Brianna Dalesandro

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Direct control of CAR T cells through small molecule-regulated antibodies.

Nat Commun. 2021 Jan 29;12(1):710

Authors: Park S, Pascua E, Lindquist KC, Kimberlin C, Deng X, Mak YSL, Melton Z, Johnson TO, Lin R, Boldajipour B, Abraham RT, Pons J, Sasu BJ, Van Blarcom TJ, Chaparro-Riggers J

Abstract
Antibody-based therapeutics have experienced a rapid growth in recent years and are now utilized in various modalities spanning from conventional antibodies, antibody-drug conjugates, bispecific antibodies to chimeric antigen receptor (CAR) T cells. Many next generation antibody therapeutics achieve enhanced potency but often increase the risk of adverse events. Antibody scaffolds capable of exhibiting inducible affinities could reduce the risk of adverse events by enabling a transient suspension of antibody activity. To demonstrate this, we develop conditionally activated, single-module CARs, in which tumor antigen recognition is directly modulated by an FDA-approved small molecule drug. The resulting CAR T cells demonstrate specific cytotoxicity of tumor cells comparable to that of traditional CARs, but the cytotoxicity is reversibly attenuated by the addition of the small molecule. The exogenous control of conditional CAR T cell activity allows continual modulation of therapeutic activity to improve the safety profile of CAR T cells across all disease indications.

PMID: 33514714 [PubMed - as supplied by publisher]

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Antibody Conjugation of a Chimeric BET Degrader Enables in vivo Activity.

ChemMedChem. 2020 01 07;15(1):17-25

Authors: Pillow TH, Adhikari P, Blake RA, Chen J, Del Rosario G, Deshmukh G, Figueroa I, Gascoigne KE, Kamath AV, Kaufman S, Kleinheinz T, Kozak KR, Latifi B, Leipold DD, Sing Li C, Li R, Mulvihill MM, O'Donohue A, Rowntree RK, Sadowsky JD, Wai J, Wang X, Wu C, Xu Z, Yao H, Yu SF, Zhang D, Zang R, Zhang H, Zhou H, Zhu X, Dragovich PS

Abstract
The ability to selectively degrade proteins with bifunctional small molecules has the potential to fundamentally alter therapy in a variety of diseases. However, the relatively large size of these chimeric molecules often results in challenging physico-chemical properties (e. g., low aqueous solubility) and poor pharmacokinetics which may complicate their in vivo applications. We recently discovered an exquisitely potent chimeric BET degrader (GNE-987) which exhibited picomolar cell potencies but also demonstrated low in vivo exposures. In an effort to improve the pharmacokinetic properties of this molecule, we discovered the first degrader-antibody conjugate by attaching GNE-987 to an anti-CLL1 antibody via a novel linker. A single IV dose of the conjugate afforded sustained in vivo exposures that resulted in antigen-specific tumor regressions. Enhancement of a chimeric protein degrader with poor in vivo properties through antibody conjugation thereby expands the utility of directed protein degradation as both a biological tool and a therapeutic possibility.

PMID: 31674143 [PubMed - indexed for MEDLINE]

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pH-responsive antibodies for therapeutic applications.

J Biomed Sci. 2021 Jan 22;28(1):11

Authors: Klaus T, Deshmukh S

Abstract
Therapeutic antibodies are instrumental in improving the treatment outcome for certain disease conditions. However, to enhance their efficacy and specificity, many efforts are continuously made. One of the approaches that are increasingly explored in this field are pH-responsive antibodies capable of binding target antigens in a pH-dependent manner. We reviewed suitability and examples of these antibodies that are functionally modulated by the tumor microenvironment. Provided in this review is an update about antigens targeted by pH-responsive, sweeping, and recycling antibodies. Applicability of the pH-responsive antibodies in the engineering of chimeric antigen receptor T-cells (CAR-T) and in improving drug delivery to the brain by the enhanced crossing of the blood-brain barrier is also discussed. The pH-responsive antibodies possess strong treatment potential. They emerge as next-generation programmable engineered biologic drugs that are active only within the targeted biological space. Thus, they are valuable in targeting acidified tumor microenvironment because of improved spatial persistence and reduced on-target off-tumor toxicities. We predict that the programmable pH-dependent antibodies become powerful tools in therapies of cancer.

PMID: 33482842 [PubMed - as supplied by publisher]

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CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells.

bioRxiv. 2021 Jan 15;:

Authors: Ma M, Badeti S, Chen CH, Pinter A, Jiang Q, Shi L, Zhou R, Xu H, Li Q, Gause W, Liu D

Abstract
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is highly contagious presenting a significant public health issue. Current therapies used to treat coronavirus disease 2019 (COVID-19) include monoclonal antibody cocktail, convalescent plasma, antivirals, immunomodulators, and anticoagulants, though the current therapeutic options remain limited and expensive. The vaccines from Pfizer and Moderna have recently been authorized for emergency use, which are invaluable for the prevention of SARS-CoV-2 infection. However, their long-term side effects are not yet to be documented, and populations with immunocompromised conditions (e.g., organ-transplantation and immunodeficient patients) may not be able to mount an effective immune response. In addition, there are concerns that wide-scale immunity to SARS-CoV-2 may introduce immune pressure that could select for escape mutants to the existing vaccines and monoclonal antibody therapies. Emerging evidence has shown that chimeric antigen receptor (CAR)- natural killer (NK) immunotherapy has potent antitumor response in hematologic cancers with minimal adverse effects in recent studies, however, the potentials of CAR-NK cells in preventing and treating severe cases of COVID-19 has not yet been fully exploited. Here, we improve upon a novel approach for the generation of CAR-NK cells for targeting SARS-CoV-2 and its D614G mutant. CAR-NK cells were generated using the scFv domain of S309 (henceforward, S309-CAR-NK), a SARS-CoV and SARS-CoV-2 neutralizing antibody that targets the highly conserved region of SARS-CoV-2 spike (S) glycoprotein, therefore would be more likely to recognize different variants of SARS-CoV-2 isolates. S309-CAR-NK cells can specifically bind to pseudotyped SARS-CoV-2 virus and its D614G mutant. Furthermore, S309-CAR-NK cells can specifically kill target cells expressing SARS-CoV-2 S protein in vitro and show superior killing activity and cytokine production, compared to that of the recently published CR3022-CAR-NK cells. Thus, these results pave the way for generating 'off-the-shelf' S309-CAR-NK cells for treatment in high-risk individuals as well as provide an alternative strategy for patients unresponsive to current vaccines.

PMID: 33469580 [PubMed]

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Development and validation of a high-throughput whole cell assay to investigate Staphylococcus aureus adhesion to host ligands.

J Biol Chem. 2020 Dec 04;295(49):16700-16712

Authors: Petrie LE, Leonard AC, Murphy J, Cox G

Abstract
Staphylococcus aureus adhesion to the host's skin and mucosae enables asymptomatic colonization and the establishment of infection. This process is facilitated by cell wall-anchored adhesins that bind to host ligands. Therapeutics targeting this process could provide significant clinical benefits; however, the development of anti-adhesives requires an in-depth knowledge of adhesion-associated factors and an assay amenable to high-throughput applications. Here, we describe the development of a sensitive and robust whole cell assay to enable the large-scale profiling of S. aureus adhesion to host ligands. To validate the assay, and to gain insight into cellular factors contributing to adhesion, we profiled a sequence-defined S. aureus transposon mutant library, identifying mutants with attenuated adhesion to human-derived fibronectin, keratin, and fibrinogen. Our screening approach was validated by the identification of known adhesion-related proteins, such as the housekeeping sortase responsible for covalently linking adhesins to the cell wall. In addition, we also identified genetic loci that could represent undescribed anti-adhesive targets. To compare and contrast the genetic requirements of adhesion to each host ligand, we generated a S. aureus Genetic Adhesion Network, which identified a core gene set involved in adhesion to all three host ligands, and unique genetic signatures. In summary, this assay will enable high-throughput chemical screens to identify anti-adhesives and our findings provide insight into the target space of such an approach.

PMID: 33453905 [PubMed - in process]

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CD27 enhances the killing effect of CAR T cells targeting trophoblast cell surface antigen 2 in the treatment of solid tumors.

Cancer Immunol Immunother. 2021 Jan 13;:

Authors: Chen H, Wei F, Yin M, Zhao Q, Liu Z, Yu B, Huang Z

Abstract
Chimeric antigen receptor (CAR) T cell therapy, a type of adoptive cell therapy, has been successfully used when treating lymphoma malignancies, but not nearly as successful in treating solid tumors. Trophoblast cell surface antigen 2 (Trop2) is expressed in various solid tumors and plays a role in tumor growth, invasion, and metastasis. In this study, a CAR targeting Trop2 (T2-CAR) was developed with different co-stimulatory intercellular domains. T2-CAR T cells demonstrated a powerful killing ability in the presence of Trop2-positive cells following an in vitro assay. Moreover, T2-CAR T cells produced multiple effector cytokines under antigen stimulation. In tumor-bearing mouse models, the CD27-based T2-CAR T cells showed a higher antitumor activity. Additionally, more CD27-based T2-CAR T cells survived in tumor-bearing mice spleens as well as in the tumor tissue. CD27-based T2-CAR T cells were also found to upregulate IL-7Rα expression, while downregulating PD-1 expression. In conclusion, the CD27 intercellular domain can enhance the T2-CAR T cell killing effect via multiple mechanisms, thus indicating that a CD27-based T2-CAR T cell approach is suitable for clinical applications.

PMID: 33439295 [PubMed - as supplied by publisher]

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Human Short Peptidoglycan Recognition Protein PGLYRP1/Tag-7/PGRP-S Inhibits Listeria monocytogenes Intracellular Survival in Macrophages.

Front Cell Infect Microbiol. 2020;10:582803

Authors: Slonova D, Posvyatenko A, Kibardin A, Sysolyatina E, Lyssuk E, Ermolaeva S, Obydennyi S, Gnuchev N, Georgiev G, Severinov K, Larin S

Abstract
PGLYRP1/Tag-7/PGRP-S is one of mammalian peptidoglycan recognition proteins (PGRPs). Here, we demonstrate that human recombinant PGLYRP1/Tag-7/PGRP-S potentiates the response of murine macrophage-like ANA-1 cells and human macrophages to facultative intracellular pathogen Listeria monocytogenes. PGLYRP1/Tag-7/PGRP-S binds to the surface of L. monocytogenes and other bacterial cells but has no effect on their growth in culture. While PGLYRP1/Tag-7/PGRP-S treatment modestly enhanced phagocytosis of bacteria by ANA-1 cells, the intracellular survival of PGLYRP1/Tag-7/PGRP-S treated L. monocytogenes was strongly inhibited 2 h after internalization. PGLYRP1/Tag-7/PGRP-S treatment of bacteria boosted oxidative burst induction and increased the level of proinflammatory cytokine IL-6 produced by ANA-1, however, these effects happened too late to be responsible for decreased intracellular survival of bacteria. Our results thus suggest that PGLYRP1/Tag-7/PGRP-S acts as a molecular sensor for detection of L. monocytogenes infection of mammalian cells that leads to increased killing through a mechanism(s) that remains to be defined.

PMID: 33425777 [PubMed - in process]

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Group A Streptococcus Cell Wall Oligosaccharide-Streptococcal C5a Peptidase Conjugates as Effective Antibacterial Vaccines.

ACS Infect Dis. 2020 02 14;6(2):281-290

Authors: Wang S, Zhao Y, Wang G, Feng S, Guo Z, Gu G

Abstract
Group A streptococcus (GAS) is one of the common Gram-positive pathogenic bacteria accounting for a variety of infectious diseases. Currently, there is no commercial vaccine for GAS. To develop efficient GAS vaccines, synthetic tri-, hexa-, and nonasaccharides of a conserved group A carbohydrate (GAC) were conjugated with an inactive mutant of group A streptococcal C5a peptidase (ScpA), ScpA193, to create bivalent conjugate vaccines, which were compared with the corresponding CRM197 and TT conjugates. Systematic evaluations of these semisynthetic conjugates demonstrated that they could induce robust and comparable T-cell-dependent immune responses in mice. It was further disclosed that antibodies provoked by the ScpA193 conjugates, especially that of hexa- and nonasaccharides, could recognize and bind to GAS cells and mediate GAS opsonophagocytosis in vitro. In vivo evaluations of the hexa- and nonasaccharide-ScpA193 conjugates using a mouse model revealed that immunizing mice with especially the latter conjugate could effectively protect the animals from GAS challenges and GAS-induced pulmonary damage and significantly increase animal survival. Further in vitro studies suggested that the two ScpA193 conjugates could function through activating CD4+ T cells and promoting helper T cells (Th) to differentiate into antigen-specific Th1 and Th2 cells. In conclusion, the nonasaccharide-ScpA193 conjugate was identified as a particularly promising GAS vaccine candidate that is worthy of further investigation and development.

PMID: 31872763 [PubMed - indexed for MEDLINE]

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Racing to build a wall: glycoconjugate assembly in Gram-positive and Gram-negative bacteria.

Curr Opin Struct Biol. 2021 Jan 08;68:55-65

Authors: Liston SD, Willis LM

Abstract
The last two years have seen major advances in understanding the structural basis of bacterial cell envelope glycoconjugate biosynthesis, including capsules, lipopolysaccharide, teichoic acid, cellulose, and peptidoglycan. The recent crystal and cryo-electron microscopy structures of proteins involved in the initial glycosyltransferase steps in the cytoplasm, the transport of large and small lipid-linked glycoconjugates across the inner membrane, the polymerization of glycans in the periplasm, and the export of molecules from the cell have shed light on the mechanisms by which cell envelope glycoconjugates are made. We discuss these recent advances and highlight remaining unanswered questions.

PMID: 33429200 [PubMed - as supplied by publisher]

Fluorescence microscopic imaging allows investigations of receptor proteins within their biological context. A key challenge is to enable site‐specific incorporation of reporter moieties into proteins without interfering with biological functions or cellular networks. Small peptide tags offer the opportunity to combine inducible labeling with small tag sizes that avoid receptor perturbation. Herein, we review the current state of live cell labeling of peptide‐tagged cell surface proteins. Considering the importance as targets in medicinal chemistry, we focus on important membrane receptors such as G protein‐coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). We discuss peptide tags that i) are subject to enzyme‐mediated modification reactions, ii) guide complementation of reporter proteins, iii) form coiled‐coil complexes, iv) interact with metal complexes, and discuss methods relying on binding of ligands with the protein of interest. Given own contributions in the field, we place emphasis on peptide‐templated labeling chemistry.

Nature Biotechnology, Published online: 11 January 2021; doi:10.1038/s41587-020-00798-z

Antibody–drug conjugates for cancer score with ROR1

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Development of Antibody-Based PROTACs for the Degradation of the Cell-Surface Immune Checkpoint Protein PD-L1.

J Am Chem Soc. 2021 Jan 04;:

Authors: Cotton AD, Nguyen DP, Gramespacher JA, Seiple IB, Wells JA

Abstract
Targeted protein degradation has emerged as a new paradigm to manipulate cellular proteostasis. Proteolysis-targeting chimeras (PROTACs) are bifunctional small molecules that recruit an E3 ligase to a target protein of interest, promoting its ubiquitination and subsequent degradation. Here, we report the development of antibody-based PROTACs (AbTACs), fully recombinant bispecific antibodies that recruit membrane-bound E3 ligases for the degradation of cell-surface proteins. We show that an AbTAC can induce the lysosomal degradation of programmed death-ligand 1 by recruitment of the membrane-bound E3 ligase RNF43. AbTACs represent a new archetype within the PROTAC field to target cell-surface proteins with fully recombinant biological molecules.

PMID: 33395526 [PubMed - as supplied by publisher]

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Crystal structure of the N-terminal domain of TagH reveals a potential drug targeting site.

Biochem Biophys Res Commun. 2020 Dec 21;536:1-6

Authors: Yang CS, Huang WC, Ko TP, Wang YC, Wang AH, Chen Y

Abstract
Bacterial wall teichoic acids (WTAs) are synthesized intracellularly and exported by a two-component transporter, TagGH, comprising the transmembrane and ATPase subunits TagG and TagH. Here the dimeric structure of the N-terminal domain of TagH (TagH-N) was solved by single-wavelength anomalous diffraction using a selenomethionine-containing crystal, which shows an ATP-binding cassette (ABC) architecture with RecA-like and helical subdomains. Besides significant structural differences from other ABC transporters, a prominent patch of positively charged surface is seen in the center of the TagH-N dimer, suggesting a potential binding site for the glycerol phosphate chain of WTA. The ATPase activity of TagH-N was inhibited by clodronate, a bisphosphonate, in a non-competitive manner, consistent with the proposed WTA-binding site for drug targeting.

PMID: 33360015 [PubMed - as supplied by publisher]

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Engineered Lysins With Customized Lytic Activities Against Enterococci and Staphylococci.

Front Microbiol. 2020;11:574739

Authors: Binte Muhammad Jai HS, Dam LC, Tay LS, Koh JJW, Loo HL, Kline KA, Goh BC

Abstract
The emergence of multidrug-resistant bacteria has made minor bacterial infections incurable with many existing antibiotics. Lysins are phage-encoded peptidoglycan hydrolases that have demonstrated therapeutic potential as a novel class of antimicrobials. The modular architecture of lysins enables the functional domains - catalytic domain (CD) and cell wall binding domain (CBD) - to be shuffled to create novel lysins. The CD is classically thought to be only involved in peptidoglycan hydrolysis whereas the CBD dictates the lytic spectrum of a lysin. While there are many studies that extended the lytic spectrum of a lysin by domain swapping, few have managed to introduce species specificity in a chimeric lysin. In this work, we constructed two chimeric lysins by swapping the CBDs of two parent lysins with different lytic spectra against enterococci and staphylococci. We showed that these chimeric lysins exhibited customized lytic spectra distinct from the parent lysins. Notably, the chimeric lysin P10N-V12C, which comprises a narrow-spectrum CD fused with a broad-spectrum CBD, displayed species specificity not lysing Enterococcus faecium while targeting Enterococcus faecalis and staphylococci. Such species specificity can be attributed to the narrow-spectrum CD of the chimeric lysin. Using flow cytometry and confocal microscopy, we found that the E. faecium cells that were treated with P10N-V12C are less viable with compromised membranes yet remained morphologically intact. Our results suggest that while the CBD is a major determinant of the lytic spectrum of a lysin, the CD is also responsible in the composition of the final lytic spectrum, especially when it pertains to species-specificity.

PMID: 33324362 [PubMed]

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Turning a pathogen protein into a therapeutic tool for sepsis.

EMBO Mol Med. 2020 Dec 17;:e13589

Authors: Vanderhaeghen T, Wallaeys C, Libert C

Abstract
Sepsis causes unacceptably high amounts of deaths worldwide. It is a huge unmet medical need, and new therapeutic interventions for sepsis and septic shock are urgently needed. By studying the mechanism by which a bacterial protein undermines the inflammatory function of macrophages, Kim et al, in the last issue of EMBO Molecular Medicine, have developed a new therapeutic protein drug, which appears to have very promising protective activities in a well-validated and aggressive polymicrobial sepsis model in mice. The chimeric protein is thought to limit macrophage inflammation while activating phagocytosis, and so, it hits two macrophage pathways at once.

PMID: 33332738 [PubMed - as supplied by publisher]

Decision tree: The cover feature depicts the decision making process for designing bioorthogonal probes for activity‐based protein profiling. Each “branch” presents its own complexities and considerations to produce a probe most appropriate for the given application. The development of selective, bioorthogonal activity‐based probes for penicillin‐binding proteins in Streptococcus pneumoniae highlights these concerns, with emphasis on linker length and environment in which the bioorthogonal reaction is applied. More information can be found in the full paper by E. E. Carlson et al.. Image created with Biorender.

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A novel flow cytometry assay based on bacteriophage-derived proteins for Staphylococcus detection in blood.

Sci Rep. 2020 04 10;10(1):6260

Authors: Costa SP, Dias NM, Melo LDR, Azeredo J, Santos SB, Carvalho CM

Abstract
Bloodstream infections (BSIs) are considered a major cause of death worldwide. Staphylococcus spp. are one of the most BSIs prevalent bacteria, classified as high priority due to the increasing multidrug resistant strains. Thus, a fast, specific and sensitive method for detection of these pathogens is of extreme importance. In this study, we have designed a novel assay for detection of Staphylococcus in blood culture samples, which combines the advantages of a phage endolysin cell wall binding domain (CBD) as a specific probe with the accuracy and high-throughput of flow cytometry techniques. In order to select the biorecognition molecule, three different truncations of the C-terminus of Staphylococcus phage endolysin E-LM12, namely the amidase (AMI), SH3 and amidase+SH3 (AMI_SH3) were cloned fused with a green fluorescent protein. From these, a higher binding efficiency to Staphylococcus cells was observed for AMI_SH3, indicating that the amidase domain possibly contributes to a more efficient binding of the SH3 domain. The novel phage endolysin-based flow cytometry assay provided highly reliable and specific detection of 1-5 CFU of Staphylococcus in 10 mL of spiked blood, after 16 hours of enrichment culture. Overall, the method developed herein presents advantages over the standard BSIs diagnostic methods, potentially contributing to an early and effective treatment of BSIs.

PMID: 32277078 [PubMed - indexed for MEDLINE]

Tumor-associated macrophages (TAMs) can have protumor properties, including suppressing immune responses, promoting vascularization and, consequently, augmenting tumor progression. To stop TAM-mediated immunosuppression, we use a novel treatment by injecting antibodies specific for scavenger receptor MARCO, which is expressed on a specific subpopulation of TAMs in the tumor. We now report...

We report the use of a chemoenzymatic glycocalyx editing strategy to create CD22‐specific ligands and sialyl Lewis X on NK cells (NK‐92MI or CIK cells) to target and eradicate B‐lymphoma efficiently.

Abstract

CD22, a member of Siglec family of sialic acid binding proteins, has restricted expression on B cells. Antibody‐based agents targeting CD22 or CD20 on B lymphoma and leukemia cells exhibit clinical efficacy for treating these malignancies, but also attack normal B cells leading to immune deficiency. Here, we report a chemoenzymatic glycocalyx editing strategy to introduce high‐affinity and specific CD22 ligands onto NK‐92MI and cytokine‐induced natural killer cells to achieve tumor‐specific CD22 targeting. These CD22‐ligand modified cells exhibited significantly enhanced tumor cell binding and killing in vitro without harming healthy B cells. For effective lymphoma cell killing in vivo, we further functionalized CD22 ligand‐modified NK‐92MI cells with the E‐selectin ligand sialyl Lewis X to promote trafficking to bone marrow. The dual‐functionalized cells resulted in the efficient suppression of B lymphoma in a xenograft model. Our results suggest that nature killer cells modified with glycan ligands to CD22 and selectins promote both targeted killing of B lymphoma cells and improved trafficking to sites where the cancer cells reside, respectively.

Natural killer (NK) cells are innate immune cells that can directly kill certain types of cancer cells. Adoptive transfer of NK cells represents a novel and promising immunotherapy for malignant tumours; however, there is a lack of methods to validate anti‐tumour activity of NK cells reaching target sites in vivo. Herein we report a new chemiluminescent probe to image in situ the granzyme B‐mediated killing activity of NK cells against cancer cells. We have optimised a granzyme B‐specific construct using an activatable phenoxydioxetane reporter so that enzymatic cleavage of the probe results in bright chemiluminescence in the visible range. The probe shows high selectivity for active granzyme B over other proteases and higher signal‐to‐noise ratios than commercial fluorophores. Finally, we demonstrate that the probe can detect activity of NK cells in preclinical mouse models, being the first chemiluminescent probe for in vivo imaging of NK cell activity in live tumours.

The transpeptidase sortase A of Staphylococcus aureus (Sa‐SrtA) is a valuable tool in protein chemistry. The native enzyme anchors surface proteins containing a highly conserved LPxTG sorting motif to a terminal glycine residue of the peptidoglycan layer in gram‐positive bacteria. This reaction is exploited for sortase‐mediated ligation (SML), allowing the site‐specific linkage of synthetic peptides and recombinant proteins by a native peptide bond. However, the moderate catalytic efficiency and specificity of Sa‐SrtA fueled the development of new biocatalysts for SML, including the screening of sortase A variants form microorganisms other than S. aureus and the directed protein evolution of the Sa‐SrtA enzyme itself. Novel display platforms and screening formats were developed to isolate sortases with altered properties from mutant libraries. This has yielded sortases with strongly enhanced catalytic activity and enzymes recognizing new sorting motifs as substrates. This mini review focuses on recent advances in the field of directed sortase evolution and applications of these tailor‐made enzymes in biochemistry

Genetic evidence is presented that MltG processes nascent peptidoglycan polymers as they are made by the different classes of cell wall polymerases.

Abstract

Bacterial cells are surrounded by a peptidoglycan (PG) cell wall. This structure is essential for cell integrity and its biogenesis pathway is a key antibiotic target. Most bacteria utilize two types of synthases that polymerize glycan strands and crosslink them: class A penicillin‐binding proteins (aPBPs) and complexes of SEDS proteins and class B PBPs (bPBPs). Although the enzymatic steps of PG synthesis are well characterized, the steps involved in terminating PG glycan polymerization remain poorly understood. A few years ago, the conserved lytic transglycosylase MltG was identified as a potential terminase for PG synthesis in Escherichia coli. However, characterization of the in vivo function of MltG was hampered by the lack of a growth or morphological phenotype in ΔmltG cells. Here, we report the isolation of MltG‐defective mutants as suppressors of lethal deficits in either aPBP or SEDS/bPBP PG synthase activity. We used this phenotype to perform a domain‐function analysis for MltG, which revealed that access to the inner membrane is important for its in vivo activity. Overall, our results support a model in which MltG functions as a terminase for both classes of PG synthases by cleaving PG glycans as they are being actively synthesized.

High‐resolution images of vertically oriented Streptococcus pneumoniae cells reveal concentric rings of PBP transpeptidase (TP) activity corresponding to the leading edge of the septal annulus (inner ring) and the midcell outer ring of elongating peptidoglycan (PG) synthesis. The rings serve as fiducial markers for assigning proteins to the septal (PBP2x and FtsZ) or elongation (PBP2b, FtsX) PG synthesis machines. In predivisional cells, PG synthesis proteins and PBP transpeptidase activity are arranged in patterns of regularly spaced nodes, whose spacing is perturbed in mutants.

Abstract

Bacterial peptidoglycan (PG) synthesis requires strict spatiotemporal organization to reproduce specific cell shapes. In ovoid‐shaped Streptococcus pneumoniae (Spn), septal and peripheral (elongation) PG synthesis occur simultaneously at midcell. To uncover the organization of proteins and activities that carry out these two modes of PG synthesis, we examined Spn cells vertically oriented onto their poles to image the division plane at the high lateral resolution of 3D‐SIM (structured‐illumination microscopy). Labeling with fluorescent D‐amino acids (FDAA) showed that areas of new transpeptidase (TP) activity catalyzed by penicillin‐binding proteins (PBPs) separate into a pair of concentric rings early in division, representing peripheral PG (pPG) synthesis (outer ring) and the leading‐edge (inner ring) of septal PG (sPG) synthesis. Fluorescently tagged PBP2x or FtsZ locate primarily to the inner FDAA‐marked ring, whereas PBP2b and FtsX remain in the outer ring, suggesting roles in sPG or pPG synthesis, respectively. Pulses of FDAA labeling revealed an arrangement of separate regularly spaced “nodes” of TP activity around the division site of predivisional cells. Tagged PBP2x, PBP2b, and FtsX proteins also exhibited nodal patterns with spacing comparable to that of FDAA labeling. Together, these results reveal new aspects of spatially ordered PG synthesis in ovococcal bacteria during cell division.