Brianna Dalesandro

ABSTRACT

Gram-negative bacteria have a tripartite cell envelope with the cytoplasmic membrane (CM), a stress-bearing peptidoglycan (PG) layer, and the asymmetric outer membrane (OM) containing lipopolysaccharide (LPS) in the outer leaflet. Cells must tightly coordinate the growth of their complex envelope to maintain cellular integrity and OM permeability barrier function. The biogenesis of PG and LPS relies on specialized macromolecular complexes that span the entire envelope. In this work, we show that Escherichia coli cells are capable of avoiding lysis when the transport of LPS to the OM is compromised, by utilizing LD-transpeptidases (LDTs) to generate 3-3 cross-links in the PG. This PG remodeling program relies mainly on the activities of the stress response LDT, LdtD, together with the major PG synthase PBP1B, its cognate activator LpoB, and the carboxypeptidase PBP6a. Our data support a model according to which these proteins cooperate to strengthen the PG in response to defective OM synthesis.

IMPORTANCE In Gram-negative bacteria, the outer membrane protects the cell against many toxic molecules, and the peptidoglycan layer provides protection against osmotic challenges, allowing bacterial cells to survive in changing environments. Maintaining cell envelope integrity is therefore a question of life or death for a bacterial cell. Here we show that Escherichia coli cells activate the LD-transpeptidase LdtD to introduce 3-3 cross-links in the peptidoglycan layer when the integrity of the outer membrane is compromised, and this response is required to avoid cell lysis. This peptidoglycan remodeling program is a strategy to increase the overall robustness of the bacterial cell envelope in response to defects in the outer membrane.

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Discovery of monoclonal antibodies cross-reactive to novel subserotypes of K. pneumoniae O3.

Sci Rep. 2017 07 26;7(1):6635

Authors: Guachalla LM, Stojkovic K, Hartl K, Kaszowska M, Kumar Y, Wahl B, Paprotka T, Nagy E, Lukasiewicz J, Nagy G, Szijártó V

Abstract
Klebsiella pneumoniae is responsible for nosocomial infections causing significant morbidity and mortality. Treatment of newly emerging multi-drug resistant strains is hampered due to severely limited antibiotic choices. Passive immunization targeting LPS O-antigens has been proposed as an alternative therapeutic option, given the limited variability of Klebsiella O-antigens. Here we report that the O3 serogroup, previously considered to have uniform O-antigen built of mannan, represents three different subtypes differing in the number of mannose residues within the O-antigen repeating units. Genetic analysis of the genes encoding mannose polymerization revealed differences that underline the observed structural alterations. The O3 variants represent antigenically different types based on the different reactivity pattern of murine monoclonal antibodies raised against a K. pneumoniae O3 strain. Typing of a collection of K. pneumoniae O3 clinical isolates showed that strains expressing the novel O3b antigen, the tri-mannose form, were more prevalent than those having the penta-mannose form, traditionally called O3, while the tetra-mannose variant, termed here O3a, seems to be rare. A monoclonal antibody cross-reacting with all three O3 sub-serogroups was also selected and shown to bind to the surface of various K. pneumoniae strains expressing different O3 subtypes and capsular antigens.

PMID: 28747785 [PubMed - indexed for MEDLINE]

Cefiderocol: A Siderophore Cephalosporin with Activity Against Carbapenem-Resistant and Multidrug-Resistant Gram-Negative Bacilli.

Drugs. 2019 Feb 02;:

Authors: Zhanel GG, Golden AR, Zelenitsky S, Wiebe K, Lawrence CK, Adam HJ, Idowu T, Domalaon R, Schweizer F, Zhanel MA, Lagacé-Wiens PRS, Walkty AJ, Noreddin A, Lynch Iii JP, Karlowsky JA

Abstract
Cefiderocol is an injectable siderophore cephalosporin discovered and being developed by Shionogi & Co., Ltd., Japan. As with other β-lactam antibiotics, the principal antibacterial/bactericidal activity of cefiderocol occurs by inhibition of Gram-negative bacterial cell wall synthesis by binding to penicillin binding proteins; however, it is unique in that it enters the bacterial periplasmic space as a result of its siderophore-like property and has enhanced stability to β-lactamases. The chemical structure of cefiderocol is similar to both ceftazidime and cefepime, which are third- and fourth-generation cephalosporins, respectively, but with high stability to a variety of β-lactamases, including AmpC and extended-spectrum β-lactamases (ESBLs). Cefiderocol has a pyrrolidinium group in the side chain at position 3 like cefepime and a carboxypropanoxyimino group in the side chain at position 7 of the cephem nucleus like ceftazidime. The major difference in the chemical structures of cefiderocol, ceftazidime and cefepime is the presence of a catechol group on the side chain at position 3. Together with the high stability to β-lactamases, including ESBLs, AmpC and carbapenemases, the microbiological activity of cefiderocol against aerobic Gram-negative bacilli is equal to or superior to that of ceftazidime-avibactam and meropenem, and it is active against a variety of Ambler class A, B, C and D β-lactamases. Cefiderocol is also more potent than both ceftazidime-avibactam and meropenem versus Acinetobacter baumannii, including meropenem non-susceptible and multidrug-resistant (MDR) isolates. Cefiderocol's activity against meropenem-non-susceptible and Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriales is comparable or superior to ceftazidime-avibactam. Cefiderocol is also more potent than both ceftazidime-avibactam and meropenem against all resistance phenotypes of Pseudomonas aeruginosa and against Stenotrophomonas maltophilia. The current dosing regimen being used in phase III studies is 2 g administered intravenously every 8 h (q8 h) using a 3-h infusion. The pharmacokinetics of cefiderocol are best described by a three-compartment linear model. The mean plasma half-life (t½) was ~ 2.3 h, protein binding is 58%, and total drug clearance ranged from 4.6-6.0 L/h for both single- and multi-dose infusions and was primarily renally excreted unchanged (61-71%). Cefiderocol is primarily renally excreted unchanged and clearance correlates with creatinine clearance. Dosage adjustment is thus required for both augmented renal clearance and in patients with moderate to severe renal impairment. In vitro and in vivo pharmacodynamic studies have reported that as with other cephalosporins the pharmacodynamic index that best predicts clinical outcome is the percentage of time that free drug concentrations exceed the minimum inhibitory concentration (%fT > MIC). In vivo efficacy of cefiderocol has been studied in a variety of humanized drug exposure murine and rat models of infection utilizing a variety of MDR and extremely drug resistant strains. Cefiderocol has performed similarly to or has been superior to comparator agents, including ceftazidime and cefepime. A phase II prospective, multicenter, double-blind, randomized clinical trial assessed the safety and efficacy of cefiderocol 2000 mg q8 h versus imipenem/cilastatin 1000 mg q8 h, both administered intravenously for 7-14 days over 1 h, in the treatment of complicated urinary tract infection (cUTI, including pyelonephritis) or acute uncomplicated pyelonephritis in hospitalized adults. A total of 452 patients were initially enrolled in the study, with 303 in the cefiderocol arm and 149 in the imipenem/cilastatin arm. The primary outcome measure was a composite of clinical cure and microbiological eradication at the test-of-cure (TOC) visit, that is, 7 days after the end of treatment in the microbiological intent-to-treat (MITT) population. Secondary outcome measures included microbiological response per pathogen and per patient at early assessment (EA), end of treatment (EOT), TOC, and follow-up (FUP); clinical response per pathogen and per patient at EA, EOT, TOC, and FUP; plasma, urine and concentrations of cefiderocol; and the number of participants with adverse events. The composite of clinical and microbiological response rates was 72.6% (183/252) for cefiderocol and 54.6% (65/119) for imipenem/cilastatin in the MITT population. Clinical response rates per patient at the TOC visit were 89.7% (226/252) for cefiderocol and 87.4% (104/119) for imipenem/cilastatin in the MITT population. Microbiological eradication rates were 73.0% (184/252) for cefiderocol and 56.3% (67/119) for imipenem/cilastatin in the MITT population. Additionally, two phase III clinical trials are currently being conducted by Shionogi & Co., Ltd., Japan. The two trials are evaluating the efficacy of cefiderocol in the treatment of serious infections in adult patients caused by carbapenem-resistant Gram-negative pathogens and evaluating the efficacy of cefiderocol in the treatment of adults with hospital-acquired bacterial pneumonia, ventilator-associated pneumonia or healthcare-associated pneumonia caused by Gram-negative pathogens. Cefiderocol appears to be well tolerated (minor reported adverse effects were gastrointestinal and phlebitis related), with a side effect profile that is comparable to other cephalosporin antimicrobials. Cefiderocol appears to be well positioned to help address the increasing number of infections caused by carbapenem-resistant and MDR Gram-negative bacilli, including ESBL- and carbapenemase-producing strains (including metallo-β-lactamase producers). A distinguishing feature of cefiderocol is its activity against resistant P. aeruginosa, A. baumannii, S. maltophilia and Burkholderia cepacia.

PMID: 30712199 [PubMed - as supplied by publisher]

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The Role of the CopA Copper Efflux System in Acinetobacter baumannii Virulence.

Int J Mol Sci. 2019 Jan 29;20(3):

Authors: Alquethamy SF, Khorvash M, Pederick VG, Whittall JJ, Paton JC, Paulsen IT, Hassan KA, McDevitt CA, Eijkelkamp BA

Abstract
Acinetobacter baumannii has emerged as one of the leading causative agents of nosocomial infections. Due to its high level of intrinsic and adapted antibiotic resistance, treatment failure rates are high, which allows this opportunistic pathogen to thrive during infection in immune-compromised patients. A. baumannii can cause infections within a broad range of host niches, with pneumonia and bacteraemia being associated with the greatest levels of morbidity and mortality. Although its resistance to antibiotics is widely studied, our understanding of the mechanisms required for dealing with environmental stresses related to virulence and hospital persistence, such as copper toxicity, is limited. Here, we performed an in silico analysis of the A. baumannii copper resistome, examining its regulation under copper stress. Using comparative analyses of bacterial P-type ATPases, we propose that A. baumannii encodes a member of a novel subgroup of P1B-1 ATPases. Analyses of three putative inner membrane copper efflux systems identified the P1B-1 ATPase CopA as the primary mediator of cytoplasmic copper resistance in A. baumannii. Using a murine model of A. baumannii pneumonia, we reveal that CopA contributes to the virulence of A. baumannii. Collectively, this study advances our understanding of how A. baumannii deals with environmental copper toxicity, and it provides novel insights into how A. baumannii combats adversities encountered as part of the host immune defence.

PMID: 30699983 [PubMed - in process]

A team of researchers have solved a 20-year riddle of how a crucial step in the biosynthesis of 'last-resort' antibiotics occurs.

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A novel cereblon modulator for targeted protein degradation.

Eur J Med Chem. 2019 Jan 17;166:65-74

Authors: Kim SA, Go A, Jo SH, Park SJ, Jeon YU, Kim JE, Lee HK, Park CH, Lee CO, Park SG, Kim P, Park BC, Cho SY, Kim S, Ha JD, Kim JH, Hwang JY

Abstract
Immunomodulatory drugs (IMiDs) exert anti-myeloma activity by binding to the protein cereblon (CRBN) and subsequently degrading IKZF1/3. Recently, their ability to recruit E3 ubiquitin ligase has been used in the proteolysis targeting chimera (PROTAC) technology. Herein, we design and synthesize a novel IMiD analog TD-106 that induces the degradation of IKZF1/3 and inhibits the proliferation of multiple myeloma cells in vitro as well as in vivo. Moreover, we demonstrate that TD-428, which comprises TD-106 linked to a BET inhibitor, JQ1 efficiently induce BET protein degradation in the prostate cancer cell line 22Rv1. Consequently, cell proliferation is inhibited due to suppressed C-MYC transcription. These results, therefore, firmly suggest that the newly synthesized IMiD analog, TD-106, is a novel CRBN modulator that can be used for targeted protein degradation.

PMID: 30684871 [PubMed - as supplied by publisher]

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Synthesis and Application of Methyl N,O-Hydroxylamine Muramyl Peptides.

Chembiochem. 2019 06 03;20(11):1369-1375

Authors: Lazor KM, Zhou J, DeMeester KE, D'Ambrosio EA, Grimes CL

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.

PMID: 30672111 [PubMed - indexed for MEDLINE]

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PROTAC-mediated crosstalk between E3 ligases.

Chem Commun (Camb). 2019 Jan 23;:

Authors: Steinebach C, Kehm H, Lindner S, Vu LP, Köpff S, López Mármol Á, Weiler C, Wagner KG, Reichenzeller M, Krönke J, Gütschow M

Abstract
Small-molecule heterobifunctional degraders can effectively control protein levels and are useful research tools. We assembled proteolysis targeting chimeras (PROTACs) from a cereblon (CRBN) and a von-Hippel-Lindau (VHL) ligase ligand and demonstrated a PROTAC-induced heterodimerization of the two E3 ligases leading to unidirectional and efficient degradation of CRBN.

PMID: 30672516 [PubMed - as supplied by publisher]

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Lysozyme Counteracts β-Lactam Antibiotics by Promoting the Emergence of L-Form Bacteria.

Cell. 2018 02 22;172(5):1038-1049.e10

Authors: Kawai Y, Mickiewicz K, Errington J

Abstract
β-lactam antibiotics inhibit bacterial cell wall assembly and, under classical microbiological culture conditions that are generally hypotonic, induce explosive cell death. Here, we show that under more physiological, osmoprotective conditions, for various Gram-positive bacteria, lysis is delayed or abolished, apparently because inhibition of class A penicillin-binding protein leads to a block in autolytic activity. Although these cells still then die by other mechanisms, exogenous lytic enzymes, such as lysozyme, can rescue viability by enabling the escape of cell wall-deficient "L-form" bacteria. This protective L-form conversion was also observed in macrophages and in an animal model, presumably due to the production of host lytic activities, including lysozyme. Our results demonstrate the potential for L-form switching in the host environment and highlight the unexpected effects of innate immune effectors, such as lysozyme, on antibiotic activity. Unlike previously described dormant persisters, L-forms can continue to proliferate in the presence of antibiotic.

PMID: 29456081 [PubMed - indexed for MEDLINE]

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Ehrlichia chaffeensis TRP32 Nucleomodulin Function and Localization Is Regulated by NEDD4L-Mediated Ubiquitination.

Front Cell Infect Microbiol. 2017;7:534

Authors: Farris TR, Zhu B, Wang JY, McBride JW

Abstract
Ehrlichia chaffeensis is an obligately intracellular bacterium that reprograms the mononuclear phagocyte through diverse effector-host interactions to modulate various host cell processes. In a previous study, we reported that the E. chaffeensis nucleomodulin TRP32 regulates transcription of host genes in several biologically relevant categories, including cell differentiation and proliferation. In this study, we investigate the effect of ubiquitination on TRP32 function and localization within the host cell. TRP32 is both mono- and polyubiquitinated on multiple lysine residues during infection and when ectopically expressed. Despite lacking a canonical PPxY motif, TRP32 interacted with, and was modified by the human HECT E3 ubiquitin (Ub) ligase NEDD4L. TRP32 ubiquitination was not by K48-linked polyUb chains, nor was it degraded by the proteasome; however, TRP32 was modified by K63-linked polyUb chains detected both in the cytosol and nucleus. HECT ligase inhibitor, heclin, altered the subnuclear localization of ectopically expressed TRP32 from a diffuse nuclear pattern to a lacy, punctate pattern with TRP32 distributed around the periphery of the nucleus and nucleoli. When a TRP32 lysine null (K-null) mutant was ectopically expressed, it exhibited a similar phenotype as single lysine mutants (K63R, K93R, and K123R). However, the K-null mutant showed increased amounts of cytoplasmic TRP32 compared to single lysine mutants or heclin-treated cells ectopically expressing TRP32. These alterations in localization corresponded to changes in TRP32 transcriptional repressor function with heclin-treated and single lysine mutants unable to repress transcription of a TRP32 target genes in a luciferase assay.

PMID: 29376035 [PubMed - indexed for MEDLINE]

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Rationally designing antisense therapy to keep up with evolving bacterial resistance.

PLoS One. 2019;14(1):e0209894

Authors: Kotil S, Jakobsson E

Abstract
Antisense molecules used as antibiotics offer the potential to keep up with acquired resistance, by redesigning the sequence of an antisense. Once bacteria acquire resistance by mutating the targeted sequence, new antisense can readily be designed by using sequence information of a target gene. However, antisense molecules require additional delivery vehicles to get into bacteria and be protected from degradation. Based on progress in the last few years it appears that, while redesigning or finding new delivery vehicle will be more difficult than redesigning the antisense cargo, it will perhaps be less difficult than finding new conventional small molecule antibiotics. In this study we propose a protocol that maximizes the combined advantages of engineered delivery vehicle and antisense cargo by decreasing the immediate growth advantage to the pathogen of mutating the entry mechanisms and increasing the advantage to the pathogen of antisense target mutations. Using this protocol, we show by computer simulation an appropriately designed antisense therapy can potentially be effective many times longer than conventional antibiotics before succumbing to resistance. While the simulations describe an in-vitro situation, based on comparison with other in-vitro studies on acquired resistance we believe the advantages of the combination antisense strategy have the potential to provide much more sustainability in vivo than conventional antibiotic therapy.

PMID: 30645595 [PubMed - in process]

Cross-lineage protection by human antibodies binding the influenza B hemagglutinin

Cross-lineage protection by human antibodies binding the influenza B hemagglutinin, Published online: 18 January 2019; doi:10.1038/s41467-018-08165-y

Immune recognition of Influenza B virus (IBV) is poorly understood. Here, Liu et al. use flow cytometry to characterize IBV-specific memory B cell responses following seasonal vaccination and show that elicited cross-reactive antibodies can protect against infection, providing a platform for vaccine design.

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Modulation of plant and bacterial polyamine metabolism during the compatible interaction between tomato and Pseudomonas syringae.

J Plant Physiol. 2018 Dec;231:281-290

Authors: Vilas JM, Romero FM, Rossi FR, Marina M, Maiale SJ, Calzadilla PI, Pieckenstain FL, Ruiz OA, Gárriz A

Abstract
The polyamines putrescine, spermidine and spermine participate in a variety of cellular processes in all organisms. Many studies have shown that these polycations are important for plant immunity, as well as for the virulence of diverse fungal phytopathogens. However, the polyamines' roles in the pathogenesis of phytopathogenic bacteria have not been thoroughly elucidated to date. To obtain more information on this topic, we assessed the changes in polyamine homeostasis during the infection of tomato plants by Pseudomonas syringae. Our results showed that polyamine biosynthesis and catabolism are activated in both tomato and bacteria during the pathogenic interaction. This activation results in the accumulation of putrescine in whole leaf tissues, as well as in the apoplastic fluids, which is explained by the induction of its synthesis in plant cells and also on the basis of its excretion by bacteria. We showed that the excretion of this polyamine by P. syringae is stimulated under virulence-inducing conditions, suggesting that it plays a role in plant colonization. However, no activation of bacterial virulence traits or induction of plant invasion was observed after the exogenous addition of putrescine. In addition, no connection was found between this polyamine and plant defence responses. Although further research is warranted to unravel the biological functions of these molecules during plant-bacterial interactions, this study contributes to a better understanding of the changes associated with the homeostasis of polyamines during plant pathogenesis.

PMID: 30342327 [PubMed - indexed for MEDLINE]

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Antibody-Mediated Killing of Carbapenem-Resistant ST258 Klebsiella pneumoniae by Human Neutrophils.

MBio. 2018 03 13;9(2):

Authors: Kobayashi SD, Porter AR, Freedman B, Pandey R, Chen L, Kreiswirth BN, DeLeo FR

Abstract
Carbapenem-resistant Klebsiella pneumoniae is a problem worldwide. A carbapenem-resistant K. pneumoniae lineage classified as multilocus sequence type 258 (ST258) is prominent in the health care setting in many regions of the world, including the United States. ST258 strains can be resistant to virtually all clinically useful antibiotics; treatment of infections caused by these organisms is difficult, and mortality is high. As a step toward promoting development of new therapeutics for ST258 infections, we tested the ability of rabbit antibodies specific for ST258 capsule polysaccharide to enhance human serum bactericidal activity and promote phagocytosis and killing of these bacteria by human neutrophils. We first demonstrated that an isogenic wzy deletion strain is significantly more susceptible to killing by human heparinized blood, serum, and neutrophils than a wild-type ST258 strain. Consistent with the importance of capsule as an immune evasion molecule, rabbit immune serum and purified IgG specific for ST258 capsule polysaccharide type 2 (CPS2) enhanced killing by human blood and serum in vitro Moreover, antibodies specific for CPS2 promoted phagocytosis and killing of ST258 by human neutrophils. Collectively, our findings suggest that ST258 CPS2 is a viable target for immunoprophylactics and/or therapeutics.IMPORTANCE Infections caused by carbapenem-resistant K. pneumoniae are difficult to treat, and mortality is high. New prophylactic approaches and/or therapeutic measures are needed to prevent or treat infections caused by these multidrug-resistant bacteria. A strain of carbapenem-resistant K. pneumoniae, classified by multilocus sequence typing as ST258, is present in many regions of the world and is the most prominent carbapenem-resistant K. pneumoniae lineage in the United States. Here we show that rabbit antibodies specific for capsule polysaccharide of ST258 significantly enhance human serum bactericidal activity and promote phagocytosis and killing of this pathogen by human neutrophils. These studies have provided strong support for the idea that development of an immunotherapy (vaccine) for carbapenem-resistant K. pneumoniae infections is feasible and has merit.

PMID: 29535199 [PubMed - indexed for MEDLINE]

Antigens reversibly conjugated to a polymeric glyco-adjuvant induce protective humoral and cellular immunity

Antigens reversibly conjugated to a polymeric glyco-adjuvant induce protective humoral and cellular immunity, Published online: 14 January 2019; doi:10.1038/s41563-018-0256-5

A vaccine platform has been developed from a polymeric glyco-adjuvant conjugated to an antigen, and is now shown to target dendritic cells via mannose-binding receptors and generate potent cellular and humoral immune response against malaria.

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E3 ubiquitin ligase Nedd4 inhibits AP-1 activity and TNF-α production through targeting p38α for polyubiquitination and subsequent degradation.

Sci Rep. 2017 07 03;7(1):4521

Authors: Liu Q, Zhang S, Chen G, Zhou H

Abstract
p38α plays an important role in many inflammatory diseases, such as skin inflammation, endotoxic shock and arthritis. Ubiquitination is a vital posttranslational modification of proteins and plays a crucial regulatory role in inflammatory cells. It has been reported that ubiquitination of Tak1 and TAB1 upstream of p38α can regulate p38α activation respectively. However, p38α ubiquitination is not yet clear. In this paper, we showed that E3 ubiquitin ligase Nedd4 is a regulatory component of the p38α pathway and is responsible for polyubiquitination of p38α through K48-linked and K63-linked polyubiquitination. The levels of p38α and its downstream target TNF-α were increased in Nedd4 deficient macrophages response to LPS compared with wild-type cells. AP-1 activity and degradation of p38α were induced by Nedd4 in a dose-dependent manner. Furthermore, we found that phosphorylation of p38α is involved in the interactions between p38α and Nedd4 and subsequently promotes polyubiquitination of p38α, especially K48-linked polyubiquitination by Nedd4. The different conformation of two p38α isoforms (p38αV1 and p38αV2) might be the cause of their different interactions with Nedd4 and their polyubiquitination sites by Nedd4. Thus, NEDD4 is a previously unknown component of the p38α signaling complex necessary for TNF-α activation.

PMID: 28674435 [PubMed - indexed for MEDLINE]

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The Role of Copper Homeostasis at the Host-Pathogen Axis: From Bacteria to Fungi.

Int J Mol Sci. 2019 Jan 05;20(1):

Authors: Li C, Li Y, Ding C

Abstract
Copper is an essential trace element participating in many vital biological processes, however it becomes a toxic agent when in excess. Thus, precise and tight regulation of copper homeostasis processes, including transport, delivery, storage, detoxification, and efflux machineries, is important, ensuring that only the amount needed to sustain basic biological functions and simultaneously prevent copper toxicity in the cell is maintained. Numerous exciting studies have revealed that copper plays an indispensable role at the microbial pathogen-host axis for entities ranging from pathogenic bacteria to deadly fungal species. Analyses of copper homeostases in bacteria and fungi extensively demonstrate that copper is utilized by the host immune system as an anti-microbial agent. The expression of copper efflux and detoxification from microbial pathogens is induced to counteract the host's copper bombardment, which in turn disrupts these machineries, resulting in the attenuation of microbial survival in host tissue. We hereby review the latest work in copper homeostases in pathogenic bacteria and fungi and focus on the maintenance of a copper balance at the pathogen-host interaction axis.

PMID: 30621285 [PubMed - in process]

Type III secretion system (T3SS) is an important virulence factor that contributes to P. aeruginosa acute infection. We demonstrate that a cysteine biosynthesis regulator CysB is critical to the ability of P. aeruginosa to modulate the expression of virulence‐associated traits and bacterial pathogenesis, which is dependent on retS expression.

Summary

Pseudomonas aeruginosa is a major pathogen that causes serious acute and chronic infections in humans. The type III secretion system (T3SS) is an important virulence factor that plays essential roles in acute infections. However, the regulatory mechanisms of T3SS are not fully understood. In this study, we found that deletion of cysB reduced T3SS gene expression and swarming motility but enhanced biofilm formation. In a mouse acute pneumonia model, mutation of cysB decreased the average bacterial load compared to that of the wild‐type strain. Further experiments demonstrated that CysB contributed to the reduced T3SS gene expression and bacterial pathogenesis by directly regulating the sensor kinase RetS. We also performed crystallographic studies of PaCysB. The overall fold of PaCysB NTD domain is similar to other LysR superfamily proteins and structural superposition revealed one possible DNA‐binding model for PaCysB. Structural comparison also revealed great flexibility of the PaCysB RD domain, which may play an important role in bending and transcriptional regulation of target DNA. Taken together, these results expand our current understanding of the complex regulatory networks of T3SS and RetS pathways. The crystal structure of CysB provides new insights for studying the function of its homologs in other bacterial species.

This article is protected by copyright. All rights reserved.

Caulobacter crescentus forms a polar stalk with peptidoglycan composition distinct from that of the cell body. The stalk is enriched in DAP‐DAP crosslinks mediated by the LD‐transpeptidase LdtD. Elevated levels of LD‐crosslinking provide resistance against lysozyme‐mediated degradation in C. crescentus stalks, and this mechanism of lysozyme resistance may apply broadly to a variety of bacterial species.

Summary

The diversity of cell shapes across the bacterial kingdom reflects evolutionary pressures that have produced physiologically important morphologies. While efforts have been made to understand the regulation of some prototypical cell morphologies such as that of rod‐shaped Escherichia coli, little is known about most cell shapes. For Caulobacter crescentus, polar stalk synthesis is tied to its dimorphic life cycle, and stalk elongation is regulated by phosphate availability. Based on the previous observation that C. crescentus stalks are lysozyme‐resistant, we compared the composition of the peptidoglycan cell wall of stalks and cell bodies and identified key differences in peptidoglycan crosslinking. Cell body peptidoglycan contained primarily DD‐crosslinks between meso‐diaminopimelic acid and D‐alanine residues, whereas stalk peptidoglycan had more LD‐transpeptidation (meso‐diaminopimelic acid‐meso‐diaminopimelic acid), mediated by LdtD. We determined that ldtD is dispensable for stalk elongation; rather, stalk LD‐transpeptidation reflects an aging process associated with low peptidoglycan turnover in the stalk. We also found that lysozyme resistance is a structural consequence of LD‐crosslinking. Despite no obvious selection pressure for LD‐crosslinking or lysozyme resistance in C. crescentus, the correlation between these two properties was maintained in other organisms, suggesting that DAP‐DAP crosslinking may be a general mechanism for regulating bacterial sensitivity to lysozyme.

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The PROTAC technology in drug development.

Cell Biochem Funct. 2019 Jan 02;:

Authors: Zou Y, Ma D, Wang Y

Abstract
Currently, a new technology termed PROTAC, proteolysis targeting chimera, has been developed for inducing the protein degradation by a targeting molecule. This technology takes advantage of a moiety of targeted protein and a moiety of recognizing E3 ubiquitin ligase and produces a hybrid molecule to specifically knock down a targeted protein. During the first decade, three pedigreed groups worked on the development of this technology. To date, this technology has been extended by different groups, aiming to develop new drugs against different diseases including cancers. This review summarizes the contributions of the groups for the development of PROTAC. SIGNIFICANCE OF THE STUDY: This review summarized the development of the PROTAC technology for readers and also presented the author's opinions on the application of the technology in tumor therapy.

PMID: 30604499 [PubMed - as supplied by publisher]

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Solid-phase synthesis for thalidomide-based proteolysis-targeting chimeras (PROTAC).

Chem Commun (Camb). 2019 Jan 02;:

Authors: Krajcovicova S, Jorda R, Hendrychova D, Krystof V, Soural M

Abstract
A preloaded resin consisting of a thalidomide moiety and an ethylene-oxy linker allows the simple and fast formation of PROTACs. The feasibility of the procedure was illustrated by conjugating different protein kinase inhibitors. The biological functionality of an ibrutinib-like conjugate was then confirmed by a cellular experiment.

PMID: 30601480 [PubMed - as supplied by publisher]

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Insight into Elongation Stages of Peptidoglycan Processing in Bacterial Cytoplasmic Membranes.

Sci Rep. 2018 Dec 07;8(1):17704

Authors: Kim S, Pires MM, Im W

Abstract
Peptidoglycan (PG) biosynthesis and assembly are needed for bacterial cell wall formation. Lipid II is the precursor in the PG biosynthetic pathway and carries a nascent PG unit that is processed by glycosyltransferases. Despite its immense therapeutic value as a target of several classes of antibiotics, the conformational ensemble of lipid II in bacterial membranes and its interactions with membrane-anchored enzymes remain elusive. In this work, lipid II and its elongated forms (lipid VI and lipid XII) were modeled and simulated in bilayers of POPE (palmitoyl-oleoyl-phosphatidyl-ethanolamine) and POPG (palmitoyl-oleoyl-phosphatidyl-glycerol) that mimic the prototypical composition of Gram-negative cytoplasmic membranes. In addition, penicillin-binding protein 1b (PBP1b) from Escherichia coli was modeled and simulated in the presence of a nascent PG to investigate their interactions. Trajectory analysis reveals that as the glycan chain grows, the non-reducing end of the nascent PG displays much greater fluctuation along the membrane normal and minimally interacts with the membrane surface. In addition, dihedral angles within the pyrophosphate moiety are determined by the length of the PG moiety and its surrounding environment. When a nascent PG is bound to PBP1b, the stem peptide remains in close contact with PBP1b by structural rearrangement of the glycan chain. Most importantly, the number of nascent PG units required to reach the transpeptidase domain are determined to be 7 or 8. Our findings complement experimental results to further understand how the structure of nascent PG can dictate the assembly of the PG scaffold.

PMID: 30531805 [PubMed - in process]

Synergistic interactions between NOD receptors and TLRs: Mechanisms and clinical implications.

J Leukoc Biol. 2018 Dec 05;:

Authors: Pashenkov MV, Murugina NE, Budikhina AS, Pinegin BV

Abstract
Interactions between pattern recognition receptors (PRRs) shape innate immune responses to particular classes of pathogens. Here, we review interactions between TLRs and nucleotide-binding oligomerization domain 1 and 2 (NOD1 and NOD2) receptors, two major groups of PRRs involved in innate recognition of bacteria. Most of experimental data both in vitro and in vivo suggest that NODs and TLRs synergize with each other at inducing the production of cytokines and antimicrobial peptides. Molecular mechanisms of this synergy remain poorly understood, although several scenarios can be proposed: (i) direct interactions of signaling pathways downstream of NODs and TLRs; (ii) mutual transcriptional regulation of unique components of NOD-dependent and TLR-dependent signaling pathways; and (iii) interactions at the post-transcriptional level. Potential practical implications of NOD-TLR synergy are dual. In sepsis, where synergistic effects probably contribute to excessive proinflammatory cytokine production, blockade of NOD1, and/or NOD2 in addition to TLR4 blockade may be required to achieve therapeutic benefit. On the other hand, synergistic combinations of relatively small doses of NOD and TLR agonists administered before infection could be used to boost innate resistance against bacterial pathogens.

PMID: 30517768 [PubMed - as supplied by publisher]

The catalytic role of water in the binding site of l,d-transpeptidase 2 within acylation mechanism: A QM/MM (ONIOM) modelling.

Tuberculosis (Edinb). 2018 Dec;113:222-230

Authors: Ibeji CU, Tolufashe GF, Ntombela T, Govender T, Maguire GEM, Lamichhane G, Kruger HG, Honarparvar B

Abstract
Mycobacterium tuberculosis is the causative agent of Tuberculosis. Formation of 3 → 3 crosslinks in the peptidoglycan layer of M. tuberculosis is catalyzed by l,d-transpeptidases. These enzymes can confer resistance against classical β-lactams that inhibit enzymes that generate 4 → 3 peptidoglycan crosslinks. The focus of this study is to investigate the catalytic role of water molecules in the acylation mechanism of the β-lactam ring within two models; 4- and 6-membered ring systems using two-layered our Own N-layer integrated Molecular Mechanics ONIOM (B3LYP/6-311++G(2d,2p): AMBER) model. The obtained thermochemical parameters revealed that the 6-membered ring model best describes the inhibition mechanism of acylation which indicates the role of water in the preference of 6-membered ring reaction pathway. This finding is in accordance with experimental data for the rate-limiting step of cysteine protease with the same class of inhibitor and binding affinity for both inhibitors. As expected, the ΔG# results also reveal that the 6-membered ring reaction pathway is the most favourable. The electrostatic potential (ESP) and the natural bond orbital analysis (NBO) showed stronger interactions in 6-membered ring transition state (TS-6) mechanism involving water in the active site of the enzyme. This study could be helpful in the development of novel antibiotics against l,d-transpeptidase.

PMID: 30514506 [PubMed - in process]

Combination of Antibodies and Antibiotics as a Promising Strategy Against Multidrug-Resistant Pathogens of the Respiratory Tract.

Front Immunol. 2018;9:2700

Authors: Domenech M, Sempere J, de Miguel S, Yuste J

Abstract
The emergence of clinical isolates associated to multidrug resistance is a serious threat worldwide in terms of public health since complicates the success of the antibiotic treatment and the resolution of the infectious process. This is of great concern in pathogens affecting the lower respiratory tract as these infections are one of the major causes of mortality in children and adults. In most cases where the respiratory pathogen is associated to multidrug-resistance, antimicrobial concentrations both in serum and at the site of infection may be insufficient and the resolution of the infection depends on the interaction of the invading pathogen with the host immune response. The outcome of these infections largely depends on the susceptibility of the pathogen to the antibiotic treatment, although the humoral and cellular immune responses also play an important role in this process. Hence, prophylactic measures or even immunotherapy are alternatives against these multi-resistant pathogens. In this sense, specific antibodies and antibiotics may act concomitantly against the respiratory pathogen. Alteration of cell surface structures by antimicrobial drugs even at sub-inhibitory concentrations might result in greater exposure of microbial ligands that are normally hidden or hardly exposed. This alteration of the bacterial envelope may stimulate opsonization by natural and/or specific antibodies or even by host defense components, increasing the recognition of the microbial pathogen by circulating phagocytes. In this review we will explain the most relevant studies, where vaccination or the use of monoclonal antibodies in combination with antimicrobial treatment has demonstrated to be an alternative strategy to overcome the impact of multidrug resistance in respiratory pathogens.

PMID: 30515172 [PubMed - in process]

by Margaret Coates, Sarah Blanchard, Amanda S. MacLeod

Insight into Elongation Stages of Peptidoglycan Processing in Bacterial Cytoplasmic Membranes

Insight into Elongation Stages of Peptidoglycan Processing in Bacterial Cytoplasmic Membranes, Published online: 07 December 2018; doi:10.1038/s41598-018-36075-y

Insight into Elongation Stages of Peptidoglycan Processing in Bacterial Cytoplasmic Membranes