Metabolic Profile of the Genome-Reduced Strain IIG-Bs-27-39: An Attractive Chassis for Recombinant Protein Production.

The Gram-positive bacterium is extensively used in the industry for the secretory production of proteins with commercial value. To further improve its performance, this microbe has been the subject of extensive genome engineering efforts, especially the removal of large genomic regions that are dispensable or even counterproductive. Here, we present the genome-reduced strain IIG-Bs-27-39, which was obtained through systematic deletion of mobile genetic elements, as well as genes for extracellular proteases, sporulation, flagella formation, and antibiotic production.

TLR4 sensing of IsdB of induces a proinflammatory cytokine response via the NLRP3-caspase-1 inflammasome cascade.

The prevalence of multidrug-resistant is of global concern, and vaccines are urgently needed. The iron-regulated surface determinant protein B (IsdB) of was investigated as a vaccine candidate because of its essential role in bacterial iron acquisition but failed in clinical trials despite strong immunogenicity. Here, we reveal an unexpected second function for IsdB in pathogen-host interaction: the bacterial fitness factor IsdB triggers a strong inflammatory response in innate immune cells via Toll-like receptor 4 and the inflammasome, thus acting as a novel pathogen-associated molecular pattern of .

Let There Be Light: Genome Reduction Enables to Produce Disulfide-Bonded Luciferase.

is a major workhorse for enzyme production in industrially relevant quantities. Compared to mammalian-based expression systems, presents intrinsic advantages, such as high growth rates, high space-time yield, unique protein secretion capabilities, and low maintenance costs. However, shows clear limitations in the production of biopharmaceuticals, especially proteins from eukaryotic origin that contain multiple disulfide bonds.

Dual Action of Eeyarestatin 24 on Sec-Dependent Protein Secretion and Bacterial DNA.

Eeyarestatin 24 (ES24) is a promising new antibiotic with broad-spectrum activity. It shares structural similarity with nitrofurantoin (NFT), yet appears to have a distinct and novel mechanism: ES24 was found to inhibit SecYEG-mediated protein transport and membrane insertion in Gram-negative bacteria. However, possible additional targets have not yet been explored.

Redirected Stress Responses in a Genome-Minimized 'midi' Strain with Enhanced Capacity for Protein Secretion.

Genome engineering offers the possibility to create completely novel cell factories with enhanced properties for biotechnological applications. In recent years, genome minimization was extensively explored in the Gram-positive bacterial cell factory Bacillus subtilis, where up to 42% of the genome encoding dispensable functions was removed. Such studies showed that some strains with minimized genomes gained beneficial features, especially for secretory protein production.

Recombinant protein secretion by Bacillus subtilis and Lactococcus lactis: pathways, applications, and innovation potential.

Secreted recombinant proteins are of great significance for industry, healthcare and a sustainable bio-based economy. Consequently, there is an ever-increasing need for efficient production platforms to deliver such proteins in high amounts and high quality. Gram-positive bacteria, particularly bacilli such as Bacillus subtilis, are favored for the production of secreted industrial enzymes.

SppI Forms a Membrane Protein Complex with SppA and Inhibits Its Protease Activity in Bacillus subtilis.

The membrane protease SppA of was first described as a signal peptide peptidase and later shown to confer resistance to lantibiotics. Here, we report that SppA forms octameric complexes with YteJ, a membrane protein of thus-far-unknown function. Interestingly, and deletion mutants exhibited no protein secretion defects.

Functional association of the stress-responsive LiaH protein and the minimal TatAyCy protein translocase in Bacillus subtilis.

The bacterial twin-arginine (Tat) pathway serves in the exclusive secretion of folded proteins with bound cofactors. While Tat pathways in Gram-negative bacteria and chloroplast thylakoids consist of conserved TatA, TatB and TatC subunits, the Tat pathways of Bacillus species and many other Gram-positive bacteria stand out for their minimalist nature with the core translocase being composed of essential TatA and TatC subunits only. Here we addressed the question whether the minimal TatAyCy translocase of Bacillus subtilis recruits additional cellular components that modulate its activity.

Relative contributions of non-essential Sec pathway components and cell envelope-associated proteases to high-level enzyme secretion by Bacillus subtilis.

Background: Bacillus subtilis is an important industrial workhorse applied in the production of many different commercially relevant proteins, especially enzymes. Virtually all of these proteins are secreted via the general secretion (Sec) pathway. Studies from different laboratories have demonstrated essential or non-essential contributions of various Sec machinery components to protein secretion in B.

Human antibody responses against non-covalently cell wall-bound Staphylococcus aureus proteins.

Human antibody responses to pathogens, like Staphylococcus aureus, are important indicators for in vivo expression and immunogenicity of particular bacterial components. Accordingly, comparing the antibody responses to S. aureus components may serve to predict their potential applicability as antigens for vaccination.

A Lactococcus lactis expression vector set with multiple affinity tags to facilitate isolation and direct labeling of heterologous secreted proteins.

The gram-positive bacterium Lactococcus lactis is a useful host for extracellular protein production. A main advantage of L. lactis over other bacterial expression systems is that lactococcal cells display low levels of autolysis and proteolysis.

Intramembrane protease RasP boosts protein production in Bacillus.

Background: The microbial cell factory Bacillus subtilis is a popular industrial platform for high-level production of secreted technical enzymes. Nonetheless, the effective secretion of particular heterologous enzymes remains challenging. Over the past decades various studies have tackled this problem, and major improvements were achieved by optimizing signal peptides or removing proteases involved in product degradation.

Versatile vector suite for the extracytoplasmic production and purification of heterologous His-tagged proteins in Lactococcus lactis.

Recent studies have shown that the Gram-positive bacterium Lactococcus lactis can be exploited for the expression of heterologous proteins; however, a versatile set of vectors suitable for inducible extracellular protein production and subsequent purification of the expressed proteins by immobilized metal affinity chromatography was so far lacking. Here we describe three novel vectors that, respectively, facilitate the nisin-inducible production of N- or C-terminally hexa-histidine (His6)-tagged proteins in L. lactis.

Active immunization with an octa-valent Staphylococcus aureus antigen mixture in models of S. aureus bacteremia and skin infection in mice.

Proteomic studies with different Staphylococcus aureus isolates have shown that the cell surface-exposed and secreted proteins IsaA, LytM, Nuc, the propeptide of Atl (pro-Atl) and four phenol-soluble modulins α (PSMα) are invariantly produced by this pathogen. Therefore the present study was aimed at investigating whether these proteins can be used for active immunization against S. aureus infection in mouse models of bacteremia and skin infection.

Low anti-staphylococcal IgG responses in granulomatosis with polyangiitis patients despite long-term Staphylococcus aureus exposure.

Chronic nasal carriage of the bacterium Staphylococcus aureus in patients with the autoimmune disease granulomatosis with polyangiitis (GPA) is a risk factor for disease relapse. To date, it was neither known whether GPA patients show similar humoral immune responses to S. aureus as healthy carriers, nor whether specific S.

Efficient production of secreted staphylococcal antigens in a non-lysing and proteolytically reduced Lactococcus lactis strain.

Cell surface-exposed and secreted proteins are attractive targets for vaccination against pathogenic gram-positive bacteria. To obtain sufficient amounts of such antigens, efficient protein production platforms are needed. In this study, a pipeline for the production and purification of surface-exposed and secreted antigens of the gram-positive bacterial pathogen Staphylococcus aureus is presented.

Degradation of extracytoplasmic catalysts for protein folding in Bacillus subtilis.

The general protein secretion pathway of Bacillus subtilis has a high capacity for protein export from the cytoplasm, which is exploited in the biotechnological production of a wide range of enzymes. These exported proteins pass the membrane in an unfolded state, and accordingly, they have to fold into their active and protease-resistant conformations once membrane passage is completed. The lipoprotein PrsA and the membrane proteins HtrA and HtrB facilitate the extracytoplasmic folding and quality control of exported proteins.

Pyruvate oxidase influences the sugar utilization pattern and capsule production in Streptococcus pneumoniae.

Pyruvate oxidase is a key function in the metabolism and lifestyle of many lactic acid bacteria and its activity depends on the presence of environmental oxygen. In Streptococcus pneumoniae the protein has been suggested to play a major role in metabolism and has been implicated in virulence, oxidative stress survival and death in stationary phase. Under semi-aerobic conditions, transcriptomic and metabolite profiling analysis of a spxB mutant grown on glucose showed minor changes compared to the wild type, apart from the significant induction of two operons involved in carbohydrate uptake and processing.

Deletion of a cation transporter promotes lysis in Streptococcus pneumoniae.

Streptococcus pneumoniae is a significant human pathogen which causes respiratory and serious invasive diseases. Mg(2+) is essential for life, and its concentration varies throughout the human body. Magnesium uptake plays an important role in the virulence of many bacterial pathogens.

Development of lactococcal GEM-based pneumococcal vaccines.

We report the development of a novel protein-based nasal vaccine against Streptococcus pneumoniae, in which three pneumococcal proteins were displayed on the surface of a non-recombinant, killed Lactococcus lactis-derived delivery system, called Gram-positive Enhancer Matrix (GEM). The GEM particles induced the production of the proinflammatory cytokine tumour necrosis factor-alpha (TNF-alpha) by macrophages as well as the maturation of dendritic cells. The pneumococcal proteins IgA1 protease (IgA1p), putative proteinase maturation protein A (PpmA) and streptococcal lipoprotein A (SlrA) were anchored in trans to the surface of the GEM particles after recombinant production of the antigens in L.

Lactococcus lactis GEM particles displaying pneumococcal antigens induce local and systemic immune responses following intranasal immunization.

The present work reports the use of non-living non-recombinant bacteria as a delivery system for mucosal vaccination. Antigens are bound to the cell-wall of pretreated Lactococcus lactis, designated as Gram-positive enhancer matrix (GEM), by means of a peptidoglycan binding domain. The influence of the GEM particles on the antigen-specific serum antibody response was studied.

Mucosal vaccine delivery of antigens tightly bound to an adjuvant particle made from food-grade bacteria.

Mucosal immunization with subunit vaccines requires new types of antigen delivery vehicles and adjuvants for optimal immune responses. We have developed a non-living and non-genetically modified gram-positive bacterial delivery particle (GEM) that has built-in adjuvant activity and a high loading capacity for externally added heterologous antigens that are fused to a high affinity binding domain. This binding domain, the protein anchor (PA), is derived from the Lactococcus lactis AcmA cell-wall hydrolase, and contains three repeats of a LysM-type cell-wall binding motif.

Novel surface display system for proteins on non-genetically modified gram-positive bacteria.

A novel display system is described that allows highly efficient immobilization of heterologous proteins on bacterial surfaces in applications for which the use of genetically modified bacteria is less desirable. This system is based on nonliving and non-genetically modified gram-positive bacterial cells, designated gram-positive enhancer matrix (GEM) particles, which are used as substrates to bind externally added heterologous proteins by means of a high-affinity binding domain. This binding domain, the protein anchor (PA), was derived from the Lactococcus lactis peptidoglycan hydrolase AcmA.