Brianna Dalesandro

Nature, Published online: 14 May 2020; doi:10.1038/s41586-020-2332-7

Proteomics of SARS-CoV-2-infected host cells reveals therapy targets

Nature Biotechnology, Published online: 11 May 2020; doi:10.1038/s41587-020-0520-5

Immunotherapy startups look to engineer macrophages to fight solid tumors.

Nature, Published online: 13 May 2020; doi:10.1038/d41586-020-01362-0

The latest science news, in brief.

DNA technology was used to precisely regulate both lateral and vertical positioning of T cell activation ligands on the membrane of red blood cells to better mimic natural antigen presenting cells, thereby providing a tool for T cell activation studies and enhanced adoptive immunotherapy.

Abstract

Artificial antigen presenting cells (aAPCs) with surface‐anchored T cell activating ligands hold great potential in adoptive immunotherapy. However, it remains challenging to precisely control the ligand positioning on those platforms using conventional bioconjugation chemistry. Utilizing DNA‐assisted bottom‐up self‐assembly, we were able to precisely control both lateral and vertical distributions of T cell activation ligands on red blood cells (RBCs). The clustered lateral positioning of the peptide‐major histocompatibility complex (pMHC) on RBCs with a short vertical distance to the cell membrane is favorable for more effective T cell activation, likely owing to their better mimicry of natural APCs. Such optimized RBC‐based artificial APCs can stimulate T cell proliferation in vivo and effectively inhibit tumor growth with adoptive immunotherapy. DNA technology is thus a unique tool to precisely engineer the cell membrane interface and tune cell–cell interactions, which is promising for applications such as immunotherapy.

Inducible MyD88/CD40 synergizes with IL-15 to enhance antitumor efficacy of CAR-NK cells.

Blood Adv. 2020 May 12;4(9):1950-1964

Authors: Wang X, Jasinski DL, Medina JL, Spencer DM, Foster AE, Bayle JH

Abstract
Natural killer (NK) cells expressing chimeric antigen receptors (CARs) are a promising anticancer immunotherapy, leveraging both innate NK cell antitumor activity and target-specific cytotoxicity. Inducible MyD88/CD40 (iMC) is a potent, rimiducid-regulated protein switch that has been deployed previously as a T-cell activator to enhance proliferation and persistence of CAR-modified T cells. In this study, iMC was extended to CAR-NK cells to enhance their growth and augment cytotoxicity against tumor cells. iMC-activated NK cells substantially increased cytokine and chemokine secretion and displayed higher levels of perforin and granzyme B degranulation. In addition, iMC activation could be coupled with ectopic interleukin-15 (IL-15) to further enhance NK cell proliferation. When coexpressed with a target-specific CAR (CD123 or BCMA), this IL-15/iMC system showed further augmented antitumor activity through enhanced CAR-NK cell expansion and cytolytic activity. To protect against potential toxicity from engineered NK cells, an orthogonal rapamycin-regulated Caspase-9 (iRC9) was included in a 4-gene, dual-switch platform. After infusion of dual-switch NK cells, pharmacologic iRC9 dimerization led to rapid elimination of a majority of expanded transduced NK cells. Thus, CAR-NK cells utilizing dual molecular switches provide an innovative and effective approach to cancer immunotherapy with controlled specificity, efficacy, and safety.

PMID: 32384544 [PubMed - as supplied by publisher]

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Cytokine Storm Drugs Move from CAR T to COVID-19.

Cancer Discov. 2020 May 05;:

Authors:

Abstract
Early reports suggest that tocilizumab, an IL6 receptor-blocking antibody used to manage toxicities associated with chimeric antigen receptor T-cell therapy, may help control cytokine storms in people infected with COVID-19. Preliminary data from randomized trials are less clear-cut.

PMID: 32371479 [PubMed - as supplied by publisher]

New directions in chimeric antigen receptor T cell [CAR-T] therapy and related flow cytometry.

Cytometry B Clin Cytom. 2020 Apr 30;:

Authors: Maryamchik E, Gallagher KME, Preffer FI, Kadauke S, Maus MV

Abstract
Chimeric antigen receptor (CAR) T cells provide a promising approach to the treatment of hematologic malignancies and solid tumors. Flow cytometry is a powerful analytical modality, which plays an expanding role in all stages of CAR T therapy, from lymphocyte collection, to CAR T cell manufacturing, to in vivo monitoring of the infused cells and evaluation of their function in the tumor environment. Therefore, a thorough understanding of the new directions is important for designing and implementing CAR T-related flow cytometry assays in the clinical and investigational settings. However, the speed of new discoveries and the multitude of clinical and preclinical trials make it challenging to keep up to date in this complex field. In this review, we summarize the current state of CAR T therapy, highlight the areas of emergent research, discuss applications of flow cytometry in modern cell therapy, and touch upon several considerations particular to CAR detection and assessing the effectiveness of CAR T therapy.

PMID: 32352629 [PubMed - as supplied by publisher]

A Chemical Switch System to Modulate Chimeric Antigen Receptor T Cell Activity through Proteolysis-Targeting Chimaera Technology.

ACS Synth Biol. 2020 Apr 30;:

Authors: Lee SM, Kang CH, Choi SU, Kim Y, Hwang JY, Jeong HG, Park CH

Abstract
Despite the excellent efficacy of chimeric antigen receptor (CAR T) cell therapy, concerns about its safety have been constantly raised. The side effects of CAR T cells result from an aberrantly upregulation of CAR T cell activity. Therefore, it is crucial to control the CAR T cell activity whenever the patient is at risk. For this purpose, the iCas9 system, which induces apoptosis in CAR T cell through caspase-9 dimerization by compound, has been invented and is currently going under clinical trial. However, the iCas9 system is irreversible, as the entire CAR T cell population is removed from the patient. Thus, CAR T cells, which are very expensive, should be reinfused to the patients after they recovered from the side-effect. Here, we propose a new CAR T cell safety strategy, which targets CAR "protein", not CAR "T cell". In this system, the CAR construct is modified to bear a bromodomain (BD). The addition of a BD in the CAR protein did not interfere with the original CAR functions, such as cytokine secretion and target cell lysis. Our data showed that the use of a proteolysis-targeting chimaera (PROTAC) compound against BD successfully degraded the BD-containing CAR protein. Moreover, the CAR expression is recovered when the PROTAC compound is removed from the cell, demonstrating that our system is reversible. In a target cell lysis assay, the PROTAC compound successfully suppressed the lytic activity of CAR T cells by degrading the CAR protein. In conclusion, we developed a new safety system in which CAR T cells can be "reversibly" controlled by a compound.

PMID: 32352759 [PubMed - as supplied by publisher]

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The challenge of intracellular antibiotic accumulation, a function of fluoroquinolone influx versus bacterial efflux.

Commun Biol. 2020 Apr 28;3(1):198

Authors: Vergalli J, Atzori A, Pajovic J, Dumont E, Malloci G, Masi M, Vargiu AV, Winterhalter M, Réfrégiers M, Ruggerone P, Pagès JM

Abstract
With the spreading of antibiotic resistance, the translocation of antibiotics through bacterial envelopes is crucial for their antibacterial activity. In Gram-negative bacteria, the interplay between membrane permeability and drug efflux pumps must be investigated as a whole. Here, we quantified the intracellular accumulation of a series of fluoroquinolones in population and in individual cells of Escherichia coli according to the expression of the AcrB efflux transporter. Computational results supported the accumulation levels measured experimentally and highlighted how fluoroquinolones side chains interact with specific residues of the distal pocket of the AcrB tight monomer during recognition and binding steps.

PMID: 32346058 [PubMed - in process]

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Mycobacterial immunotherapy for prostate cancer: where can we go from here?

Nat Rev Urol. 2020 04;17(4):189-190

Authors: Stavrinides V, Dalgleish A, Copier JP, Moore CM

PMID: 31996816 [PubMed - indexed for MEDLINE]

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Antibody-PROTAC conjugates enable HER2-dependent targeted protein degradation of BRD4.

ACS Chem Biol. 2020 Apr 27;:

Authors: Maneiro M, Forte N, Shchepinova MM, Kounde CS, Chudasama V, Baker JR, Tate EW

Abstract
Targeting protein degradation with Proteolysis-Targeting Chimeras (PROTACs) is an area of great current interest in drug discovery. Nevertheless, although the high effectiveness of PROTACs against a wide variety of targets has been established, most degraders reported to date display limited intrinsic tissue selectivity, and do not discriminate between cells of different types. Here we describe a strategy for selec-tive protein degradation in a specific cell type. We report the design and synthesis of a trastuzumab-PROTAC conjugate (Ab PROTAC 3) in which E3 ligase-directed degrader activity is caged with an antibody linker which can be hydrolyzed following antibody-PROTAC internal-ization, releasing the active PROTAC and inducing catalytic protein degradation. We show that 3 selectively targets bromodomain-containing protein 4 (BRD4) for degradation only in HER2 positive breast cancer cell lines, whilst sparing HER2 negative cells. Using live cell confocal microscopy, we show internalization and lysosomal trafficking of the conjugate specifically in HER2 positive cells, leading to release of active PROTAC in quantities sufficient to induce potent BRD4 degradation. These studies demonstrate proof-of-concept for tissue-specific BRD4 degradation, overcoming limitations of PROTAC selectivity, with significant potential for application to novel targets.

PMID: 32338867 [PubMed - as supplied by publisher]

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An alternative penicillin-binding protein involved in Salmonella relapses following ceftriaxone therapy.

EBioMedicine. 2020 Apr 25;55:102771

Authors: Castanheira S, López-Escarpa D, Pucciarelli MG, Cestero JJ, Baquero F, García-Del Portillo F

Abstract
BACKGROUND: Salmonella causes intracellular infections in humans. Besides quinolones, third generation cephalosporins are first line drugs used for salmonellosis therapy. An unresolved anomaly of this practice involves high relapse rates associated to quinolone- or cephalosporin-susceptible Salmonella isolates in patients that are discharged clinically following initial recovery. Reduced drug accessibility to intracellular locations has been hypothesized to impair pathogen eradication although supporting evidence is lacking in vivo. Here, we uncover a novel penicillin-binding protein as the first Salmonella factor likely contributing to relapse following beta-lactam, mainly ceftriaxone, therapy.
METHODS: We used Salmonella enterica serovar Typhimurium mutants lacking the alternative penicillin-binding proteins PBP2SAL or PBP3SAL. Affinity of PBP2SAL and PBP3SAL for beta-lactam antibiotics was tested. Relapse after ceftriaxone therapy was analysed in the murine typhoid model.
FINDINGS: S. Typhimurium does not express PBP2SAL or PBP3SAL in the Mueller-Hinton medium used for susceptibility testing. The pathogen produces these PBPs in response to acidic pH and nutrient limitation, conditions found in phagosomes of mammalian cells. PBP3SAL has low affinity for beta-lactams, even at acidic pH. In vitro susceptibility to ceftriaxone at low pH is strongly reduced. S. Typhimurium lacking PBP3SAL was unable to cause relapse in mice following ceftriaxone therapy.
INTERPRETATION: The reduced capacity of ceftriaxone to clear S. Typhimurium in vivo is favoured by a switch in beta-lactam targets. This switch, involving production of the less-susceptible PBP3SAL, remains invisible for standard procedures used in clinical therapy. We conclude that eradication of salmonellosis will be possible only upon targeting of PBP3SAL with novel drugs.

PMID: 32344200 [PubMed - as supplied by publisher]

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Enterotoxins can support CAR T cells against solid tumors.

Proc Natl Acad Sci U S A. 2019 12 10;116(50):25229-25235

Authors: von Scheidt B, Wang M, Oliver AJ, Chan JD, Jana MK, Ali AI, Clow F, Fraser JD, Quinn KM, Darcy PK, Kershaw MH, Slaney CY

Abstract
Responses of solid tumors to chimeric antigen receptor (CAR) T cell therapy are often minimal. This is potentially due to a lack of sustained activation and proliferation of CAR T cells when encountering antigen in a profoundly immunosuppressive tumor microenvironment. In this study, we investigate if inducing an interaction between CAR T cells and antigen-presenting cells (APCs) in lymphoid tissue, away from an immunosuppressive microenvironment, could enhance solid-tumor responses. We combined CAR T cell transfer with the bacterial enterotoxin staphylococcal enterotoxin-B (SEB), which naturally links a proportion of T cell receptor (TCR) Vβ subtypes to MHC-II, present on APCs. CAR T cell proliferation and function was significantly enhanced by SEB. Solid tumor-growth inhibition in mice was increased when CAR T cells were administered in combination with SEB. CAR T cell expansion in lymphoid tissue was demonstrated, and inhibition of lymphocyte egress from lymph nodes using FTY720 abrogated the benefit of SEB. We also demonstrate that a bispecific antibody, targeting a c-Myc tag on CAR T cells and cluster of differentiation 40 (CD40), could also enhance CAR T cell activity and mediate increased antitumor activity of CAR T cells. These model systems serve as proof-of-principle that facilitating the interaction of CAR T cells with APCs can enhance their ability to mediate antitumor activity.

PMID: 31767744 [PubMed - indexed for MEDLINE]

In vivo detection of d-amino acid oxidase with hyperpolarized d-[1-13 C]alanine.

NMR Biomed. 2020 Apr 23;:e4303

Authors: Radaelli A, Gruetter R, Yoshihara HAI

Abstract
d-amino acid oxidase (DAO) is a peroxisomal enzyme that catalyzes the oxidative deamination of several neutral and basic d-amino acids to their corresponding α-keto acids. In most mammalian species studied, high DAO activity is found in the kidney, liver, brain and polymorphonuclear leukocytes, and its main function is to maintain low circulating d-amino acid levels. DAO expression and activity have been associated with acute and chronic kidney diseases and with several pathologies related to N-methyl-d-aspartate (NMDA) receptor hypo/hyper-function; however, its precise role is not completely understood. In the present study we show that DAO activity can be detected in vivo in the rat kidney using hyperpolarized d-[1-13 C]alanine. Following a bolus of hyperpolarized d-alanine, accumulation of pyruvate, lactate and bicarbonate was observed only when DAO activity was not inhibited. The measured lactate-to-d-alanine ratio was comparable to the values measured when the l-enantiomer was injected. Metabolites downstream of DAO were not observed when scanning the liver and brain. The conversion of hyperpolarized d-[1-13 C]alanine to lactate and pyruvate was detected in blood ex vivo, and lactate and bicarbonate were detected on scanning the blood pool in the heart in vivo; however, the bicarbonate-to-d-alanine ratio was significantly lower compared with the kidney. These results demonstrate that the specific metabolism of the two enantiomers of hyperpolarized [1-13 C]alanine in the kidney and in the blood can be distinguished, underscoring the potential of d-[1-13 C]alanine as a probe of d-amino acid metabolism.

PMID: 32325540 [PubMed - as supplied by publisher]

Commensal bacteria stimulate antitumor responses via T cell cross-reactivity.

JCI Insight. 2020 Apr 23;5(8):

Authors: Bessell CA, Isser A, Havel JJ, Lee S, Bell DR, Hickey JW, Chaisawangwong W, Glick Bieler J, Srivastava R, Kuo F, Purohit T, Zhou R, Chan TA, Schneck JP

Abstract
Recent studies show gut microbiota modulate antitumor immune responses; one proposed mechanism is cross-reactivity between antigens expressed in commensal bacteria and neoepitopes. We found that T cells targeting an epitope called SVYRYYGL (SVY), expressed in the commensal bacterium Bifidobacterium breve (B. breve), cross-react with a model neoantigen, SIYRYYGL (SIY). Mice lacking B. breve had decreased SVY-reactive T cells compared with B. breve-colonized mice, and the T cell response was transferable by SVY immunization or by cohousing mice without Bifidobacterium with ones colonized with Bifidobacterium. Tumors expressing the model SIY neoantigen also grew faster in mice lacking B. breve compared with Bifidobacterium-colonized animals. B. breve colonization also shaped the SVY-reactive TCR repertoire. Finally, SVY-specific T cells recognized SIY-expressing melanomas in vivo and led to decreased tumor growth and extended survival. Our work demonstrates that commensal bacteria can stimulate antitumor immune responses via cross-reactivity and how bacterial antigens affect the T cell landscape.

PMID: 32324171 [PubMed - as supplied by publisher]

Neuropeptides in gut-brain axis and their influence on host immunity and stress.

Comput Struct Biotechnol J. 2020;18:843-851

Authors: Wei P, Keller C, Li L

Abstract
In recent decades, neuropeptides have been found to play a major role in communication along the gut-brain axis. Various neuropeptides are expressed in the central and peripheral nervous systems, where they facilitate the crosstalk between the nervous systems and other major body systems. In addition to being critical to communication from the brain in the nervous systems, neuropeptides actively regulate immune functions in the gut in both direct and indirect ways, allowing for communication between the immune and nervous systems. In this mini review, we discuss the role of several neuropeptides, including calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activating polypeptide (PACAP), corticotropin-releasing hormone (CRH) and phoenixin (PNX), in the gut-brain axis and summarize their functions in immunity and stress. We choose these neuropeptides to highlight the diversity of peptide communication in the gut-brain axis.

PMID: 32322366 [PubMed]

In nature, bacteria often form complex communities where interactions between different species have important roles in shaping the overall behavior of the microbial community. Here, we show that GlcNAc‐induced inhibition of ClpXP causes an activation of the Pqs and Rhl quorum‐sensing systems in Pseudomonas aeruginosa, resulting in an increase in the antimicrobial activity of this important human pathogen. Thus, the chemical‐induced alteration of protein homeostasis may represent a new mechanism for interspecies interactions.

Abstract

Intracellular protein degradation is essential for the survival of all organisms, but its role in interspecies interaction is unknown. Here, we show that the ClpXP protease of Pseudomonas aeruginosa suppresses its antimicrobial activity against Staphylococcus aureus, a common pathogen co‐isolated with P. aeruginosa from polymicrobial human infections. Using proteomic, biochemical, and molecular genetic approaches, we found that this effect is due to the inhibitory effects of ClpXP on the quorum sensing (QS) of P. aeruginosa, mainly by degrading proteins (e.g., PhnA, PhnB, PqsR, and RhlI) which are critical for the production of QS signal molecules PQS and C4‐HSL. We provide evidence that co‐culturing with S. aureus induces a decrease in the activity of ClpXP in P. aeruginosa, an effect which was also achieved by the treatment of P. aeruginosa with N‐acetylglucosamine (GlcNAc), a widespread chemical present on the surface of diverse cell types from bacteria to humans. These findings extend the range of biological events governed by proteolytic machinery to microbial community structure, thus also suggesting that a chemical‐induced alteration of protein homeostasis is a mechanism for interspecies interactions.

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Gut microbiota mediate the protective effects on endometritis induced by Staphylococcus aureus in mice.

Food Funct. 2020 Apr 20;:

Authors: Hu X, Mu R, Xu M, Yuan X, Jiang P, Guo J, Cao Y, Zhang N, Fu Y

Abstract
Endometritis, the inflammation of the endometrial lining caused by bacterial pathogens, is associated with reproductive failure. Recent studies have shown that gut microbiota play an important role in infectious diseases. However, the roles of the gut microbiota in endometritis remain unclear. Here, we assessed the effects and mechanisms of the gut microbiota during endometritis induced by Staphylococcus aureus (S. aureus). A mouse gut microbiota-dysbiosis model was established by a mixture of antibiotics (Abx) and subsequently, a model of endometritis was established by the uterine perfusion of S. aureus. Fecal microbiota transplantation (FMT) was performed to evaluate the relationship between gut microbiota and endometritis. The results showed that the mice with gut microbiota-dysbiosis developed uterine inflammation, while this inflammatory response of the uterus was alleviated in mice with FMT to gut microbiota-dysbiosis. In addition, S. aureus-induced endometritis was greater in severity in the mice with gut dysbiosis as compared to the untreated mice. Moreover, these effects were reversed in mice with FMT to the gut microbiota-dysbiosis. GC-MS analysis demonstrated that the levels of short-chain fatty acids (SCFAs) in the feces of mice with gut microbiota-dysbiosis significantly decreased and pretreatment with sodium butyrate or sodium propionate increased the concentrations of butyrate or propionate in both the circulation and uterine tissues, thereby reducing the severity of endometritis induced by S. aureus. In addition, the increased pathogen load in the uteri of the mice with gut microbiota-dysbiosis was associated with a reduction in the phagocytic ability and responsiveness of neutrophils. In conclusion, the gut microbiota offer a protective effect against S. aureus-induced endometritis by regulating the levels of SCFAs and maintaining the phagocytic ability and responsiveness of neutrophils.

PMID: 32307472 [PubMed - as supplied by publisher]

Nature, Published online: 20 April 2020; doi:10.1038/d41586-020-01105-1

The mammalian gut must defend against a variety of infectious agents. Neurons, cells not usually thought of as first-responders during infection, are now found to aid the gut’s barrier function and stop bacteria from spreading elsewhere.

Nature, Published online: 21 April 2020; doi:10.1038/d41586-020-01163-5

Despite uncertainties, some scientists are betting that blood tests will help end lockdowns and get people back to work.

Antibiotic resistance is a growing health issue that is now rendering humans vulnerable once again to infections that have been treatable for decades. Various approaches have been proposed to overcome this threat and effectively treat bacterial infections. DNA nanostructures, functionalized with aptamers, were used as a vehicle for delivering the antibacterial enzyme lysozyme in a specific and efficient manner, to destroy bacterial targets.

Abstract

We report the use of DNA origami nanostructures, functionalized with aptamers, as a vehicle for delivering the antibacterial enzyme lysozyme in a specific and efficient manner. We test the system against Gram‐positive (Bacillus subtilis ) and Gram‐negative (Escherichia coli ) targets. We use direct stochastic optical reconstruction microscopy (d STORM) and atomic force microscopy (AFM) to characterize the DNA origami nanostructures and structured illumination microscopy (SIM) to assess the binding of the origami to the bacteria. We show that treatment with lysozyme‐functionalized origami slows bacterial growth more effectively than treatment with free lysozyme. Our study introduces DNA origami as a tool in the fight against antibiotic resistance, and our results demonstrate the specificity and efficiency of the nanostructure as a drug delivery vehicle.

Nature Biotechnology, Published online: 16 April 2020; doi:10.1038/s41587-020-0513-4

SARS-CoV-2 in patient samples is detected in under an hour using a CRISPR-based lateral flow assay.

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Cultivation-Free Typing of Bacteria Using Optical DNA Mapping.

ACS Infect Dis. 2020 Apr 15;:

Authors: Müller V, Nyblom M, Johnning A, Wrande M, Dvirnas A, Kk S, Giske CG, Ambjornsson T, Sandegren L, Kristiansson E, Westerlund F

Abstract
A variety of pathogenic bacteria can infect humans, and rapid species identification is crucial for the correct treatment. However, the identification process can often be time-consuming and depend on the cultivation of the bacterial pathogen(s). Here we present a standalone, enzyme-free, optical DNA mapping assay capable of species identification by matching intensity profiles of large DNA molecules to a database of fully assembled bacterial genomes (>10 000). The assay includes a new data analysis strategy as well as a general DNA extraction protocol for both Gram-negative and Gram-positive bacteria. We demonstrate that the assay is capable of identifying bacteria directly from uncultured clinical urine samples, as well as in mixtures, with the potential to be discriminative even at the subspecies level. We foresee that the assay has applications both within research labs and in clinical settings, where the time-consuming step of cultivation can be minimized or even completely avoided.

PMID: 32294378 [PubMed - as supplied by publisher]

Scientific Reports, Published online: 15 April 2020; doi:10.1038/s41598-020-63475-w

Gut metagenomic and short chain fatty acids signature in hypertension: a cross-sectional study

ABSTRACT

Staphylococcus aureus is a major concern in human health care, mostly due to the increasing prevalence of antibiotic resistance. Intracellular localization of S. aureus plays a key role in recurrent infections by protecting the pathogens from antibiotics and immune responses. Peptidoglycan hydrolases (PGHs) are highly specific bactericidal enzymes active against both drug-sensitive and -resistant bacteria. However, PGHs able to effectively target intracellular S. aureus are not yet available. To overcome this limitation, we first screened 322 recombineered PGHs for staphylolytic activity under conditions found inside eukaryotic intracellular compartments. The most active constructs were modified by fusion to different cell-penetrating peptides (CPPs), resulting in increased uptake and enhanced intracellular killing (reduction by up to 4.5 log units) of various S. aureus strains (including methicillin-resistant S. aureus [MRSA]) in different tissue culture infection models. The combined application of synergistic PGH-CPP constructs further enhanced their intracellular efficacy. Finally, synergistically active PGH-CPP cocktails reduced the total S. aureus by more than 2.2 log units in a murine abscess model after peripheral injection. Significantly more intracellular bacteria were killed by the PGH-CPPs than by the PGHs alone. Collectively, our findings show that CPP-fused PGHs are effective novel protein therapeutics against both intracellular and drug-resistant S. aureus.

IMPORTANCE The increasing prevalence of antibiotic-resistant bacteria is one of the most urgent problems of our time. Staphylococcus aureus is an important human pathogen that has acquired several mechanisms to evade antibiotic treatment. In addition, S. aureus is able to invade and persist within human cells, hiding from the immune response and antibiotic therapies. For these reasons, novel antibacterial strategies against these pathogens are needed. Here, we developed lytic enzymes which are able to effectively target drug-resistant and intracellular S. aureus. Fusion of these so-called enzybiotics to cell-penetrating peptides enhanced their uptake and intracellular bactericidal activity in cell culture and in an abscess mouse model. Our results suggest that cell-penetrating enzybiotics are a promising new class of therapeutics against staphylococcal infections.