Engineering of chimeric enzymes with expanded tolerance to ionic strength.

Antimicrobial resistance poses a significant global threat, reaching dangerously high levels as reported by the World Health Organization. The emergence and rapid spread of new resistance mechanisms, coupled with the absence of effective treatments in recent decades, have led to thousands of deaths annually from infections caused by drug-resistant microorganisms. Consequently, there is an urgent need for the development of new compounds capable of combating antibiotic-resistant bacteria. A promising class of molecules exhibiting potent bactericidal effects is peptidoglycan hydrolases. Previously, we cloned and characterized the biochemical properties of the M23 catalytic domain of the EnpA (EnpACD) protein from Enterococcus faecalis. Unlike other enzymes within the M23 family, EnpACD demonstrates broad specificity. However, its activity is constrained under low ionic strength conditions. In this study, we present the engineering of three chimeric enzymes comprising EnpACD fused with three distinct SH3b cell wall-binding domains. These chimeras exhibit enhanced tolerance to environmental conditions and sustained activity in bovine and human serum. Furthermore, our findings demonstrate that the addition of SH3b domains influences the activity of the chimeric enzymes, thereby expanding their potential applications in combating antimicrobial resistance.IMPORTANCEThese studies demonstrate that the addition of the SH3b-binding domain to the EnpACD results in generation of chimeras with a broader tolerance to ionic strength and pH values, enabling them to remain active over a wider range of conditions. Such approach offers a relatively straightforward method for obtaining antibacterial enzymes with tailored properties and emphasizes the potential for proteins' engineering with enhanced functionality, contributing to the ongoing efforts to address antimicrobial resistance effectively.

An essential protease, FtsH, influences daptomycin resistance acquisition in Enterococcus faecalis.

Daptomycin is a last-line antibiotic commonly used to treat vancomycin-resistant Enterococci, but resistance evolves rapidly and further restricts already limited treatment options. While genetic determinants associated with clinical daptomycin resistance (DAPR) have been described, information on factors affecting the speed of DAPR acquisition is limited. The multiple peptide resistance factor (MprF), a phosphatidylglycerol-modifying enzyme involved in cationic antimicrobial resistance, is linked to DAPR in pathogens such as methicillin-resistant Staphylococcus aureus. Since Enterococcus faecalis encodes two paralogs of mprF and clinical DAPR mutations do not map to mprF, we hypothesized that functional redundancy between the paralogs prevents mprF-mediated resistance and masks other evolutionary pathways to DAPR. Here, we performed in vitro evolution to DAPR in mprF mutant background. We discovered that the absence of mprF results in slowed DAPR evolution and is associated with inactivating mutations in ftsH, resulting in the depletion of the chaperone repressor HrcA. We also report that ftsH is essential in the parental, but not in the ΔmprF, strain where FtsH depletion results in growth impairment in the parental strain, a phenotype associated with reduced extracellular acidification and reduced ability for metabolic reduction. This presents FtsH and HrcA as enticing targets for developing anti-resistance strategies.

Targeting Enterococcus faecalis HMG-CoA reductase with a non-statin inhibitor.

HMG-CoA reductase (HMGR), a rate-limiting enzyme of the mevalonate pathway in Gram-positive pathogenic bacteria, is an attractive target for development of novel antibiotics. In this study, we report the crystal structures of HMGR from Enterococcus faecalis (efHMGR) in the apo and liganded forms, highlighting several unique features of this enzyme. Statins, which inhibit the human enzyme with nanomolar affinity, perform poorly against the bacterial HMGR homologs. We also report a potent competitive inhibitor (Chembridge2 ID 7828315 or compound 315) of the efHMGR enzyme identified by a high-throughput, in-vitro screening. The X-ray crystal structure of efHMGR in complex with 315 was determined to 1.27 Å resolution revealing that the inhibitor occupies the mevalonate-binding site and interacts with several key active site residues conserved among bacterial homologs. Importantly, 315 does not inhibit the human HMGR. Our identification of a selective, non-statin inhibitor of bacterial HMG-CoA reductases will be instrumental in lead optimization and development of novel antibacterial drug candidates.

Identification of HPr kinase/phosphorylase inhibitors: novel antimicrobials against resistant Enterococcus faecalis.

Enterococcus faecalis, a gram-positive bacterium, is among the most common nosocomial pathogens due to its limited susceptibility to antibiotics and its reservoir of the genes coding for virulence factors. Bacterial enzymes such as kinases and phosphorylases play important roles in diverse functions of a bacterial cell and, thus, are potential antibacterial drug targets. In Gram-positive bacteria, HPr Kinase/Phosphorylase (HPrK/P), a bifunctional enzyme is involved in the regulation of carbon catabolite repression by phosphorylating/dephosphorylating the histidine-containing phosphocarrier protein (HPr) at Ser46 residue. Deficiencies in HPrK/P function leads to severe defects in bacterial growth. This study aimed at identifying novel inhibitors of E. faecalis HPrK/P from a commercial compound library using structure-based virtual screening. The hit molecules were purchased and their effect on enzyme activity and growth of resistant E. faecalis was evaluated in vitro. Furthermore, docking and molecular dynamics simulations were performed to study the interactions of the hit compounds with HPrK/P. Among the identified hit molecules, two compounds inhibited the phosphorylation of HPr as well as significantly reduced the growth of resistant E. faecalis in vitro. These identified potential HPrK/P inhibitors open new research avenues towards the development of novel antimicrobials against resistant Gram-positive bacteria.

Molecular basis for substrate recognition and septum cleavage by AtlA, the major N-acetylglucosaminidase of Enterococcus faecalis.

The cleavage of septal peptidoglycan at the end of cell division facilitates the separation of the two daughter cells. The hydrolases involved in this process (called autolysins) are potentially lethal enzymes that can cause cell death; their activity, therefore, must be tightly controlled during cell growth. In Enterococcus faecalis, the N-acetylglucosaminidase AtlA plays a predominant role in cell separation. atlA mutants form long cell chains and are significantly less virulent in the zebrafish model of infection. The attenuated virulence of atlA mutants is underpinned by a limited dissemination of bacterial chains in the host organism and a more efficient uptake by phagocytes that clear the infection. AtlA has structural homologs in other important pathogens, such as Listeria monocytogenes and Salmonella typhimurium, and therefore represents an attractive model to design new inhibitors of bacterial pathogenesis. Here, we provide a 1.45 Å crystal structure of the E. faecalis AtlA catalytic domain that reveals a closed conformation of a conserved ß-hairpin and a complex network of hydrogen bonds that bring two catalytic residues to the ideal distance for an inverting mechanism. Based on the model of the AtlA-substrate complex, we identify key residues critical for substrate recognition and septum cleavage during bacterial growth. We propose that this work will provide useful information for the rational design of specific inhibitors targeting this enterococcal virulence factor and its orthologs in other pathogens.

Engineering acetyl-CoA supply and ERG9 repression to enhance mevalonate production in Saccharomyces cerevisiae.

Mevalonate is a key precursor in isoprenoid biosynthesis and a promising commodity chemical. Although mevalonate is a native metabolite in Saccharomyces cerevisiae, its production is challenged by the relatively low flux toward acetyl-CoA in this yeast. In this study we explore different approaches to increase acetyl-CoA supply in S. cerevisiae to boost mevalonate production. Stable integration of a feedback-insensitive acetyl-CoA synthetase (Se-acsL641P) from Salmonella enterica and the mevalonate pathway from Enterococcus faecalis results in the production of 1,390 ± 10 mg/l of mevalonate from glucose. While bifid shunt enzymes failed to improve titers in high-producing strains, inhibition of squalene synthase (ERG9) results in a significant enhancement. Finally, increasing coenzyme A (CoA) biosynthesis by overexpression of pantothenate kinase (CAB1) and pantothenate supplementation further increased production to 3,830 ± 120 mg/l. Using strains that combine these strategies in lab-scale bioreactors results in the production of 13.3 ± 0.5 g/l, which is ∼360-fold higher than previously reported mevalonate titers in yeast. This study demonstrates the feasibility of engineering S. cerevisiae for high-level mevalonate production.

In silico prediction, molecular docking and binding studies of acetaminophen and dexamethasone to Enterococcus faecalis diaminopimelate epimerase.

Enterococcus faecalis (E. faecalis) is a Gram-positive coccoid, non-sporulating, facultative anaerobic, multidrug resistance bacterium responsible for almost 65% to 80% of all enterococcal nosocomial infections. It usually causes infective endocarditis, urinary tract and surgical wound infections. The increase in E. faecalis resistance to conventionally available antibiotic has rekindled intense interest in developing useful antibacterial drugs. In E. faecalis, diaminopimelate epimerase (DapF) is involved in the lysine biosynthetic pathway. The product of this pathway is precursors of peptidoglycan synthesis, which is a component of bacterial cell wall. Also, because mammals lack this enzyme, consequently E. faecalis diaminopimelate epimerase (EfDapF) represents a potential target for developing novel class of antibiotics. In this regard, we have successfully cloned, overexpressed the gene encoding DapF in BL-21(DE3) and purified with Ni-NTA Agarose resin. In addition to this, binding studies were performed using fluorescence spectroscopy in order to confirm the bindings of the identified lead compounds (acetaminophen and dexamethasone) with EfDapF. Docking studies revealed that acetaminophen found to make hydrogen bonds with Asn72 and Asn13 while dexamethasone interacted by forming hydrogen bonds with Asn205 and Glu223. Thus, biochemical studies indicated acetaminophen and dexamethasone, as potential inhibitors of EfDapF and eventually can reduce the catalytic activity of EfDapF.

Inactivation of GalU Leads to a Cell Wall-Associated Polysaccharide Defect That Reduces the Susceptibility of Enterococcus faecalis to Bacteriolytic Agents.

Enterococcal plasmid-encoded bacteriolysin Bac41 is a selective antimicrobial system that is considered to provide a competitive advantage to Enterococcus faecalis cells that carry the Bac41-coding plasmid. The Bac41 effector consists of the secreted proteins BacL1 and BacA, which attack the cell wall of the target E. faecalis cell to induce bacteriolysis. Here, we demonstrated that galU, which encodes UTP-glucose-1-phosphate uridylyltransferase, is involved in susceptibility to the Bac41 system in E. faecalis Spontaneous mutants that developed resistance to the antimicrobial effects of BacL1 and BacA were revealed to carry a truncation deletion of the C-terminal amino acid (aa) region 288 to 298 of the translated GalU protein. This truncation resulted in the depletion of UDP-glucose, leading to a failure to utilize galactose and produce the enterococcal polysaccharide antigen (EPA), which is expressed abundantly on the cell surface of E. faecalis This cell surface composition defect that resulted from galU or EPA-specific genes caused an abnormal cell morphology, with impaired polarity during cell division and alterations of the limited localization of BacL1 Interestingly, these mutants had reduced susceptibility to beta-lactams besides Bac41, despite their increased susceptibility to other bacteriostatic antimicrobial agents and chemical detergents. These data suggest that a complex mechanism of action underlies lytic killing, as exogenous bacteriolysis induced by lytic bacteriocins or beta-lactams requires an intact cell physiology in E. faecalisIMPORTANCE Cell wall-associated polysaccharides of bacteria are involved in various physiological characteristics. Recent studies demonstrated that the cell wall-associated polysaccharide of Enterococcus faecalis is required for susceptibility to bactericidal antibiotic agents. Here, we demonstrated that a galU mutation resulted in resistance to the enterococcal lytic bacteriocin Bac41. The galU homologue is reported to be essential for the biosynthesis of species-specific cell wall-associated polysaccharides in other Firmicutes In E. faecalis, the galU mutant lost the E. faecalis-specific cell wall-associated polysaccharide EPA (enterococcal polysaccharide antigen). The mutant also displayed reduced susceptibility to antibacterial agents and an abnormal cell morphology. We demonstrated here that galU was essential for EPA biosynthesis in E. faecalis, and EPA production might underlie susceptibility to lytic bacteriocin and antibiotic agents by undefined mechanisms.

Exploring the selenium-over-sulfur substrate specificity and kinetics of a bacterial selenocysteine lyase.

Selenium is a vital micronutrient in many organisms. While traces are required for microbial utilization, excess amounts are toxic; thus, selenium can be regarded as a biological double-edged sword. Selenium is chemically similar to the essential element sulfur, but curiously, evolution has selected the former over the latter for a subset of oxidoreductases. Enzymes involved in sulfur metabolism are less discriminate in terms of preventing selenium incorporation; however, its specific incorporation into selenoproteins reveals a highly discriminate process that is not completely understood. We have identified SclA, a NifS-like protein in the nosocomial pathogen, Enterococcus faecalis, and characterized its enzymatic activity and specificity for l-selenocysteine over l-cysteine. It is known that Asp-146 is required for selenocysteine specificity in the human selenocysteine lyase. Thus, using computational biology, we compared the bacterial and mammalian enzymes and identified His-100, an Asp-146 ortholog in SclA, and generated site-directed mutants in order to study the residue's potential role in the l-selenocysteine discrimination mechanism. The proteins were overexpressed, purified, and characterized for their biochemical properties. All mutants exhibited varying Michaelis-Menten behavior towards l-selenocysteine, but His-100 was not found to be essential for this activity. Additionally, l-cysteine acted as a competitive inhibitor of all enzymes with higher affinity than l-selenocysteine. Finally, we discovered that SclA exhibited low activity with l-cysteine as a poor substrate regardless of mutations. We conclude that His-100 is not required for l-selenocysteine specificity, underscoring the inherent differences in discriminatory mechanisms between bacterial NifS-like proteins and mammalian selenocysteine lyases.

Realgar increases defenses against infection by Enterococcus faecalis in Caenorhabditis elegans.

ETHNOPHARMACOLOGICAL RELEVANCE: Realgar has been used in traditional remedies for a long history in China and India. It is clinically used to treat diverse cancers, especially acute promyelocytic leukemia (APL), chronic myelogenous leukemia (CML) in China. However, paradoxic roles of realgar to increase or decrease immunity are reported. It is urgent to address this question, due to immune depression can be strongly benefit to cancer development, but detrimental to patients. AIM OF THE STUDY: This present work is to explore whether realgar promote or suppress immune responses, and shed light on its mode of action. Our results should provide cues for rational strategy to explore realgar for clinical use. MATERIAL AND METHODS: Infection model in vivo was established by using Enterococcus faecalis to attack Caenorhabditis elegans, then realgar was used to treat the infected worms to investigate its effects on infectivity and the underlying mechanism. Killing analysis was carried out to test whether realgar can mitigate worm infection. Thermotolerance resistance analysis was used to evaluate if realgar functions hormetic effect. Quantification of live E. faecalis in nematode intestine was employed to ascertain if realgar alleviate the bacterial load in worm gut. Quantitative real-time PCR (qRT-PCR) was used to test the expression of antibacterial effectors. Western blot was used to test the effect of realgar on the expressions of p38 and phospho-p38 in worms infected by E. faecalis. RESULTS: Realgar alleviated the infected worms in strains of N2, glp-4, and daf-2, but failed in sek-1, glp-4; sek-1, and daf-2; daf-16 when p38 MAPK or daf-16 was blocked or inactivated. Western blot assay demonstrated that realgar increased the expression of phosph-p38. Thermotolerance assay showed that realgar played a hormetic role on nemtodes, triggered protective response and reduced bacterial load after realgar treatment for 120 h qRT-PCR demonstrated that realgar significantly increased antibacterial effectors, thus leading to pathogen elimination. CONCLUSION: Realgar increased defenses against E. faecalis in C. elegans by inducing both immune responses and protective responses. It was regulated by p38 MAPK pathway and DAF-16.

Group II Introns Generate Functional Chimeric Relaxase Enzymes with Modified Specificities through Exon Shuffling at Both the RNA and DNA Level.

Group II introns are large self-splicing RNA enzymes with a broad but somewhat irregular phylogenetic distribution. These ancient retromobile elements are the proposed ancestors of approximately half the human genome, including the abundant spliceosomal introns and non-long terminal repeat retrotransposons. In contrast to their eukaryotic derivatives, bacterial group II introns have largely been considered as harmful selfish mobile retroelements that parasitize the genome of their host. As a challenge to this view, we recently uncovered a new intergenic trans-splicing pathway that generates an assortment of mRNA chimeras. The ability of group II introns to combine disparate mRNA fragments was proposed to increase the genetic diversity of the bacterial host by shuffling coding sequences. Here, we show that the Ll.LtrB and Ef.PcfG group II introns from Lactococcus lactis and Enterococcus faecalis respectively can both use the intergenic trans-splicing pathway to catalyze the formation of chimeric relaxase mRNAs and functional proteins. We demonstrated that some of these compound relaxase enzymes yield gain-of-function phenotypes, being significantly more efficient than their precursor wild-type enzymes at supporting bacterial conjugation. We also found that relaxase enzymes with shuffled functional domains are produced in biologically relevant settings under natural expression levels. Finally, we uncovered examples of lactococcal chimeric relaxase genes with junctions exactly at the intron insertion site. Overall, our work demonstrates that the genetic diversity generated by group II introns, at the RNA level by intergenic trans-splicing and at the DNA level by recombination, can yield new functional enzymes with shuffled exons, which can lead to gain-of-function phenotypes.

Visualizing the enzyme mechanism of mevalonate diphosphate decarboxylase.

Mevalonate diphosphate decarboxylases (MDDs) catalyze the ATP-dependent-Mg2+-decarboxylation of mevalonate-5-diphosphate (MVAPP) to produce isopentenyl diphosphate (IPP), which is essential in both eukaryotes and prokaryotes for polyisoprenoid synthesis. The substrates, MVAPP and ATP, have been shown to bind sequentially to MDD. Here we report crystals in which the enzyme remains active, allowing the visualization of conformational changes in Enterococcus faecalis MDD that describe sequential steps in an induced fit enzymatic reaction. Initial binding of MVAPP modulates the ATP binding pocket with a large loop movement. Upon ATP binding, a phosphate binding loop bends over the active site to recognize ATP and bring the molecules to their catalytically favored configuration. Positioned substrates then can chelate two Mg2+ ions for the two steps of the reaction. Closure of the active site entrance brings a conserved lysine to trigger dissociative phosphoryl transfer of γ-phosphate from ATP to MVAPP, followed by the production of IPP.

Prevalence of High-Level Aminoglycoside Resistance and Genes Encoding Aminoglycoside-Modifying Enzymes in Enterococcus faecalis and Enterococcus faecium Isolated in a University Hospital in Tokyo.

High-level aminoglycoside resistance (HLAR) limits treatment options for invasive enterococcal infections. We examined the prevalence of HLAR, carriage of genes encoding aminoglycoside-modifying enzymes, and production of ß-lactamase using the disk diffusion method, polymerase chain reaction, and a nitrocefin-based test, respectively, in Enterococcus faecalis and Enterococcus faecium isolated from patients at a university hospital in Tokyo in 2010. Of the 100 E. faecalis isolates analyzed, 30 isolates had high-level resistance (HLR) to gentamicin, and 22 isolates had HLR to streptomycin. Of the 40 E. faecium isolates analyzed, 9 isolates had HLR to gentamicin, and 9 isolates had HLR to streptomycin. Of the 39 gentamicin-HLR enterococcal isolates, 24 isolates were non-HLR to streptomycin. All 39 isolates with HLR to gentamicin as well as 19 of 101 without HLR carried aac(6')-Ie-aph(2'')-Ia. Carriage of ant(6')-Ia was confirmed in 25 of 31 streptomycin-HLR isolates. Production of ß-lactamase was documented in none of the E. faecalis and E. faecium isolates. Whole-genome sequencing analysis revealed that all but one E. faecalis isolate that carried aac(6')-Ie-aph(2'')-Ia and ant(6')-Ia belonged to sequence type (ST) 4 (n = 8), ST16 (n = 4), or ST179 (n = 9). Nevertheless, most of the pairs of isolates had > 10 single-nucleotide polymorphisms even among the isolates of the same ST.

Real-time observation of CRISPR spacer acquisition by Cas1-Cas2 integrase.

Cas1 integrase associates with Cas2 to insert short DNA fragments into a CRISPR array, establishing nucleic acid memory in prokaryotes. Here we applied single-molecule FRET methods to the Enterococcus faecalis (Efa) Cas1-Cas2 system to establish a kinetic framework describing target-searching, integration, and post-synapsis events. EfaCas1-Cas2 on its own is not able to find the CRISPR repeat in the CRISPR array; it only does so after prespacer loading. The leader sequence adjacent to the repeat further stabilizes EfaCas1-Cas2 contacts, enabling leader-side integration and subsequent spacer-side integration. The resulting post-synaptic complex (PSC) has a surprisingly short mean lifetime. Remarkably, transcription effectively resolves the PSC, and we predict that this is a conserved mechanism that ensures efficient and directional spacer integration in many CRISPR systems. Overall, our study provides a complete model of spacer acquisition, which can be harnessed for DNA-based information storage and cell lineage tracing technologies.

Crystal structure of the Enterococcus faecalis α-N-acetylgalactosaminidase, a member of the glycoside hydrolase family 31.

Glycoside hydrolases catalyze the hydrolysis of glycosidic linkages in carbohydrates. The glycoside hydrolase family 31 (GH31) contains α-glucosidase, α-xylosidase, α-galactosidase, and α-transglycosylase. Recent work has expanded the diversity of substrate specificity of GH31 enzymes, and α-N-acetylgalactosaminidases (αGalNAcases) belonging to GH31 have been identified in human gut bacteria. Here, we determined the first crystal structure of a truncated form of GH31 αGalNAcase from the human gut bacterium Enterococcus faecalis. The enzyme has a similar fold to other reported GH31 enzymes and an additional fibronectin type 3-like domain. Additionally, the structure in complex with N-acetylgalactosamine reveals that conformations of the active site residues, including its catalytic nucleophile, change to recognize the ligand. Our structural analysis provides insight into the substrate recognition and catalytic mechanism of GH31 αGalNAcases.

Comparative genetics of Enterococcus faecalis intestinal tissue isolates before and after surgery in a rat model of colon anastomosis.

We have recently demonstrated that collagenolytic Enterococcus faecalis plays a key and causative role in the pathogenesis of anastomotic leak, an uncommon but potentially lethal complication characterized by disruption of the intestinal wound following segmental removal of the colon (resection) and its reconnection (anastomosis). Here we hypothesized that comparative genetic analysis of E. faecalis isolates present at the anastomotic wound site before and after surgery would shed insight into the mechanisms by which collagenolytic strains are selected for and predominate at sites of anastomotic disruption. Whole genome optical mapping of four pairs of isolates from rat colonic tissue obtained following surgical resection (herein named "pre-op" isolates) and then 6 days later from the anastomotic site (herein named "post-op" isolates) demonstrated that the isolates with higher collagenolytic activity formed a distinct cluster. In order to perform analysis at a deeper level, a single pair of E. faecalis isolates (16A pre-op and 16A post-op) was selected for whole genome sequencing and assembled using a hybrid assembly algorithm. Comparative genomics demonstrated absence of multiple gene clusters, notably a pathogenicity island in the post-op isolate. No differences were found in the fsr-gelE-sprE genes (EF1817-1822) responsible for regulation and production of collagenolytic activity. Analysis of unique genes among the 16A pre-op and post-op isolates revealed the predominance of transporter systems-related genes in the pre-op isolate and phage-related and hydrolytic enzyme-encoding genes in the post-op isolate. Despite genetic differences observed between pre-op and post-op isolates, the precise genetic determinants responsible for their differential expression of collagenolytic activity remains unknown.

Detection of Bacterial Alkaline Phosphatase Activity by Enzymatic In Situ Self-Assembly of the AIEgen-Peptide Conjugate.

Abnormal levels of alkaline phosphatase (ALP) activity are associated with various diseases, and many ALP probes have been developed to date. However, the development of ALP-sensitive probes for living cells, especially for the detection of bacterial ALP, remains challenging because of the complex and dynamic context. In this study, we constructed the first fluorescent probe (TPEPy-pY) for sensing bacterial ALP activity. TPEPy-pY is an AIEgen-peptide conjugate with property of aggregation-induced emission (AIE) and could turn on its fluorescence by ALP-catalyzed in situ self-assembly of the probe. The probe shows excellent selectivity and sensitivity for ALP activity, with a detection limit of 6.6 × 10-3 U mL-1. TPEPy-pY performs well in detection and in situ imaging of bacterial ALP activity against E. coli. Also, the detection does not require tedious washing steps and takes approximately 1 h, which is advantageous over commercial ALP kits. Therefore, the proposed strategy paved a new avenue for bacterial ALP detection, and we envision that more self-assembling fluorescent probes will be designed with higher sensitivity in the near future.

An in vitro tissue model for screening sustained release of phosphate-based therapeutic attenuation of pathogen-induced proteolytic matrix degradation.

Tissue response to intestinal injury or disease releases pro-inflammatory host stress signals triggering microbial shift to pathogenic phenotypes. One such phenotype is increased protease production resulting in collagen degradation and activation of host matrix metalloproteinases contributing to tissue breakdown. We have shown that surgical injury depletes local intestinal phosphate concentration triggering bacterial virulence and that polyphosphate replenishment attenuates virulence and collagenolytic activity. Mechanistic studies of bacterial and host protease expression contributing to tissue breakdown are difficult to achieve in vivo necessitating the development of novel in vitro tissue models. Common techniques for screening in vitro protease activity, including gelatin zymography or fluorogenic protease-sensitive substrate kits, do not readily translate to 3D matrix degradation. Here, we report the application of an in vitro assay in which collagenolytic pathogens are cultured in the presence of a proteolytically degradable poly(ethylene) glycol scaffold and a non-degradable phosphate and/or polyphosphate nanocomposite hydrogel matrix. This in vitro platform enables quantification of pathogen-induced matrix degradation and screening of sustained release of phosphate-based therapeutic efficacy in attenuating protease expression. To evaluate matrix degradation as a function of bacterial enzyme levels secreted, we also present a novel method to quantify hydrogel degradation. This method involves staining protease-sensitive hydrogels with Sirius red dye to correlate absorbance of the degraded gel solution with hydrogel weight. This assay enables continuous monitoring and greater accuracy of hydrogel degradation kinetics compared to gravimetric measurements. Combined, the proposed in vitro platform and the presented degradation assay provide a novel strategy for screening efficacy of therapeutics in attenuating bacterial protease-induced matrix degradation.

Degradation of the Incretin Hormone Glucagon-Like Peptide-1 (GLP-1) by Enterococcus faecalis Metalloprotease GelE.

Metabolic diseases, including type 2 diabetes and obesity, have become increasingly prevalent global health concerns. Studies over the past decade have established connections between the gastrointestinal microbiota and host metabolism, but the mechanisms behind these connections are only beginning to be understood. We were interested in identifying microbes that have the ability to modulate the levels of the incretin hormone glucagon-like peptide-1 (GLP-1). Using a human-derived cell line that is capable of secreting GLP-1 in response to stimulatory ligands (NCI-H716), we identified supernatants from several bacterial isolates that were capable of decreasing GLP-1 levels, including several strains of Enterococcus faecalis We further identified the secreted protease GelE, an established virulence factor from E. faecalis, as being responsible for GLP-1 inhibition via direct cleavage of GLP-1 by GelE. Finally, we demonstrated that E. faecalis supernatants can disrupt a colonic epithelial monolayer and cleave GLP-1 in a gelE-dependent manner. This work suggests that a secreted factor from an intestinal microbe can traverse the epithelial barrier and impact levels of an important intestinal hormone.IMPORTANCE Humans have a complex and interconnected relationship with their gastrointestinal microbiomes, yet our interest in the microbiome tends to focus on overt pathogenic or probiotic activities, leaving the roles that commensal species may have on host physiology and metabolic processes largely unexplored. Commensal organisms in the microbiome produce and secrete many factors that have an opportunity to interact with the gastrointestinal tract and host biology. Here, we show that a secreted protease from E. faecalis, GelE, is able to degrade the gastrointestinal hormone GLP-1, which is responsible for regulating glucose homeostasis and appetite in the body. The disruption of natural GLP-1 signaling by GelE may have significant consequences for maintaining healthy blood glucose levels and in the development of metabolic disease. Furthermore, this work deepens our understanding of specific host-microbiome interactions.

Integrated approach for obtaining bioactive peptides from whey proteins hydrolysed using a new proteolytic lactic acid bacteria.

In this study, we investigated the ability of Enterococcus faecalis 2/28, isolated from artisan cheese, to release biopeptides from whey proteins. We used an in silico approach for predicting the bioactivities of peptides generated by E. faecalis. The results of the in vitro study showed that the whey protein hydrolysates (WPHs) obtained had angiotensin-I-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory activities, with inhibition of ACE being stronger than that of DPP-IV. To identify peptides that may be potential inhibitors of ACE, WPH with the highest ACE inhibitory activity was analysed using Sephadex G-75 gel filtration chromatography, Superdex peptide 10/300 GL size exclusion chromatography, and liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS). Among the identified peptides were ACE-inhibitory peptides (LDAQSAPLR, LKGYGGVSLPEW, and LKALPMH), antimicrobial peptides (AASDISLLDAQSAPLR, IIAEKTKIPAVF, IDALNENK, and VLVLDTDYK), DPP-IV-inhibitory peptides (LKALPMH, LKPTPEGDLEIL, LKGYGGVSLPE, LKPTPEGDLE, ILDKVGINY, and VLVLDTDYK), proliferation stimulating peptide (IDALNENK), and cytotoxic peptide (LIVTQTMK).