scholarly journals Non-Lytic Antibacterial Peptides That Translocate Through Bacterial Membranes to Act on Intracellular Targets

2019 ◽  
Vol 20 (19) ◽  
pp. 4877 ◽  
Author(s):  
Marlon H. Cardoso ◽  
Beatriz T. Meneguetti ◽  
Bruna O. Costa ◽  
Danieli F. Buccini ◽  
Karen G. N. Oshiro ◽  
...  
Keyword(s):  
Cell Wall ◽  
Protein Synthesis ◽  
Pathogenic Bacteria ◽  
Biological Properties ◽  
Antibacterial Peptides ◽  
Bacterial Cells ◽  
Bacterial Membranes ◽  
Intracellular Targets

The advent of multidrug resistance among pathogenic bacteria has attracted great attention worldwide. As a response to this growing challenge, diverse studies have focused on the development of novel anti-infective therapies, including antimicrobial peptides (AMPs). The biological properties of this class of antimicrobials have been thoroughly investigated, and membranolytic activities are the most reported mechanisms by which AMPs kill bacteria. Nevertheless, an increasing number of works have pointed to a different direction, in which AMPs are seen to be capable of displaying non-lytic modes of action by internalizing bacterial cells. In this context, this review focused on the description of the in vitro and in vivo antibacterial and antibiofilm activities of non-lytic AMPs, including indolicidin, buforin II PR-39, bactenecins, apidaecin, and drosocin, also shedding light on how AMPs interact with and further translocate through bacterial membranes to act on intracellular targets, including DNA, RNA, cell wall and protein synthesis.

scholarly journals TSPphg Lysin from the Extremophilic Thermus Bacteriophage TSP4 as a Potential Antimicrobial Agent against Both Gram-Negative and Gram-Positive Pathogenic Bacteria

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 192 ◽  
Author(s):  
Feng Wang ◽  
Xinyu Ji ◽  
Qiupeng Li ◽  
Guanling Zhang ◽  
Jiani Peng ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Pathogenic Bacteria ◽  
Infection Model ◽  
Bacterial Cells ◽  
Skin Damage ◽  
Gram Positive ◽  
Antibiotic Resistant ◽  
Gram Negative

New strategies against antibiotic-resistant bacterial pathogens are urgently needed but are not within reach. Here, we present in vitro and in vivo antimicrobial activity of TSPphg, a novel phage lysin identified from extremophilic Thermus phage TSP4 by sequencing its whole genome. By breaking down the bacterial cells, TSPphg is able to cause bacteria destruction and has shown bactericidal activity against both Gram-negative and Gram-positive pathogenic bacteria, especially antibiotic-resistant strains of Klebsiella pneumoniae, in which the complete elimination and highest reduction in bacterial counts by greater than 6 logs were observed upon 50 μg/mL TSPphg treatment at 37 °C for 1 h. A murine skin infection model further confirmed the in vivo efficacy of TSPphg in removing a highly dangerous and multidrug-resistant Staphylococcus aureus from skin damage and in accelerating wound closure. Together, our findings may offer a therapeutic alternative to help fight bacterial infections in the current age of mounting antibiotic resistance, and to shed light on bacteriophage-based strategies to develop novel anti-infectives.

scholarly journals Structural basis of peptidoglycan endopeptidase regulation

2020 ◽  
Vol 117 (21) ◽  
pp. 11692-11702 ◽  
Author(s):  
Jung-Ho Shin ◽  
Alan G. Sulpizio ◽  
Aaron Kelley ◽  
Laura Alvarez ◽  
Shannon G. Murphy ◽  
...  
Keyword(s):  
Cell Wall ◽  
Structural Basis ◽  
Bacterial Cells ◽  
Cell Wall Degradation ◽  
Open Conformation ◽  
Poor Understanding ◽  
A Cell ◽  
Inhibitory Domain

Most bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over; the latter process is mediated by PG cleavage enzymes, for example, the endopeptidases (EPs). EPs themselves are essential for growth but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., β-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogenVibrio cholerae. Our data suggest that ShyA assumes two drastically different conformations: a more open form that allows for substrate binding and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogensNeisseria gonorrheaeandEscherichia coli. Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain 1 from the M23 active site, likely through conformational rearrangement in vivo.

scholarly journals Conserved mechanism of cell-wall synthase regulation revealed by the identification of a new PBP activator inPseudomonas aeruginosa

2018 ◽  
Vol 115 (12) ◽  
pp. 3150-3155 ◽  
Author(s):  
Neil G. Greene ◽  
Coralie Fumeaux ◽  
Thomas G. Bernhardt
Keyword(s):  
Cell Wall ◽  
Drug Targets ◽  
Bacterial Cells ◽  
Acinetobacter Baylyi ◽  
Gram Negative ◽  
E Coli ◽  
Outer Membrane Lipoprotein ◽  
Synthase Regulation

Penicillin-binding proteins (PBPs) are synthases required to build the essential peptidoglycan (PG) cell wall surrounding most bacterial cells. The mechanisms regulating the activity of these enzymes to control PG synthesis remain surprisingly poorly defined given their status as key antibiotic targets. Several years ago, the outer-membrane lipoproteinEcLpoB was identified as a critical activator ofEscherichia coliPBP1b (EcPBP1b), one of the major PG synthases of this organism. Activation ofEcPBP1b is mediated through the association ofEcLpoB with a regulatory domain onEcPBP1b called UB2H. Notably,Pseudomonas aeruginosaalso encodes PBP1b (PaPBP1b), which possesses a UB2H domain, but this bacterium lacks an identifiable LpoB homolog. We therefore searched for potentialPaPBP1b activators and identified a lipoprotein unrelated to LpoB that is required for the in vivo activity ofPaPBP1b. We named this protein LpoP and found that it interacts directly withPaPBP1b in vitro and is conserved in many Gram-negative species. Importantly, we also demonstrated thatPaLpoP-PaPBP1b as well as an equivalent protein pair fromAcinetobacter baylyican fully substitute forEcLpoB-EcPBP1b inE. colifor PG synthesis. Furthermore, we show that amino acid changes inPaPBP1b that bypass thePaLpoP requirement map to similar locations in the protein as changes promotingEcLpoB bypass inEcPBP1b. Overall, our results indicate that, although different Gram-negative bacteria activate their PBP1b synthases with distinct lipoproteins, they stimulate the activity of these important drug targets using a conserved mechanism.

scholarly journals A Toxin Involved inSalmonellaPersistence Regulates Its Activity by Acetylating Its Cognate Antitoxin, a Modification Reversed by CobB Sirtuin Deacetylase

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Chelsey M. VanDrisse ◽  
Anastacia R. Parks ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Protein Synthesis ◽  
Pathogenic Bacteria ◽  
Bacterial Toxin ◽  
Host Defenses ◽  
Mobility Shift ◽  
Reverse Transcription Pcr ◽  
Acetylation State ◽  
Host Infection

ABSTRACTBacterial toxin-antitoxin systems trigger the onset of a persister state by inhibiting essential cellular processes. The TacT toxin ofSalmonella entericais known to induce a persister state in macrophages through the acetylation of aminoacyl-tRNAs. Here, we show that the TacT toxin and the TacA antitoxin work as a complex that modulates TacT activity via the acetylation state of TacA. TacT acetylates TacA at residue K44, a modification that is removed by the NAD+-dependent CobB sirtuin deacetylase. TacA acetylation increases the activity of TacT, downregulating protein synthesis. TacA acetylation altered binding to its own promoter, although this did not changetacATexpression levels. These claims are supported by results fromin vitroprotein synthesis experiments used to monitor TacT activity,in vivogrowth analyses, electrophoretic mobility shift assays, and quantitative reverse transcription-PCR (RT-qPCR) analysis. TacT is the first example of a Gcn5-relatedN-acetyltransferase that modifies nonprotein and protein substrates.IMPORTANCEDuring host infection, pathogenic bacteria can modulate their physiology to evade host defenses. Some pathogens use toxin-antitoxin systems to modulate a state of self-toxicity that can decrease their cellular activity, triggering the onset of a persister state. The lower metabolic activity of persister cells allows them to escape host defenses and antibiotic treatments. Hence a better understanding of the mechanisms used by pathogens to ingress and egress the persister state is of relevance to human health.

scholarly journals Structural basis of peptidoglycan endopeptidase regulation

2019 ◽  
Author(s):  
Jung-Ho Shin ◽  
Alan G. Sulpizio ◽  
Aaron Kelley ◽  
Laura Alvarez ◽  
Shannon G. Murphy ◽  
...  
Keyword(s):  
Cell Wall ◽  
Vibrio Cholerae ◽  
Active Site ◽  
Normal Growth ◽  
Structural Basis ◽  
Bacterial Cells ◽  
Cell Wall Digestion ◽  
Novel Antibiotics

AbstractMost bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over, the latter process is mediated by PG cleavage enzymes, for example the endopeptidases (EPs). EPs themselves are essential for growth, but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., β-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogen Vibrio cholerae. Our data suggest that ShyA assumes two drastically different conformations; a more open form that allows for substrate binding, and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogens Neisseria gonorrheae and Escherichia coli. Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain1 from the M23 active site, likely through conformational re-arrangement in vivo.SignificanceBacteria digest their cell wall following exposure to antibiotics like penicillin. The endopeptidases (EPs) are among the proteins that catalyze cell wall digestion processes after antibiotic exposure, but we do not understand how these enzymes are regulated during normal growth. Herein, we present the structure of the major EP from the diarrheal pathogen Vibrio cholerae. Surprisingly, we find that EPs from this and other pathogens appear to be produced as a largely inactive precursor that undergoes a conformational shift exposing the active site to engage in cell wall digestion. These results enhance our understanding of how EPs are regulated and could open the door for the development of novel antibiotics that overactivate cell wall digestion processes.

scholarly journals Antibacterial efficacy of Jackfruit rag extract against clinically important pathogens and validation of its antimicrobial activity in Shigella dysenteriae infected Drosophila melanogaster infection model

2020 ◽  
Author(s):  
NV Dhwani ◽  
Gayathri Raju ◽  
Sumi E Mathew ◽  
Gaurav Baranwal ◽  
Shivakumar B Shivaram ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacterial Cell ◽  
Antibacterial Property ◽  
Mouse Fibroblast ◽  
Bacterial Cell Wall ◽  
Shigella Dysenteriae ◽  
Infection Model ◽  
Bacterial Cells

AbstractThe aim of this study was to determine the antibacterial property of extract derived from a part of the Jackfruit called ‘rag’, that is generally considered as fruit waste. Morpho-physical characterization of the Jackfruit rag extract (JFRE) was performed using gas-chromatography, where peaks indicative of furfural; pentanoic acid; and hexadecanoic acid were observed. In vitro biocompatibility of JFRE was performed using the MTT assay, which showed comparable cellular viability between extract-treated and untreated mouse fibroblast cells. Agar well disc diffusion assay exhibited JFRE induced zones of inhibition for a wide variety of laboratory and clinical strains of gram-positive and gram-negative bacteria. Analysis of electron microscope images of bacterial cells suggests that JFRE induces cell death by disintegration of the bacterial cell wall and precipitating intracytoplasmic clumping. The antibacterial activity of the JFREs was further validated in vivo using Shigella dysenteriae infected fly model, where JFRE pre-fed flies infected with S. dysenteriae had significantly reduced mortality compared to controls. JFRE demonstrates broad antibacterial property, both in vitro and in vivo, possibly by its activity on bacterial cell wall. This study highlights the importance of exploring alternative sources of antibacterial compounds, especially from plant-derived waste, that could provide economical and effective solutions to current challenges in antimicrobial therapy.

scholarly journals Production, purification, characterization, and biological properties of Rhodosporidium paludigenum polysaccharide

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0246148
Author(s):  
Mengjian Liu ◽  
WenJu Zhang ◽  
Jun Yao ◽  
Junli Niu
Keyword(s):  
Pathogenic Bacteria ◽  
Inhibitory Concentration ◽  
Biological Properties ◽  
Molecular Weights ◽  
Fermentation Parameters ◽  
Main Components ◽  
Image Position ◽  
G Ratio

The yield of marine red yeast polysaccharide (MRYP) obtained from Rhodosporidium paludigenum was increased by optimizing fermentation conditions, and the pure polysaccharide was extracted by column chromatography. The molecular weight of pure MRYP and the ratio of mannose to glucose in components of MRYP were determined. Antioxidant and antibacterial abilities of MRYP were investigated in vitro and in vivo. The optimal fermentation parameters were as follows: Medium 4, pH = 6.72, temperature = 30.18°C, blades speed = 461.36 r/min; the optimized yield reached 4323.90 mg/L, which was 1.31 times the original yield. The sequence of factors that affected the MRYP yield was the blades speed>pH>temperature. The main components of MRYP were MYH-1 and MYH-2. The molecular weights of MYH-1 and MYH-2 were 246.92 kDa and 21.88 kDa, respectively; they accounted for 53.60% and 28.75% of total polysaccharide. In MYH-1 and MYH-2, the proportion of glucose and mannose accounted for 46.94%, 38.46%, and 67.10%, 7.17%, respectively. In vitro, the ability of scavenging DPPH•, •OH, and •O2− radical was 32.26%, 24.34%, and 22.09%; the minimum inhibitory concentration (MIC) of MRYP was 480 μg/mg. In vivo, MRYP improved the lambs’ body weight, antioxidant enzyme activity, and the number of probiotics, but it reduced the feed/gain (F/G) ratio and the number of pathogenic bacteria in 60-days-old lambs.

scholarly journals Mechanism of action of oxazolidinones: effects of linezolid and eperezolid on translation reactions.

1997 ◽  
Vol 41 (10) ◽  
pp. 2132-2136 ◽  
Author(s):  
D L Shinabarger ◽  
K R Marotti ◽  
R W Murray ◽  
A H Lin ◽  
E P Melchior ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Pathogenic Bacteria ◽  
Coli Strain ◽  
In Vitro Translation ◽  
E Coli ◽  
Initiation Phase ◽  
New Class ◽  
Bacterial Translation

The oxazolidinones are a new class of synthetic antibiotics with good activity against gram-positive pathogenic bacteria. Experiments with a susceptible Escherichia coli strain, UC6782, demonstrated that in vivo protein synthesis was inhibited by both eperezolid (formerly U-100592) and linezolid (formerly U-100766). Both linezolid and eperezolid were potent inhibitors of cell-free transcription-translation in E. coli, exhibiting 50% inhibitory concentrations (IC50s) of 1.8 and 2.5 microM, respectively. The ability to demonstrate inhibition of in vitro translation directed by phage MS2 RNA was greatly dependent upon the amount of RNA added to the assay. For eperezolid, 128 microg of RNA per ml produced an IC50 of 50 microM whereas a concentration of 32 microg/ml yielded an IC50 of 20 microM. Investigating lower RNA template concentrations in linezolid inhibition experiments revealed that 32 and 8 microg of MS2 phage RNA per ml produced IC50s of 24 and 15 microM, respectively. This phenomenon was shared by the translation initiation inhibitor kasugamycin but not by streptomycin. Neither oxazolidinone inhibited the formation of N-formylmethionyl-tRNA, elongation, or termination reactions of bacterial translation. The oxazolidinones appear to inhibit bacterial translation at the initiation phase of protein synthesis.

scholarly journals Covariance of Complementary rRNA Loop Nucleotides Does Not Necessarily Represent Functional Pseudoknot Formation In Vivo

2000 ◽  
Vol 182 (20) ◽  
pp. 5671-5675 ◽  
Author(s):  
Natalya S. Chernyaeva ◽  
Emanuel J. Murgola
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Cell Growth ◽  
Site Directed Mutagenesis ◽  
23S Rrna ◽  
Directed Mutagenesis ◽  
Bacterial Cells ◽  
Domain I

ABSTRACT We examined mutationally a two-hairpin structure (nucleotides 57 to 70 and 76 to 110) in a region of domain I ofEscherichia coli 23S rRNA that has been implicated in specific functions in protein synthesis by other studies. On the basis of the observed covariance of several nucleotides in each loop inBacteria, Archaea, and chloroplasts, the two hairpins have been proposed to form a pseudoknot. Here, appropriate loop changes were introduced in vitro by site-directed mutagenesis to eliminate any possibility of base pairing between the loops. The bacterial cells containing each cloned mutant rRNA operon were then examined for cell growth, termination codon readthrough, and assembly of the mutant rRNAs into functional ribosomes. The results show that, under the conditions examined, the two hairpins do not form a pseudoknot structure that is required for the functioning of the ribosome in vivo and therefore that sequence covariance does not necessarily indicate the formation of a functional pseudoknot.

scholarly journals Containment of Phytoplasma-Associated Plant Diseases by Antibiotics and Other Antimicrobial Molecules

Antibiotics ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1398
Author(s):  
Assunta Bertaccini
Keyword(s):  
Cell Wall ◽  
Pathogenic Bacteria ◽  
Plant Diseases ◽  
Ribosome Inactivating Proteins ◽  
A Cell ◽  
Microbial Strains ◽  
Resistance Inducers ◽  
Plasma Activated Water

Phytoplasmas are plant-pathogenic bacteria that infect many important crops and environmentally relevant plant species, causing serious economic and environmental losses worldwide. These bacteria, lacking a cell wall, are sensitive to antibiotics such as tetracyclines that affect protein synthesis mechanisms. Phytoplasma cultivation in axenic media has not been achieved for many strains; thus, the screening of antimicrobials must be performed using mainly in vivo materials. Some studies have investigated using in vitro phytoplasma-infected shoots, and several antimicrobials, including tetracyclines, have been tested. The screening of phytoplasma antimicrobials is important for the sustainable control of phytoplasma-associated diseases. The use of molecules with different modes of action such as ribosome inactivating proteins, plant hormones, and resistance inducers such as plasma-activated water, is advised, to avoid the use of antibiotics in agriculture and the possible emergence of resistant microbial strains.

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