The prepilin peptidase is required for protein secretion by and the virulence of the intracellular pathogen Legionella pneumophila

Prepilin peptidases cleave, among other substrates, the leader sequences from prepilin-like proteins that are required for type II protein secretion in Gram-negative bacteria. To begin to assess the importance of type II secretion for the virulence of an intracellular pathogen, we examined the effect of inactivating the prepilin peptidase (pilD) gene of Legionella pneumophila. Although the pilD mutant and its parent grew similarly in bacteriological media, they did differ in colony attributes and recoverability from late stationary phase. Moreover, at least three proteins were absent from the mutant's supernatant, indicating that PilD is necessary for the secretion of Legionella proteins. The absence of both the major secreted protein and a haemolytic activity from the mutant signalled that the L. pneumophila zinc metalloprotease is excreted via type II secretion. Most interestingly, the pilD mutant was greatly impaired in its ability to grow within Hartmannella vermiformis amoebae and the human macrophage-like U937 cells. As reintroduction of pilD into the mutant restored inefectivity and as a mutant lacking type IV pilin replicated like wild type, these data suggested that the intracellular growth of L. pneumophila is promoted by proteins secreted via a type II pathway. Intratracheal inoculation of guinea pigs revealed that the LD50 for the pilD mutant is at least 100-fold greater than that for its parent, and the culturing of bacteria from infected animals showed a rapid clearance of the mutant from the lungs. This is the first study to indicate a role for PilD and type II secretion in intracellular parasitism.     

A recombinant metalloprotease antigen of Vibrio vulnificus elicits protective antibodies in a murine model

Aims: To investigate whether Vibrio vulnificus metalloprotease (VvpE) can induce the production of specific anti‐VvpE antibody to confer effective protection against Vibrio vulnificus infection and to evaluate the possibility of VvpE as a potential vaccine candidate against disease caused by V. vulnificus. Methods and Results: The gene encoding the 65‐kDa VvpE of V. vulnificus was amplified by PCR and cloned into the expression vector pET21(b). The recombinant VvpE of V. vulnificus was expressed in Escherichia coli BL21(DE3). This His₆‐tagged VvpE was purified and injected intramuscularly into mice to evaluate its ability to stimulate immune response. Specific antibody levels were measured by ELISA. The 75% protective efficacy of recombinant VvpE was evaluated by active immunization and intraperitoneal challenge with V. vulnificus in mice. Conclusions: The recombinant His₆‐tagged VvpE of V. vulnificus is capable of inducing high antibody response in mice to confer effective protection against lethal challenge with V. vulnificus. VvpE might be a potential vaccine candidate to against V. vulnificus infection. Significance and Impact of the Study: This study uses His₆‐tagged VvpE to act as vaccine that successfully induces effective and specific anti‐VvpE antibody and offers an option for the potential vaccine candidate against V. vulnificus infection.     

Vibrio vulnificus metalloprotease VvpE is essentially required for swarming

Bacterial swarming constitutes a good in vitro model for surface adherence and colonization, and is accompanied by expressions of virulence factors related to invasiveness. In this study, it was determined that Vibrio vulnificus swarming was abolished by mutation of the vvpE gene encoding a metalloprotease VvpE and this swarming defect was recovered by complementation of the vvpE gene. Expression of the vvpE gene began simultaneously with the beginning of swarming and increased along with expression of the luxS gene encoding the synthase of the precursor of quorum-sensing signal molecule autoinducer 2, and this increased vvpE expression was decreased by mutation of the luxS gene. Moreover, VvpE destroyed IgA and lactoferrins, which are responsible for mucosal immunity. These results suggest that VvpE may play important roles in the surface adherence and colonization of V. vulnificus by facilitating swarming and in the mucosal invasion of V. vulnificus by destroying IgA and lactoferrin.     

Identification and characterization of a novel serine protease, VvpS, that contains two functional domains and is essential for autolysis of Vibrio vulnificus

Little is known about the molecular mechanism for autolysis of Gram-negative bacteria. In the present study, we identified the vvpS gene encoding a serine protease, VvpS, from Vibrio vulnificus, a Gram-negative food-borne pathogen. The amino acid sequence predicted that VvpS consists of two functional domains, an N-terminal protease catalytic domain (PCD) and a C-terminal carbohydrate binding domain (CBD). A null mutation of vvpS significantly enhanced viability during stationary phase, as measured by enumerating CFU and differentially staining viable cells. The vvpS mutant reduced the release of cytoplasmic β-galactosidase and high-molecular-weight extracellular chromosomal DNA into the culture supernatants, indicating that VvpS contributes to the autolysis of V. vulnificus during stationary phase. VvpS is secreted via a type II secretion system (T2SS), and it exerts its effects on autolysis through intracellular accumulation during stationary phase. Consistent with this, a disruption of the T2SS accelerated intracellular accumulation of VvpS and thereby the autolysis of V. vulnificus. VvpS also showed peptidoglycan-hydrolyzing activity, indicating that the autolysis of V. vulnificus is attributed to the self-digestion of the cell wall by VvpS. The functions of the VvpS domains were assessed by C-terminal deletion analysis and demonstrated that the PCD indeed possesses a proteolytic activity and that the CBD is required for hydrolyzing peptidoglycan effectively. Finally, the vvpS mutant exhibited reduced virulence in the infection of mice. In conclusion, VvpS is a serine protease with a modular structure and plays an essential role in the autolysis and pathogenesis of V. vulnificus.     

The type IV pre-pilin leader peptidase of Xanthomonas campestris pv. campestris is functional without conserved cysteine residues

Type IV pre-pilin leader peptidase was demonstrated to be required for protein secretion, in addition to its involvement in biogenesis of type IV pili. The type IV pre-pilin leader peptidase gene of Xanthomonas campestris pv. campestris was located on a 3 kb Acc l fragment on account of its hybridization with the DNA fragment containing the type IV pre-pilin leader-peptidase gene pilD/xcpA of Pseudomonas aeruginosa . Sequencing of the cloned fragment revealed an open reading frame (ORF) (designated xpsO ) of 287 amino acid residues. A protein with an apparent molecular mass of approximately 32.5 kDa was synthesized in vitro from a DNA fragment containing the xpsO gene. The amino acid sequence shares 50% identity with that of PilD throughout the entire sequence. Among other type IV pre-pilin leader peptidases, XpsO is unique in not having the two conserved -CXXC- motifs in a cytoplasmic domain. Instead, new motifs were noted when the protein was compared with XpsE, which is another member of the extracellular protein-secretion machinery. When the xpsO gene was introduced into the pilD mutant of P. aeruginosa , both the sensitivity against infection with the pilus-specific phage PO4 and the ability to secrete extracellular protein were recovered. Furthermore, immunoblot analysis indicated that the P. aeruginosa pilin was apparently processed in vivo by the xpsO gene product.     

Cloning of an Aeromonas hydrophila type IV pilus biogenesis gene cluster: complementation of pilus assembly functions and characterization of a type IV leader peptidase/N-methyltransferase required for extracellular protein secretion

Aeromonas hydrophila secretes several extracellular proteins that are associated with virulence including an enterotoxin, a protease, and the hole-forming toxin, aerolysin. These degradative enzymes and toxins are exported by a conserved pathway found in many Gram-negative bacteria. In Pseudomonas aeruginosa this export pathway and type IV pilus biogenesis are dependent on the product of the pilD gene. PilD is a bifunctional enzyme that processes components of the extracellular secretory pathway as well as a type IV prepilin. An A. hydrophila genomic library was transferred into a P. aeruginosa pilD mutant that is defective for type IV pilus biogenesis. The A. hydrophila pilD homologue, tapD , was identified by its ability to complement the pilD mutation in P. aeruginosa . Transconjugants containing tapD were sensitive to the type IV pilus-specific phage, PO4. Sequence data revealed that tapD is part of a cluster of genes ( tapABCD ) that are homologous to P. aeruginosa type IV pilus biogenesis genes ( pilABCD ). We showed that TapB and TapC are functionally homologous to P. aeruginosa PilB and PilC, the first such functional complementation of pilus assembly demonstrated between bacteria that express type IV pili. In vitro studies revealed that TapD has both endopeptidase and N -methyltransferase activities using P. aeruginosa prepilin as substrate. Furthermore, we show that tapD is required for extracellular secretion of aerolysin and protease, indicating that tapD may play an important role in the virulence of A. hydrophila     

Cysteine peptidases in the tomato trypanosomatid Phytomonas serpens: influence of growth conditions, similarities with cruzipain and secretion to the extracellular environment

We have characterized the cysteine peptidase production by Phytomonas serpens, a tomato trypanosomatid. The parasites were cultivated in four distinct media, since growth conditions could modulate the synthesis of bioactive molecules. The proteolytic profile has not changed qualitatively regardless the media, showing two peptidases of 38 and 40 kDa; however, few quantitative changes were observed including a drastic reduction (around 70%) on the 40 and 38 kDa peptidase activities when parasites were grown in yeast extract and liver infusion trypticase medium, respectively, in comparison with parasites cultured in Warren medium. The time-span of growth did not significantly alter the protein and peptidase expression. The proteolytic activities were blocked by classical cysteine peptidase inhibitors (E-64, leupeptin, and cystatin), being more active at pH 5.0 and showing complete dependence to reducing agents (dithiothreitol and l-cysteine) for full activity. The cysteine peptidases were able to hydrolyze several proteinaceous substrates, including salivary gland proteins from Oncopeltus fasciatus, suggesting broad substrate utilization. By means of agglutination, fluorescence microscopy, flow cytometry and Western blotting analyses we showed that both cysteine peptidases produced by P. serpens share common epitopes with cruzipain, the major cysteine peptidase of Trypanosoma cruzi. Moreover, our data suggest that the 40 kDa cysteine peptidase was located at the P. serpens cell surface, attached to membrane domains via a glycosylphosphatidylinositol anchor. The 40 kDa peptidase was also detected in the cell-free culture supernatant, in an active form, which suggests secretion of this peptidase to the extracellular environment.     

Identification of the Vibrio cholerae type 4 prepilin peptidase required for cholera toxin secretion and pilus formation

Cholera toxin secretion is dependent upon the extracellular protein secretion apparatus encoded by the eps gene locus of Vibrio cholerae . Although the eps gene locus encodes several type four prepilin-like proteins, the peptidase responsible for processing these proteins has not been identified. This report describes the identification of a prepilin peptidase from the V. cholerae genomic database by virtue of its homology with the PilD prepilin peptidase of Pseudomonas aeruginosa . Plasmid disruption or deletion of this peptidase gene in either El Tor or classical V. cholerae O1 biotype strains results in a dramatic decrease in cholera toxin secretion. In the case of the El Tor biotype mutants, surface expression of the type 4 pilus responsible for mannose-sensitive haemagglutination is abolished. The cloned V. cholerae peptidase processes either EpsI or MshA preproteins when co-expressed in E. coli . Mutation of the V. cholerae peptidase gene also results in a defect in virulence and decreased levels of OmpU. The V. cholerae peptidase gene sequence shows 80% homology with the Vibrio vulnificus VvpD type 4 prepilin peptidase required for pilus assembly and cytolysin secretion in V. vulnificus . Accordingly, the V. cholerae type 4 prepilin peptidase required for pilus assembly and cholera toxin secretion has been designated VcpD.     

Biology of type II secretion

The type II secretion pathway or the main terminal branch of the general secretion pathway, as it has also been referred to, is widely distributed among Proteobacteria, in which it is responsible for the extracellular secretion of toxins and hydrolytic enzymes, many of which contribute to pathogenesis in both plants and animals. Secretion through this pathway differs from most other membrane transport systems, in that its substrates consist of folded proteins. The type II secretion apparatus is composed of at least 12 different gene products that are thought to form a multiprotein complex, which spans the periplasmic compartment and is specifically required for translocation of the secreted proteins across the outer membrane. This pathway shares many features with the type IV pilus biogenesis system, including the ability to assemble a pilus-like structure. This review discusses recent findings on the organization of the secretion apparatus and the role of its various components in secretion. Different models for pilus-mediated secretion through the gated pore in the outer membrane are also presented, as are the possible properties that determine whether a protein is recognized and secreted by the type II pathway.     

Overexpression of Methanococcus voltae Flagellin Subunits in Escherichia coli and Pseudomonas aeruginosa: a Source of Archaeal Preflagellin

Methanococcus voltae is a flagellated member of the Archaea. Four highly similar flagellin genes have previously been cloned and sequenced, and the presence of leader peptides has been demonstrated. While the flagellins of M. voltae are predicted from their gene sequences to be approximately 22 to 25 kDa, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified flagella revealed flagellin subunits with apparent molecular masses of 31 and 33 kDa. Here we describe the expression of a M. voltae flagellin in the bacteria Escherichia coli and Pseudomonas aeruginosa. Both of these systems successfully generated a specific expression product with an apparently uncleaved leader peptide migrating at approximately 26.5 kDa. This source of preflagellin was used to detect the presence of preflagellin peptidase activity in the membranes of M. voltae. In addition to the native flagellin, a hybrid flagellin gene containing the sequence encoding the M. voltae FlaB2 mature protein fused to the P. aeruginosa pilin (PilA) leader peptide was constructed and transformed into both wild-type P. aeruginosa and a prepilin peptidase (pilD) mutant of P. aeruginosa. Based on migration in SDS-PAGE, the leader peptide appeared to be cleaved in the wild-type cells. However, the archaeal flagellin could not be detected by immunoblotting when expressed in the pilD mutant, indicating a role of the peptidase in the ultimate stability of the fusion product. When the +5 position of the mature flagellin portion of the pilin-flagellin fusion was changed from glycine to glutamic acid (as in the P. aeruginosa pilin) and expressed in both wild-type and pilD mutant P. aeruginosa, the product detected by immunoblotting migrated slightly more slowly in the pilD mutant, indicating that the fusion was likely processed by the prepilin peptidase present in the wild type. Potential assembly of the cleaved fusion product by the type IV pilin assembly system in a P. aeruginosa PilA-deficient strain was tested, but no filaments were noted on the cell surface by electron microscopy.     

Expression and processing of Vibrio anguillarum zinc-metalloprotease in Escherichia coli

The extracellular zinc-metalloprotease of Vibrio anguillarum is a secreted virulence factor. It is synthesized from the empA gene as a 611-residue preproprotease and processed to the active mature protease (EmpA) with concomitant secretion via the type II secretion pathway. Active EmpA has been found only in the V. anguillarum culture supernatant and the process of the activation seems to vary depending on strains analyzed. To better understand the mechanism of EmpA export and processing, the empA gene was cloned and expressed in Escherichia coli strains. Expression of empA did not have toxic effect on bacterial growth. Rupturing E. coli TOP10 cells by heating in gel-loading buffer resulted in activation of EmpA and severe proteolysis of the samples. In contrast, the same treatment of the E. coli MC4100A strain did not lead to the general proteolysis. In this strain, EmpA was exported into the periplasm via the Sec pathway. The periplasmic EmpA was detected in two active conformations. Therefore, in E. coli processing of EmpA precursor to an active enzyme did not require secretion to the media and the help of other V. anguillarum protein. Like in V. anguillarum, heterologous expression of empA in E. coli showed strain-specific activation process.     

The role of calcium in the secretion of surfactant by rat alveolar type II cells

Beta adrenergic agonists, tetradecanoylphorbol acetate, and the ionophore A23187 all stimulate surfactant secretion in type II cells isolated from rats. We found that combinations of these agonists cause augmented secretion, suggesting that the agonists may effect different steps in the secretory process. Previous studies have shown that cAMP is likely to be an intracellular 'second messenger' in type II cells. A23187, which has been reported to increase cAMP in some cell systems, did not increase the cAMP content of type II cells. We investigated the possible role of Ca2+ as another 'second messenger' by studying cellular 45Ca fluxes and the effect of extracellular calcium depletion on secretion. Depletion of extracellular calcium for as long as 3 h did not alter stimulated secretion, although basal secretion was increased. Secretagogues did not stimulate 45Ca influx from extracellular sources. A23187 and, to a lesser extent, terbutaline caused an acceleration of 45Ca efflux from type II cells. The addition of terbutaline or tetradecanoylphorbol acetate to A23187 further accelerated 45Ca efflux, suggesting that these agonists may act on separate calcium pools or by different mechanisms on the same calcium pool. Although secretion from type II cells is not inhibited by extracellular calcium depletion, the studies on 45Ca efflux suggest that Ca2+ plays a role in the regulation of surfactant secretion from isolated type II cells.     

Characterization of type IV pilus genes in plant growth-promoting Pseudomonas putida WCS358.

In a search for factors that could contribute to the ability of the plant growth-stimulating Pseudomonas putida WCS358 to colonize plant roots, the organism was analyzed for the presence of genes required for pilus biosynthesis. The pilD gene of Pseudomonas aeruginosa, which has also been designated xcpA, is involved in protein secretion and in the biogenesis of type IV pili. It encodes a peptidase that processes the precursors of the pilin subunits and of several components of the secretion apparatus. Prepilin processing activity could be demonstrated in P. putida WCS358, suggesting that this nonpathogenic strain may contain type IV pili as well. A DNA fragment containing the pilD (xcpA) gene of P. putida was cloned and found to complement a pilD (xcpA) mutation in P. aeruginosa. Nucleotide sequencing revealed, next to the pilD (xcpA) gene, the presence of two additional genes, pilA and pilC, that are highly homologous to genes involved in the biogenesis of type IV pili. The pilA gene encodes the pilin subunit, and pilC is an accessory gene, required for the assembly of the subunits into pili. In comparison with the pil gene cluster in P. aeruginosa, a gene homologous to pilB is lacking in the P. putida gene cluster. Pili were not detected on the cell surface of P. putida itself, not even when pilA was expressed from the tac promoter on a plasmid, indicating that not all the genes required for pilus biogenesis were expressed under the conditions tested. Expression of pilA of P. putida in P. aeruginosa resulted in the production of pili containing P. putida PilA subunits.     

Cleavage, methylation, and localization of the Pseudomonas aeruginosa export proteins XcpT, -U, -V, and -W.

Four components of the apparatus of extracellular protein secretion of Pseudomonas aeruginosa, Xcpt, -U, -V, and -W (XcpT-W), are synthesized as precursors with short N-terminal leader peptides that share sequence similarity with the pilin subunit of this organism. A specialized leader peptidase/methylase, product of the pilD gene, has been shown to cleave the leader peptide from prepilin and to methylate the N-terminal phenylalanine of the mature pilin. Antibodies were prepared against XcpT-W and used to purify each of these proteins. Sequence analysis of XcpT-W has shown that these proteins, like mature pilin, contain N-methylphenylalanine as the N-terminal amino acid. Analysis of cellular fractions from wild-type and pilD mutant strains of P. aeruginosa showed that the precursor forms of XcpT-W are located predominantly in the bacterial inner membrane, and their localization is not altered after PilD-mediated removal of the leader sequence. These studies demonstrate that the biogenesis of the apparatus of extracellular protein secretion and that of type IV pili share a requirement for PilD. This bifunctional enzyme, acting in the inner membrane, cleaves the leader peptides from precursors of pilins and XcpT-W and subsequently methylates the amino group of the N-terminal phenylalanine of each of its substrates.     

Characterization of a xylanolytic complex from Streptomyces sp.

Streptomyces sp. DSM 41796 produced four major extracellular xylanases with Mr of 145, 120, 60 and 45 kDa. Those of 145 and 60 kDa formed a heterodimer. All xylanases, except that of 120 kDa, were induced by xylose, d-arabinose or sucrose, while commercial xylans induced the 60 kDa xylanase in a major proportion than others, and sugar-cane bagasse pith or lemon peel induced predominantly the 45 kDa xylanase.