine-coated glassmicroscope slides (Sigma). The immobilized spores were thentreated with 1% bovine serum albumin to block nonspecific bindingsites and washed three times with 1 ml of cold (4°C) phosphate-bufferedsaline (PBS) (8) containing 0.5% Tween 20 (Sigma). A 30-µldrop of SA26 (5 µg/ml in PBS) was placed on each sporesample, and the slides were incubated for 1 h at 4°C ina humid chamber. The spores were washed as described above.We then placed on each spore sample a 30-µl drop containing(in PBS) Alexa 488-labeled goat anti-mouse IgG (2 µg/ml)(Molecular Probes) and 400 nM peptide (ATYPLPIR)-phycoerythrinconjugate that binds specifically to B. anthracis spores (13).(The peptide ligand was included only to help visualize spores[see below].) The samples were incubated and washed as describedabove and examined by phase-contrast and fluorescence microscopy.

Examination of spores in preparation 3, which contained highlevels of EA1, was most revealing (Fig. 2). Many bright sporeswere observed by phase-contrast microscopy (Fig. 2A), and allspores were fluorescently (red) labeled with the peptide-phycoerythrinligand (Fig. 2B). In contrast, no spores were fluorescently(green) labeled with SA26 (as detected with Alexa-labeled anti-mouseIgG) (Fig. 2C). Instead, SA26 bound to irregularly shaped particulatematerial typically much larger than a spore. This material waspresent in large amounts in spore preparation 3 and in low amountsin spore preparation 2 and was undetectable in spore preparation1 (data not shown). These results and the observed reactivitywith SA26 indicated that the particulate material containedlarge aggregates of EA1, presumably derived from vegetativecells. The particulate material could also be observed in liquidsuspension by phase-contrast microscopy, although not as wellas when dried. In suspension, the particulate material formedsmaller clusters and was essentially devoid of vegetative cells(data not shown). Note that, in control experiments, we confirmedthat the reactivity of SA26 and its detection with anti-mouseIgG were the same in the presence and in the absence of thepeptide-phycoerythrin conjugate (data not shown).

View larger version (39K):
[in this window]
[in a new window]
FIG. 2. Binding of an anti-EA1 MAb (SA26) to contaminating material in spore preparations but not to spores. The figure shows spores and contaminating material in spore preparation 3, which contains high levels of EA1. The sample was examined by phase-contrast microscopy (A) and by fluorescence microscopy under conditions that detect the fluorescence of either phycoerythrin (PE) (B) or Alexa 488 (C) (12, 13).

Determining the source of EA1 in spore preparations.

Top

Determining the source of...

Our results clearly demonstrated that EA1 was present in ourspore preparations as a contaminant. To understand how thiscontamination occurred and could be avoided, we monitored EA1levels at four stages of the standard procedure for the preparationof highly purified spores (1, 5). EA1 levels were measured afterspores were harvested from 30 ml of culture, after the sporeshad been washed with 30 ml of cold (4°C) water each dayfor 3 days, after the spores were sedimented through 50% Renografin,and after the Renografin-purified spores had been washed threetimes with 10 ml of cold water. A sample containing 3 x 10⁸spores was removed from each fraction and treated as if to extractspore surface proteins as described above. Proteins in eachsample were separated by SDS-PAGE on a 4 to 15% polyacrylamidegradient gel and visualized by staining with Coomassie brilliantblue (Fig. 3). The results show that a large amount of EA1,apparently contained in vegetative cell debris, was collectedwith the spores upon initial harvesting by centrifugation (lane1). Extensive washing of the spores removed a negligible amountof EA1 (lane 2). However, sedimentation through 50% Renografinremoved all but trace amounts of EA1 (lane 3). This small amountof EA1 was removed by additional washing (lane 4). These resultsindicate that contamination of spore preparations with EA1 occurs(occasionally) because of the large amount of this protein thatis collected with the spores and the persistence of this proteinthroughout most steps of the purification protocol. In addition,the results suggest that a high level of EA1 contamination ismost likely due to a problem with the Renografin purificationstep.

View larger version (35K):
[in this window]
[in a new window]
FIG. 3. Levels of contaminating EA1 at selected stages of purification of B. anthracis spores. For each stage, a sample containing the same number of spores was treated to extract spore surface proteins, and these proteins were separated by SDS-PAGE and visualized by Coomassie blue staining. EA1 levels were measured after spores were harvested (lane 1), after spores were washed three times (lane 2), after spores were sedimented through 50% Renografin (lane 3), and after spores were washed three more times (lane 4). The position of EA1 in the gel is marked with a filled arrowhead.

Concluding remarks.

Top

Concluding remarks.

Our results demonstrate that EA1 is not present on the surfaceof B. anthracis spores. The presence of this protein in sporepreparations is due to contamination. This result is consistentwith the observation that inactivation of the eag gene has noapparent effect on sporulation or spore structure (11). However,the presence of contaminating EA1 in spore preparations is apotentially important problem for the detection of pathogenicspores of B. anthracis. Many detectors of B. anthracis sporesrely on antispore antibodies, especially MAbs, as sensors. Theseantibodies are raised against spores that are typically nothighly purified. Thus, these preparations are likely to be contaminatedwith highly antigenic EA1, and antibodies raised against thesepreparations are likely to react (primarily) with EA1. The incorporationof anti-EA1 antibodies into spore detectors would lead to thedetection of a nonpathogenic contaminant and not spores. Thissituation could result in the failure to detect highly purifiedspores or to accurately estimate spore number. Both failurescould have catastrophic consequences.

ACKNOWLEDGMENTS

Protein sequencing was performed in the UAB Cancer Center SharedFacility for Protein Analysis.

Sequencing of the B. anthracis genome by TIGR was accomplishedwith support from ONR, DOE, NIAID, and DERA. D.D.W. was supportedby the Medical Scientist Training Program at UAB. This workwas supported by NIH grant AI50566 and DARPA grant MDA972-01-1-0030.

FOOTNOTES

* Corresponding author. Mailing address: UAB Department of Microbiology, BBRB 409, 1530 3rd Ave. South, Birmingham, AL 35294-2170. Phone: (205) 934-6289. Fax: (205) 975-5479. E-mail: ChuckT{at}uab.edu.

REFERENCES

Top