Cyclophilin and Trigger Factor from Bacillus subtilis Catalyze in Vitro Protein Folding and Are Necessary for Viability under Starvation Conditions

Göthel, Sven F.; Schölz, Christian; Schmid, Franz X.; Marahiel, Mohamed A.

doi:10.1021/bi981253w

Cited by 63 publications

(45 citation statements)

References 45 publications

(70 reference statements)

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“…Similarly, two out of four PPIases have been deleted from Bacillus subtilis, with no phenotypic consequence in rich medium or under several stress conditions tested. The double mutant showed a strongly retarded growth behavior only in the absence of all amino acids except tryptophan and phenylalanine (65). In Erwinia chrysanthemi, disruption of rotA caused no change in cell morphology, cell viability, growth rate, or stability of the extracellular and periplasmic proteins (66).…”

Section: Discussionmentioning

confidence: 97%

The Periplasmic Escherichia coli Peptidylprolyl cis,trans-Isomerase FkpA

Bothmann¹,

Plückthun

2000

Journal of Biological Chemistry

155

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The production of recombinant proteins in the periplasm of Escherichia coli can be limited by folding problems, leading to periplasmic aggregates. We used a selection system for periplasmic chaperones based on the coexpression of an E. coli library with a poorly expressing antibody single-chain Fv (scFv) fragment displayed on filamentous phage (Bothmann, H., and Plü ckthun, A. (1998) Nature Biotechnol. 16, 376 -380). By selection for a functional antibody, the protein Skp had been enriched previously and shown to improve periplasmic expression of a wide range of scFv fragments. This selection strategy was now repeated with a library constructed from the genomic DNA of an skpdeficient strain, leading to enrichment of the periplasmic peptidylprolyl cis,trans-isomerase ( An important strategy for the production of recombinant disulfide-containing proteins is their secretion to the periplasmic space of Escherichia coli, as the periplasm is the location of the disulfide-forming machinery (1, 2). Antibodies are one class of particularly important disulfide-containing proteins that have frequently been expressed in this compartment, and this is probably the most convenient method to obtain engineered antibodies for research and medical or technological applications (3-5). Unfortunately, folding problems of antibodies and other heterologous proteins in the periplasm are frequently observed, and the nature and existence of molecular chaperones in this compartment are still only poorly understood (2, 6, 7).It has been previously shown (8 -12) that the functional periplasmic expression of antibody fragments can be limited by the sequence-dependent, moderate efficiency of periplasmic folding, which can furthermore be accompanied by cell lysis, as has also been observed for other proteins (13). Typically, the actual membrane transport is not limiting (8), and large amounts of correctly processed, but precipitated protein are available in the periplasm. It is still unclear which factors, if any, might play a role in preventing the aggregation and in guiding the correct folding of functional antibody fragments or other periplasmic proteins in E. coli, and it is certainly conceivable that different factors play a role for different proteins.We have recently developed a selection system for the identification of novel factors that may help the functional periplasmic expression of a substrate protein in question (14). It was based on the assumption that the folding of soluble periplasmic protein occurs in contact with the same machinery and in the same environment as that of a protein displayed on filamentous phage. Fusion proteins with the minor coat protein gene-3 protein (g3p) 1 of filamentous phage are transiently produced in a state anchored to the inner membrane, before they are incorporated into the phage coat (15). Thus, the folding of the fusion protein occurs in the periplasm and should be influenced by the same factors as that of a soluble periplasmic protein.The selection system works by displaying a poorly expressing...

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Section: Discussionmentioning

confidence: 97%

The Periplasmic Escherichia coli Peptidylprolyl cis,trans-Isomerase FkpA

Bothmann¹,

Plückthun

2000

Journal of Biological Chemistry

155

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“…One known example of a cytoplasmic cyclophilin is the PpiB protein from B. subtilis (39). Deletion of the ppiB gene has no effect on bacterial growth/viability in rich medium; however, a growth defect was observed under starvation conditions (40). The reason for this growth defect is unknown, and no effect on protein secretion was demonstrated.…”

Section: Discussionmentioning

confidence: 99%

An Intracellular Peptidyl-Prolyl cis / trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease

et al. 2017

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Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus.IMPORTANCE Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently, once outside the cell, they must refold into their active form. This step often requires the assistance of bacterial folding proteins, such as PPIases. In this work, we investigate the role of PPIases in S. aureus and uncover a cyclophilin-type enzyme that assists in the folding/refolding of staphylococcal nuclease.KEYWORDS cyclophilin, Nuc, PI-PLC, PPIase, parvulin, PpiB, PrsA, Staphylococcus aureus, nuclease, protease T he proline peptide bond is unique in nature in that both the cis and trans forms can occur in vivo, with the cis conformation existing approximately 6.5% of the time (1). In contrast, for all other naturally occurring amino acids, steric hindrance between side chains precludes the cis form and overwhelmingly favors the trans form (2). The presence of both the cis and trans forms of proline peptide bonds has important consequences for protein tertiary structure. In certain cases, the isomerization state of

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“…In addition, recent results indicate that secretory proteins are directed into the SecB-SecA-mediated posttranslational targeting pathway by means of their preferential recognition by TF (15). In B. subtilis, it was shown that TF, together with a second cytoplasmic PPIase (cyclophilin, also called PpiB), ac- counts for the entire PPIase activity in the cytoplasm (107). A direct involvement of TF or cyclophilin in protein secretion by B. subtilis has not been reported.…”

Section: Cytosolic Chaperonesmentioning

confidence: 99%

Signal Peptide-Dependent Protein Transport in Bacillus subtilis : a Genome-Based Survey of the Secretome

Tjalsma

Bolhuis

Jongbloed

et al. 2000

Microbiol Mol Biol Rev

748

768

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SUMMARY One of the most salient features of Bacillus subtilis and related bacilli is their natural capacity to secrete a variety of proteins into their environment, frequently to high concentrations. This has led to the commercial exploitation of bacilli as major “cell factories” for secreted enzymes. The recent sequencing of the genome of B. subtilis has provided major new impulse for analysis of the molecular mechanisms underlying protein secretion by this organism. Most importantly, the genome sequence has allowed predictions about the composition of the secretome, which includes both the pathways for protein transport and the secreted proteins. The present survey of the secretome describes four distinct pathways for protein export from the cytoplasm and approximately 300 proteins with the potential to be exported. By far the largest number of exported proteins are predicted to follow the major “Sec” pathway for protein secretion. In contrast, the twin-arginine translocation “Tat” pathway, a type IV prepilin-like export pathway for competence development, and ATP-binding cassette transporters can be regarded as “special-purpose” pathways, through which only a few proteins are transported. The properties of distinct classes of amino-terminal signal peptides, directing proteins into the various protein transport pathways, as well as the major components of each pathway are discussed. The predictions and comparisons in this review pinpoint important differences as well as similarities between protein transport systems in B. subtilis and other well-studied organisms, such as Escherichia coli and the yeast Saccharomyces cerevisiae. Thus, they may serve as a lead for future research and applications.

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Cyclophilin and Trigger Factor from Bacillus subtilis Catalyze in Vitro Protein Folding and Are Necessary for Viability under Starvation Conditions

Cited by 63 publications

References 45 publications

The Periplasmic Escherichia coli Peptidylprolyl cis,trans-Isomerase FkpA

The Periplasmic Escherichia coli Peptidylprolyl cis,trans-Isomerase FkpA

An Intracellular Peptidyl-Prolyl cis / trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease

Signal Peptide-Dependent Protein Transport in Bacillus subtilis : a Genome-Based Survey of the Secretome

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