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Variability of S-layer proteins in Lactobacillus helveticus strains
... acidophilus homology group (including Lb. acidophilus, Lb. helveticus, Lb. crispatus, Lb. amylovorus, Lb. gallinarum): slpA, slpB, and slpX, being slpA and slpB in opposite orientation to each other. Interestingly, the presence or not of secondary slp genes in strains of Lb. helveticus showed high variability by comparative genomic analyses (Waśko et al., 2014) and consequently, they could be used for strain typing in dairy products (Moser et al., 2017). Also, Fontana et al. (2019) found an increased number of slp genes in isolates from natural whey cultures compared to other Lb. ...
... In contrast, the C-terminal, involved in the cell wall binding, appeared conserved between the species of the genus. The anchoring domain or cell wall-binding domain (CWBD) has been proposed for these species due to the high similarity found between the Cterminal region (Table 3) (Hynönen et al., 2014;Waśko et al., 2014). When SLPs from Levilactobacillus and Lentilactobacillus were aligned (Figure 3), it was evidenced that the more conserved N-terminal part of the protein was involved in the cell wall anchoring, while the C-terminal variable region was associated with self-assembly (Table 2). ...
... This binding activity is specifically thought to mediate bacterial colonization of the gut, contributing to the probiotic's interaction with the host tissues and other factors, such as cell surface hydrophobicity, auto-aggregation, mucin-and fibronectin-binding proteins interplay. Different authors have proved that SLPs extracted from probiotic Lactobacillus have the ability to in vitro bind host cells and extracellular matrix proteins (Carasi et al., 2014;Prado-Acosta et al., 2010;Waśko et al., 2014;Zhang et al., 2017;Zhu et al., 2016). ...
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.
... S-layer proteins have various functions in cell protection, formation of cellspecific topology, adhesion and aggregation. It is also suggested that it has protective role against digestive enzymes and acids [14][15][16][17]. It is thought that some lactic acid bacteria that play an active role in maintaining microbial balance in human intestines may have an S-layer, which gives them an advantage in passage through the gastrointestinal tract and adherence to epithelium. ...
... LiCl was removed by washing with distilled water. The number of viable cells was determined by serial plating on MRS agar before and after LiCl treatment [2, 17,18]. ...
... Table 3 shows the changes in the adhesion ability of strains before and after LiCl treatment. [17] reported that amino acid composition, the secondary structure, and the physical properties of these proteins were found to be quite similar to those of S-layer proteins from other lactobacilli, in different strains of Lactobacillus helveticus. All ingested microorganisms expose harsh stress conditions in gastrointestinal system, including low pH, proteolytic enzymes, digestive juices, therefore, researchers investigated how this conditions affect different lactic acid bacteria. ...
S-layer proteins in Lactic acid bacteria are not the only cell surface structures used for aggregation, but also plays significant role for intestinal tissue adhesion along with some other functional elements. In addition, it was determined that the properties of S-layer proteins differs not only between species but also the strains which belong to same species. In this work, presence and some functions of S-layer in lactic acid bacteria were determined, its effect on resistance to gastrointestinal enzymes, aggregation and adhesion ability were investigated as well. For this purpose S-layers of microorganisms were removed by 5 M LiCl treatment and size of the proteins were determined by SDS-PAGE analysis. The removal of S-layer proteins caused a change in the resistance of microorganisms to GIS enzymes. After the S-layer removal, two strains considerably lost their resistance to GIS enzymes. The strains mostly lost their aggregation ability in the absence of S-layer. The results showed that S-layer proteins are not the only structures involved in aggregation processes but, is a major mediator in Lactobacilli. Removal of S-layer had no effect on adhesion ability of W. cibaria DA28, the effect on L. casei DA4, L. coryniformis DA263 and L. plantarum DA140 was moderate, but the effect was high on L. plantarum DA100. The study showed that S-layer proteins play limited protection against GIS enzymes. In addition, absence of S-layer adversely affected aggregation and adhesion ability of strains.
... Surface layer proteins constitute the outermost structure of the cell envelope, which is an array of single non-covalently bound proteins. 3 The biological functions of surface layer proteins are cell protection, determination of cell shape, molecular and ion trapping, and adhesion to surfaces. 4 Cell adhesion and aggregation is well documented for gut-associated lactobacilli as is their protective role against digestive enzymes and acids. ...
... 4 Cell adhesion and aggregation is well documented for gut-associated lactobacilli as is their protective role against digestive enzymes and acids. 3 These surface proteins give the cell a hydrophobic character and play a role in specific interactions with intestinal epithelium cells and with other bacteria. 4 Surface layer associated with moonlighting proteins could act as adherence factors. ...
... 4 Surface layer associated with moonlighting proteins could act as adherence factors. 3 Lb. helveticus T159 has the high capability of adhesion, auto and coaggregation. 4 This protein used to detected in Lb. helveticus strain. ...
... The measurement of the zeta-potential as a function of pH of each heat-inactivated bacteria was assessed in order to determine the values of isoelectric point (pI) ( Figure 1B). L. helveticus containing an S-layer in its cell wall [34] has a pI of 2.2, while L. plantarum has a pI of 3.7. The interpretation of the zeta-potential in terms of surface charge densities is difficult for microorganisms, but it can be assumed that bacterial outer cell wall components mainly contribute to the surface charge, which can lead to a qualitative description of the main outer component present on the bacteria [35]. ...
... Gram-positive cell wall lactic acid bacteria are composed of an inner layer of peptidoglycan and an outer layer The measurement of the zeta-potential as a function of pH of each heat-inactivated bacteria was assessed in order to determine the values of isoelectric point (pI) ( Figure 1B). L. helveticus containing an S-layer in its cell wall [34] has a pI of 2.2, while L. plantarum has a pI of 3.7. The interpretation of the zeta-potential in terms of surface charge densities is difficult for microorganisms, but it can be assumed that bacterial outer cell wall components mainly contribute to the surface charge, which can lead to a qualitative description of the main outer component present on the bacteria [35]. ...
It is generally accepted that microbes play a critical role in maintaining gut barrier function , making them ideal to target in order to mitigate the effects of intestinal diseases such as inflam-matory bowel disease with specialist supplementations such as probiotic or postbiotic preparations. In this study, specific strains of Lactobacillus helvictus both live and inactivated and Lactobacillus plantarum inactivated were fed to zebrafish at an inclusion level of 6 × 10 6 cells/g in order to assess the effects on gut barrier function and protection. Taken together, our results indicate that dietary administration of pro-or postbiotics strengthens the gut barrier function and innate immunity of healthy zebrafish in a strain-specific and process-dependent way. With some differences in the response intensity, the three treatments led to increased intestinal villi length and proportion of IELs, reinforcement of the GC population and up-regulated expression of biomarkers of AMP production and tight junction zona-occludin 2a (zo-2a). In addition, LPPost had an impact on the adaptive immune response, and we hypothesized that it conferred the potential to drive Th17/ILC3 immunity, as suggested by its effect on the gene expression of il22, of different AMPs, and the expression of zo2a. Moreover, LPPost showed the potential to drive Th1/ILC1-like immunity, with a higher percentage of CD8 + cells and higher ifnγ gene expression. In summary, the use of inactivated Lactobacilli species in this study represented a promising strategy for improving barrier function and regulating the immune fate of the intestinal mucosa in a strain-specific way.
... The nucleotide sequence heterogeneity of this locus in L. helveticus has already been described by other researchers (Ventura et al., 2000;Gatti et al., 2005;Waśko et al., 2014). Since Waśko et al. (2014) reported they could not detect the slpH gene in five L. helveticus strains, the question arises if this gene is ubiquitous in L. helveticus. ...
... The nucleotide sequence heterogeneity of this locus in L. helveticus has already been described by other researchers (Ventura et al., 2000;Gatti et al., 2005;Waśko et al., 2014). Since Waśko et al. (2014) reported they could not detect the slpH gene in five L. helveticus strains, the question arises if this gene is ubiquitous in L. helveticus. Unfortunately, the authors neither explained how the species of these strains were determined nor did they show experimental evidence for the lack of the slpH gene in their paper. ...
The advent of massive parallel sequencing technologies has opened up possibilities for the study of the bacterial diversity of ecosystems without the need for enrichment or single strain isolation. By exploiting 78 genome data-sets from Lactobacillus helveticus strains, we found that the slpH locus that encodes a putative surface layer protein displays sufficient genetic heterogeneity to be a suitable target for strain typing. Based on high-throughput slpH gene sequencing and the detection of single-base DNA sequence variations, we established a culture-independent method to assess the biodiversity of the L. helveticus strains present in fermented dairy food. When we applied the method to study the L. helveticus strain composition in 15 natural whey cultures (NWCs) that were collected at different Gruyère, a protected designation of origin (PDO) production facilities, we detected a total of 10 sequence types (STs). In addition, we monitored the development of a three-strain mix in raclette cheese for 17 weeks.
... Significant researches have been dedicated to describe the location and ultrastructure of S-layer lattices of a great number of Bacteria and Archaea by electron microscopy of thin-sectioned, freeze-etched, freeze-dried and shadowed, negatively stained or frozen hydrated preparations (Thornley et al., 1974;Sleytr and Glauert, 1975;Beveridge, 1994;Sleytr et al., 1999;Györvary et al., 2003;Toca-Herrera et al., 2004;Dordic et al., 2012;Shin et al., 2012;Wasko et al., 2014). More recently, atomic force microscopy (AFM) Tang et al., 2008;Chung et al., 2010;Lopez et al., 2011) and small-angle X-ray scattering (SAXS) (Toca-Herrera et al., 2004;Horejs et al., 2010;Kontro et al., 2014) provided the information of lattice constants and crystallite size averaged over the sample. ...
... SDS-PAGE migration pattern of LiCl-extracted Slps from Lactobacillus helveticus CGMCC1.1877 was shown in Fig. 1. There was a single protein band with a molecular mass of around 48.5 kDa, which was of similar molecular weight as reported in a previous work (Wasko et al., 2014). ...
S-layer proteins (Slps) are crystalline arrays of protein on bacterial cell surface layers. Owning to their capability to reassemble on the surface of lipid layers, Slps have been employed to modify liposomes for various profits. But the interaction information between Slps and positively charged liposomes are destitute, especially the gastrointestinal adhesion of Slps-coating liposomes is rarely reported. In the present work, the Slps extracted from Lactobacillus helveticus were reassembled on the surface of novel positively charged liposomes composed of soybean lecithin, Eudragit®RL100 and cholesterol. The particle size and remarkable changes of Zeta potential with various Slps/lipid weight ratios were determined by dynamic light scattering and phase analysis light scattering. Significant difference in fluorescence dequenching percentage of liposomes decorated by ODA-FITC confirmed Slps self-reassemble on the surface of liposomes. A higher integrity of vesicular membrane after the addition of Triton X-100 solution demonstrated the stability enhancement of Slps-coating liposomes. Fourier transform infrared (FTIR) spectroscopy illustrated the interaction came from non-covalent bond. The mucoadhesion of Slps-coating liposomes was evaluated by the resident FITC-LP on the gastric and intestinal tract of mice at 7 h and 12 h after intragastrical administration, which proved that the Slps-coating improved the gastrointestinal adhesion significantly.
... Bacterial cultures were adjusted to the same optical density OD 600 of 1.8. Isolation of surface proteins and detection was performed according to the method described previously [16]. ...
... It was expected that these proteins had an appreciable effect on cell wall properties. We had previously described that S-layer proteins in Lb. helveticus T159 are highly basic, with an isoelectric point above pH ¼ 9, and they fully cover the cell wall [16]. The hydrophobic moieties of surface proteins are one of the factors that lead to extensive adhesion and aggregation of bacteria [4]. ...
The goal of this study was to identify moonlighting proteins in Lactobacillus helveticus that play an important role in adhesion and aggregation. The label-free method was used for identification and analysis of expression of cellular proteins. The analysis revealed the presence of eight moonlighting proteins in the cell envelope of Lb. helveticus. The tested strains mainly differed with respect to the presence of S-layer proteins and the level of expression of moonlighting proteins in Lb. helveticus strain T159. These surface proteins give the cell a hydrophobic character and play a role in specific interactions with intestinal epithelium cells and with other bacteria. In Lb. helveticus T159, the S-layer associated with moonlighting proteins could act as adherence factors, which was evidenced by the high capability of adhesion, auto- and coaggregation. The hydrophobicity, adhesion and aggregation abilities provide biological activities in food products and they are regarded as an important criterion for probiotic selection.
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... The strains of L. helveticus are extensively used in food industry, particularly as lactic acid starter cultures in fermented products, and some of them have been characterized as probiotics. The presence of Slayer in L. helveticus from different sources has been reported with differences in the amino acid sequence among strains (Waśko et al., 2014;Suzuki et al., 2019). The role of the S-layer proteins in the probiotic properties of L. helveticus has been assessed for different strains such as R0052 (Johnson-Henry et al., 2007), M92 (Beganović et al., 2011), and koumiss-isolated NS8 (Rong et al., 2015). ...
In bacteria, as in other microorganisms, surface compounds interact with different pattern recognition receptors expressed by host cells, which usually triggers a variety of cellular responses that result in immunomodulation. The S-layer is a two-dimensional macromolecular crystalline structure formed by (glyco)-protein subunits that covers the surface of many species of Bacteria and almost all Archaea. In Bacteria, the presence of S-layer has been described in both pathogenic and non-pathogenic strains. As surface components, special attention deserves the role that S-layer proteins (SLPs) play in the interaction of bacterial cells with humoral and cellular components of the immune system. In this sense, some differences can be predicted between pathogenic and non-pathogenic bacteria. In the first group, the S-layer constitutes an important virulence factor, which in turn makes it a potential therapeutic target. For the other group, the growing interest to understand the mechanisms of action of commensal microbiota and probiotic strains has prompted the studies of the role of the S-layer in the interaction between the host immune cells and bacteria bearing this surface structure. In this review, we aim to summarize the main latest reports and the perspectives of bacterial SLPs as immune players, focusing on those from pathogenic and commensal/probiotic most studied species.
... Protective functions of Slps in these bacteria provide resistance to colonization of host tissues by pathogenic microorganisms [5]. The variability of the sequences and the functions of the surface layer (S-layer) in lactobacilli depend not only on the species, but also on the strain [6,7]. This variability is one of the reasons for the significant strain differences in the biological properties of lactobacilli. ...
We have previously demonstrated the ability of the human vaginal strain Lactobacillus crispatus 2029 (LC2029) for strong adhesion to cervicovaginal epithelial cells, expression of the surface layer protein 2 (Slp2), and antagonistic activity against urogenital pathogens. Slp2 forms regular two-dimensional structure around the LC2029 cells, is secreted into the medium and inhibits intestinal pathogen-induced activation of caspase-9 and caspase-3 in the human intestinal Caco-2 cells. Here, we elucidated the effects of soluble Slp2 on adhesion of proteobacteria pathogens inducing necrotizing enterocolitis (NEC), such as Escherichia coli ATCC E 2348/69, E. coli ATCC 31705, Salmonella enteritidis ATCC 13076, Campylobacter jejuni ATCC 29428, and Pseudomonas aeruginosa ATCC 27853 to Caco-2 cells, as well as on growth promotion, differentiation, vascular endothelial growth factor (VEGF) production, and intestinal barrier function of Caco-2 cell monolayers. Slp2 acts as anti-adhesion agent for NEC-inducing proteobacteria, promotes growth of immature Caco-2 cells and their differentiation, and enhances expression and functional activity of sucrase, lactase, and alkaline phosphatase. Slp2 stimulates VEGF production, decreases paracellular permeability, and increases transepithelial electrical resistance, strengthening barrier function of Caco-2 cell monolayers. These data support the important role of Slp2 in the early postnatal development of the human small intestine enterocytes.
... To characterize the new strain L. hilgardii FLUB, we also tested its protein profile with SDS-PAGE as surface proteins are crucial to survival in the harsh environmental conditions of alcoholic beverages. Protein profiles and outer surface proteins were isolated according to Waśko et al. [91] and visualized using SDS-PAGE. ...
The production of mead holds great value for the Polish liquor industry, which is why the bacterium that spoils mead has become an object of concern and scientific interest. This article describes, for the first time, Lactobacillus hilgardii FLUB newly isolated from mead, as a mead spoilage bacteria. Whole genome sequencing of L. hilgardii FLUB revealed a 3 Mbp chromosome and five plasmids, which is the largest reported genome of this species. An extensive phylogenetic analysis and digital DNA-DNA hybridization confirmed the membership of the strain in the L. hilgardii species. The genome of L. hilgardii FLUB encodes 3043 genes, 2871 of which are protein coding sequences, 79 code for RNA, and 93 are pseudogenes. L. hilgardii FLUB possesses three clustered regularly interspaced short palindromic repeats (CRISPR), eight genomic islands (44,155 bp to 6345 bp), and three (two intact and one incomplete) prophage regions. For the first time, the characteristics of the genome of this species were described and a pangenomic analysis was performed. The concept of the pangenome was used not only to establish the genetic repertoire of this species, but primarily to highlight the unique characteristics of L. hilgardii FLUB. The core of the genome of L. hilgardii is centered around genes related to the storage and processing of genetic information, as well as to carbohydrate and amino acid metabolism. Strains with such a genetic constitution can effectively adapt to environmental changes. L. hilgardii FLUB is distinguished by an extensive cluster of metabolic genes, arsenic detoxification genes, and unique surface layer proteins. Variants of MRS broth with ethanol (10-20%), glucose (2-25%), and fructose (2-24%) were prepared to test the strain's growth preferences using Bioscreen C and the PYTHON script. L. hilgardii FLUB was found to be more resistant than a reference strain to high concentrations of alcohol (18%) and sugars (25%). It exhibited greater preference for fructose than glucose, which suggests it has a fructophilic nature. Comparative genomic analysis supported by experimental research imitating the conditions of alcoholic beverages confirmed the niche specialization of L. hilgardii FLUB to the mead environment.
... In the present study, we examined surface hydrophobicity and autoaggregation properties, and they play roles in bacterial adhesion to host components, but surface layer (S-layer) proteins that many lactobacilli possess are also very important for adhesion Sengupta et al., 2013;Waśko, Polak-Berecka, Kuzdraliński, & Skrzypek, 2014). S-layer proteins bind to the cell wall in a non-covalent manner, and their biological functions include determination of cell shape, protection, and molecular and ion capture to surfaces, as well as the attachment of a bacterial cell to the mucus layer (Alp et al., 2020;Hynönen & Palva, 2013;Sara & Sleytr, 2000;Sleytr & Beveridge, 1999). ...
Honey is a health promoter that is used to treat in various diseases. Probiotics are live microorganisms that exert health benefits on the host, when administered in adequate amounts. With other diet constituents, probiotic bacteria can alter the composition of microbiota and exert more health benefits. This study aimed to investigate the effects of lime honey on well-known probiotic bacteria Lactobacillus acidophilus LA-5 and Lactobacillus rhamnosus GG, and in vitro cytotoxic effects of the combination on breast and colon cancer cells MCF-7 and Caco-2, respectively. The results showed that lime honey enhanced the probiotic properties of Lactobacillus acidophilus and Lactobacillus rhamnosus, including auto-aggregation and surface hydrophobicity. Furthermore, the addition of lime honey to the growth of lactobacilli enhanced cytotoxicity in breast and colon cancer cells, while it did not significantly affect healthy cells. In conclusion, honey may have not only direct effects on human health, but also indirect benefits mediated by beneficial microorganisms.
... Even though the biological functions of the S-layer include determination of cell shape, protection, and molecular and ion capture to surfaces, it is thought to be responsible for the attachment of a bacterial cell to the mucus layer [35,[40][41][42]. Thus, not only hydrophobicity and aggregation play a role in good adhesion ability, but the S-layer proteins also play a key role [43,44]. ...
Chestnut honey has been used as ethnomedicine. Probiotics are defined as live microorganisms that can provide a health benefit, impeding the development of several health conditions and diseases, including cancer. This study aims to investigate the effects of chestnut honey on probiotic bacteria and the in vitro cytotoxic effects of the combination of probiotics and chestnut honey on cancer cells. First, the effects of chestnut honey on the growth of bacteria were examined, followed by its effects on the probiotic properties of Lactobacillus acidophilus LA-5 and Lactobacillus rhamnosus GG. Once the bacteria had grown on chestnut honey, the in vitro cytotoxic effects on breast and colon cancer cell lines, MCF-7 and Caco-2, respectively, and a non-cancerous breast epithelial cell line, MCF-10A, were investigated. Chestnut honey positively affected the probiotic bacteria by increasing the growth and modulating probiotic properties such as autoaggregation and surface hydrophobicity. Furthermore, probiotics grown on chestnut honey had more cytotoxic effects on the cancer cell lines than probiotics or honey alone. The present study showed that new combinations of honey and probiotics have the potential to formulate new nutraceuticals.
... Previous reports describe the nucleotide sequence heterogeneity of the slpH gene, which encodes an L. helveticus S-layer protein (Gatti et al., 2005;Ventura et al., 2000;Waśko et al., 2014). Moser et al. (2017b) recently confirmed the high number of nucleotide polymorphisms in this gene by analyzing the genomic sequences of 79 L. helveticus strains, and proposed a method for L. helveticus strain typing in dairy products based on high-throughput amplicon sequencing of the slpH locus. ...
Monitoring L. helveticus strain dynamics in natural whey starters is of great interest at the industrial level due to the key role that this bacterial population plays in Grana Padano cheese production. In this study, we aimed to develop a PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) assay based on the slpH locus, in parallel with performing culture-dependent analysis of whey samples using optimized media to maximize the number of isolated strains. We designed new primers targeting the slpH locus to amplify a gene region that would be suitable for PCR-DGGE analysis and discriminating strains. Our results confirmed that the developed PCR-DGGE method was rapid and reliable for monitoring the L. helveticus population in whey starter cultures. All sequences of bands detected in the PCR-DGGE profiles from whey samples showed high similarity to S-layer genes of L. helveticus, and perfectly matched with the slpH locus sequences of dominant strains. Overall, our findings indicated that the target region of the slpH locus was sufficiently heterologous to discriminate L. helveticus strains, and that our PCR-DGGE analysis provided a more accurate picture of the population composition of whey starters compared to culture-dependent techniques that often fail to isolate the most abundant strains.
... Alignment of the primary structures of Slp2 (slp2-2029) of vaginal L. crispatus LC2029, SlpA (slpa-ZJ001) of L. crispatus ZJ001 isolated from pig intestines [41], and CbsA (cbsa-5810) of L. crispatus JCM 5810 isolated from chicken intestines [43] is shown in Fig. 2. The amino acid sequence identity between SlpA and CbsA was as high as 84%, whilst the identity levels of these sequences with that of Slp2 were only 49% and 50% (respectively). The C-terminal regions of the three proteins appear to be more conserved than the N-terminal regions (Fig. 2), which is similar to Slps L. helveticus and L. acidophilus [39,44], but different from Slps of L. brevis with N-terminal regions being more conserved [45]. N-terminal regions of Slps are responsible for binding of bacteria to epithelial cells or pericellular tissue components in specific niches. ...
We have previously demonstrated that human vaginal Lactobacillus crispatus 2029 (LC2029) strain is highly adhesive to cervicovaginal epithelial cells, exhibits antagonistic activity against genitourinary pathogens and expresses surface-layer protein (Slp). The aims of the present study were elucidation of Slp structural and immunomodulatory characteristics and its roles in protective properties of the whole vaginal LC2029 bacteria against foodborne pathogens. Enteric Caco-2 and colon HT-29 cell lines were used as the in vitro models of the human intestinal epithelial layer. LC2029 strain has two homologous surface-layer (S-layer) genes, slp1 and slp2. Whilst we found no evidence for the expression of slp1 under the growth conditions used, a very high level of expression of the slp2 gene was detected. C-terminal part of the amino sequence of Slp2 protein was found to be highly similar to that of the conserved C-terminal region of SlpA protein of L. crispatus Zj001 isolated from pig intestines and CbsA protein of L. crispatus JCM5810 isolated from chicken intestines, and was substantially variable at the N-terminal and middle regions. The amino acid sequence identity between SlpA and CbsA was as high as 84%, whilst the identity levels of these sequences with that of Slp2 were only 49% and 50% (respectively). LC2029 strain was found to be both acid and bile tolerant. Survival in simulated gastric and intestinal juices of LC2029 cells unable to produce Slp2 was reduced by 2-3 logs. Vaginal L. crispatus 1385 (LC1385) strain not expressing Slp was also very sensitive to gastric and intestinal stresses. Slp2 was found to be non-covalently bound to the surface of the bacterium, acting as an adhesin and facilitating interaction of LC2029 lactobacilli with the host immature or fully differentiated Caco-2 cells, as well as HT-29 cells. No toxicity to or damage of Caco-2 or HT-29 epithelial cells were detected after 24 h of colonization by LC2029 lactobacilli. Both Slp2 protein and LC2029 cells induced NF-kB activation in Caco-2 and HT-29 cells, but did not induce expression of innate immunity mediators Il-8, Il-1β, and TNF-α. Slp2 and LC2029 inhibited Il-8 production in Caco-2 and HT-29 cells induced by MALP-2 and increased production of anti-inflammatory cytokine Il-6. Slp2 inhibited production of CXCL1 and RANTES by Caco-2 cells during differentiation and maturation process within 15 days. Culturing Caco-2 and HT-29 cells in the presence of Slp2 increased adhesion of bifidobacteria BLI-2780 to these enterocytes. Upon binding to Caco-2 and HT-29 cells, Slp2 protein and LC2029 lactobacilli were recognized by toll-like receptors (TLR) 2/6. It was shown that LC2029 strain is a strong co-aggregator of foodborne pathogens Campylobacter jejuni, Salmonella enteritidis, and Escherichia coli O157:H used in this study. The Slp2 was responsible for the ability of LC2029 to co-aggregate these enteropathogens. Slp2 and intact LC2029 lactobacilli inhibited foodborne pathogen-induced activation of caspase-9 and caspase-3 as apoptotic biomarkers in Caco-2 and HT-29 cells. In addition, Slp2 and Slp2-positive LC2029 strain reduced adhesion of tested pathogenic bacteria to Caco-2 and HT-29 cells. Slp2-positive LC2029 strain but not Slp2 alone provided bactericidal effect on foodborne pathogens. These results suggest a range of mechanisms involved in inhibition of growth, viability, and cell-adhesion properties of pathogenic Proteobacteria by the Slp2 producing LC2029, which may be useful in treatment of necrotizing enterocolitis (NEC) in newborns and foodborne infectious diseases in children and adults, increasing the colonization resistance and maintaining the intestinal homeostasis.
... S layer is formed by non-covalently bonded protein chain which consist of acidic and hydrophobic amino acids. The most common amino acids are lysine, arginine, histidine and methionine ( Wasko et al. 2014;Meng et al. 2017). It is suggested that one of the structures responsible for adherence of bacterial cells to the intestinal epithelium is the S layer and its ability to bind is believed to based on its repeating microstructures ( Goh and Klaenhammer 2010;Ossowski et al. 2011;Denkova et al. 2014). ...
Adhesion ability is a primary criterion for the selection of probiotic microorganisms. Lactic acid bacteria contribute the majority of microorganisms with probiotic properties. They have several important mechanisms for intestinal epithelial cell adhesion. In order to adhere to the intestinal cells, they generally use various structures such as flagella, pili, S layer proteins, lipoteichoic acid, exopolysaccharides and mucus binding proteins. Various in vitro experiments were designed or study models were developed to reveal the mechanisms they utilize for binding to the intestinal cells, yet, the mechanisms for their adhesion are not fully explained. The major disadvantage of conventional models is the lack of layers forming the intestinal mucosa. Besides, these models omit the presence of natural microbiota, digestive conditions and the presence of a food matrix. Because of the disadvantages of existing models, natural tissues or novel applications like 3D organ cultures, which are better able to mimic in vivo conditions, are preferred.
... 6 However, the details of the interactions between liposomes and Slp remain unclear. In recent years, a series of electron microscopy technologies, such as the thinsectioned, freeze-dried, and freeze-etched methods, 30,[71][72][73][74][75][76][77][78] have been adopted to elucidate the location and ultrastructure of Slp lattices. In addition, information on lattice constants, such as average size, was provided by atomic force microscopy 31,79-83 and small-angle X-ray scattering. ...
Slp forms a crystalline array of proteins on the outermost envelope of bacteria and archaea with a molecular weight of 40–200 kDa. Slp can self-assemble on the surface of liposomes in a proper environment via electrostatic interactions, which could be employed to functionalize liposomes by forming Slp-coated liposomes for various applications. Among the molecular characteristics, the stability, adhesion, and immobilization of biomacromolecules are regarded as the most meaningful. Compared to plain liposomes, Slp-coated liposomes show excellent physicochemical and biological stabilities. Recently, Slp-coated liposomes were shown to specifically adhere to the gastrointestinal tract, which was attributed to the “ligand–receptor interaction” effect. Furthermore, Slp as a “bridge” can immobilize functional biomacromol-ecules on the surface of liposomes via protein fusion technology or intermolecular forces, endowing liposomes with beneficial functions. In view of these favorable features, Slp-coated liposomes are highly likely to be an ideal platform for drug delivery and biomedical uses. This review aims to provide a general framework for the structure and characteristics of Slp and the interactions between Slp and liposomes, to highlight the unique properties and drug delivery as well as the biomedical applications of the Slp-coated liposomes, and to discuss the ongoing challenges and perspectives.
... La souche (ATCC 12046) utilisée pour l'étude présentait une protéine de 52 kDa sur la couche la plus externe de la paroi. Bien que pour cette souche et la majorité des lactobacilles, aucune glycosylation n'a été détectée sur leurs protéines S-layer, de récents travaux sur des souches de L. helveticus ont présenté six Slayers glycosylées (Wasko et al., 2014). La glycosylation et les différentes protéines associées à la S-layer peuvent influencer la charge du manteau de S-layer et ainsi créer un environnement physicochimique spécifique à chaque souche. ...
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... crispatus is involved in adherence to these matrix components [71]. To date, some surface layer proteins from probiotics have been identified, for example, SlpA, SlpB, and SlpX from Lac. acidophilus [72]; Cbp from Lac. plantarum [73]; and SlpH from Lac. helveticus [74]. In B. longum subsp. ...
... Recently, their potential for antigen delivery as live oral vaccines due to adhesive and immunomodulation properties has also been recognized (Hyn€ onen & Palva, 2013;Lebeer, Vanderleyden, & De Keersmacker, 2008). Besides that, Slayer proteins might have a technologically important function in commercial starter strains because of their ability to anchor proteolytic enzymes to the cell surface (Scolari, Vescovo, Zacconi, & Vescovi, 2006; Wa sko, Polak-Berecka, Kuzdrali nski, & Skrzypek, 2014). ...
An autochthonous strain isolated from traditionally produced smoked fresh cheese, Lactobacillus brevis D6, carries a 45-kDa S-layer protein. In this study, the functional role of S-layer proteins of L. brevis D6 was investigated to define this strain as probiotic starter culture. Presence of S-layer proteins and microencapsulation of L. brevis D6 cells in casein increased survival during freeze-drying, which is important prerequisite for exertion of probiotic properties. This strain displayed the highest autoaggregation ability among all the Lactobacillus strains tested, which was decreased by the removal of S-layer proteins using 5M guanidine hydrochloride. The capacity of the L. brevis D6 to adhere to extracellular matrix proteins and Caco-2 intestinal epithelial cell line in vitro was assayed, where the removal of S-layer proteins negatively affected adhesion properties. Exclusion of Salmonella Typhimurium FP1 by L. brevis D6 and the survival of D6 in simulated conditions of the gastrointestinal tract, were better before removal of S-layer from the cell surface. Additionally, cytokine production in monocyte-derived dendritic cells (moDC) and moDC maturation in response to purified S-layer proteins of L. brevis D6 were determined by ELISA and FACS, respectively. Interleukin production remained unaltered, while a slight induction of TNF-α production in moDC was observed.
... The electron microscopy images indicate that the viscous layer on the outer surface of the acclimated variant strain L. salivarius UCO_979C-2 fits to EPS and they are not the S-layer found in other bacteria. In line with our results, Wasko et al. [22] have shown that the S-layer proteins present different morphology by electron micrographs. The S-layer is less dense and more translucent than the EPS. ...
Background: Bacterial acclimation involves cellular changes permitting the survival of a microorganism to prolonged acid pH exposure. The general aim of this work is to support this idea by determining the effect of pH in the survival of the human gastric derived probiotic strain Lactobacillus salivarius UCO_979C-1 (wild type) and L. salivarius UCO_979C-2 (acclimation to pH 2.6), which possesses anti-Helicobacter pylori properties.
Results: To assess this aim, the exopolysaccharide production through the phenol-sulfuric acid method was evaluated. Moreover, morphological and structural changes by transmission and scanning electron microscopy were observed. The bacterial survival was measured by viable count. The results showed that the acclimated variant strain synthesized higher levels of exopolysaccharide (690 ± 0.03 mg/L) more than the wild type (450 ± 0.12 mg/L). In addition, the acclimated variant preserved the viable count at pH 2.6 for 48 h, whereas the wild type strain decreases after 6 h and was non-viable at 24 h.
Conclusion: The results suggest that the acid stress acclimation of the strain L. salivarius UCO_979C-1 modified some cellular properties making this strain potentially useful as a gastric probiotic.
... Comparative genomic studies have found that L. helveticus is rather closely related to the gut organism L. acidophilus although these two lactobacilli occupy different environmental niches [20]. However, L. helveticus DPC4571 was reported to possess a frame-shifted nonfunctional bile salt hydrolase gene, which suggested genetic adaption to the dairy environment might occur during the evolution [9]. ...
Koumiss is a traditionally fermented mare’s milk described with health-promoting potentials for decades. However, only a few studies focused on the probiotic strains isolated from koumiss. In this study, we collected koumiss samples from Inner Mongolian pasturing area of China and selected a promising strain of Lactobacillus helveticus, isolate NS8, based on the survival abilities in gastrointestinal tract (GIT) and adhesion to intestinal endothelial cells in vitro. As the ability to positively modulate host immune response is a feature of increasing importance in measuring the probiotic potential of a bacterial strain, our study mainly focus on the immunomodulatory properties of L. helveticus NS8 by using in vivo and ex vivo analyses.
L. helveticus NS8 was identified by molecular-typing methods, both at genus and species levels. As a typical food niche-specific bacteria, NS8 showed a moderate survival ability in GIT environment in vitro. However, an excellent binding capacity to the human intestinal epithelial cells, along with significant autoaggregation and cell-surface hydrophobicity was observed. Additionally, the presence of S-layer protein was responsible for the cell surface properties of this strain. NS8 was found to be rather protective against TNBS (2,4,6-trinitrobenzene sulfonic acid)-induced murine colitis. In the meantime, co-culture with NS8 induced an increased level of secretion of anti-inflammatory cytokine IL-10 in peripheral blood mono-nuclear cells (PBMCs). Furthermore, NS8 was also able to diminish the proinflammatory effects of lipopolysaccharide (LPS) in mouse macrophage cell line RAW264.7 by inducing higher levels of IL-10. Specially, adding of the purified S-layer protein didn’t influence the production of IL-10. The specific ligand-host receptor interactions on the NS8 specific immune responses need to be learned further.
In summary, L. helveticus NS8 exhibited good probiotic and particularly immunomodulatory properties, with a potential for development of functional food commercially or therapeutic adjuvant for inflammatory diseases.
Paraprobiotics and postbiotics represent a valid alternative to probiotic strains for ameliorating and preserving a healthy intestinal epithelial barrier (IEB). The present study investigated the effects of surface layer proteins (S-layer) of the dairy strain Lactobacillus helveticus ATCC® 15009™ (Lb ATCC® 15009™), as paraprobiotic, on the morpho-functional modulation of IEB in comparison to live or heat-inactivated Lb ATCC® 15009™ in an in vitro co-culture of Caco-2/HT-29 70/30 cells. Live or heat-inactivated Lb ATCC® 15009™ negatively affected transepithelial electrical resistance (TEER) and paracellular permeability, and impaired the distribution of Claudin-1, a tight junction (TJ) transmembrane protein, as detected by immunofluorescence (IF). Conversely, the addition of the S-layer improved TEER and decreased permeability in physiological conditions in co-cultures with basal TEER lower than 50 ohmcm2, indicative of a more permeable physiological IEB known as leaky gut. Transmission electron microscopy (TEM) and IF analyses suggested that the S-layer induces a structural TJ rearrangement and desmosomes' formation. S-layer also restored TEER and permeability in the presence of LPS, but not of a mixture of pro-inflammatory cytokines (TNF-α plus IFN-γ). IF analyses showed an increase in Claudin-1 staining when LPS and S-layer were co-administered with respect to LPS alone; in addition, the S-layer counteracted the reduction of alkaline phosphatase detoxification activity and the enhancement of pro-inflammatory interleukin-8 release both induced by LPS. Altogether, these data corroborate a paraprobiotic role of S-layer from Lb ATCC® 15009™ as a possible candidate for therapeutic and prophylactic uses in conditions related to gastrointestinal health and correlated with extra-intestinal disorders.
Aims:
To characterize S-layer proteins produced by four lactobacilli isolated from Romanian artisan fermented products.
Methods and results:
Four lactobacilli strains have been shown to produce S-layer proteins, both under optimal and stressfull conditions. The presence of S-layer proteins was confirmed by transmission electron microscopy. Removal of S-layer proteins caused a loss of the bacterial resistance to stress conditions and of the autoaggregation ability. Liquid chromatography-mass spectrometry analysis identified peptides corresponding to Slp M sequence in case of Levilactobacillus brevis 403, and peptides corresponding to Slp A sequence in case of Lactobacillus helveticus 34.9. The analysis confirmed molecular masses of ~51 and 48 kDa, respectively, for the two proteins, and gave information about their pI, of about 9.4-9.6. A specific PCR amplification was obtained for the genome of Lact. helveticus 34.9 with slpA primers, and the amplicon sequence was 95.31% identical to slpA gene.
Conclusions:
Our findings indicate that certain environmental stress conditions can induce the S-layer production, which helps the producing cells to survive under unfavorable conditions.
This article describes the development of a novel liposome nanocarrier system. Carvacrol (Car) is first embedded in β-cyclodextrin (β-CD) by the freeze-drying method to form the β-cyclodextrin-carvacrol inclusion compound (β-CD–Car), and then β-CD–Car liposomes (β-CD–Car-LPs) and β-CD–Car liposomes coated with S-layer proteins (SLPs) from Lactobacillus buchneri 20023 (SLP/β-CD–Car-LPs) were prepared. The liposomes were characterized, and their stabilities, in vitro release characteristics, and antibacterial activities were investigated. Results showed that the fabricated liposome SLP/β-CD–Car-LPs was nanosized, oval and homogenous, with the particle size of 229.1± 6.81 nm, the polydispersity index of 0.139, and the zeta potential of 27.9 mV. Measurements based on Triton X-100 resistance indicated that the SLP-coated liposomes were more stable than naked liposomes. The in vitro release study results showed that the rate of release from SLP-coated liposomes was much lower than that from uncoated liposomes. The minimum inhibitory activity (MIC) of SLP/β-CD–Car-LPs (0.05 mg/ml) was 6.4 times higher than that of the free carvacrol (0.32 mg/ml) and was twice that of β-CD–Car-LPs (0.1 mg/ml). In general, the stability, antibacterial activity, and sustained release effect of β-CD–Car-LPs modified with SLPs were improved. Findings suggested that SLP-coated liposomes could be developed as a favorable delivery system for potential applications in the food industry.
The present study investigated whether the surface layer (S‐layer), which is known to have a varying effect from strain to strain on aggregation, adhesion ability, also has an effect on the resistance of bacteria to digestive enzymes, phenol, lysozymes. The effect of S‐layers on the resistance against various enzymes, aggregation and adhesion abilities, and strain specificity were determined of eight Lactiplantibacillus plantarum strains. Strains were treated with 5 M lithium chloride (LiCl) to extract the S‐layers, the presence of this layer in those microorganisms was demonstrated by polyacrylamide gel electrophoresis. Scanning electron microscopy was used to visualize the separation of the S‐layer, which surrounds the microorganism, from the microorganism by the LiCl. The images were taken three times, once at the beginning, once 30 min later, and once at the end of this process, which took 2 h in total. The effect against enzymes varied depending on the strain, but it was determined that all the tested strains had a serious loss of viability against phenol in the absence of an S‐layer. Lpb. plantarum DA100 showed a maximum decrease against gastrointestinal system enzymes after the LiCl (96.48 ± 0.03% before and 66.46 ± 0.01% after LiCl). Lpb. plantarum DA255 showed a significant decrease against lysozyme (99.11 ± 0.00% before and 62.80 ± 0.0% after LiCl). Removal of the S‐layer greatly affected the adhesion ability of some strains, while for others there was hardly any change. The results showed that the role of the S‐layer may be strain‐specific, the rate of effect can vary. The primary function of S‐layer proteins is thought to contribute to the adhesion ability of bacteria. There are limited studies that have reported the protective property of this layer against various enzymes, however, our results showed that S‐layer could be one of the resistance strategies developed by bacteria against enzymes.
In this study, the surface layer protein (SLP) from Lactobacillus kefiri HBA20 was characterized. The SLP was extracted by 5 M LiCl. The molecular mass of the SLP was approximately 64 kDa as analyzed via SDS-PAGE. The surface morphology and the adhesion potential of L. kefiri HBA20 in the absence and presence of SLP were measured by AFM. Moreover, the protein secondary structure was evaluated by using circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR), respectively. SLP had high β-sheet contents and low content of α-helix. Thermal analysis of SLP of Lactobacillus kefiri HBA20 exhibited one transition peak at 129.64 °C. Furthermore, SEM measurements were showed that after the SLP were removed from the cell surface, the coaggregation ability with Saccharomyces cerevisiae Y8 of the strain was significantly reduced. In conclusion, the SLP of Lactobacillus kefiri HBA20 has a stable structure and the ability of adhesion to yeast. Molecular docking study revealed that mannan bind with the hydrophobic residues of SLP. Our results will help further understanding of the new surface layer protein and the interaction between L. kefiri and S. cerevisiae.
Antimicrobial peptides that contribute to innate immunity are among the most important protective measures against infection in many organisms. Several substances are known to regulate the expression of antimicrobial peptides. In this study, we investigated the factors in lactic acid bacteria (LAB) that induce antimicrobial peptide expression in the host. We found that Lactobacillus helveticus SBT2171 (LH2171) induced the expression of human β-defensin (hBD)2 in Caco-2 human colonic epithelial cells. Specifically, surface layer protein (SLP) of LH2171 stimulated hBD2 expression by activating c-Jun N-terminal kinase (JNK) signaling via Toll-like receptor (TLR)2 in Caco-2 cells. SLPs extracted from other lactobacilli similarly increased hBD2 expression, suggesting that this stimulatory effect is common feature of Lactobacillus SLPs. Interestingly, Lactobacillus strains that strongly induced hBD2 expression also potently activated JNK signaling. Thus, upregulation of hBD2 induced by TLR2–JNK signaling contributes to protection of the host against infection.
Background:
Surface-layer proteins (SLP) have been found in the outermost layer of the cell wall in many kinds of lactobacillus, which is considered an important factor to intestinal immune.
Results:
This study compared the effects of SLP extracted by different concentrations of LiCl and carbamide, and then identified by SDS-PAGE, circular dichroism (CD)and differential scanning calorimetry (DSC). Furthermore, RAW 264.7 cells were used to evaluate the immuno-modulatory effects of SLP. SLP derived from L. acidophilus CICC6074 with a molecular weight of 46 kDa, which consisted of 16.9% α-helix, 42.3% β-sheet, 20.8% β-turns, and 22.5% random coils. SLP promoted NO secretion and that higher quantities of NO were produced as SLP concentrations increased. SLP concentrations over 50 µg/mL significantly decreased the amount of TNF-α secreted by RAW264.7 cells.
Conclusion:
SLP can trigger immuno-modulatory effects in RAW 264.7 cells. This will provide crucial information to enable the further use of L. acidophilus in food, medicine and other products.
The S-layer is a proteinaceous envelope constituted by subunits that self-assemble to form a two-dimensional lattice that covers the surface of different species of Bacteria and Archaea, and it could be involved in cell recognition of microbes among other several distinct functions. In this work, both proteomic and genomic approaches were used to gain knowledge about the sequences of the S-layer protein (SLPs) encoding genes expressed by six aggregative and sixteen non-aggregative strains of potentially probiotic Lactobacillus kefiri. Peptide mass fingerprint (PMF) analysis confirmed the identity of SLPs extracted from L. kefiri, and based on the homology with phylogenetically related species, primers located outside and inside the SLP-genes were employed to amplify genomic DNA. The O-glycosylation site SASSAS was found in all L. kefiri SLPs. Ten strains were selected for sequencing of the complete genes. The total length of the mature proteins varies from 492 to 576 amino acids, and all SLPs have a calculated pI between 9.37 and 9.60. The N-terminal region is relatively conserved and shows a high percentage of positively charged amino acids. Major differences among strains are found in the C-terminal region. Different groups could be distinguished regarding the mature SLPs and the similarities observed in the PMF spectra. Interestingly, SLPs of the aggregative strains are 100% homologous, although these strains were isolated from different kefir grains. This knowledge provides relevant data for better understanding of the mechanisms involved in SLPs functionality and could contribute to the development of products of biotechnological interest from potentially probiotic bacteria.
Surface layer (S-layer) glycoproteins have been described on many archaeobacteria. Among eubacteria their presence has only been reported for the Bacillaceae (for a review see Messner and Sleytr, 1992). Lactic bacteria often produce extracellular polymers such as slimes and capsules (for example those described by Nakajima et al., 1990 and Racine et al., 1991). Most of these polymers are polysaccharides but a few reports claim the presence of glycoproteins in lactic streptococci (Macura and Townsley, 1984) and lactobacilli (Garcia-Garibay and Marshall, 1991). These observations have encouraged us to extend our survey on glycosylated S-layer proteins to lactobacilli.
In this research, changes were observed in cell surface proteins of a typical strain of the Lactobacillus casei was investigated in response to acidic growth conditions. Two dimensional electrophoresis and Western blot analyses were carried out to detect changes in relative abundance of proteins at the cell surface. The identity of the differentially expressed proteins extracted by LiCl, a chemical routinely used to extract surface proteins of lactic acid bacteria, was determined by MALDI-TOF/TOF MS. Many enzymes involved in glycolysis were up-regulated in the cell surface fraction following growth at low pH, including enolase, lactate dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase. Several of these proteins were also related to adhesion and generalized stress responses. It is demonstrated that growth of L. casei under acidic conditions caused molecular changes at the cell surface to develop an adaptive strategy corresponding to slower growth at low pH.
Lactobacillus helveticus CNRZ 892 contains a surface layer (S-layer) composed of protein monomers of 43 kDa organized in regular arrays. The gene encoding this protein (slpH) has been cloned in Escherichia coli and sequenced. slpH consists of 440 codons and is preceded by a ribosome-binding site (RBS) and followed by a putative rho-independent terminator. Indeed, Northern analysis revealed that slpH is a monocistronic gene. The gene is preceded by a possible promotor of which the -35 and -10 hexanucleotides are separated by 17 nt. By primer extension analysis the transcription start site was mapped at 7 nt downstream of the -10 sequence while the deduced amino acid sequence of SlpH shows a leader peptide of 30 aa. The slpH gene has been amplified by PCR and the fragment, carrying the complete gene from the RBS to the stop codon, has been cloned in a lactococcal gene expression vector downstream of promoter P32. Lactococcus lactis MG1363 carrying the resulting plasmid produced and secreted an S-layer monomer with the same molecular mass as the authentic L. helveticus CNRZ 892 SlpH, as judged by SDS-PAGE. Immunoelectron microscopy revealed that SlpH was bound to the lactococcal cell walls in small clumps and accumulated in the growth medium as small sheets.
Comparative analysis of molecular sequence data is essential for reconstructing the evolutionary histories of species and inferring the nature and extent of selective forces shaping the evolution of genes and species. Here, we announce the release of Molecular Evolutionary Genetics Analysis version 5 (MEGA5), which is a user-friendly software for mining online databases, building sequence alignments and phylogenetic trees, and using methods of evolutionary bioinformatics in basic biology, biomedicine, and evolution. The newest addition in MEGA5 is a collection of maximum likelihood (ML) analyses for inferring evolutionary trees, selecting best-fit substitution models (nucleotide or amino acid), inferring ancestral states and sequences (along with probabilities), and estimating evolutionary rates site-by-site. In computer simulation analyses, ML tree inference algorithms in MEGA5 compared favorably with other software packages in terms of computational efficiency and the accuracy of the estimates of phylogenetic trees, substitution parameters, and rate variation among sites. The MEGA user interface has now been enhanced to be activity driven to make it easier for the use of both beginners and experienced scientists. This version of MEGA is intended for the Windows platform, and it has been configured for effective use on Mac OS X and Linux desktops. It is available free of charge from http://www.megasoftware.net.
Since the presence of S-layer protein conditioned the autoaggregation capacity of some strains of Lactobacillus kefir, S-layer proteins from aggregating and non-aggregating L. kefir strains were characterized by immunochemical reactivity, MALDI-TOF spectrometry and glycosylation analysis. Two anti-S-layer monoclonal antibodies (Mab5F8 and Mab1F8) were produced; in an indirect enzyme-linked immunosorbent assay Mab1F8 recognized S-layer proteins from all L. kefir tested while Mab5F8 recognized only S-layer proteins from aggregating strains. Periodic Acid-Schiff staining of proteins after polyacrylamide gel electrophoresis under denaturing conditions revealed that all L. kefir S-layer proteins tested were glycosylated. Growth of bacteria in the presence of the N-glycosylation inhibitor tunicamycin suggested the presence of glycosydic chains O-linked to the protein backbone. MALDI-TOF peptide map fingerprint for S-layer proteins from 12 L. kefir strains showed very similar patterns for the aggregating strains, different from those for the non-aggregating ones. No positive match with other protein spectra in MSDB Database was found. Our results revealed a high heterogeneity among S-layer proteins from different L. kefir strains but also suggested a correlation between the structure of these S-layer glycoproteins and the aggregation properties of whole bacterial cells.
Lactobacillus helveticus is a homofermentative thermophilic lactic acid bacterium that is used in the manufacture of Swiss type and long-ripened Italian
cheeses, such as Emmental, Grana, and Provolone cheeses. Substantial differences in several technologically important characteristics
are found among L. helveticus strains isolated from natural dairy starter cultures. In the present study we investigated the genotypic diversity of 74
strains isolated from different dairy cultures used for manufacturing Grana and Provolone cheeses and six collection strains.
A restriction fragment length polymorphism analysis of both total genomic DNA and the 16S rRNA gene (ribotyping) was used
as genotypic fingerprinting. A multivariate statistical analysis of the data enabled us to identify significant genotypic
heterogeneity inL. helveticus. We found that genotypic fingerprinting could be used to distinguish strains; in particular, it was possible to associate
the presence of specific strain genotypes with dairy ecosystem sources (e.g., Grana or Provolone cheese). Our data contribute
to the description of microbial heterogeneity in L. helveticus and provide a more solid basis for understanding the functional and ecological significance of the presence of differentL. helveticus biotypes in natural dairy starter cultures.
We have identified and sequenced the genes encoding the aggregation-promoting factor (APF) protein from six different strains
of Lactobacillus johnsonii and Lactobacillus gasseri. Both species harbor two apf genes, apf1 and apf2, which are in the same orientation and encode proteins of 257 to 326 amino acids. Multiple alignments of the deduced amino
acid sequences of these apf genes demonstrate a very strong sequence conservation of all of the genes with the exception of their central regions. Northern
blot analysis showed that both genes are transcribed, reaching their maximum expression during the exponential phase. Primer
extension analysis revealed that apf1 and apf2 harbor a putative promoter sequence that is conserved in all of the genes. Western blot analysis of the LiCl cell extracts
showed that APF proteins are located on the cell surface. Intact cells of L. johnsonii revealed the typical cell wall architecture of S-layer-carrying gram-positive eubacteria, which could be selectively removed
with LiCl treatment. In addition, the amino acid composition, physical properties, and genetic organization were found to
be quite similar to those of S-layer proteins. These results suggest that APF is a novel surface protein of the Lactobacillus acidophilus B-homology group which might belong to an S-layer-like family.
Competition, competitive exclusion and displacement of eight strains of Escherichia coli and Salmonella spp. by Lactobacillus rhamnosus GG and Lactobacillus casei Shirota from adhesion on human intestinal mucus glycoproteins and Caco-2 cell surfaces were studied. Lactobacilli were able to compete with, exclude and displace pathogenic gastrointestinal (GI) bacteria when they were incubated together, but the degree of inhibition of adhesion was bacterial strain-dependent. Competition and exclusion profiles of GI bacteria by lactobacilli were similar. Displacement profiles of GI bacteria were different from those of competition and exclusion and the process was relatively slow: displacement equilibrium took more than 2 h. These findings are important for development, selection and in vitro assessment of target- and function-specific probiotics.
In the present study we report for the first time the presence of S-layer proteins in Lactobacillus kefir and Lactobacillus parakefir isolated from kefir grains. Soluble whole-cell protein profile obtained either by mechanical disruption (X-press) or by a combined treatment with lysozyme and SDS on whole cells, showed a significant band of apparent molecular mass of 66-71 kDa as measured by SDS-PAGE. The intensity of this band was considerably reduced when cells were treated with 5 M-LiCl. The above mentioned proteins were recovered in the LiCl extracts. After dialysis and concentration, the proteins extracted were able to reassemble in a regular array. Negative staining of these protein preparations were analysed by transmission electron microscopy and a paracrystalline arrangement was seen. Thin sections of bacteria analysed by transmission electron micrographs showed an outermost layer over the bacterial cell wall, that was lost after the LiCl treatment. The production of this surface structure under different culture conditions was also evaluated. Finally, the relationship between the presence of S-layer proteins and surface properties (e.g. adhesion to Caco-2 cells, autoaggregation, and hemagglutination) was investigated.
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.
The S-layer-encoding genes of 21 Lactobacillus helveticus strains were characterized. Phylogenetic analysis based on the identified S-layer genes revealed two main clusters, one which includes a sequence similar to that of the slpH1 gene of L. helveticus CNRZ 892 and a second cluster which includes genes similar to that of prtY. These results were further confirmed by Southern blot hybridization. This study demonstrates S-layer gene variability in the species L. helveticus. Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Thirty-eight isolates of Lactobacillus gallinarum cultured from the crops of broiler chickens were screened for the presence of genes encoding S-layer proteins. All of the
isolates had two S-protein genes, which were designated Lactobacillus gallinarum S-protein (lgs) genes. One gene in each isolate was either lgsA or lgsB. The Lactobacillus isolates were further characterized by pulsed-field gel electrophoresis of DNA digests, which grouped the isolates into 17
genotypes (strains). The second gene in each of eight representative strains was sequenced and shown to differ among strains
(lgsC, lgsD, lgsE, lgsF, lgsG, lgsH, and lgsI). The genome of each strain thus encoded a common S-protein (encoded by either lgsA or lgsB) and a strain-specific S-protein. The extraction of cell surface proteins from cultures of the eight strains showed that
each strain produced a single S-protein that was always encoded by the strain-specific lgs gene. Two of the strains were used to inoculate chickens maintained in a protected environment which were Lactobacillus-free prior to inoculation. DNAs and RNAs extracted from the digesta of the chickens were used for PCR and reverse transcription-PCR,
respectively, to demonstrate the presence and transcription of lgs genes in vivo. In both cases, only the strain-specific gene was transcribed. Both of the strains adhered to the crop epithelium,
consistent with published data predicting that S-proteins of lactobacilli are adhesins. The results of this study provide
a basis for the investigation of gene duplication and sequence variation as mechanisms by which bacterial strains of the same
species can share the same habitat.
Strain R0052, isolated from a North American dairy starter culture, was initially identified as Lactobacillus acidophilus based on phenotypic analyses. However, upon sequencing the 16S rRNA gene, it became clear that the isolate was very highly related to Lactobacillus suntoryeus, Lactobacillus helveticus and Lactobacillus gallinarum, as similarities ranging from 99.3 to 99.8 % were observed. As an initial screening test to investigate the relatedness of strain R0052 and reference strains of L. suntoryeus, L. helveticus and L. gallinarum, the partial sequences for the genes encoding the alpha subunit of ATP synthase (atpA), RNA polymerase alpha subunit (rpoA), phenylalanyl-tRNA synthase alpha subunit (pheS), the translational elongation factor Tu (tuf), a surface-layer protein (slp) and the Hsp60 chaperonins (groEL) were determined and they revealed high relatedness between all of the strains. The determination of the 16S-23S rRNA internally transcribed spacer (ITS) sequences revealed 98.3-100% similarity between L. suntoryeus and L. helveticus strains. SDS-PAGE of whole-cell proteins did not distinguish between these species. Fluorescent amplified fragment length polymorphism (FAFLP) could distinguish between these taxa, but they still constituted a single cluster within the L. acidophilus group. Finally, DNA-DNA hybridization experiments between strain R0052 and the type strains of L. helveticus and L. suntoryeus yielded reassociation values above 70% and confirmed that these names are synonyms.
The interest of probiotics as remedy for a broad number of infectious diseases has gained wide interest over the last few years, but little is known about their underlying mechanism of action. In this study, five selected Lactobacillus isolated from human intestinal and ferment milk were preliminarily identified by 16S rDNA gene sequencing and assessed the ability to inhibit the adhesion of enteropathogens using HT-29 cells model through a process which may be related to specific components of the bacterial surface. Surface layer proteins are located in a paracrystalline layer outside the bacterial cell wall and are thought to play a role in tissue adherence. Removal of S-layer proteins from the Lactobacillus (treated with 5 M LiCl) reduced inhibition activity as revealed in exclusion, competition and displacement assays, which suggested that S-layer proteins had involved in the adhesion of probiotics. SDS-PAGE analysis confirmed the presence of S-layer proteins with dominant band which was approximately 60 kDa. Further analysis of S-layer proteins revealed that the hydrophobic amino acids accounted for 43.2 % of the total amino acid for the Lactobacillus paracasei M7. So, these probiotics could be used in the health-promoting food products, which could prevent the diarrhea caused by pathogens.
In a previous study, electron microscopic examinations of thin sections of Lactobacillus helveticus ATCC 12046 revealed a three-layered structure of the cell wall. The outermost component was identified as a layer of a non-glycosylated 52 kDa protein. Freeze-etched preparations of intact cells have now demonstrated that this protein layer is an oblique surface layer (S-layer) lattice (a = 4.5nm, b = 9.6nm, y = 77°) which completely covers the cell surface. Treatment with 5 M-LiCl extracted the S-layer protein from intact cells efficiently and selectively. Viability did not decrease significantly. Moreover, the S-layer reappeared when treated cells were allowed to grow again. In vitro self-assembly products obtained upon aggregation of isolated S-layer subunits exhibited the same oblique S-layer symmetry as observed on intact cells in vivo. The purified S-layer protein had a high content (44%) of hydrophobic amino acids. The N-terminal sequence was mainly composed of alanine, threonine, asparagine and aspartic acid.
The presence of regular arrays (RAs) in the cell walls of strains of the genus Lactobacillus was examined by electron microscopy. The RAs were found in 6 species including L. bulgaricus, L. helveticus, L. acidophilus, L. fermentum, L. brevis and L. buchneri. The RAs were composed of a protein with an apparent Mr ranging from about 41000 to 55000, depending on the species upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amino acid composition of the RA proteins was shown to be acidic and hydrophobic. The antigenicity of the RA protein from L. buchneri appeared to be specific but not common among the RA proteins from the other lactobacilli.
The paper describes two methods that allow rapid and accurate identification of Lactobacillus helveticus strains based on the nucleotide sequence of the gene coding for the surface layer (S-layer) protein. The first method is based on a polymerase chain reaction amplification using primers targeting a specific fragment of the S-layer gene. The second method involved a portion of the S-layer gene of L. helveticus as a probe in a hybridisation test. The specificity of these protocols was checked for DNA samples isolated from various Lactobacillus strains.
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.
Forty-two Lactobacillus helveticus strains isolated from two whey starter cultures and three cheeses were typed by multilocus restriction typing (MLRT), analyzing the restriction fragment-length polymorphism of PCR products generated from eight loci of housekeeping genes: β-galactosidase, lactose permease, UDP-galactose-4 epimerase, β-galactosidase transcriptional regulator, D-lactate dehydrogenase, peptidase N, stress-inducible protein and S-layer protein. MLRT analysis indicated wide strain heterogeneity. Strains were grouped according to their source of isolation. The low intra-species polymorphism detected in the 16S rRNA gene did not allow grouping of the strains with the same sensitivity reached by MLRT of protein-coding genes. This is the first demonstration of MLRT as a technique for the discrimination of Lb. helveticus strains; this technique is a promising tool to evaluate genetic diversity of related strains from different ecological niches.
The strain Lactobacillus helveticus DPC4571 has emerged as a promising flavor adjunct culture for Cheddar cheese given that it is consistently associated with improved flavor. The availability of the complete genome sequence of Lb. helveticus DPC4571 has enabled the search for the presence or absence of specific genes on the genome, in particular those of technological interest. Indeed, this analysis has facilitated a greater understanding into the functioning of lactic acid bacteria as a whole. The biochemical pathways of Lb. helveticus responsible for producing flavor compounds during cheese ripening are poorly understood but now with the availability of a complete genomic sequence are ripe for exploitation. Bioinformatic analysis of the genome of Lb. helveticus DPC4571 has revealed a plethora of genes with industrial potential including those responsible for key metabolic functions that contribute to cheese flavor development such as proteolysis, lipolysis, and cell lysis. In addition, it has been demonstrated that Lb. helveticus has the potential to produce bioactive peptides such as angiotensin converting enzyme inhibitory activity in fermented dairy products, demonstrating the therapeutic value of this species. A most intriguing feature of the genome of Lb. helveticus DPC4571 is the remarkable similarity in gene content with many intestinal lactobacilli, although originating from considerably different environments. Bioinformatic analysis demonstrated that 65 to 75% of genes were conserved between the commensal and dairy lactobacilli, which allowed key niche-specific gene sets to be described. This review focuses on the isolation, characterization, and exploitation of the Lb. helveticus species with particular emphesis on taking into consideration recent genome sequence data for Lb. helveticus and other Lactobacillus species.
The surface properties of lactobacilli are of significant technological importance as they determine the interaction of the bacterial cells with the gastrointestinal mucosa, and therefore influence their location in the gut and their functionality. Studying the surface of the bacteria is critical for understanding the adhesion process better. This review compiles the knowledge from studies on the characterization Lactobacillus surfaces and evaluates the potential relationship between the cells' physicochemical characteristics and their adhesive abilities. It also discusses the effect that the production processes, such as fermentation and drying, can exert on the surface properties and adhesion abilities of lactobacilli.
The most-dominant surface-exposed protein in many bacterial species is the S-protein. This protein crystallises into a regular monolayer on the outside surface of the bacteria: the S-layer. Lactobacillus acidophilus harbours two S-protein-encoding genes, slpA and slpB, only one of which (slpA) is expressed. In this study, we show by polymerase chain reaction (PCR) analysis that slpA and slpB are located on a 6 kb chromosomal segment, in opposite orientations. In a small fraction of the bacterial population, this segment is inverted. The inversion reads to interchanging of the expressed and silent S-protein-encoding genes, and places the formerly silent gene behind the S-promoter which is located outside the inverted segment. A 26 bp sequence showing a high degree of similarity with the consensus sequence recognized by the Din family of invertases is present in the region where recombination occurs. Expression of the slpA gene seems to be favoured under laboratory growth conditions because 99.7% of the chromosomes of an L. acidophilus ATCC 4356 broth culture had the slpA gene present at the sip expression site.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
An S-layer (surface regular array) was found in the cell wall from six out of ten strains of Lactobacillus acidophilus examined by electron microscopic observations. All of the six strains which were shown to carry the S-layers belonged to the deoxyribonucleic acid (DNA) homology group A, but not to B, which had been classified by Johnson et al (Int. J. Syst. Bacteriol. 30: 53-68, 1980). On the other hand, the other four strains which possessed no S-layers were in the homology group B. The apparent molecular weights of the S-layer proteins ranged from 41 to 49 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of the S-layer proteins from the six strains, three were susceptible to chemical cleavage with N-chlorosuccinimide, giving different peptide maps. All of the six S-layer proteins were fragmented by limited proteolysis with Staphylococcus aureus V8 protease, and gave markedly different peptide patterns by the subsequent peptide mapping analysis, except that the peptide maps of the S-layer proteins from the two strains which were in the same subgroup were identical.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
The presence of S-layer proteins in Lactobacillus brevis was examined by SDS-PAGE analysis. Thirty six out of a total of 41 L. brevis strains possessed S-layer proteins of molecular masses ranging from 38 to 55 kDa. Western blot analysis using antisera raised against whole cells of S-layer protein-carrying strains demonstrated the heterogeneity of L. brevis S-layer proteins. No clear relationship was observed between the presence of S-layer proteins or their immunological characteristics and the physiological activity of L. brevis as a beer spoilage organism.
The most-dominant surface-exposed protein in many bacterial species is the S-protein. This protein crystallises into a regular monolayer on the outside surface of the bacteria: the S-layer. Lactobacillus acidophilus harbours two S-protein-encoding genes, slpA and slpB, only one of which (slpA) is expressed. In this study, we show by polymerase chain reaction (PCR) analysis that slpA and slpB are located on a 6 kb chromosomal segment, in opposite or orientations. In a small fraction of the bacterial population, this segment is inverted. The inversion leads to interchanging of the expressed and silent S-protein-encoding genes, and places the formerly silent gene behind the S-promoter which is located outside the inverted segment. A 26 bp sequence showing a high degree of similarity with the consensus sequence recognized by the Din family of invertases is present in the region where recombination occurs. Expression of the slpA gene seems to be favoured under laboratory growth conditions because 99.7% of the chromosomes of an L. acidophilus ATCC 4356 broth culture had the slpA gene present at the slp expression site.
The paper describes two methods that allow rapid and accurate identification of Lactobacillus helveticus strains based on the nucleotide sequence of the gene coding for the surface layer (S-layer) protein. The first method is based on a polymerase chain reaction amplification using primers targeting a specific fragment of the S-layer gene. The second method involved a portion of the S-layer gene of L. helveticus as a probe in a hybridisation test. The specificity of these protocols was checked for DNA samples isolated from various Lactobacillus strains.
Surface (S-) layers are crystalline arrays of proteinaceous subunits present as the outermost component of cell wall in several species of the genus Lactobacillus, as well as in many other bacteria and Archaea. Despite the high similarity of the amino acid composition of all known S-layer proteins, the overall sequence similarity is, however, surprisingly small even between the Lactobacillus S-layer proteins. In addition, the typical characteristics of Lactobacillus S-layer proteins, distinguishing them from other S-layer proteins, are small size and high-predicted pI value. Several lactobacilli possess multiple S-layer protein genes, which can be differentially or simultaneously expressed. To date, the characterized functions of Lactobacillus S-layers are involved in mediating adhesion to different host tissues. A few applications for the S-layer proteins of lactobacilli already exist, including their use as antigen delivery vehicles.
Adherence of intestinal pathogens, including Escherichia coli O157:H7, to human intestinal epithelial cells is a key step in pathogenesis. Probiotic bacteria, including Lactobacillus helveticus R0052 inhibit the adhesion of E. coli O157:H7 to epithelial cells, a process which may be related to specific components of the bacterial surface. Surface-layer proteins (Slps) are located in a paracrystalline layer outside the bacterial cell wall and are thought to play a role in tissue adherence. However, the ability of S-layer protein extract derived from probiotic bacteria to block adherence of enteric pathogens has not been investigated. Human epithelial (HEp-2 and T84) cells were treated with S-layer protein extract alone, infected with E. coli O157:H7, or pretreated with S-layer protein extract prior to infection to determine their importance in the inhibition of pathogen adherence. The effects of S-layer protein extracts were characterized by phase-contrast and immunofluorescence microscopy and measurement of the transepithelial electrical resistance of polarized monolayers. Pre-treatment of host epithelial cells with S-layer protein extracts prior to E. coli O157:H7 infection decreased pathogen adherence and attaching-effacing lesions in addition to preserving the barrier function of monolayers. These in vitro studies indicate that a non-viable constituent derived from a probiotic strain may prove effective in interrupting the infectious process of an intestinal pathogen.
Proteolytic activities were extracted from a dairy Lactobacillus helveticus strain and partially characterized. A first cell envelope proteinase (CEP) was extracted using a high ionic strength buffer, both in the presence and in the absence of Ca2+. Moreover, cell treatment by 5 M LiCl allowed for the selective removal of the S-layer protein and CEP, suggesting an enzyme ionic linkage to the cell envelope similar to that observed for the Slayer structure. The enzyme specificity against alpha(s1)-CN (f1-23) showed unusual activity on the Lys3-His4 bond compared with other proteinases of the same species. A second proteinase appeared to be linked to the cell membrane because it was extractable only after membrane disgregation by detergents. Its specificity against CN fractions and alpha(s1)-CN (f1-23) was different from that of the first CEP; moreover, the measured activity was lower than that of CEP.
Character-ization of the S-layer glycoproteins of two lactobacilli Advances in bacterial paracrystalline surface layers
- A Möschl
- C Schӓffer
- Sleytr Ub
- Kristian P R Messner
- Schulz
Möschl A, Schӓffer C, Sleytr UB, Messner P, Kristian R, Schulz G. Character-ization of the S-layer glycoproteins of two lactobacilli. In: Beveridge TJ, Koval SF, editors. Advances in bacterial paracrystalline surface layers. New York: Plenum Press; 1993. pp. 281e4.
PCR detection of slp genes. Lane: M: molecular weight marker; 1: Lb. helveticus 80
- Fig
Fig. 5. PCR detection of slp genes. Lane: M: molecular weight marker; 1: Lb. helveticus
80; 2: Lb. helveticus T104; 3: Lb. helveticus T105; 4: Lb. helveticus T159; 5: Lb. helveticus
T141; 6: Lb. helveticus B734; 7: Lb. helveticus T80.
Characterization of the S-layer glycoproteins of two lactobacilli
- A Möschl
- C Schӓffer
- U B Sleytr
- P Messner
- R Kristian
- G Schulz
Möschl A, Schӓffer C, Sleytr UB, Messner P, Kristian R, Schulz G. Characterization of the S-layer glycoproteins of two lactobacilli. In: Beveridge TJ,
Koval SF, editors. Advances in bacterial paracrystalline surface layers. New
York: Plenum Press; 1993. pp. 281e4.