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

The Gram-positive bacterium Bacillus subtilis 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 B. subtilis 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. Different from previously characterized genome-reduced B. subtilis strains, the IIG-Bs-27-39 strain was still able to grow on minimal media. We used this feature to benchmark strain IIG-Bs-27-39 against its parental strain 168 with respect to heterologous protein production and metabolic parameters during bioreactor cultivation. The IIG-Bs-27-39 strain presented superior secretion of difficult-to-produce staphylococcal antigens, as well as higher specific growth rates and biomass yields. At the metabolic level, changes in byproduct formation and internal amino acid pools were observed, whereas energetic parameters such as the ATP yield, ATP/ADP levels, and adenylate energy charge were comparable between the two strains. Intriguingly, we observed a significant increase in the total cellular NADPH level during all tested conditions and increases in the NAD+ and NADP(H) pools during protein production. This indicates that the IIG-Bs-27-39 strain has more energy available for anabolic processes and protein production, thereby providing a link between strain physiology and production performance. On this basis, we conclude that the genome-reduced strain IIG-Bs-27-39 represents an attractive chassis for future biotechnological applications.

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

IMPORTANCE: The prevalence of multidrug-resistant Staphylococcus aureus is of global concern, and vaccines are urgently needed. The iron-regulated surface determinant protein B (IsdB) of S. aureus 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 S. aureus. Our discovery contributes to a better understanding of how S. aureus modulates the immune response, which is necessary for vaccine development against the sophisticated pathogen.

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

Bacillus subtilis is a major workhorse for enzyme production in industrially relevant quantities. Compared to mammalian-based expression systems, B. subtilis presents intrinsic advantages, such as high growth rates, high space-time yield, unique protein secretion capabilities, and low maintenance costs. However, B. subtilis shows clear limitations in the production of biopharmaceuticals, especially proteins from eukaryotic origin that contain multiple disulfide bonds. In the present study, we deployed genome minimization, signal peptide screening, and coexpression of recombinant thiol oxidases as strategies to improve the ability of B. subtilis to secrete proteins with multiple disulfide bonds. Different genome-reduced strains served as the chassis for expressing the model protein Gaussia Luciferase (GLuc), which contains five disulfide bonds. These chassis lack extracellular proteases, prophages, and key sporulation genes. Importantly, compared to the reference strain with a full-size genome, the best-performing genome-minimized strain achieved over 3000-fold increased secretion of active GLuc while growing to lower cell densities. Our results show that high-level GLuc secretion relates, at least in part, to the absence of major extracellular proteases. In addition, we show that the thiol-disulfide oxidoreductase requirements for disulfide bonding have changed upon genome reduction. Altogether, our results highlight genome-engineered Bacillus strains as promising expression platforms for proteins with 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. Moreover, its activity was notably better against Gram-positive bacteria, for which its mechanism of action had not yet been investigated. We have used transcriptomic stress response profiling, phenotypic assays, and protein secretion analyses to investigate the mode of action of ES24 in comparison with NFT using the Gram-positive model bacterium Bacillus subtilis and have compared our findings to Gram-negative Escherichia coli. Here, we show the inhibition of Sec-dependent protein secretion in B. subtilis and additionally provide evidence for DNA damage, probably caused by the generation of reactive derivatives of ES24. Interestingly, ES24 caused a gradual dissipation of the membrane potential, which led to delocalization of cytokinetic proteins and subsequent cell elongation in E. coli. However, none of those effects were observed in B. subtilis, thereby suggesting that ES24 displays distinct mechanistic differences with respect to Gram-positive and Gram-negative bacteria. Despite its structural similarity to NFT, ES24 profoundly differed in our phenotypic analysis, which implies that it does not share the NFT mechanism of generalized macromolecule and structural damage. Importantly, ES24 outperformed NFT in vivo in a zebrafish embryo pneumococcal infection model. Our results suggest that ES24 not only inhibits the Sec translocon, but also targets bacterial DNA and, in Gram-negative bacteria, the cell membrane.

Redirected Stress Responses in a Genome-Minimized 'midiBacillus' 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. However, strains with the most minimal genomes displayed growth defects. This focused our attention on strains with less extensive genomic deletions that display close-to-wild-type growth properties while retaining the acquired beneficial traits in secretory protein production. A strain of this category is B. subtilis IIG-Bs27-47-24, here referred to as midiBacillus, which lacks 30.95% of the parental genome. To date, it was unknown how the altered genomic configuration of midiBacillus impacts cell physiology in general, and protein secretion in particular. The present study bridges this knowledge gap through comparative quantitative proteome analyses with focus on protein secretion. Interestingly, the results show that the secretion stress responses of midiBacillus, as elicited by high-level expression of the immunodominant staphylococcal antigen A, are completely different from secretion stress responses that occur in the parental strain 168. We further show that midiBacillus has an increased capacity for translation and that a variety of critical Sec secretion machinery components is present at elevated levels. Altogether, our observations demonstrate that high-level protein secretion has different consequences for wild-type and genome-engineered Bacillus strains, dictated by the altered genomic and proteomic configurations. IMPORTANCE Our present study showcases a genome-minimized nonpathogenic bacterium, the so-called midiBacillus, as a chassis for the development of future industrial strains that serve in the production of high-value difficult-to-produce proteins. In particular, we explain how midiBacillus, which lacks about one-third of the original genome, effectively secretes a protein of the major human pathogen Staphylococcus aureus that cannot be produced by the parental Bacillus subtilis strain. This is important, because the secreted S. aureus protein is exemplary for a range of targets that can be implemented in future antistaphylococcal immunotherapies. Accordingly, we anticipate that midiBacillus chassis will contribute to the development of vaccines that protect both humans and livestock against diseases caused by S. aureus, a bacterial pathogen that is increasingly difficult to fight with antibiotics, because it has accumulated resistances to essentially all antibiotics that are currently in clinical practice.

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. Nevertheless, recombinant protein production in the B. subtilis cell factory can be very challenging due to bottlenecks in the general (Sec) secretion pathway as well as this bacterium's intrinsic capability to secrete a cocktail of highly potent proteases. This has placed another Gram-positive bacterium, Lactococcus lactis, in the focus of attention as an alternative, non-proteolytic, cell factory for secreted proteins. Here we review our current understanding of the secretion pathways exploited in B. subtilis and L. lactis to deliver proteins from their site of synthesis, the cytoplasm, into the fermentation broth. An advantage of this cell factory comparison is that it identifies opportunities for protein secretion pathway engineering to remove or bypass current production bottlenecks. Noteworthy new developments in cell factory engineering are the mini-Bacillus concept, highlighting potential advantages of massive genome minimization, and the application of thus far untapped 'non-classical' protein secretion routes. Altogether, it is foreseen that engineered lactococci will find future applications in the production of high-quality proteins at the relatively small pilot scale, while engineered bacilli will remain a favored choice for protein production in bulk.

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

The membrane protease SppA of Bacillus subtilis 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, sppA and yteJ deletion mutants exhibited no protein secretion defects. However, these mutant strains differed significantly in their resistance to antimicrobial peptides. In particular, sppA mutant cells displayed increased sensitivity to the lantibiotics nisin and subtilin and the human lysozyme-derived cationic antimicrobial peptide LP9. Importantly, YteJ was shown to antagonize SppA activity both in vivo and in vitro, and this SppA-inhibitory activity involved the C-terminal domain of YteJ, which was therefore renamed SppI. Most likely, SppI-mediated control is needed to protect B. subtilis against the potentially detrimental protease activity of SppA since a mutant overexpressing sppA by itself displayed defects in cell division. Altogether, we conclude that the SppA-SppI complex of B. subtilis has a major role in protection against antimicrobial peptides.IMPORTANCE Our study presents new insights into the molecular mechanism that regulates the activity of SppA, a widely conserved bacterial membrane protease. We show that the membrane proteins SppA and SppI form a complex in the Gram-positive model bacterium B. subtilis and that SppI inhibits SppA protease activity in vitro and in vivo Furthermore, we demonstrate that the C-terminal domain of SppI is involved in SppA inhibition. Since SppA, through its protease activity, contributes directly to resistance to lantibiotic peptides and cationic antibacterial peptides, we propose that the conserved SppA-SppI complex could play a major role in the evasion of bactericidal peptides, including those produced as part of human innate immune defenses.

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. To this end, TatAyCy was purified by affinity- and size exclusion chromatography, and interacting co-purified proteins were identified by mass spectrometry. This uncovered the cell envelope stress responsive LiaH protein as an accessory subunit of the TatAyCy complex. Importantly, our functional studies show that Tat expression is tightly trailed by LiaH induction, and that LiaH itself determines the capacity and quality of TatAyCy-dependent protein translocation. In contrast, LiaH has no role in high-level protein secretion via the general secretion (Sec) pathway. Altogether, our observations show that protein translocation by the minimal Tat translocase TatAyCy is tightly intertwined with an adequate bacterial response to cell envelope stress. This is consistent with a critical need to maintain cellular homeostasis, especially when the membrane is widely opened to permit passage of large fully-folded proteins via Tat.

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. subtilis. However, a systematic comparison of the impact of each individual Sec machinery component under conditions of high-level protein secretion was so far missing. RESULTS: In the present study, we have compared the contributions of non-essential Sec pathway components and cell envelope-associated proteases on the secretion efficiency of three proteins expressed at high level. This concerned the α-amylases AmyE from B. subtilis and AmyL from Bacillus licheniformis, and the serine protease BPN' from Bacillus amyloliquefaciens. We compared the secretion capacity of mutant strains in shake flask cultures, and the respective secretion kinetics by pulse-chase labeling experiments. The results show that secDF, secG or rasP mutations severely affect AmyE, AmyL and BPN' secretion, but the actual effect size depends on the investigated protein. Additionally, the chaperone DnaK is important for BPN' secretion, while AmyE or AmyL secretion are not affected by a dnaK deletion. Further, we assessed the induction of secretion stress responses in mutant strains by examining AmyE- and AmyL-dependent induction of the quality control proteases HtrA and HtrB. Interestingly, the deletion of certain sip genes revealed a strong differential impact of particular signal peptidases on the magnitude of the secretion stress response. CONCLUSIONS: The results of the present study highlight the importance of SecDF, SecG and RasP for protein secretion and reveal unexpected differences in the induction of the secretion stress response in different mutant strains.

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. The present study was aimed at assessing immunoglobulin G (IgG) responses elicited by non-covalently cell surface-bound proteins of S. aureus, which thus far received relatively little attention. To this end, we applied plasma samples from patients with the genetic blistering disease epidermolysis bullosa (EB) and healthy S. aureus carriers. Of note, wounds of EB patients are highly colonized with S. aureus and accordingly these patients are more seriously exposed to staphylococcal antigens than healthy individuals. Ten non-covalently cell surface-bound proteins of S. aureus, namely Atl, Eap, Efb, EMP, IsaA, LukG, LukH, SA0710, Sle1 and SsaA2, were selected by bioinformatics and biochemical approaches. These antigens were recombinantly expressed, purified and tested for specific IgG responses using human plasma. We show that high exposure of EB patients to S. aureus is mirrored by elevated IgG levels against all tested non-covalently cell wall-bound staphylococcal antigens. This implies that these S. aureus cell surface proteins are prime targets for the human immune system.

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. Previously, we developed a set of vectors for nisin-inducible extracellular production of N- or C-terminally hexa-histidine (His6)-tagged proteins. The present study was aimed at expanding our portfolio of L. lactis expression vectors for protein purification and site-specific labeling. Specifically, we present two new groups of vectors allowing N- or C-terminal provision of proteins with a Strep-tag II or AVI-tag. Vectors for AVI-tagging encode an additional His6-tag for protein purification. Another set of vectors allows removal of N-terminal Strep- or His6-tags from expressed proteins with the tobacco etch virus protease. Two possible applications of the developed vectors are presented. First, we show that Strep-tagged LytM of Staphylococcus aureus in the growth medium of L. lactis can be directly bound to microtiter plates coated with an affinity reagent and used for enzyme-linked immunosorbent assays. Second, we show that the AVI-tagged Sle1 protein from S. aureus produced in L. lactis can be directly biotinylated and fluorescently labeled. The fluorescently labeled Sle1 was successfully applied for S. aureus re-binding studies, allowing subcellular localization by fluorescence microscopy. In conclusion, we have developed a set of expression vectors that enhances the versatility of L. lactis as a system for production of proteins with tags that can be used for affinity purification and site-specific protein labeling.

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. On the other hand, serious bottlenecks in the protein export process per se remained enigmatic, especially for protein secretion at commercially significant levels by cells grown to high density. The aim of our present study was to assess the relevance of the intramembrane protease RasP for high-level protein production in B. subtilis. RESULTS: Deletion of the rasP gene resulted in reduced precursor processing and extracellular levels of the overproduced α-amylases AmyE from B. subtilis and AmyL from Bacillus licheniformis. Further, secretion of the overproduced serine protease BPN' from Bacillus amyloliquefaciens was severely impaired in the absence of RasP. Importantly, overexpression of rasP resulted in threefold increased production of a serine protease from Bacillus clausii, and 2.5- to 10-fold increased production of an AmyAc α-amylase from Paenibacillus curdlanolyticus, depending on the culture conditions. Of note, growth defects due to overproduction of the two latter enzymes were suppressed by rasP-overexpression. CONCLUSION: Here we show that an intramembrane protease, RasP, sets a limit to high-level production of two secreted heterologous enzymes that are difficult to produce in the B. subtilis cell factory. This finding was unexpected and suggests that proteolytic membrane sanitation is key to effective enzyme production in Bacillus.

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. One of these vectors also encodes a tobacco etch virus (TEV) protease cleavage site allowing removal of the N-terminal His6-tag from expressed proteins. Successful application of the developed vectors for protein expression, purification and/or functional studies is exemplified with six different cell wall-bound or secreted proteins from Staphylococcus aureus. The results show that secretory production of S. aureus proteins is affected by the position, N- or C-terminal, of the His6-tag. This seems to be due to an influence of the His6-tag on protein stability. Intriguingly, the S. aureus IsdB protein, which is phosphorylated in S. aureus, was also found to be phosphorylated when heterologously produced in L. lactis, albeit not on the same Tyr residue. This implies that this particular post-translational protein modification is to some extent conserved in S. aureus and L. lactis. Altogether, we are confident that the present vector set combined with the L. lactis expression host has the potential to become a very useful tool in optimization of the expression, purification and functional analysis of extracytoplasmic bacterial proteins.

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. aureus types are associated with GPA. Therefore, this study was aimed at assessing humoral immune responses of GPA patients against S. aureus antigens in relation to the genetic diversity of their nasal S. aureus isolates. A retrospective cohort study was conducted, including 85 GPA patients and 18 healthy controls (HC). Humoral immune responses against S. aureus were investigated by determining serum IgG levels against 59 S. aureus antigens. Unexpectedly, patient sera contained lower anti-staphylococcal IgG levels than sera from HC, regardless of the patients' treatment, while total IgG levels were similar or higher. Furthermore, 210 S. aureus isolates obtained from GPA patients were characterized by different typing approaches. This showed that the S. aureus population of GPA patients is highly diverse and mirrors the general S. aureus population. Our combined findings imply that GPA patients are less capable of mounting a potentially protective antibody response to S. aureus than healthy individuals.

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. To this end, recombinant His-tagged fusions of IsaA, LytM, Nuc and pro-Atl were isolated from Lactococcus lactis or Escherichia coli, while the PSMα1-4 peptides were chemically synthesized. Importantly, patients colonized by S. aureus showed significant immunoglobulin G (IgG) responses against all eight antigens. BALB/cBYJ mice were immunized subcutaneously with a mixture of the antigens at day one (5 µg each), and boosted twice (25 µg of each antigen) with 28 days interval. This resulted in high IgG responses against all antigens although the response against pro-Atl was around one log lower compared to the other antigens. Compared to placebo-immunized mice, immunization with the octa-valent antigen mixture did not reduce the S. aureus isolate P load in blood, lungs, spleen, liver, and kidneys in a bacteremia model in which the animals were challenged for 14 days with a primary load of 3 × 10(5) CFU. Discomfort scores and animal survival rates over 14 days did not differ between immunized mice and placebo-immunized mice upon bacteremia with S. aureus USA300 (6 × 10(5) CFU). In addition, this immunization did not reduce the S. aureus isolate P load in mice with skin infection. These results show that the target antigens are immunogenic in both humans and mice, but in the used animal models do not result in protection against S. aureus infection.

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. Cytoplasmic or extracellular production of S. aureus antigens was achieved using the Lactococcus lactis strain PA1001, which lacks the major extracellular protease HtrA and the autolysin AcmA to minimize proteolysis and cell lysis, respectively. For most tested S. aureus antigens, secretory production directed by the signal peptide of the major secreted protein Usp45 of L. lactis resulted in higher yields than intracellular production without a signal peptide. Additionally, secretory production of His-tagged antigens allowed their facile one-step purification from the growth medium by metal affinity chromatography. For three of the purified antigens, biological activity was confirmed through enzyme activity assays. We, furthermore, show that the present pipeline can be used to produce staphylococcal antigens with an N-terminal AVI-tag for site-specific labeling with biotin or a C-terminal cell wall-binding domain for cell surface display. We conclude that our L. lactis-based pipeline allows the efficient production of S. aureus antigens and their subsequent purification in one step.

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. Among the native exported proteins of B. subtilis are at least 10 proteases that have previously been implicated in the degradation of heterologous secreted proteins. Recently, we have shown that these proteases also degrade many native membrane proteins, lipoproteins, and secreted proteins. The present studies were therefore aimed at assessing to what extent these proteases also degrade extracytoplasmic catalysts for protein folding. To this end, we employed a collection of markerless protease mutant strains that lack up to 10 different extracytoplasmic proteases. The results show that PrsA, HtrA, and HtrB are indeed substrates of multiple extracytoplasmic proteases. Thus, improved protein secretion by multiple-protease-mutant strains may be related to both reduced proteolysis and improved posttranslocational protein folding and quality control.

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. This induction leads to a change in the sugar utilization capabilities of the bacterium, as indicated by the analysis of the growth profiles of the D39 parent and spxB mutant on alternative carbohydrates. Metabolic analysis and growth experiments showed that inactivation of SpxB has no effect on the glucose fermentation pattern, except under aerobic conditions. More importantly, we show that mutation of spxB results in the production of increased amounts of capsule, the major virulence factor of S. pneumoniae. Part of this increase can be attributed to induction of capsule operon (cps) transcription. Therefore, we propose that S. pneumoniae utilizes pyruvate oxidase as an indirect sensor of the oxygenation of the environment, resulting in the adaption of its nutritional capability and the amount of capsule to survive in the host.

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. To study the Mg(2+) uptake of S. pneumoniae strain D39, a mutant was generated in SPD1383, a P-type ATPase with homology to the Salmonella Mg(2+) transporter MgtA, which has also been shown to be a Ca(2+) exporter in strain TIGR4. Under low-Ca(2+) conditions, mutation led to a growth defect in complex medium and the gene was nearly essential for growth under low-Mg(2+) conditions. Addition of Mg(2+) restored the normal growth of the mutant in all cases, but the addition of other divalent cations had no effect. Addition of Ca(2+), Mn(2+), and Zn(2+) in the presence of high Mg(2+) concentrations inhibited restoration of growth. The mutant was unable to proliferate in blood, which was also alleviated by the addition of Mg(2+). The protein was located in the membrane and produced in various S. pneumoniae strains and pathogenic streptococcal species. Surprisingly, mutation of the gene led to an elevated toxicity for endothelial cells. This was caused by an increased amount of pneumolysin in the medium, mediated by elevated lysis of the mutant. Thus, in this study, we uncovered a role for SPD1383 in Mg(2+) uptake and hypothesize that the protein is a Mg(2+/)Ca(2+) antiporter. Furthermore, a disturbance in Mg(2+) homeostasis seems to promote lysis of S. pneumoniae.

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. lactis as hybrids with a lactococcal cell wall binding domain, named Protein Anchor domain (PA). Intranasal immunisation with the SlrA-IgA1p or trivalent vaccine combinations without additional adjuvants showed significant protection against fatal pneumococcal pneumonia in mice. The GEM-based trivalent vaccine is a potential pneumococcal vaccine candidate that is expected to be easy to administer, safe and affordable to produce.