Probiotic lactobacillar and bifidobacterial strains (ingredients of probiotics) used in our work
On the one hand, probiotics as microbial cellular preparations of usefulness for human include a lot of examples of successful applications supporting healthy status of organism. Majority of probiotics are represented by lactobacilli, bifidobacteria, and their mixtures . Among them Acilact (consortium Lactobacillus acidophilus NK1 + 100ash + K3III24), Lactobacterin (L. plantarum 8RA-3), Bifidin (Bifidobacterium adolescentis MC-42), Bifidumbacterin (B. bifidum N1), Biovestin (B. adolescentis + B. bifidum) and others are well-known probiotics produced and used in Russia (Table 1). These probiotics are based on probiotic strains from healthy adults gut (Collection of microorganism at G.N. Gabrichevsky Research Institute for Epidemiology & Microbiology ). However being of live cell origin, survival and metabolism of probiotics could not be reliably controlled, and theoretically in some cases originally probiotic bacteria have some risk to be changed towards decreasing useful activities and revealing negative features similarly to some relative pathogens. So search of non-cellular types of natural agents imitating probiotics is really important.
On the other hand, lectins as carbohydrate-binding/recognizing/sensitive proteins of non-immunoglobulin nature are multifunctional and multidomain (at least one type domain is CRD: carbohydrate binding at the level of aminoacid sequence), widely occur in nature [3 - 9], and can be specifically assembled to different soluble or not glycans, polysaccharides or glycoconjugates (GC) [glycoproteins, glycolipids, other glycol-non-proteins, any targets with exposed GC] in selected directions especially on solid or cell surfaces [10 - 15]. During assembling, lectin complexes: a) increase their multivalent and multifunctional recognition (more CBS: carbohydrate binding sites [CRD or epitopes in space], appearance of new types of CBS and new targets are reached), b) form a dynamic partially reversible net system of lectin associates revealing carbohydrate recognition (the relatively changeable vector of resulting recognition by such a system can be evaluated by ordering a panel of carbohydrate targets according to their affinity to lectins). As a result, any lectin molecule in biological surroundings can be theoretically represented as: a) a lectin system (LS) of complexes and ensembles, b) a cascade of the directed assembling reactions, and c) a cascade system . For example, complexes or oligomers of lectins or lectin-GC may be able to reveal new or modified carbohydrate/GC specificity, for example, in locations between subunits . So lectin type cascades involving changeable originally the same molecules of lectins are possible.
Lectins are represented by more than 20 families and large groups involving in regulation of metabolism and widely used in biotechnology [5, 7, 8, 13, 15, 17]. Symbiotic microbial lectins are important regulators of relationships between microbes and eukaryotic macroorganisms . However, among symbiotic lectins, PBL are the least studied recognition factors [8, 16, 18].
In 2004 probiotic bacterial lectins (PBL) including lactobacillar and bifidobacterial lectins (LL and BL) of human origin were firstly identified and preliminarily characterized by us . The present study extend our knowledge concerning PBL as new class of natural symbiotic compounds. Such lectins may play important role in human superorganism in the regulation of inter- and intrapopulation relationships between bacteria and between bacteria and the host . The data concerning lectins allow evaluation of important potential of PBL as cofunctioning factors produced by probiotics. The aim was to review our current study of PBL in aspects of their prospects for biotechnology and medicine.
2. Isolation and characterization of PBL
Criteria of choice of bacterial sources of PBL were:: a) probiotic lactobacilli and bifidobacteria, b) industrial strains, and c) consortium variants of increased antagonistic activities against reference microbial diagnosticums. Acilact corresponded to all these criteria. So LL isolated were represented as a combination of lectins of all ingredient strains of Acilact. Analogously, BL isolated included combination of lectins of strains MC-42 and N1. We studied lectins from probiotical lactobacilli and bifidobacteria, originally isolated from the healthy adults gut (Table 1).
Identification of PBL  was performed using a panel of biotinylated artificial polymeric linear water-soluble GC (www.lectinity.com). Advantages of such GC were homogenecity, multiple carbohydrate residues in side exposed positions (on polyacrylamide chain) similar to mucin glycan clusters or to simple carbohydrate antigen organization, and increased affinity of interaction due to polyvalent carbohydrate targets. The combined scheme of identification and isolation of PBL is presented in Fig. 1. The critical step of identification is isoelectric focusing of protein fractions in the slab of polyacrylamide gel followed by gel electric blotting to membrane. Immobilized lectins treated with biotinyl-GC were visualized by streptavidin-peroxidase conjugate in the presence of chemiluminescent substrate of peroxidase using Dark Room of the BioChemi System (UVP, Calif.). Chemiluminescence kinetics was registered to optimise regime of PBL registration. The main positions of PBL were established (see Table 2). Lectins revealed in acidic region (within pI 4-4.5) of pH gradient were combined as acidic PBL (preparations aLL and aBL), and lectins revealed in basic region (within pI 7.6-8) were combined as basic PBL (preparations bLL or bBL). Additional PBL were identified as slightly acidic (within pI 5.1-6)  or approximately neutral. Artificial Mannan [GC as polyMan]- or (Mucin-like[GC as polyGalNAc])-binding PBL were rerpresented by LL (preferentially Mucin-like binding) and BL (preferentially Mannan-like binding). Combined preparations of LL (aLL or bLL) of Acilact were represented by contributions of the corresponding aLL or bLL of Acilact ingredient strains. Similarly, combined LB of strains MC-42 and N1 completed each other.
PBL were localized on the surface of bacteria (lactobacilli) within complexes which can be simply desorbed in the presence of LiCl (not NaCl). System of cell surface LL (as more protected) was represented by more extended panel of forms compared to secreted LL (as more dissociated and available to hydrolases of surrounding) into cultural fluid. Maximal forms of LL were obtained when boiled in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (ME) (Table 3).
2.1. Isolation of PBL
Scheme of isolation of PBL is presented in Fig. 1. Being on the bacterial cell surface in complexes, PBL can be esially desorpted in vitro or in cultural fluids in the presence of chaotropic agents in combinations with surfactants (endogenic or exogenic) and chelate compounds. The way of isolation of active PBL is protected by the patent (in process). Procedure of PBL isolation needed approximately 3 days. As a result, PBL preparations were characterized as uncolor, transparent fluids, without smell, resistant to freezing.
|No||Species*, strains||Previous names||Probiotics in Russia including strain as ingredient|
|1||L. helveticus NK1||L. acidophilus NK1||Acilact, Normospectrum, Polybacterin|
|L. acidophilus K3III24||Acilact, Normospectrum|
|3||L. helveticus 100ash||L. acidophilus 100ash||Acilact|
|4||L. plantarum 8RA-3||L. plantarum 8RA-3||Lactobacterin|
|5||B. longum MC-42||B. adolescentis MC-42||Bifidin|
|6||B. bifidum N1||B. bifidum N1||Bifidok, Bifidumbacterin|
Original localization in ordered complexes within cell surface layers; facilitated desorption into surroundings
Molecular masses within 52-80 kD
System forms: acidic [a] (within pI 3.7-4.5), slowly acidic (within рI 5.1-6), neutral (within рI 6.5-7.5) and basic [b] (pI 7.6-8)
Contain exposed aromatic aminoacids: Tyr (partially masked in different erxtent in aLL and aBL), Trp (preferentially in BL, Phe (some differences between aLL and aBL)
Aggregation state (preferentially for aL)
Sensitivity to detergents (preferentially for bL)
Capability to adhesion on hydrophobic surfaces like polysterene and immobillon P (aL "/> bL)
Contain ions Ca, Mg
major 58-59 kD
minors 60-62 and 53-55 kD
рI 3.8-4 (2 bands)
(D350 – D400)/D240= 46.3
Majors and minors 56-57, 53-54, 60-64 kD
рI 3.7-4.2 (1 band + 2 dublet bands)
(D350 – D400)/D240 = 66.7
62-80 kD , рI 7.6-8
(D350 – D400)/D240 = 33.8
58-62; 52-54 kD; рI 7.6-8
(D350 – D400)/D240 = 33.2
|Sources of PBL||Specificity to polymeric GC||Positions of PBL bands, pI*||Intensity of PBL**|
|В. adolescentis MC-42||GalNAc-|
|В. gallinarum GB***||GalNAc-|
|В. bifidum N1||GalNAc-|
|LiCl-cell surface extracted protein concentrates|
|L. acidophilus 100ash||GalNAc-|
|L. acidophilus NK1||GalNAc-|
|(SDS+МE)-treated proteins of concentrate fractions|
|L. acidophilus 100ash||GalNAc-|
|L. acidophilus NK1||GalNAc-|
The main physicochemical and biochemical properties of PBL are presented in Table 2. As it can be seen from the Table 2, PBL are relatively hydrophobic proteins and can be presented in aggregated forms with partially exposed aromatic aminoacid residues (especially controlled for Tyr and Trp). Protein stability of PBL needed the presence of cocktail of protease inhibitors (“Complete”, R & D). Increased disappearance of bBL upon storing in glass tubes (compared to polypropylene tubes) for a long time was observed (increased sorption on glass walls is possible). PBL contained cations of metals. For example, major forms of PBL of L. helveticus NK1 (strain as dominated contributors of LL into Acilact) contain approximately 2 Ca2+ in molecule. Fluorescent properties of PBL (especially in case of BL) are increased in PBL complexes including endogenic exopolymers.
Aforementioned data allow preliminary classifications of PBL [5, 7, 16]. Currently, PBL can be considered as: originally surface proteins of recognition, Ca2+ (and other metal cation)-containing and binding proteins, relatively (random structure)-organized (decreasing of randomly ordered structure in complexes as refolding recognition process), preferentially originally mono- or bivalent (one CBS in polypeptide) low sensitive haemagglutinins (similar to pan-agglutinins), with capability to create complexes, oligomeres and aggregated particles, members of functional superfamilies.
2.2. Biological properties of PBL
PBL imitate the following general main activities of probiotics: antimicrobial, immunocorrecting, ssupporting consortium, stabilizing healthy status in communicative directions “Microbes - Microbes” and “Microbes - Host”. In addition, PBL reveal unique properties which complete probiotics to synbiotics and extend spectrum of useful activities in combinations “Probiotics + PBL” (see below).
PBL are represented by four LS (Table 1). Among them LL and LB (acidic and basic) were isolated and studied by us in detail. In addition, in case of slowly acidic LL it was suggested their potential cofunctioning to oxidase-reductase system within potential lactobacillus consortium of Acilact strains and L. plantarum 8RA-3 . The role of such LL may be in regulation of protection of probiotic consortium in biotopes against peroxide stress. Examples of regulation of oxidoreductases with lectins are well documented . Mean time, the role of neutral LL is still unclear.
2.2.1. Interactions between PBL and GC [14, 17, 19, 20, 22-24]
Major forms of soluble PBL are represented mainly as molecules and their complexes with one CBS. Such PBL forms needed hydrolase treated red cells for visualization of haemagglutination reaction. In haemagglutination reaction (Clostridium perfringens sialidase-treated human AII-blood group erythrocytes) interaction between PBL and GC was as approximately equimolar (1 : 1, M/M).
We identified different lectins secreted by lactobacilli and bifidobacteria using a panel of GC and mainly three methods including: a) dot-blotted supernatant concentrates on Immobillon-P membrane (Millipore), b) proteins blotted after isoelectric focusing supernatant concentrated protein fractions in polyacrylamide plate, c) proteins sorpted on sialidase (or trypsin)-treated human AII-red cells [5, 22 - 25].
For identification of lectins among extended panel of lactobacilli and bifidobacteria strains we used GC (0.5-5 mkg/ml, PBS) containing multiply exposed side carbohydrate residues on biotynylated (b) or not polyacrylamide (PAA) chain (www.lectinity.com):
Gal(3-Sulfate)β1- [3-HSO3Galβ1- ;β-D-Galactan-3-Sulfate polymer],
GaNAcα1- [Tn-like antigen containing polymer],
GalNAcα1,3Galβ1- [Adi as (AII-blood group substance)-like containing polymer],
GalNAcα1,3GalNAcβ1- [Fs as (Forssman antigen)-like containing polymer],
Galα1,3GalNAcα1- [Tαα-like antigen containing polymer],
GalNAcβ1- [desialylated Mucin-like],
Galβ1,4GlcNAcβ1- [poly(LacNAc)-containing mucin-like],
GlcNAcβ1- [soluble linear Chitin-like],
Man(6-phosphate)α1- [6-H2PO3Manα1-polymer; α-D-PhosphoMannan],
(MurNAc-L-Ala-D-isoGln)β1- [MDP-; Muramyldipeptide containing polymer; bacterial Peptidoglycan-like],
The whole resulted chemiluminescent pictures of LL and BL separated by isoelectric focusing followed by blotting were distinct and needed individual optimized regimes of registration. It is seen from the Table 3 that: a) the pictures of PBL are unigue and depended on strain origin, b) dominated PBL types are revealed as mucin- and/or Mannan-binding; b) PBL of probiotic consortium include PBL of ingredient strains. Mannan-binding lectins of L. plantarum 8RA-3 possessed increased intensities of chemiluminescence . These data were supported by study of PBL specificity to GC in haemagglutination reaction [5, 23]. Dissociation of PBL-(hydrolase-treated human AII-red cells) agglutinates was observed in the presence of 0.5-1 mkg/ml of GC. Effectivenes of GC was decreased in the order: poly(GalNAc) or Mannan > Galactan >> Chitin-like polymer (no influence).
In other seria of experiments we extended panel of probiotic bacteria and extended panel of GC to identify new PBL types using dot-blotting technique [24, 25]. It was shown that PBL of lactobacilli and bifidobacteria are capable to discriminate GalNAc-containing GC (GalNAc residues as exposed, internal/masked, or dublicated) glycoantigens Adi-, Fs-, or Tn- depending on strain origin. No binding of PBL to Tαα was observed. PBL also discriminated artificial peptidoglycan, mannans and mucins. Due to PBL revealing as LS  when two or more PBL forms (major and minor ones) vary on specificity, similarity (identical part of mosaic of the same specificity) and differences (the whole mosaic as unique, ranging intensity of components with the same specificity, some components which simultaneously recognize two types of target GC) between recognizing potential of species and genus of lactobacilli and bifidobacteria can be established.
Using dot-blotting technique, at least 7 types of LS were identified for extended panel of lactobacilli and bifidobacteria which occur in human gut. Among these, LS were represented by lectins which especially significantly recognized α–D-Mannan (phosphorylated or not; yeast-like), α-L-Fucan (algal-like), peptidoglycan (bacterial-like), mucins (mammalian gut-like); antigens Тn and Forssman, blood group AII substance. Such lectins were identified as mosaic within bacterial mainly acidic protein massive.
Aforementioned data on interaction between PBL and GC indicate that PBL may serve as additional important functional characteristic. The latter can serve the basis to study biotope metabolic relationships involving probiotic bacteria as antagonistic to opportunistic microorganisms in keeping healthy biotope status; and to construct cofunctioning systems of PBL together with yeast and higher plant ingredients.
Involving biodegradation (proteolysis) (LL > BL);
Synergistic action (LL + BL: against staphylococci [effectiveness: LL > BL]; BL + LL: against microfungi [effectiveness: BL > LL]; BL + antibiotics: against Candida species [possibility to decrease effective work doses of antibiotics]);
Action as cascades (action of aPBL followed by action of bPBL);
Concurent use of resources of pathogens during their different live cycle steps (wrong essembling of biofilms of pathogens, choice and switching of metabolome nets, increased degradation of pathogen constructions including their lysis).
The following general comments on antimicrobial action of PBL should be noted. The action of PBL is directed against colorectal and urogenital clinical strains from human biotopes. PBL act as the members of new class of biofilm destructors . Anti-Staphylococcus and anti-Candida action reveal multistep synergism in space (different regions of action of aLL and bLL, aBL and bBL, aLL and aBL, antibiotic-like and lytic actions) and in time (earlier action as antibiotic-like, later lytic action of aPBL followed by lysis by bPBL). It takes place multisynergism of anti-Candida action between PBL and antibiotics (azoles, amphotericin B, nystatin). Taken together, PBL imitate anti-Staphylococcus and anti-Candida activities of probiotic lactobacilli and bifidobacteria [33, 34] and can be potentially used for treatment of candidoses and staphylococcoses.
It should be also noted that PBL possess advantages compared to other antimicrobials: prolonged action; cascade synergistic action, low subcytoagglutinating doses; non-dependence on antibiotic types (upon therapy) [probiotics delivered can be inactivated by some antibiotics]. In addition, ketokonazol and some other antibiotics are poorly soluble in PBS that decreases their effectiveness and control.
2.2.2. Activities of PBL in respect of potential probiotic compartment of biotope [35, 37, 38]
PBL reveal a spectrum of activities in respect of populations of lactobacilli isolated from the same biotope. Results indicate that LL support healthy status of normoflora in biotope due to realization of supervisor signal functions of PBL. It is expected that when delivered, PBL increase synbiotic compartment of biotope against potential pathogenic compartment (in addition to other positive events in direction “Microbiocenoses - Host”).
2.2.3. Other biological activities of PBL
Inducing production of TNF-α by human periphery blood lymphocytes;
Modulation of peritoneal macrophage migration in manner which is differed from action of GC;
Predicted PBL activities based on similarities to symbiotic bacterial lectins , other lectins and probiotics:
As possible ingredients of drug forms in cases of colorectal alterations (potential effectiveness: BL > LL), or urogenital alterations (potential effectiveness: LL > BL).
Direct antitumor action: against changed human cell systems similarly to PBL action against eukaryotic pathogens as xenoagents in organism (potential effectiveness: BL > LL); througph increased affinity of PBL to Poly(LacNac) as potential tumor antigens  (potential effectiveness: LL > BL);
Against protozoan pathogens (like action against another type of eukaryotic pathogens - microfungi);
Against viruses (like Acilact action against rotavirus infection of children; similar to Mannan-binding phytolectins possessing activity against HIV-1);
Intracellular sorting into organells and vesicules [due to capability of PBL to recognize poly(Man-6-P) within targets, similar to animal Man-6-P-binding lectins)];
Biocompatibility and synergism of LL and/or BL together with other probiotic microbial lectins as antimicrobials;
Biocompatibility and synergism of PBL to other type antimicrobials possessing distinct mechanisms of action;
The possible forming additional antimicrobial pool as PBL fragments in the presence of host and microbial hydrolases of surroundings in biotopes.
All aforementioned data support wide potential of PBL for industrial and medical biotechnology.
The following main prospects of applications of PBL can be underlined:
For cell cultures [autostimulators, supporting probiotic bacterial cultures: mixed or not, in the presence of pathogen, etc.],
In constructing bioadditives, anti-infectives and drug forms [system drugs of synergistic and selective action as antipathogenic agents, and as factors supporting probiotic compartment in biotopes];
In constructing of cascade biosensors based on LS-organization of PBL [for monitoring biotope healthy balance, for screening strains and their mixtures especially on solid surfaces like sensibilized membranes, polysterol or polypropelene;
keeping metabolome status;
switching (on/off) microbial nets and cascades;
controlling microbiocenosis functioning; providing cell teaching;
factors in constructing of beneficial microbiocenoses;
directed antipathogen action by changing ontogenesis of pathogens) ;
helpers in building cell and cytokine-like gradients ;
synergistic and synbiotic factors in mixed cultures of microorganisms or in host biotopes ;
synergism of PBL signaling and signal proteinases/oligopeptidases and (oligo)peptides of surrounding;
screening, selection and typing of strains;
in recombinant lectin technologies ;
ingredients of functional bioadditives ;
In system drug therapy when added PBL (LL and/or BL) will modulate whole spectrum of system drug activities;
In landscape microecology and architectuire of microbiocenoses (PBL as the direct participants and organizers of landscapes) .
It is clear that solid or cell surfaces are of preferential importance for any directed assembling initiated by PBL (increased accumulated interphased concentrations of reactants, initiating or triggering assembling on immobilized first components of cascades, achievement of maximally long and asymmetric products). That is why PBL within pore PAA hydrophilic gels or membranes (Durapore membranes as [multi]layer microaccumulators), immobilized PBL on PVDF membranes (Immobillon P) or polysterene microplates and latex particles are of especial perspectiveness.
List of abbreviations
a – acidic
b – basic
CBD – carbohydrate binding domain
CBS – carbohydrate binding site
D – optical density
BL bifidobacterial lectins
LL lactobacillar lectins
LS lectin system(s)
PBL – probiotic bacterial lectins
GC – glycoconjugate(s)
PBS – phosphate buffer saline pH 7