Structure-function analysis of PrsA reveals roles for the parvulin-like and flanking N- and C-terminal domains in protein folding and secretion in Bacillus subtilis

The PrsA protein of Bacillus subtilis is an essential membrane-bound lipoprotein that is assumed to assist post-translocational folding of exported proteins and stabilize them in the compartment between the cytoplasmic membrane and cell wall. This folding activity is consistent with the homology of a segment of PrsA with parvulin-type peptidyl-prolyl cis/trans isomerases (PPIase). In this study, molecular modeling showed that the parvulin-like region can adopt a parvulin-type fold with structurally conserved active site residues. PrsA exhibits PPIase activity in a manner dependent on the parvulin-like domain. We constructed deletion, peptide insertion, and amino acid substitution mutations and demonstrated that the parvulin-like domain as well as flanking N- and C-terminal domains are essential for in vivo PrsA function in protein secretion and growth. Surprisingly, none of the predicted active site residues of the parvulin-like domain was essential for growth and protein secretion, although several active site mutations reduced or abolished the PPIase activity or the ability of PrsA to catalyze proline-limited protein folding in vitro. Our results indicate that PrsA is a PPIase, but the essential role in vivo seems to depend on some non-PPIase activity of both the parvulin-like and flanking domains.     

Posttranslocation chaperone PrsA2 regulates the maturation and secretion of Listeria monocytogenes proprotein virulence factors

PrsA2 is a conserved posttranslocation chaperone and a peptidyl prolyl cis-trans isomerase (PPIase) that contributes to the virulence of the Gram-positive intracellular pathogen Listeria monocytogenes. One of the phenotypes associated with a prsA2 mutant is decreased activity of the broad-range phospholipase C (PC-PLC). PC-PLC is made as a proenzyme whose maturation is mediated by a metalloprotease (Mpl). The proforms of PC-PLC and Mpl accumulate at the membrane-cell wall interface until a decrease in pH triggers their maturation and rapid secretion into the host cell. In this study, we examined the mechanism by which PrsA2 regulates the activity of PC-PLC. We observed that in the absence of PrsA2, the proenzymes are secreted at physiological pH and do not mature upon a decrease in pH. The sensitivity of the prsA2 mutant to cell wall hydrolases was modified. However, no apparent changes in cell wall porosity were detected. Interestingly, synthesis of PC-PLC in the absence of its propeptide lead to the secretion of a fully active enzyme in the cytosol of host cells independent of PrsA2, indicating that neither the propeptide of PC-PLC nor PrsA2 is required for native folding of the catalytic domain, although both influence secretion of the enzyme. Taken together, these results suggest that PrsA2 regulates compartmentalization of Mpl and PC-PLC, possibly by influencing cell wall properties and interacting with the PC-PLC propeptide. Moreover, the ability of these proproteins to respond to a decrease in pH during intracellular growth depends on their localization at the membrane-cell wall interface.     

Optimization of the cell wall microenvironment allows increased production of recombinant Bacillus anthracis protective antigen from B. subtilis.

The stability of heterologous proteins secreted by gram-positive bacteria is greatly influenced by the microenvironment on the trans side of the cytoplasmic membrane, and secreted heterologous proteins are susceptible to rapid degradation by host cell proteases. In Bacillus subtilis, degradation occurs either as the proteins emerge from the presecretory translocase and prior to folding into their native conformation or after the native conformation has been reached. The former process generally involves membrane- and/or cell wall-bound proteases, while the latter involves proteases that are released into the culture medium. The identification and manipulation of factors that influence the folding of heterologous proteins has the potential to improve the yield of secreted heterologous proteins. Recombinant anthrax protective antigen (rPA) has been used as a model secreted heterologous protein because it is sensitive to proteolytic degradation both before and after folding into its native conformation. This paper describes the influence of the microenvironment on the trans side of the cytoplasmic membrane on the stability of rPA. Specifically, we have determined the influence of net cell wall charge and its modulation by the extent to which the anionic polymer teichoic acid is D-alanylated on the secretion and stability of rPA. The potential role of the dlt operon, responsible for D-alanylation, was investigated using a Bacillus subtilis strain encoding an inducible dlt operon. We show that, in the absence of D-alanylation, the yield of secreted rPA is increased 2.5-fold. The function of D-alanylation and the use of rPA as a model protein are evaluated with respect to the optimization of B. subtilis for the secretion of heterologous proteins.     

Secretion of heterologous proteins in Bacillus subtilis can be improved by engineering cell components affecting post-translocational protein folding and degradation

AIMS: To explore the potential to enhance secretion of heterologous proteins in Bacillus subtilis by engineering cell factors affecting extracytoplasmic protein folding and degradation. METHODS AND RESULTS: Bottleneck components affecting the extracytoplasmic phase of protein secretion were genetically engineered and their effects on the secretion of 11 industrially interesting heterologous proteins were studied by Western blotting and enzymatic assays. Overproduction of PrsA lipoprotein enhanced the secretion of alpha-amylase of Bacillus stearothermophilus (fourfold) and pneumolysin (1.5-fold). Increasing the net negative charge of the cell wall because of lack of the d-alanine substitution of anionic cell wall polymers enhanced the secretion of pneumolysin c. 1.5-fold. Decreasing the level of HtrA-type quality control proteases caused harmful effects on growth and did not enhance secretion. Pertussis toxin subunit, S1 was found to be a substrate for HtrA-type proteases and its secretion was dependent on these proteases. CONCLUSIONS: Secretion of heterologous proteins can be enhanced by engineering components involved in late stages of secretion in a protein-dependent manner. SIGNIFICANCE AND IMPACT OF THE STUDY: The study revealed both possibilities and limitations of modulating the post-translocational phase of secretion as a means to improve the yield of heterologous proteins.     

Simultaneous inactivation of the wprA and dltB genes of Bacillus subtilis reduces the yield of alpha-amylase

AIMS: In Gram-positive bacteria, signal peptide-bearing secretory proteins are translocated through the cytoplasmic membrane and fold into their native conformation on the outside of the cell. The products of the Bacillus subtilis wprA and dltB genes separately influence post-translocational stages of the secretion process by mediating proteolytic degradation and folding of secretory proteins. Inactivation of either wprA or dltB in B. subtilis increases the yield of secretory proteins released into the culture medium in an intact and biologically active conformation. The aim of this work was to study the combined influence of these genes. METHODS AND RESULTS: A wprA/dltB double mutant was constructed, but did not have an additive effect on secretion and caused a significant reduction in the yield of alpha-amylase. CONCLUSIONS AND SIGNIFICANCE: The activities of the wprA gene and the dlt operon interact in a negative way to influence the growth cycle and protein secretion. The mechanism by which this may occur, and its potential significance for the secretion of native and non-native proteins from B. subtilis and related bacteria, is discussed.     

Role of the cell wall microenvironment in expression of a heterologous SpaP-S1 fusion protein by Streptococcus gordonii

The charge density in the cell wall microenvironment of Gram-positive bacteria is believed to influence the expression of heterologous proteins. To test this, the expression of a SpaP-S1 fusion protein, consisting of the surface protein SpaP of Streptococcus mutans and a pertussis toxin S1 fragment, was studied in the live vaccine candidate bacterium Streptococcus gordonii. Results showed that the parent strain PM14 expressed very low levels of SpaP-S1. By comparison, the dlt mutant strain, which has a mutation in the dlt operon preventing d-alanylation of the cell wall lipoteichoic acids, and another mutant strain, OB219(pPM14), which lacks the LPXTG major surface proteins SspA and SspB, expressed more SpaP-S1 than the parent. Both the dlt mutant and the OB219(pPM14) strain had a more negatively charged cell surface than PM14, suggesting that the negative charged cell wall played a role in the increase in SpaP-S1 production. Accordingly, the addition of Ca(2+), Mg(2+), and K(+), presumably increasing the positive charge of the cell wall, led to a reduction in SpaP-S1 production, while the addition of bicarbonate resulted in an increase in SpaP-S1 production. The level of SpaP-S1 production could be correlated with the level of PrsA, a peptidyl-prolyl cis/trans isomerase, in the cells. PrsA expression appears to be regulated by the cell envelope stress two-component regulatory system LiaSR. The results collectively indicate that the charge density of the cell wall microenvironment can modulate heterologous SpaP-S1 protein expression in S. gordonii and that this modulation is mediated by the level of PrsA, whose expression is regulated by the LiaSR two-component regulatory system.     

Bacillus subtilis PrsA is required in vivo as an extracytoplasmic chaperone for secretion of active enzymes synthesized either with or without pro-sequences

In prsA (protein secretion) mutants of Bacillus subtilis, decreased levels of exoproteins, including α-amylase and subtilisins, are found extracellularly. The effect of prsA on subtilisin secretion is elaborated here. Extracytoplasmic folding and secretion of active subtilisin is assisted by the N-terminal pro-sequence of its precursor. In this paper we present evidence that the product of the prsA gene is additionally required for these processes in vivo. We examined inducible expression of different subtilisin-alkaline phosphatase fusion genes in the prsA3 mutant. We found massive degradation of the fusion proteins, and a lack of enzymatic activity in the protein secreted. We suggest that PrsA is a novel chaperone with a predicted extracytoplasmic location, and is important in vivo for the proper conformation of various exoproteins, including those with pro-sequence (like subtilisin) and those without (like α-amylase).     

Degradation of Extracytoplasmic Catalysts for Protein Folding in Bacillus subtilis

The general protein secretion pathway of Bacillus subtilis has a high capacity for protein export from the cytoplasm, which is exploited in the biotechnological production of a wide range of enzymes. These exported proteins pass the membrane in an unfolded state, and accordingly, they have to fold into their active and protease-resistant conformations once membrane passage is completed. The lipoprotein PrsA and the membrane proteins HtrA and HtrB facilitate the extracytoplasmic folding and quality control of exported proteins. Among the native exported proteins of B. subtilis are at least 10 proteases that have previously been implicated in the degradation of heterologous secreted proteins. Recently, we have shown that these proteases also degrade many native membrane proteins, lipoproteins, and secreted proteins. The present studies were therefore aimed at assessing to what extent these proteases also degrade extracytoplasmic catalysts for protein folding. To this end, we employed a collection of markerless protease mutant strains that lack up to 10 different extracytoplasmic proteases. The results show that PrsA, HtrA, and HtrB are indeed substrates of multiple extracytoplasmic proteases. Thus, improved protein secretion by multiple-protease-mutant strains may be related to both reduced proteolysis and improved posttranslocational protein folding and quality control.     

Quantitation of the capacity of the secretion apparatus and requirement for PrsA in growth and secretion of alpha-amylase in Bacillus subtilis

Regulated expression of AmyQ alpha-amylase of Bacillus amyloliquefaciens was used to examine the capacity of the protein secretion apparatus of B. subtilis. One B. subtilis cell was found to secrete maximally 10 fg of AmyQ per h. The signal peptidase SipT limits the rate of processing of the signal peptide. Another limit is set by PrsA lipoprotein. The wild-type level of PrsA was found to be 2 x 10(4) molecules per cell. Decreasing the cellular level of PrsA did not decrease the capacity of the protein translocation or signal peptide processing steps but dramatically affected secretion in a posttranslocational step. There was a linear correlation between the number of cellular PrsA molecules and the number of secreted AmyQ molecules over a wide range of prsA and amyQ expression levels. Significantly, even when amyQ was expressed at low levels, overproduction of PrsA enhanced its secretion. The finding is consistent with a reversible interaction between PrsA and AmyQ. The high cellular level of PrsA suggests a chaperone-like function. PrsA was also found to be essential for the viability of B. subtilis. Drastic depletion of PrsA resulted in altered cellular morphology and ultimately in cell death.     

Penicillin‐binding protein folding is dependent on the PrsA peptidyl‐prolyl cis‐trans isomerase in Bacillus subtilis

The PrsA protein is a membrane‐anchored peptidyl‐prolyl cis‐trans isomerase in Bacillus subtilis and most other Gram‐positive bacteria. It catalyses the post‐translocational folding of exported proteins and is essential for normal growth of B. subtilis. We studied the mechanism behind this indispensability. We could construct a viable prsA null mutant in the presence of a high concentration of magnesium. Various changes in cell morphology in the absence of PrsA suggested that PrsA is involved in the biosynthesis of the cylindrical lateral wall. Consistently, four penicillin‐binding proteins (PBP2a, PBP2b, PBP3 and PBP4) were unstable in the absence of PrsA, while muropeptide analysis revealed a 2% decrease in the peptidoglycan cross‐linkage index. Misfolded PBP2a was detected in PrsA‐depleted cells, indicating that PrsA is required for the folding of this PBP either directly or indirectly. Furthermore, strongly increased uniform staining of cell wall with a fluorescent vancomycin was observed in the absence of PrsA. We also demonstrated that PrsA is a dimeric or oligomeric protein which is localized at distinct spots organized in a helical pattern along the cell membrane. These results suggest that PrsA is essential for normal growth most probably as PBP folding is dependent on this PPIase.     

A gene (prsA) of Bacillus subtilis involved in a novel, late stage of protein export

A gene locus of Bacillus subtilis identified by mutations (prs) conferring a defect in protein secretion was cloned from a lambdaGEM-11 expression library. The sites of three closely linked prs mutations (prs-3, prs-29 and prs-40) were found to reside in a 5.3 kb DNA fragment, which also complemented the secretion defect in prs-3 and prs-29 mutants. Partial sequencing of the fragment showed that these three mutations affect one distinct gene (prsA) encoding a putative protein of 292 amino acids (33 kDa). Sequence analysis indicated the PrsA protein to be a lipoprotein located outside the cytoplasmic membrane. Thirty percent identity was shown to the PrtM protein of Lactococcus lactis, which is involved in the maturation of an exported proteinase. The phenotypes of prsA mutants and the structural similarity of PrsA with PrtM suggest that PrsA may have a novel function at a late phase in protein export.     

Production of Bacillus anthracis protective antigen is dependent on the extracellular chaperone,PrsA

Protective antigen (PA) is a component of the Bacillus anthracis lethal and edema toxins and the basis of the current anthrax vaccine. In its heptameric form, PA targets host cells and internalizes the enzymatically active components of the toxins, namely lethal and edema factors. PA and other toxin components are secreted from B. anthracis using the Sec-dependent secretion pathway. This requires them to be translocated across the cytoplasmic membrane in an unfolded state and then to be folded into their native configurations on the trans side of the membrane, prior to their release from the environment of the cell wall. In this study we show that recombinant PA (rPA) requires the extracellular chaperone PrsA for efficient folding when produced in the heterologous host, B. subtilis; increasing the concentration of PrsA leads to an increase in rPA production. To determine the likelihood of PrsA being required for PA production in its native host, we have analyzed the B. anthracis genome sequence for the presence of genes encoding homologues of B. subtilis PrsA. We identified three putative B. anthracis PrsA proteins (PrsAA, PrsAB, and PrsAC) that are able to complement the activity of B. subtilis PrsA with respect to cell viability and rPA secretion, as well as that of AmyQ, a protein previously shown to be PrsA-dependent.     

The peptidyl-prolyl isomerase motif is lacking in PmpA,the PrsA-like protein involved in the secretion machinery of Lactococcus lactis

The prsA-like gene from Lactococcus lactis encoding its single homologue to PrsA, an essential protein triggering the folding of secreted proteins in Bacillus subtilis, was characterized. This gene, annotated pmpA, encodes a lipoprotein of 309 residues whose expression is increased 7- to 10-fold when the source of nitrogen is limited. A slight increase in the expression of the PrsA-like protein (PLP) in L. lactis removed the degradation products previously observed with the Staphylococcus hyicus lipase used as a model secreted protein. This shows that PmpA either triggers the folding of the secreted lipase or activates its degradation by the cell surface protease HtrA. Unlike the case for B. subtilis, the inactivation of the gene encoding PmpA reduced only slightly the growth rate of L. lactis in standard conditions. However, it almost stopped its growth when the lipase was overexpressed in the presence of salt in the medium. Like PrsA of B. subtilis and PrtM of L. lactis, the L. lactis PmpA protein could thus have a foldase activity that facilitates protein secretion. These proteins belong to the third family of peptidyl-prolyl cis/trans-isomerases (PPIases) for which parvulin is the prototype. Almost all PLP from gram-positive bacteria contain a domain with the PPIase signature. An exception to this situation was found only in Streptococcaceae, the family to which L. lactis belongs. PLP from Streptococcus pneumoniae and Enterococcus faecalis possess this signature, but those of L. lactis, Streptococcus pyogenes, and Streptococcus mutans do not. However, secondary structure predictions suggest that the folding of PLP is conserved over the entire length of the proteins, including the unconserved signature region. The activity associated with the expression of PmpA in L. lactis and these genomic data show that either the PPIase motif is not necessary for PPIase activity or, more likely, PmpA foldase activity does not necessarily require PPIase activity.     

Functional analysis of the Listeria monocytogenes secretion chaperone PrsA2 and its multiple contributions to bacterial virulence

As an organism that has evolved to live in environments ranging from soil to the cytosol of mammalian cells, Listeria monocytogenes must regulate the secretion and activity of protein products that promote survival within these habitats. The post-translocation chaperone PrsA2 has been adapted to assist in the folding and activity of L. monocytogenes secreted proteins required for bacterial replication within host cells. Here we present the first structure/function investigation of the contributions of PrsA2 to protein secretion and activity as well as to bacterial virulence. Domain swap experiments with the closely related L. monocytogenes PrsA1 protein combined with targeted mutagenesis indicate distinct functional roles for the PrsA2 peptidyl-prolyl isomerase (PPIase) and the N- and C-terminal domains in pathogenesis. In contrast to other PrsA-like proteins described thus far in the literature, an absolute in vivo requirement for PrsA2 PPIase activity is evident in mouse infection models. This work illustrates the diversity of function associated with L. monocytogenes PrsA2 that serves to promote bacterial life within the infected host.     

单增李斯特菌prsA1基因的截短表达、纯化及多克隆抗体的制备

单增李斯特菌（Listeria monocytogenes）是一种常见的食源性致病菌,能够引发李斯特菌病,对食品安全构成巨大威胁。prsA1具有保守性和特异性,利用SignalP 4.1 Server程序、TMHMM Server V.2.0程序和SEPPA 2.0程序预测了PrsA1的信号肽段、跨膜区域及空间抗原表位,预测结果显示PrsA1的N端含有信号肽段及跨膜区且该蛋白具有良好抗原表位结构,因而可作为检测靶标。在此基础上,采用PCR法获得prsA1的非跨膜区序列即Δ84prsA1,构建重组质粒pET30a-Δ84prsA1并转入到大肠杆菌中诱导表达Δ28PrsA1,Ni-IDA柱亲和纯化重组蛋白Δ28PrsA1,以纯化的Δ28PrsA1为抗原制备多克隆抗体。间接ELISA检测多克隆抗体的效价,高达1∶128 000。Western blotting分析结果显示该多克隆抗体能够识别从单增李斯特菌中提取的PrsA1蛋白。利用生物信息学筛选检测靶标并分析抗原表位结构,最后成功制备了多克隆抗体,为单增李斯特菌检测靶标的筛选和免疫学检测提供了实践基础。     

The plant apoplasm is an important recipient compartment for nematode secreted proteins

Similarly to microbial pathogens, plant-parasitic nematodes secrete into their host plants proteins that are essential to establish a functional interaction. Identifying the destination of nematode secreted proteins within plant cell compartment(s) will provide compelling clues on their molecular functions. Here the fine localization of five nematode secreted proteins was analysed throughout parasitism in Arabidopsis thaliana. An immunocytochemical method was developed that preserves both the host and the pathogen tissues, allowing the localization of nematode secreted proteins within both organisms. One secreted protein from the amphids and three secreted proteins from the subventral oesophageal glands involved in protein degradation and cell wall modification were secreted in the apoplasm during intercellular migration and to a lower extent by early sedentary stages during giant cell formation. Conversely, another protein produced by both subventral and dorsal oesophageal glands in parasitic stages accumulated profusely at the cell wall of young and mature giant cells. In addition, secretion of cell wall-modifying proteins by the vulva of adult females suggested a role in egg laying. The study shows that the plant apoplasm acts as an important destination compartment for proteins secreted during migration and during sedentary stages of the nematode.     

Bacillus subtilis alpha-amylase: interactions of a partially folded conformer with small unilamellar vesicles

We studied the interactions between conformers of exocellular alpha-amylase and small unilamellar vesicles (SUV) composed of the major membrane lipids of Bacillus subtilis under physiological conditions of pH, temperature and ionic strength. Using fluorescence spectroscopy, surface plasmon resonance (SPR) and phase separation, we show that the native alpha-amylase has no affinity for the SUV, whereas a partially folded form, displaying structural properties in common with the competent state for secretion, binds to the vesicles (KA approximately 10(5) M(-1)). This association prevented its subsequent folding. The complex was destabilized in the presence of PrsA, a major peripheric lipoprotein of B. subtilis which displays a strong affinity for SUV (KA approximately 1.5x10(8) M(-1)). Vesicles coated with PrsA lost their ability to bind the partially folded conformer. The approach in vitro, in which our aim was to mimic the last stage of alpha-amylase translocation, indicates that PrsA possibly helps, in vivo, the secreted protein to acquire its native conformation by modulating the interaction between the latter and the lipid polar heads on the trans side of the cytoplasmic membrane.     

The Peptidyl-Prolyl Isomerase Motif Is Lacking in PmpA, the PrsA-Like Protein Involved in the Secretion Machinery of Lactococcus lactis

The prsA-like gene from Lactococcus lactis encoding its single homologue to PrsA, an essential protein triggering the folding of secreted proteins in Bacillus subtilis, was characterized. This gene, annotated pmpA, encodes a lipoprotein of 309 residues whose expression is increased 7- to 10-fold when the source of nitrogen is limited. A slight increase in the expression of the PrsA-like protein (PLP) in L. lactis removed the degradation products previously observed with the Staphylococcus hyicus lipase used as a model secreted protein. This shows that PmpA either triggers the folding of the secreted lipase or activates its degradation by the cell surface protease HtrA. Unlike the case for B. subtilis, the inactivation of the gene encoding PmpA reduced only slightly the growth rate of L. lactis in standard conditions. However, it almost stopped its growth when the lipase was overexpressed in the presence of salt in the medium. Like PrsA of B. subtilis and PrtM of L. lactis, the L. lactis PmpA protein could thus have a foldase activity that facilitates protein secretion. These proteins belong to the third family of peptidyl-prolyl cis/trans-isomerases (PPIases) for which parvulin is the prototype. Almost all PLP from gram-positive bacteria contain a domain with the PPIase signature. An exception to this situation was found only in Streptococcaceae, the family to which L. lactis belongs. PLP from Streptococcus pneumoniae and Enterococcus faecalis possess this signature, but those of L. lactis, Streptococcus pyogenes, and Streptococcus mutans do not. However, secondary structure predictions suggest that the folding of PLP is conserved over the entire length of the proteins, including the unconserved signature region. The activity associated with the expression of PmpA in L. lactis and these genomic data show that either the PPIase motif is not necessary for PPIase activity or, more likely, PmpA foldase activity does not necessarily require PPIase activity.