Zymographic Techniques for the Analysis of Bacterial Cell Wall in Bacillus

Fukushima, Tatsuya; Sekiguchi, Junichi

doi:10.1007/978-1-4939-3676-2_7

Cited by 18 publications

(14 citation statements)

References 16 publications

(5 reference statements)

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“…Peptidoglycan from P. acnes cells was isolated according to Fukushima and Sekiguchi, (2016) [77]. Following purification, the isolated peptidoglycan was washed five times with Milli-Q water, lyophilized, and stored at −20 • C. Turbidity reduction assays using isolated peptidoglycan were performed as described above (using cell suspension), with the only exception that the absorbance of the peptidoglycan suspension was measured at 540 nm.…”

Section: Turbidity Reduction Assays Using Isolated Peptidoglycanmentioning

confidence: 99%

Characterization and Engineering Studies of a New Endolysin from the Propionibacterium acnes Bacteriophage PAC1 for the Development of a Broad-Spectrum Artilysin with Altered Specificity

Varotsou

Premetis

Labrou

2023

IJMS

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The emergence of multidrug-resistant (MDR) bacteria has risen rapidly, leading to a great threat to global public health. A promising solution to this problem is the exploitation of phage endolysins. In the present study, a putative N-acetylmuramoyl-L-alanine type-2 amidase (NALAA-2, EC 3.5.1.28) from Propionibacterium bacteriophage PAC1 was characterized. The enzyme (PaAmi1) was cloned into a T7 expression vector and expressed in E. coli BL21 cells. Kinetics analysis using turbidity reduction assays allowed the determination of the optimal conditions for lytic activity against a range of Gram-positive and negative human pathogens. The peptidoglycan degradation activity of PaAmi1 was confirmed using isolated peptidoglycan from P. acnes. The antibacterial activity of PaAmi1 was investigated using live P. acnes cells growing on agar plates. Two engineered variants of PaAmi1 were designed by fusion to its N-terminus two short antimicrobial peptides (AMPs). One AMP was selected by searching the genomes of Propionibacterium bacteriophages using bioinformatics tools, whereas the other AMP sequence was selected from the antimicrobial peptide databases. Both engineered variants exhibited improved lytic activity towards P. acnes and the enterococci species Enterococcus faecalis and Enterococcus faecium. The results of the present study suggest that PaAmi1 is a new antimicrobial agent and provide proof of concept that bacteriophage genomes are a rich source of AMP sequences that can be further exploited for designing novel or improved endolysins.

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Section: Turbidity Reduction Assays Using Isolated Peptidoglycanmentioning

confidence: 99%

Characterization and Engineering Studies of a New Endolysin from the Propionibacterium acnes Bacteriophage PAC1 for the Development of a Broad-Spectrum Artilysin with Altered Specificity

Varotsou

Premetis

Labrou

2023

IJMS

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“…Purification of A. baumannii cell wall peptidoglycan and labelling with Remazol Brilliant Blue-R Peptidoglycan from A. baumannii cells was isolated as previously described [62] with some minor modifications. Immediately after reaching the stationary phase, the culture was subjected to thermal inactivation (121 °C, 30 min).…”

Section: Differential Scanning Fluorimetry (Dsf)mentioning

confidence: 99%

Characterization of a glycoside hydrolase endolysin from Acinetobacter baumannii phage AbTZA1 with high antibacterial potency and novel structural features

et al. 2022

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The classification of Acinetobacter baumannii by WHO as 'priority 1' antibiotic-resistant pathogen underlines the urgent need for novel antimicrobial agents towards this pathogen. In this work, screening of the A. baumannii phage AbTZA1 genome allowed the identification of a putative endolysin (AbLys1, EC3.2.1.17) that belongs to the glycoside hydrolase family 24 (GH24). The sequence of AbLys1 was cloned, expressed in E. coli and purified. The lytic activity and specificity of AbLys1 were evaluated against a range of Gram-positive and Gram-negative human pathogens. AbLys1 was found to display a high selectivity towards A. baumannii. Kinetic analysis was carried out to characterize the dependence of its lytic activity on pH. The enzyme shows its maximal activity at pH values 7-8. The structure of AbLys1 was determined by X-ray crystallography to 1.82 Å resolution. The overall structure revealed two helical domains: a small, antenna-like, N-terminal domain and a larger C-terminal domain with six α-helices and a β-hairpin. Both the antenna-like and β-hairpin regions contain short sequences (AMseq1 and AMseq2) with predicted antimicrobial activity. Engineering studies revealed a key role of AMseq1 and AMseq2 on the enzyme's lytic activity towards A. baumannii cells but not towards purified peptidoglycan. This suggests that both sequences affect the destabilization of the outer membrane, thus providing access of the catalytic domain to the peptidoglycan. In addition, the deletion of AMseq1 enhanced the enzyme stability, whereas the deletion of AMseq2 diminished it. The results suggest that AbLys1 is a promising new enzybiotic with efficient lytic and antimicrobial activity.

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“…After electrophoresis, the gel was shaken at 37°C for 16 h in 50 ml of 25 mM Tris-HCl (pH 8.0) solution containing 1% (vol/ vol) Triton X-100 to allow protein renaturation. Clear bands resulting from lytic activity were visualized after staining with 1% (wt/vol) methylene blue (Sigma)-0.01% (wt/vol) KOH and subsequent destaining with distilled water (57,79). The PG was further separated from PSII by hydrofluoric acid treatment (29).…”

Section: Control Of Cwla Localization By Phosphorylationmentioning

confidence: 99%

Ser/Thr Kinase-Dependent Phosphorylation of the Peptidoglycan Hydrolase CwlA Controls Its Export and Modulates Cell Division in Clostridioides difficile

García-García

Poncet

Cuenot

et al. 2021

mBio

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Cell growth and division require a balance between synthesis and hydrolysis of the peptidoglycan (PG). Inhibition of PG synthesis or uncontrolled PG hydrolysis can be lethal for the cells, making it imperative to control peptidoglycan hydrolase (PGH) activity. The synthesis or activity of several key enzymes along the PG biosynthetic pathway is controlled by the Hanks-type serine/threonine kinases (STKs). In Gram-positive bacteria, inactivation of genes encoding STKs is associated with a range of phenotypes, including cell division defects and changes in cell wall metabolism, but only a few kinase substrates and associated mechanisms have been identified. We previously demonstrated that STK-PrkC plays an important role in cell division, cell wall metabolism, and resistance to antimicrobial compounds in the human enteropathogen Clostridioides difficile. In this work, we characterized a PG hydrolase, CwlA, which belongs to the NlpC/P60 family of endopeptidases and hydrolyses cross-linked PG between daughter cells to allow cell separation. We identified CwlA as the first PrkC substrate in C. difficile. We demonstrated that PrkC-dependent phosphorylation inhibits CwlA export, thereby controlling hydrolytic activity in the cell wall. High levels of CwlA at the cell surface led to cell elongation, whereas low levels caused cell separation defects. Thus, we provided evidence that the STK signaling pathway regulates PGH homeostasis to precisely control PG hydrolysis during cell division. IMPORTANCE Bacterial cells are encased in a PG exoskeleton that helps to maintain cell shape and confers physical protection. To allow bacterial growth and cell separation, PG needs to be continuously remodeled by hydrolytic enzymes that cleave PG at critical sites. How these enzymes are regulated remains poorly understood. We identify a new PG hydrolase involved in cell division, CwlA, in the enteropathogen C. difficile. Lack or accumulation of CwlA at the bacterial surface is responsible for a division defect, while its accumulation in the absence of PrkC also increases susceptibility to antimicrobial compounds targeting the cell wall. CwlA is a substrate of the kinase PrkC in C. difficile. PrkC-dependent phosphorylation controls the export of CwlA, modulating its levels and, consequently, its activity in the cell wall. This work provides a novel regulatory mechanism by STK in tightly controlling protein export.

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Zymographic Techniques for the Analysis of Bacterial Cell Wall in Bacillus

Cited by 18 publications

References 16 publications

Characterization and Engineering Studies of a New Endolysin from the Propionibacterium acnes Bacteriophage PAC1 for the Development of a Broad-Spectrum Artilysin with Altered Specificity

Characterization and Engineering Studies of a New Endolysin from the Propionibacterium acnes Bacteriophage PAC1 for the Development of a Broad-Spectrum Artilysin with Altered Specificity

Characterization of a glycoside hydrolase endolysin from Acinetobacter baumannii phage AbTZA1 with high antibacterial potency and novel structural features

Ser/Thr Kinase-Dependent Phosphorylation of the Peptidoglycan Hydrolase CwlA Controls Its Export and Modulates Cell Division in Clostridioides difficile

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