Brianna Dalesandro

Nature Communications, Published online: 17 January 2020; doi:10.1038/s41467-019-13654-9

Antibodies expressed in the cytosol often form insoluble aggregates, which makes it hard to target intracellular proteins. Here the authors engineer an ultra-stable cytoplasmic antibody (STAND) with a low isoelectric point that can be used in vivo.

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Synthesis of glycocluster-containing conjugates for a vaccine against cholera.

Org Biomol Chem. 2019 04 17;17(16):4049-4060

Authors: Pfister HB, Kelly M, Qadri F, Ryan ET, Kováč P

Abstract
Glycoclusters displaying synthetic fragments of the O-specific polysaccharide (OSP) of Vibrio cholerae O1 serotype Inaba on a carbohydrate platform were prepared by Cu(i)-catalysed azide alkyne cycloaddition (CuAAC, click chemistry). The clusters were subsequently conjugated to BSA via squaric acid chemistry. Their immunoreactivity was compared with those of similar conventional conjugates, i.e. made from single oligosaccharides presented in non cluster form, using plasma of patients recovering from cholera. The results showed that the conjugates were displayed in immunologically relevant manners and that the immunoreactivity of hexasaccharide-cluster conjugates was similar to that of a conjugate displaying OSP isolated from wild type V. cholerae, further supporting the immunologic relevance of antigens made from synthetic oligosaccharides.

PMID: 30950473 [PubMed - indexed for MEDLINE]

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Dynamic post-translational modification profiling of M. tuberculosis-infected primary macrophages.

Elife. 2020 Jan 17;9:

Authors: Budzik JM, Swaney DL, Jimenez-Morales D, Johnson JR, Garelis NE, Repasy T, Roberts AW, Popov LM, Parry TJ, Pratt D, Ideker T, Krogan NJ, Cox JS

Abstract
Macrophages are highly plastic cells with critical roles in immunity, cancer, and tissue homeostasis, but how these distinct cellular fates are triggered by environmental cues is poorly understood. To uncover how primary murine macrophages respond to bacterial pathogens, we globally assessed changes in post-translational modifications of proteins during infection with Mycobacterium tuberculosis, a notorious intracellular pathogen. We identified hundreds of dynamically regulated phosphorylation and ubiquitylation sites, indicating that dramatic remodeling of multiple host pathways, both expected and unexpected, occurred during infection. Most of these cellular changes were not captured by mRNA profiling, and included activation of ubiquitin-mediated autophagy, an evolutionarily ancient cellular antimicrobial system. This analysis also revealed that a particular autophagy receptor, TAX1BP1, mediates clearance of ubiquitylated Mtb and targets bacteria to LC3-positive phagophores. These studies provide a new resource for understanding how macrophages shape their proteome to meet the challenge of infection.

PMID: 31951200 [PubMed - as supplied by publisher]

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Outer membrane protein A (OmpA) as a potential therapeutic target for Acinetobacter baumannii infection.

J Biomed Sci. 2020 Jan 18;27(1):26

Authors: Nie D, Hu Y, Chen Z, Li M, Hou Z, Luo X, Mao X, Xue X

Abstract
Acinetobacter baumannii (A. baumannii) is an important opportunistic pathogen causing serious nosocomial infections, which is considered as the most threatening Gram-negative bacteria (GNB). Outer membrane protein A (OmpA), a major component of outer membrane proteins (OMPs) in GNB, is a key virulence factor which mediates bacterial biofilm formation, eukaryotic cell infection, antibiotic resistance and immunomodulation. The characteristics of OmpA in Escherichia coli (E. coli) have been extensively studied since 1974, but only in recent years researchers started to clarify the functions of OmpA in A. baumannii. In this review, we summarized the structure and functions of OmpA in A. baumannii (AbOmpA), collected novel therapeutic strategies against it for treating A. baumannii infection, and emphasized the feasibility of using AbOmpA as a potential therapeutic target.

PMID: 31954394 [PubMed - in process]

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Suppression of Staphylococcus aureus superantigen-independent interferon gamma response by a probiotic polysaccharide.

Infect Immun. 2020 Jan 13;:

Authors: Paik W, Alonzo F, Knight KL

Abstract
Staphylococcus aureus is a gram-positive opportunistic pathogen that causes a variety of diseases. Bloodstream infection is the most severe, with mortality rates reaching 20-50%. Exopolysaccharide (EPS) from the probiotic, Bacillus subtilis, reduces bacterial burden and inflammation during S. aureus bloodstream infection in mice. Protection is due, in part, to hybrid macrophages that restrict S. aureus growth through reactive oxygen species, and to limiting superantigen-induced T cell activation, and IFN-γ production during infection. The decrease in IFN-γ production was observed within 24 hr after infection, and here, we investigated how EPS abrogates its production. We discovered that S. aureus uses a rapid, superantigen-independent mechanism to induce host IFN-γ and that this is mediated by IL-12 activation of NK cells. Furthermore, we found that EPS limits IFN-γ production by modulating host immunity in a TLR4-dependent manner, a signaling pathway that is required for EPS-mediated protection from S. aureus infection in vivo We conclude that EPS protects hosts from acute bloodstream S. aureus infection not only by inducing macrophages that restrict S. aureus growth and inhibit superantigen-activated T cells, but also by limiting NK cell production of IFN-γ after S. aureus infection, in a TLR4-dependent manner.

PMID: 31932326 [PubMed - as supplied by publisher]

Antibiotic-resistant bacteria are increasingly the source of deadly infections. A team of scientists from the Technical University of Munich (TUM) and the Helmholtz Center for Infection Research (HZI) in Braunschweig have now modified an approved cancer drug to develop an active agent against multidrug-resistant pathogens.

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Identification of drug-resistance determinants in a clinical isolate of Pseudomonas aeruginosa by high-density transposon mutagenesis.

Antimicrob Agents Chemother. 2019 Dec 09;:

Authors: Sonnabend MS, Klein K, Beier S, Angelov A, Kluj R, Mayer C, Groß C, Hofmeister K, Beuttner A, Willmann M, Peter S, Oberhettinger P, Schmidt A, Autenrieth IB, Schütz M, Bohn E

Abstract
With the aim to identify potential new targets to restore antimicrobial susceptibility of multidrug-resistant (MDR) Pseudomonas aeruginosa (Pa), we generated a high-density transposon (Tn) insertion mutant library in a MDR Pa bloodstream isolate (ID40). The depletion of Tn insertion mutants upon exposure to cefepime or meropenem was measured in order to determine the common resistome for these clinically important antipseudomonal β-lactam antibiotics. The approach was validated by clean deletions of genes involved in peptidoglycan synthesis/recycling such as the lytic transglycosylase MltG, the murein endopeptidase MepM1, the MurNAc/GlcNAc-kinase AmgK and the uncharacterized protein YgfB that all were identified in our screen as playing a decisive role for survival of treatment with cefepime or meropenem. We found that the antibiotic resistance of Pa can be overcome by targeting usually non-essential genes that turn essential in the presence of therapeutic concentrations of antibiotics. For all validated genes, we demonstrated that their deletion leads to the reduction of ampC expression, resulting in a significant decrease of β-lactamase activity and consequently these mutants partly or completely lost resistance against cephalosporins, carbapenems and acylaminopenicillins. In summary, the determined resistome may comprise promising targets for developing drugs that could be used to restore the sensitivity towards existing antibiotics specifically in MDR strains of Pa.

PMID: 31818817 [PubMed - as supplied by publisher]

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Molecular Nanomachines Disrupt Bacterial Cell Wall Increasing Sensitivity of Extensively Drug Resistant Klebsiella pneumoniae to Meropenem.

ACS Nano. 2019 Dec 09;:

Authors: Galbadage T, Liu D, Alemany LB, Pal R, Tour JM, Gunasekera RS, Cirillo JD

Abstract
Multidrug-resistance in pathogenic bacteria is an increasing problem in patient care and public health. Molecular nanomachines (MNM) have the ability to open cell membranes using nanomechanical action. We hypothesized that MNM could be used as antibacterial agents by drilling into bacterial cell walls and increasing susceptibility of drug resistant bacteria to recently ineffective antibiotics. We exposed extensively drug resistant K. pneumoniae to light-activated MNM and found that MNM increase susceptibility to meropenem. MNM with meropenem can effectively kill K. pneumoniae that are considered meropenem resistant. We examined the mechanisms of MNM action using permeability assays and transmission electron microscopy, finding that MNM disrupt the cell wall of extensively drug resistant K. pneumoniae, exposing the bacteria to meropenem. These observations suggest that MNM could be used to make conventional antibiotics more efficacious against multidrug-resistant pathogens.

PMID: 31815423 [PubMed - as supplied by publisher]

Nanopipettes with zwitterionic membranes may offer improved monitoring of changes in pH surrounding living cells, which can indicate traits of invasive cancer cells and their response to treatment, report researchers at Kanazawa University in Nature Communications.

Nature Reviews Microbiology, Published online: 04 December 2019; doi:10.1038/s41579-019-0310-6

Two recent studies report new compounds that could be useful in combatting antibiotic resistance.

Imagine a sugar that has only 38 percent of the calories of traditional table sugar, is safe for diabetics, and will not cause cavities. Now add that this dream sweetener is not an artificial substitute but a real sugar found in nature and it tastes like, well, sugar. You'd probably want to use that in your next cup of coffee, right?

Research led by scientists from McMaster University has yielded a potent antimicrobial that works against the toughest infectious disease strains. The find could be the beginning of developing new therapeutics to combat drug-resistant infections.

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Metabolic Incorporation of N-Acetyl Muramic Acid Probes into Bacterial Peptidoglycan.

Curr Protoc Chem Biol. 2019 Dec;11(4):e74

Authors: DeMeester KE, Liang H, Zhou J, Wodzanowski KA, Prather BL, Santiago CC, Grimes CL

Abstract
Bacterial cells utilize small carbohydrate building blocks to construct peptidoglycan (PG), a highly conserved mesh-like polymer that serves as a protective coat for the cell. PG production has long been a target for antibiotics, and its breakdown is a source for human immune recognition. A key component of bacterial PG, N-acetyl muramic acid (NAM), is a vital element in many synthetically derived immunostimulatory compounds. However, the exact molecular details of these structures and how they are generated remain unknown due to a lack of chemical probes surrounding the NAM core. A robust synthetic strategy to generate bioorthogonally tagged NAM carbohydrate units is implemented. These molecules serve as precursors for PG biosynthesis and recycling. Escherichia coli cells are metabolically engineered to incorporate the bioorthogonal NAM probes into their PG network. The probes are subsequently modified using copper-catalyzed azide-alkyne cycloaddition to install fluorophores directly into the bacterial PG, as confirmed by super-resolution microscopy and high-resolution mass spectrometry. Here, synthetic notes for key elements of this process to generate the sugar probes as well as streamlined user-friendly metabolic labeling strategies for both microbiology and immunological applications are described. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of peracetylated 2-azido glucosamine Basic Protocol 2: Synthesis of 2-azido and 2-alkyne NAM Basic Protocol 3: Synthesis of 3-azido NAM methyl ester Basic Protocol 4: Incorporation of NAM probes into bacterial peptidoglycan Basic Protocol 5: Confirmation of bacterial cell wall remodeling by mass spectrometry.

PMID: 31763799 [PubMed - in process]

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Mapping Epitopes of a Novel Peptidoglycan Cross-Linking Enzyme Cwp22 Recognized by Human Sera Obtained from Patients with Clostridioides difficile Infection and Cord Blood.

Microorganisms. 2019 Nov 14;7(11):

Authors: Razim A, Pacyga K, Martirosian G, Szuba A, Gamian A, Myc A, Górska S

Abstract
Clostridioides difficile (CD) cause a severe diarrhea which can lead to pseudomembranous colitis and even patient death. CD infection (CDI) is connected mainly with changes in intestinal microbiota as a consequence of antibiotic treatment. The growing resistance to antibiotics, justifies the search for new methods of combating CD. Despite of ongoing research on the immunity against the pathogen, there is still lack of any reliable vaccine. Most recently, Cwp22, that is a cross-linking enzyme involved in the production of CD peptidoglycan, seems to be a promising target to prevent CDI in high-risk patients. In this paper, the Cwp22 protein polypeptide-specific epitopes were mapped in silico and using PEPSCAN procedure. They were recognized not only by antibodies from CDI patients' but also by umbilical cord blood sera. We identified three epitopes 54EFRVAT59, 201KVNGKM206 and 268WQEKNGKKYY277 of Cwp22 protein. Since Cwp22 protein has key functionality and the described above epitopes are also recognized by umbilical cord blood serum, we postulate that they could have important protective properties. In this paper, we propose Cwp22 protein as a good antigen candidate for CDI preventive vaccine. Our results open the possibility to use 54EFRVAT59, 201KVNGKM206 and 268WQEKNGKKYY277, epitopes as suitable anti-CD vaccine antigens.

PMID: 31739602 [PubMed]

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Formation of a β-barrel membrane protein is catalyzed by the interior surface of the assembly machine protein BamA.

Elife. 2019 11 14;8:

Authors: Lee J, Tomasek D, Santos TM, May MD, Meuskens I, Kahne D

Abstract
The β-barrel assembly machine (Bam) complex in Gram-negative bacteria and its counterparts in mitochondria and chloroplasts fold and insert outer membrane β-barrel proteins. BamA, an essential component of the complex, is itself a β-barrel and is proposed to play a central role in assembling other barrel substrates. Here, we map the path of substrate insertion by the Bam complex using site-specific crosslinking to understand the molecular mechanisms that control β-barrel folding and release. We find that the C-terminal strand of the substrate is stably held by BamA and that the N-terminal strands of the substrate are assembled inside the BamA β-barrel. Importantly, we identify contacts between the assembling β-sheet and the BamA interior surface that determine the rate of substrate folding. Our results support a model in which the interior wall of BamA acts as a chaperone to catalyze β-barrel assembly.

PMID: 31724945 [PubMed - indexed for MEDLINE]

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A novel antimicrobial peptide acting via formyl peptide receptor 2 shows therapeutic effects against rheumatoid arthritis.

Sci Rep. 2018 10 02;8(1):14664

Authors: Park YJ, Park B, Lee M, Jeong YS, Lee HY, Sohn DH, Song JJ, Lee JH, Hwang JS, Bae YS

Abstract
In oriental medicine, centipede Scolopendra subspinipes mutilans has long been used as a remedy for rheumatoid arthritis (RA), a well-known chronic autoimmune disorder. However, the molecular identities of its bioactive components have not yet been extensively investigated. We sought to identify bioactive molecules that control RA with a centipede. A novel antimicrobial peptide (AMP) (scolopendrasin IX) was identified from Scolopendra subspinipes mutilans. Scolopendrasin IX markedly activated mouse neutrophils, by enhancing cytosolic calcium increase, chemotactic cellular migration, and generation of superoxide anion in neutrophils. As a target receptor for scolopendrasin IX, formyl peptide receptor (FPR)2 mediates neutrophil activation induced by the AMP. Furthermore, scolopendrasin IX administration strongly blocked the clinical phenotype of RA in an autoantibody-injected model. Mechanistically, the novel AMP inhibited inflammatory cytokine synthesis from the joints and neutrophil recruitment into the joint area. Collectively, we suggest that scolopendrasin IX is a novel potential therapeutic agent for the control of RA via FPR2.

PMID: 30279454 [PubMed - indexed for MEDLINE]

ABSTRACT

All evolutionary processes are underpinned by a cellular capacity to mutate DNA. To identify factors affecting mutagenesis, it is necessary to compare mutation rates between different strains and conditions. Drug resistance-based mutation reporters are used extensively to measure mutation rates, but they are suitable only when the compared strains have identical drug tolerance levels—a condition that is not satisfied under many "real-world" circumstances, e.g., when comparing mutation rates among a series of environmental or clinical isolates. Candida glabrata is a fungal pathogen that shows a high degree of genetic diversity and fast emergence of antifungal drug resistance. To enable meaningful comparisons of mutation rates among C. glabrata clinical isolates, we developed a novel fluorescence-activated cell sorting-based approach to measure the mutation rate of a chromosomally integrated GFP gene. We found that in Saccharomyces cerevisiae this approach recapitulated the reported mutation rate of a wild-type strain and the mutator phenotype of a shu1 mutant. In C. glabrata, the GFP reporter captured the mutation rate increases caused either by a genotoxic agent or by deletion of DNA mismatch repair gene MSH2, as well as the specific mutational signature associated with msh2. Finally, the reporter was used to measure the mutation rates of C. glabrata clinical isolates carrying different alleles of MSH2. Together, these results show that fluorescence-based mutation reporters can be used to measure mutation rates in microbes under conditions of unequal drug susceptibility to reveal new insights about drivers of mutagenesis.

IMPORTANCE Measurements of mutation rates—i.e., how often proliferating cells acquire mutations in their DNA—are essential for understanding cellular processes that maintain genome stability. Many traditional mutation rate measurement assays are based on detecting mutations that cause resistance to a particular drug. Such assays typically work well for laboratory strains but have significant limitations when comparing clinical or environmental isolates that have various intrinsic levels of drug tolerance, which confounds the interpretation of results. Here we report the development and validation of a novel method of measuring mutation rates, which detects mutations that cause loss of fluorescence rather than acquisition of drug resistance. Using this method, we measured the mutation rates of clinical isolates of fungal pathogen Candida glabrata. This assay can be adapted to other organisms and used to compare mutation rates in contexts where unequal drug sensitivity is anticipated.

Reduction of systemic inflammation without immune system inhibition: A photoswitchable ligand for regulation of immune activation through optical control of the heterodimerization of toll‐like receptors 1 and 2 (TLR1/2) has been developed, offering the potential to regulate immune activation and inflammation.

Abstract

The activation of toll‐like receptors (TLRs) plays important roles in the immune response. The ability to control the activities of TLRs could be usable as a switch for immune response. Here we have rationally designed and synthesized a photoswitchable Pam₃CSK₄ derivative—P10—to control the activation of TLR1/2. The ground‐state trans‐P10 was able to stimulate and activate antigen‐presenting cells (APCs) by promoting TLR1/2 heterodimerization. However, cis‐P10, derived from UV irradiation of trans‐P10, reduced the activities of APCs by impeding the TLR1/2 heterodimerization. In the absence of UV radiation, the cis‐P10 slowly returned to its ground trans state, restoring the activities of the APCs stimulation. Our results indicated that optical control of TLR1/2 heterodimerization mediated by the photoswitchable P10 offers the potential to regulate immune activation and inflammation.

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Targeted cancer immunotherapy with genetically engineered oncolytic Salmonella typhimurium.

Cancer Lett. 2019 Oct 24;:

Authors: Guo Y, Chen Y, Liu X, Min JJ, Tan W, Zheng JH

Abstract
Conventional chemotherapies have some limitations, including the lack of selectivity, high toxicity to normal tissues, multidrug resistance, and tumor relapse. Recently, great progress was made in immunotherapies for anticancer research, with bacteria-mediated cancer therapy one of the most promising approaches among them. Attenuated Salmonella have very specific targeting to various solid cancers, making them ideal vectors for the delivery and expression of immunostimulators. They have native bacterial immunogenicity and induce strong anticancer immunity in vivo. In this review, the recent advances in Salmonella-mediated cancer immunotherapies and the related mechanisms of Salmonella-based cancer therapies are summarized.

PMID: 31666180 [PubMed - as supplied by publisher]

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Disrupting the transmembrane domain-mediated oligomerization of protein tyrosine phosphatase receptor J inhibits EGFR-driven cancer cell phenotypes.

J Biol Chem. 2019 12 06;294(49):18796-18806

Authors: Bloch E, Sikorski EL, Pontoriero D, Day EK, Berger BW, Lazzara MJ, Thévenin D

Abstract
Receptor protein tyrosine phosphatases (RPTPs) play critical regulatory roles in mammalian signal transduction. However, the structural basis for the regulation of their catalytic activity is not fully understood, and RPTPs are generally not therapeutically targetable. This knowledge gap is partially due to the lack of known natural ligands or selective agonists of RPTPs. Contrary to what is known from structure-function studies of receptor tyrosine kinases (RTKs), RPTP activities have been reported to be suppressed by dimerization, which may prevent RPTPs from accessing their RTK substrates. We report here that homodimerization of protein tyrosine phosphatase receptor J (PTPRJ, also known as DEP-1) is regulated by specific transmembrane (TM) residues. We found that disrupting these interactions destabilizes homodimerization of full-length PTPRJ in cells, reduces the phosphorylation of the known PTPRJ substrate epidermal growth factor receptor (EGFR) and of other downstream signaling effectors, antagonizes EGFR-driven cell phenotypes, and promotes substrate access. We demonstrate these observations in human cancer cells using mutational studies and identified a peptide that binds to the PTPRJ TM domain and represents the first example of an allosteric agonist of RPTPs. The results of our study provide fundamental structural and functional insights into how PTPRJ activity is tuned by TM interactions in cells. Our findings also open up opportunities for developing peptide-based agents that could be used as tools to probe RPTPs' signaling mechanisms or to manage cancers driven by RTK signaling.

PMID: 31676686 [PubMed - indexed for MEDLINE]