Brianna Dalesandro

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Functional analysis of Clostridium difficile sortase B reveals key residues for catalytic activity and substrate specificity.

J Biol Chem. 2020 Jan 31;:

Authors: Kang CY, Huang IH, Chou CC, Wu TY, Chang JC, Hsiao YY, Cheng CH, Tsai WJ, Hsu KC, Wang S

Abstract
Most of Gram-positive bacteria anchor surface proteins to the peptidoglycan cell wall by sortase, a cysteine transpeptidase that targets proteins displaying a cell wall sorting signal. Unlike other bacteria, Clostridium difficile, the major human pathogen responsible for antibiotic-associated diarrhea, has only a single functional sortase (SrtB). Sortase's vital importance in bacterial virulence has been long recognized, and C. difficile sortase B (Cd-SrtB) has become an attractive therapeutic target for managing C. difficile infection (CDI). A better understanding of the molecular activity of Cd-SrtB may help spur the development of effective agents against CDI. In this study, using site-directed mutagenesis, biochemical and biophysical tools, LC-MS/MS, and crystallographic analyses, we identified key residues essential for Cd-SrtB catalysis and substrate recognition. To the best of our knowledge, we report first evidence that a conserved serine residue near the active site participates in the catalytic activity of Cd-SrtB and also SrtB from Staphylococcus aureus The serine residue indispensable for SrtB activity may be involved in stabilizing a thioacyl-enzyme intermediate because it is neither a nucleophilic residue nor a substrate-interacting residue, based on the LC-MS/MS data and available structural models of SrtB-substrate complexes. Furthermore, we also demonstrated that residues 163-168 located on the β6/β7 loop of Cd-SrtB dominate specific recognition of the peptide substrate PPKTG. The results of this work reveal key residues with roles in catalysis and substrate specificity of Cd-SrtB.

PMID: 32005667 [PubMed - as supplied by publisher]

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Functional characterization of a broad and potent neutralizing monoclonal antibody directed against outer membrane protein (OMP) of Salmonella typhimurium.

Appl Microbiol Biotechnol. 2020 Jan 29;:

Authors: Reddy PN, Makam SS, Kota RK, Yatung G, Urs RM, Batra H, Tuteja U

Abstract
In the present study, we have generated a murine monoclonal antibody (mAb) named Sal-06 by using the crude outer membrane protein preparation of Salmonella enteric subsp. enterica serovar Typhimurium ATCC 14028 strain as antigen. Sal-06mAb belonging to IgG1 isotype demonstrated broad cross-reactivity to standard and isolated strains of genus Salmonella and others such as Escherichia coli, Klebsiella pneumonia, and Proteus mirabilis. Cross-reactivity across several bacterial genera indicated that the epitopes reactive to Sal-06mAb are conserved among these members. Neutralizing effects of Sal-06mAb on Salmonella growth and survival was evaluated in vitro using bacteriostatic and bactericidal activity with and without complement and bacterial invasion inhibition assay. Sal-06mAb demonstrated a bacteriostatic effect on the growth of S. typhimurium ATCC 14028 strain which is both time and concentration (of mAb) dependent. It was also found that the bacterial growth inhibition was complement independent. When the bacterial cells were preincubated with Sal-06mAb, it reduced the adherence and invasion of bacterial cells into A549 epithelial cell line. This was confirmed by CFU count analysis, phase contrast, and fluorescence microscopy. Scanning electron microscope (SEM) imaging confirmed the antimicrobial effects of Sal-06mAb on S. typhimurium ATCC 14028. The development of broadly reactive and cross protective Sal-06mAb opens new possibilities for immunotherapy of sepsis caused by Gram-negative Enterobacteriaceae members.

PMID: 31997109 [PubMed - as supplied by publisher]

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Nucleotide-induced folding of cell division protein FtsZ from Staphylococcus aureus.

FEBS J. 2020 Jan 29;:

Authors: Huecas S, Canosa-Valls AJ, Araújo-Bazán L, Ruiz FM, Laurents DV, Fernández-Tornero C, Andreu JM

Abstract
The essential bacterial division protein FtsZ uses GTP binding and hydrolysis to assemble into dynamic filaments that treadmill around the Z-ring, guiding septal wall synthesis and cell division. FtsZ is a structural homolog of tubulin and a target for discovering new antibiotics. Here, using FtsZ from the pathogen S aureus (SaFtsZ), we reveal that, prior to assembly, FtsZ monomers require nucleotide binding for folding; this is possibly relevant to other mesophilic FtsZs. Apo-SaFtsZ is essentially unfolded, as assessed by nuclear magnetic resonance and circular dichroism. Binding of GTP (≥ 1mM) dramatically shifts the equilibrium towards the active folded protein. Supportingly, SaFtsZ refolded with GDP crystallizes in a native structure. Apo-SaFtsZ also folds with 3.4 M glycerol, enabling high-affinity GTP binding (KD 20 nM determined by isothermal titration calorimetry) similar to thermophilic stable FtsZ. Other stabilizing agents that enhance nucleotide binding include ethylene glycol, trimethylamine N-oxide (TMAO), and several bacterial osmolytes. High salt stabilizes SaFtsZ without bound nucleotide in an inactive twisted conformation. We identified a cavity behind the SaFtsZ-GDP nucleotide-binding pocket that harbors different small compounds, which is available for extended nucleotide-replacing inhibitors. Furthermore, we devised a competition assay to detect any inhibitors that overlap the nucleotide site of SaFtsZ, or Escherichia coli FtsZ, employing osmolyte-stabilized apo-FtsZs and the specific fluorescence anisotropy change of mant-GTP upon dissociation from the protein. This robust assay provides a basis to screening for high affinity GTP-replacing ligands, which combined with structural studies and phenotypic profiling should facilitate development of a next generation of FtsZ-targeting antibacterial inhibitors.

PMID: 31997533 [PubMed - as supplied by publisher]

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TRIM21-mediated proteasomal degradation of SAMHD1 regulates its antiviral activity.

EMBO Rep. 2020 Jan 07;21(1):e47528

Authors: Li Z, Huan C, Wang H, Liu Y, Liu X, Su X, Yu J, Zhao Z, Yu XF, Zheng B, Zhang W

Abstract
SAMHD1 possesses multiple functions, but whether cellular factors regulate SAMHD1 expression or its function remains not well characterized. Here, by investigating why cultured RD and HEK293T cells show different sensitivity to enterovirus 71 (EV71) infection, we demonstrate that SAMHD1 is a restriction factor for EV71. Importantly, we identify TRIM21, an E3 ubiquitin ligase, as a key regulator of SAMHD1, which specifically interacts and degrades SAMHD1 through the proteasomal pathway. However, TRIM21 has no effect on EV71 replication itself. Moreover, we prove that interferon production stimulated by EV71 infection induces increased TRIM21 and SAMHD1 expression, whereas increasing TRIM21 overrides SAMHD1 inhibition of EV71 in cells and in a neonatal mouse model. TRIM21-mediated degradation of SAMHD1 also affects SAMHD1-dependent restriction of HIV-1 and the regulation of interferon production. We further identify the functional domains in TRIM21 required for SAMHD1 binding and the ubiquitination site K622 in SAMHD1 and show that phosphorylation of SAMHD1 at T592 also blocks EV71 restriction. Our findings illuminate how EV71 overcomes SAMHD1 inhibition via the upregulation of TRIM21.

PMID: 31797533 [PubMed - in process]

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Fluorescence Assessment of the AmpR-Signaling Network of Pseudomonas aeruginosa to Exposure to β-Lactam Antibiotics.

ACS Chem Biol. 2020 Jan 28;:

Authors: Dik DA, Kim C, Madukoma CS, Fisher JF, Shrout JD, Mobashery S

Abstract
Gram-negative bacteria have evolved an elaborate pathway to sense and respond to exposure to β-actam antibiotics. The β-actam antibiotics inhibit penicillin-binding proteins, whereby the loss of their activities alters/damages the cell-wall peptidoglycan. Bacteria sense this damage and remove the affected peptidoglycan into complex recycling pathways. As an offshoot of these pathways, muropeptide chemical signals generated from the cell-wall recycling manifest the production of a class C β-actamase, which hydrolytically degrades the β-lactam antibiotic as a resistance mechanism. We disclose the use of a fluorescence probe that detects the activation of the recycling system by the formation of the key muropeptides involved in signaling. This same probe additionally detects natural-product cell-wall-active antibiotics that are produced in situ by cohabitating bacteria.

PMID: 31990176 [PubMed - as supplied by publisher]

Nature, Published online: 29 January 2020; doi:10.1038/d41586-020-00197-z

Researchers are investigating how the community of microbes living in the gut might help people with multiple sclerosis, lupus and type 1 diabetes.

Nature, Published online: 29 January 2020; doi:10.1038/d41586-020-00199-x

Targeting the microbiome could hold the key to combating a range of malignant diseases.

Nature Chemistry, Published online: 29 January 2020; doi:10.1038/s41557-019-0415-6

Monoclonal antibodies have shown tremendous success in cancer treatment; however, humanization for clinical applications is expensive and not straightforward. Now, molecularly imprinted polymer nanogels have been developed that can block cell-surface proteins and disrupt tumour spheroids.

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Uncovering the activities, biological roles, and regulation of bacterial cell wall hydrolases and tailoring enzymes.

J Biol Chem. 2020 Jan 23;:

Authors: Do T, Page JE, Walker S

Abstract
Bacteria account for a thousand-fold more biomass than humans. They vary widely in shape and size. The morphological diversity of bacteria is due largely to the different peptidoglycan-based cell wall structures that encase bacterial cells. Although the basic structure of peptidoglycan is highly conserved, consisting of long glycan strands that are crosslinked by short peptide chains, the mature cell wall is chemically diverse. Peptidoglycan hydrolases and cell wall tailoring enzymes that regulate glycan strand length, degree of crosslinking, and addition of other modifications to peptidoglycan are central in determining the final architecture of the bacterial cell wall. Historically, it has been difficult to biochemically characterize these enzymes that act on peptidoglycan because suitable peptidoglycan substrates were inaccessible. In this review, we discuss fundamental aspects of bacterial cell wall synthesis, describe the regulation and diverse biochemical and functional activities of peptidoglycan hydrolases, and highlight recently developed methods to make and label defined peptidoglycan substrates. We also review how access to these substrates has now enabled biochemical studies that deepen our understanding of how bacterial cell wall enzymes cooperate to build a mature cell wall. Such improved understanding is critical to the development of new antibiotics that disrupt cell wall biogenesis, a process essential to the survival of bacteria.

PMID: 31974163 [PubMed - as supplied by publisher]

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Development of chimeric peptides to facilitate the neutralisation of lipopolysaccharides during bactericidal targeting of multidrug-resistant Escherichia coli.

Commun Biol. 2020 Jan 23;3(1):41

Authors: Wang Z, Liu X, Da Teng, Mao R, Hao Y, Yang N, Wang X, Li Z, Wang X, Wang J

Abstract
Pathogenic Escherichia coli can cause fatal diarrheal diseases in both animals and humans. However, no antibiotics or antimicrobial peptides (AMPs) can adequately kill resistant bacteria and clear bacterial endotoxin, lipopolysaccharide (LPS) which leads to inflammation and sepsis. Here, the LPS-targeted smart chimeric peptides (SCPs)-A6 and G6 are generated by connecting LPS-targeting peptide-LBP14 and killing domain-N6 via different linkers. Rigid and flexible linkers retain the independent biological activities from each component. SCPs-A6 and G6 exert low toxicity and no bacterial resistance, and they more rapidly kill multiple-drug-resistant E. coli and more effectively neutralize LPS toxicity than N6 alone. The SCPs can enhance mouse survival more effectively than N6 or polymyxin B and alleviate lung injuries by blocking mitogen-activated protein kinase and nuclear factor kappa-B p65 activation. These findings uniquely show that SCPs-A6 and G6 may be promising dual-function candidates as improved antibacterial and anti-endotoxin agents to treat bacterial infection and sepsis.

PMID: 31974490 [PubMed - in process]

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Novel short peptide tag from a bacterial toxin for versatile applications.

J Immunol Methods. 2020 04;479:112750

Authors: Lee TH, Kim KS, Kim JH, Jeong JH, Woo HR, Park SR, Sohn MH, Lee HJ, Rhee JH, Cha SS, Hwang JH, Chung KM

Abstract
The specific recognition between a monoclonal antibody (mAb) and its epitope can be used in a tag system that has proved valuable in a wide range of biological applications. Herein, we describe a novel tag called RA-tag that is composed of a seven amino acid sequence (DIDLSRI) and recognized by a highly specific mAb, 47RA, against the bacterial toxin Vibrio vulnificus RtxA1/MARTXVv. By using recombinant proteins with the RA-tag at the N-terminal, C-terminal, or an internal site, we demonstrated that the tag system could be an excellent biological system for both protein purification and protein detection in enzyme-linked immunosorbent, Western blot, flow cytometry, and immunofluorescence staining analyses in Escherichia coli, mammalian cell lines, yeast, and plant. In addition, our RA-tag/47RA mAb combination showed high sensitivity and reliable affinity (KD = 5.90 × 10-8 M) when compared with conventional tags. Overall, our results suggest that the RA-tag system could facilitate the development of a broadly applicable tag system for biological research.

PMID: 31981564 [PubMed - indexed for MEDLINE]

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Transient Membrane-Linked FtsZ Assemblies Precede Z-Ring Formation in Escherichia coli.

Curr Biol. 2020 Jan 20;:

Authors: Walker BE, Männik J, Männik J

Abstract
During the early stages of cytokinesis, FtsZ protofilaments form a ring-like structure, the Z-ring, in most bacterial species. This cytoskeletal scaffold recruits downstream proteins essential for septal cell wall synthesis. Despite progress in understanding the dynamic nature of the Z-ring and its role in coordinating septal cell wall synthesis, the early stages of protofilament formation and subsequent assembly into the Z-ring are still not understood. Here we investigate a sequence of assembly steps that lead to the formation of the Z-ring in Escherichia coli using high temporal and spatial resolution imaging. Our data show that formation of the Z-ring is preceded by transient membrane-linked FtsZ assemblies. These assemblies form after attachment of short cytosolic protofilaments, which we estimate to be less than 20 monomers long, to the membrane. The attachments occur at random locations along the length of the cell. The filaments treadmill and show periods of rapid growth and shrinkage. Their dynamic properties imply that protofilaments are bundled in these assemblies. Furthermore, we establish that the size of assemblies is sensitively controlled by the availability of FtsZ molecules and by the presence of ZapA proteins. The latter has been implicated in cross-linking the protofilaments. The likely function of these dynamic FtsZ assemblies is to sample the cell surface for the proper location for the Z-ring.

PMID: 31978334 [PubMed - as supplied by publisher]

Release of extracellular vesicles (EVs) is a common feature among eukaryotes, archaea, and bacteria. However, the biogenesis and downstream biological effects of EVs released from gram-positive bacteria remain poorly characterized. Here, we report that EVs purified from a community-associated methicillin-resistant Staphylococcus aureus strain were internalized into human macrophages in vitro...

Surface plasmon resonance microscopy has emerged as a versatile platform for single‐molecule sensing and single‐cell imaging with high spatiotemporal resolution. This Minireview highlights the recent advances in the SPRM‐based analysis of single entities. Future challenges and their limitations as well as potential research directions are discussed.

Abstract

Surface plasmon resonance microscopy (SPRM) is a versatile platform for chemical and biological sensing and imaging. Great progress in exploring its applications, ranging from single‐molecule sensing to single‐cell imaging, has been made. In this Minireview, we introduce the principles and instrumentation of SPRM. We also summarize the broad and exciting applications of SPRM to the analysis of single entities. Finally, we discuss the challenges and limitations associated with SPRM and potential solutions.

Ok, take a bite: Responsive exosome nano‐bioconjugates were constructed by engineering M1 exosomes with aCD47 and aSIRPα linked with a pH‐sensitive bond. After systemic administration, the synergism of specific targeting by aCD47, blocking of “don't eat me” signaling by aCD47 and aSIRPα, and M2 reprogramming by M1 exosomes resulted in a potent anticancer effect.

Abstract

Exosomes hold great potential in therapeutic development. However, native exosomes usually induce insufficient effects in vivo and simply act as drug delivery vehicles. Herein, we synthesize responsive exosome nano‐bioconjugates for cancer therapy. Azide‐modified exosomes derived from M1 macrophages are conjugated with dibenzocyclooctyne‐modified antibodies of CD47 and SIRPα (aCD47 and aSIRPα) through pH‐sensitive linkers. After systemic administration, the nano‐bioconjugates can actively target tumors through the specific recognition between aCD47 and CD47 on the tumor cell surface. In the acidic tumor microenvironment, the benzoic‐imine bonds of the nano‐bioconjugates are cleaved to release aSIRPα and aCD47 that can, respectively, block SIRPα on macrophages and CD47, leading to abolished “don't eat me” signaling and improved phagocytosis of macrophages. Meanwhile, the native M1 exosomes effectively reprogram the macrophages from pro‐tumoral M2 to anti‐tumoral M1.

A covalently linked paclitaxel–methylene blue conjugate (PTX‐MB) is used for real‐time monitoring of drug release from nanocarriers via photoacoustic imaging. The PTX‐MB remained in acoustically silent form when encapsulated in poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles. After release, PTX‐MB instantly oxidized to the photoacoustically active form to report quantity and biodistribution.

Abstract

We report a new approach to monitor drug release from nanocarriers via a paclitaxel–methylene blue conjugate (PTX‐MB) with redox activity. This construct is in a photoacoustically silent reduced state inside poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles (PTX‐MB@PLGA NPs). During release, PTX‐MB is spontaneously oxidized to produce a concentration‐dependent photoacoustic signal. An in vitro drug‐release study showed an initial burst release (25 %) between 0–24 h and a sustained release between 24–120 h with a cumulative release of 40.6 % and a 670‐fold increase in photoacoustic signal. An in vivo murine drug release showed a photoacoustic signal enhancement of up to 649 % after 10 hours. PTX‐MB@PLGA NPs showed an IC₅₀ of 78 μg mL⁻¹ and 44.7±4.8 % decrease of tumor burden in an orthotopic model of colon cancer via luciferase‐positive CT26 cells.

ABSTRACT

Group D and group B Salmonella enterica serovars differ in their susceptibility to colistin with the former frequently intrinsically resistant (MIC > 2 μg/ml); however, the mechanism has not been described. Here, we show that the O-antigen epitope in group D Salmonella governs the levels of colistin susceptibility. Substitution of the rfbJ gene in a group B Salmonella with the rfbSE genes from a group D Salmonella conferred a decrease in susceptibility to colistin. The presence of dideoxyhexose, abequose, and the deoxymannose, tyvelose, differentiate the Salmonella group B and group D O antigens, respectively. We hypothesize that the subtle difference between abequose and tyvelose hinders the colistin molecule from reaching its target. Whole-genome sequencing also revealed that increased colistin susceptibility in a group D Salmonella veterinary isolate was due to a defect in the O-antigen polymerase protein, Rfc. This study shows that two different mechanisms that influence the presence and composition of O antigens affect colistin susceptibility in Salmonella enterica.

IMPORTANCE Some serovars of Salmonella, namely, those belonging to group D, appear to show a degree of intrinsic resistance to colistin. This observed intrinsic colistin resistance is of concern since this last-resort drug might no longer be effective for treating severe human infections with the most common Salmonella serovar, Salmonella enterica serovar Enteritidis. Here, we show that the O-antigen epitope in group D Salmonella governs the levels of colistin susceptibility. Using whole-genome sequencing, we also revealed that increased colistin susceptibility in a group D Salmonella veterinary isolate was due to a defect in the O-antigen polymerase protein, Rfc. In summary, we show that two different mechanisms that influence the presence and composition of O antigens affect colistin susceptibility in Salmonella enterica.

Take your vitamins: The alpha isoform of folate receptor (FR‐α) is overexpressed in ovarian and endometrial cancer cells. Existing NIR fluorescent probes targeting FR‐α show high non‐specific tissue adsorption. An NIR fluorescent probe, FolateSiR‐1 was designed and synthesized. This probe exhibits very low background fluorescence and afforded a tumor‐to‐background ratio of up to 83 in FR‐expressing tumor‐bearing mice within 30 min after the injection.

Abstract

Folate receptors (FRs) are membrane proteins involved in folic acid uptake, and the alpha isoform (FR‐α) is overexpressed in ovarian and endometrial cancer cells. For fluorescence imaging of FRs in vivo, the near‐infrared (NIR) region (650–900 nm), in which tissue penetration is high and autofluorescence is low, is optimal, but existing NIR fluorescent probes targeting FR‐α show high non‐specific tissue adsorption, and require prolonged washout to visualize tumors. We have designed and synthesized a new NIR fluorescent probe, FolateSiR‐1, utilizing a Si‐rhodamine fluorophore having a carboxy group at the benzene moiety, coupled to a folate ligand moiety through a negatively charged tripeptide linker. This probe exhibits very low background fluorescence and afforded a tumor‐to‐background ratio (TBR) of up to 83 in FR‐expressing tumor‐bearing mice within 30 min. Thus, FolateSiR‐1 has the potential to contribute to the research in the field of biology and the clinical medicine.

Scientific Reports, Published online: 22 January 2020; doi:10.1038/s41598-020-57776-3

The antibiotic vancomycin induces complexation and aggregation of gastrointestinal and submaxillary mucins

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Crystallographic analysis of Staphylococcus aureus LcpA, the primary wall teichoic acid ligase.

J Biol Chem. 2020 Jan 22;:

Authors: Li FKK, Rosell FI, Gale RT, Simorre JP, Brown ED, Strynadka NCJ

Abstract
Gram-positive bacteria, including major clinical pathogens such as Staphylococcus aureus, are becoming increasingly drug-resistant. Their cell wall is composed of a thick layer of peptidoglycan (PG) modified by the attachment of wall teichoic acid (WTA), an anionic glycopolymer that is linked to pathogenicity and regulation of cell division and PG synthesis. The transfer of WTA from lipid carriers to PG, catalyzed by the LytR-CpsA-Psr (LCP) enzyme family, offers a unique extracellular target for the development of new anti-infective agents. Inhibitors of LCP enzymes have the potential to manage a wide range of bacterial infections as the target enzymes are implicated in the assembly of many other bacterial cell wall polymers including capsular polysaccharide of streptococcal species and arabinogalactan of mycobacterial species. In this study, we present the first crystal structure of S. aureus LcpA with bound substrate at 1.9 Å resolution and those of Bacillus subtilis LCP enzymes, TagT, TagU and TagV, in the apo form at 1.6-2.8 Å resolution. The structures of these WTA transferases provide new insight into the binding of lipid-linked WTA and enable assignment of the catalytic roles of conserved active site residues. Furthermore, we identified potential subsites for binding the saccharide core of PG using computational docking experiments, and multi-angle light scattering experiments disclosed novel oligomeric states of the LCP enzymes. The crystal structures and modeled substrate-bound complexes of the LCP enzymes reported here provide insights into key features linked to substrate binding and catalysis and may aid the structure-guided design of specific LCP inhibitors.

PMID: 31969390 [PubMed - as supplied by publisher]

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Anti-PcrV IgY antibodies protect against Pseudomonas aeruginosa infection in both acute pneumonia and burn wound models.

Mol Immunol. 2019 12;116:98-105

Authors: Ranjbar M, Behrouz B, Norouzi F, Mousavi Gargari SL

Abstract
Pseudomonas aeruginosa is a common nosocomial pathogen in burn patients, and rapidly acquires antibiotic resistance; thus, developing an effective therapeutic approach is the most promising strategy for combating infection. Type III secretion system (T3SS) translocates bacterial toxins into the cytosol of the targeted eukaryotic cells, which plays important roles in the virulence of P. aeruginosa infections in both acute pneumonia and burn wound models. The PcrV protein, a T3SS translocating protein, is required for T3SS function and is a well-validated target in animal models of immunoprophylactic strategies targeting P. aeruginosa. In the present study, we evaluated the protective efficacy of chicken egg yolk antibodies (IgY) raised against recombinant PcrV (r-PcrV) in both acute pneumonia and burn wound models. R-PcrV protein was generated by expressing the pcrV gene (cloned in pET-28a vector) in E. coli BL-21. Anti-PcrV IgY was obtained by immunization of hen. Anti-PcrV IgY induced greater protection in P. aeruginosamurine acute pneumonia and burn wound models than control IgY (C-IgY) and PBS groups. Anti-PcrV IgY improved opsonophagocytic killing and inhibition of bacterial invasion of host cells. Taken together, our data provide evidence that anti-PcrV IgY can be a promising therapeutic candidate for combating P. aeruginosa infections.

PMID: 31634816 [PubMed - indexed for MEDLINE]

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Mechanisms of substrate recognition by a typhoid toxin secretion-associated muramidase.

Elife. 2020 Jan 20;9:

Authors: Geiger T, Lara-Tejero M, Xiong Y, Galán JE

Abstract
Typhoid toxin is a virulence factor for the bacterial pathogen Salmonella Typhi, which causes typhoid fever in humans. After its synthesis by intracellular bacteria, typhoid toxin is secreted into the lumen of the Salmonella-containing vacuole by a secretion mechanism strictly dependent on TtsA, a specific muramidase that facilitates toxin transport through the peptidoglycan layer. Here we show that substrate recognition by TtsA depends on a discrete domain within its carboxy terminus, which targets the enzyme to the bacterial poles to recognize YcbB-edited peptidoglycan. Comparison of the atomic structures of TtsA bound to its substrate and that of a close homolog with different specificity identified specific determinants involved in substrate recognition. Combined with structure-guided mutagenesis and in vitro and in vivo crosslinking experiments, this study provides an unprecedented view of the mechanisms by which a muramidase recognizes its peptidoglycan substrate to facilitate protein secretion.

PMID: 31958059 [PubMed - as supplied by publisher]

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Oxidization of TGFβ-activated kinase by MPT53 is required for immunity to Mycobacterium tuberculosis.

Nat Microbiol. 2019 08;4(8):1378-1388

Authors: Wang L, Liu Z, Wang J, Liu H, Wu J, Tang T, Li H, Yang H, Qin L, Ma D, Chen J, Liu F, Wang P, Zheng R, Song P, Zhou Y, Cui Z, Wu X, Huang X, Liang H, Zhang S, Cao J, Wu C, Chen Y, Su D, Chen X, Zeng G, Ge B

Abstract
Mycobacterium tuberculosis (Mtb)-derived components are usually recognized by pattern recognition receptors to initiate a cascade of innate immune responses. One striking characteristic of Mtb is their utilization of different type VII secretion systems to secrete numerous proteins across their hydrophobic and highly impermeable cell walls, but whether and how these Mtb-secreted proteins are sensed by host immune system remains largely unknown. Here, we report that MPT53 (Rv2878c), a secreted disulfide-bond-forming-like protein of Mtb, directly interacts with TGF-β-activated kinase 1 (TAK1) and activates TAK1 in a TLR2- or MyD88-independent manner. MPT53 induces disulfide bond formation at C210 on TAK1 to facilitate its interaction with TRAFs and TAB1, thus activating TAK1 to induce the expression of pro-inflammatory cytokines. Furthermore, MPT53 and its disulfide oxidoreductase activity is required for Mtb to induce the host inflammatory responses via TAK1. Our findings provide an alternative pathway for host signalling proteins to sense Mtb infection and may favour the improvement of current vaccination strategies.

PMID: 31110366 [PubMed - indexed for MEDLINE]

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Harnessing bacterial interactions to manage infections: a review on the opportunistic pathogen Pseudomonas aeruginosa as a case example.

J Med Microbiol. 2020 Jan 21;:

Authors: Rezzoagli C, Granato ET, Kümmerli R

Abstract
During infections, bacterial pathogens can engage in a variety of interactions with each other, ranging from the cooperative sharing of resources to deadly warfare. This is especially relevant in opportunistic infections, where different strains and species often co-infect the same patient and interact in the host. Here, we review the relevance of these social interactions during opportunistic infections using the human pathogen Pseudomonas aeruginosa as a case example. In particular, we discuss different types of pathogen-pathogen interactions, involving both cooperation and competition, and elaborate on how they impact virulence in multi-strain and multi-species infections. We then review evolutionary dynamics within pathogen populations during chronic infections. We particuarly discuss how local adaptation through niche separation, evolutionary successions and antagonistic co-evolution between pathogens can alter virulence and the damage inflicted on the host. Finally, we outline how studying bacterial social dynamics could be used to manage infections. We show that a deeper appreciation of bacterial evolution and ecology in the clinical context is important for understanding microbial infections and can inspire novel treatment strategies.

PMID: 31961787 [PubMed - as supplied by publisher]

Researchers from the University of Sheffield have, for the first time, manufactured 3-D printed parts that show resistance to common bacteria. This could stop the spread of infections such as MRSA in hospitals and care homes, saving the lives of vulnerable patients.