scholarly journals Production, Secretion, and Cell Surface Display of Recombinant Sporosarcina ureae S-Layer Fusion Proteins in Bacillus megaterium

2011 ◽  
Vol 78 (2) ◽  
pp. 560-567 ◽  
Author(s):  
Denise Knobloch ◽  
Kai Ostermann ◽  
Gerhard Rödel
Keyword(s):  
Cell Surface ◽  
Bacillus Megaterium ◽  
Fusion Proteins ◽  
Shuttle Vector ◽  
Surface Display ◽  
Fluorescent Labeling ◽  
Content Type ◽  
Sporosarcina Ureae

ABSTRACTMonomolecular crystalline bacterial cell surface layers (S-layers) have broad application potential in nanobiotechnology due to their ability to generate functional supramolecular structures. Here, we report thatBacillus megateriumis an excellent host organism for the heterologous expression and efficient secretion of hemagglutinin (HA) epitope-tagged versions of the S-layer protein SslA fromSporosarcina ureaeATCC 13881. Three chimeric proteins were constructed, comprising the precursor, C-terminally truncated, and N- and C-terminally truncated forms of the S-layer SslA protein tagged with the human influenza hemagglutinin epitope. For secretion of fusion proteins, the open reading frames were cloned into theEscherichia coli-Bacillus megateriumshuttle vector pHIS1525. After transformation of the respective plasmids intoBacillus megateriumprotoplasts, the recombinant genes were successfully expressed and the proteins were secreted into the growth medium. The isolated S-layer proteins are able to assemblein vitrointo highly ordered, crystalline, sheetlike structures with the fused HA tag accessible to antibody. We further show by fluorescent labeling that the secreted S-layer fusion proteins are also clustered on the cell envelope ofBacillus megaterium, indicating that the cell surface can servein vivoas a nucleation point for crystallization. Thus, this system can be used as a display system that allows the dense and periodic presentation of S-layer proteins or the fused tags.

scholarly journals Use of an Endogenous Plasmid Locus for Stable intransComplementation in Borrelia burgdorferi

2014 ◽  
Vol 81 (3) ◽  
pp. 1038-1046 ◽  
Author(s):  
Irene N. Kasumba ◽  
Aaron Bestor ◽  
Kit Tilly ◽  
Patricia A. Rosa
Keyword(s):  
Borrelia Burgdorferi ◽  
Shuttle Vector ◽  
Targeted Mutagenesis ◽  
Multiple Cloning Site ◽  
Stable Gene ◽  
Content Type ◽  
Shuttle Vectors ◽  
Stable Maintenance

ABSTRACTTargeted mutagenesis and complementation are important tools for studying genes of unknown function in the Lyme disease spirocheteBorrelia burgdorferi. A standard method of complementation is reintroduction of a wild-type copy of the targeted gene on a shuttle vector. However, shuttle vectors are present at higher copy numbers thanB. burgdorferiplasmids and are potentially unstable in the absence of selection, thereby complicating analyses in the mouse-tick infectious cycle.B. burgdorferihas over 20 plasmids, with some, such as linear plasmid 25 (lp25), carrying genes required by the spirochetein vivobut relatively unstable duringin vitrocultivation. We propose that complementation on an endogenous plasmid such as lp25 would overcome the copy number andin vivostability issues of shuttle vectors. In addition, insertion of a selectable marker on lp25 could ensure its stable maintenance by spirochetes in culture. Here, we describe the construction of a multipurpose allelic-exchange vector containing a multiple-cloning site and either of two selectable markers. This suicide vector directs insertion of the complementing gene into thebbe02locus, a site on lp25 that was previously shown to be nonessential during bothin vitroandin vivogrowth. We demonstrate the functional utility of this strategy by restoring infectivity to anospCmutant through complementation at this site on lp25 and stable maintenance of theospCgene throughout mouse infection. We conclude that this represents a convenient and widely applicable method for stable gene complementation inB. burgdorferi.

scholarly journals Dissecting the Polyhydroxyalkanoate-Binding Domain of the PhaF Phasin: Rational Design of a Minimized Affinity Tag

2020 ◽  
Vol 86 (12) ◽  
Author(s):  
Aranzazu Mato ◽  
Francisco G. Blanco ◽  
Beatriz Maestro ◽  
Jesús M. Sanz ◽  
Jesús Pérez-Gil ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Rational Design ◽  
Complex Structure ◽  
Helical Conformation ◽  
Affinity Tag ◽  
Binding Properties ◽  
Content Type ◽  
Pha Granule

ABSTRACT Phasin PhaF from Pseudomonas putida consists of a modular protein whose N-terminal domain (BioF) has been demonstrated to be responsible for binding to the polyhydroxyalkanoate (PHA) granule. BioF has been exploited for biotechnological purposes as an affinity tag in the functionalization of PHA beads with fusion proteins both in vivo and in vitro. The structural model of this domain suggests an amphipathic α-helical conformation with the hydrophobic residues facing the PHA granule. In this work, we analyzed the mean hydrophobicity and the hydrophobic moment of the native BioF tag to rationally design shorter versions that maintain affinity for the granule. Hybrid proteins containing the green fluorescent protein (GFP) fused to the BioF derivatives were studied for in vivo localization on PHA, stability on the surface of the PHA granule against pH, temperature, and ionic strength, and their possible influence on PHA synthesis. Based on the results obtained, a minimized BioF tag for PHA functionalization has been proposed (MinP) that retains similar binding properties but possesses an attractive biotechnological potential derived from its reduced size. The MinP tag was further validated by analyzing the functionality and stability of the fusion proteins MinP–β-galactosidase and MinP-CueO from Escherichia coli. IMPORTANCE Polyhydroxyalkanoates (PHAs) are biocompatible, nontoxic, and biodegradable biopolymers with exceptional applications in the industrial and medical fields. The complex structure of the PHA granule can be exploited as a toolbox to display molecules of interest on their surface. Phasins, the most abundant group of proteins on the granule, have been employed as anchoring tags to obtain functionalized PHA beads for high-affinity bioseparation, enzyme immobilization, diagnostics, or cell targeting. Here, a shorter module based on the previously designed BioF tag has been demonstrated to maintain the affinity for the PHA granule, with higher stability and similar functionalization efficiency. The use of a 67% shorter peptide, which maintains the binding properties of the entire protein, constitutes an advantage for the immobilization of recombinant proteins on the PHA surface both in vitro and in vivo.

Separation of GvH- and GvL-Immune Responses in Allogeneic Stem Cell Transplantation by CD95-Mediated Depletion of Alloreactive T-Cells Using CD178 Based Proapoptotic Chimeric Molecules.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3053-3053
Author(s):  
Marcus Kaltwasser ◽  
Matthias Lehne ◽  
Harald Wajant ◽  
Christoph Huber ◽  
Udo F. Hartwig
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Cell Surface ◽  
Stem Cell Transplantation ◽  
Fusion Proteins ◽  
Alloreactive T Cells ◽  
Donor T Cells ◽  
Chimeric Molecules

Abstract Depletion of donor T cells (TCD) from the graft effectively ameliorates graft-vs-host (GvH) disease (GvHD) in allogeneic stem cell transplantation (SCT). TCD, however, is associated with impaired engraftment, immunosuppression, and abrogation of the beneficial graft-vs-leukemia (GvL) effect. Thus, based on previous results (Hartwig et al., Blood 2002) to eliminate alloreactive specificities but preserve oligoclonal antiviral- and GvL- immunity we explored proapoptotic chimeric molecules composed of the extracellular domains of human CD178 (FasL) and CD28 or RANK to selectively deplete GvH-reactive T cells by AICD in vitro and in vivo. As these fusion proteins are expressed as soluble Flag-tagged trimers and reconstitute bioactivity only upon binding to well defined cell-surface molecules (Samel et al., J. Biol. Chem., 2003), they can be specifically targeted onto e.g. recipient derived DC or donor T cells via the molecules CD80/86 or RANKL/TRANCE, respectively. Using an MHC-mismatched murine BMT model proliferative responses of purified CD90+ alloreactive T cells from C57BL/6 (H-2b) mice were strongly reduced following stimulation with DC from F1 (C57BL/6xBalb/c; H-2bxd) mice in vitro in the presence of CD178-CD28 or CD178-RANK to levels comparably obtained with agonistic anti-CD95 mAb (Jo2). Depletion was specific to alloantigen since H-2d restricted Hemagglutinin (HA)-peptide specific T cells titrated into the system persisted among retained T cells. In addition, adoptive transfer experiments of H-2b derived CD90+ T cells depleted of alloreactivity in vitro prior to injection into irradiated F1 recipients confirmed the in vitro results as these mice did not develop clinical signs of GvHD as compared to controls. Moreover, in current studies the CD178-fusion proteins are applied in vivo in our BMT model by injecting 2.5 to 5.0 x 106 syngeneic (H-2bxd) DC preloaded with CD178-CD28 into lethally irradiated F1 mice reconstituted with 1 x 107 TCD H-2b donor marrow cells. Alternatively, soluble CD178-fusion proteins are applied i.v. following BMT. Preliminary results obtained in first experiments suggest that host DC expressing cell surface bound CD178 fusion proteins confer protection to acute GvH-reactions in recipients challenged with purified CD90+ T cells of H-2b origin as these mice show prolonged survival compared to controls. Furthermore, studies on evaluating GvL-reactivity in this model using donor T cells from HA-primed Balb/c mice and A20 cells expressing HA as a defined tumor surrogate antigen are in progress, and their results will be discussed. Taken together, these results suggest that CD178-based proapoptotic fusion proteins devoid of systemic toxicity due to cell-surface antigen-restricted activation might provide a promising tool to separate GvH- and GvL-responses in vivo without inducing severe side effects on CD95-mediated apoptosis in sensitive tissues.

scholarly journals Assembly of Synthetic Functional Cellulosomal Structures onto the Cell Surface ofLactobacillus plantarum, a Potent Member of the Gut Microbiome

2018 ◽  
Vol 84 (8) ◽  
Author(s):  
Johanna Stern ◽  
Sarah Moraïs ◽  
Yonit Ben-David ◽  
Rachel Salama ◽  
Melina Shamshoum ◽  
...  
Keyword(s):  
Cell Surface ◽  
Lactobacillus Plantarum ◽  
Lignocellulosic Biomass ◽  
Bacterial Cell ◽  
Enzyme Stability ◽  
Surface Display ◽  
Extracellular Polysaccharide ◽  
Genetically Engineered ◽  
Content Type

ABSTRACTHeterologous display of enzymes on microbial cell surfaces is an extremely desirable approach, since it enables the engineered microbe to interact directly with the plant wall extracellular polysaccharide matrix. In recent years, attempts have been made to endow noncellulolytic microbes with genetically engineered cellulolytic capabilities for improved hydrolysis of lignocellulosic biomass and for advanced probiotics. Thus far, however, owing to the hurdles encountered in secreting and assembling large, intricate complexes on the bacterial cell wall, only free cellulases or relatively simple cellulosome assemblies have been introduced into live bacteria. Here, we employed the “adaptor scaffoldin” strategy to compensate for the low levels of protein displayed on the bacterial cell surface. That strategy mimics natural elaborated cellulosome architectures, thus exploiting the exponential features of their Lego-like combinatorics. Using this approach, we produced several bacterial consortia ofLactobacillus plantarum, a potent gut microbe which provides a very robust genetic framework for lignocellulosic degradation. We successfully engineered surface display of large, fully active self-assembling cellulosomal complexes containing an unprecedented number of catalytic subunits all producedin vivoby the cell consortia. Our results demonstrate that the enzyme stability and performance of the cellulosomal machinery, which are superior to those seen with the equivalent secreted free enzyme system, and the high cellulase-to-xylanase ratios proved beneficial for efficient degradation of wheat straw.IMPORTANCEThe multiple benefits of lactic acid bacteria are well established in health and industry. Here we present an approach designed to extensively increase the cell surface display of proteins via successive assembly of interactive components. Our findings present a stepping stone toward proficient engineering ofLactobacillus plantarum, a widespread, environmentally important bacterium and potent microbiome member, for improved degradation of lignocellulosic biomass and advanced probiotics.

scholarly journals Role and Function of LitR, an Adenosyl B12-Bound Light-Sensitive Regulator of Bacillus megaterium QM B1551, in Regulation of Carotenoid Production

2015 ◽  
Vol 197 (14) ◽  
pp. 2301-2315 ◽  
Author(s):  
Hideaki Takano ◽  
Kou Mise ◽  
Kenta Hagiwara ◽  
Naoya Hirata ◽  
Shoko Watanabe ◽  
...  
Keyword(s):  
Bacillus Megaterium ◽  
Biochemical Properties ◽  
Binding Activity ◽  
Negative Regulator ◽  
Gram Positive ◽  
Content Type ◽  
Carotenoid Production ◽  
Dna Binding Activity

ABSTRACTThe LitR/CarH family of proteins is a light-sensitive MerR family of transcriptional regulators that contain an adenosyl B12(coenzyme B12or AdoB12)-binding domain at the C terminus. The genes encoding these proteins are found in phylogenetically diverse bacterial genera; however, the biochemical properties of these proteins from Gram-positive bacteria remain poorly understood. We performed genetic and biochemical analyses of a homolog of the LitR protein fromBacillus megateriumQM B1551, a Gram-positive endospore-forming soil bacterium. Carotenoid production was induced by illumination in this bacterium.In vivoanalysis demonstrated that LitR plays a central role in light-inducible carotenoid production and serves as a negative regulator of the light-inducible transcription ofcrtandlitRitself. Biochemical evidence showed that LitR in complex with AdoB12binds to the promoter regions oflitRand thecrtoperon in a light-sensitive manner.In vitrotranscription experiments demonstrated that AdoB12-LitR inhibited the specific transcription of thecrtpromoter generated by a σA-containing RNA polymerase holoenzyme under dark conditions. Collectively, these data indicate that the AdoB12-LitR complex serves as a photoreceptor with DNA-binding activity inB. megateriumQM B1551 and that its function as a transcriptional repressor is fundamental to the light-induced carotenoid production.IMPORTANCEMembers of the LitR/CarH family are AdoB12-based photosensors involved in light-inducible carotenoid production in nonphototrophic Gram-negative bacteria. Our study revealed thatBacillusLitR in complex with AdoB12also serves as a transcriptional regulator with a photosensory function, which indicates that the LitR/CarH family is generally involved in the light-inducible carotenoid production of nonphototrophic bacteria.

scholarly journals Generation of a Stable Plasmid forIn VitroandIn VivoStudies of Staphylococcus Species

2016 ◽  
Vol 82 (23) ◽  
pp. 6859-6869 ◽  
Author(s):  
Christina N. Krute ◽  
Kelsey L. Krausz ◽  
Mary A. Markiewicz ◽  
Jason A. Joyner ◽  
Srijana Pokhrel ◽  
...  
Keyword(s):  
Shuttle Vector ◽  
Antibiotic Usage ◽  
Open Reading Frames ◽  
Antibiotic Selection ◽  
Content Type ◽  
Plasmid Maintenance ◽  
Genetic Tools ◽  
Reading Frames

ABSTRACTA major shortcoming to plasmid-based genetic tools is the necessity of using antibiotics to ensure plasmid maintenance. While selectable markers are very powerful, their use is not always practical, such as duringin vivomodels of bacterial infection. During previous studies, it was noted that the uncharacterized LAC-p01 plasmid inStaphylococcus aureusUSA300 isolates was stable in the absence of a known selection and therefore could serve as a platform for new genetic tools forStaphylococcusspecies. LAC-p01 was genetically manipulated into anEscherichia coli-S. aureusshuttle vector that remained stable for at least 100 generations without antibiotic selection. The double- and single-stranded (dsoandsso) origins were identified and found to be essential for plasmid replication and maintenance, respectively. In contrast, deletion analyses revealed that none of the four LAC-p01 predicted open reading frames were necessary for stability. Subsequent to this, the shuttle vector was used as a platform to generate two plasmids. The first plasmid, pKK22, contains all genes native to the plasmid for use inS. aureusUSA300 strains, while the second, pKK30, lacks the four predicted open reading frames for use in non-USA300 isolates. pKK30 was also determined to be stable inStaphylococcus epidermidis. Moreover, pKK22 was maintained for 7 days postinoculation during a murine model ofS. aureussystemic infection and successfully complemented anhlamutant in a dermonecrosis model. These plasmids that eliminate the need for antibiotics during bothin vitroandin vivoexperiments are powerful new tools for studies ofStaphylococcus.IMPORTANCEPlasmid stability has been problematic in bacterial studies, and historically antibiotics have been used to ensure plasmid maintenance. This has been a major limitation duringin vivostudies, where providing antibiotics for plasmid maintenance is difficult and has confounding effects. Here, we have utilized the naturally occurring plasmid LAC-p01 from anS. aureusUSA300 strain to construct stable plasmids that obviate antibiotic usage. These newly modified plasmids retain stability over a multitude of generationsin vitroandin vivowithout antibiotic selection. With these plasmids, studies requiring genetic complementation, protein expression, or genetic reporter systems would not only overcome the burden of antibiotic usage but also eliminate the side effects of these antibiotics. Thus, our plasmids can be used as a powerful genetic tool for studies ofStaphylococcusspecies.

scholarly journals Regulatory Role of Glycerol in Candida albicans Biofilm Formation

mBio ◽  
2013 ◽  
Vol 4 (2) ◽  
Author(s):  
Jigar V. Desai ◽  
Vincent M. Bruno ◽  
Shantanu Ganguly ◽  
Ronald J. Stamper ◽  
Kaitlin F. Mitchell ◽  
...  
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Cell Surface ◽  
Biofilm Formation ◽  
Regulatory Role ◽  
Content Type ◽  
Upregulated Genes ◽  
Implanted Devices

ABSTRACTBiofilm formation byCandida albicanson medically implanted devices poses a significant clinical challenge. Here, we compared biofilm-associated gene expression in two clinicalC. albicansisolates, SC5314 and WO-1, to identify shared gene regulatory responses that may be functionally relevant. Among the 62 genes most highly expressed in biofilms relative to planktonic (suspension-grown) cells, we were able to recover insertion mutations in 25 genes. Twenty mutants had altered biofilm-related properties, including cell substrate adherence, cell-cell signaling, and azole susceptibility. We focused on one of the most highly upregulated genes in our biofilm proles,RHR2, which specifies the glycerol biosynthetic enzyme glycerol-3-phosphatase. Glycerol is 5-fold-more abundant in biofilm cells than in planktonic cells, and anrhr2Δ/Δ strain accumulates 2-fold-less biofilm glycerol than does the wild type. Underin vitroconditions, therhr2Δ/Δ mutant has reduced biofilm biomass and reduced adherence to silicone. Therhr2Δ/Δ mutant is also severely defective in biofilm formationin vivoin a rat catheter infection model. Expression profiling indicates that therhr2Δ/Δ mutant has reduced expression of cell surface adhesin genesALS1,ALS3, andHWP1, as well as many other biofilm-upregulated genes. Reduced adhesin expression may be the cause of therhr2Δ/Δ mutant biofilm defect, because overexpression ofALS1,ALS3, orHWP1restores biofilm formation ability to the mutantin vitroandin vivo. Our findings indicate that internal glycerol has a regulatory role in biofilm gene expression and that adhesin genes are among the main functional Rhr2-regulated genes.IMPORTANCECandida albicansis a major fungal pathogen, and infection can arise from the therapeutically intractable biofilms that it forms on medically implanted devices. It stands to reason that genes whose expression is induced during biofilm growth will function in the process, and our analysis of 25 such genes confirms that expectation. One gene is involved in synthesis of glycerol, a small metabolite that we find is abundant in biofilm cells. The impact of glycerol on biofilm formation is regulatory, not solely metabolic, because it is required for expression of numerous biofilm-associated genes. Restoration of expression of three of these genes that specify cell surface adhesins enables the glycerol-synthetic mutant to create a biofilm. Our findings emphasize the significance of metabolic pathways as therapeutic targets, because their disruption can have both physiological and regulatory consequences.

scholarly journals Exogenous Alginate Protects Staphylococcus aureus from Killing by Pseudomonas aeruginosa

2019 ◽  
Vol 202 (8) ◽  
Author(s):  
Courtney E. Price ◽  
Dustin G. Brown ◽  
Dominique H. Limoli ◽  
Vanessa V. Phelan ◽  
George A. O’Toole
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Physical Space ◽  
Late Time ◽  
Protective Effects ◽  
Content Type ◽  
Alginate Production ◽  
The Impact

ABSTRACT Cystic fibrosis (CF) patients chronically infected with both Pseudomonas aeruginosa and Staphylococcus aureus have worse health outcomes than patients who are monoinfected with either P. aeruginosa or S. aureus. We showed previously that mucoid strains of P. aeruginosa can coexist with S. aureus in vitro due to the transcriptional downregulation of several toxic exoproducts typically produced by P. aeruginosa, including siderophores, rhamnolipids, and HQNO (2-heptyl-4-hydroxyquinoline N-oxide). Here, we demonstrate that exogenous alginate protects S. aureus from P. aeruginosa in both planktonic and biofilm coculture models under a variety of nutritional conditions. S. aureus protection in the presence of exogenous alginate is due to the transcriptional downregulation of pvdA, a gene required for the production of the iron-scavenging siderophore pyoverdine as well as the downregulation of the PQS (Pseudomonas quinolone signal) (2-heptyl-3,4-dihydroxyquinoline) quorum sensing system. The impact of exogenous alginate is independent of endogenous alginate production. We further demonstrate that coculture of mucoid P. aeruginosa with nonmucoid P. aeruginosa strains can mitigate the killing of S. aureus by the nonmucoid strain of P. aeruginosa, indicating that the mechanism that we describe here may function in vivo in the context of mixed infections. Finally, we investigated a panel of mucoid clinical isolates that retain the ability to kill S. aureus at late time points and show that each strain has a unique expression profile, indicating that mucoid isolates can overcome the S. aureus-protective effects of mucoidy in a strain-specific manner. IMPORTANCE CF patients are chronically infected by polymicrobial communities. The two dominant bacterial pathogens that infect the lungs of CF patients are P. aeruginosa and S. aureus, with ∼30% of patients coinfected by both species. Such coinfected individuals have worse outcomes than monoinfected patients, and both species persist within the same physical space. A variety of host and environmental factors have been demonstrated to promote P. aeruginosa-S. aureus coexistence, despite evidence that P. aeruginosa kills S. aureus when these organisms are cocultured in vitro. Thus, a better understanding of P. aeruginosa-S. aureus interactions, particularly mechanisms by which these microorganisms are able to coexist in proximal physical space, will lead to better-informed treatments for chronic polymicrobial infections.

scholarly journals In Vitro Synergy and In Vivo Activity of Tigecycline-Ciprofloxacin Combination Therapy against Vibrio vulnificus Sepsis

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Seong Eun Kim ◽  
Hee Kyung Kim ◽  
Su-Mi Choi ◽  
Yohan Yu ◽  
Uh Jin Kim ◽  
...  
Keyword(s):  
Survival Rate ◽  
Mortality Rate ◽  
Combination Therapy ◽  
Combination Treatment ◽  
Vibrio Vulnificus ◽  
Antibiotic Regimen ◽  
Content Type ◽  
Time Kill

ABSTRACT The mortality rate associated with Vibrio vulnificus sepsis remains high. An in vitro time-kill assay revealed synergism between tigecycline and ciprofloxacin. The survival rate was significantly higher in mice treated with tigecycline plus ciprofloxacin than in mice treated with cefotaxime plus minocycline. Thus, combination treatment with tigecycline-ciprofloxacin may be an effective novel antibiotic regimen for V. vulnificus sepsis.

scholarly journals ZN148 Is a Modular Synthetic Metallo-β-Lactamase Inhibitor That Reverses Carbapenem Resistance in Gram-Negative Pathogens In Vivo

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Ørjan Samuelsen ◽  
Ove Alexander Høgmoen Åstrand ◽  
Christopher Fröhlich ◽  
Adam Heikal ◽  
Susann Skagseth ◽  
...  
Keyword(s):  
Active Site ◽  
Therapeutic Potential ◽  
Treatment Options ◽  
Carbapenem Resistance ◽  
Functional Space ◽  
Bactericidal Effect ◽  
Gram Negative ◽  
Content Type

ABSTRACT Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (β-lactamases able to inactivate carbapenems) have been identified in both serine β-lactamase (SBL) and metallo-β-lactamase (MBL) families. The recent introduction of SBL carbapenemase inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited. Here, we present ZN148, a zinc-chelating MBL-inhibitor capable of restoring the bactericidal effect of meropenem and in vitro clinical susceptibility to carbapenems in >98% of a large international collection of MBL-producing clinical Enterobacterales strains (n = 234). Moreover, ZN148 was able to potentiate the effect of meropenem against NDM-1-producing Klebsiella pneumoniae in a murine neutropenic peritonitis model. ZN148 showed no inhibition of the human zinc-containing enzyme glyoxylase II at 500 μM, and no acute toxicity was observed in an in vivo mouse model with cumulative dosages up to 128 mg/kg. Biochemical analysis showed a time-dependent inhibition of MBLs by ZN148 and removal of zinc ions from the active site. Addition of exogenous zinc after ZN148 exposure only restored MBL activity by ∼30%, suggesting an irreversible mechanism of inhibition. Mass-spectrometry and molecular modeling indicated potential oxidation of the active site Cys221 residue. Overall, these results demonstrate the therapeutic potential of a ZN148-carbapenem combination against MBL-producing Gram-negative pathogens and that ZN148 is a highly promising MBL inhibitor that is capable of operating in a functional space not presently filled by any clinically approved compound.

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