Brianna Dalesandro

ABSTRACT

The keystone oral pathogen Porphyromonas gingivalis is associated with severe periodontitis. Intriguingly, this bacterium is known to secrete large amounts of an enzyme that converts peptidylarginine into citrulline residues. The present study was aimed at identifying possible functions of this citrullinating enzyme, named Porphyromonas peptidylarginine deiminase (PPAD), in the periodontal environment. The results show that PPAD is detectable in the gingiva of patients with periodontitis, and that it literally neutralizes human innate immune defenses at three distinct levels, namely bacterial phagocytosis, capture in neutrophil extracellular traps (NETs), and killing by the lysozyme-derived cationic antimicrobial peptide LP9. As shown by mass spectrometry, exposure of neutrophils to PPAD-proficient bacteria reduces the levels of neutrophil proteins involved in phagocytosis and the bactericidal histone H2. Further, PPAD is shown to citrullinate the histone H3, thereby facilitating the bacterial escape from NETs. Last, PPAD is shown to citrullinate LP9, thereby restricting its antimicrobial activity. The importance of PPAD for immune evasion is corroborated in the infection model Galleria mellonella, which only possesses an innate immune system. Together, the present observations show that PPAD-catalyzed protein citrullination defuses innate immune responses in the oral cavity, and that the citrullinating enzyme of P. gingivalis represents a new type of bacterial immune evasion factor.

IMPORTANCE Bacterial pathogens do not only succeed in breaking the barriers that protect humans from infection, but they also manage to evade insults from the human immune system. The importance of the present study resides in the fact that protein citrullination is shown to represent a new bacterial mechanism for immune evasion. In particular, the oral pathogen P. gingivalis employs this mechanism to defuse innate immune responses by secreting a protein-citrullinating enzyme. Of note, this finding impacts not only the global health problem of periodontitis, but it also extends to the prevalent autoimmune disease rheumatoid arthritis, which has been strongly associated with periodontitis, PPAD activity, and loss of tolerance against citrullinated proteins, such as the histone H3.

Structure and function of L,D- and D,D-transpeptidase family enzymes from Mycobacterium tuberculosis.

Curr Med Chem. 2018 12 03;:

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

Abstract
Peptidoglycan, the exoskeleton of bacterial cell and an essential barrier that protects the cell, is synthesized by a pathway whose final steps are catalysed by transpeptidases, including M.tuberculosis. Knowledge of the structure and function of these vital enzymes that generate this macromolecule in M. tuberculosis could facilitate the development of potent lead compounds against tuberculosis. This review summarizes the experimental and computational studies to date on these aspects of transpeptidases in M. tuberculosis that have been identified and validated. The reported structures of L,D- and D,D-transpeptidases, as well as their functionalities, are reviewed and the proposed enzymatic mechanisms for L,D-transpeptidases are summarized. In addition, we provide bioactivities of known M. tuberculosis drugs against these enzymes based on both experimental and computational approaches. Advancing knowledge about these prominent targets in M. tuberculosis supports the development of new drugs with novel inhibition mechanisms overcoming to address current need for new drugs against tuberculosis.

PMID: 30501595 [PubMed - as supplied by publisher]

Bacterial defence molecules target viral DNA

Bacterial defence molecules target viral DNA, Published online: 05 December 2018; doi:10.1038/d41586-018-07576-7

Bacteria can use specific protein-based strategies to defend individual cells against viruses. Evidence that bacterial small molecules also target viruses provides fresh insights into how bacteria thwart viral infection.

The silent epidemic killing more people than HIV, malaria or TB

The silent epidemic killing more people than HIV, malaria or TB, Published online: 05 December 2018; doi:10.1038/d41586-018-07592-7

Viral hepatitis is on the rise. Tackling hepatitis B in Africa is key to fighting back.

Seeking a selectivity solution: We have worked towards an extracellular version of the antibody–drug conjugate (ADC) modality by conjugating a nonselective protease inhibitor to a high‐affinity antibody targeting a single protease. Detailed studies demonstrated that antibody binding kinetics could overcome poor small‐molecule selectivity, thus enabling simultaneous and selective binding to the target.

Abstract

Antibody–drug conjugates (ADCs) are a growing class of therapeutics that harness the specificity of antibodies and the cell‐killing potency of small‐molecule drugs. Beyond cytotoxics, there are few examples of the application of an ADC approach to difficult drug discovery targets. Here, we present the initial development of a non‐internalising ADC, with a view to selectively inhibiting an extracellular protein. Employing the wellinvestigated matrix metalloproteinase‐9 (MMP‐9) as our model, we adapted a broad‐spectrum, nonselective MMP inhibitor for conjugation and linked this to a MMP‐9‐targeting antibody. The resulting ADC fully inhibits MMP‐9, and ELISA results suggest antibody targeting can direct a nonselective inhibitor.

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MDM2-recruiting PROTAC Offers Superior, Synergistic Anti-proliferative Activity via Simultaneous Degradation of BRD4 and Stabilization of p53.

Cancer Res. 2018 Nov 01;:

Authors: Hines J, Lartigue S, Dong H, Qian Y, Crews CM

Abstract
While the number of proteins effectively targeted for post-translational degradation by PROTAC has grown steadily, the number of E3 ligases successfully exploited to accomplish this has been limited to the few for which small molecule ligands have been discovered. Although the E3 ligase MDM2 is bound by the nutlin class of small molecule ligands, there are few nutlin-based PROTAC. Since a nutlin-based PROTAC should both knockdown its target protein and upregulate the tumor suppressor, p53, we examined the ability of such a PROTAC to decrease cancer cell viability. A nutlin-based, BRD4-degrading PROTAC, A1874, was able to degrade its target protein by 98% with nanomolar potency. Given the complementary ability of A1874 to stabilize p53, we discovered that the nutlin-based PROTAC was more effective in inhibiting proliferation of many cancer cell lines with wild type p53 than was a corresponding VHL-utilizing PROTAC with similar potency and efficacy to degrade BRD4. This is the first report of a PROTAC in which the E3 ligase ligand and targeting warhead combine to exert a synergistic antiproliferative effect. Our study highlights the untapped potential that may be unlocked by expanding the repertoire of E3 ligases that can be recruited by PROTAC.

PMID: 30385614 [PubMed - as supplied by publisher]

Bacterial cytoplasmic membrane serves as the primary site of critical membrane‐related functions such as protein secretion, lipid biogenesis and energy production that are generally performed by intracellular organelles in eukaryotes. Here, we show that two small cytoplasmic membrane proteins, YciB and DcrB, play a synergistic role in maintaining the integrity of the bacterial cell envelope. Broadly, our study identifies a previously unknown essential pathway which is a potential target for development of novel antibacterial compounds.

Summary

The bacterial cytoplasmic membrane is a principal site of protein translocation, lipid and peptidoglycan biogenesis, signal transduction, transporters and energy generating components of the respiratory chain. Although 25–30% of bacterial proteomes consist of membrane proteins, a comprehensive understanding of their influence on fundamental cellular processes is incomplete. Here, we show that YciB and DcrB, two small cytoplasmic membrane proteins of previously unknown functions, play an essential synergistic role in maintaining cell envelope integrity of Escherichia coli. Lack of both YciB and DcrB results in pleiotropic cell defects including increased levels of lipopolysaccharide, membrane vesiculation, dynamic shrinking and extension of the cytoplasmic membrane accompanied by lysis and cell death. The stalling of an abundant outer membrane lipoprotein, Lpp, at the periplasmic face of the inner membrane leads to lethal inner membrane–peptidoglycan linkages. Additionally, the periplasmic chaperone Skp contributes to yciB dcrB mutant cell death by possibly mistargeting stalled porins into the inner membrane. Consistent with the idea of a compromised envelope in the yciB dcrB mutant, multiple envelope stress response systems are induced, with Cpx signal transduction being required for growth. Taken together, our results suggest a fundamental role for YciB and DcrB in cell envelope biogenesis.

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Ubiquitination mediates Kv1.3 endocytosis as a mechanism for protein kinase C-dependent modulation.

Sci Rep. 2017 02 10;7:42395

Authors: Martínez-Mármol R, Styrczewska K, Pérez-Verdaguer M, Vallejo-Gracia A, Comes N, Sorkin A, Felipe A

Abstract
The voltage-dependent potassium channel Kv1.3 plays essential physiological functions in the immune system. Kv1.3, regulating the membrane potential, facilitates downstream Ca2+ -dependent pathways and becomes concentrated in specific membrane microdomains that serve as signaling platforms. Increased and/or delocalized expression of the channel is observed at the onset of several autoimmune diseases. In this work, we show that adenosine (ADO), which is a potent endogenous modulator, stimulates PKC, thereby causing immunosuppression. PKC activation triggers down-regulation of Kv1.3 by inducing a clathrin-mediated endocytic event that targets the channel to lysosomal-degradative compartments. Therefore, the abundance of Kv1.3 at the cell surface decreases, which is clearly compatible with an effective anti-inflammatory response. This mechanism requires ubiquitination of Kv1.3, catalyzed by the E3 ubiquitin-ligase Nedd4-2. Postsynaptic density protein 95 (PSD-95), a member of the MAGUK family, recruits Kv1.3 into lipid-raft microdomains and protects the channel against ubiquitination and endocytosis. Therefore, the Kv1.3/PSD-95 association fine-tunes the anti-inflammatory response in leukocytes. Because Kv1.3 is a promising multi-therapeutic target against human pathologies, our results have physiological relevance. In addition, this work elucidates the ADO-dependent PKC-mediated molecular mechanism that triggers immunomodulation by targeting Kv1.3 in leukocytes.

PMID: 28186199 [PubMed - indexed for MEDLINE]

Lipids as activators of innate immunity in peptide vaccine delivery.

Curr Med Chem. 2018 Oct 25;:

Authors: Bartlett S, Skwarczynski M, Toth I

Abstract
BACKGROUND: Innate immune system plays an important role in pathogen detection and the recognition of vaccines, mainly through pattern recognition receptors (PRRs) that identify pathogen components (danger signals). One of the typically recognised bacterial components are lipids in conjugation with peptides, proteins and saccharides. Lipidic compounds are readily recognised by the immune system, and thus are ideal candidates for peptide-based vaccine delivery. Thus, bacterial or synthetic lipids mixed with, or conjugated to, antigens have shown adjuvanting properties. These systems have many advantages over traditional adjuvants, including low toxicity and good efficacy for stimulating mucosal and systemic immune responses.
METHODS: The most recent literature on the role of lipids in stimulation of immune responses was selected for this review. The vast majority of reviewed papers were published in the last decade. Older but significant findings are also cited.
RESULTS: This review focuses on development of lipopeptide vaccine systems including application of palmitic acid, bacterial lipopeptides, glycolipids and the lipid core peptide and their routes of administration. The use of liposomes as a delivery system that incorporates lipopeptides is discussed. The review also includes a brief description of immune system in relation to vaccinology and discussion on vaccine delivery routes.
CONCLUSION: Lipids and their conjugates are an ideal frontrunner in the development of safe and efficient vaccines for different immunisation routes.

PMID: 30362416 [PubMed - as supplied by publisher]

Rhodamine‐labelled Ub (RhoUb) was delivered into live cells through the preparation of a RhoUb conjugate containing the disulfide‐linked dimeric C‐TAT peptide at its C terminus. This reagent enters cells by endocytosis and escapes from the endosomes into the cytoplasm, where it encounters DUB‐mediated cleavage. The released RhoUb is then incorporated into various Ub conjugates through the endogenous Ub machinery.

Abstract

Proteins and other macromolecules can be delivered into live cells by noninvasive techniques using cell‐penetrating peptides. These peptides are easily synthesised by solid‐phase peptide synthesis and can be conjugated onto cargo molecules to mediate cellular delivery. We designed a TAT‐based cell‐penetrating ubiquitin (Ub) reagent by conjugating a dimeric disulfide‐linked TAT peptide to the C terminus of a rhodamine‐labelled Ub (RhoUb) protein. This reagent efficiently enters the cell by endocytosis and escapes from endosomes into the cytoplasm. Once the conjugate is inside the cytoplasm, the delivery vehicle is proteolytically removed by endogenous deubiquitinases (DUBs), at which point the intrinsic ubiquitination machinery is able to incorporate the RhoUb into ubiquitin conjugates. Our approach enables the controlled delivery of labelled or mutant Ub derivatives into cells, increasing our options for studying the ubiquitin system.

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Quercetin impairs Streptococcus pneumoniae biofilm formation by inhibiting sortase A activity.

J Cell Mol Med. 2018 Oct 18;:

Authors: Wang J, Song M, Pan J, Shen X, Liu W, Zhang X, Li H, Deng X

Abstract
Biofilm formation mediated by sortase A (srtA) is important for bacterial colonisation and resistance to antibiotics. Thus, the inhibitor of SrtA may represent a promising agent for bacterial infection. The structure of Streptococcus pneumoniae D39 srtA has been characterised by crystallisation. Site-directed mutagenesis was used for the determination of the key residues for the activity of S. pneumoniae D39 srtA. An effective srtA inhibitor, quercetin, and its mechanism was further identified using srtA activity inhibition assay and molecular modelling. In this study, the crystal structure of S. pneumoniae D39 srtA has been solved and shown to contain a unique domain B. Additionally, its transpeptidase activity was evaluated in vitro. Based on the structure, we identified Cys207 as the catalytic residue, with His141 and Arg215 serving as binding sites for the peptide substrate. We found that quercetin can specifically compete with the natural substrate, leading to a significant decrease in the catalytic activity of this enzyme. In cells co-cultured with this small molecule inhibitor, NanA cannot anchor to the cell wall effectively, and biofilm formation and biomass decrease significantly. Interestingly, when we supplemented cultures with sialic acid, a crucial signal for pneumococcal coloniation and the invasion of the host in the co-culture system, biofilm loss did not occur. This result indicates that quercetin inhibits biofilm formation by affecting sialic acid production. In conclusion, the inhibition of pneumococcal srtA by the small molecule quercetin offers a novel strategy for pneumococcal preventative therapy.

PMID: 30334338 [PubMed - as supplied by publisher]

Related Articles

A central role for PBP2 in the activation of peptidoglycan polymerization by the bacterial cell elongation machinery.

PLoS Genet. 2018 Oct 18;14(10):e1007726

Authors: Rohs PDA, Buss J, Sim SI, Squyres GR, Srisuknimit V, Smith M, Cho H, Sjodt M, Kruse AC, Garner EC, Walker S, Kahne DE, Bernhardt TG

Abstract
Cell elongation in rod-shaped bacteria is mediated by the Rod system, a conserved morphogenic complex that spatially controls cell wall assembly by the glycan polymerase RodA and crosslinking enzyme PBP2. Using Escherichia coli as a model system, we identified a PBP2 variant that promotes Rod system function when essential accessory components of the machinery are inactivated. This PBP2 variant hyperactivates cell wall synthesis in vivo and stimulates the activity of RodA-PBP2 complexes in vitro. Cells with the activated synthase also exhibited enhanced polymerization of the actin-like MreB component of the Rod system. Our results define an activation pathway governing Rod system function in which PBP2 conformation plays a central role in stimulating both glycan polymerization by its partner RodA and the formation of cytoskeletal filaments of MreB to orient cell wall assembly. In light of these results, previously isolated mutations that activate cytokinesis suggest that an analogous pathway may also control cell wall synthesis by the division machinery.

PMID: 30335755 [PubMed - as supplied by publisher]

Traditional teaching in immunology tells us that antigen-specific recall responses are the realm of adaptive immunity. However, over the past decade, there has been increasing evidence and interest in the concept of innate immune memory. Natural killer (NK) cells are the founding member of the innate lymphocyte family, initially recognized...

Angewandte Chemie International Edition, EarlyView.

Silencing Staphylococcus aureus with probiotics

Silencing <i>Staphylococcus aureus</i> with probiotics, Published online: 16 October 2018; doi:10.1038/s41579-018-0111-3

This study reports that the consumption of probiotic Bacillus bacteria abolishes Staphylococcus aureus gut colonization by interfering with S. aureus quorum-sensing.

Infections with Salmonella bacteria, often caused by eating or handling undercooked meat or eggs, affect about 100 million people a year worldwide. The suffering the infection causes—abdominal cramps, fever and diarrhea—is the result of an extremely precise set of molecular interactions between the bacterium and the infected human's cells. In a new study published in the Journal of Biological Chemistry, researchers at Imperial College London and the Francis Crick Institute report some of the details of how Salmonella shuts down an immune pathway after infection.

The peptidoglycan (PG) layer stabilizes the bacterial cell envelope to maintain the integrity and shape of the cell. Penicillin-binding proteins (PBPs) synthesize essential 4–3 cross-links in PG and are inhibited by β-lactam antibiotics. Some clinical isolates and laboratory strains of Enterococcus faecium and Escherichia coli achieve high-level β-lactam resistance by...

ChemBioChem, Volume 19, Issue 20, Page 2120-2120, October 18, 2018.

The bacteria Pseudomonas aeruginosa can cause serious and difficult to treat infections. The infection process involves the activation of toxic substances from the bacteria by a common protein in our cells. Researchers at Karolinska Institutet in Sweden now show how this happens and that the activation can be stopped with drug-like molecules. The results are presented in Nature Communications.

Scientists at The University of Western Australia, in collaboration with researchers in Canada, have developed a new compound that can combat antibiotic-resistant bacteria.

Detection of high-risk carbapenem-resistant Klebsiella pneumoniae and Enterobacter cloacae isolates using volatile molecular profiles

Detection of high-risk carbapenem-resistant <i>Klebsiella pneumoniae</i> and <i>Enterobacter cloacae</i> isolates using volatile molecular profiles, Published online: 05 September 2018; doi:10.1038/s41598-018-31543-x

Detection of high-risk carbapenem-resistant Klebsiella pneumoniae and Enterobacter cloacae isolates using volatile molecular profiles

Structural basis of cell wall peptidoglycan amidation by the GatD/MurT complex of Staphylococcus aureus

Structural basis of cell wall peptidoglycan amidation by the GatD/MurT complex of <i>Staphylococcus aureus</i>, Published online: 28 August 2018; doi:10.1038/s41598-018-31098-x

Structural basis of cell wall peptidoglycan amidation by the GatD/MurT complex of Staphylococcus aureus

No association between resistance mutations, empiric antibiotic, and mortality in ceftriaxone-resistant Escherichia coli and Klebsiella pneumoniae bacteremia

No association between resistance mutations, empiric antibiotic, and mortality in ceftriaxone-resistant <i>Escherichia coli</i> and <i>Klebsiella pneumoniae</i> bacteremia, Published online: 24 August 2018; doi:10.1038/s41598-018-31081-6

No association between resistance mutations, empiric antibiotic, and mortality in ceftriaxone-resistant Escherichia coli and Klebsiella pneumoniae bacteremia

The outer membrane (OM) of the diderm “gram-negative” class of bacteria is an essential organelle and a robust permeability barrier that prevents many antibiotics from reaching their intracellular targets (1). The OM is a unique asymmetrical lipid bilayer (Fig. 1): The inner leaflet is composed of phospholipids (PLs), and the...