Calcium-induced Folding and Stabilization of the Pseudomonas aeruginosa Alkaline Protease

Zhang, Liang; Conway, James F.; Thibodeau, Patrick H.

doi:10.1074/jbc.m111.310300

Cited by 54 publications

(108 citation statements)

References 43 publications

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“…Protein was expressed at 37°C for 4 to 6 h. The SlpB protein was purified from the insoluble material under denaturing conditions by following protocols previously described for PrtS and AprA (23). In vitro protein refolding and protease activity were assessed using a fluorescent peptide and casein substrates, as previously described (42). The AprI protease inhibitor was expressed and purified as previously described (23).…”

Section: Slpb and Apri Purification And Analysismentioning

confidence: 99%

Identification of SlpB, a Cytotoxic Protease from Serratia marcescens

et al. 2015

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The Gram-negative bacterium and opportunistic pathogen Serratia marcescens causes ocular infections in healthy individuals. Secreted protease activity was characterized from 44 ocular clinical isolates, and a higher frequency of protease-positive strains was observed among keratitis isolates than among conjunctivitis isolates. A positive correlation between protease activity and cytotoxicity to human corneal epithelial cells in vitro was determined. Deletion of prtS in clinical keratitis isolate K904 reduced, but did not eliminate, cytotoxicity and secreted protease production. This indicated that PrtS is necessary for full cytotoxicity to ocular cells and implied the existence of another secreted protease(s) and cytotoxic factors. Bioinformatic analysis of the S. marcescens Db11 genome revealed three additional open reading frames predicted to code for serralysin-like proteases noted here as slpB, slpC, and slpD. Induced expression of prtS and slpB, but not slpC and slpD, in strain PIC3611 rendered the strain cytotoxic to a lung carcinoma cell line; however, only prtS induction was sufficient for cytotoxicity to a corneal cell line. Strain K904 with deletion of both prtS and slpB genes was defective in secreted protease activity and cytotoxicity to human cell lines. PAGE analysis suggests that SlpB is produced at lower levels than PrtS. Purified SlpB demonstrated calcium-dependent and AprI-inhibited protease activity and cytotoxicity to airway and ocular cell lines in vitro. Lastly, genetic analysis indicated that the type I secretion system gene, lipD, is required for SlpB secretion. These genetic data introduce SlpB as a new cytotoxic protease from S. marcescens. Microbial keratitis (MK) is a blinding disease with poor visual outcomes even with effective antibiotics and antifungal agents (1-3). In addition to being a major cause of hospital-acquired infections, such as ventilator-associated pneumonia (4, 5), Serratia marcescens is a common cause of MK (1, 2, 6-8), yet the virulence factors involved in this process are poorly understood. In general, bacterial secreted factors, including hemolysins and proteases, contribute to the pathogenesis of bacterial corneal infections (9-12). There are several studies characterizing the importance of proteases from S. marcescens isolates in ocular infections, most recently reviewed by Matsumoto (13). Serralysin is a cytotoxic factor capable of killing mammalian cells in vitro (14,15). Purified serralysin is sufficient to cause keratitis when injected into rabbit eyes and promotes the spread of bacteria through the corneal stroma (16-18). Additionally, serralysin can degrade components of the human immune system, such as PAR-2, in vitro, and this may impact corneal pathogenesis (13,(19)(20)(21). Serralysin, and serralysin family metalloproteases, such as alkaline protease from Pseudomonas aeruginosa, can proteolyze mammalian cell surface proteins, thereby modulating cell physiology. A recent example is the protease-mediated activation of the epithelial sodium channel, lea...

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Section: Slpb and Apri Purification And Analysismentioning

confidence: 99%

Identification of SlpB, a Cytotoxic Protease from Serratia marcescens

et al. 2015

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“…5) from these proteins are found in relevant functional motifs observed in characterized AprA (Zhang et al, 2012), LipA (Duong et al, 1994), and ExoU (PriceWhelan et al, 2007) proteins from model Pseudomonas strains (right panels; Fig. 5).…”

Section: Discussionmentioning

confidence: 99%

Candidate nematicidal proteins in a new <i>Pseudomonas veronii</i> isolate identified by its antagonistic properties against <i>Xiphinema index</i>

Canchignia

Altimira

Montes

et al. 2017

J. Gen. Appl. Microbiol.

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“…It was deduced that several factors play important role to gain protein flexibility, including reduction of electrostatic non-covalent weak interactions (salt bridges, polar interactions between aromatic side chains, hydrogen bonds) and decrease of hydrophobicity [113,114]. Also, molecular adaptation appears to have induced looser surface loops, more polar surfaces exposed to solvent, lower affinity of Ca 2+ binding sites and less stabilized α-helices [71,111,115].…”

Section: Alkaline Proteases From Psychrophiles/psychrotrophsmentioning

confidence: 99%

“…The alkaline proteases of Pseudomonas sp. are generally Zn 2+ dependent metallozymes with a consensus zinc binding sequence at their N-terminal proteolytic domain [115,146,147]. Another well described zinc dependent metalloprotease is a 56 kDa aminopeptidase of by P. aeruginosa.…”

Section: Alkaline Proteases From Alkaliphilesmentioning

confidence: 99%

Extreme Environments: Goldmine of Industrially Valuable Alkali-stable Proteases

Garg¹,

Singh²

2015

Enz Eng

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Since the advent and release of Bio 40 (the first detergent with bacterial alkaline protease) in 1959, exploration of the microbial alkaline proteases has been exploited beyond expectations. The inventory of microbial alkaline proteases is difficult to draft as it is increasing daily. Most of these proteases are the result of studies in which one or a few isolates were targeted for an alkali-stable enzyme. Although, the worldwide research on microbial alkaline proteases has been widely performed, we still need an improved enzyme capable of catalyzing reactions under extreme conditions (also called as extremozymes), e.g., high alkalinity and salinity, anhydrous environment, cold and hot environment, etc. As the search for extremozymes is in progress, it is imperative to explore the extreme environments to get some novel microbial strains (extremophiles) for alkali-stable proteases. This review article is an effort to compile the scattered information on some extremophiles and their alkali-stable proteases, which may have better specific industrial applications. It includes: (i) various extreme environments relevant to industrial biocatalysts, (ii) strategies of extremophilic microbes and enzymes which make them able to tolerate such conditions, and (iii) an overview of some important work done so far to explore industrially relevant extremophilic enzymes from extremophiles.

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Calcium-induced Folding and Stabilization of the Pseudomonas aeruginosa Alkaline Protease

Cited by 54 publications

References 43 publications

Identification of SlpB, a Cytotoxic Protease from Serratia marcescens

Identification of SlpB, a Cytotoxic Protease from Serratia marcescens

Candidate nematicidal proteins in a new <i>Pseudomonas veronii</i> isolate identified by its antagonistic properties against <i>Xiphinema index</i>

Extreme Environments: Goldmine of Industrially Valuable Alkali-stable Proteases

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