Brianna Dalesandro

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Updates on antibody functions in Mycobacterium tuberculosis infection and their relevance for developing a vaccine against tuberculosis.

Curr Opin Immunol. 2018 08;53:30-37

Authors: Achkar JM, Prados-Rosales R

Abstract
A more effective vaccine to control tuberculosis (TB), a major global public health problem, is urgently needed. Current vaccine candidates focus predominantly on eliciting cell-mediated immunity but other arms of the immune system also contribute to protection against TB. We review here recent studies that enhance our current knowledge of antibody-mediated functions against Mycobacterium tuberculosis. These findings, which contribute to the increasing evidence that antibodies have a protective role against TB, include demonstrations that firstly distinct human antibody Fc glycosylation patterns, found in latent M. tuberculosis infection but not in active TB, influence the efficacy of the host to control M. tuberculosis infection, secondly antibody isotype influences human antibody functions, and thirdly that antibodies targeting M. tuberculosis surface antigens are protective. We discuss these findings in the context of TB vaccine development and highlight the need for further research on antibody-mediated immunity in M. tuberculosis infection.

PMID: 29656063 [PubMed - indexed for MEDLINE]

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Large-scale chemical-genetics yields new M. tuberculosis inhibitor classes.

Nature. 2019 Jun 19;:

Authors: Johnson EO, LaVerriere E, Office E, Stanley M, Meyer E, Kawate T, Gomez JE, Audette RE, Bandyopadhyay N, Betancourt N, Delano K, Da Silva I, Davis J, Gallo C, Gardner M, Golas AJ, Guinn KM, Kennedy S, Korn R, McConnell JA, Moss CE, Murphy KC, Nietupski RM, Papavinasasundaram KG, Pinkham JT, Pino PA, Proulx MK, Ruecker N, Song N, Thompson M, Trujillo C, Wakabayashi S, Wallach JB, Watson C, Ioerger TR, Lander ES, Hubbard BK, Serrano-Wu MH, Ehrt S, Fitzgerald M, Rubin EJ, Sassetti CM, Schnappinger D, Hung DT

Abstract
New antibiotics are needed to combat rising levels of resistance, with new Mycobacterium tuberculosis (Mtb) drugs having the highest priority. However, conventional whole-cell and biochemical antibiotic screens have failed. Here we develop a strategy termed PROSPECT (primary screening of strains to prioritize expanded chemistry and targets), in which we screen compounds against pools of strains depleted of essential bacterial targets. We engineered strains that target 474 essential Mtb genes and screened pools of 100-150 strains against activity-enriched and unbiased compound libraries, probing more than 8.5 million chemical-genetic interactions. Primary screens identified over tenfold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insights. We identified over 40 compounds that target DNA gyrase, the cell wall, tryptophan, folate biosynthesis and RNA polymerase, as well as inhibitors that target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating the ability of PROSPECT to yield inhibitors against targets that would have eluded conventional drug discovery.

PMID: 31217586 [PubMed - as supplied by publisher]

Nature Chemical Biology, Published online: 17 June 2019; doi:10.1038/s41589-019-0312-8

The limited availability of small-molecule ligands for E3 ubiquitin ligases stymies the development of next-generation degraders. Two recent papers report the identification of novel, covalent and PROTAC-compatible ligands that hijack the previously untargeted ligases RNF114 and DCAF16.

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PhiA, a peptidoglycan hydrolase inhibitor of Brucella involved in the virulence process.

Infect Immun. 2019 Jun 10;:

Authors: Del Giudice MG, Romani AM, Ugalde JE, Czibener C

Abstract
The peptidoglycan in gram-negative bacteria is a dynamic structure in constant remodeling. This dynamism, achieved through synthesis and degradation, is essential because the peptidoglycan is necessary to maintain the structure of the cell but has to have enough plasticity to allow the transport and assembly of macromolecular complexes in the periplasm and outer membrane. Additionally, this remodeling has to be coordinated with the division process. Among the multiple mechanisms bacteria have to degrade the peptidoglycan are the lytic transglycosidases, enzymes of the lysozyme family that cleave the glycan chains generating gaps in the mesh structure increasing its permeability. Because these enzymes can act as autolysins their activity has to be tightly regulated and one of the mechanisms bacteria have evolved is the synthesis of membrane bound or periplasmic inhibitors. In the present manuscript we identify a periplasmic lytic transglycosidase inhibitor (PhiA) in Brucella abortus and demonstrate that it inhibits the activity of SagA, a lytic transglycosidase we have previously shown is involved in the assembly of the type IV secretion system. A deletion mutant in phiA results in a strain with the incapacity to synthesize a complete LPS but has a faster replication rate compared to the wild type parental strain suggesting a link between peptidoglycan remodeling and speed of multiplication.

PMID: 31182616 [PubMed - as supplied by publisher]

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Proteomics of Bordetella pertussis whole-cell and acellular vaccines.

BMC Res Notes. 2019 Jun 10;12(1):329

Authors: Möller J, Kraner ME, Burkovski A

Abstract
OBJECTIVES: Bordetella pertussis is the etiological agent of whooping cough, a bacterial infection of especially children, which may be fatal without treatment. In frame of studies to investigate putative effects of vaccination on host-pathogen interaction and clonal distribution of strains, in addition to Corynebacterium diphtheriae and Clostridium tetani toxoid vaccines, also whole-cell and acellular pertussis vaccines were analyzed by mass spectrometry.
DATA DESCRIPTION: LC-MS/MS spectra were generated and analyzed using B. pertussis genome data and proteins present in whole-cell and acellular pertussis vaccines were identified. Subcellular localization of proteins and presence of signal peptides was determined bioinformatically.

PMID: 31182148 [PubMed - in process]

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TRIM21 Mitigates Human Lung Microvascular Endothelial Cells' Inflammatory Responses to LPS.

Am J Respir Cell Mol Biol. 2019 12;61(6):776-785

Authors: Li L, Wei J, Mallampalli RK, Zhao Y, Zhao J

Abstract
Endothelial cell (EC) inflammation is regarded as an important pathogenic feature of many inflammatory diseases, including acute lung injury and sepsis. An increase in EC inflammation results in neutrophil infiltration from the blood to the site of inflammation, further promoting EC permeability. The ubiquitin E3 ligase TRIM21 has been implicated in human disorders; however, the roles of TRIM21 in endothelial dysfunction and acute lung injury have not been reported. Here, we reveal an antiinflammatory property of TRIM21 in a mouse model of acute lung injury and human lung microvascular ECs. Overexpression of TRIM21 by lentiviral vector infection effectively dampened LPS-induced neutrophil infiltration, cytokine release, and edema in mice. TRIM21 inhibited human lung microvascular endothelial cell inflammatory responses as evidenced by attenuation of the NF-κB pathway, release of IL-8, expression of intercellular adhesion molecules, and adhesion of monocytes to ECs. Furthermore, we demonstrated that TRIM21 was predominantly degraded by an increase in its monoubiquitination and lysosomal degradation after inflammatory stimuli. Thus, inhibition of vascular endothelial inflammation by TRIM21 provides a novel therapeutic target to lessen pulmonary inflammation.

PMID: 31184939 [PubMed - indexed for MEDLINE]

Nature Chemistry, Published online: 10 June 2019; doi:10.1038/s41557-019-0278-x

Modulating particular ubiquitin chains using binding molecules is challenging given the diversity of chain lengths and linkages found in vivo. Now, tight binding modulators that are specific to K48-linked ubiquitin chains have been found by combining protein synthesis and screening of macrocyclic peptide ligands.

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Drug resistance in Mycobacterium tuberculosis and targeting the l,d-transpeptidase enzyme.

Drug Dev Res. 2019 02;80(1):11-18

Authors: Gokulan K, Varughese KI

Abstract
Tuberculosis (TB) is a disease that has afflicted mankind for thousands of years, but in the last seven decades, much progress has been made in anti-TB therapy. Early drugs, such as para-aminosalicylic acid, streptomycin, isoniazid, and rifamycins were very effective in combatting the disease, giving rise to the hope that TB would be eradicated from the face of the earth by 2010. Despite that optimism, TB continues to kill more than a million people annually worldwide. A major reason for our inability to contain TB is the emergence drug resistance in Mycobacterium tuberculosis. This commentary is based on our recent publication on the structure of l,d-transpeptidase enzyme, relevant to drug resistance. As a background, we briefly outline the history and development of anti-TB therapy. Based on the crystal structure, we suggest a potential direction for designing more potent drugs against TB.

PMID: 30312987 [PubMed - indexed for MEDLINE]

Dap on target: By introducing the non‐proteinogenic amino acid 2,3‐diaminopropionic acid (Dap), a pH‐responsive anticancer peptide has been developed. The Dap peptide exerts over tenfold increased toxicity at pH 6.0 specific to cancer tissues compared with that at pH 7.4. This strategy will lead to a new mechanism of cancer tissue targeting to enhance cancer selectivity.

Abstract

Endowment of pH responsivity to anticancer peptides is a promising approach to achieve better selectivity to cancer tissues. In this research, a template peptide was designed based on magainin 2, an antimicrobial peptide with anticancer activity, and a series of peptides were designed by replacing different numbers of lysine with the unnatural amino acid, 2,3diaminopropionic acid (Dap), which has a positive charge at weakly acidic pH in cancer tissues, but is neutral at physiological pH 7.4. These Dap‐containing peptides are expected to interact more strongly with tumor cells than with normal cells because 1) weakly acidic conditions form in tumors, and 2) the membrane of tumor cells is more anionic than that of normal cells. Although all examined peptides showed potent cytotoxicities to multidrug‐resistant cancer cells at a weakly acidic pH (ED₅₀≈5 μm), the toxicity decreased with an increase in the number of Dap at pH 7.4 (8 Dap residues resulted in ED₅₀≈60 μm). Furthermore, the introduction of Dap reduced cytotoxicity against normal cells. Thus, Dap led to significantly improved cancer targeting due to a pH‐dependent charge shift. Fluorescence imaging and model membrane experiments supported this charge‐shift model.

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Chemical Inactivation of the E3 Ubiquitin Ligase Cereblon by Pomalidomide-based Homo-PROTACs.

J Vis Exp. 2019 May 15;(147):

Authors: Lindner S, Steinebach C, Kehm H, Mangold M, Gütschow M, Krönke J

Abstract
The immunomodulatory drugs (IMiDs) thalidomide and its analogs, lenalidomide and pomalidomide, all FDA approved drugs for the treatment of multiple myeloma, induce ubiquitination and degradation of the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) via the cereblon (CRBN) E3 ubiquitin ligase for proteasomal degradation. IMiDs have recently been utilized for the generation of bifunctional proteolysis targeting chimeras (PROTACs) to target other proteins for ubiquitination and proteasomal degradation by the CRBN E3 ligase. We designed and synthesized pomalidomide-based homobifunctional PROTACs and analyzed their ability to induce self-directed ubiquitination and degradation of CRBN. Here, CRBN serves as both, the E3 ubiquitin ligase and the target at the same time. The homo-PROTAC compound 8 degrades CRBN with a high potency with only minimal remaining effects on IKZF1 and IKZF3. CRBN inactivation by compound 8 had no effect on cell viability and proliferation of different multiple myeloma cell lines. This homo-PROTAC abrogates the effects of IMiDs in multiple myeloma cells. Therefore, our homodimeric pomalidomide-based compounds may help to identify CRBN's endogenous substrates and physiological functions and investigate the molecular mechanism of IMiDs.

PMID: 31157769 [PubMed - in process]

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LPS-neutralizing peptides reduce outer membrane vesicle-induced inflammatory responses.

Biochim Biophys Acta Mol Cell Biol Lipids. 2019 Jun 01;:

Authors: Pfalzgraff A, Correa W, Heinbockel L, Schromm AB, Lübow C, Gisch N, Martinez-de-Tejada G, Brandenburg K, Weindl G

Abstract
Outer membrane vesicles (OMVs) are secreted by Gram-negative bacteria and induce a stronger inflammatory response than pure LPS. After endocytosis of OMVs by macrophages, lipopolysaccharide (LPS) is released from early endosomes to activate its intracellular receptors followed by non-canonical inflammasome activation and pyroptosis, which are critically involved in sepsis development. Previously, we could show that the synthetic anti-endotoxin peptide Pep19-2.5 neutralizes inflammatory responses induced by intracellular LPS. Here, we aimed to investigate whether Pep19-2.5 is able to suppress cytoplasmic LPS-induced inflammation under more physiological conditions by using OMVs which naturally transfer LPS to the cytosol. Isothermal titration calorimetry revealed an exothermic reaction between Pep19-2.5 and Escherichia coli OMVs and the Limulus Amebocyte Lysate assay indicated a strong endotoxin blocking activity. In THP-1 macrophages and primary human macrophages Pep19-2.5 and polymyxin B reduced interleukin (IL)-1β and tumor necrosis factor (TNF) release as well as pyroptosis induced by OMVs, while the Toll-like receptor 4 signaling inhibitor TAK-242 suppressed OMV-induced TNF and IL-1β secretion, but not pyroptosis. Internalization of Pep19-2.5 was at least partially mediated by the P2X7 receptor in macrophages but not in monocytes. Additionally, a cell-dependent difference in the neutralization efficiency of Pep19-2.5 became evident in macrophages and monocytes, indicating a critical role for peptide-mediated IL-1β secretion via the P2X7 receptor. In conclusion, we provide evidence that LPS-neutralizing peptides inhibit OMV-induced activation of the inflammasome/IL-1 axis and give new insights into the mechanism of peptide-mediated neutralization of cytoplasmic LPS suggesting an essential and cell-type specific role for the P2X7 receptor.

PMID: 31163264 [PubMed - as supplied by publisher]

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Resident bacteria in breast cancer tissue: pathogenic agents or harmless commensals?

Discov Med. 2018 09;26(142):93-102

Authors: O'Connor H, MacSharry J, Bueso YF, Lindsay S, Kavanagh EL, Tangney M, Clyne M, Saldova R, McCann A

Abstract
Breast cancer is the second most common cancer in women. Recent evidence identifies a unique microbiome in breast tissue; a site previously thought to be sterile. The identification that this microbiome varies considerably from healthy subjects to cancer patients has prompted investigations into the role of specific bacterial species in oncogenesis. Indeed, certain bacteria have been shown to aid cancer development in vitro by promoting genomic instability, invasion, and chemotherapy resistance. However, the in vivo role of the breast microbiome in cancer appears to be more complex, involving numerous interactions between its constituent species and host cells. As such, reduced abundances of species which exert a protective effect against oncogenesis have come into focus and there is an emerging consensus that states of microbial dysbiosis, in which the normal balance of bacterial species is altered, can contribute to the development of cancer. This review summarizes the findings to date from the available literature pertaining to the microbiome in breast cancer and outlines areas worthy of further investigation.

PMID: 30399327 [PubMed - indexed for MEDLINE]

Ubiquitination regulates many essential cellular processes in eukaryotes. This post-translational modification (PTM) is typically achieved by E1, E2, and E3 enzymes that sequentially catalyze activation, conjugation, and ligation reactions, respectively, leading to covalent attachment of ubiquitin, usually to lysine residues of substrate proteins. Ubiquitin can also be successively linked to one of the seven lysine residues on ubiquitin to form distinctive forms of polyubiquitin chains, which, depending upon the lysine used and the length of the chains, dictate the fate of substrate proteins. Recent discoveries revealed that this ubiquitin code is further expanded by PTMs such as phosphorylation, acetylation, deamidation, and ADP-ribosylation, on ubiquitin, components of the ubiquitination machinery, or both. These PTMs provide additional regulatory nodes to integrate development or insulting signals with cellular homeostasis. Understanding the precise roles of these PTMs in the regulation of ubiquitin signaling will provide new insights into the mechanisms and treatment of various human diseases linked to ubiquitination, including neurodegenerative diseases, cancer, infection, and immune disorders.

Nature, Published online: 27 May 2019; doi:10.1038/d41586-019-01659-9

Two newfound genes help to shield bacteria from tigecycline, which doctors use to treat drug-resistant infections.

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Inflammatory production of reactive oxygen species by Drosophila hemocytes activates cellular immune defenses.

Biochem Biophys Res Commun. 2018 11 02;505(3):726-732

Authors: Myers AL, Harris CM, Choe KM, Brennan CA

Abstract
The production of reactive oxygen species (ROS) is a prominent response to infection among innate immune cells such as macrophages and neutrophils. To better understand the relationship between antimicrobial and regulatory functions of blood cell ROS, we have characterized the ROS response to infection in Drosophila hemocytes. Using fluorescent probes, we find a biphasic hemocyte ROS response to bacterial infection. In the first hour, virtually all hemocytes generate a transient ROS signal, with nonphagocytic cells including prohemocytes and crystal cells displaying exceptionally strong responses. A distinct, and more delayed ROS response starting at 90 min is primarily within cells that have engulfed bacteria, and is sustained for several hours. The early response has a clear regulatory function, as dampening or intensifying the intracellular ROS level has profound effects on plasmatocyte activation. In addition, ROS are necessary and sufficient to activate JNK signalling in crystal cells, and to promote JNK-dependent crystal cell rupture. These findings indicate that Drosophila will be a promising model in which to dissect the mechanisms of ROS stimulation of immune activation.

PMID: 30292413 [PubMed - indexed for MEDLINE]

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Peptidoglycan editing by a specific LD-transpeptidase controls the muramidase-dependent secretion of typhoid toxin.

Nat Microbiol. 2018 11;3(11):1243-1254

Authors: Geiger T, Pazos M, Lara-Tejero M, Vollmer W, Galán JE

Abstract
Protein secretion mechanisms are essential for the virulence of most bacterial pathogens. Typhoid toxin is an essential virulence factor for Salmonella Typhi, the cause of typhoid fever in humans. This toxin is unique in that it is only produced within mammalian cells, and it must be trafficked to the extracellular space before intoxicating target cells. An essential and poorly understood aspect of this transport pathway is the secretion of typhoid toxin from the bacterium into the S. Typhi-containing vacuole. We show here that typhoid toxin secretion requires its translocation to the trans side of the peptidoglycan layer at the bacterial poles for subsequent release through the outer membrane. This translocation process depends on a specialized muramidase, the activity of which requires the localized editing of peptidoglycan by a specific ld-transpeptidase. These studies describe a protein export mechanism that is probably conserved in other bacterial species.

PMID: 30250245 [PubMed - indexed for MEDLINE]

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Enterococcus faecalis escapes complement-mediated killing via recruitment of complement factor H.

J Infect Dis. 2019 May 06;:

Authors: Ali YM, Sim RB, Schwaeble W, Sshabaan MA

Abstract
Enterococcus faecalis (E. faeclais) is considered the most important species of enterococci responsible for blood stream infections in critically ill patients. In blood, the Complement System is activated via the classical (CP), the lectin (LP) or the alternative (AP) pathways and plays a critical role in opsonophagocytosis of bacteria including E. faecalis. Our results show a strong C3b deposition on E. faecalis via both the CP and the LP but not through the AP. We demonstrate that complement recognition molecules C1q, CL-11 and murine ficolin-A bind the enterococcus and drive the CP and the LP in human and mouse. We further describe that E. faecalis evades the AP by recruitment of FH on its surface. In a mouse model of enterococcus peritonitis, BALB-C mice were challenged with a high dose of E. faecalis 12 h after i.p administration of anti-FH antibodies or isotype control. Four hours later, control mice developed higher bacterial burden in blood and organs compared to mice treated with anti-FH antibodies. These findings indicate that E.faecalis avoids the complement phagocytosis by the AP via sequestering complement FH from the host blood.

PMID: 31058287 [PubMed - as supplied by publisher]

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Tryptophan fluorescence quenching in β-lactam-interacting proteins is modulated by the structure of intermediates and final products of the acylation reaction.

ACS Infect Dis. 2019 May 06;:

Authors: Triboulet S, Edoo Z, Compain F, Ourghanlian C, Dupuis A, Dubée V, Sütterlin L, Atze H, Etheve-Quelquejeu M, Hugonnet JE, Arthur M

Abstract
In most bacteria, β-lactam antibiotics inhibit the last cross-linking step of peptidoglycan synthesis by acylation of the active-site Ser of D,D-transpeptidases belonging to the penicillin-binding protein (PBP) family. In mycobacteria, cross-linking is mainly ensured by L,D-transpeptidases (LDTs), which are promising targets for the development of β-lactam-based therapies for multidrug-resistant tuberculosis. For this purpose, fluorescence spectroscopy is used to investigate the efficacy of LDT inactivation by β-lactams but the basis for fluorescence quenching during enzyme acylation remains unknown. In contrast to what has been reported for PBPs, we show here using a model L,D-transpeptidase (Ldtfm) that fluorescence quenching of Trp residues does not depend upon direct hydrophobic interaction between Trp residues and β-lactams. Rather, Trp fluorescence was quenched by the drug covalently bound to the active-site Cys residue of Ldtfm. Fluorescence quenching was not quantitatively determined by the size of the drug and was not specific of the thioester link connecting the β-lactam carbonyl to the catalytic Cys as quenching was also observed for acylation of the active-site Ser of β-lactamase BlaC from M. tuberculosis. Fluorescence quenching was extensive for reaction intermediates containing an amine anion and for acylenzymes containing an imine stabilized by mesomeric effect, but not for acylenzymes containing a protonated β-lactam nitrogen. Together, these results indicate that the extent of fluorescence quenching is determined by the status of the β-lactam nitrogen. Thus, fluorescence kinetics can provide information not only on the efficacy of enzyme inactivation but also on the structure of the covalent adducts responsible for enzyme inactivation.

PMID: 31056908 [PubMed - as supplied by publisher]

Novel recognition sequences of a microbial transglutaminase derived from natural substrates were evaluated for fast, efficient, and site‐specific production of antibody–drug conjugates. An optimized sequence displayed superior conjugation efficiency in the context of an antibody fusion compared to previously used recognition tags.

Abstract

Microbial transglutaminase (mTG) has recently emerged as a powerful tool for antibody engineering. In nature, it catalyzes the formation of amide bonds between glutamine side chains and primary amines. Being applied to numerous research fields from material sciences to medicine, mTG enables efficient site‐specific conjugation of molecular architectures that possess suitable recognition motifs. In monoclonal antibodies, the lack of native transamidation sites is bypassed by incorporating specific peptide recognition sequences. Herein, we report a rapid and efficient mTG‐catalyzed bioconjugation that relies on a novel recognition motif derived from its native substrate Streptomyces papain inhibitor (SPI_P). Improved reaction kinetics compared to commonly applied sequences were demonstrated for model peptides and for biotinylation of Her2‐targeting antibody trastuzumab variants. Moreover, an antibody–drug conjugate assembled from trastuzumab that was C‐terminally tagged with the novel recognition sequence revealed a higher payload‐antibody ratio than the reference antibody.

Immunotherapeutic potential of an N-formylated peptide of Listeria monocytogenes in experimental tuberculosis.

Immunopharmacol Immunotoxicol. 2019 May 03;:1-7

Authors: Mir SA, Sharma S

Abstract
OBJECTIVE: The current therapeutic regimens for tuberculosis (TB) are complex and involve the prolonged use of multiple antibiotics with diverse side effects that lead to therapeutic failure and bacterial resistance. The standard appliance of immunotherapy may aid as a powerful tool to combat the ensuing threat of TB. We have earlier reported the immunotherapeutic potential of N-formylated peptides of two secretory proteins of Mycobacterium tuberculosis H37Rv. Here, we investigated the immunotherapeutic effect of an N-formylated peptide from Listeria monocytogenes in experimental TB.
METHODS: The N-terminally formylated listerial peptide with amino acid sequence 'f-MIGWII' was tested for its adjunctive therapeutic efficacy in combination with anti-tuberculosis drugs (ATDs) in the mouse model of TB. In addition, its potential to generate reactive oxygen species (ROS) in murine neutrophils was also evaluated.
RESULTS: The LemA peptide (f-MIGWII) induced a significant increase in the intracellular ROS levels of mouse neutrophils (p ≤ .05). The ATD treatment reduced the colony forming units (CFU) in lungs and spleen of infected mice by 2.39 and 1.67 log10 units, respectively (p < .001). Treatment of the infected mice with combination of ATDs and LemA peptide elicited higher therapeutic efficacy over ATDs alone. The histopathological changes in the lungs of infected mice also correlated well with the CFU data.
CONCLUSIONS: Our results clearly indicate that LemA peptide conferred an additional therapeutic effect when given in combination with the ATDss (p < .01) and hence can be used as adjunct to the conventional chemotherapy against TB.

PMID: 31046503 [PubMed - as supplied by publisher]

Usnic acid modifies modifies MRSA drug resistance through down-regulation of proteins involved in peptidoglycan and fatty acid biosynthesis.

FEBS Open Bio. 2019 May 03;:

Authors: Sinha S, Gupta VK, Kumar P, Kumar R, Joshi R, Pal A, Darokar MP

Abstract
Multidrug-resistant Staphylococcus aureus infections place a huge burden on the healthcare sector and the wider community. An increasing rate of infections caused by MRSA has necessitated the development of alternative agents. We previously reported that usnic acid (UA) has activity against MRSA; here, we report the effect of UA in combination with norfloxacin on the drug resistance of MRSA clinical isolates. We observed that the combination of UA-norfloxacin significantly reduces bacterial burden in mouse models infected with S. aureus, without causing any detectable associated toxicity. Proteomic analysis indicated that UA-norfloxacin induces oxidative stress within cells, which leads to membrane damage and inhibits metabolic activity and biosynthesis of peptidoglycan and fatty acids. Collectively, this study provides evidence that UA in combination with norfloxacin may be a potential candidate for development into a resistance-modifying agent for the treatment of invasive MRSA infections.

PMID: 31050202 [PubMed - as supplied by publisher]

Bright shot! Optical control of enzymatic activity by using visible‐light photoswitchable protein–protein modulators. The synthesis and characterization of two visible‐light‐responsive peptide backbone photoswitches, based on azobenzene derivatives, are reported.

Abstract

Life relies on a myriad of carefully orchestrated processes, in which proteins and their direct interplay ultimately determine cellular function and disease. Modulation of this complex crosstalk has recently attracted attention, even as a novel therapeutic strategy. Herein, we describe the synthesis and characterization of two visible‐light‐responsive peptide backbone photoswitches based on azobenzene derivatives, to exert optical control over protein–protein interactions (PPI). The novel peptidomimetics undergo fast and reversible isomerization with low photochemical fatigue under alternatively blue‐/green‐light irradiation cycles. Both bind in the nanomolar range to the protein of interest. Importantly, the best peptidomimetic displays a clear difference between isomers in its protein‐binding capacity and, in turn, in its potential to inhibit enzymatic activity through PPI disruption. In addition, crystal structure determination, docking and molecular dynamics calculations allow a molecular interpretation and open up new avenues in the design and synthesis of future photoswitchable PPI modulators.

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A Mycobacterium tuberculosis surface protein recruits ubiquitin to trigger host xenophagy.

Nat Commun. 2019 Apr 29;10(1):1973

Authors: Chai Q, Wang X, Qiang L, Zhang Y, Ge P, Lu Z, Zhong Y, Li B, Wang J, Zhang L, Zhou D, Li W, Dong W, Pang Y, Gao GF, Liu CH

Abstract
Ubiquitin-mediated xenophagy, a type of selective autophagy, plays crucial roles in host defense against intracellular pathogens including Mycobacterium tuberculosis (Mtb). However, the exact mechanism by which host ubiquitin targets invaded microbes to trigger xenophagy remains obscure. Here we show that ubiquitin could recognize Mtb surface protein Rv1468c, a previously unidentified ubiquitin-binding protein containing a eukaryotic-like ubiquitin-associated (UBA) domain. The UBA-mediated direct binding of ubiquitin to, but not E3 ubiquitin ligases-mediated ubiquitination of, Rv1468c recruits autophagy receptor p62 to deliver mycobacteria into LC3-associated autophagosomes. Disruption of Rv1468c-ubiquitin interaction attenuates xenophagic clearance of Mtb in macrophages, and increases bacterial loads in mice with elevated inflammatory responses. Together, our findings reveal a unique mechanism of host xenophagy triggered by direct binding of ubiquitin to the pathogen surface protein, and indicate a diplomatic strategy adopted by Mtb to benefit its persistent intracellular infection through controlling intracellular bacterial loads and restricting host inflammatory responses.

PMID: 31036822 [PubMed - in process]

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Isolation, Purification and Characterization of L,D-transpeptidase 2 from Mycobacterium tuberculosis.

Acta Naturae. 2019 Jan-Mar;11(1):23-28

Authors: Baldin SM, Shcherbakova TA, Švedas VK

Abstract
L,D-transpeptidase 2 from Mycobacterium tuberculosis plays a key role in the formation of nonclassical 3-3 peptidoglycan cross-links in a pathogen's cell wall making it resistant to a broad range of penicillin antibiotics. The conditions of cultivation, isolation, and purification of recombinant L,D-transpeptidase 2 from M. tuberculosis have been optimized in this study. Oxidation of the free SH groups of catalytic cysteine Cys354 is an important factor causing the inactivation of the enzyme, which occurs during both the expression and storage of enzyme preparations. The biochemical characteristics of purified L,D-transpeptidase 2 and L,D-transpeptidase 2 lacking domain A were determined; the kinetic constants of enzyme-catalyzed nitrocefin transformation were evaluated.

PMID: 31024745 [PubMed]