Brianna Dalesandro

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SNAC-tag for sequence-specific chemical protein cleavage.

Nat Methods. 2019 04;16(4):319-322

Authors: Dang B, Mravic M, Hu H, Schmidt N, Mensa B, DeGrado WF

Abstract
Site-specific protein cleavage is essential for many protein-production protocols and typically requires proteases. We report the development of a chemical protein-cleavage method that is achieved through the use of a sequence-specific nickel-assisted cleavage (SNAC)-tag. We demonstrate that the SNAC-tag can be inserted before both water-soluble and membrane proteins to achieve fusion protein cleavage under biocompatible conditions with efficiency comparable to that of enzymes, and that the method works even when enzymatic cleavages fail.

PMID: 30923372 [PubMed - indexed for MEDLINE]

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Do the Microbiota Influence Vaccines and Protective Immunity to Pathogens? If So, Is There Potential for Efficacious Microbiota-Based Vaccines?

Cold Spring Harb Perspect Biol. 2018 02 01;10(2):

Authors: Littman DR

Abstract
The gut-resident constituents of the microbiota protect the mucosa from invasive pathogens through engagement of both innate and adaptive branches of the immune system. They are also likely to provide systemic protection from pathogens, by enhancing host robustness and tolerance to the invasive microbes and by inducing immune responses that prevent their growth. These properties of commensal microbiota, particularly the capacity of some bacteria to induce diverse types of antigen-specific immune responses, raises the prospect that they could be deployed as vaccine vectors to generate effective local and systemic immunity to viral and bacterial pathogens.

PMID: 28432131 [PubMed - indexed for MEDLINE]

AJICAP™, a method for the affinity‐peptide mediated‐regiodivergent functionalization of proteins that enables the synthesis of antibody–drug conjugates (ADCs) from native IgG antibodies is reported.

Abstract

The need for atom‐precise biomolecule modification, and particularly the irreversible formation of covalent bonds to specific amino acids in proteins, has become an essential issue in the fields of pharmaceuticals and chemical biology. For example, antibody–drug conjugates (ADCs) are increasingly common entries into the clinical oncology pipeline. Herein, we report a new method of affinity peptide mediated regiodivergent functionalization (AJICAP™) that enables the synthesis of ADCs from native IgG antibodies. We succeeded in introducing thiol functional groups onto three lysine residues in IgGs using Fc affinity peptide reagents without antibody engineering. A cytotoxic molecule was then connected to the newly introduced thiol group, and both a surface plasmon resonance binding assay and in vivo xenograft mouse model results showed that the resulting ADC could selectively target and kill HER2‐positive cells. Our strategy provides a new approach for constructing complex antibody‐derived biomolecules.

Protein homeostasis is tightly regulated, and multiple cellular mechanisms are in place to dispose of misfolded or no-longer-needed proteins. One of the key players is the ubiquitin (Ub)-proteasome system (UPS), in which a variety of specific ligases mark substrate proteins with a Ub “flag” to be recognized by the proteolytic...

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In Vitro Adhesion, Invasion, and Transcytosis of Streptococcus pneumoniae with Host Cells.

Methods Mol Biol. 2019;1968:137-146

Authors: Brissac T, Orihuela CJ

Abstract
Physical interactions of bacteria with host cells are often a principal aspect of bacterial pathogenesis. In the case of Streptococcus pneumoniae (Spn), which does not produce a secreted toxin, adhesion to and/or invasion of host cells is necessary for colonization of the nasopharynx and subsequently to cause opportunistic disease in its human host. Knowledge of how pneumococci interact with host cells thereby helps to explain its biology and may identify potential targets for intervention. One of the simplest, yet powerful, assays that can be leveraged to dissect the molecular basis of this vital host-pathogen interaction is the in vitro adhesion and invasion assay. Among many key results, this assay has been used to discover the bacterial and host determinants involved in bacterial attachment, identify host signaling networks required for uptake of the bacteria into an endosome, and the characterization of the intracellular trafficking machinery that is subverted by Spn during development of bacteremia and meningitis. These assays have also been used to characterize the epithelial, endothelial, and/or immune cell response to these bacteria, and to learn how pneumococci disperse from an established biofilm to a planktonic phenotype to colonize another niche and/or transmit. Herein, we will review this protocol, highlighting how simple changes in the bacterial strain or host cell line can elucidate the underlying molecular mechanisms for Spn virulence.

PMID: 30929212 [PubMed - in process]

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Immune Evasion by Staphylococcus aureus.

Microbiol Spectr. 2019 Mar;7(2):

Authors: de Jong NWM, van Kessel KPM, van Strijp JAG

Abstract
Staphylococcus aureus has become a serious threat to human health. In addition to having increased antibiotic resistance, the bacterium is a master at adapting to its host by evading almost every facet of the immune system, the so-called immune evasion proteins. Many of these immune evasion proteins target neutrophils, the most important immune cells in clearing S. aureus infections. The neutrophil attacks pathogens via a plethora of strategies. Therefore, it is no surprise that S. aureus has evolved numerous immune evasion strategies at almost every level imaginable. In this review we discuss step by step the aspects of neutrophil-mediated killing of S. aureus, such as neutrophil activation, migration to the site of infection, bacterial opsonization, phagocytosis, and subsequent neutrophil-mediated killing. After each section we discuss how S. aureus evasion molecules are able to resist the neutrophil attack of these different steps. To date, around 40 immune evasion molecules of S. aureus are known, but its repertoire is still expanding due to the discovery of new evasion proteins and the addition of new functions to already identified evasion proteins. Interestingly, because the different parts of neutrophil attack are redundant, the evasion molecules display redundant functions as well. Knowing how and with which proteins S. aureus is evading the immune system is important in understanding the pathophysiology of this pathogen. This knowledge is crucial for the development of therapeutic approaches that aim to clear staphylococcal infections.

PMID: 30927347 [PubMed - in process]

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Coordination of capsule assembly and cell wall biosynthesis in Staphylococcus aureus.

Nat Commun. 2019 03 29;10(1):1404

Authors: Rausch M, Deisinger JP, Ulm H, Müller A, Li W, Hardt P, Wang X, Li X, Sylvester M, Engeser M, Vollmer W, Müller CE, Sahl HG, Lee JC, Schneider T

Abstract
The Gram-positive cell wall consists of peptidoglycan functionalized with anionic glycopolymers, such as wall teichoic acid and capsular polysaccharide (CP). How the different cell wall polymers are assembled in a coordinated fashion is not fully understood. Here, we reconstitute Staphylococcus aureus CP biosynthesis and elucidate its interplay with the cell wall biosynthetic machinery. We show that the CapAB tyrosine kinase complex controls multiple enzymatic checkpoints through reversible phosphorylation to modulate the consumption of essential precursors that are also used in peptidoglycan biosynthesis. In addition, the CapA1 activator protein interacts with and cleaves lipid-linked CP precursors, releasing the essential lipid carrier undecaprenyl-phosphate. We further provide biochemical evidence that the subsequent attachment of CP is achieved by LcpC, a member of the LytR-CpsA-Psr protein family, using the peptidoglycan precursor native lipid II as acceptor substrate. The Ser/Thr kinase PknB, which can sense cellular lipid II levels, negatively controls CP synthesis. Our work sheds light on the integration of CP biosynthesis into the multi-component Gram-positive cell wall.

PMID: 30926919 [PubMed - indexed for MEDLINE]

As current antibiotics dwindle in effectiveness against multidrug-resistant pathogens, researchers are seeking potential replacements in some unlikely places. Now a team has identified bacteria with promising antibiotic activity against known pathogens—even dangerous organisms, such as the microbe that causes MRSA infections—in the protective mucus that coats young fish.

Development of a fluorescence-based method for the rapid determination of Zika virus polymerase activity and the screening of antiviral drugs

Development of a fluorescence-based method for the rapid determination of Zika virus polymerase activity and the screening of antiviral drugs, Published online: 01 April 2019; doi:10.1038/s41598-019-41998-1

Development of a fluorescence-based method for the rapid determination of Zika virus polymerase activity and the screening of antiviral drugs

The human macrophage galactose-type lectin, MGL, recognizes the outer core of E. coli lipooligosaccharide.

Chembiochem. 2019 Mar 28;:

Authors: Maalej MM, Forgione RE, Marchetti R, Bulteau FB, Thepaut MT, Lanzetta R, Laguri C, Simorre JP, Fieschi F, Molinaro A, Silipo A

Abstract
Carbohydrate-lectin interactions intervene in and mediate most biological processes, including a crucial modulation of immune responses to pathogens. Despite the growing interest in investigating the association between host receptor lectins and exogenous glycan ligands, the molecular mechanisms underlying bacterial recognition by human lectins are still not fully understood. Here we describe a novel molecular interaction between the human macrophage galactose-type lectin (MGL) and the lipooligosaccharide (LOS) of E. coli strain R1. Saturation Transfer Difference (STD) NMR analysis, supported by computational studies, demonstrated that MGL bound to the purified deacylated LOSR1 mainly through the recognition of its outer core and established crucial interactions with the terminal Galα(1,2)Gal epitope. These results assess the ability of MGL to recognize glycan moieties exposed on Gram-negative bacterial surfaces.

PMID: 30919527 [PubMed - as supplied by publisher]

Virus tricks the immune system into ignoring bacterial infections

Virus tricks the immune system into ignoring bacterial infections, Published online: 28 March 2019; doi:10.1038/d41586-019-00991-4

The finding could explain why the body tolerates some microbes ― and lead to better treatments for chronic infections.

Prioritization of potential vaccine targets using comparative proteomics and designing of the chimeric multi-epitope vaccine against Pseudomonas aeruginosa

Prioritization of potential vaccine targets using comparative proteomics and designing of the chimeric multi-epitope vaccine against <i>Pseudomonas aeruginosa</i>, Published online: 27 March 2019; doi:10.1038/s41598-019-41496-4

Prioritization of potential vaccine targets using comparative proteomics and designing of the chimeric multi-epitope vaccine against Pseudomonas aeruginosa

CAR T cell trogocytosis and cooperative killing regulate tumour antigen escape

CAR T cell trogocytosis and cooperative killing regulate tumour antigen escape, Published online: 27 March 2019; doi:10.1038/s41586-019-1054-1

Chimeric antigen receptors (CARs) promote antigen loss in tumour cells by trogocytosis, which results in T cell fratricide killing and exhaustion but can be counteracted by cooperative killing and combinatorial targeting.

Synthetic substitute: An approach based on click chemistry for the targeted synthesis of artificially linked ubiquitin dimers has been explored. Structural and dynamic characterisation by means of NMR spectroscopy, accompanied by biochemical analysis, reveals the native‐like conformation and functionality of the dimers.

Abstract

As one of the most prevalent post‐translational modifications in eukaryotic cells, ubiquitylation plays vital roles in many cellular processes, such as protein degradation, DNA metabolism, and cell differentiation. Substrate proteins can be tagged by distinct types of polymeric ubiquitin (Ub) chains, which determine the eventual fate of the modified protein. A facile, click chemistry based approach for the efficient generation of linkage‐defined Ub chains, including Ub dimers, was recently established. Within these chains, individual Ub moieties are connected through a triazole linkage, rather than the natural isopeptide bond. Herein, it is reported that the conformation of an artificially K48‐linked Ub dimer resembles that of the natively linked dimer, with respect to structural and dynamic characteristics, as demonstrated by means of high‐resolution NMR spectroscopy. Thus, it is proposed that artificially linked Ub dimers, as generated by this approach, represent potent tools for studying the inherently different properties and functions of distinct Ub chains.

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Spermine and oxacillin stress response on the cell wall synthesis and the global gene expression analysis in Methicillin-resistance Staphylococcus aureus.

Genes Genomics. 2019 01;41(1):43-59

Authors: Pawar S, Yao X, Lu CD

Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) is a rapidly emerging bacteria causing infection, which has developed resistance to most of the beta-lactam antibiotics because of newly acquired low-affinity penicillin-binding protein (PBP2a), which can continue to build the cell wall when beta-lactams block other PBPs. Exogenous spermine exerts a dose-dependent inhibition effect on the growth of Escherichia coli, Salmonella enterica serovar, and S. aureus. Selection of an MRSA Mu50 derivative which harbors mutation on PBP2 gene (named as MuM) showing spermine resistance and which confers a complete abolishment of spermine-beta-lactam synergy was identified. To further investigate the gene expression changes, a transcriptome profiling of MuM against Mu50 (wild-type) without any treatment, MuM and Mu50 in response to high dose spermine and Mu50 in response to spermine-beta-lactam synergy at 15, 30 and 60 min time points was performed. Functional annotation was further performed to delineate the metabolic pathways associated with the significant genes. A significant down-regulation in the iron regulatory system, potassium channel uptake and polyamine transport system with an up-regulation in general stress response sigB dependent operon in MuM strain at 15, 30 and 60 min time points with spermine treatment compared to Mu50 strain was observed. Analysis of spermine-dependent synergy with beta-lactams on cell wall synthesis revealed that it significantly reduces the degree of cross-linkage on cell wall with no change in trypsin digestion pattern of purified PBPs and without affecting PBPs expression or PBPs acylation by Bocillin. A strong relation between PBP2 protein and general stress sigB response, iron, potassium and polyamine transport systems was observed. SigB regulon should be activated on stress, which was not seen in some of our previous studies where it was down-regulated in wild-type Mu50 strain with spermine stress. Here, an intriguing finding is made where there seems to be a correction of this abnormal response of no SigB induction to a significant induction by PBP2 mutation. In MuM strain, a significant down-regulation of KdpABC operon genes at 15, 30 and 60 min time points on spermine stress is seen, which seems to be absent without spermine treatment. Since KCL has been found to protect the cell against spermine stress in wild-type strain by induction of KdpABC operon, it fails to do so in MuM strain underlying the importance of PBP2 protein in spermine stress. Analysis of spermine-dependent synergy with beta-lactams on cell wall synthesis revealed that it significantly reduces the degree of cross-linkage on cell wall with no change in trypsin digestion patterns of purified PBPs and without affecting PBPs expression or PBPs acylation by Bocillin. Furthermore, spermine does not help in enhancing the binding of beta-lactams to PBPs and binding of spermine to PBPs does not cause conformational changes to PBPs, as tested with trypsin digestion patterns. Future studies on the molecular mechanism of spermine interactions with these systems hold great potential for the development of new therapeutics for MRSA infections.

PMID: 30229508 [PubMed - indexed for MEDLINE]

His‐tags reloaded: Protein labeling demands high specificity, rapid as well as efficient conjugation, while maintaining low concentration and biocompatibility under physiological conditions. In this context, recent progress in the recognition of multivalent chelator heads with the small, genetically engineered His‐tag fused to proteins is highly relevant.

Abstract

With the advent of single‐molecule methods, chemoselective and site‐specific labeling of proteins evolved to become a central aspect in chemical biology as well as cell biology. Protein labeling demands high specificity, rapid as well as efficient conjugation, while maintaining low concentration and biocompatibility under physiological conditions. Generic methods that do not interfere with the function, dynamics, subcellular localization of proteins, and crosstalk with other factors are crucial to probe and image proteins in vitro and in living cells. Alternatives to enzyme‐based tags or autofluorescent proteins are short peptide‐based recognition tags. These tags provide high specificity, enhanced binding rates, bioorthogonality, and versatility. Here, we report on recent applications of multivalent chelator heads, recognizing oligohistidine‐tagged proteins. The striking features of this system has facilitated the analysis of protein complexes by single‐molecule approaches.

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Characterization of putative DD-carboxypeptidase-encoding genes in Mycobacterium smegmatis.

Sci Rep. 2019 Mar 26;9(1):5194

Authors: Ealand CS, Asmal R, Mashigo L, Campbell L, Kana BD

Abstract
Penicillin binding proteins (PBPs) are the target of numerous antimicrobial agents that disrupt bacterial cell wall synthesis. In mycobacteria, cell elongation occurs through insertion of nascent cell wall material in the sub-polar region, a process largely driven by High Molecular Weight PBPs. In contrast, the function of DD-carboxypeptidases (DD-CPases), which are Low Molecular Weight Class 1C PBPs, in mycobacteria remains poorly understood. Mycobacterium smegmatis encodes four putative DD-CPase homologues, which display homology to counterparts in Escherichia coli. Herein, we demonstrate that these are expressed in varying abundance during growth. Deletion of MSMEG_1661, MSMEG_2433 or MSMEG_2432, individually resulted in no defects in growth, cell morphology, drug susceptibility or spatial incorporation of new peptidoglycan. In contrast, deletion of MSMEG_6113 (dacB) was only possible in a merodiploid strain expressing the homologous M. tuberculosis operon encoding Rv3627c (dacB), Rv3626c, Rv3625c (mesJ) and Rv3624c (hpt), suggestive of essentiality. To investigate the role of this operon in mycobacterial growth, we depleted gene expression using anhydrotetracycline-responsive repressors and noted reduced bipolar peptidoglycan synthesis. These data point to a possible role for this four gene operon, which is highly conserved across all mycobacterial species, in regulating spatial localization of peptidoglycan synthesis.

PMID: 30914728 [PubMed - in process]

Selective toxicity: Peptide‐stabilized, monodisperse platinum nanoparticles were developed that selectively kill liver cancer cells over other cancer and non‐cancerous cells. The peptide was identified in a combinatorial screening; it renders the PtNPs highly stable and enables their cellular uptake as well as the release of toxic Pt^II ions in an oxidative environment.

Abstract

Peptide‐stabilized platinum nanoparticles (PtNPs) were developed that have significantly greater toxicity against hepatic cancer cells (HepG2) than against other cancer cells and non‐cancerous liver cells. The peptide H‐Lys‐Pro‐Gly‐dLys‐NH₂ was identified by a combinatorial screening and further optimized to enable the formation of water‐soluble, monodisperse PtNPs with average diameters of 2.5 nm that are stable for years. In comparison to cisplatin, the peptide‐coated PtNPs are not only more toxic against hepatic cancer cells but have a significantly higher tumor cell selectivity. Cell viability and uptake studies revealed that high cellular uptake and an oxidative environment are key for the selective cytotoxicity of the peptide‐coated PtNPs.

A sulfonium moiety was attached to vancomycin, enhancing its antibacterial activity against vancomycin‐resistant bacteria both in vitro and in vivo. Further evaluation, including toxicity, stability, and pharmacokinetic studies, demonstrate the good drugability of these sulfonium derivatives. This strategy is beneficial for combating drug‐resistant bacterial infection, and advances the knowledge of sulfonium derivatives for drug development.

Abstract

In the antibiotics arsenal, vancomycin is a last resort for the treatment of intractable infections. However, this situation is under threat because of the increasing appearance of vancomycin‐resistant bacteria (VRB). Herein, we report a series of novel vancomycin derivatives carrying a sulfonium moiety. The sulfonium–vancomycin derivatives exhibited enhanced antibacterial activity against VRB both in vitro and in vivo. These derivatives also exhibited activity against some Gram‐negative bacteria. The sulfonium modification enhanced the interaction of vancomycin with the bacterial cell membrane and disrupts membrane integrity. Furthermore, the in vivo pharmacokinetic profile, stability, and toxicity of these derivatives demonstrated good druggability of the sulfonium–vancomycin analogues. This work provides a promising strategy for combating drug‐resistant bacterial infection, and advances the knowledge on sulfonium derivatives for structural optimization and drug development.

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Aspartate deficiency limits peptidoglycan synthesis and sensitizes cells to antibiotics targeting cell wall synthesis in Bacillus subtilis.

Mol Microbiol. 2018 09;109(6):826-844

Authors: Zhao H, Roistacher DM, Helmann JD

Abstract
Peptidoglycan synthesis is an important target for antibiotics and relies on intermediates derived from central metabolism. As a result, alterations of metabolism may affect antibiotic sensitivity. An aspB mutant is auxotrophic for aspartate (Asp) and asparagine (Asn) and lyses when grown in Difco sporulation medium (DSM), but not in LB medium. Genetic and physiological studies, supported by amino acid analysis, reveal that cell lysis in DSM results from Asp limitation due to a relatively low Asp and high glutamate (Glu) concentrations, with Glu functioning as a competitive inhibitor of Asp uptake by the major Glu/Asp transporter GltT. Lysis can be specifically suppressed by supplementation with 2,6-diaminopimelate (DAP), which is imported by two different cystine uptake systems. These studies suggest that aspartate limitation depletes the peptidoglycan precursor meso-2,6-diaminopimelate (mDAP), inhibits peptidoglycan synthesis, upregulates the cell envelope stress response mediated by σM and eventually leads to cell lysis. Aspartate limitation sensitizes cells to antibiotics targeting late steps of PG synthesis, but not steps prior to the addition of mDAP into the pentapeptide sidechain. This work highlights the ability of perturbations of central metabolism to sensitize cells to peptidoglycan synthesis inhibitors.

PMID: 29995990 [PubMed - indexed for MEDLINE]

Assessing the viability of transplanted gut microbiota by sequential tagging with D-amino acid-based metabolic probes

Assessing the viability of transplanted gut microbiota by sequential tagging with D-amino acid-based metabolic probes, Published online: 21 March 2019; doi:10.1038/s41467-019-09267-x

The mechanisms underlying the success or failure of fecal microbiota transplantation (FMT) are unclear. Here, Wang et al. use a mouse model of FMT to show that fluorescent D-amino acids can be used to track the transplanted microbiota in the recipient animals.

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Pomalidomide hybrids act as proteolysis targeting chimeras: Synthesis, anticancer activity and B-Raf degradation.

Bioorg Chem. 2019 Mar 19;87:191-199

Authors: Chen H, Chen F, Pei S, Gou S

Abstract
As the first intracellular signaling molecule and the most frequently mutated oncogene, B-Raf represents an important target in cancer therapy. Here we report several pomalidomide hybrids acting as proteolysis targeting chimeras (PROTACs) for the degradation of B-Raf. Due to its high expression of B-Raf, MCF-7 cells are sensitive to these compounds. Among them, compound 2 can effectively kill cancer cells via inducing cells apoptosis. As a B-Raf degrader, compound 2 can accelerate the degradation of B-Raf by recruiting ubiquitin-proteasome system, and further affects the expression of Mcl-1, a downstream protein of B-Raf. The anticancer mechanism of compound 2 is quite different from its mother compound and cancer cells seem to be more sensitive to the degrader, hinting that degradation of B-Raf by PROTAC is a potential way for cancer treatment.

PMID: 30901674 [PubMed - as supplied by publisher]

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Drug repurposing against arabinosyl transferase (EmbC) of Mycobacterium tuberculosis: Essential dynamics and free energy minima based binding mechanics analysis.

Gene. 2019 Apr 20;693:114-126

Authors: Singh A, Somvanshi P, Grover A

Abstract
Arabinosyl tranferases (embA, embB, embC) are the key enzymes responsible for biogenesis of arabinan domain of arabinogalactan (AG) and lipoarabinomannan (LAM), two major heteropolysaccharide constituents of the peculiar mycobacterial cell envelope. EmbC is predominantly responsible for LAM synthesis and has been commonly associated with Ethambutol resistance. We have screened the FDA library against EmbC to reposition a drug better than Ethambutol with higher binding affinity to Embc. High throughput virtual screening followed by extra precision docking using Glide gave two best leads i.e. Terlipressin and Amikacin with docking score of -11.39 kcal/mol and -10.71 kcal/mol, respectively. Binding mechanics of the selected drugs was elucidated through long range molecular dynamics simulations (100 ns) using binding free energy rescoring, essential dynamics and free energy minima based approaches, thus revealing the most stable binding modes of Terlipressin with EmbC. Our study establishes the EmbC binding potential of the repurposed drugs Terlipressin and Amikacin.

PMID: 30716439 [PubMed - indexed for MEDLINE]

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Functional mimetic of the G-protein coupled receptor CXCR4 on a soluble antibody scaffold.

MAbs. 2019 Mar 22;:

Authors: Koksal AC, Pennini ME, Marelli M, Xiao X, Dall'Acqua WF

Abstract
G-protein coupled receptors (GPCRs) constitute major drug targets due to their involvement in critical biological functions and pathophysiological disorders. The leading challenge in their structural and functional characterization has been the need for a lipid environment to accommodate their hydrophobic cores. Here, we report an antibody scaffold mimetic (ASM) platform where we have recapitulated the extracellular functional domains of the GPCR, C-X-C chemokine receptor 4 (CXCR4) on a soluble antibody framework. The engineered ASM molecule can accommodate the N-terminal loop and all three extracellular loops of CXCR4. These extracellular features are important players in ligand recruitment and interaction for allostery and signal transduction. Our study shows that ASMCXCR4 can be recognized by the anti-CXCR4 antibodies, MEDI3185, 2B11, and 12G5, and that ASMCXCR4 can bind the HIV-1 glycoprotein ligand gp120, and the natural chemokine ligand SDF-1α. Further, we show that ASMCXCR4 can competitively inhibit the SDF-1α signaling pathway, and be used as an immunogen to generate CXCR4 specific antibodies. This platform will be useful in the study of GPCR biology in a soluble receptor context for evaluating its extracellular ligand interactions.

PMID: 30900513 [PubMed - as supplied by publisher]

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Lipoproteins and Their Trafficking to the Outer Membrane.

EcoSal Plus. 2019 Mar;8(2):

Authors: Grabowicz M

Abstract
Lipoproteins are produced by both Gram-positive and Gram-negative bacteria. Once secreted, lipoproteins are quickly acylated, anchoring them into the plasma membrane. Recent work has shown that Gram-positive bacteria are able to generate considerable diversity in the acylation of their lipoproteins, though the mechanisms involved are only just beginning to emerge. In Gram-negative organisms, most lipoproteins are subsequently trafficked to the outer membrane (OM). Lipoprotein trafficking is an essential pathway in these bacteria. At least one OM lipoprotein component is required by each of the essential machines that assemble the OM (such as the Bam and Lpt machines) and build the peptidoglycan cell wall (Lpo-penicillin-binding protein complexes). The Lol pathway has been the paradigm for OM lipoprotein trafficking: a complex of LolCDE extracts lipoproteins from the plasma membrane, LolA shuttles them through the periplasmic space, and LolB anchors them into the OM. The peptide signals responsible for OM-targeting via LolCDE have long been known for Escherichia coli. Remarkably, production of novel lipoprotein acyl forms in E. coli has reinforced the idea that lipid signals also contribute to OM targeting via LolCDE. Moreover, recent work has shown that lipoprotein trafficking can occur in E. coli without either LolA or LolB. Therefore, current evidence suggests that at least one additional, LolAB-independent route for OM lipoprotein trafficking exists. This chapter reviews the posttranslocation modifications of all lipoproteins, with a focus on the trafficking of lipoproteins to the OM of Gram-negative bacteria.

PMID: 30900542 [PubMed - in process]

A different (pheno)type of probe: A toolkit of clickable and fluorescent activity‐based triazole urea probes enables the selective manipulation and visualization of different serine hydrolases in live S. aureus cells. These probes were used to dissect phenotypic differences among bacterial cells at the population and single‐cell level.

Abstract

Phenotypically distinct cellular (sub)populations are clinically relevant for the virulence and antibiotic resistance of a bacterial pathogen, but functionally different cells are usually indistinguishable from each other. Herein, we introduce fluorescent activity‐based probes as chemical tools for the single‐cell phenotypic characterization of enzyme activity levels in Staphylococcus aureus. We screened a 1,2,3‐triazole urea library to identify selective inhibitors of fluorophosphonate‐binding serine hydrolases and lipases in S. aureus and synthesized target‐selective activity‐based probes. Molecular imaging and activity‐based protein profiling studies with these probes revealed a dynamic network within this enzyme family involving compensatory regulation of specific family members and exposed single‐cell phenotypic heterogeneity. We propose the labeling of enzymatic activities by chemical probes as a generalizable method for the phenotyping of bacterial cells at the population and single‐cell level.

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Host-directed kinase inhibitors act as novel therapies against intracellular Staphylococcus aureus.

Sci Rep. 2019 Mar 19;9(1):4876

Authors: Bravo-Santano N, Stölting H, Cooper F, Bileckaja N, Majstorovic A, Ihle N, Mateos LM, Calle Y, Behrends V, Letek M

Abstract
Host-directed therapeutics are a promising anti-infective strategy against intracellular bacterial pathogens. Repurposing host-targeted drugs approved by the FDA in the US, the MHRA in the UK and/or regulatory equivalents in other countries, is particularly interesting because these drugs are commercially available, safe doses are documented and they have been already approved for other clinical purposes. In this study, we aimed to identify novel therapies against intracellular Staphylococcus aureus, an opportunistic pathogen that is able to exploit host molecular and metabolic pathways to support its own intracellular survival. We screened 133 host-targeting drugs and found three host-directed tyrosine kinase inhibitors (Ibrutinib, Dasatinib and Crizotinib) that substantially impaired intracellular bacterial survival. We found that Ibrutinib significantly increased host cell viability after S. aureus infection via inhibition of cell invasion and intracellular bacterial proliferation. Using phosphoproteomics data, we propose a putative mechanism of action of Ibrutinib involving several host factors, including EPHA2, C-JUN and NWASP. We confirmed the importance of EPHA2 for staphylococcal infection in an EPHA2-knock-out cell line. Our study serves as an important example of feasibility for identifying host-directed therapeutics as candidates for repurposing.

PMID: 30890742 [PubMed - in process]

Reaching new depths: Polymer–peptide conjugates (PPCs) designed to undergo an acid‐induced increase in hydrophobicity with a narrow pH‐response range (from pH 7.4 to 6.5) underwent in vivo self‐assembly in the tumor microenvironment (see picture). The PPCs in single‐chain form can penetrate deeply into the tumor and self‐assemble into nanoaggregates at molecular concentrations around the IC₅₀ values of the PPCs for enhanced cancer therapy.

Abstract

In cancer treatment, the unsatisfactory solid‐tumor penetration of nanomaterials limits their therapeutic efficacy. We employed an in vivo self‐assembly strategy and designed polymer–peptide conjugates (PPCs) that underwent an acid‐induced hydrophobicity increase with a narrow pH‐response range (from 7.4 to 6.5). In situ self‐assembly in the tumor microenvironment at appropriate molecular concentrations (around the IC₅₀ values of PPCs) enabled drug delivery deeper into the tumor. A cytotoxic peptide KLAK, decorated with the pH‐sensitive moiety cis‐aconitic anhydride (CAA), and a cell‐penetrating peptide TAT were conjugated onto poly(β‐thioester) backbones to produce PT‐K‐CAA, which can penetrate deeply into solid tumors owing to its small size as a single chain. During penetration in vivo, CAA responds to the weak acid, leading to the self‐assembly of PPCs and the recovery of therapeutic activity. Therefore, a deep‐penetration ability for enhanced cancer therapy is provided by this in vivo assembly strategy.

Make a change: A facile approach to specifically modify fragments of bacterial peptidoglycan (PG) and its derived immunostimulatory molecules is presented. N,O‐hydroxylamine linkers are incorporated onto a synthetic PG derivative, muramyl dipeptide (MDP). This methodology provides rapid access to PG probes in one step.

Abstract

The innate immune system's interaction with bacterial cells plays a pivotal role in a variety of human diseases. Carbohydrate units derived from a component of bacterial cell wall, peptidoglycan (PG), are known to stimulate an immune response. Nonetheless, access to modified late‐stage peptidoglycan intermediates is limited due to their synthetic complexity. A method to rapidly functionalize PG fragments is needed to better understand the natural host–PG interactions. Here methyl N,O‐hydroxylamine linkers are incorporated onto a synthetic PG derivative, muramyl dipeptide (MDP). The modification of MDP maintained the ability to stimulate a nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) immune response dependent on the expression of nucleotide‐binding oligomerization domain‐containing protein 2 (Nod2). Intrigued by this modification's maintenance of biological activity, several applications were explored. Methyl N,O‐hydroxylamine MDP was amendable to N‐hydroxylsuccinimide (NHS) chemistry for bioconjugation to fluorophores as well as a self‐assembled monolayer for Nod2 surface plasmon resonance analysis. Finally, linker incorporation was applicable to larger PG fragments, both enzymatically generated from Escherichia coli or chemically synthesized. This methodology provides rapid access to PG probes in one step and allows for the installation of a variety of chemical handles to advance the molecular understanding of PG and the innate immune system.