Brianna Dalesandro

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A Vancomycin-Arginine Conjugate Inhibits Growth of Carbapenem-resistant E. coli and Targets Cell-Wall Synthesis.

ACS Chem Biol. 2019 Sep 03;:

Authors: Antonoplis A, Zang X, Wegner T, Wender PA, Cegelski L

Abstract
The emergence of multidrug-resistant Gram-negative bacteria, including carbapenem-resistant Enterobacteriaceae, is a major health problem that necessitates the development of new antibiotics. Vancomycin inhibits cell wall synthesis in Gram-positive bacteria, but is generally ineffective against Gram-negative bacteria and unable to penetrate the outer membrane barrier. In an effort to determine whether vancomycin and other antibiotics effective against Gram-positive bacteria could, through modification, be rendered effective against Gram-negative bacteria, we discovered that covalent attachment of a single arginine to vancomycin yielded conjugates with order-of-magnitude improvements in activity against Gram-negative bacteria, including pathogenic E. coli. The vancomycin-arginine conjugate (V-R) exhibited efficacy against actively-growing bacteria, induced loss of rod cellular morphology, and resulted in intracellular accumulation of peptidoglycan precursors, all consistent with cell-wall synthesis disruption as its mechanism of action. Membrane permeabilization studies demonstrated enhanced outer membrane permeability of V-R as compared to vancomycin. The conjugate exhibited no mammalian cell toxicity or hemolytic activity in MTT and hemolysis assays. Our study introduces a new vancomycin derivative effective against Gram-negative bacteria and underscores the broader potential of generating new antibiotics through combined mode-of-action and synthesis-informed design studies.

PMID: 31479234 [PubMed - as supplied by publisher]

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Design, synthesis and biological evaluation of immunostimulating mannosylated desmuramyl peptides.

Beilstein J Org Chem. 2019;15:1805-1814

Authors: Ribić R, Stojković R, Milković L, Antica M, Cigler M, Tomić S

Abstract
Muramyl dipeptide is the minimal structure of peptidoglycan with adjuvant properties. Replacement of the N-acetylmuramyl moiety and increase of lipophilicity are important approaches in the preparation of muramyl dipeptide analogues with improved pharmacological properties. Mannose receptors present on immunocompetent cells are pattern-recognition receptors and by mannose ligands binding they affect the immune system. Here we present the design, synthesis and biological evaluation of novel mannosylated desmuramyl peptide derivatives. Mannose was coupled to dipeptides containing a lipophilic adamantane on N- or C-terminus through a glycolyl or hydroxyisobutyryl linker. Adjuvant activities of synthesized compounds were investigated in the mouse model using ovalbumin as an antigen. Their activities were compared to the previously described mannosylated adamantane-containing desmuramyl peptide and peptidoglycan monomer. Tested compounds exhibited adjuvant activity and the strongest enhancement of IgG production was stimulated by compound 21 (Man-OCH2-ᴅ-(1-Ad)Gly-ʟ-Ala-ᴅ-isoGln).

PMID: 31467600 [PubMed]

Finding hidden bacteria: An AIEgen‐peptide‐based fluorescent bioprobe was designed as a phototheranostic agent for phagosome‐entrapped bacteria. Activation of the probe by caspase‐1 in infected macrophages leads to a fluorescent signal. Moreover, the AIEgen can serve as a photosensitizer for generation of reactive oxygen species, which induces highly efficient killing of the bacteria.

Abstract

The detection and elimination of intracellular bacteria remain a major challenge. In this work, we report an aggregation‐induced emission (AIE) bioprobe that can detect bacterial infection and kill bacteria surviving inside macrophages through a dynamic process, notably specific molecular tailoring of the probe by caspase‐1 activation in infected macrophages and accumulation of the residue on phagosomes containing bacteria, leading to light‐up fluorescent signals. Moreover, the AIEgen can serve as a photosensitizer for generation of reactive oxygen species (ROS); and the average ROS indicator fluorescent signal intensity per unit area in the bacterial phagosomes is approximately 2.7‐fold higher than that in the cytoplasm. This, in turn, induces bacteria killing with high efficiency and minimal cytotoxicity towards macrophages. We envision that this specific light‐up bioprobe may provide a new approach for selective and sensitive detection and eradication of intracellular bacterial infections.

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Complement mediated Klebsiella pneumoniae capsule changes.

Microbes Infect. 2019 Aug 29;:

Authors: Jensen TS, Opstrup KV, Christiansen G, Rasmussen PV, Thomsen ME, Justesen DL, Schønheyder HC, Lausen M, Birkelund S

Abstract
The Gram-negative bacterium Klebsiella pneumoniae is an opportunistic pathogen, which can cause life-threatening infections such as sepsis. Worldwide, emerging multidrug resistant K. pneumoniae infections are challenging to treat, hence leading to increased mortality. Therefore, understanding the interactions between K. pneumoniae and the immune system is important to develop new treatment options. We characterized ten clinical K. pneumoniae isolates obtained from blood of bacteremia patients. The interaction of the isolates with human serum was investigated to elucidate how K. pneumoniae escapes the host immune system, and how complement activation by K. pneumoniae changed the capsule structure. All K. pneumoniae isolates activated the alternative complement pathway despite serum resistance of seven isolates. One serum sensitive isolate activated two or all three pathways, and this isolate was lysed and had numerous membrane attack complexes in the outer membrane. However, we also found deposition of complement components in the capsule of serum resistant isolates resulting in morphological capsule changes and capsule shedding. These bacteria did not lyse, and no membrane attack complex was observed despite deposition of C5b-9 within the capsule, indicating that the capsule of serum resistant K. pneumoniae isolates is a defense mechanism against complement-mediated lysis.

PMID: 31473336 [PubMed - as supplied by publisher]

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Peptidoglycan associated cyclic lipopeptide disrupts viral infectivity.

J Virol. 2019 Aug 28;:

Authors: Johnson BA, Hage A, Kalveram B, Mears M, Plante JA, Rodriguez SE, Ding Z, Luo X, Bente D, Bradrick SS, Freiberg AN, Popov V, Rajsbaum R, Rossi S, Russell WK, Menachery VD

Abstract
Enteric viruses exploit bacterial components including lipopolysaccharides (LPS) and peptidoglycan (PG) to facilitate infection in humans. With origins in the bat enteric system, we wondered if severe acute respiratory syndrome-coronavirus (SARS-CoV) or Middle East respiratory syndrome-CoV (MERS-CoV) also use bacterial components to modulate infectivity. To test this question, we incubated CoVs with LPS and PG and evaluated infectivity finding no change following LPS treatment. However, PG from B. subtilis reduced infection >10,000-fold while PG from other bacterial species failed to recapitulate this. Treatment with an alcohol solvent transferred inhibitory activity to the wash and mass spectrometry revealed surfactin, a cyclic lipopeptide antibiotic, as the inhibitory compound. This antibiotic had robust dose- and temperature-dependent inhibition of CoV infectivity. Mechanistic studies indicated that surfactin disrupts CoV virion integrity and surfactin treatment of the virus inoculum ablated infection in vivo Finally, similar cyclic lipopeptides had no effect on CoV infectivity and the inhibitory effect of surfactin extended broadly to enveloped viruses including influenza, Ebola, Zika, Nipah, Chikungunya, Una, Mayaro, Dugbe, and Crimean-Congo hemorrhagic fever viruses. Overall, our results indicate that peptidoglycan-associated surfactin has broad virucidal activity and suggest bacteria byproducts may negatively modulate virus infection.IMPORTANCE In this manuscript, we considered a role for bacteria in shaping coronavirus infection. Taking cues from studies of enteric viruses, we initially investigated how bacterial surface components might improve CoV infection. Instead, we found that peptidoglycan-associated surfactin is a potent viricidal compound that disrupts virion integrity with broad activity against enveloped viruses. Our results indicate that interactions with commensal bacterial may improve or disrupt viral infections highlighting the importance of understanding these microbial interactions and their implications for viral pathogenesis and treatment.

PMID: 31462558 [PubMed - as supplied by publisher]

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Redundant and Distinct Roles of Secreted Protein Eap and Cell Wall-Anchored Protein SasG in Biofilm Formation and Pathogenicity of Staphylococcus aureus.

Infect Immun. 2019 04;87(4):

Authors: Yonemoto K, Chiba A, Sugimoto S, Sato C, Saito M, Kinjo Y, Marumo K, Mizunoe Y

Abstract
Chronic and fatal infections caused by Staphylococcus aureus are sometimes associated with biofilm formation. Secreted proteins and cell wall-anchored proteins (CWAPs) are important for the development of polysaccharide-independent biofilms, but functional relationships between these proteins are unclear. In the present study, we report the roles of the extracellular adherence protein Eap and the surface CWAP SasG in S. aureus MR23, a clinical methicillin-resistant isolate that forms a robust protein-dependent biofilm and accumulates a large amount of Eap in the extracellular matrix. Double deletion of eap and sasG, but not single eap or sasG deletion, reduced the biomass of the formed biofilm. Mutational analysis demonstrated that cell wall anchorage is essential for the role of SasG in biofilm formation. Confocal laser scanning microscopy revealed that MR23 formed a rugged and thick biofilm; deletion of both eap and sasG reduced biofilm ruggedness and thickness. Although sasG deletion did not affect either of these features, eap deletion reduced the ruggedness but not the thickness of the biofilm. This indicated that Eap contributes to the rough irregular surface structure of the MR23 biofilm and that both Eap and SasG play roles in biofilm thickness. The level of pathogenicity of the Δeap ΔsasG strain in a silkworm larval infection model was significantly lower (P < 0.05) than those of the wild type and single-deletion mutants. Collectively, these findings highlight the redundant and distinct roles of a secreted protein and a CWAP in biofilm formation and pathogenicity of S. aureus and may inform new strategies to control staphylococcal biofilm infections.

PMID: 30670553 [PubMed - indexed for MEDLINE]

Photoacoustic imaging of copper(II) in the brain: Small‐molecule probe RPS1 can selectively bind to Cu²⁺ to form a radical with a turn‐on photoacoustic response in the near‐infrared region. RPS1 can effectively cross the blood–brain barrier and image copper(II) in the brain of a mouse suffering form Alzheimer's disease.

Abstract

Copper enrichment in the brain is highly related to Alzheimer's disease (AD) pathogenesis, but in vivo tracing of Cu²⁺ in the brain by imaging techniques is still a great challenge. In this work, we developed a series of activatable photoacoustic (PA) probes with low molecular weights (less than 438 Da), RPS1–RPS4, which can specifically chelate with Cu²⁺ to form radicals with turn‐on PA signals in the near‐infrared (NIR) region. Introducing the electron‐donating group N,N‐dimethylaniline into the probe was found to significantly enhance the radical stability and PA intensity. The best probe in the series, RPS1, showed a fast response (within seconds) to Cu²⁺ with high selectivity and a low PA detection limit of 90.9 nm. Owing to the low molecular weight and amphiphilic structure, RPS1 could effectively cross the blood–brain barrier (BBB) and thus allowed us, for the first time, to visualize Cu²⁺ in vivo via PA imaging in the brains of AD mice.

An organic semiconducting pro‐nanostimulant was developed for synergetic cancer therapy. Only upon near‐infrared (NIR) irradiation is the pro‐nanostimulant activated to simultaneously exert phototherapy and checkpoint blockade immunotherapy, inhibiting the growth of primary/distant tumors and significantly suppressing the lung metastasis without causing any obvious in vivo toxicity.

Abstract

In this study, an organic semiconducting pro‐nanostimulant (OSPS) with a near‐infrared (NIR) photoactivatable immunotherapeutic action for synergetic cancer therapy is presented. OSPS comprises a semiconducting polymer nanoparticle (SPN) core and an immunostimulant conjugated through a singlet oxygen (¹O₂) cleavable linkers. Upon NIR laser irradiation, OSPS generates both heat and ¹O₂ to exert combinational phototherapy not only to ablate tumors but also to produce tumor‐associated antigens. More importantly, NIR irradiation triggers the cleavage of ¹O₂‐cleavable linkers, triggering the remote release of the immunostimulants from OSPS to modulate the immunosuppressive tumor microenvironment. Thus, the released tumor‐associated antigens in conjunction with activated immunostimulants induce a synergistic antitumor immune response after OSPS‐mediated phototherapy, resulting in the inhibited growth of both primary/distant tumors and lung metastasis in a mouse xenograft model, which is not observed for sole phototherapy.

Trapped: The metalloprotease class of deubiquitinases (DUBs) is vital to many cellular processes, but not much is known about this class because of the lack of specific probes. These metalloenzymes have zinc in their active site that can be trapped with a chelation‐based activity probe. Described here is a chemically synthesized Ub‐based probe containing a zinc chelator that can bind to and pull‐down metalloprotease DUBs from mammalian cell lysates.

Abstract

Deubiquitinases (DUBs) are a family of enzymes that regulate the ubiquitin signaling cascade by removing ubiquitin from specific proteins in response to distinct signals. DUBs that belong to the metalloprotease family (metalloDUBs) contain Zn²⁺ in their active sites and are an integral part of distinct cellular protein complexes. Little is known about these enzymes because of the lack of specific probes. Described here is a Ub‐based probe that contains a ubiquitin moiety modified at its C‐terminus with a Zn²⁺ chelating group based on 8‐mercaptoquinoline, and a modification at the N‐terminus with either a fluorescent tag or a pull‐down tag. The probe is validated using Rpn11, a metalloDUB found in the 26S proteasome complex. This probe binds to metalloDUBs and efficiently pulled down overexpressed metalloDUBs from a HeLa cell lysate. Such probes may be used to study the mechanism of metalloDUBs in detail and allow better understanding of their biochemical processes.

Beat tryptophan! The 4‐aminophthalimido system as an amino acid side chain component is isosteric with the tryptophan indole moiety but shows much more powerful and polarity‐sensitive fluorescence. With the aid of amino acids incorporating this moiety it was possible to use fluorescence readout to examine the local polarity in the α‐helical transmembrane fragment of the human epidermal growth factor receptor.

Abstract

Fluorescence probing of transmembrane (TM) peptides is needed to complement state‐of‐the art methods—mainly oriented circular dichroism and solid‐state NMR spectroscopy—and to allow imaging in living cells. Three new amino acids incorporating the solvatofluorescent 4‐aminophthalimide in their side chains were synthesized in order to examine the local polarity in the α‐helical TM fragment of the human epidermal growth factor receptor. It was possible to distinguish their locations, either in the hydrophobic core of the lipid bilayer or at the membrane surface, by fluorescence readout, including blue shift and increased quantum yield. An important feature is the small size of the 4‐aminophthalimide chromophore. It makes one of the new amino acids approximately isosteric to tryptophan, typically used as a very small fluorescent amino acid in peptides and proteins. In contrast to the only weakly fluorescent indole system in tryptophan, the 4‐aminophthalimide moiety produces a significantly more informative fluorescence readout and is selectively excited outside the biopolymer absorption range.

Related Articles

Design and characterization of SGK3-PROTAC1, an isoform specific SGK3 kinase PROTAC degrader.

ACS Chem Biol. 2019 Aug 28;:

Authors: Tovell H, Testa A, Zhou H, Shpiro N, Crafter C, Ciulli A, Alessi DR

Abstract
SGK3 is a PX domain containing protein kinase activated at endosomes downstream of Class 1 and 3 PI3K family members by growth factors and oncogenic mutations. SGK3 plays a key role in mediating resistance of breast cancer cells to Class 1 PI3K or Akt inhibitors, by substituting for loss of Akt activity and restoring proliferative pathways such as mTORC1 signaling. It is therefore critical to develop tools to potently target SGK3, and obstruct its role in inhibitor resistance. Here we describe the development of SGK3-PROTAC1, a PROTAC conjugate of the 308-R SGK inhibitor with the VH032 VHL binding ligand, targeting SGK3 for degradation. 0.3 µM SGK3-PROTAC1 induced 50% degradation of endogenous SGK3 within 2 hours, with maximal 80% degradation observed within 8 hours, accompanied by a loss of phosphorylation of NDRG1, an SGK3 substrate. SGK3-PROTAC1 did not degrade closely related SGK1 and SGK2 isoforms that are nevertheless engaged and inhibited by 308-R. Proteomic analysis revealed that SGK3 was the only cellular protein whose cellular levels were significantly reduced following treatment with SGK3-PROTAC1. Low doses of SGK3-PROTAC1 (0.1-0.3 µM) restored sensitivity of SGK3 dependent ZR-75-1 and CAMA-1 breast cancer cells to Akt (AZD5363) and PI3K (GDC0941) inhibitors, whereas the cis epimer analogue incapable of binding to the VHL E3 ligase had no impact. SGK3-PROTAC1 suppressed proliferation of ZR-75-1 and CAMA-1 cancer cell lines treated with a PI3K inhibitor (GDC0941) more effectively than could be achieved by a conventional SGK isoform inhibitor (14H). This work underscores the benefit of the PROTAC approach in targeting protein kinase signaling pathways with greater efficacy and selectivity than can be achieved with conventional inhibitors. SGK3-PROTAC1 will be an important reagent to explore the roles of the SGK3 pathway.

PMID: 31461270 [PubMed - as supplied by publisher]

Nature Immunology, Published online: 12 August 2019; doi:10.1038/s41590-019-0454-6

Aifantis and colleagues identify the E3 ubiquitin ligase SPOP as a negative regulator of ‘emergency hematopoiesis’ in bone marrow hematopoietic stem progenitor cells.

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Novel Assay To Characterize Neutrophil Responses to Oral Biofilms.

Infect Immun. 2019 02;87(2):

Authors: Oveisi M, Shifman H, Fine N, Sun C, Glogauer N, Senadheera D, Glogauer M

Abstract
Neutrophils, the most numerous leukocytes, play an important role in maintaining oral health through interactions with oral microbial biofilms. Both neutrophil hyperactivity and the bacterial subversion of neutrophil responses can cause inflammation-mediated tissue damage like that seen in periodontal disease. We describe here an assay that assesses neutrophil activation responses to monospecies biofilm bacteria in vitro based on the surface expression of cluster of differentiation (CD) markers associated with various neutrophil functions. Most of what we know about neutrophil responses to bacteria is based on in vitro assays that use planktonic bacteria and isolated/preactivated neutrophils, which makes interpretation of the neutrophil responses to bacteria a challenge. An understanding of how neutrophils differentially interact with and respond to commensal and pathogenic oral bacteria is necessary in order to further understand the neutrophil's role in maintaining oral health and the pathogenesis of periodontal disease. In this study, a flow cytometry-based in vitro assay was developed to characterize neutrophil activation states based on CD marker expressions in response to oral monospecies bacterial biofilms. Using this approach, changes in CD marker expressions in response to specific prominent oral commensal and pathogenic bacteria were assayed. Several functional assays, including assays for phagocytosis, production of reactive oxygen species, activation of the transcription factor Nrf2, neutrophil extracellular trap formation, and myeloperoxidase release, were also performed to correlate neutrophil function with CD marker expression. Our results demonstrate that neutrophils display bacterial species-specific responses. This assay can be used to characterize how specific biofilms alter specific neutrophil pathways associated with their activation.

PMID: 30455195 [PubMed - indexed for MEDLINE]

Development of opsonic mouse monoclonal antibodies against multidrug resistant enterococci.

Infect Immun. 2019 Jul 08;:

Authors: Kalfopoulou E, Laverde D, Miklic K, Romero-Saavedra F, Malic S, Carboni F, Adamo R, Lenac Rovis T, Jonjic S, Huebner J

Abstract
Multidrug-resistant Enterococci are major causes of hospital-acquired infections. Immunotherapy with monoclonal antibodies (mAbs) targeting bacterial antigens would be a valuable treatment option in this setting. Here we describe the development of two mAbs through hybridoma technology that target antigens from the most clinically relevant enterococcal species. Diheteroglycan (DHG), a well-characterized capsular polysaccharide of E. faecalis, and the secreted antigen A (SagA), an immunogenic protein from E. faecium, are both immunogens that have been proven to raise opsonic and cross-reactive antibodies against enterococcal strains. For this purpose, a conjugated form of the native DHG with SagA was used to raise the antibodies in mice, while ELISA and opsonophagocytic assay were combined in the selection process of hybridoma cells producing immunoreactive and opsonic antibodies targeting the selected antigens. From this process two highly specific IgG1 kappa mAbs were obtained: one against the polysaccharide (DHG.01) and one against the protein (SagA.01). Both mAbs exhibited good opsonic killing against the target bacterial strains: DHG.01 showed 90% killing against E. faecalis Type 2 and SagA.01 showed 40% killing against E. faecium 11231/6. In addition, both mAbs showed cross-reactivity towards other E. faecalis and E. faecium strains. The sequences from the variable regions of the heavy and light chain were reconstructed in expression vectors, and the activity of the mAbs upon expression in eukaryotic cells was confirmed with the same immunological assays. In summary, we identified two opsonic mAbs against enterococci which could be used for therapeutic or prophylactic approaches against enterococcal infections.

PMID: 31285252 [PubMed - as supplied by publisher]

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The essential role of complement in antibody-mediated resistance to Salmonella.

Immunology. 2019 01;156(1):69-73

Authors: Rossi O, Coward C, Goh YS, Claassens JWC, MacLennan CA, Verbeek SJ, Mastroeni P

Abstract
Vaccines can serve as essential tools to prevent bacterial diseases via the induction of long-lasting IgG responses. The efficacy of such vaccines depends on the effector mechanisms triggered by IgG. The complement system and Fc-gamma receptors (FcγRs) can potentially play a crucial role in IgG-mediated immunity against bacterial diseases. However, their relative importance in vivo is unclear, and has been the object of controversy and debate. In this brief study, we have used gene-targeted mice lacking either FcγRI, II, II and IV or the C3 complement component as well as a novel mouse strain lacking both C3 and FcγRs to conclusively show the essential role of complement in antibody-mediated host resistance to Salmonella enterica systemic infection. By comparing the effect of IgG2a antibodies against Salmonella O-antigen in gene-targeted mice, we demonstrate that the complement system is essential for the IgG-mediated reduction of bacterial numbers in the tissues.

PMID: 30179254 [PubMed - indexed for MEDLINE]

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Linear and Rationally Designed Stapled Peptides Abrogate TLR4 Pathway and Relieve Inflammatory Symptoms in Rheumatoid Arthritis Rat Model.

J Med Chem. 2019 Jul 08;:

Authors: Achek A, Shah M, Seo JY, Kwon HK, Gui X, Shin HJ, Cho EY, Lee BS, Kim DJ, Lee SH, Yoo TH, Kim MS, Choi S

Abstract
A mounting evidence exists for the despicable role of the aberrant immune response in the pathogenesis of rheumatoid arthritis (RA), where toll-like receptor 4 (TLR4) can activate synovial fibroblasts that lead to the chronic inflammation and joint destruction, thus making TLR4 a potent drug target in RA. We report that novel TLR4-antagonizing peptide, PIP2, inhibits the induction of inflammatory biomarkers in vitro as well as in vivo. Systemically, PIP2 inhibits the lipopolysaccharide (LPS)-elicited TNF-α, IL-6, and IL-12p40 in a mouse model. The rationally designed cyclic derivative, cPIP2, is capable of inhibiting LPS-induced proinflammatory cytokines at significantly lower concentration as compared to PIP2 (PIP2 IC50 = 20 μM, cPIP2 IC50 = 5 μM). Finally, cPIP2 was able to relieve the inflammatory symptoms and synovial tissue destruction in the RA rat model. Cumulatively, these data suggest that PIP2 and cPIP2 hold strong promise for the development of peptide-based immunotherapeutics that could be of great value in curbing TLR-related immune complications including RA.

PMID: 31283217 [PubMed - as supplied by publisher]

Nature Communications, Published online: 09 July 2019; doi:10.1038/s41467-019-10865-y

Human cytomegalovirus (HCMV) can persist for the life of a host in the face of robust immune responses owing to a wide range of immune evasion strategies. Here Liu and colleagues show that HCMV evades the IgG-mediated response by the endoplasmic reticulum-associated degradation of the neonatal Fc receptor for IgG.

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Broad-spectrum capture of clinical pathogens using engineered Fc-mannose-binding lectin enhanced by antibiotic treatment.

F1000Res. 2019;8:108

Authors: Seiler BT, Cartwright M, Dinis ALM, Duffy S, Lombardo P, Cartwright D, Super EH, Lanzaro J, Dugas K, Super M, Ingber DE

Abstract
Background: Fc-mannose-binding lectin (FcMBL), an engineered version of the blood opsonin MBL that contains the carbohydrate recognition domain (CRD) and flexible neck regions of MBL fused to the Fc portion of human IgG1, has been shown to bind various microbes and pathogen-associated molecular patterns (PAMPs). FcMBL has also been used to create an enzyme-linked lectin sorbent assay (ELLecSA) for use as a rapid (<1 h) diagnostic of bloodstream infections. Methods: Here we extended this work by using the ELLecSA to test FcMBL's ability to bind to more than 190 different isolates from over 95 different pathogen species. Results: FcMBL bound to 85% of the isolates and 97 of the 112 (87%) different pathogen species tested, including bacteria, fungi, viral antigens and parasites. FcMBL also bound to PAMPs including, lipopolysaccharide endotoxin (LPS) and lipoteichoic acid (LTA) from Gram-negative and Gram-positive bacteria, as well as lipoarabinomannan (LAM) and phosphatidylinositol mannoside 6 (PIM 6) from Mycobacterium tuberculosis. Conclusions: The efficiency of pathogen detection and variation between binding of different strains of the same species could be improved by treating the bacteria with antibiotics, or mechanical disruption using a bead mill, prior to FcMBL capture to reveal previously concealed binding sites within the bacterial cell wall. As FcMBL can bind to pathogens and PAMPs in urine as well as blood, its broad-binding capability could be leveraged to develop a variety of clinically relevant technologies, including infectious disease diagnostics, therapeutics, and vaccines.

PMID: 31275563 [PubMed - in process]

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Testing a Human Antimicrobial RNase Chimera Against Bacterial Resistance.

Front Microbiol. 2019;10:1357

Authors: Prats-Ejarque G, Li J, Ait-Ichou F, Lorente H, Boix E

Abstract
The emergence of bacterial resistance to the most commonly used antibiotics encourages the design of novel antimicrobial drugs. Antimicrobial proteins and peptides (AMPs) are the key players in host innate immunity. They exert a rapid and multifaceted action that reduces the development of bacterial adaptation mechanisms. Human antimicrobial RNases belonging to the vertebrate specific RNase A superfamily participate in the maintenance of tissue and body fluid sterility. Among the eight human canonical RNases, RNase 3 stands out as the most cationic and effective bactericidal protein against Gram-negative species. Its enhanced ability to disrupt the bacterial cell wall has evolved in detriment of its catalytic activity. Based on structure-functional studies we have designed an RNase 3/1 hybrid construct that combines the high catalytic activity of RNase 1 with RNase 3 bactericidal properties. Next, we have explored the ability of this hybrid RNase to target the development of bacterial resistance on an Acinetobacter baumannii cell culture. Synergy assays were performed in combination with colistin, a standard antimicrobial peptide used as an antibiotic to treat severe infections. Positive synergism was observed between colistin and the RNase 3/1 hybrid protein. Subsequently, using an in vitro experimental evolution assay, by exposure of a bacterial culture to colistin at incremental doses, we demonstrated the ability of the RNase 3/1 construct to reduce the emergence of bacterial antimicrobial resistance. The results advance the potential applicability of RNase-based drugs as antibiotic adjuvants.

PMID: 31275278 [PubMed]

ABSTRACT

Mannitol-1-phosphate dehydrogenase (M1PDH) is a key enzyme in Staphylococcus aureus mannitol metabolism, but its roles in pathophysiological settings have not been established. We performed comprehensive structure-function analysis of M1PDH from S. aureus USA300, a strain of community-associated methicillin-resistant S. aureus, to evaluate its roles in cell viability and virulence under pathophysiological conditions. On the basis of our results, we propose M1PDH as a potential antibacterial target. In vitro cell viability assessment of mtlD knockout and complemented strains confirmed that M1PDH is essential to endure pH, high-salt, and oxidative stress and thus that M1PDH is required for preventing osmotic burst by regulating pressure potential imposed by mannitol. The mouse infection model also verified that M1PDH is essential for bacterial survival during infection. To further support the use of M1PDH as an antibacterial target, we identified dihydrocelastrol (DHCL) as a competitive inhibitor of S. aureus M1PDH (SaM1PDH) and confirmed that DHCL effectively reduces bacterial cell viability during host infection. To explain physiological functions of SaM1PDH at the atomic level, the crystal structure of SaM1PDH was determined at 1.7-Å resolution. Structure-based mutation analyses and DHCL molecular docking to the SaM1PDH active site followed by functional assay identified key residues in the active site and provided the action mechanism of DHCL. Collectively, we propose SaM1PDH as a target for antibiotic development based on its physiological roles with the goals of expanding the repertory of antibiotic targets to fight antimicrobial resistance and providing essential knowledge for developing potent inhibitors of SaM1PDH based on structure-function studies.

IMPORTANCE Due to the shortage of effective antibiotics against drug-resistant Staphylococcus aureus, new targets are urgently required to develop next-generation antibiotics. We investigated mannitol-1-phosphate dehydrogenase of S. aureus USA300 (SaM1PDH), a key enzyme regulating intracellular mannitol levels, and explored the possibility of using SaM1PDH as a target for developing antibiotic. Since mannitol is necessary for maintaining the cellular redox and osmotic potential, the homeostatic imbalance caused by treatment with a SaM1PDH inhibitor or knockout of the gene encoding SaM1PDH results in bacterial cell death through oxidative and/or mannitol-dependent cytolysis. We elucidated the molecular mechanism of SaM1PDH and the structural basis of substrate and inhibitor recognition by enzymatic and structural analyses of SaM1PDH. Our results strongly support the concept that targeting of SaM1PDH represents an alternative strategy for developing a new class of antibiotics that cause bacterial cell death not by blocking key cellular machinery but by inducing cytolysis and reducing stress tolerance through inhibition of the mannitol pathway.

Vibrio cholerae mutants lacking lytic transglycosylases MltC and RlpA are defective for daughter cell separation. Our results suggest that lytic transglycosylases at the division septum serve as a back‐up mechanism to cleave peptidoglycan strands that cannot be cleared by highly‐regulated amidase activity, and to clear peptidoglycan debris that may block the completion of outer‐membrane invagination.

Summary

The cell wall is a crucial structural feature in the vast majority of bacteria and comprises a covalently closed network of peptidoglycan (PG) strands. While PG synthesis is important for survival under many conditions, the cell wall is also a dynamic structure, undergoing degradation and remodeling by ‘autolysins’, enzymes that break down PG. Cell division, for example, requires extensive PG remodeling, especially during separation of daughter cells, which depends heavily upon the activity of amidases. However, in Vibrio cholerae, we demonstrate that amidase activity alone is insufficient for daughter cell separation and that lytic transglycosylases RlpA and MltC both contribute to this process. MltC and RlpA both localize to the septum and are functionally redundant under normal laboratory conditions; however, only RlpA can support normal cell separation in low‐salt media. The division‐specific activity of lytic transglycosylases has implications for the local structure of septal PG, suggesting that there may be glycan bridges between daughter cells that cannot be resolved by amidases. We propose that lytic transglycosylases at the septum cleave PG strands that are crosslinked beyond the reach of the highly regulated activity of the amidase and clear PG debris that may block the completion of outer membrane invagination.

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Complex formation between Mur enzymes from Streptococcus pneumoniae.

Biochemistry. 2019 Jul 02;:

Authors: Miyachiro MM, Granato D, Trindade DM, Ebel C, Paes Leme AF, Dessen A

Abstract
Peptidoglycan is one of the major components of the bacterial cell wall, being responsible for shape and stability. Due to its essential nature, its biosynthetic pathway is the target for major antibiotics, and proteins involved in its biosynthesis continue to be targeted for inhibitor studies. The biosynthesis of its major building block, Lipid II, is initiated in the bacterial cytoplasm with the sequential reactions catalyzed by Mur enzymes, which have been suggested to form a multi-protein complex in order to facilitate shuttling of the building blocks towards the inner membrane. In this work, we purified MurC, MurD, MurE, MurF and MurG from the human pathogen Streptococcus pneumoniae and characterized their interactions using chemical cross-linking, mass spectrometry, analytical ultracentrifugation, and microscale thermophoresis. Mur enzymes interact strongly as binary complexes, with interaction regions mapping mostly to loop regions. Interestingly, MurC, MurD and MurE display ten times higher affinity for each other than for MurF and MurG, suggesting that Mur ligases that catalyze the initial reactions in the peptidoglycan biosynthesis pathway could form a sub-complex that could be important to facilitate Lipid II biosynthesis. The interface between Mur proteins could represent a yet unexplored target for new inhibitor studies that could lead to the development of novel antimicrobials.

PMID: 31264408 [PubMed - as supplied by publisher]

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AraR, an L-Arabinose-Responding Transcription Factor, Negatively Regulates Resistance of Mycobacterium smegmatis to Isoniazid.

Biochemistry (Mosc). 2019 May;84(5):540-552

Authors: Zhou L, He ZG, Li W

Abstract
L-Arabinose is an important component of mycobacterial cell wall. L-Arabinose is involved in the synthesis of arabinogalactan, lipoarabinomannan, and other sugar compounds, which suggests that it can modulate cell wall permeability and drug resistance. However, whether L-arabinose affects mycobacterial antibiotic resistance and the underlying regulatory mechanism remains unclear. In this study, we characterized a new transcription factor of Mycobacterium smegmatis, AraR, that responds to L-arabinose and regulates mycobacterial sensitivity to isoniazid (INH). AraR specifically recognizes two conserved 15-bp motifs within the upstream regulatory region of the arabinose (araR) operon. AraR functions as a transcriptional repressor that negatively regulates araR expression. In contrast to the effect of AraR, overexpression of the araR operon contributes to the mycobacterial INH resistance. L-arabinose acts as an effector and derepresses transcriptional inhibition by AraR. The araR-deficient strain is more resistant to INH than the wild-type strain, whereas the araR-overexpressing strain is more sensitive to INH. Addition of L-arabinose to the medium can significantly increase the resistance to INH of the wild-type strain, but not of the araR knockout strain. Therefore, we identified a new L-arabinose-responding transcription factor and revealed its effect on the bacterial antibiotic resistance. These findings can provide new insights in the regulatory mechanisms mediated by sugar molecules and their relationship with drug resistance in mycobacteria.

PMID: 31234768 [PubMed - in process]

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Stationary phase persister formation in Escherichia coli can be suppressed by piperacillin and PBP3 inhibition.

BMC Microbiol. 2019 Jun 24;19(1):140

Authors: Aedo SJ, Orman MA, Brynildsen MP

Abstract
BACKGROUND: Persisters are rare phenotypic variants within a bacterial population that are capable of tolerating lethal antibiotic concentrations. Passage through stationary phase is associated with the formation of persisters (type I), and a major physiological response of Escherichia coli during stationary phase is cell wall restructuring. Given the concurrence of these processes, we sought to assess whether perturbation to cell wall synthesis during stationary phase impacts type I persister formation.
RESULTS: We tested a panel of cell wall inhibitors and found that piperacillin, which primarily targets penicillin binding protein 3 (PBP3 encoded by ftsI), resulted in a significant reduction in both β-lactam (ampicillin, carbenicillin) and fluoroquinolone (ofloxacin, ciprofloxacin) persister levels. Further analyses showed that piperacillin exposure through stationary phase resulted in cells with more ATP, DNA, RNA, and protein (including PBPs) than untreated controls; and that their physiology led to more rapid resumption of DNA gyrase supercoiling activity, translation, and cell division upon introduction into fresh media. Previously, PBP3 inhibition had been linked to antibiotic efficacy through the DpiBA two component system; however, piperacillin suppressed persister formation in ΔdpiA to the same extent as it did in wild-type, suggesting that DpiBA is not required for the phenomenon reported here. To test the generality of PBP3 inhibition on persister formation, we expressed FtsI Ser307Ala to genetically inhibit PBP3, and suppression of persister formation was also observed, although not to the same magnitude as that seen for piperacillin treatment.
CONCLUSIONS: From these data we conclude that stationary phase PBP3 activity is important to type I persister formation in E. coli.

PMID: 31234796 [PubMed - in process]

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Massive antibody discovery used to probe structure-function relationships of the essential outer membrane protein LptD.

Elife. 2019 Jun 25;8:

Authors: Storek KM, Chan J, Vij R, Chiang N, Lin Z, Bevers J, Koth CM, Vernes JM, Meng YG, Yin J, Wallweber H, Dalmas O, Shriver S, Tam C, Schneider K, Seshasayee D, Nakamura G, Smith PA, Payandeh J, Koerber JT, Comps-Agrar L, Rutherford ST

Abstract
Outer membrane proteins (OMPs) in Gram-negative bacteria dictate permeability of metabolites, antibiotics, and toxins. Elucidating the structure-function relationships governing OMPs within native membrane environments remains challenging. We constructed a diverse library of >3000 monoclonal antibodies to assess the roles of extracellular loops (ECLs) in LptD, an essential OMP that inserts lipopolysaccharide into the outer membrane of Escherichia coli. Epitope binning and mapping experiments with LptD-loop-deletion mutants demonstrated that 7 of the 13 ECLs are targeted by antibodies. Only ECLs inaccessible to antibodies were required for the structure or function of LptD. Our results suggest that antibody-accessible loops evolved to protect key extracellular regions of LptD, but are themselves dispensable. Supporting this hypothesis, no α-LptD antibody interfered with essential functions of LptD. Our experimental workflow enables structure-function studies of OMPs in native cellular environments, provides unexpected insight into LptD, and presents a method to assess the therapeutic potential of antibody targeting.

PMID: 31237236 [PubMed - in process]

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Enhancing the antibacterial activity of polymyxins using a nonantibiotic drug.

Infect Drug Resist. 2019;12:1393-1405

Authors: Krishnamurthy M, Lemmon MM, Falcinelli EM, Sandy RA, Dootz JN, Mott TM, Rajamani S, Schaecher KE, Duplantier AJ, Panchal RG

Abstract
Purpose: The rapid emergence of multidrug-resistant (MDR) bacteria and the lack of new therapies to eliminate them poses a major threat to global health. With the alarming rise in antimicrobial resistance (AMR), focus has now shifted to the use of the polymyxin class of antibiotics as the last line of defense for treatment of Gram-negative infections. Unfortunately, the growing resistance of bacteria against polymyxins is threatening the treatment of MDR infections, necessitating the need for novel strategies. The objective of this study was to determine if combination of polymyxin (polymyxin B or colistin) with a nonantibiotic small molecule AR-12, a celecoxib derivative that is devoid of cyclooxygenase 2 (COX-2) inhibitory activities, can be an effective strategy against polymyxin-resistant MDR bacteria. Methods: Growth inhibition studies, time-kill assays and permeability assays were conducted to investigate the effect of AR-12 on the antibacterial activity of polymyxins. Results: Growth studies were performed on a panel of polymyxin-resistant MDR strains using the combination of AR-12 with either colistin or polymyxin B. The combination treatment had no effect on strains that have inherent polymyxin resistance; however, AR-12 was effective in lowering the minimal inhibitory concentration (MIC) of polymyxins by 4-60-fold in several strains that had acquired polymyxin resistance. Time-kill assays using the combination of AR-12 and colistin with select MDR strains suggest rapid killing and bactericidal activity, while the permeability assays using fluorescently labeled dansylated polymyxin and 1-N-phenylnaphthylamine (NPN) in these MDR strains suggest that AR-12 can potentiate the antibacterial activity of polymyxins by possibly altering the bacterial outer membrane via modification of lipopolysaccharide and thereby improving the uptake of polymyxins. Conclusion: Our studies indicate that the combination of AR-12 and polymyxin is effective in targeting select Gram-negative bacteria that have acquired polymyxin resistance. Further understanding of the mechanism of action of AR-12 will provide new avenues for developing narrow-spectrum antibacterials to target select Gram-negative MDR bacteria. Importantly, our studies show that the use of nonantibiotic small molecules in combination with polymyxins is an attractive strategy to counter the growing resistance of bacteria to polymyxins.

PMID: 31239720 [PubMed]