The addition of arginine deiminase potentiates Mithramycin A-induced cell death in patient-derived glioblastoma cells via ATF4 and cytochrome C.

BACKGROUND: Arginine auxotrophy constitutes a shortcoming for ~ 30% of glioblastoma multiforme (GBM). Indeed, arginine-depleting therapy using arginine deiminase from Streptococcus pyogenes (SpyADI) has proven activity against GBM in preclinical studies. The good safety profile of SpyADI renders this agent an ideal combination partner for cytostatic therapy. METHODS: In this study, we combined the antineoplastic antibiotic Mithramycin A (MitA) with SpyADI to boost single-agent activity and analyzed underlying response mechanisms in-depth. RESULTS: MitA monotherapy induced a time- and dose-dependent cytotoxicity in eight patient-derived GBM cell lines and had a radiosensitizing effect in all but one cell line. Combination treatment boosted the effects of the monotherapy in 2D- and 3D models. The simultaneous approach was superior to the sequential application and significantly impaired colony formation after repetitive treatment. MitA monotherapy significantly inhibited GBM invasiveness. However, this effect was not enhanced in the combination. Functional analysis identified SpyADI-triggered senescence induction accompanied by increased mitochondrial membrane polarization upon mono- and combination therapy. In HROG63, induction of lysosomes was seen after both monotherapies, indicative of autophagy. These cells seemed swollen and had a more pronounced cortically formed cytoskeleton. Also, cytochrome C and endoplasmatic reticulum-stress-associated proteins ATF4 and Calnexin were enhanced in the combination, contributing to apoptosis. Notably, no significant increases in glioma-stemness marker were seen. CONCLUSIONS: Therapeutic utilization of a metabolic defect in GBM along with cytostatic therapy provides a novel combination approach. Whether this SpyADI/MitA regimen will provide a safe alternative to combat GBM, will have to be addressed in subsequent (pre-)clinical trials.

Implementation of a combined CDK inhibition and arginine-deprivation approach to target arginine-auxotrophic glioblastoma multiforme cells.

Constitutive activation of cyclin-dependent kinases (CDKs) or arginine auxotrophy are hallmarks of Glioblastoma multiforme (GBM). The latter metabolic defect renders tumor cells vulnerable to arginine-depleting substances, such as arginine deiminase from Streptococcus pyogenes (SpyADI). Previously, we confirmed the susceptibility of patient-derived GBM cells towards SpyADI as well as CDK inhibitors (CDKis). To improve therapeutic effects, we here applied a combined approach based on SpyADI and CDKis (dinaciclib, abemaciclib). Three arginine-auxotrophic patient-derived GBM lines with different molecular characteristics were cultured in 2D and 3D and effects of this combined SpyADI/CDKi approach were analyzed in-depth. All CDKi/SpyADI combinations yielded synergistic antitumoral effects, especially when given sequentially (SEQ), i.e., CDKi in first-line and most pronounced in the 3D models. SEQ application demonstrated impaired cell proliferation, invasiveness, and viability. Mitochondrial impairment was demonstrated by increasing mitochondrial membrane potential and decreasing oxygen consumption rate and extracellular acidification rate after SpyADI/abemaciclib monotherapy or its combination regimens. The combined treatment even induced autophagy in target cells (abemaciclib/SpyADI > dinaciclib/SpyADI). By contrast, the unfolded protein response and p53/p21 induced senescence played a minor role. Transmission electron microscopy confirmed damaged mitochondria and endoplasmic reticulum together with increased vacuolization under CDKi mono- and combination therapy. SEQ-abemaciclib/SpyADI treatment suppressed the DSB repair system via NHEJ and HR, whereas SEQ-dinaciclib/SpyADI treatment increased γ-H2AX accumulation and induced Rad51/Ku80. The latter combination also activated the stress sensor GADD45 and ß-catenin antagonist AXIN2 and induced expression changes of genes involved in cellular/cytoskeletal integrity. This study highlights the strong antitumoral potential of a combined arginine deprivation and CDK inhibition approach via complex effects on mitochondrial dysfunction, invasiveness as well as DNA-damage response. This provides a good starting point for further in vitro and in vivo proof-of-concept studies to move forward with this strategy.

The Non-phosphorylating Glyceraldehyde-3-Phosphate Dehydrogenase GapN Is a Potential New Drug Target in Streptococcus pyogenes.

The strict human pathogen Streptococcus pyogenes causes infections of varying severity, ranging from self-limiting suppurative infections to life-threatening diseases like necrotizing fasciitis or streptococcal toxic shock syndrome. Here, we show that the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase GapN is an essential enzyme for S. pyogenes. GapN converts glyceraldehyde 3-phosphate into 3-phosphoglycerate coupled to the reduction of NADP to NADPH. The knock-down of gapN by antisense peptide nucleic acids (asPNA) significantly reduces viable bacterial counts of S. pyogenes laboratory and macrolide-resistant clinical strains in vitro. As S. pyogenes lacks the oxidative part of the pentose phosphate pathway, GapN appears to be the major NADPH source for the bacterium. Accordingly, other streptococci that carry a complete pentose phosphate pathway are not prone to asPNA-based gapN knock-down. Determination of the crystal structure of the S. pyogenes GapN apo-enzyme revealed an unusual cis-peptide in proximity to the catalytic binding site. Furthermore, using a structural modeling approach, we correctly predicted competitive inhibition of S. pyogenes GapN by erythrose 4-phosphate, indicating that our structural model can be used for in silico screening of specific GapN inhibitors. In conclusion, the data provided here reveal that GapN is a potential target for antimicrobial substances that selectively kill S. pyogenes and other streptococci that lack the oxidative part of the pentose phosphate pathway.

All Driven by Energy Demand? Integrative Comparison of Metabolism of Enterococcus faecalis Wildtype and a Glutamine Synthase Mutant.

Lactic acid bacteria (LAB) play a significant role in biotechnology, e.g., food industry and also in human health. Many LAB genera have developed a multidrug resistance in the past few years, causing a serious problem in controlling hospital germs worldwide. Enterococcus faecalis accounts for a large part of the human infections caused by LABs. Therefore, studying its adaptive metabolism under various environmental conditions is particularly important to promote the development of new therapeutic approaches. In this study, we investigated the effect of glutamine auxotrophy (ΔglnA mutant) on metabolic and proteomic adaptations of E. faecalis in response to a changing pH in its environment. Changing pH values are part of the organism's natural environment in the human body and play a role in the food industry. We compared the results with those of the wildtype. Using a genome-scale metabolic model constrained by metabolic and proteomic data, our integrative method allows us to understand the bigger picture of the adaptation strategies of this bacterium. The study showed that energy demand is the decisive factor in adapting to a new environmental pH. The energy demand of the mutant was higher at all conditions. It has been reported that ΔglnA mutants of bacteria are energetically less effective. With the aid of our data and model we are able to explain this phenomenon as a consequence of a failure to regulate glutamine uptake and the costs for the import of glutamine and the export of ammonium. Methodologically, it became apparent that taking into account the nonspecificity of amino acid transporters is important for reproducing metabolic changes with genome-scale models because it affects energy balance. IMPORTANCE The integration of new pH-dependent experimental data on metabolic uptake and release fluxes, as well as of proteome data with a genome-scale computational model of a glutamine synthetase mutant of E. faecalis is used and compared with those of the wildtype to understand why glutamine auxotrophy results in a less efficient metabolism and how-in comparison with the wildtype-the glutamine synthetase knockout impacts metabolic adjustments during acidification or simply exposure to lower pH. We show that forced glutamine auxotrophy causes more energy demand and that this is likely due to a disregulated glutamine uptake. Proteome changes during acidification observed for the mutant resemble those of the wildtype with the exception of glycolysis-related genes, as the mutant is already energetically stressed at a higher pH and the respective proteome changes were in effect.

PEGylation increases antitumoral activity of arginine deiminase of Streptococcus pyogenes.

Arginine auxotrophy is a metabolic defect that renders tumor cells vulnerable towards arginine-depleting substances, such as arginine deiminase (ADI) from Streptococcus pyogenes (SpyADI). Previously, we confirmed SpyADI susceptibility on patient-derived glioblastoma multiforme (GBM) models in vitro and in vivo. For application in patients, serum half-life of the enzyme has to be increased and immunogenicity needs to be reduced. For this purpose, we conjugated the S. pyogenes-derived SpyADI with 20 kDa polyethylene glycol (PEG20) moieties, achieving a PEGylation of seven to eight of the 26 accessible primary amines of the SpyADI. The PEGylation reduced the overall activity of the enzyme by about 50% without affecting the Michaelis constant for arginine. PEGylation did not increase serum stability of SpyADI in vitro, but led to a longer-lasting reduction of plasma arginine levels in mice. Furthermore, SpyADI-PEG20 showed a higher antitumoral capacity towards GBM cells in vitro than the native enzyme. KEY POINTS: • PEGylation has no effect on the affinity of SpyADI for arginine • PEGylation increases the antitumoral effects of SpyADI on GBM in vitro • PEGylation prolongs plasma arginine depletion by SpyADI in mice.

Protein cost allocation explains metabolic strategies in Escherichia coli.

In-depth understanding of microbial growth is crucial for the development of new advances in biotechnology and for combating microbial pathogens. Condition-specific proteome expression is central to microbial physiology and growth. A multitude of processes are dependent on the protein expression, thus, whole-cell analysis of microbial metabolism using genome-scale metabolic models is an attractive toolset to investigate the behaviour of microorganisms and their communities. However, genome-scale models that incorporate macromolecular expression are still inhibitory complex: the conceptual and computational complexity of these models severely limits their potential applications. In the need for alternatives, here we revisit some of the previous attempts to create genome-scale models of metabolism and macromolecular expression to develop a novel framework for integrating protein abundance and turnover costs to conventional genome-scale models. We show that such a model of Escherichia coli successfully reproduces experimentally determined adaptations of metabolism in a growth condition-dependent manner. Moreover, the model can be used as means of investigating underutilization of the protein machinery among different growth settings. Notably, we obtained strongly improved predictions of flux distributions, considering the costs of protein translation explicitly. This finding in turn suggests protein translation being the main regulation hub for cellular growth.

The colonic mucosa-associated microbiome in SIV infection: shift towards Bacteroidetes coincides with mucosal CD4+ T cell depletion and enterocyte damage.

The intesinal microbiome is considered important in human immunodeficiency virus (HIV) pathogenesis and therefore represents a potential therapeutic target to improve the patients' health status. Longitudinal alterations in the colonic mucosa-associated microbiome during simian immunodeficiency virus (SIV) infection were investigated using a 16S rRNA amplicon approach on the Illumina sequencing platform and bioinformatics analyses. Following SIV infection of six animals, no alterations in microbial composition were observed before the viral load peaked in the colon. At the time of acute mucosal SIV replication, the phylum Bacteroidetes including the Bacteroidia class as well as the phylum Firmicutes and its families Ruminococcaceae and Eubacteriaceae became more abundant. Enrichment of Bacteroidetes was maintained until the chronic phase of SIV infection. The shift towards Bacteroidetes in the mucosa-associated microbiome was associated with the extent of SIV infection-induced mucosal CD4+ T cell depletion and correlated with increasing rates of enterocyte damage. These observations suggest that Bacteroidetes strains increase during virus-induced mucosal immune destruction. As Bacteroidetes belong to the lipopolysaccharide- and short chain fatty acids-producing bacteria, their rapid enrichment may contribute to inflammatory tissue damage and metabolic alterations in SIV/HIV infection. These aspects should be considered in future studies on therapeutic interventions.

Comparison of Inflammatory Effects in THP-1 Monocytes and Macrophages after Exposure to Metal Ions.

Monocytes and macrophages are the first barrier of the innate immune system, which interact with abrasion and corrosion products, leading to the release of proinflammatory mediators and free reactive molecules. The aim of this study was to understand inflammation-relevant changes in monocytes and macrophages after exposure to corrosion products. To do this, the THP-1 cell line was used to analyze the effects of metal ions simultaneously in monocytes and differentiated macrophages. Cells were stimulated with several concentrations of metal salts (CoCl2, NiCl2, CrCl3 × 6H2O) to analyze viability, gene expression, protein release and ROS production. Untreated cells served as negative controls. While exposure to Cr(3+) did not influence cell viability in both cell types, the highest concentration (500 µM) of Co(2+) and Ni(2+) showed cytotoxic effects mirrored by significantly reduced metabolism, cell number and a concomitant increase of ROS. The release of IL-1ß, IL-8, MCP-1 and M-CSF proteins was mainly affected in macrophages after metal ion exposure (100 µM), indicating a higher impact on pro-inflammatory activity. Our results prove that monocytes and macrophages react very sensitively to corrosion products. High concentrations of bivalent ions lead to cell death, while lower concentrations trigger the release of inflammatory mediators, mainly in macrophages.

Analysis of Cellular Activity Short-Term Exposure to Cobalt and Chromium Ions in Mature Human Osteoblasts.

In aseptic loosening of endoprosthetic implants, metal particles, as well as their corrosion products, have been shown to elicit a biological response. Due to different metal alloy components, the response may vary depending on the nature of the released corrosion product. Our study aimed to compare the biological effects of different ions released from metal alloys. In order to mimic the corrosion products, different metal salts (CoCl2, NiCl2 and CrCl3 × 6H2O) were dissolved and allowed to equilibrate. Human osteoblasts were incubated with concentrations of 10 µM to 500 µM metal salt solutions under cell culture conditions, whereas untreated cells served as negative controls. Cells exposed to CoCr28Mo6 particles served as positive controls. The cell activity and expression of osteogenic differentiation and pro-osteolytic mediators were determined. Osteoblastic activity revealed concentration- and material-dependent influences. Collagen 1 synthesis was reduced after treatment with Co(2+) and Ni(2+). Additionally, exposure to these ions (500 µM) resulted in significantly reduced OPG protein synthesis, whereas RANKL as well as IL-6 and IL-8 secretion were increased. TLR4 mRNA was significantly induced by Co(2+) and CoCr28Mo6 particles. The results demonstrate the pro-osteolytic capacity of metal ions in osteoblasts. Compared to CoCr28Mo6 particles, the results indicated that metal ions intervene much earlier in inflammatory processes.

The 5'-nucleotidase S5nA is dispensable for evasion of phagocytosis and biofilm formation in Streptococcus pyogenes.

5'-nucleotidases are widespread among all domains of life. The enzymes hydrolyze phosphate residues from nucleotides and nucleotide derivatives. In some pathobiontic bacteria, 5'-nucleotidases contribute to immune evasion by dephosphorylating adenosine mono-, di-, or tri-phosphates, thereby either decreasing the concentration of pro-inflammatory ATP or increasing the concentration of anti-inflammatory adenosine, both acting on purinergic receptors of phagocytic cells. The strict human pathogen Streptococcus pyogenes expresses a surface-associated 5'-nucleotidase (S5nA) under infection conditions that has previously been discussed as a potential virulence factor. Here we show that deletion of the S5nA gene does not significantly affect growth in human blood, evasion of phagocytosis by neutrophils, formation of biofilms and virulence in an infection model with larvae of the greater wax moth Galleria mellonella in S. pyogenes serotypes M6, M18 and M49. Hence, the surface-associated 5'-nucleotidase S5nA seems dispensable for evasion of phagocytosis and biofilm formation in S. pyogenes.

Arginine-Depleting Enzymes - An Increasingly Recognized Treatment Strategy for Therapy-Refractory Malignancies.

Arginine auxotrophy occurs in certain tumor types and is usually caused by the silencing of argininosuccinate synthetase 1 or arginine lyase genes. Such tumors are often associated with an intrinsic chemoresistance and thus a poor prognosis. Arginine auxotrophy however renders these tumors vulnerable to treatment with arginine-degrading enzymes. Among the most frequently applied arginine-degrading agents are bacterial arginine deiminases (ADI). The anti-cancerous effects of ADI derived from different bacteria were extensively studied in numerous preclinical cell culture and xenograft models. Mycoplasma-derived ADI-PEG20 is most commonly used and is currently under clinical investigation as a single agent therapeutic as well as in combination with different antineoplastic compounds. Mechanistically, ADI is capable of reducing metabolic activity in tumor cells, contributing to autophagy, senescence and apoptosis in arginine auxotrophic cells. Although clinical trials are promising, the resistance development upon initial treatment response is an increasing challenge. Furthermore, interference of ADI with the tumor microenvironment is poorly understood. In the present review, we outline recent experimental ADI-based treatment approaches and their translation into the clinic. Furthermore, we summarize new insights into the molecular mechanisms underlying the anti-cancer effects of ADI that might facilitate the refinement of ADI-based combination therapy approaches.

Adipose-derived mesenchymal stem cells release microvesicles with procoagulant activity.

Extracellular vesicles are produced by a number of different cell types, among them mesenchymal stromal/stem cells (MSC) of different sources. It has been shown that extracellular vesicles of MSC exert similar therapeutic effects as the cells themselves. Here, we isolated and characterized extracellular vesicles produced by adipose-derived MSC (adMSC) in vitro upon stimulation with the proinflammatory substances lipopolysaccharide (LPS) and tumor necrosis factor (TNF). We found that the number of vesicles produced by adMSC does not change upon stimulation of the cells with LPS and TNF. Furthermore, adMSC-derived extracellular vesicles exert procoagulant activity independent of previous stimulation with LPS or TNF. We found evidence that the vesicles induce coagulation via both the intrinsic and the extrinsic pathway of coagulation.

Deciphering molecular mechanisms of arginine deiminase-based therapy - Comparative response analysis in paired human primary and recurrent glioblastomas.

Arginine auxotrophy constitutes the Achilles' heel for several tumors, among them glioblastoma multiforme (GBM). Hence, arginine-depleting enzymes such as arginine deiminase (ADI) from Streptococcus pyogenes are promising for treatment of primary and maybe even refractory GBM. Based on our previous study in which ADI-susceptibility was shown on a panel of patient-derived GBM cell lines, we here aimed at deciphering underlying molecular mechanisms of ADI-mediated growth inhibition. We found that ADI (35 mU/mL) initially induces a cellular stress-response that is characterized by upregulation of genes primarily belonging to the heat-shock protein family. In addition to autophagocytosis, we show for the first time that senescence constitutes another cellular response mechanism upon ADI-treatment and that this bacterial enzyme is able to act as radiosensitizer (» cases). Long-term treatment schedules revealed no resistance development, with treated cells showing morphological signs of cell stress. Next, several combination strategies were employed to optimize ADI-based treatment. Simultaneous and sequential S. pyogenes ADI-based combinations included substances acting at different molecular pathways (curcumin, resveratrol, quinacrine, and sorafenib, 2 × 72 h treatment). Adding drugs to GBM cell lines (n = 4, including a matched pair of primary and recurrent GBM in one case) accelerated and potentiated ADI-mediated cytotoxicity. Autophagy was identified as the main cause of tumor growth inhibition. Of note, residual cells again showed classical signs of senescence in most combinations. Our results suggest an alternative treatment regimen for this fatal cancer type which circumvents many of the traditional barriers. Using the metabolic defect in GBM thus warrants further (pre-) clinical evaluation.

Deletion of the L-Lactate Dehydrogenase Gene ldh in Streptococcus pyogenes Leads to a Loss of SpeB Activity and a Hypovirulent Phenotype.

Streptococcus pyogenes uses lactic acid fermentation for the generation of ATP. Here, we analyzed the impact of a deletion of the L-lactate dehydrogenase gene ldh on the virulence of S. pyogenes M49. While the ldh deletion does not cause a general growth deficiency in laboratory media, the growth in human blood and plasma is significantly hampered. The ldh deletion strain is furthermore less virulent in a Galleria mellonella infection model. We show that the ldh deletion leads to a decrease in the activity of the cysteine protease SpeB, an important secreted virulence factor of S. pyogenes. The reduced SpeB activity is caused by a hampered autocatalytic activation of the SpeB zymogen into the mature SpeB. The missing SpeB activity furthermore leads to increased plasmin activation and a reduced activation of the contact system on the surface of S. pyogenes. All these effects can be reversed when ldh is reintroduced into the mutant via a plasmid. The results demonstrate a previously unappreciated role for LDH in modulation of SpeB maturation.

Lipopolysaccharide induces proliferation and osteogenic differentiation of adipose-derived mesenchymal stromal cells in vitro via TLR4 activation.

Multipotent mesenchymal stromal cells (MSC) are capable of multi-lineage differentiation and support regenerative processes. In bacterial infections, resident MSC can come intocontact with and need to react to bacterial components. Lipopolysaccharide (LPS), a typical structure of Gram-negative bacteria, increases the proliferation and osteogenic differentiation of MSC. LPS is usually recognized by the toll-like receptor (TLR) 4 and induces pro-inflammatory reactions in numerous cell types. In this study, we quantified the protein expression of TLR4 and CD14 on adipose-derived MSC (adMSC) in osteogenic differentiation and investigated the effect of TLR4 activation by LPS on NF-κB activation, proliferation and osteogenic differentiation of adMSC. We found that TLR4 is expressed on adMSC whereas CD14 is not, and that osteogenic differentiation induced an increase of the amount of TLR4 protein whereas LPS stimulation did not. Moreover, we could show that NF-κB activation via TLR4 occurs upon LPS treatment. Furthermore, we were able to show that competitive inhibition of TLR4 completely abolished the stimulatory effect of LPS on the proliferation and osteogenic differentiation of adMSC. In addition, the inhibition of TLR4 leads to the complete absence of osteogenic differentiation of adMSC, even when osteogenically stimulated. Thus, we conclude that LPS induces proliferation and osteogenic differentiation of adMSC in vitro through the activation of TLR4 and that the TLR4 receptor seems to play a role during osteogenic differentiation of adMSC.

Phosphoglycerate Kinase-A Novel Streptococcal Factor Involved in Neutrophil Activation and Degranulation.

BACKGROUND: Neutrophils have been proposed as important contributors to the hyperinflammatory responses that are associated with severe invasive Streptococcus pyogenes infections. In particular, streptococcal surface proteins have been implicated as potent neutrophil activators. Here we explore the impact of streptococcus-secreted factors on neutrophil activation and degranulation. METHODS: Primary human neutrophils were exposed to supernatants prepared from cultures of invasive S. pyogenes strains of varying serotypes in the stationary growth phase. Neutrophil activation was assessed by measurement of secreted resistin, an azurophilic granule marker, and by determination of the secretome profile, using mass spectrometry. RESULTS: Marked variation in resistin release and the neutrophil secretome profile were observed following exposure to different strains. A high resistin response was triggered exclusively by SpeB-negative strains, suggesting that at least 1 stimulatory factor is susceptible to SpeB proteolytic degradation. Further analysis, including proteomics and stimulation analyses, identified phosphoglycerate kinase as a stimulatory factor for neutrophils. CONCLUSIONS: Taken together, results of this study reveal a novel secreted streptococcal factor that, in the absence of SpeB, can trigger neutrophil activation and degranulation. This finding is of interest in light of reports of hypervirulent SpeB-negative S. pyogenes variants present during invasive infections.

Genome-scale reconstruction of the Streptococcus pyogenes M49 metabolic network reveals growth requirements and indicates potential drug targets.

Genome-scale metabolic models comprise stoichiometric relations between metabolites, as well as associations between genes and metabolic reactions and facilitate the analysis of metabolism. We computationally reconstructed the metabolic network of the lactic acid bacterium Streptococcus pyogenes M49. Initially, we based the reconstruction on genome annotations and already existing and curated metabolic networks of Bacillus subtilis, Escherichia coli, Lactobacillus plantarum and Lactococcus lactis. This initial draft was manually curated with the final reconstruction accounting for 480 genes associated with 576 reactions and 558 metabolites. In order to constrain the model further, we performed growth experiments of wild type and arcA deletion strains of S. pyogenes M49 in a chemically defined medium and calculated nutrient uptake and production fluxes. We additionally performed amino acid auxotrophy experiments to test the consistency of the model. The established genome-scale model can be used to understand the growth requirements of the human pathogen S. pyogenes and define optimal and suboptimal conditions, but also to describe differences and similarities between S. pyogenes and related lactic acid bacteria such as L. lactis in order to find strategies to reduce the growth of the pathogen and propose drug targets.

Analysis of migration rate and chemotaxis of human adipose-derived mesenchymal stem cells in response to LPS and LTA in vitro.

Mesenchymal stem cells (MSC) are able to stimulate the regeneration of injured tissue. Since bacterial infections are common complications in wound healing, bacterial pathogens and their components come into direct contact with MSC. The interaction with bacterial structures influences the proliferation, differentiation and migratory activity of the MSC, which might be of relevance during regeneration. Studies on MSC migration in response to bacterial components have shown different results depending on the cell type. Here, we analyzed the migration rate and chemotaxis of human adipose-derived MSC (adMSC) in response to the basic cell-wall components lipopolysaccharide (LPS) of Gram-negative bacteria and lipoteichoic acid (LTA) of Gram-positive bacteria in vitro. To this end, we used transwell and scratch assays, as well as a specific chemotaxis assay combined with live-cell imaging. We found no significant influence of LPS or LTA on the migration rate of adMSC in transwell or scratch assays. Furthermore, in the µ-slide chemotaxis assay, the stimulation with LPS did not exert any chemotactic effect on adMSC.

Integrating highly quantitative proteomics and genome-scale metabolic modeling to study pH adaptation in the human pathogen Enterococcus faecalis.

Genome-scale metabolic models represent the entirety of metabolic reactions of an organism based on the annotation of the respective genome. These models commonly allow all reactions to proceed concurrently, disregarding the fact that at no point all proteins will be present in a cell. The metabolic reaction space can be constrained to a more physiological state using experimentally obtained information on enzyme abundances. However, high-quality, genome-wide protein measurements have been challenging and typically transcript abundances have been used as a surrogate for protein measurements. With recent developments in mass spectrometry-based proteomics, exemplified by SWATH-MS, the acquisition of highly quantitative proteome-wide data at reasonable throughput has come within reach. Here we present methodology to integrate such proteome-wide data into genome-scale models. We applied this methodology to study cellular changes in Enterococcus faecalis during adaptation to low pH. Our results indicate reduced proton production in the central metabolism and decreased membrane permeability for protons due to different membrane composition. We conclude that proteomic data constrain genome-scale models to a physiological state and, in return, genome-scale models are useful tools to contextualize proteomic data.

Arginine deprivation by arginine deiminase of Streptococcus pyogenes controls primary glioblastoma growth in vitro and in vivo.

Arginine auxotrophy constitutes a weak point of several tumors, among them glioblastoma multiforme (GBM). Hence, those tumors are supposed to be sensitive for arginine-depleting substances, such as arginine deiminase (ADI). Here we elucidated the sensitivity of patient-individual GBM cell lines toward Streptococcus pyogenes-derived ADI. To improve therapy, ADI was combined with currently established and pre-clinical cytostatic drugs. Additionally, effectiveness of local ADI therapy was determined in xenopatients. Half of the GBM cell lines tested responded well toward ADI monotherapy. In those cell lines, viability decreased significantly (up to 50%). Responding cell lines were subjected to combination therapy experiments to test if any additive or even synergistic effects may be achieved. Such promising results were obtained in 2/3 cases. In cell lines HROG02, HROG05 and HROG10, ADI and Palomid 529 combinations were most effective yielding more than 70% killing after 2 rounds of treatment. Comparable boosted antitumoral effects were observed after adding chloroquine to ADI (>60% killing). Apoptosis, as well as cell cycle dysregulation were found to play a minor role. In some, but clearly not all cases, (epi-) genetic silencing of arginine synthesis pathway genes (argininosuccinate synthetase 1 and argininosuccinate lyase) explained obtained results. In vivo, ADI as well as the combination of ADI and SAHA efficiently controlled HROG05 xenograft growth, whereas adding Palomid 529 to ADI did not further increase the strong antitumoral effect of ADI. The cumulative in vitro and in vivo results proved ADI as a very promising candidate therapeutic, especially for development of adjuvant GBM combination treatments.