An <i>in vivo</i> protease activity assay for investigating the functions of the <i>Escherichia coli</i> membrane protease HtpX

Yoshitani, Kohei; Hizukuri, Yohei; Akiyama, Yoshinori

doi:10.1002/1873-3468.13368

Cited by 15 publications

(17 citation statements)

References 38 publications

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“…We also observed an upregulation of genes that are involved in phage shock protein operon (pspA, pspB, pspC, and pspE). Yoshitani et al (2019) found that htpX along with ftsH lyse the abnormal IM proteins of EHEC (Yoshitani et al, 2019). Besides, Shimohata et al (2002) reported that the accumulation of misfolded proteins in the IM may occur due to the inefficiency or loss of htpX or ftsH which further activates Cpx stress response proteins located in periplasmic space (Shimohata et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

Antagonistic Mechanism of Metabolites Produced by Lactobacillus casei on Lysis of Enterohemorrhagic Escherichia coli

et al. 2020

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Enhancing extracellular metabolic byproducts of probiotics is one of the promising strategies to improve overall host health as well as to control enteric infections caused by various foodborne pathogens. However, the underlying mechanism of action of those metabolites and their effective concentrations are yet to be established. In this study, we determined the antibacterial potential of the metabolites in the cell-free culture supernatant (CFCS) collected from wild-type Lactobacillus casei (LCwt) and genetically modified LC to overexpress linoleate isomerase (LCCLA). We also evaluated the mechanism of action of CFCSs collected from the culture of LCwt in the presence or absence of 0.5% peanut flour (CFCSwt and CFCSwt+PF, respectively) and LCCLA alone (CFCSCLA) against enterohemorrhagic Escherichia coli (EHEC). The metabolites present in CFCSwt+PF and CFCSCLA eliminated EHEC within 24 and 48 h, respectively. Whereas CFCSwt failed to eliminate EHEC but reduced their growth by 6.7 logs (p < 0.05) as compared to the control. Significant downregulation of the expression of cell division gene, ftsZ, supported the observed degree of bactericidal and bacteriostatic properties of the collected CFCSs. Upregulation of EHEC genes related to maintaining cell membrane integrity, DNA damage repair, and molecular chaperons indicated an intensive stress condition imposed by the total metabolites present in CFCSs on EHEC growth and cellular structures. A range of deviated morphological features provoked by the metabolites indicated a membrane-targeted action, in general, to compromise the membrane permeability of EHEC. The information obtained from this study may contribute to a more efficient prevention of EHEC related infections.

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Section: Discussionmentioning

confidence: 99%

Antagonistic Mechanism of Metabolites Produced by Lactobacillus casei on Lysis of Enterohemorrhagic Escherichia coli

et al. 2020

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“…Proteins were visualized by a Lumino image analyzer LAS-4000 mini (Cytiva) using ECL (enhanced chemiluminescence) or ECL Prime Western Blotting Detection Reagents (Cytiva). Rabbit polyclonal anti–hemagglutinin (HA) [HA-probe (Y-11), Santa Cruz Biotechnology], anti–maltose-binding protein (MBP) ( 57 ), anti- Ec RseP ( 19 ), and anti-SecB ( 21 ) antibodies and rat monoclonal anti-PA antibodies ( 58 ) were used for immunoblotting. Also, for detection of His-tagged proteins, anti-His antibodies from the Penta-His HRP Conjugate Kit (Qiagen) were used.…”

Section: Methodsmentioning

confidence: 99%

Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP

et al. 2022

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Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including Escherichia coli RseP. Structure-based chemical modification and cross-linking experiments indicated that the RseP domains surrounding the active center undergo conformational changes to expose the substrate-binding site, suggesting that RseP has a gating mechanism to regulate substrate entry. Furthermore, mutational analysis suggests that a conserved electrostatic linkage between the transmembrane and peripheral membrane-associated domains mediates the conformational changes. In vivo cleavage assays also support that the substrate transmembrane helix is unwound by strand addition to the intramembrane β sheet of RseP and is clamped by a conserved asparagine residue at the active center for efficient cleavage. This mechanism underlying the substrate binding, i.e., unwinding and clamping, appears common across distinct families of intramembrane proteases that cleave transmembrane segments.

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“…Proteins were visualized by Lumino image analyzer LAS-4000 mini (Cytiva) using ECL or ECL Prime Western Blotting Detection Reagents (Cytiva). Rabbit polyclonal anti-HA (HA-probe (Y-11), Santa Cruz Biotechnology), anti-MBP ( 62 ), anti- Ec RseP ( 19 ) and anti-SecB ( 21 ) antibodies, and rat monoclonal anti-PA antibodies ( 63 ) were used for immunoblotting. Also, for detection of His-tagged proteins, anti-His antibodies from the Penta-His HRP Conjugate Kit (Qiagen) were used.…”

Section: Methodsmentioning

confidence: 99%

Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP

Imaizumi

Takanuki

Miyake

et al. 2022

Preprint

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Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including E. coli RseP. Our observations are consistent with a rearrangement of the RseP domains surrounding the active center to expose the substrate-binding site where a conserved electrostatic linkage between the transmembrane and membrane-associated domains mediates the conformational changes, suggesting that RseP has a gating mechanism to regulate substrate entry. Mutational analysis also supports that the substrate transmembrane helix is unwound by strand addition to the intramembrane β sheet and is clamped at the active center for efficient cleavage. Furthermore, this substrate accommodation mechanism appears to be common across distinct intramembrane proteases.

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An in vivo protease activity assay for investigating the functions of the Escherichia coli membrane protease HtpX

Cited by 15 publications

References 38 publications

Antagonistic Mechanism of Metabolites Produced by Lactobacillus casei on Lysis of Enterohemorrhagic Escherichia coli

Antagonistic Mechanism of Metabolites Produced by Lactobacillus casei on Lysis of Enterohemorrhagic Escherichia coli

Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP

Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP

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