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GlycoconjugatesfortherecognitionofBacillusspores

ARTICLEinCARBOHYDRATERESEARCH·JANUARY2005

ImpactFactor:1.93·DOI:10.1016/j.carres.2004.10.008·Source:PubMed

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KalleLevon

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GLYCOCONJUGATES FOR RECOGNITION OF BACILLUS SPORES

Olga Tarasenko, Sharmin Islam, David Paquiot, and Kalle Levon*

The Othmer Department of Chemical and Biological Sciences and Engineering,

Polytechnic University, Brooklyn, NY, USA.

* Corresponding author:

Kalle Levon, Ph.D., Professor

The Othmer Department of Chemical and

Biological Sciences and Engineering,

Polytechnic University

Six MetroTech Center

Brooklyn, NY, 11201

USA

Tel.: +1 718-260-3339

Fax: +1 718-260-3125

E-mail: klevon@poly.edu

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Abstract

Carbohydrates act as ligands in many biological processes, including the folding and

secretion of proteins, cell-cell recognition, adhesion, and sporulation in the Bacillus genus. To

elucidate the mechanism of bacterial spore recognition, fluorescent labeled disaccharide

glycoconjugates as a model system have been applied to evaluate binding to bacterial spores,

thus addressing key issues in the cell surface carbohydrate recognition field. Our study has

shown that specific recognition of bacterial spores is based on interactions between units of the

disaccharide glycoconjugates that act as ligands and monosaccharide units expressed on the

exterior of bacterial spores exterior. Using fluorophore assisted carbohydrate electrophoresis

(FACE) the carbohydrates that comprised the receptor recognition sites on the spores’ exterior

were enumerated. The findings have an impact on how carbohydrates function as recognition

signals in nature, as well as offering a basis for sensor development or inhibition of the bacterial

spores.

Keywords: B. cereus, B. thuringiensis, B. pumilus, B. subtilis spores, glycoconjugate, FACE,

ELISA.

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1. Introduction

The problems associated with the Bacilli genus are as diverse as the environments where

they are found. The bacterial spore is the cause of problems such as food spoilages and food-

borne illnesses 1. Spores can withstand a wide range of assaults including heat, UV radiation,

oxidizing agents, desiccation, and physical damage 2-6. This makes them suitable for

incorporation into bioweapons and for concealment in terrorist weapons 7.

Given the severity of the threat posed by biological weapons, detection of potentially

harmful pathogens needs to be rapid and extremely sensitive since the presence of a single

pathogen can lead to infection. Furthermore, very selective detection methods are required

because pathogenic bacteria exist alongside non-pathogenic organisms in their environment.

There are several detection systems currently on the market: (i) Anthrax Bio-Threat Alert

(BTA) test strips (Tetracore, Gaithersburg, MD), (ii) BioWarfare Agent Detection Devices

(BADD) (Osborne Scientific, Lakeside, AZ), and (iii) Anthrax (spore) SMART II (New

Horizons Diagnostics, Columbia, MD) 8; immunomagnetic assay system 9-10; PCR-based assays 11-14 or traditional phenotyping of cultured bacteria 15-17. These systems are complex, expensive,

time consuming, and require highly qualified technical staffs. Other less complex and more

portable systems are based on antibody binding to surface antigens 18-19. Unfortunately these

simpler systems are flawed. Current antibody-based detectors suffer from a short shelf life, lack

of accuracy, and inadequate sensitivity. These deficiencies results in an unacceptably high level

of false-positive and false-negative responses, according to federal government trials 20 and

independent tests 8.

This lack of accuracy and sensitivity in the antibody-based detectors is compounded by

the presence of other spore-forming bacteria in the environment. Particularly problematic are

spores of the B. cereus group, including both the human pathogen B. cereus and the insect

pathogen B. thuringiensis, species which, based on genome sequence comparisons, are the most

similar to B. anthracis 21. Due to the limitations previously cited, all currently available systems

for detecting B. anthracis are inadequate for frontline use by first response units: firefighters,

police officers, soldiers, hospital personnel, and hazardous materials teams [HAZMAT] 22-23.

There is an urgent need for a quick and accurate biosensor, especially one specific to B.

anthracis. While working toward that goal, it was discovered that short peptides ligands bind

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selectively to spores of B. anthracis 24. The Asn-His-Phe-Leu-Pro peptide has been identified

that selectively binds to spores of closely related species of B. anthracis 25. We recall that B.

cereus, genetically almost indistinguishable from B. anthracis 24. Our group has focused on the

synthesis and development of enhanced ligands for selective recognition of closely related

species of the B. cereus group to be used for bacterial spore recognition. Enhanced ligands,

which include peptides, polypeptides, dendrimers 26-28, lectins, antibodies 29-30, liposomes 31, 32,

and glycoconjugates 33-35, have been designed and have proved to be effective tools for selective

recognition of bacterial spores.

In our previous publications, we have shown that macromolecular conjugates allow for

the simultaneous binding of multiple ligands to biological entities such as spores 26, 29, 31-33, 35.

This binding was confirmed by fluorescence-based assays and the results clearly showed that the

affinity of the biomolecules was enhanced to a great extent by the polyvalent effect 36-40. These

polyvalent interactions can be collectively stronger than the corresponding monovalent ligands

as previously reported 36-40. The main focus of our work has been an electrochemical sensor that

uses potentiometry for biorecognition of bacterial spores 42, 43. This approach has allowed the

identification of specific ionic interactions between the spore’s surface 42, 43 and selected ligands 26-28, 43 and enables quick selective recognition of spores of the B. cereus group 41.

Until recently, the explanation of cell recognition has focused mostly on proteins while

marginalizing the role played by carbohydrates. Although carbohydrates are ubiquitously present

both in prokaryotic and eukaryotic cells, an appreciation of their diverse biochemical roles has

began only recently 44, 45. The mammalian and bacterial cellular surfaces are littered with

complex carbohydrate structures such as glycoproteins, glycolipids, glycosaminoglycans and

proteoglycans.

In all biological systems, glycoconjugates are involved with the recognition and signaling

processes intrinsic to biochemical functions in both healthy and diseased cells. They play a

central role in cell-to-cell adhesion and in subsequent recognition, receptor activation, and in

growth and in the maturation of living organisms 46-49. The host receptors that play a role in

microbial recognition are in fact glycoconjugates 49, and the various antigenic structures

identified by the host receptors, are also constituted in part by glycoconjugates 50.

The difficulty in understanding the relationship between structure and function in

glycoconjugates is compounded not only by the diversity of carbohydrate structures, and also by

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the number of different cellular environments in which they can be found. Furthermore,

glycoconjugates have been found to often exhibit relatively low binding affinities for their

binding partners 37. It is therefore is difficulty to locate the specific function in such a broad

range of structures.

The carbohydrate’s role in recognition has only been studied in the case of endogenous

lectins: proteins that form stable complexes upon binding carbohydrates 51-53. Carbohydrate

groups can assist in protein folding or improve its stability 54-56. It is now known that N-

glycosylation of an Fc fragment of immunoglobulin influences the conformation of the Fc

peptide backbone 54. Different glycoforms stabilize different conformations and can exhibit

significantly different functional behaviors in vivo 54-56. Biochemically, carbohydrates tend to

further the binding initially begun by protein structures. For example, in the case of

immunoglobulin binding, the protein structures form an initial complex with the antigen 54-56.

However, it is the carbohydrates present that allow binding into a tighter complex. Given the

numerous binding sites on a carbohydrate (a result of the many sites available for chain

elongation and their structural flexibility) it is not surprising that structural heterogeneity and

conformational multiplicity are characteristic features of glycoproteins 55. The complex

geometries of the immunoglobulin domains and of the oligosaccharides mean that even relatively

small changes in glycan structure can leads to functionally significant changes in protein

structure 56.

A model system was needed in order to better study the specifics of carbohydrate-

carbohydrate interactions. This model system needed to have ligands and receptors with

corresponding glycoconjugate counterparts that illustrate carbohydrate-carbohydrate interaction.

B. cereus, B. anthracis, B. thuringiensis species, along with B. mycoides, comprise the

phylogenetically similar B. cereus group 21. The different Bacilli are however, quite distinct in

terms of chemical composition and morphology 57, 58. For this study, the spore’s specific

composition and morphology dictate its use as a model system.

A spore’s morphological structure typically includes the following components:

appendages (hair- or pilus-like filaments located on the outer spore surface), an exosporium, an

outer coat, an inner coat, a cortex, and a core. For each species of spore, the morphology can be

slightly different. It has been reported that B. cereus and B. thuringiensis spores possess

appendages 59, originating from an exterior basal membrane of the exosporium 59-61.

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Appendages, though present on a variety of Bacillus spores, have not been observed in B. subtilis 62, 63.

Biochemical analysis of appendages and of the exosporium of B. cereus spores has been

reported 57, 58. Since lacking appendages 62, 63, the exosporium of B. subtilis has been found to be

composed of mostly proteins with minor amounts of carbohydrates and lipids 64, 65. Several major

proteins in the exosporium of B. anthracis Sterne strain have been identified 18, 39. A collagen-

like glycoprotein BclA 39 was recently identified that appears to be a structural component of the

exosporium hair-like nap 18. Two other unique proteins, BxpA and BxpB, were also identified18.

In addition, BxpB appears to be glycosylated or associated with glycosylated material 18.

The exosporium and appendages might in fact be the regions where chemical recognition

occurs. Considerable efforts have been directed toward understanding the chemical recognition

process in spores and toward recreating it in-vitro. Biomolecules such as polysaccharides,

glycoproteins or lipoproteins embedded in the exosporium or the appendages could, in fact, play

a role in the recognition process.

Glycoconjugates have been extensively used with the purpose of studying carbohydrate-

binding sites in histochemical and cytochemical experiments 37, 66-72. Their ability to bind

carbohydrate epitopes was established for a number of important cell surface proteins, such as

clusters of differentiation and adhesion factors 37, 66- 72. Furthermore, certain cells are able to

selectively bind to mono- or oligosaccharides present on the glycoconjugate probes 73. Attempts

have been made to develop an effective system to mimic spore recognition 24-35. Receptor-

specific sites of Bacillus species have not yet been characterized, but identification of specific

sites is indispensable for understanding the recognition events.

For our studies, the glycoconjugate-polyacrylamide-fluorescein (glycoconjugate PAA-

flu) polymers proved to be the most convenient synthetic ligands because they could be

immobilized on a solid-phase 74. The glycoconjugate-PAA-flu probe binds non-specifically with

cellular components 67-72, 74, 75. Both the fluorescent label and the polymer backbone affect this

interaction minimally 45, 69, 70. PAA-flu has certain properties that make it advantageous as the

polymer backbone of this glycoconjugate probe. It is hydrophilic and shows low binding affinity

and flexibility. In addition, its structure allows for the attachment of shorter ligands. The function

of the fluorescent label is to be an efficient marker for different techniques: spectroscopic

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measurements, fluorescence and confocal microscopy, microspectroscopy on cells and tissue

sections 67-69, 74, 75.

The aim of the present study is two-fold: first, to elucidate the mechanism of Bacillus

spore recognition based on the interaction between the glycoconjugate-PAA-flu and spores as a

model, and second, to identify receptors expressed on the spore surface that are involved in this

interaction.

2. Results and Discussions

Often, recognition can easily be accomplished using surface-immobilized molecules that

interact with other species such as analytes in solution. Specific interaction can lead to the

binding of analytes to the immobilized ligands. The function of an assay is to detect binding

events, to convert them to registration signals, and to extract information. In pursuit of the

recognition of bacterial spores (analyte), we have developed (i) a glycoconjugate assay using the

polystyrene microplates, (ii) a novel sensing concept based on a synthetic ligand like

glycoconjugates, and (iii) we introduce synthetic ligands into ELISA as described in the

Experimental section. Binding events were converted into registration signals to produce binding

curves based on the average signal intensity measured at optical density (OD) 405 nm.

In our system, fluoresceinated glycoconjugates served as the synthetic ligands of our

hypothetical system to recognize and potentially capture the spores of B. cereus, B. subtilis, B.

pumilus and finally B. thuringiensis. The glycoconjugate-PAA-flu group is comprised of a

soluble PAA backbone of approximately 30 kDA with various carbohydrate side groups and a

fluorescent molecule. All the ten glycoconjugates used: Fucα1-4 GlcNacβ–PAA-flu (1), Fucα1-3

GlcNacβ–PAA-flu (2), Galβ1-3 GalNacβ–PAA-flu (3),Galβ1-3 GalNacα –PAA-flu (4), Galβ1-3

Galβ–PAA-flu (5), Galβ1-4 Glcβ–PAA-flu (6), Galα1-3 GalNacα–PAA-flu (7), GalNacβ1-3

GalNacα–PAA-flu (8), GalNacβ1-6 GalNac-PAA-flu (9), and GlcNacβ1-4 GlcNacβ–PAA-flu

(10) have the same molecular weight. In the microtiter plate we utilized each well and coated it

with a specific glycoconjugate of the ten. After coating each well, glycoconjugates served as the

synthetic ligands of our hypothetical system to mimic the spore recognition used to capture

spores. Next, the spores were introduced to bind with glycoconjugates. An anti-mouse (IgM

+IgG) horseradish peroxidase (HRP) labeled conjugate was added, leading to the creation of the

spore-glycoconjugate complex formation. Concerning the complex formed in the wells, the more

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enzyme present, the more the HRP-labeled conjugate attached itself to the ligand-target complex.

Therefore, the amount of HRP-labeled conjugate present is determined by the amount of target

available. Finally, because glycoconjugates bind to an antigen (spore), the more target antigen

that is accessible, the more the ligand-target complex will be retained. The measure of OD values

therefore, reflects the amount of ligand-target complex initially present. The formation of this

multivalent complex was a critical step in establishing the high avidity binding. The overall

strength of binding ligands to spores (antigens) is called its avidity 76, 37-40 and was determined by

finding the magnitude of the optical density of the complex in each well. The OD values are a

measure of how tightly the synthetic ligands bind spores. A greater OD value indicates both a

stronger overall binding and a greater number of spores attached to the studied glycoconjugates.

OD analysis was performed using a SpectraMax® Plus384 spectrophotometer.

We evaluated glycoconjugates as a tool to discriminate related Bacilli species including

B. cereus and B. subtilis (Graph 1), B. thuringiensis and B. pumilus (Graph 3); B. cereus and B.

thuringiensis (Graph 5); B. subtilis and B. pumilus (Graph 7); B. subtilis and B. thuringiensis

(Graph 9); B. cereus and B. pumilus (Graph 11). Graphs 2, 4, 6, 8, and 10 present the influence

of the concentration of the ligands during spore recognition. The Y-axis represents OD values for

different spotted glycoconjugate ligands (X-axis) obtained at a wavelength of 405nm. Two sets

of experiments were carried out for each of the 10 sets of glycoconjugates. The results of both

experiments were nearly identical.

Graph 1. Patterns of B. cereus and B. subtilis

spore recognition using glycoconjugates. The

glycoconjugates used are: Fucα1-4 GlcNacβ–

PAA-flu (1), Fucα1-3 GlcNacβ–PAA-flu (2),

Galβ1-3 GalNacβ–PAA-flu (3),Galβ1-3

GalNacα–PAA-flu (4), Galβ1-3Galβ–PAA-

flu (5), Galβ1-4 Glcβ–PAA-flu (6), Galα1-3

GalNacα–PAA-flu (7), GalNacβ1-3 GalNacα–PAA-flu (8), GalNacβ1-6 GalNac–PAA-flu (9),

and GlcNacβ1-4 GlcNacβ–PAA-flu (10). The Y-axis represents the OD binding values for each

glycoconjugate ligands (X-axis) obtained at a wavelength of 405nm (here and thereafter).

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Graph 1 displays the signal intensity for each glycoconjugate-spore binding event of B.

cereus and B. subtilis spore recognition. The average signal intensity value for each

glycoconjugate was plotted to illustrate the glycoconjugates that bound more strongly to a

particular spore. It can be clearly seen that i) on the whole, the glycoconjugates utilized in this

experiment were binding to B. cereus with higher OD value (Graph 1); ii) glycoconjugates

Galβ1-4 Glcβ–PAA-flu (6), and Galα1-3 GalNacα–PAA-flu (7), have a marked affinity for B.

cereus; iii) all glycoconjugates bound to B. subtilis with extremely low OD values compare with

B. cereus spores.

In order to examine the concentration effects of the ligands during spore recognition,

solutions of the glycoconjugates used were prepared according to the manufacturer’s protocol.

Than solutions were diluted ×10, ×100, ×1000 and used in recognition of B. cereus and B.

subtilis spores (Graph 2A-C). In Graph 2 the

curves of both sets of experiments almost

completely overlap. Our results demonstrate

that the dilution of glycoconjugates does not

impede spore recognition as seen in Graph

2. We would like to point out that due to

dilution, the recognition ability of B. cereus

using Galβ1-4 Glcβ–PAA-flu (6) has

increased, while the recognition ability of B.

cereus using Galα1-3 GalNacα–PAA-flu (7)

has observed with approximately same OD

values (Graph 2A-C).

Graph 2. Role of dilution effect of

glycoconjugates on recognition abilities of

B. cereus and B. subtilis spores. There are

glycoconjugate dilution factors ×10 (A);

×100 (B); ×1000 (C).

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The recognition ability of B. subtilis using Galβ1-3Galβ–PAA-flu (5), and GlcNacβ1-4

GlcNacβ–PAA-flu (10) has increased using ×10, ×100 dilutions (Graph 2A, B). Presumably,

different units of the glycoconjugates recognized different receptor units expressed on the

exterior (Figure 5) of B. cereus and B. subtilis spores (Figure 3).

Graph 3 evaluates the binding of glycoconjugates with B. thuringiensis and B. pumilus

spores. There is a greater OD value for all glycoconjugates bonded to B. thuringiensis spores

than those bonded to B. pumilus as shown in the graph. Particularly, and Fucα1-4 GlcNacβ–

PAA-flu (1) have higher average signal intensity for the spores of B. thuringiensis. Value of

Galβ1-3 GalNacα–PAA-flu (4) with B. thuringiensis spores was high as well. In the case of B.

pumilus, Galα1-3 GalNacα–PAA-flu (7) had the highest OD value, follow up by Galβ1-4 Glcβ–

PAA-flu (6), and Galβ1-3 GalNacα–PAA-flu (4). However, these values were significantly

lower than the B. thuringiensis values. Since glycoconjugates bind more strongly to B.

thuringiensis than to B. pumilus the two spores can be differentiated.

Graph 3. Patterns of B. thuringiensis and B.

pumilus spore recognition using

glycoconjugates.

Graph 4 shows the dilution effect of glycoconjugates on recognition abilities of B.

thuringiensis, and B. pumilus spores. Recognition of B thuringiensis using GlcNacβ1-4

GlcNacβ–PAA-flu (10) has increased using ×10, ×100, ×1000 dilutions (Graph 4A-C). Values of

Fucα1-4 GlcNacβ–PAA-flu (1) and Galβ1-3 GalNacα–PAA-flu (4) glycoconjugates interactions

with B. thuringiensis spores were approximately same as shown at Graph 3. As a result of

dilution ×10, ×100, ×1000 (Graph 5A-C), the OD value of Galβ1-3 GalNacα–PAA-flu (4),

Galβ1-4 Glcβ–PAA-flu (6) increased in the case of B. pumilus as well (Graph 4A-B). Possibly

the presence of GlcNacβ in the structures of glycoconjugates Fucα1-4 GlcNacβ–PAA-flu (1) and

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GlcNacβ1-4 GlcNacβ–PAA-flu (10)

facilitates binding to B. thuringiensis

spores. The presence of Galβ in the ligands

increases the binding ability of

glycoconjugates in the case of B. pumilus.

Diluted glycoconjugates bind more

strongly to B. thuringiensis than to B.

pumilus the two spores can be

differentiated

Graph 4. Role of dilution effect of

glycoconjugates on recognition abilities of

B. thuringiensis and B. pumilus spores.

There are glycoconjugate dilution factors

×10 (A); ×100 (B); ×1000 (C).

Graph 5. Patterns of B. cereus and

B. thuringiensis spore recognition.

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Graph 5 illustrates the average signal intensity values for the spores that have had the

highest average signal intensity: B. cereus and B. thuringiensis. The data illustrates that i) despite

being the most similar species of spore morphology, with both appendages and a developed

exosporium 59, they can still be differentiated by these synthetic ligands; ii) glycoconjugates

Galβ1-4 Glcβ–PAA-flu (6) and Galα1-3 GalNacα–PAA-flu (7) have a marked affinity for both

species of spores. The OD values for glycoconjugates GlcNacβ1-4 GlcNacβ–PAA-flu (10), and

Fucα1-4 GlcNacβ–PAA-flu (1) which bound to both B. thuringiensis and B. cereus spores, were

greater in the case of B. thuringiensis than of B. cereus as shown by Graph 5.

As a result of dilution ×10, ×100, ×1000 (Graph 6A-C), the OD value for Galβ1-4 Glcβ–

PAA-flu (6) increased with respect to B.

cereus, while decreased in the case of B.

thuringiensis. The OD value for

glycoconjugate GlcNacβ1-4 GlcNacβ–

PAA-flu (10) increased with respect to B.

thuringiensis and decreased in the case of B.

cereus (Graph 6A-C). Diluted ×1000

glycoconjugates differentiated B. cereus and

B. thuringiensis (Graph 6C).

Graph 6. Role of dilution effect of

glycoconjugates on recognition abilities of

B. cereus and B. thuringiensis spores. There

are glycoconjugate dilution factors ×10 (A);

×100 (B); ×1000 (C).

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Graph 7. Patterns of B. subtilis and B.

pumilus spores recognition.

We compared the average signal

intensity values for B. subtilis and B. pumilus spores as well (Graph 7). These species have a

different morphology (Fig.3 B-C). B. subtilis has only an exosporium and B. pumilus has only

appendages as previously reported 59, 61-63. Here the data shows that i) both B. pumilus and B.

subtilis have a low affinity binding with the ten glycoconjugates in question. No average signal

intensity is greater than 0.5. ii) Glycoconjugates Galα1-3 GalNacα–PAA-flu (7) Galβ1-4 Glcβ–

PAA-flu (6), Galβ1-3 GalNacα–PAA-flu (4), Fucα1-4 GlcNacβ–PAA-flu (1) and GlcNacβ1-4

GlcNacβ–PAA-flu (10) showed the highest average signal intensity for B. pumilus. iii) Used

glycoconjugates have noticeably lower average signal intensities with B. subtilis than with B.

pumilus (Graph 7). iv) Glycoconjugates Galα1-3 GalNacα–PAA-flu (7), Galβ1-3Galβ–PAA-flu

(5), Galβ1-4 Glcβ–PAA-flu (6), and GlcNacβ1-4 GlcNacβ–PAA-flu (10) have bound to both

spores, but stronger to B. pumilus than to B. subtilis spores. Graph 8 shows the effects of dilution

for recognition of B. subtilis and B. pumilus. On the whole dilutions decreased binding of

glycoconjugates to B. subtilis and B. pumilus (Graph 8A-C).

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Graph 8. Role of dilution effect of

glycoconjugates on recognition abilities of

B. subtilis and B. pumilus pores. There are

glycoconjugate dilution factors ×10 (A);

×100 (B); ×1000 (C).

In trial A and B, only glycoconjugates

Galβ1-3 GalNacβ–PAA-flu (3), and Galβ1-4

Glcβ–PAA-flu (6) allowed both species of

spores to be distinguished (Graph 8A, B). At

×1000 with the exception of glycoconjugate

Galβ1-3 GalNacβ–PAA-flu (3) and Galβ1-

3Galβ–PAA-flu (5), the OD values for both

species of spores are quite similar (Graph

8C).

Graph 9. Patterns of B. subtilis and B.

thuringiensis spore recognition.

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Graph 9 shows that glycoconjugates can recognize and distinguish B. subtilis and B.

thuringiensis spores. Even when diluted,

glycoconjugates were still able to recognize

and distinguish both B. subtilis and B.

thuringiensis spores as shown in Graph 10.

Graph 10. Role of dilution effect of

glycoconjugates on recognition abilities of

B. subtilis and B. thuringiensis

spores. There are glycoconjugate dilution

factors ×10 (A); ×100 (B); ×1000 (C).

Graph 11. Patterns of B. cereus and B. pumilus

spore recognition.

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Next, we compared B. cereus and B. pumilus spore recognition (Graph 11). B. cereus and

B. pumilus possess appendages (Fig.3 A, C).

In Graph 11, with the exception of glycoconjugates Galβ1-3 GalNacβ–PAA-flu (3), and

GalNacβ1-3 GalNacα–PAA-flu (8), the OD values were significantly higher for B. cereus than

B. pumilus. The interaction of Galβ1-4 Glcβ–PAA-flu (6) and Galα1-3 GalNacα–PAA-flu (7)

with B. cereus and B. pumilus had the highest OD values.

Like Graph 11, Graph 12 shows that further dilution markedly increases the OD value for

glycoconjugate Galβ1-4 Glcβ–PAA-flu (6) in the case of B. cereus. B. pumilus showed lower

overall OD values.

Graph 12. Role of dilution effect of

glycoconjugates on recognition abilities of

B. cereus and B. pumilus spores

recognition. There are glycoconjugate

dilution factors ×10 (A); ×100 (B); ×1000

(C).

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Figure 1. Glycoconjugate binding to B. subtilis, B. pumilus,

B. cereus, B. thuringiensis spores. There are stock glycoconjugate solutions

(A), ×10 (B), ×100 (C), ×1000 diluted (D). Interactions with higher OD values presented. There

are Galβ1-4 Glcβ–PAA-flu (6), Galα1-3 GalNacα–PAA-flu (7) for B. cereus; Galα1-3 GalNacα–

PAA-flu (7) for B. subtilis; Fucα1-4 GlcNacβ–PAA-flu (1), GlcNacβ1-4 GlcNacβ–PAA-flu (10)

for B. thuringiensis; Galβ1-4 Glcβ–PAA-flu (6), Galα1-3 GalNacα–PAA-flu (7), GlcNacβ1-4

GlcNacβ–PAA-flu (10) for B. pumilus.

Figure 1 shows glycoconjugate-spore interactions with higher OD values. Galβ1-4 Glcβ–

PAA-flu (6) was capable recognize of B. cereus (Fig. 1 A-D). Recognition ability of B. cereus

has increased with dilutions of glycoconjugate Galβ1-4 Glcβ–PAA-flu (6) (Fig. 1 B-D).

Although, Galβ1-4 Glcβ–PAA-flu (6) was among glycoconjugates with OD values that bound to

B. pumilus spores. ×10, ×100, ×1000 diluted glycoconjugates did not increase binding of Galβ1-

4 Glcβ–PAA-flu (6) with B. pumilus spores (Fig. 1 B-D). Galα1-3 GalNacα–PAA-flu (7) has

shown the binding with B. cereus, B. subtilis, and B. pumilus, but OD values were lower with B.

subtilis, and B. pumilus spores. Dilutions of Galα1-3 GalNacα–PAA-flu (7) did not change

binding ability (Fig. 1 B-D). A high OD value was observed for interaction of GlcNacβ1-4

GlcNacβ–PAA-flu (10) glycoconjugate with B. thuringiensis and with B. pumilus spores.

Recognition of B. thuringiensis using GlcNacβ1-4 GlcNacβ–PAA-flu (10) has increased using

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×10, and ×100 dilutions (Fig. 1B-C), while the recognition ability of B. thuringiensis using

Fucα1-4 GlcNacβ–PAA-flu (1) has decreased. GlcNacβ1-4 GlcNacβ–PAA-flu (10) was capable

of binding to B. pumilus spores, but the OD value was lower than for B. thuringiensis. A high

OD value was observed for interaction of Fucα1-4 GlcNacβ–PAA-flu (1) glycoconjugate and B.

thuringiensis. The OD value of ×10, ×100 dilutions of Fucα1-4 GlcNacβ–PAA-flu (1)

glycoconjugate and B. thuringiensis interaction was approximately the same. In summary, using

glycoconjugates as synthetic ligands allowed us to recognize and distinguish four species of

bacteria spores from each other (Graphs 1, 3, 5, 7, 9, 11, Fig. 1). Changes in the concentration of

glycoconjugates did not hinder the recognition (Graphs 2, 4, 6, 8, 10, 12, Fig. 1 B-D). Diluted

glycoconjugates were still capable of recognizing and distinguishing individual spores.

According to recent research, the avidity of binding glycoconjugates to the surface of bacterial

spores that display multiple receptor sites depends on both the structure of the antigen and on

that of the ligands 77, 37-40. Though both the antigen and the ligands may display different

bimolecular units in various arrangements; glycoconjugates afford us the chance to mimic the

spores’ recognition.

Nikon Eclipse E400 Pol, a polarizing optical microscope, was used to visually confirm

the time-scale for ligand-target complex presence. Images were taken at constant intervals prior

to (Fig. 2A) and after ELISA completion (Fig. 2 B, C). Figure 2 A shows spores only. The

glycoconjugate-spore complexes have appeared under optical microscopy as shown at the Figure

2 B.

Figure 2. Spores as observed under optical microcopy as a control (A). Interaction of

glycoconjugate-spore complexes under optical microcopy (B) and fluorescent microscopy (C).

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Figure 2 C depicts the changes in fluorescence intensities that characterize the interaction with

the help of fluorescein-labeled glycoconjugates. This method appears to be a convenient method

to reveal the presence of interacted glycoconjugate-spores complexes.

Scanning Electron Microscopy (SEM) was performed for B. cereus, B. subtilis, and B.

pumilus (Fig. 3 A-B). SEM has revealed the actual shape of bacterial spores as well as their

morphological structure. The filamentous appendages of B. cereus and B. pumilus have been

clearly depicted under the SEM (Fig. 3 A, C). Our data has shown the difference in lengths of the

appendages of B. cereus, and B. pumilus compare with previously reported data using

transmission electron microscopy 77 -78. This observed difference may be due to different

techniques applied. In the case of B. subtilis, no

appendages were observed (Fig. 3 B). Earlier

morphological observations have shown that

filamentous structures have not been found in the

B. subtilis group 63-64, 77-79, and only an

exosporium-like structure was found tightly

associated with the main body 63. Based on SEM

observations, we have come to the conclusion

that morphological structures of the studied

spores are noticeably different from one another

(Fig. 3). B. cereus and B. thuringiensis (data not

shown) belong to a class of spores bearing both

appendages and an exosporium 59, 63, 77-79.

Furthermore, we consider B. subtilis as belonging

to a class with only an exosporium and B.

pumilus to a class with only appendages 99.

Figure 3. Scanning Electron Microscopy image

of B. cereus (A), B. subtilis (B), and B. pumilus

(C) spores. B. thuringiensis did not show due to

similar morphology to B. cereus. A (white color)

represents appendages, E- an exosporium.

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Given the varied structure present in all four spores used in this experiment, it is our belief that

glycoconjugates can account for their varied morphology (Fig. 3). Varied morphology here not

only refers to the many different receptors

expressed on the spore surface and their

arrangement (Fig. 3) but it also refers to the

existence of filamentous appendages on B.

cereus, B. thuringiensis (data not shown)

and B. pumilus (Fig. 3 A, C), and the lack of

an exosporium in the case of B. subtilis (Fig.

3B) 99. These results, at first glance, allude to

the role played by structure in avidity.

Furthermore, for a number of

bacterial genera, including Bacillus, the

presence of extracellular polysaccharides in

bacterial and fungal spores has previously

been demonstrated 57-58, 64-66, 81.

In order to identify receptors

expressed on the spore surface that involved

glycoconjugate-spore interaction, the

exterior layers of bacterial spore strains have

been isolated using physico-chemical

treatment as described in the Experimental

section.

Figure 4. Tapping Mode AFM images

showing spore prior to (A) and after

treatment (B, C). Mixture of isolated spore’s

exterior (B) and remaining inner shell (C) of

B. cereus ATCC 11778 spore: a- height and

b- amplitude.

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The process of isolation consists of reducing the disulfide bonds in a protein, by using a

sulfhydryl agent such as 2% 2-mercaptoethanol. Successful isolation of the exterior layer was

confirmed by AFM (Fig. 4). Figure 3 shows B. cereus ATCC 11778 spore prior to (Fig. 3 A) and

after physico-chemical treatment (Fig. B, C).

Then, the content of the exterior of four Bacillus spore strains was analyzed using FACE

(Fig. 5). FACE allows the examination of monosaccharides from neutral and amine sugars

hydrolysis. FACE electrophoresis analysis reveals the presence of several monosaccharides,

namely N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), galactose, mannose,

fucose as well as glucose.

Figure 5. Monosaccharide composition of (2- 9) spores’ exterior and AMAC–labeled

monosaccharide standard (1, 10); lines 2, 4, 6, 8 represent monosaccharides from neutral sugar

hydrolysis; lines 3, 5, 7, 9 represent monosaccharides from amino sugar hydrolysis. Lines 2, 3

represent monosaccharide composition of B. cereus; lines 4, 5 represent monosaccharide

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composition of B. thuringiensis spores; lines 6, 7 represent monosaccharide composition of B.

subtilis; lines 8, 9 represent monosaccharide composition of B. pumilus spores.

Both, B. cereus and B. thuringiensis contained neutral and amine profiles. In contrast, B.

subtilis and B. pumilus spore’s exterior exclusively exhibit neutral sugar profiles. Galactose has

been identified in the B. cereus spores exterior through neutral and amine sugar profiles. A

neutral sugar profile of B. thuringiensis appendages exhibited two monosaccharides: glucose and

galactose, whereas the amine sugar profile contained only galactose. Mannose, fucose, and

galactose were detected on the B. subtilis spore’s exterior. B. pumilus spores contained galactose

and GlcNAc. FACE has proved to be a powerful tool in identification of monosaccharide units of

studied spores. B. cereus 81, as well B. subtilis typically contain high levels of galactose 81, 82.

Glucose is considered the principal neutral carbohydrate found in B. anthracis, B. thuringiensis

and B. subtilis spores 58. Using FACE, the carbohydrates that comprised the receptor recognition

sites on the spore’ exterior were enumerated 33. It was shown that BxpB protein appears to be

glycosylated or associated with glycosylated material 18.

In addition, carbohydrates are recognized as differentiation markers and as antigenic

determinants. Recent evidence suggests that carbohydrate determinants may serve as binding

molecules and are essential for recognition through the interaction between their carbohydrate

moieties 83-88, 91-94. The crucial biological processes are either mediated by oligosaccharide

sequences, by common terminal sequences, or even by further modifications of the sugars

themselves. Such monosaccharide units of oligosaccharides/ glycoproteins/ glycolipids are also

more likely to be targets for recognition by pathogenic toxins and microorganisms 44, 80 or other

molecules. Being used primarily as a substrate for recognition, fluoresceinated glycoconjugates

played a major role in our study. As synthetic ligands they helped to establish the relevancy of

carbohydrate-carbohydrate interaction. The carbohydrates located on the spores’ exterior surface

created the potential multivalent displays of receptor/s (Fig. 5) to be recognized by carbohydrates

units of disaccharide glycoconjugates (Graphs 1-12, Fig. 1). The technique presented in this

study may be valuable in finding and isolating future spore receptor recognition sites and may be

valuable in discovering of the inhibitors that are essential for the bacterial spores’ survival.

Detecting carbohydrate-binding sites is a suitable application since most pathogens possess

unique cell-surface carbohydrates. The specific carbohydrate structures differentially expressed

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in each cell type at each stage of development are assumed to be recognized by complementary

molecule(s) expressed on the surface of the counterpart interacting cell 89, 90. Previously reported

experimental data directly shows complex carbohydrates in the recognition processes, including

adhesion between cells, adhesion of cells to the extracellular matrix, and specific recognition of

cells by one another 46-49, 67-72. It was shown that carbohydrate-carbohydrate interactions play an

important role for complimentary binding of glycosphingolipids 91- 93.

The major advantage of our approach is based on glycoconjugates binding capabilities to

the studied bacterial spores. Furthermore, we applied our method to detect carbohydrate binding

and to investigate the selectivity of glycoconjugates for various Bacillus spores. Our results

demonstrated that disaccharide glycoconjugates were able to recognize and to distinguish the

Bacilli group including B. cereus, B. thuringiensis, B. pumilus, and B. subtilis spores (Graphs 1-

12, Fig. 1). Furthermore, our results demonstrated that the dilution of glycoconjugates does not

impede spore recognition. The advantage associated with using glycoconjugates as recognition

tools is they contain a full array of carbohydrate determinants that are essential for recognition.

This recognition may be critical to optimal binding. In the future, the experimental evidence can

shed some light on the function of complex carbohydrates in these recognition processes and

adhesion between cells, adhesion of cells to the extracellular matrix, and specific recognition of

cells by one another 83-88. Another advantage of the proposed platform becomes clearly evident

when the chromogenic reaction product is quantitatively determined using a plate reader upon

completion of the immuno- and enzymatic reactions. This approach has also dramatically

increased the potential of rapidly determining the presence of specific carbohydrate epitopes.

Most importantly, there is a high surface-area to-volume ratio in the immunosorbent.

Furthermore, it was shown that molecular interaction based on carbohydrate-carbohydrate

interaction is a rapid process (1-5 min) 95. While a single molecular force between a pair of

carbohydrates is weak, the strength of interaction between clusters of carbohydrates is

comparable to that of protein-protein interactions 45, 88. Secondly, the U-shaped wells ensure

improved contact between the sample and solid-phase glycoconjugates, resulting in an increased

antigen-antibody encounter rate. Moreover, immunobinding is quantitatively achieved during the

relatively short time of immunoassays.

Moreover, glycoconjugates were observed to interact with a monosaccharide epitope(s)

expressed on the exterior layer of spores (Graphs 1-12, Fig. 5). This observation also suggests

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that our approach is of value when it comes to discovering unexpected, but biologically relevant,

bacterial species. The detection of carbohydrate-binding epitopes is considered useful for this

type of application since most pathogens possess unique cell-surface carbohydrates. Taken

together, the major advantage of our approach lies in the glycoconjugate-specificity and

selectivity to spore species, reproducibility, and the magnitude of interaction. The multiple

aspects of carbohydrate-carbohydrate recognition lead to applications like the detection of

specific bacterial spores. In conclusion, the this study provides further evidence that specific

recognition of bacterial spores is based on multivalent carbohydrate-carbohydrate interactions

between disaccharide glycoconjugates that act as ligands and monosaccharide units expressed on

the exterior of bacterial spores.

3. Experimental

3.1 Materials.

Multivalent disaccharide-PAA-flu glycoconjugates were purchased from GlycoTech Inc.

(Rockville, MD, USA). Tween 20, 30% Albumin bovine solution, water W3500 tissue culture

grade, 2,2’–azino–bis(3–ethylbenzthiazoline–6–sulfonic acid) liquid substrate system for ELISA,

proteins standards were purchased from Sigma- Aldrich (St. Louis, MO, USA). Anti-mouse

(IgG+IgM)-horseradish peroxidase (HRP) conjugate was purchased Roche (Indianapolis, IN,

USA). Sterile bacterial spore suspensions, namely B. cereus ATCC 11778, B. thuringiensis

ATCC 29730, B. subtilis ATCC 9372, and B. pumilus ATCC 27142 were purchased from Raven

Biological Laboratories Inc. (Omaha, Nebraska, USA). The fluorophore assisted carbohydrate

electrophoresis FACE® monosaccharides composition kit was obtained from Glyko Inc.,

(Novato, CA, USA). Monosaccharides were obtained from Aldrich (Milwaukee, WI, USA).

3.2 Isolation of Spores’ Exterior Layer

The isolation of the exterior layer of spores was performed as described earlier 62. Briefly, a 500

µl spore suspension with a concentration of 2×106/100µl was mixed with 2% solution of 2-

mercaptoethanol (1mM carbonate - bicarbonate buffer, pH 10.0), vortexed for 5 min, and

incubated at 37oC for 120 min. After exposure to the reagent, the mixture was centrifuged at

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4,000 × g for 20 min. The fractions, containing appendages (supernatant) and spore inner

constituents (pellets), were washed four times with deionized water. Successful isolation of

fractions was confirmed by AFM (Fig. 4) prior to further use.

3.3 Scanning Electron Microscopy (SEM)

Spore samples were deposited on the AFM mica disks for SEM observations and desiccated

during seven days. The surface of the specimen for SEM must be electron conducting. This

requires, in most cases, metal layer coating by sputtering of the specimen surface 96. Normally, in

order to satisfyingly scan spore samples using SEM, each sample is to be coated individually by

conductive materials 97- 98 such as gold and/or palladium, due to transparency. Samples were

coated with a thin film of evaporated gold, approximately 10 nm in thickness, for 60 sec and then

examined with a scanning electron microscope S-570 by Hitachi (44 Old Ridenburg Road,

Danbury, CT 06810) at an accelerating voltage of 15-20 kV.

3.4 Atomic Force Microscopy (AFM)

Solutions of untreated spores and purified spore’ exterior layer were immobilized on mica discs

(Digital Instruments, Inc., Santa Barbara, CA, USA) using sterile syringes, then dried in air at

room temperature. The prepared samples were later mounted on an AFM sample holder for

imaging. All AFM observations were carried out at room temperature (20°C), using a Nano

Scope® IIIa controller as well as a MultiModeTM microscope (Digital Instruments, Inc., Santa

Barbara, CA, USA) operating in tapping mode (amplitude) together with an E scanner. A 125

µm silicon Nanoprobe (Digital Instruments, Inc.) was employed. The calculated spring constant

was 0.3 N/m, the resonance frequency remained in the range of 240-280 kHz, the radius of

curvature was approx. 10 nm, and the scan rate was of 1 µm/s. Flattening and high-pass filtering

of the image data were performed in order to remove the substrate slope from images as well as

high-frequency noise strikes, otherwise more pronounced in the high-resolution tapping mode

imaging.

3.5 FACE of Bacterial Spores’ Exterior Layer

FACE was performed with the FACE® monosaccharide composition kit according to

manufacturer’s instructions (Glyko, Inc., Novato, CA, USA). The kit allows for the analysis of

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constituent neutral and amine monosaccharides. Briefly, monosaccharides originating in the

exterior layers of the spore’ strains were hydrolyzed. Neutral monosaccharides were hydrolyzed

by dissolving in 2 M trifluoroacetic acid (TFA) at 100°C for 5 h, whereas amino sugars were

hydrolyzed by dissolving isolated mixture of the exterior layers in 100 µl of 4 M HCl at 100°C

for 3 h. Following hydrolysis, the mixture was dried under reduced pressure. The dried

monosaccharides from the amino hydrolysis reaction were re-N-acetylated by the addition of a

re-acetylation reagent M3 according to the manufacturer’s protocol. The dried monosaccharide

from both hydrolyses were labeled with a fluorescent tag (AMAC, see Scheme 1 for labeling

reaction) and incubated overnight at 37°C.

Scheme 1. FACE labeling chemistry. Fluorophore labeling reaction was carried out with AMAC

forming the Schiff base. Reduction occurred with NaBH3CN.

Separation of the fluorophore labeled monosaccharides was done through polyacrylamide gel

electrophoresis. Upon comparison with the standard (the standard mixture of monosaccharides,

consists of 100 pmol each of AMAC-labeled N-acetylglucosamine (GlcNAc), N-

acetylgalactosamine (GalNAc), galactose, mannose, fucose, and glucose, was then loaded into a

lane adjacent to the lane of amine and neutral hydrolysis reaction) the resulting band patterns

illustrated the monosaccharide composition of the starting material which. Electrophoresis was

performed at 5°C with a constant electric current per gel for 75 min.

3. 6 Enzyme Linked Immunosorbent Assay (ELISA)

The utilization of ELISA for the study bacterial spores with glycoconjugates was performed as

follows: each of the wells of microplate (Nunc, MaxiSorp) was coated with 20 µl disaccharide

glycoconjugate-PAA-flu overnight at 4 oC. The plates were washed three times with 50 µl per

well phosphate-buffered saline (PBS) containing 0.1% Tween 20. Then the plates blocked with

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100 µl /well of 3% BSA in PBS at 37 oC for 1 h or 2 hrs at room temperature followed by a

washing step. Later, spores were added at a 7.5 µl /well rate and incubated for 1-2 hrs at room

temperature with continuous shaking. The plate was washed three times with PBS containing

0.1% Tween-20, after which 12.5 µl anti-mouse (IgG + IgM) - horseradish peroxidase(HRP)-

labeled conjugate (1:4000 dilution) (Roche Diagnostics Corp., Indianapolis, IN) was added and

incubated at 37 oC for 1 h. After washing, a substrate solution 2,2’–azino–bis(3–

ethylbenzthiazoline–6–sulfonic acid) was added and the colorimetric reaction was terminated by

addition of 50 µl /well of 1 M H2SO4. Spectrophotometric measurements were carried out with a

microplate reader SpectraMax® Plus384 (Molecular Devices Corp., Sunnyvale, CA, USA) at 405

nm. SpectraMax® Plus384 which allows carrying out a standard spectrophotometric measurement

in seconds. PathCheck Sensor of SpectraMax® Plus384 was used to measure the depth (optical

pathlength) of samples within the microplate. SOFTmax® PRO software was used to

automatically normalize the well absorbance with an equivalent path length of 1 cm 100. Blank

readings were subtracted from the optical density of the final reaction in order to obtain the

corrected absorbance value.

3. 7 Study of the Dilution Effect

Solutions of disaccharide glycoconjugates were prepared according to the manufacture’s

protocol. The solutions were then diluted ×10, ×100, ×1000 times into a phosphate-buffered

saline (PBS) / 0.2% NaH3 buffer. The ELISA platform was used in same manner as described

above (3.6 of Experimental section). Recognition was accomplished using surface-immobilized

molecules such as native and diluted glycoconjugates as ligands, which interact with other

species such as analytes (spores) in solution based on binding curves (Graphs 2, 4, 6, 8, 10, 12).

3. 8 Conventional Optical and Fluorescent Microscopy

Visual confirmation of the time-scale of glycoconjugate-spore complexes presence was

performed prior and after completion of ELISA. The glycoconjugate-spores complexes were

visually examined under fluorescent microscopy using a Nikon Eclipse E400 POL, at a

magnification of 400 times. Digital pictures of spores and of interactions of glycoconjugate-

spore complex were taken in real-time (Fig. 2).

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Acknowledgments

This research was supported by grant 0660076225, originating from the Defense Advanced

Research Projects Agency (DARPA). We express our deepest gratitude to Joany Jackman,

Senior Scientist, Applied Physics Laboratory, Johns Hopkins University, Laurel, MD for

valuable discussions during this study. We would also like to thank Prof. Mary Cowman, Othmer

Department of Chemical and Biological Sciences and Engineering, Polytechnic University, NY

for allowing us to use her atomic force microscope. We express our appreciation to Professor

Said Nourbakhsh, Department of Mechanical Engineering for technical assistance during

scanning electron microscopy, Prof. Arnost Reiser and Prof. Edward Weil, Othmer Department

of Chemical and Biological Sciences and Engineering, Polytechnic University, NY for critical

reading of the manuscript.

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