CRISPR/Cas9-mediated genomic insertion of functional genes into WCFS1.

Unlabelled: a natural inhabitant of the human body, is a promising candidate vehicle for vaccine delivery. An obstacle in developing bacterial delivery vehicles is generating a production strain that lacks antibiotic resistance genes and contains minimal foreign DNA. To deal with this obstacle, we have constructed a finetuned, inducible two-plasmid CRISPR/Cas9-system for chromosomal gene insertion in .

Antigen surface display in two novel whole genome sequenced food grade strains, Lactiplantibacillus pentosus KW1 and KW2.

Background: Utilization of commensal bacteria for delivery of medicinal proteins, such as vaccine antigens, is an emerging strategy. Here, we describe two novel food-grade strains of lactic acid bacteria, Lactiplantibacillus pentosus KW1 and KW2, as well as newly developed tools for using this relatively unexplored but promising bacterial species for production and surface-display of heterologous proteins.

Results: Whole genome sequencing was performed to investigate genomic features of both strains and to identify native proteins enabling surface display of heterologous proteins.

Lactiplantibacillus plantarum as a novel platform for production and purification of integral membrane proteins using RseP as the benchmark.

The present study describes a detailed procedure for expressing and purifying the integral membrane protein RseP using the pSIP system and Lactiplantibacillus plantarum as an expression host. RseP is a membrane-bound site-2-protease and a known antibacterial target in multiple human pathogens. In the present study, we screened five RseP orthologs from Gram-positive bacteria and found RseP from Enterococcus faecium (EfmRseP) to yield the highest protein levels.

The role of site-2-proteases in bacteria: a review on physiology, virulence, and therapeutic potential.

Site-2-proteases are a class of intramembrane proteases involved in regulated intramembrane proteolysis. Regulated intramembrane proteolysis is a highly conserved signaling mechanism that commonly involves sequential digestion of an anti-sigma factor by a site-1- and site-2-protease in response to external stimuli, resulting in an adaptive transcriptional response. Variation of this signaling cascade continues to emerge as the role of site-2-proteases in bacteria continues to be explored.

The extracellular domain of site-2-metalloprotease RseP is important for sensitivity to bacteriocin EntK1.

Enterocin K1 (EntK1), a bacteriocin that is highly potent against vancomycin-resistant enterococci, depends on binding to an intramembrane protease of the site-2 protease family, RseP, for its antimicrobial activity. RseP is highly conserved in both EntK1-sensitive and EntK1-insensitive bacteria, and the molecular mechanisms underlying the interaction between RseP and EntK1 and bacteriocin sensitivity are unknown. Here, we describe a mutational study of RseP from EntK1-sensitive Enterococcus faecium to identify regions of RseP involved in bacteriocin binding and activity.

Comparison of the Immunogenic Properties of Carrying the Mycobacterial Ag85B-ESAT-6 Antigen at Various Cellular Localizations.

The bacille Calmette-Guèrin (BCG) vaccine has been used for a century; nonetheless, tuberculosis (TB) remains one of the deadliest diseases in the world. Thus, new approaches to developing a new, more efficient vaccine are desirable. Mucosal vaccines are of particular interest, considering that first enters the body through the mucosal membranes.

Heterologous Protein Production in Lactobacillus (plantarum) Using pSIP Vectors.

While lactobacilli are not generally regarded as efficient cell factories for heterologous proteins, these food-grade Gram-positive bacteria are attractive as expression hosts for medicinal proteins. Furthermore, tools have been developed not only to secrete the protein of interest, but also to anchor the protein to the cell membrane or the cell wall. Research efforts aimed at the production and surface display of complex vaccine proteins have shown that lactobacilli are capable of producing heterologous proteins that are otherwise difficult to produce in soluble form.

The Fish Pathogen Aliivibrio salmonicida LFI1238 Can Degrade and Metabolize Chitin despite Gene Disruption in the Chitinolytic Pathway.

The fish pathogen () LFI1238 is thought to be incapable of utilizing chitin as a nutrient source, since approximately half of the genes representing the chitinolytic pathway are disrupted by insertion sequences. In the present study, we combined a broad set of analytical methods to investigate this hypothesis. Cultivation studies revealed that grew efficiently on acetylglucosamine (GlcNAc) and chitobiose [(GlcNAc)], the primary soluble products resulting from enzymatic chitin hydrolysis.

A bacteriocin-based treatment option for Staphylococcus haemolyticus biofilms.

Bacteriocins are ribosomally-synthesized antimicrobial peptides, showing great potential as novel treatment options for multidrug-resistant pathogens. In this study, we designed a novel hybrid bacteriocin, Hybrid 1 (H1), by combing the N-terminal part and the C-terminal part of the related bacteriocins enterocin K1 (K1) and enterocin EJ97 (EJ97), respectively. Like the parental bacteriocins, H1 used the membrane-bound protease RseP as receptor, however, it differed from the others in the inhibition spectrum.

Efficient Secretion and Recombinant Production of a Lactobacillal α-amylase in WCFS1: Analysis and Comparison of the Secretion Using Different Signal Peptides.

Lactic acid bacteria (LAB) have been used as starter cultures and producers of enzymes, antimicrobial peptides or metabolites that contribute to the flavor, texture and safety of food products. , one of the best-studied LAB, is considered as safe and effective cell factory for food applications. In this study, our aim was to use as the producer for high levels of a food-grade lactobacillal α-amylase, which has potential applications in food, fermentation and feed industries.

Quantitative comparison of the biomass-degrading enzyme repertoires of five filamentous fungi.

The efficiency of microorganisms to degrade lignified plants is of great importance in the Earth's carbon cycle, but also in industrial biorefinery processes, such as for biofuel production. Here, we present a large-scale proteomics approach to investigate and compare the enzymatic response of five filamentous fungi when grown on five very different substrates: grass (sugarcane bagasse), hardwood (birch), softwood (spruce), cellulose and glucose. The five fungi included the ascomycetes Aspergillus terreus, Trichoderma reesei, Myceliophthora thermophila, Neurospora crassa and the white-rot basidiomycete Phanerochaete chrysosporium, all expressing a diverse repertoire of enzymes.

Anchoring of heterologous proteins in multiple Lactobacillus species using anchors derived from Lactobacillus plantarum.

Members of the genus Lactobacillus have a long history in food applications and are considered as promising and safe hosts for delivery of medically interesting proteins. We have assessed multiple surface anchors derived from Lactobacillus plantarum for protein surface display in multiple Lactobacillus species, using a Mycobacterium tuberculosis hybrid antigen as test protein. The anchors tested were a lipoprotein anchor and two cell wall anchors, one non-covalent (LysM domain) and one covalent (sortase-based anchoring using the LPXTG motif).

Cell Wall Anchoring of a Bacterial Chitosanase in Using a Food-Grade Expression System and Two Versions of an LP TG Anchor.

Lactic acid bacteria (LAB) have attracted increasing interest recently as cell factories for the production of proteins as well as a carrier of proteins that are of interest for food and therapeutic applications. In this present study, we exploit a lactobacillal food-grade expression system derived from the pSIP expression vectors using the (alanine racemase) gene as the selection marker for the expression and cell-surface display of a chitosanase in using two truncated forms of a LP × TG anchor. CsnA, a chitosanase from 168 (ATCC23857), was fused to two different truncated forms (short-S and long-L anchors) of an LP × TG anchor derived from Lp_1229, a key-protein for mannose-specific adhesion in WCFS1.

Immobilization of β-Galactosidases on the Cell Surface Using the Peptidoglycan-Binding Motif LysM.

Lysin motif (LysM) domains are found in many bacterial peptidoglycan hydrolases. They can bind non-covalently to peptidoglycan and have been employed to display heterologous proteins on the bacterial cell surface. In this study, we aimed to use a single LysM domain derived from a putative extracellular transglycosylase Lp_3014 of WCFS1 to display two different lactobacillal β-galactosidases, the heterodimeric LacLM-type from and the homodimeric LacZ-type from subsp.

Comparison of eight Lactobacillus species for delivery of surface-displayed mycobacterial antigen.

Lactobacillus spp. comprise a large group of Gram-positive lactic acid bacteria with varying physiological, ecological and immunomodulatory properties that are widely exploited by mankind, primarily in food production and as health-promoting probiotics. Recent years have shown increased interest in using lactobacilli for delivery of vaccines, mainly due to their ability to skew the immune system towards pro-inflammatory responses.

Comparison of three seemingly similar lytic polysaccharide monooxygenases from suggests different roles in plant biomass degradation.

Many fungi produce multiple lytic polysaccharide monooxygenases (LPMOs) with seemingly similar functions, but the biological reason for this multiplicity remains unknown. To address this question, here we carried out comparative structural and functional characterizations of three cellulose-active C4-oxidizing family AA9 LPMOs from the fungus , LPMO9A (NCU02240), LPMO9C (NCU02916), and LPMO9D (NCU01050). We solved the three-dimensional structure of copper-bound LPMO9A at 1.

Inactivated Carrying a Surface-Displayed Ag85B-ESAT-6 Fusion Antigen as a Booster Vaccine Against Infection.

Vaccination is considered the most effective strategy for controlling tuberculosis (TB). The existing vaccine, the Bacille Calmette-Guérin (BCG), although partially protective, has a number of limitations. Therefore, there is a need for developing new TB vaccines and several strategies are currently exploited including the use of viral and bacterial delivery vectors.

CRISPR Interference for Rapid Knockdown of Essential Cell Cycle Genes in .

Studies of essential genes in bacteria are often hampered by the lack of accessible genetic tools. This is also the case for , a key species in food and health applications. Here, we develop a clustered regularly interspaced short palindromic repeat interference (CRISPRi) system for knockdown of gene expression in The two-plasmid CRISPRi system, in which a nuclease-inactivated Cas9 (dCas9) and a gene-specific single guide RNA (sgRNA) are expressed on separate plasmids, allows efficient knockdown of expression of any gene of interest.

Over 2000-Fold Increased Production of the Leaderless Bacteriocin Garvicin KS by Increasing Gene Dose and Optimization of Culture Conditions.

The leaderless bacteriocin Garvicin KS (GarKS) is a potent antimicrobial, being active against a wide range of important pathogens. GarKS production by the native producer KS1546 is, however, relatively low (80 BU/ml) under standard laboratory growth conditions (batch culture in GM17 at 30°C). To improve the production, we systematically evaluated the impact of different media and media components on bacteriocin production.

Profiling of microorganism-binding serum antibody specificities in professional athletes.

The goal of this work was to elucidate similarities between microorganisms from the perspective of the humoral immune system reactivity in professional athletes. The reactivity of serum IgG of 14 young, individuals was analyzed to 23 selected microorganisms as antigens by use of the in house ELISA. Serum IgM and IgA reactivity was also analyzed and a control group of sex and age matched individuals was used for comparison.

Lactobacillus plantarum producing a Chlamydia trachomatis antigen induces a specific IgA response after mucosal booster immunization.

Mucosal immunity is important for the protection against a wide variety of pathogens. Traditional vaccines administered via parenteral routes induce strong systemic immunity, but they often fail to generate mucosal IgA. In contrast, bacteria-based vaccines comprise an appealing strategy for antigen delivery to mucosal sites.

From by-product to valuable components: Efficient enzymatic conversion of lactose in whey using β-galactosidase from .

β-Galactosidase from was overexpressed in a food-grade organism, WCFS1. Laboratory cultivations yielded 11,000 U of β-galactosidase activity per liter of culture corresponding to approximately 170 mg of enzyme. Crude cell-free enzyme extracts obtained by cell disruption and subsequent removal of cell debris showed high stability and were used for conversion of lactose in whey permeate.

Immunogenic Properties of Lactobacillus plantarum Producing Surface-Displayed Mycobacterium tuberculosis Antigens.

Unlabelled: Tuberculosis (TB) remains among the most deadly diseases in the world. The only available vaccine against tuberculosis is the bacille Calmette-Guérin (BCG) vaccine, which does not ensure full protection in adults. There is a global urgency for the development of an effective vaccine for preventing disease transmission, and it requires novel approaches.