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Murein hydrolase activity of surface layer proteins from Lactobacillus acidophilus against Escherichia coli
Abstract
The aim of this study was to investigate the murein hydrolase activities of the surface layer proteins (SLPs) from two strains of Lactobacillus acidophilus using zymography. The influence of these hydrolase activities on Escherichia coli ATCC 43893 was also evaluated by analysing their growth curve, cell morphology and physiological state. After the incubation of E. coli with SLPs, growth was inhibited, the number of viable cells was significantly reduced, examination by transmission electron microscopy showed that the cell wall was damaged and flow cytometry results indicated that the majority of the cells were sublethally injured. All of these results suggested that the SLPs of both L. acidophilus strains possessed murein hydrolase activities that were sublethal to E. coli cells.
Copyright © 2015. Published by Elsevier B.V.
... Murein hydrolases are able to hydrolyze bacterial CW components and cause cell death 35 ; this activity generally causes rapid lysis of bacterial cells. Previous studies have indicated that the S-layer protein SA of L. acidophilus causes CW peptidoglycan hydrolysis, therefore enhancing passage of nisin into the cell membrane by enabling it to cross the CW 16,36 . This action caused the rapid lysis of Salmonella cells. ...
... This action caused the rapid lysis of Salmonella cells. Additionally, SA alone cannot hydrolyze the CW of Gram-positive bacteria, but when combined with nisin results in cell lysis 16,36 . The authors speculated that nisin provides the sudden ion-nonspecific dissipation of the proton motive force required to enhance the S-layer murein hydrolase activity. ...
... However, whether this damage on CM linked to CW rupture remains to be investigated. Acosta et al. and Meng et al. postulated that nisin provides the sudden ion-nonspecific dissipation of the proton motive force that is required to enhance S-layer endopeptidase activity 16,36 . In this study, sudden dissipation of Δψ was observed in the nisin + SlpB treatment, whereas no cell lysis was observed. ...
SlpB, a surface layer protein isolated from Lactobacillus crispatus, has the potential to enhance the antimicrobial activity of nisin. Previous research indicated that, when combined with nisin, SlpB acted synergistically to inhibit Staphylococcus saprophyticus growth, thus extending the shelf life of chicken meat. In order to understand how SlpB enhances the antibacterial activity of nisin, electron microscopy, confocal laser scanning microscopy, flow cytometry and transmembrane electrical potential analysis were used to study cell wall organization and cell membrane integrity. No remarkable bacteriolytic effects were observed, indicating that cell death could not be attributed to cell lysis, although SlpB caused dramatic modifications of cell wall, thereby altering cell shape. The combination of SlpB and nisin also induced the release of ATP or UV-absorbing materials, as well as sudden dissipation of the transmembrane electrical potential by compromising membrane integrity. Considering that SlpB led to structural disorganization of the cell wall, and nisin access is enhanced to form a stable pore, cell death is a predictable outcome. SlpB significantly enhanced the effect of nisin at half of the minimum inhibitory concentration, which resulted in cell death by destroying the cell wall and cell membrane, therefore providing a new, feasible approach in food preservation.
... It has also been demonstrated that S-layer proteins inhibit the growth of some pathogens. According to Meng et al. (2015) [106], the SLP derived from Lactobacillus acidophilus damaged the cell walls and membranes of Escherichia coli, inhibiting their growth. A study by Sun et al. (2017) [73] aimed to determine the effect of the combination of SLP and nisin against the foodborne spoilage bacterium S. saprophyticus P2 in chicken meat and to clarify how SLP acted synergistically with nisin. ...
... It has also been demonstrated that S-layer proteins inhibit the growth of some pathogens. According to Meng et al. (2015) [106], the SLP derived from Lactobacillus acidophilus damaged the cell walls and membranes of Escherichia coli, inhibiting their growth. A study by Sun et al. (2017) [73] aimed to determine the effect of the combination of SLP and nisin against the foodborne spoilage bacterium S. saprophyticus P2 in chicken meat and to clarify how SLP acted synergistically with nisin. ...
The control of spoilage microorganisms and foodborne pathogens in meat and meat products is a challenge for food producers, which potentially can be overcome through the combined use of biopreservatives, in the form of a mix of various microbial hurdles. The objective of this work is to systematically review the available knowledge to reveal whether various microbial hurdles applied in combination can pose an effective decontamination strategy for meat and meat products. PubMed, Web of Science, and Scopus were utilized to identify and evaluate studies through February 2023. Search results yielded 45 articles that met the inclusion criteria. The most common meat biopreservatives were combinations of various starter cultures (24 studies), and the use of mixtures of non-starter protective cultures (13 studies). In addition, studies evaluating antimicrobial combinations of bacteriocins with other bacteriocins, BLIS (bacteriocin-like inhibitory substance), non-starter protective cultures, reuterin, and S-layer protein were included in the review (7 studies). In one study, a biopreservative mixture comprised antifungal protein PgAFP and protective cultures. The literature search revealed a positive effect, in most of the included studies, of the combination of various bacterial antimicrobials in inhibiting the growth of pathogenic and spoilage bacteria in meat products. The main advantages of the synergistic effect achieved were: (1) the induction of a stronger antimicrobial effect, (2) the extension of the spectrum of antibacterial action, and (3) the prevention of the regrowth of undesirable microorganisms. Although further research is required in this area, the combination of various microbial hurdles can pose a green and valuable biopreservation approach for maintaining the safety and quality of meat products.
... We have previously postulated that the S-layer protein of L. acidophilus acts as anti-viral and anti-bacterial candidate (Prado Acosta et al. 2008Prado-Acosta et al. 2010;Martínez et al. 2012), as pre-treatment of the bacterial cells with purified S-layers not only reduced viability but also prevented infection of host cells (Martínez et al. 2012;Prado Acosta et al. 2016). Moreover, previous work has also shown that different Slayer proteins play a role both in the phenomena of exclusion of enteric pathogens (Hynönen et al. 2014;Meng et al. 2015;Zhu et al. 2016) and in the stimulation of the immune response via interactions with some intestinal epithelial cell components (Ashida et al. 2011;Carasi et al. 2014;Hymes and Klaenhammer 2016). Moreover, S-layer protein encoded by J. Fina Martin and M. M. Palomino contributed equally to this work. ...
... Agglutination was also evident with Gram-positive B. cereus probably based on the interaction with cell wall polymers. Notably, bacterial agglutination increased the number of dead cells (showed by red fluorescence using Live-Dead fluorophore), which correlates with the anti-microbial activity reported for the S-layer protein ( Fig. 1) (Prado Acosta et al. 2008;Martínez et al. 2012;Hynönen et al. 2014;Meng et al. 2015;Prado Acosta et al. 2016;Zhu et al. 2016). ...
The surface layer (S-layer) protein of Lactobacillus acidophilus is a crystalline array of self-assembling, proteinaceous subunits non-covalently bound to the outmost bacterial cell wall envelope and is involved in the adherence of bacteria to host cells. We have previously described that the S-layer protein of L. acidophilus possesses anti-viral and anti-bacterial properties. In this work, we extracted and purified S-layer proteins from L. acidophilus ATCC 4356 cells to study their interaction with cell wall components from prokaryotic (i.e., peptidoglycan and lipoteichoic acids) and eukaryotic origin (i.e., mucin and chitin), as well as with viruses, bacteria, yeast, and blood cells. Using chimeric S-layer fused to green fluorescent protein (GFP) from different parts of the protein, we analyzed their binding capacity. Our results show that the C-terminal part of the S-layer protein presents lectin-like activity, interacting with different glycoepitopes. We further demonstrate that lipoteichoic acid (LTA) serves as an anchor for the S-layer protein. Finally, a structure for the C-terminal part of S-layer and possible binding sites were predicted by a homology-based model.
... Особливу увагу приділено пробіотичним мікроорганізмам виду Lactobacillus асіdophilus. Мікроорганізми даного виду синтезують вітаміни , що стабілізують нервову систему; виробляють антибіотики, які перешкоджають дії шкідливих мікроорганізмів , наприклад кишкової палички [2]. Крім того, ацидофільна паличка метаболізує речовини, що відіграють роль імуномодуляторів у системі будь-якого організму людини [3]; знижує рівень холестерину у плазмі крові [4]; накопичує молочну кислоту, яка запобігає розвитку гнилісних мікроорганізмів [5]. ...
... Ринок цієї групи товарів в Україні також швидко розвивається: його обсяг наразі складає 81 млн. грн. [2]. Таке помітне зростання продажів харчових продуктів для спортсменів зумовлене , насамперед, розширенням кола покупців. ...
The effect of polysaccharide thickeners - xanthan and guar gum - on the colony-forming ability of probiotic microorganisms of the species Lactobacillus acidophilus (LA-5) was investigated. The spectrum of inorganic salts, which in the given media can be a source of free calcium ions for further Lactobacillus acidophilus encapsulation in shells based on sodium alginate was considered. According to the results of microbiological studies, it was found that the polysaccharide components do not reduce the activity of the probiotic microorganisms, and inorganic salts introduced in concentrations that provide an osmotic pressure on the Lactobacillus acidophilus cells, inhibit metabolism processes. Based on the data obtained, qualitative and quantitative composition of the medium, the components of which do not reduce the viability of microorganisms and contribute to the encapsulation process for increasing the shelf life of food products based on the systems considered was determined.
... brevis KM3 and brevis KM7) was shown in Fig. 2. Two strains were shown same dominant protein bands approximately around 48 kDa. These results are in agreement with previous studies that researchers reported molecular masses of 43-52 kDa for S-layer proteins of different lactobacillus strains [13,20]. Concentration of extracted S-layer protein in total cell proteins of two strains was lower than GuHCL extractions. ...
In this work, some important characteristics of surface layer (S-layer) proteins extracted from two new and native Lactobacillus strains, L.brevis KM3 and L.brevis KM7, were investigated. The presence of S-layer on the external surface of L.brevis KM3 was displayed by thin sectioning and negative staining. SDS-PAGE analysis were shown same dominant protein bands approximately around 48 kDa for both S-layer proteins. Moreover, the S-layer reappeared when LiCl treated cells were allowed to grow again. Protein secondary structure and thermal behavior was evaluated by using circular dichroism (CD) and differential scanning calorimetry (DSC), respectively. Both S-layer proteins had high content of β-sheet and low amount of α-helix. The thermograms of lyophilized S-layer proteins of L.brevis KM3 and L.brevis KM7 showed one transition peak at 67.9 °C and 59.14 °C, respectively. To determine monodispersity of extracted S-layer proteins, dynamic light scattering (DLS) was used. The results indicated that the main population of S-layer molecules in two tested lactobacillus strains were composed of monomer with expected diameter close to 10 nm. Furthermore, Zeta potential measurements were showed positive potential for both S-layer proteins, as expected. Our results could be used as the basis for biotechnological applications of these two new S-layer proteins.
... Among Lactobacillaceae, Lactiplantibacillus plantarum was the most frequently reported species, with 36 instances of growth inhibiting, bactericidal, or anti-biofilm properties against at least one ESKAPEE pathogen . Lactobacillus fermentum [21,22,28,30,31,43,[55][56][57][58][59][60][61][62][63][64][65] and Lacticaseibacillus rhamnosus [19,21,25,27,31,[66][67][68][69][70][71][72][73][74][75][76][77] were both reported 17 times, Lactobacillus acidophilus was reported 16 times [21,28,55,67,[78][79][80][81][82][83][84][85][86][87][88][89]. The next most common species were Lactobacillus paracasei (11 times) [19,20,58,73,74,77,[90][91][92][93][94], Lactobacillus casei (9 times) [88,[95][96][97][98][99][100][101][102], Limosilactobacillus reuteri (6 times) [19,[103][104][105][106][107], Levilactobacillus brevis (6 times) [19,25,29,[108][109][110], Lactobacillus salivarius [50, [111][112][113] and Lactobacillus helveticus [40, 102,114,115] (4 times), Lactobacillus delbrueckii [19,116,117] and Lactobacillus crispatus [65,118,119] (3 times), Lactobacillus pentosus [26,120], Lactobacillus gasseri [119,121], and Lactobacillus curvatus [51,102] (2 times). ...
Combatting the rapidly growing threat of antimicrobial resistance and reducing prevalence and transmission of ESKAPEE pathogens in healthcare settings requires innovative strategies, one of which is displacing these pathogens using beneficial microorganisms. Our review comprehensively examines the evidence of probiotic bacteria displacing ESKAPEE pathogens, with a focus on inanimate surfaces. A systematic search was conducted using the PubMed and Web of Science databases on 21 December 2021, and 143 studies were identified examining the effects of Lactobacillaceae and Bacillus spp. cells and products on the growth, colonization, and survival of ESKAPEE pathogens. While the diversity of study methods limits evidence analysis, results presented by narrative synthesis demonstrate that several species have the potential as cells or their products or supernatants to displace nosocomial infection-causing organisms in a variety of in vitro and in vivo settings. Our review aims to aid the development of new promising approaches to control pathogen biofilms in medical settings by informing researchers and policymakers about the potential of probiotics to combat nosocomial infections. More targeted studies are needed to assess safety and efficacy of different probiotic formulations, followed by large-scale studies to assess utility in infection control and medical practice.
... Several authors reported the SLP's role in antimicrobial properties and immune activation. Lb. acidophilus ATCC 4356 SLP C-terminal region presented murein hydrolase activity by zymogram against the cell wall of Salmonella enterica serovar Newport (Prado et al., 2008) and E. coli (Meng et al., 2015). Indeed, Lb. acidophilus ATCC 4356 possesses an additional mechanism against Gram-positive bacterial pathogens, such as Staphylococcus aureus and Bacillus cereus, provided by its SLP . ...
The S-layer or surface layer protein (SLP) is the most ancient biological envelope, highly conserved in several Bacteria and Archaea. In lactic acid bacteria (LAB), SLP is only found in species belonging to the Lactobacillaceae family, many of them considered probiotic microorganisms. New reclassification of members within the Lactobacillaceae family (International Journal of Systematic and Evolutionary Microbiology, 2020, 70, 2782) and newly sequenced genomes demands an updated revision on SLP genes and domain organization. There is growing information concerning SLP occurrence, molecular biology, biophysical properties, and applications. Here, we focus on the prediction of slp genes within the Lactobacillaceae family, and specifically, on the neat interconnection between the two different modular SLP domain organizations and the new reclassified genera. We summarize the results in a concise tabulated manner to review the present knowledge on SLPs and discuss the most relevant and updated concepts regarding SLP sequence clustering. Our assessment is based on sequence alignments considering the new genera classification and protein domain definition with post-translational modifications. We analyse the difficulties encountered to resolve the SLPs 3D structure, describing the need for structure prediction approaches and the relation between protein structure and its anchorage mechanism to the cell wall. Finally, we enumerate new SLP applications regarding heterologous display, pathogen exclusion, immunostimulation, and metal binding.
... However, a few studies showed that substances on the surface of Lactobacillus also performed antibacterial and antibiofilm inhibition activities [15,16]. For example, surface proteins containing cytoplasmic hydrolases from L. acidophilus inhibited E. coli growth by damaging the cell wall [17]. Jung et al. [18] showed lipophosphatidic acid of L. plantarum inhibited the biofilm formation of Enterococcus faecalis in a dose-dependent manner. ...
Background
Foodborne pathogens and spoilage bacteria survived in the biofilm pose a serious threat to food safety and human health. It is urgent to find safe and effective methods to control the planktonic bacteria as well as the biofilm formation. Substances with antibacterial and antibiofilm activity found in lactic acid bacteria were mainly metabolites secreted in the cell-free supernatant. Previously, Lacticaseibacillus rhamnosus YT was isolated because its cell pellets displayed distinguished antibacterial activity under neutral conditions. This study aimed to investigate the antibacterial and antibiofilm properties of the L. rhamnosus YT cells and its crude cell-surface extract.
Results
The antibacterial activity of the L. rhamnosus YT cells constantly increased with cells growth and reached the peak value after the cells grew into stationary phase. After cocultivation with the L. rhamnosus YT cells, the biofilm formation of B. subtilis and S. enterica was reduced. The antibacterial activity of the L. rhamnosus YT cells was varied along with various culture conditions (carbon sources, nitrogen sources, medium pH and cultural temperatures) and the antibacterial intensity (antibacterial activity per cell) was disproportional to the biomass. Furthermore, the cell-surface extract was isolated and displayed broad antimicrobial spectrum with a bacteriostatic mode of action. The antibiofilm activity of the extract was concentration-dependent. In addition, the extract was stable to physicochemical treatments (heat, pH and protease). The extract performed favorable emulsifying property which could reduce the water surface tension from 72.708 mN/m to 51.011 mN/m and the critical micelle concentration (CMC) value was 6.88 mg/mL. Besides, the extract was also able to emulsify hydrocarbon substrates with the emulsification, index (E24) ranged from 38.55% (for n-hexane) to 53.78% (for xylene). The E24 for xylene/extract emulsion was merely decreased by 5.77% after standing for 120 h. The main components of the extract were polysaccharide (684.63 μg/mL) and protein (120.79 μg/mL).
Conclusion
The properties of the extract indicated that it might be a kind of biosurfactant. These data suggested that L. rhamnosus YT and the cell-surface extract could be used as an alternative antimicrobial and antibiofilm agent against foodborne pathogens and spoilage bacteria in food industry.
... The role of S-layer proteins as carriers for the display of therapeutic biomolecules has opened interesting perspectives due to their GRAS status, intrinsic adjuvant properties and immunomodulatory effects. Moreover, the C-terminal region of L. acidophilus ATCC 4356 has murein hydrolase activity against the cell wall ofthe Gram-negative Salmonella enterica serovar Newport (Prado-Acosta et al. 2008), E. coli (Meng et al. 2015) and Gram-positive pathogens such as Staphylococcus aureus and Bacillus cereus (Prado-Acosta et al. 2010). Furthermore, in silico and experimental analyses have revealed that S-layer proteins present two carbohydrate recognition domains localized in the C-terminus domain. ...
The surface-layer (S-layer) protein of Lactobacillus acidophilus is a crystalline array of self-assembling subunits, non-covalently bound to the most outer cell wall envelope, which constitutes up to 20% of the total cell protein content. These attributes make S-layer proteins an excellent anchor for the development of microbial cell-surface display systems. In L. acidophilus, the S-layer is formed predominantly by the protein SlpA. We have previously shown that the C-terminal domain of SlpA is responsible for the cell wall anchorage on L. acidophilus ATCC 4356. In the present study, we evaluated the C-terminal domain of SlpA of L. acidophilus ATCC 4356 as a potential anchor domain to display functional proteins on the surface of non-genetically modified lactic acid bacteria (LAB). To this end, green fluorescent protein (GFP)-CTSlpA was firstly produced in Escherichia coli and the recombinant proteins were able to spontaneously bind to the cell wall of LAB in a binding assay. GFP was successfully displayed on the S-layer stripped surface of L. acidophilus. Both the binding stability and cell survival of L. acidophilus decorated with the recombinant protein were then studied in simulated gastrointestinal conditions. Furthermore, NaCl was tested as a safer alternative to LiCl for S-layer removal. This study presents the development of a protein delivery platform involving L. acidophilus, a microorganism generally regarded as safe, which utilizes the contiguous, non-covalently attached S-layer at the cell surface of the bacterium without introducing any genetic modification.
Graphic abstract
... Similar to L. kefiri, L. acidophilus strains have surface layer associated protein that demonstrates im-munomodulatory effect (Johnson, Selle, O'Flaherty, Goh, & Klaenhammer, 2013). Meng, Gao, Zhang, & Lu (2015) determined that the murein hydrolase activity of surface layer proteins from different subspecies of L. acidophilus inhibited the E. coli. ...
Probiotic microorganisms are defined as living microorganisms that provide health benefits on the host when administered in adequate amounts. The benefits include improvement of microbial balance immune system and oral health, provision of cholesterol-lowering effect, and antimicrobial activity against a wide variety of bacteria and some fungi. Kefir microbiota contains active living microorganisms. Many researches were carried out that potential probiotic bacteria such as Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus kefir, Lactobacillus kefiranofaciens, Leuconostoc mesenteroides, or yeasts like microorganisms such as Saccharomyces cerevisiae, Kluyveromyces lactis, and Kluyveromyces marxianus were isolated from kefir grains. This chapter presents the data both on the probiotic bacteria isolated from kefir grains or kefir and the probiotic properties of kefir produced with these microorganisms.
... Moreover, Meng et al. demonstrated that L. acidophilus strains possessed murein hydrolase activities that are sublethal to E. coli cells [33]. Lipocalin-2 (Lcn2), a multifunctional innate immune protein derived from L. lactis inhibited the growth of E. coli and reduced the bioactivity of enterobactin (E. ...
Synbiotics are a combination of probiotic bacteria and the growth promoting prebiotic ingredients that purport “synergism”. Probiotics are well known for their ability to restore a healthful balance of commensal microbes supporting the repair of hyper-permeable epithelial barriers and interfering with infection. The present research was designed to evaluate the anti-virulence role of cell-free supernatants of synbiotic cultures. The effect of prebiotics fructo-oligosaccharide, inulin and isomaltose on probiotic Lactobacillus acidophilus, Lactococcus lactis, Lactobacillus casei, Lactobacillus reuteri and Saccharomyces boulardii growth, fermentation products has been investigated. Cell-free supernatants of synbiotics cultures have been studied for their antimicrobial, antibiofilm and anti-adhesion properties. Results highlight that prebiotics can enhance the antimicrobial and anti-virulence activities of probiotics against potential pathogens Staphylococcus aureus and Escherichia coli. The present research supports, for the first time, the value of anti-virulence activity of metabolites produced by synbiotics suggesting their use as a suitable adjuvant in antibacterial treatment.
... Recently, the adaptation of probiotics to stress conditions has been analyzed (Khaleghi 2010; and reviewed (Mills et al. 2011), and results have shown that general stress response, DNA repair, and energy metabolism favor increased survival during exposure to stressful conditions. We propose, for biotechnological applications, that pre-growth of lactobacilli in high-salt conditions would result in an advantage for the probiotic nature of cells: the increased production and release of the S-layer might be appropriate for their antimicrobial capacity (Prado-Acosta et al. 2008Martínez et al. 2012;Meng et al. 2015). Also, the reduction of the LTA content needs to be evaluated for possible anti-inflammatory (Kim et al. 2014;Kaji et al. 2010) or inflammatory effects (Lebeer et al. 2012;Mohamadzadeh et al. 2011;Zadeh et al. 2012). ...
In this work, we studied the role of surface layer (S-layer) proteins in the adaptation of Lactobacillus acidophilus ATCC 4356 to the osmotic stress generated by high salt. The amounts of the predominant and the auxiliary S-layer proteins SlpA and SlpX were strongly influenced by the growth phase and high-salt conditions (0.6 M NaCl). Changes in gene expression were also observed as the mRNAs of the slpA and slpX genes increased related to the growth phase and presence of high salt. A growth stage-dependent modification on the S-layer protein profile in response to NaCl was observed: while in control conditions, the auxiliary SlpX protein represented less than 10 % of the total S-layer protein, in high-salt conditions, it increased to almost 40 % in the stationary phase. The increase in S-layer protein synthesis in the stress condition could be a consequence of or a way to counteract the fragility of the cell wall, since a decrease in the cell wall thickness and envelope components (peptidoglycan layer and lipoteichoic acid content) was observed in L. acidophilus when compared to a non-S-layer-producing species such as Lactobacillus casei. Also, the stationary phase and growth in high-salt medium resulted in increased release of S-layer proteins to the supernatant medium. Overall, these findings suggest that pre-growth in high-salt conditions would result in an advantage for the probiotic nature of L. acidophilus ATCC 4356 as the increased amount and release of the S-layer might be appropriate for its antimicrobial capacity.
Lactic acid bacteria (LAB) which produce diverse imperative antimicrobial metabolites have an immense number of applications in food industry. Here, human-derived strain YT was isolated due to its cell free supernatant (CFS-YT) and cells (Cs-YT) respectively performed obvious inhibitory ring to Gram positive and negative spoilage bacteria. Strain YT was identified as Lacticaseibacillus rhamnosus YT by 16s rDNA sequence and morphology. The antibacterial activity of CFS-YT was demonstrated to be growth-dependent, pH sensitive, broad thermostable and proteases insensitive. Cs-YT displayed a broad antibacterial spectrum with the action mode of bacteriostatic. The antibacterial activity of Cs-YT was due to substances located at the cell surface which was sensitive to heat, stable at broad pH gradients and sensitive to specific proteases. These data suggested that L. rhamnosus YT could be used as alternative antimicrobial agents against foodborne spoilage bacteria in food industry.
The synergetic biological effect of scaffolds with biomimetic properties including the ECM micro-architecture and intestinal macro-mechanical properties on intestinal models in vitro remains unclear. Here, we investigate the profitable role of biomimetic scaffolds on 3D intestinal epithelium models. Gelatin/bacterial cellulose nanofiber composite scaffolds crosslinked by the Maillard reaction are tuned to mimic the chemical component, nanofibrous network, and crypt architecture of intestinal ECM collagen and the stability and mechanical properties of intestinal tissue. In particular, scaffolds with comparable elasticity and viscoelasticity of intestinal tissue possess the highest biocompatibility and best cell proliferation and differentiation ability, which makes the intestinal epithelium models closest to their counterpart intestinal tissues. The constructed duodenal epithelium models and colon epithelium models are utilized to assess the immunobiotics-host interactions, and both of them can sensitively respond to foreign microorganisms, but the secretion levels of cytokines are intestinal cell specific. The results demonstrate that probiotics alleviate the inflammation and cell apoptosis induced by Escherichia coli, indicating that probiotics can protect the intestinal epithelium from damage by inhibiting the adhesion and invasion of E. coli to intestinal cells. The designed biomimetic scaffolds can serve as powerful tools to construct in vitro intestinal epithelium models, providing a convenient platform to screen intestinal anti-inflammatory components and even to assess other physiological functions of the intestine.
Various multi-drug-resistant microorganisms have appeared while a single antibacterial agent is increasingly no longer adequate for dealing with these resistant microorganisms. Herein, commercially purchased 50 nm-average-diameter silver nanoparticles (AgNPs) and Lactobacillus buchneri-isolated surface-layer proteins (SLPs) as a capping agent were used to fabricate a hybrid antibacterial agent (SLP-AgNPs) with enhanced antibacterial activity, and the possible synergistic antibacterial mechanism was explored. Characterization results revealed that SLP-AgNPs were uniformly surrounded by protein corona provided from SLP, and the formulations were mainly mediated by the electrostatic interactions and hydrogen bonding, which was evidenced by the results of Fourier transform infrared spectroscopy. According to the antibacterial tests, the minimum inhibitory concentration of SLP-AgNPs against Salmonella enterica (0.010 mg/mL) and Staphylococcus aureus (0.005 mg/mL) was 5–10 times lower than that of bare AgNPs, and while SLP-AgNPs showed a higher antibiofilm activity. Furthermore, bacterial cells exposed to SLP-AgNPs exhibited higher cell membrane permeability and stronger inhibition of respiratory-chain dehydrogenase activity, resulting in more severe cell death compared with bare AgNPs. The synergistic effect of SLP on AgNPs was probably carried out by enhanced function of adhesion to bacteria and antibacterial ability of SLP and SLP’s supramolecular lattice structure on the sustained release of silver ion.
Surface layer proteins (SLPs) are crystalline arrays in the outermost layer of cell envelope in many archaea and bacteria. SLPs subunits have the ability to reassemble on the surface of lipid layers. In this work, the SLP from Lactobacillus acidophilus ATCC 4356 was extracted and reassembled on the surface of positively charged liposomes composed of dipalmitoyl phosphatidylcholine, cholesterol and octadecylamine. Zeta potentials and particle size were determined to describe the adsorption process of SLP on liposomes. The liposomes completely coated with SLP were observed by transmission electron microscope. To investigate the stabilizing effects of SLP on liposomes, carboxyfluorescein (CF) was encapsulated and its leakage was determined as an evaluation index. The results showed that the L. acidophilus ATCC 4356 SLP significantly (P < 0.05) increased the stability of the liposomes in the course of thermal challenge. Furthermore, SLP was able to reduce the aggregation of liposomes in serum. Storage stability of liposomes was performed at 25 °C, 4 °C and −20 °C for 90 days. And the SLP-coated liposomes released less CF than the control liposomes during storage at the three evaluated temperatures. Our findings extended the application field of Lactobacillus SLPs and introduced a novel nanocarrier system with good chemical stability.
The objective of this study was to evaluate the bactericidal effects of surface-layer protein (Slp) derived from Lactobacillus acidophilus NCFM, L. acidophilus CGMCC1.1878 in combination with nisin against Staphylococcus aureus and investigate their antibacterial mechanism. Compared with the nisin (MIC, 300 IU/mL) alone treatment, the Slps (50 μg/mL) + nisin (1/2 MIC, 150 IU/mL) treatment effectively inhibited S. aureus growth and significantly enhanced its cell membrane permeability (P < 0.05). This combination treatment did not evidently accelerate the leakage of nucleic acids in the cytoplasm, but flow cytometry results showed that the proportion of damaged bacterial cells (including dead and sublethally injured cells) was almost 96.8%, and this combination inactivated intracellular enzymes and decreased the cell membrane potential. Our results showed that Slps enhanced the effect of nisin (1/2 MIC) and reduced the dosage of nisin required for inhibiting S. aureus growth. These results indicate that Slp is a promising combined agent that enhances the antibacterial actions of nisin. The Slps + nisin combination can also be used as a potential antibacterial treatment for food preservation.
Lactobacillus acidophilus NCFM, a probiotic generally regarded as safe, carries a proteinaceous surface (S) layer, composed of numerous identical subunits (surface layer protein, Slp). S-layer proteins have been confirmed to possess multiple biological properties, but their role in maintaining the intestinal epithelial barrier is not fully known. We investigated the effects of Slp on tumor necrosis factor (TNF)-α-elicited intestinal barrier dysfunction and explored the underlying molecular mechanism. TNF-α administration markedly induced intestinal epithelial injury and inflammation in Caco-2 cells. Preincubation of Caco-2 cells with Slp at concentrations ranging from 50 to 100 μg/mL for 6 h improved intestinal epithelial cell integrity and permeability, restored ZO-1 and Occludin protein expressions (P < 0.05) and reduced the secretion of interleukin 8 by a maximum of 47.8%. Furthermore, the addition of Slp to Caco-2 cell monolayers attenuated cell apoptosis and inhibited nuclear factor-κB (NF-κB) p65 nucleus translocation by suppressing the activation of NF-κB. Collectively, the ability of Slp to attenuate dysfunction of the intestinal epithelial barrier stimulated by TNF-α and to exert anti-inflammatory effects supports its potential use in the development of functional foods and in the prevention of inflammatory bowel diseases.
The objective of our research was to evaluate the molecular mechanism of the anti-inflammatory effects of surface layer protein (Slp) derived from Lactobacillus acidophilus NCFM in lipopolysaccharide-induced RAW264.7 cells. Our results presented that Slp, with an apparent size of 46 kDa, attenuated the production of TNF-α, IL-1β, and reactive oxygen species (ROS), by inhibiting the MAPK and NF-κB signaling pathways. In addition, 10 μg mL-1 of Slp significantly inhibited NO and PGE2 production (P < 0.001) through down-regulating the expression levels of iNOS and COX-2 protein. Furthermore, Slp was found to inhibit NF-κB p65 translocation into the nucleus to activate inflammatory gene transcription. These findings suggest that Slp is a potential immune-modulating bioactive protein derived from probiotics and holds promise for use as an additive in functional foods.
To identify and investigate the role of surface layer proteins (SLPs) on the probiotic properties of Lactobacillus strains, SLPs were extracted from Lactobacillus bulgaricus fb04, L. rhamnosus fb06, L. gasseri fb07, and L. acidophilus NCFM by 5 mol/L lithium chloride. The molecular masses of the four SLPs were approximately 45–47 kDa as analyzed by SDS-PAGE. Hydrophobic amino acids were the main components of the four SLPs. The secondary structure content of the four SLPs showed extensive variability among different strains. After the SLPs were removed from the cell surface, the autoaggregation ability, coaggregation ability, and gastrointestinal tolerability of the four lactobacilli were significantly reduced as compared with the intact cells (P < 0.05). When exposed to bile salt stress, L. rhamnosus fb06, L. gasseri fb07, and L. acidophilus NCFM expressed more SLPs as determined by Bradford method. In conclusion, the four lactobacilli all possessed functional SLPs, which had positive contributions to the probiotic properties of the four Lactobacillus strains. This research could reveal the biological contributions of SLPs from Lactobacillus strains and offer a theoretical basis for the application of lactobacilli and their SLPs in food and pharmaceutical industries.
The objective of this study was to explore the antioxidant effect of the surface layer proteins (SLPs) and their mechanism. We investigated four SLPs which were extracted from L. casei zhang, L. rhamnosus, L. gasseri and L. acidophilus NCFM respectively using LiCl. The protective effect of SLPs on H2O2-induced HT-29 cells oxidative injury was investigated. As results, SLPs (100?g/mL) could significantly mitigate HT-29 cells cytotoxicity, improve the activities of total antioxidant capacity (T-AOC), catalase (CAT) and superoxide dismutase (SOD), decrease the contents of malondialdehyde (MDA) and lactate dehydrogenase (LDH), compared with H2O2-induced group (P<0.05). Furthermore, SLPs were also shown to attenuate the apoptosis rate (10.94-24.03%, P<0.01), suppress the elevation of intracellular reactive oxygen species (ROS) and calcium levels, restore mitochondrial membrane potential (MMP) and block the activation of apoptosis-related proteins of caspase-3 and caspase-9 (P<0.05). Considering all the parameters analysed, we concluded that Lactobacillus SLPs play an essential role in the antioxidant capacity of HT-29 cells induced by H2O2, and the mechanism could be attributed to SLPs' ability to enhance the activity of the intracellular antioxidant enzyme system, reduce ROS accumulation and to inhibit apoptosis by regulating mitochondrial pathway.
Currently, screening of microbial biosurfactants (BSs) is based on their equilibrium surface tension values obtained using static surface tension measurement. However, a good surfactant should not only have a low equilibrium surface tension, but its dynamic surface tension (DST) should also decrease rapidly with time. In this study, screening of BSs produced by Lactobacillus plantarum subsp. plantarum PTCC 1896 (probiotic) was performed based on their DST values measured by Wilhelmy plate tensiometry. The relationship between DST and structural and functional properties (anti-adhesive activity) of the BSs was investigated. The results showed that the changes in the yield, productivity and structure of the BSs were growth medium and incubation time dependent (p < 0.05). Structurally different BSs produced exhibited identical equilibrium surface tension values. However, differences among the structure/yield of the BSs were observed through the measurement of their DST. Structurally different BSs produced exhibited identical equilibrium surface tension values. However, differences among the structure/yield of the BSs were observed through the measurement of their DST to be positively correlated with DST. The results suggest that the DST measurement could serve as an efficient method for the clever screening of BSs producer/production condition, and consequently, for the investigation of probiotic features of bacteria, since the anti-adhesive activity is an important criterion of probiotics.
Intestinal pathogens have been proposed to adhere to epithelial cells and cause apoptosis. This study was to investigate the inhibitory effects of surface layer protein (SLP, 46kDa) from Lactobacillus acidophilus NCFM on Escherichia coli and Salmonella-induced apoptosis in HT-29 cells and the mechanism of the inhibition was also studied. The SLP could alleviate the chromatin condensation caused by intestinal pathogens as observed under fluorescent microscope. Flow cytometry analysis showed that the SLP decreased E. coli and Salmonella-induced apoptosis by 46% and 48%, respectively. The SLP could also inhibit the mitochondrial membrane potential reduction and Ca(2+) level increase in HT-29 cells. Furthermore, the activation of caspase-9 and caspase-3 induced by E. coli and Salmonella was significantly decreased by the addition of SLP. These results suggested that L. acidophilus NCFM SLP could protect HT-29 cells against intestinal pathogen-induced apoptosis through a mitochondria-mediated pathway. These findings may reveal a new method for the treatment of intestinal infection and provide a theoretical basis for the practical application of SLP in food, biological and pharmaceutical fields.
While it may appear difficult to analyze complex microflora (400-800 species) and their interactions with gut cells in the mature intestine, this is now made feasible with the availability of new techniques. Fluorescent
in situ hybridization utilizing bacterial rRNA can identify and quantify major genera of bacteria, even if they are non-culturable in stools[65,66]. Bacterial microarray chips developed during the last year can identify thousands of bacterial species in stools in one experiment[67,68]. Denaturing gradient gel electrophoresis can be utilized to monitor changes in microflora pattern[69,70] over time and after administration of probiotic supplements. Live colonocytes can be isolated from stool samples and used to examine the expression of genes and proteins during different experimental and/or disease states[71,72]. At this juncture, there is a need for the scientific community to engage in careful evaluation of probiotic strains in in vitro and in vivo systems prior to initiation of clinical trials. With the new non-invasive tools at hand, such preclinical endeavors, coupled with concurrent examination of changes in the gut flora and host responses during clinical trials, hold great promise in discerning the difference between "snake oil" and "magic bullets" when it comes to the role of probiotic therapy in human medicine.
Bacterial surface (S-) layers are crystalline arrays of self-assembling, proteinaceous subunits called S-layer proteins (Slps), with molecular masses ranging from 40 to 200 kDa. The S-layer forming bacterium, Lactobacillus acidophilus NCFM expresses three major surface layer proteins: SlpA (46 kDa), SlpB (47 kDa) and SlpX (51 kDa). SlpA has a demonstrated role in adhesion to Caco-2 intestinal epithelial cells in vitro, and has been shown to modulate dendritic cell (DC) and T-cell functionalities with murine DCs. In this study, a modification of a standard lithium chloride (LiCl) S-layer extraction revealed 37 proteins were solubilized from the S-layer wash fraction. Of these, 30 have predicted cleavage sites for secretion; 24 are predicted to be extracellular; 6 are lipid-anchored; 3 have N-terminal hydrophobic membrane spanning regions; and 4 are intracellular, potentially moonlighting proteins. Some of these proteins, designated Surface-Layer Associated Proteins (SLAPs), may be loosely associated with or embedded within the bacterial S-layer complex. Lba-1029, a putative SLAP, was deleted from the chromosome of L. acidophilus. Phenotypic characterization of the deletion mutant demonstrated that the SLAP LBA1029 contributes to a pro-inflammatory TNF-α response from murine DCs. This study identified novel extracellular proteins and putative SLAPs of L. acidophilus NCFM using liquid chromatography-tandem mass spectrometry (LC-MS/MS). SLAPs appear to impart important surface display features and immunological properties to microbes that are coated by S-layers.
Cell walls are an important structural component of prokaryotic organisms and essential for many aspects of their life. Particularly, the diverse structures of the outermost boundary layers strongly reflect adaptations of organisms to specific ecological and environmental conditions (6).
Bacterial surface (S) layers are the outermost proteinaceous cell envelope structures found on members of nearly all taxonomic groups of bacteria and Archaea. They are composed of numerous identical subunits forming a symmetric, porous, lattice-like layer that completely covers the cell surface. The subunits are held together and attached to cell wall carbohydrates by non-covalent interactions, and they spontaneously reassemble in vitro by an entropy-driven process. Due to the low amino acid sequence similarity among S-layer proteins in general, verification of the presence of an S-layer on the bacterial cell surface usually requires electron microscopy. In lactobacilli, S-layer proteins have been detected on many but not all species. Lactobacillus S-layer proteins differ from those of other bacteria in their smaller size and high predicted pI. The positive charge in Lactobacillus S-layer proteins is concentrated in the more conserved cell wall binding domain, which can be either N- or C-terminal depending on the species. The more variable domain is responsible for the self-assembly of the monomers to a periodic structure. The biological functions of Lactobacillus S-layer proteins are poorly understood, but in some species S-layer proteins mediate bacterial adherence to host cells or extracellular matrix proteins or have protective or enzymatic functions. Lactobacillus S-layer proteins show potential for use as antigen carriers in live oral vaccine design because of their adhesive and immunomodulatory properties and the general non-pathogenicity of the species.
A set of isogenic mutants of Bacillus subtilis 168, insertionally inactivated in the genes encoding a number of lytic enzymes and a sigma factor (sigma D, which controls the expression of a number of autolysins) was constructed. Phenotypic analysis of the mutants determined the individual and combined roles of the autolysins in vegetative growth. The major vegetative autolysins of B. subtilis, LytC (50 kDa amidase) and LytD (90 kDa glucosaminidase), were shown to have roles in cell separation, cell wall turnover, antibiotic-induced lysis and motility. LytC was also shown to have a role in general cell lysis induced by sodium azide. Renaturing SDS-PAGE of cell-wall-binding protein extracts of the mutant strains revealed the presence of a novel autolysin that was previously masked by LytC. This 49 kDa enzyme was shown to be sigma D-controlled and was identified as a candidate cell separation and cell wall turnover enzyme. A multiple mutant strain, lacking LytC, LytD and the 49 kDa enzyme, retained at least ten bands of autolytic activity. These may correspond to individual or proteolytically processed novel autolysins, the functions of which are unknown. The multiple mutant strains facilitate the study of these, and other lytic enzymes, to determine their cellular functions.
In many conditions, bacterial surface properties are changed as a result of variation in growth medium and conditions. This study examined the influence of bile salt concentrations (0-0.1%) on colony morphotype, hydrophobicity, H2O2 concentration, S-layer protein production and slpA gene expression in Lactobacillus acidophilus ATCC 4356. It was observed that two types of colonies (R and S) were in the control group and the stress condition. When the bile level increased in the medium, the amount of S type was more than the R. A stepwise increment in the bile concentration resulted in a stepwise decline in the maximum growth rate. The results showed that hydrophobicity was increased in 0.01%-0.02% bile but it was decreased in 0.1% bile. Treatment by bile (0.01%- 0.1%) profoundly decreased H2O2 formation. S-layer protein and slpA gene expression was also altered by stress condition. S-protein expression was increased in stress condition. slpA gene expression increased in 0.01%-0.05% bile and it decreased in 0.1% bile. However, we found that different of bile salt concentrations influence on morphology and some surface properties of L. acidophilus ATCC 4356. These changes were very different in the 0.1% bile. It appears that the bacteria respond abruptly to 0.1% bile.
We have previously described a murein hydrolase activity for the surface layer (S-layer) of Lactobacillus acidophilus ATCC 4356. Here we show that, in combination with nisin, this S-layer acts synergistically to inhibit the growth of pathogenic
Gram-negative Salmonella enterica and potential pathogenic Gram-positive bacteria, Staphylococcus aureus and Bacillus cereus. In addition, bacteriolytic effects were observed for the Gram-positive species tested. We postulate that the S-layer enhances
the access of nisin into the cell membrane by enabling it to cross the cell wall, while nisin provides the sudden ion-nonspecific
dissipation of the proton motive force required to enhance the S-layer murein hydrolase activity.
It is increasingly recognized that Lactobacillus plantarum (L. plantarum) has the ability to protect against Enteropathogenic Escherichia coli (EPEC)-induced damage of the epithelial monolayer barrier function by preventing changes in host cell morphology, attaching/effacing (A/E) lesion formation, monolayer resistance, and macromolecular permeability. However, the cellular mechanism involved in this protective effect still remained to be clarified.
This study was to investigate the effect of L. plantarum on the changes of Caco-2 cells responding to Enteroinvasive Escherichia coli (EIEC), the permeability of cell monolayer and the transmissivity of dextran, and the distribution and expression of the tight junction (TJ) proteins, such as Claudin-1, Occludin, JAM-1 and ZO-1 were examined when infected with EIEC or adhesived of L. plantarum after infection by confocal laser scanning microscopy (CLSM), immunohistochemistry and Western blotting, the cytoskeleton protein F-actin were observed with FITC-phalloidin.
This study demonstrated that the transepithelial electrical resistance (TER) step down and dextran integrated intensity (DII) step up with time after infected with EIEC, but after treating with L. plantarum, the changes of TER and DII were improved as compared with EIEC group. L. plantarum prevented the damage of expression and rearrangement of Claudin-1, Occludin, JAM-1 and ZO-1 proteins induced by EIEC, and could ameliorate the injury of cytoskeleton protein F-actin infected with EIEC.
L. plantarum exerted a protective effect against the damage to integrity of Caco-2 monolayer cells and the structure and distribution of TJ proteins by EIEC infection.
Dendritic cells (DCs) are antigen-presenting cells that play an essential role in mucosal tolerance. They regularly encounter beneficial intestinal bacteria, but the nature of these cellular contacts and the immune responses elicited by the bacteria are not entirely elucidated. Here, we examined the interactions of Lactobacillus acidophilus NCFM and its cell surface compounds with DCs. L. acidophilus NCFM attached to DCs and induced a concentration-dependent production of IL-10, and low IL-12p70. We further demonstrated that the bacterium binds to DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a DC- specific receptor. To identify the DC-SIGN ligand present on the bacterium, we took advantage of a generated array of L. acidophilus NCFM mutants. A knockout mutant of L. acidophilus NCFM lacking the surface (S) layer A protein (SlpA) was significantly reduced in binding to DC-SIGN. This mutant incurred a chromosomal inversion leading to dominant expression of a second S layer protein, SlpB. In the SlpB-dominant strain, the nature of the interaction of this bacterium with DCs changed dramatically. Higher concentrations of proinflammatory cytokines such as IL-12p70, TNFalpha, and IL-1beta were produced by DCs interacting with the SlpB-dominant strain compared with the parent NCFM strain. Unlike the SlpA-knockout mutant, T cells primed with L. acidophilus NCFM stimulated DCs produced more IL-4. The SlpA-DC-SIGN interaction was further confirmed as purified SlpA protein ligated directly to the DC-SIGN. In conclusion, the major S layer protein, SlpA, of L. acidophilus NCFM is the first probiotic bacterial DC-SIGN ligand identified that is functionally involved in the modulation of DCs and T cells functions.
We describe a new enzymatic functionality for the surface layer (S-layer) of Lactobacillus acidophilus ATCC 4356, namely, an endopeptidase activity against the cell wall of Salmonella enterica serovar Newport, assayed via zymograms and identified by Western blotting. Based on amino acid sequence comparisons, the
hydrolase activity was predicted to be located at the C terminus. Subsequent cloning and expression of the C-terminal domain
in Bacillus subtilis resulted in the functional verification of the enzymatic activity.
Lysins are highly evolved enzymes produced by bacteriophage (phage for short) to digest the bacterial cell wall for phage progeny release. In Gram-positive bacteria, small quantities of purified recombinant lysin added externally results in immediate lysis causing log-fold death of the target bacterium. Lysins have been used successfully in a variety of animal models to control pathogenic antibiotic resistant bacteria found on mucosal surfaces and infected tissues. The advantages over antibiotics are their specificity for the pathogen without disturbing the normal flora, the low chance of bacterial resistance to lysins, and their ability to kill colonizing pathogens on mucosal surfaces, a capacity previously unavailable. Thus, lysins may be a much needed anti-infective in an age of mounting antibiotic resistance.
Pal and Cpl-1, two purified bacteriophage lytic enzymes, were tested for their in vitro activity, alone and in combination,
against several serotypes of Streptococcus pneumoniae, including penicillin-resistant strains. The enzymes demonstrated synergism in their ability to cleave the bacterial peptidoglycan
and thus may be more efficient for the prevention and elimination of pneumococcal colonization.
To investigate the functional role of surface layer proteins (S-layer) in probiotic strain Lactobacillus acidophilus M92, especially its influence on adhesiveness to mouse ileal epithelial cells.
Sodium dodecyl sulphate polyacrylamide gel electrophoresis of cell surface proteins revealed the presence of potential surface layer (S-layer) proteins, ca at 45 kDa in L. acidophilus M92. Southern blot with pBK1 plasmid, containing slpA gene, gave a positive signal, suggesting that L. acidophilus M92 has a slpA gene coding for the S-layer proteins. S-layer proteins of this strain are present during all phases of growth. The S-layer proteins appeared when cells treated with 5 mol l(-1) LiCl were allowed to grow again. Removal of the S-layer proteins reduced adhesion of L. acidophilus M92 to mouse ileal epithelial cells. Furthermore, the viability of cells without S-layer were reduced in simulated gastric juice at low pH range (2, 2.5, 3) and simulated pancreatic juice with bile salts (1.5 and 3 g l(-1)). S-layer proteins of L. acidophilus M92 were resistant to pepsin and pancreatin, in contrast, the treatment with proteinase K led to a significant proteolysis of the S-layer proteins.
These results demonstrated functional role of S-layer; it is responsible for adhesiveness of Lactobacillus acidophilus M92 to mouse ileal epithelial cells and has a protective role for this strain.
S-layer proteins have an important role in the establishment of probiotic strain Lactobacillus acidophilus M92 in the gastrointestinal tract.
Lactobacilli are major inhabitants of the normal microflora of the gastrointestinal tract, and some select species have been used extensively as probiotic cultures. One potentially important property of these organisms is their ability to interact with epithelial cells in the intestinal tract, which may promote retention and host-bacterial communication. However, the mechanisms by which they attach to intestinal epithelial cells are unknown. The objective of this study was to investigate cell surface proteins in Lactobacillus acidophilus that may promote attachment to intestinal tissues. Using genome sequence data, predicted open reading frames were searched against known protein and protein motif databases to identify four proteins potentially involved in adhesion to epithelial cells. Homologous recombination was used to construct isogenic mutations in genes encoding a mucin-binding protein, a fibronectin-binding protein, a surface layer protein, and two streptococcal R28 homologs. The abilities of the mutants to adhere to intestinal epithelial cells were then evaluated in vitro. Each strain was screened on Caco-2 cells, which differentiate and express markers characteristic of normal small-intestine cells. A significant decrease in adhesion was observed in the fibronectin-binding protein mutant (76%) and the mucin-binding protein mutant (65%). A surface layer protein mutant also showed reduction in adhesion ability (84%), but the effect of this mutation is likely due to the loss of multiple surface proteins that may be embedded in the S-layer. This study demonstrated that multiple cell surface proteins in L. acidophilus NCFM can individually contribute to the organism's ability to attach to intestinal cells in vitro.
The autolysins of Lactobacillus helveticus ISLC5 were detected and partially characterized by renaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels (zymogram). By using lyophilized Micrococcus luteus cells or heated whole cells of L. helveticus ISLC5 (0.2% [wt/vol]) as a substrate, several lytic activities were detected in the whole-cell SDS extract of strain ISLC5 (i) one activity at 42.4 kDa, which was named autolysin A, and (ii) six other activities having very similar molecular weights (29.1, 29.6, 30, 30.8, 31.7, and 32.8 kDa), which were named autolysins B (B1 through B6, respectively). As regards the temporal distribution of the enzymes, autolysins A and B were detected in the cells harvested from the beginning of the exponential growth phase. Autolysin A appeared to be associated only with viable cells, whereas the autolysins B remained associated with the cell envelope several days after the complete loss of culture viability. When SDS-treated walls of L. helveticus ISLC5 were used as a substrate, a supplementary lytic activity appeared at 37.5 kDa; it was considered a peptidoglycan hydrolase, since it was not able to induce lysis of whole-cell substrate. The autolysins of 30 other strains of L. helveticus from various geographical origins were also analyzed by zymogram; all the activity profiles obtained were similar to that of strain ISLC5 in terms of the number of lytic bands and their apparent molecular weights. Only the relative intensities of the lytic bands corresponding to autolysins A and B were variable depending on the strains. This observation suggested that autolysins are highly conserved enzymes. A concentrated crude lysate of the virulent bacteriophage 832-B1 infecting L. helveticus was also analyzed by zymogram; one lytic activity with an apparent molecular weight of 31.7 kDa, very close to the weights of the autolysins B, was observed. Finally, the autolysins of L. helveticus ISLC5 were successfully extracted from whole cells by using a 1 M lithium chloride solution; they were partially purified by precipitation, selective resolubilization, and gel filtration chromatography, which led to a 20-fold increase in specific activity.
The objective of this study was the characterisation of the surface layer proteins (SLPs) and their functional role in the probiotic activity of L. helveticus fb213, L. acidophilus fb116 and L. acidophilus fb214. SLPs were extracted and identified by SDS-PAGE, circular dichroism spectra and LC-MS analysis. The results revealed that the molecular masses of the three proteins were 49.7kDa, 46.0kDa and 44.6kDa, respectively. The secondary structures and amino acid compositions of the three proteins were found to be similar. After removing SLPs, the survival of the three lactobacilli in simulated gastric and intestinal juices was reduced by 2 to 3 log as compared with survival of the intact cells. And the adhesion ability of the three strains to HT-29 cells decreased by 61%, 65% and 92%, respectively. SLPs also inhibited the adhesion and invasion of Escherichia. coli ATCC 43893 to HT-29 cells.These results suggest that SLPs are advantageous barriers for lactobacilli in the gastrointestinal tract, and these proteins help make it possible for lactobacilli to serve their probiotic functions.Keywords: surface layer protein; Lactobacillus; secondary structure; amino acid composition; probiotic function.
FTIR spectroscopy was used to structurally characterize the interaction of S-layer proteins extracted from two strains of Lactobacillus kefir (the aggregating CIDCA 8348 and the non-aggregating JCM 5818) with metal ions (Cd+2, Zn+2, Pb+2 and Ni+2). The infrared spectra indicate that the metal/protein interaction occurs mainly through the carboxylate groups of the side chains of Asp and Glut residues, with some contribution of the NH groups belonging to the peptide backbone. The frequency separation between the νCOO− anti-symmetric and symmetric stretching vibrations in the spectra of the S-layers in presence of the metal ions was found to be ca. 190 cm−1 for S-layer CIDCA 8348 and ca. 170 cm−1 for JCM 5818, denoting an unidentate coordination in both cases. Changes in the secondary structures of the S-layers induced by the interaction with the metal ions were also noticed: a general trend to increase the amount of β-sheet structures and to reduce the amount of α-helices was observed. These changes allow the proteins to adjust their structure to the presence of the metal ions at minimum energy expense, and accordingly, these adjustments were found to be more important for the bigger ions.
The methods employed in most previous studies to investigate the non-thermal action of pulsed electric field (PEF) on microbial cells are only capable of revealing the obvious morphological changes of microbial cells induced by PEF, or only differentiate between culturable and not culturable cells. They are totally incapable of real time detection of sublethally injured cells under stress of PEF. In this study, flow cytometry (FCM) in combination with fluorescent techniques was used for quantitative and real time detection of PEF induced damage on Escherichia coli cells and sublethally injured microbial cells. The present work confirmed the thesis that electropermeabilisation was not an all-or-nothing-event. FCM sorting of double stained cells put forward direct evidence for the occurrence of sublethally injured microbial cells under stress of PEF. This work provided quantitative and real time analysis of sublethally injured microbial cells in PEF treatment, and further revealed the underlying mechanism on synergistic effect of moderate heat and PEF on microbial inactivation.
To gain insight into the mechanism of Lactobacillus S-layer protein in antimicrobial activity, we examined how the Lactobacillus acidophilus ATCC 4356 S-layer protein inhibited the adhesion and invasion of Salmonella enterica subsp. enterica serovar Typhimurium SL1344 (Salmonella Typhimurium SL1344) in vitro in cultured Caco-2 cells. The results showed that S-layer protein reduced Salmonella Typhimurium SL1344 association in Caco-2 cells by 69–88% in the adhesive experiments (competition, exclusion, and displacement
assays). Although the antagonistic activity was demonstrated in the exclusion assay (preincubated with the S-layer protein),
a greater effect for the S-layer protein on Salmonella Typhimurium was observed when Salmonella Typhimurium was coincubated with the S-layer protein. The S-layer protein could be bound directly to the Caco-2 cell line
or be associated with Salmonella Typhimurium surface, thereby blocking Salmonella attachment. The data support the antimicrobial mechanisms of Lactobacillus S-layer protein, which are involved not only in competition for binding sites on the surface of host epithelial cells, but
also in a direct interaction between this protein and Salmonella Typhimurium surface.
It has been previously described that S-layer binds to the C-type lectin DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN, CD209). It was also shown that DC-SIGN is a cell-surface adhesion factor that enhances viral entry of several virus families. Among those, Junin virus (JUNV) entry is enhanced in cells expressing DC-SIGN and for that reason surface-layer protein (S-layer) of Lactobacillus acidophilus ATCC 4365 was evaluated as a possible JUNV inhibitor. Experiments using 3T3 cells stably expressing DC-SIGN, showed an almost complete inhibition of JUNV infection when they were treated with S-layer in a similar extend as the inhibition shown by mannan. However no inhibition effect was observed in 3T3 wild type cells or in 3T3 cells expressing liver/lymph node-specific ICAM-3 grabbing nonintegrin (L-SIGN or DC-SIGNR or CD209L). Treatments with S-layer during different times in the infection demonstrated that inhibition was only observed when S-layer was presented in early stages of the viral infection. This inhibition does not involve the classic recognition of mannose by this C-type lectin as the S-layer showed no evidence to be glycosylated. In fact, the highly basic nature of the S-layer (pI>9.5) seems to be involved in electrostatic interactions between DC-SIGN and S-layer, since high pH abolished the inhibitory effect on infection cause by the S-layer. In silico analysis predicts a Ca(2+)-dependant carbohydrate recognition domain in the SlpA protein. This novel characteristic of the S-layer, a GRAS status protein, contribute to the pathogen exclusion reported for this probiotic strain and may be applied as an antiviral agent to inhibit several kinds of viruses.
The autolysins of Bacillus subtilis 168 were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels. Four bands of vegetative autolytic activity of 90, 50, 34, and 30 kDa (bands A1 to A4) were detected in SDS and LiCl extracts and in native cell walls by using B. subtilis 168 vegetative cell walls as the substrate incorporated in the gel. The four enzyme activities showed different substrate specificities and sensitivities to various chemical treatments. The autolysin profile was not medium dependent and remained constant during vegetative growth. During sporulation, band A4 greatly increased in activity just prior to mother-cell lysis. No germination-associated changes in the profile were observed, although a soluble 41-kDa endospore-associated cortex-lytic enzyme was found. By using insertionally inactivated mutants, bands A1 and A2 were positively identified as the previously characterized 90-kDa glucosaminidase and 50-kDa amidase, respectively. The common filamentous phenotype of various regulatory mutants could not be correlated to specific changes in the autolysin profile.
The cell surfaces of several Lactobacillus species are covered by a regular layer composed of a single species of protein, the S-protein. The 43-kDa S-protein of the neotype strain Lactobacillus acidophilus ATCC 4356, which originated from the pharynx of a human, was purified. Antibodies generated against purified S-protein were used to screen a lambda library containing chromosomal L. acidophilus ATCC 4356 DNA. Several phages showing expression of this S-protein in Escherichia coli were isolated. A 4.0-kb DNA fragment of one of those phages hybridized to a probe derived from an internal tryptic fragment of the S-protein. The slpA gene, coding for the surface layer protein, was located entirely on the 4.0-kb fragment as shown by deletion analysis. The nucleotide sequence of the slpA gene was determined and appeared to encode a protein of 444 amino acids. The first 24 amino acids resembled a putative secretion signal, giving rise to a mature S-protein of 420 amino acids (44.2 kDa). The predicted isoelectric point of 9.4 is remarkably high for an S-protein but is in agreement with the data obtained during purification. The expression of the entire S-protein or of large, C-terminally truncated S-proteins is unstable in E. coli.
Peptidoglycan (PG), a component of the bacterial cell wall, has various immunomodulating activities, including the capacity to induce delayed-type hypersensitivity reactions to antigens administered in Freund's adjuvant. We report that PG induces interleukin-12 (IL-12) mRNA production and IL-12 secretion by mouse macrophages. The capacity of PG to induce IL-12 production, like its previously reported immunomodulating activities, was dependent on the structure of its peptide subunit. PG from Bacillus megaterium and Staphylococcus aureus induced IL-12 production, whereas PG from Micrococcus luteus and Corynebacterium poinsettiae did not. The ability of most bacterial PGs to induce IL-12 production suggests that they play an important role in triggering host defense mechanisms against bacterial infections.
The structure of the crystallisation domain, SAN, of the SA-protein of Lactobacillus acidophilus ATCC 4356 was analysed by insertion and deletion mutagenesis, and by proteolytic treatment. Mutant SA-protein synthesised in Escherichia coli with 7–13 amino acid insertions near the N terminus or within regions of sequence variation in SAN (amino acid position 7, 45, 114, 125, 193), or in the cell wall-binding domain (position 345) could form crystalline sheets, whereas insertions in conserved regions or in regions with predicted secondary structure elements (positions 30, 67, 88 and 156) destroyed this capacity. FACscan analysis of L. acidophilus synthesising three crystallising and one non-crystallising SA-protein c-myc (19 amino acid residues) insertion mutant was performed with c-myc antibodies. Fluorescence was most pronounced for insertions at positions 125 and 156, less for position 45 and severely reduced for position 7. By cytometric flow sorting a transformant harbouring the mutant SA-protein gene (position 125) was isolated that showed an increased fluorescense signal. Immunofluorescence microscopy suggested that the transformant synthesized mutant SA-protein only. PCR analysis of the transformant grown in the absence of selection pressure indicated that the mutant allele was stably integrated in the chromosome. Proteolytic treatment of SA-protein indicated that only sites near the middle of SAN are susceptible, although potential cleavage sites are present through the entire molecule. Expression in E. coli of DNA sequences encoding the two halves of SAN yielded peptides that could oligomerize. Our results indicate that SAN consists of a ∼12 kDa N and a ∼18 kDa C-terminal subdomain linked by a surface exposed loop. The capacity of SA-protein of L. acidophilus to present epitopes, up to ∼19 amino acid residues in length, at the bacterial surface in a genetically stable form, makes the system, in principle, suitable for application as an oral delivery vehicle.
Recent studies have shown that the Staphylococcus aureus cidABC and lrgAB operons are involved in the regulation of cell death and lysis. The transcription of cidABC and lrgAB was shown to be induced by acetic acid and was dependent on the cidR gene encoding a new member of the LysR-type transcription regulator (LTTR) family of proteins. In the study presented here, we examined the phenotypic and regulatory effects of disrupting a cidR homologue in Bacillus anthracis. As in S. aureus, the cidR mutation affected expression of the B. anthracis cid and lrg homologues, murein hydrolase activity and cell viability in stationary phase. Interestingly, the predominant murein hydrolase affected was an 85 kDa protein that was identified as Sap, a primary constituent of the S-layer in B. anthracis. The ability of Sap, as well as its counterpart EA1, to exhibit murein hydrolase activity was confirmed by cloning their respective genes in Escherichia coli and showing that the overexpressed proteins contained this activity. Northern blot analyses revealed that the cidR mutation caused reduced transcription of the genes encoding Sap and EA1, as well as CsaB involved in the attachment of the S-layer proteins to the cell wall. The results of these studies not only establish the existence of the cid and lrg murein hydrolase regulatory network in B. anthracis, but also help to define the function and regulation of the S-layer proteins.
Extensive research has been conducted on the development of three groups of naturally occurring antimicrobials as novel alternatives to antibiotics: bacteriophages (phages), bacterial cell wall hydrolases (BCWH), and antimicrobial peptides (AMP). Phage therapies are highly efficient, highly specific, and relatively cost-effective. However, precautions have to be taken in the selection of phage candidates for therapeutic applications as some phages may encode toxins and others may, when integrated into host bacterial genome and converted to prophages in a lysogenic cycle, lead to bacterial immunity and altered virulence. BCWH are divided into three groups: lysozymes, autolysins, and virolysins. Among them, virolysins are the most promising candidates as they are highly specific and have the capability to rapidly lyse antibiotic-resistant bacteria on a generally species-specific basis. Finally, AMP are a family of natural proteins produced by eukaryotic and prokaryotic organisms or encoded by phages. AMP are of vast diversity in term of size, structure, mode of action, and specificity and have a high potential for clinical therapeutic applications.