RESUMEN
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.
Asunto(s)
Proteínas Bacterianas , Glicoproteínas de Membrana , Proteínas Bacterianas/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/metabolismo , Lactobacillaceae/metabolismoRESUMEN
S-layers are bacterial structures present on the surface of several Gram-positive and Gram-negative bacteria that play a role in bacterial protection. In Lactobacillus acidophilus (L. acidophilus ATCC 4356), the S-layer is mainly composed of the protein SlpA. A tandem of two copies of the protein domain SLP-A (pfam: 03217) was identified at the C-terminal of SlpA, being this double SLP-A protein domain (in short dSLP-A) necessary and sufficient for the association of the protein to the L. acidophilus cell wall. A variety of proteins fused to the dSLP-A domain were able to spontaneously associate with high affinity to the cell wall of L. acidophilus and Bacillus subtilis var. natto, in a process that we termed decoration. Binding of dSLP-A-containing-proteins to L. acidophilus was stable at conditions that mimic the gastrointestinal transit in terms of pH, proteases, and bile salts. To evaluate if protein decoration of L. acidophilus can be adapted to generate an oral vaccine platform, a chimeric antigen derived from the bacterial pathogen Shiga-toxin-producing Escherichia coli (STEC) was constructed by fusing the sequences encoding the polypeptides EspA36-192, Intimin653-953, Tir240-378, and H7 flagellin352-374 (EITH7) to the dSLP-A domain (EITH7-dSLP-A). Recombinantly expressed EITH7-dSLP-A protein was affinity purified and combined with L. acidophilus cultures to allow the association of the chimeric antigen to the bacterial surface. EITH7-decorated L. acidophilus was orally administered to BALB/c mice and the induction of anti-EITH7 specific antibodies in sera and feces determined by ELISA. Mice presenting significantly higher anti-EITH7 antibodies titers were able to control more efficiently an experimental STEC infection than mice that received the non-decorated L. acidophilus carrier, indicating that antigen-decorated L. acidophilus can be adapted as a mucosal immunization delivery platform to elicit a protective immune response for vaccine purposes.
RESUMEN
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.
Asunto(s)
Membrana Celular/química , Lactobacillales/metabolismo , Lactobacillus acidophilus/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Pared Celular/química , Clonación Molecular , Medios de Cultivo/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Viabilidad Microbiana , Microscopía Electrónica de Transmisión , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Lactobacillus acidophilus/metabolismo , Lectinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Adhesión Bacteriana , Proteínas Bacterianas/genética , Sitios de Unión , Proteínas Fluorescentes Verdes/genética , Glicoproteínas de Membrana/aislamiento & purificación , Unión ProteicaRESUMEN
Lactobacillus helveticus is a lactic acid bacterium used traditionally in the dairy industry, especially in the manufacture of cheeses. We present here the 2,141,841-bp draft genome sequence of L. helveticus strain ATCC 12046, a potential starter strain for improving cheese production.
RESUMEN
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.
Asunto(s)
Expresión Génica , Lactobacillus acidophilus/genética , Lactobacillus acidophilus/metabolismo , Glicoproteínas de Membrana/metabolismo , Presión Osmótica , Lactobacillus acidophilus/efectos de los fármacos , Cloruro de Sodio/metabolismoRESUMEN
We present the 1,956,699-bp draft genome sequence of Lactobacillus acidophilus strain ATCC 4356. Comparative genomic analysis revealed 99.96% similarity with L. acidophilus NCFM NC_006814.3 and 99.97% with La-14 NC_021181.2 genomes.
RESUMEN
We previously observed that Bacillus subtilis spores from sspE mutants presented a lower germination capacity in media containing high salt concentrations (0.9 M NaCl). This deficiency was attributed to the absence of SASP-E (gamma-type small-acid-soluble protein), rich in osmocompatible amino acids released by degradation. Herein we observed that, in addition, this mutant spore presented a reduced capacity to use L-alanine as germinant (L-ala pathway), required longer times to germinate in calcium dipicolinate (Ca(2+)-DPA), but germinated well in asparagine, glucose, fructose, and potassium chloride (AGFK pathway). Moreover, mild sonic treatment of mutant spores partially recovered their germination capacity in L-ala. Spore qualities were also altered, since sporulating colonies from the sspE mutant showed a pale brownish color, a higher adherence to agar plates, and lower autofluorescence, properties related to their spore coat content. Furthermore, biochemical analysis showed a reduced partition in hexadecane and a higher content of Ca(2+)-DPA when compared with its isogenic wild-type control. Coat protein preparations showed a different electrophoretic pattern, in particular when detected with antibodies against CotG and CotE. The complementation with a wild-type sspE gene in a plasmid allowed for recovering the wild-type coat phenotype. This is the first report of a direct involvement of SASP-E in the spore coat assembly during the differentiation program of sporulation.
Asunto(s)
Bacillus subtilis/fisiología , Proteínas Bacterianas/metabolismo , Multimerización de Proteína , Esporas Bacterianas/fisiología , Bacillus subtilis/crecimiento & desarrollo , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Eliminación de Gen , Prueba de Complementación Genética , Esporas Bacterianas/crecimiento & desarrollo , Esporas Bacterianas/metabolismoRESUMEN
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.
Asunto(s)
Antivirales/farmacología , Proteínas Bacterianas/farmacología , Virus Junin/efectos de los fármacos , Lactobacillus acidophilus , Glicoproteínas de Membrana/farmacología , Internalización del Virus/efectos de los fármacos , Células 3T3 , Secuencia de Aminoácidos , Animales , Antivirales/química , Antivirales/aislamiento & purificación , Infecciones por Arenaviridae , Proteínas Bacterianas/aislamiento & purificación , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Chlorocebus aethiops , Secuencia de Consenso , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/aislamiento & purificación , Ratones , Datos de Secuencia Molecular , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Células VeroRESUMEN
Bacillus species have been involved in metal association as biosorbents, but there is not a clear understanding of this chelating property. In order to evaluate this metal chelating capacity, cultures and spores from Grampositive bacteria of species either able or unable to produce surface layer proteins (S-layers) were analyzed for their capacity of copper biosorption. Only those endowed of S-layers, like Bacillus sphaericus and B. thuringiensis, showed a significant biosorption capacity. This capacity (nearly 50%) was retained after heating of cultures, thus supporting that structural elements of the envelopes are responsible for such activity. Purified Slayers from two Bacillus sphaericus strains had the ability to biosorb copper. Copper biosorption parameters were determined for strain B. sphaericus 2362, and after analyses by means of the Langmuir model, the affinity and capacity were shown to be comparable to other bacterial biosorbents. A competitive effect of Ca2+ and Zn2+, but not of Cd2+, was also observed, thus indicating that other cations may be biosorbed by this protein. Spores that have been shown to be proficient for copper biosorption were further analyzed for the presence of Slayer content. The retention of S-layers by these spores was clearly observed, and after extensive treatment to eliminate the S-layers, the biosorption capacity of these spores was significantly reduced. For the first time, a direct correlation between S-layer protein content and metal biosorption capacity is shown. This capacity is linked to the retention of S-layer proteins attached to Bacillus spores and cells.
Asunto(s)
Bacillus/metabolismo , Glicoproteínas de Membrana/metabolismo , Metales/metabolismo , Cationes Bivalentes/metabolismo , Quelantes/metabolismo , Unión Proteica , Esporas Bacterianas/metabolismoRESUMEN
We here describe a new method for electroporation of Lactobacillus species, obligately homofermentative and facultatively heterofermentative, based on the cell-wall weakening resulting from growth in high-salt media. For L. casei, optimum transformation efficiency of up to 10(5) transformants per microgram of plasmid DNA was achieved following growth in the presence of 0.9 M NaCl. Plasmids of different sizes and replication origins were also similarly transformed. These competent cells could be used either directly or stored frozen, up to 1 month, for future use, with similar efficiency. This protocol was assayed with different Lactobacillus species: L. delbrueckii subsp. lactis, L. paracasei, L. plantarum and L. acidophilus, and it was found that they were transformed with similar efficiency.
Asunto(s)
Medios de Cultivo/química , Electroporación/métodos , Lactobacillus/genética , Sales (Química)/metabolismo , Criopreservación/métodos , Lactobacillus/crecimiento & desarrollo , Viabilidad Microbiana , PlásmidosRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Conservantes de Alimentos/farmacología , Lactobacillus acidophilus/enzimología , N-Acetil Muramoil-L-Alanina Amidasa/farmacología , Nisina/farmacología , Bacillus cereus/efectos de los fármacos , Bacillus cereus/crecimiento & desarrollo , Membrana Celular/efectos de los fármacos , Pared Celular/efectos de los fármacos , Recuento de Colonia Microbiana , Sinergismo Farmacológico , Microbiología de Alimentos , Genes Bacterianos/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/crecimiento & desarrollo , Calor , Pruebas de Sensibilidad Microbiana , Permeabilidad , Polilisina/farmacología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Tensoactivos/farmacologíaRESUMEN
The study was focused on the role of the penicillin binding protein PBP4* of Bacillus subtilis during growth in high salinity rich media. Using pbpE-lacZ fusion, we found that transcription of the pbpE gene is induced in stationary phase and by increased salinity. This increase was also corroborated at the translation level for PBP4* by western blot. Furthermore, we showed that a strain harboring gene disruption in the structural gene (pbpE) for the PBP4* endopeptidase resulted in a salt-sensitive phenotype and increased sensitivity to cell envelope active antibiotics (vancomycin, penicillin and bacitracin). Since the pbpE gene seems to be part of a two-gene operon with racX, a racX::pRV300 mutant was obtained. This mutant behaved like the wild-type strain with respect to high salt. Electron microscopy showed that high salt and mutation of pbpE resulted in cell wall defects. Whole cells or purified peptidoglycan from WT cultures grown in high salt medium showed increased autolysis and susceptibility to mutanolysin. We demonstrate through zymogram analysis that PBP4* has murein hydrolyze activity. All these results support the hypothesis that peptidoglycan is modified in response to high salt and that PBP4* contributes to this modification.
Asunto(s)
Bacillus subtilis/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/fisiología , Proteínas de Unión a las Penicilinas/fisiología , Salinidad , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/fisiología , Antibacterianos/farmacología , Bacillus subtilis/crecimiento & desarrollo , Bacillus subtilis/ultraestructura , Bacitracina/farmacología , Bacteriólisis , Pared Celular/efectos de los fármacos , Pared Celular/ultraestructura , Microscopía Electrónica de Transmisión , N-Acetil Muramoil-L-Alanina Amidasa/deficiencia , Penicilina G/farmacología , Proteínas de Unión a las Penicilinas/deficiencia , Peptidoglicano/metabolismo , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/deficiencia , Transcripción Genética , Vancomicina/farmacologíaRESUMEN
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.
Asunto(s)
Lactobacillus acidophilus/enzimología , Glicoproteínas de Membrana/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Secuencia de Aminoácidos , Bacillus subtilis/enzimología , Pared Celular/metabolismo , Clonación Molecular , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
The role of glutamate as osmoprotector was investigated through the study of a mutation in its biosynthetic pathway. A glt::Tn917-lacZ-cat insertion mutant (N1) conferring glutamate auxotrophy and enhanced beta-galactosidase expression on high-salt media was selected. Co-transformation experiments and PCR analysis allowed locating the insertion into the gltB gene corresponding to the small unit of the glutamate synthase (GOGAT). The N1 mutant strain presented a glutamate requirement for growth and a tenfold decrease in GOGAT activity. Transcriptional activity of GOGAT, measured as beta-galactosidase from the transposon fusion, correlated with enzymatic activity; expression was enhanced at the stationary phase and in high-ionic-strength media. However, osmotolerance of cultures of N1 mutant were as wild-type (wt), at least in semi-rich medium. In contrast, sporulation was slightly reduced (75% of wt), and spores were less resistant to UV, heat, and osmolarity, properties linked to the content of small, acid-soluble proteins (SASP). The content of these proteins was, in fact, reduced, in particular the SASP-gamma type. The peptidoglycan-cortex, however, was not impaired since spores maintained lysozyme resistance. Addition of glutamate during sporulation partially rescued spore resistance, but germination and outgrowth remained impaired. Deficiencies in germination and outgrowth were also observed with spores from a gltA mutant strain. Taken together, these results pointed to the importance of GOGAT activity during sporulation, in particular for the synthesis SASPs.