Exoprotein Secretion Research Articles

Ciprofloxacin binding to GyrA causes global changes in the proteome of Pseudomonas aeruginosa.

Ciprofloxacin is one of the most widely-used antibiotics, and has proven especially effective at controlling infections associated with the opportunistic human pathogen, Pseudomonas aeruginosa. In this work, we show that sub-inhibitory concentrations of ciprofloxacin induce discrete changes in the intracellular proteome. Central metabolism and cell envelope-associated functions are particularly affected. In spite of the low magnitude of the intracellular proteomic changes, we found that sub-lethal concentrations of ciprofloxacin had substantial effects on motility and exoprotein secretion. Crucially, the proteomic and phenotypic modulations that we observed were absolutely dependent upon the presence of wild-type GyrA; an isogenic strain of P. aeruginosa carrying a ciprofloxacin-insensitive form of GyrA (a T83→I mutant) did not display ciprofloxacin-dependent changes unless complemented with wild-type gyrA in trans. These results show that the diverse effects of sub-inhibitory ciprofloxacin on the cell are routed through its primary target in the cell, DNA gyrase.

Zinc coordination is essential for the function and activity of the type II secretion ATPase EpsE.

The type II secretion system Eps in Vibrio cholerae promotes the extracellular transport of cholera toxin and several hydrolytic enzymes and is a major virulence system in many Gram‐negative pathogens which is structurally related to the type IV pilus system. The cytoplasmic ATPase EpsE provides the energy for exoprotein secretion through ATP hydrolysis. EpsE contains a unique metal‐binding domain that coordinates zinc through a tetracysteine motif (CXXCX29CXXC), which is also present in type IV pilus assembly but not retraction ATPases. Deletion of the entire domain or substitution of any of the cysteine residues that coordinate zinc completely abrogates secretion in an EpsE‐deficient strain and has a dominant negative effect on secretion in the presence of wild‐type EpsE. Consistent with the in vivo data, chemical depletion of zinc from purified EpsE hexamers results in loss of in vitro ATPase activity. In contrast, exchanging the residues between the two dicysteines with those from the homologous ATPase XcpR from Pseudomonas aeruginosa does not have a significant impact on EpsE. These results indicate that, although the individual residues in the metal‐binding domain are generally interchangeable, zinc coordination is essential for the activity and function of EpsE.

Substrate recognition by the bacterial type II secretion system: more than a simple interaction.

Type II secretion system (T2SS) is a multiprotein trans-envelope complex that translocates fully folded proteins through the outer membrane of Gram-negative bacteria. Although T2SS is extensively studied in several bacteria pathogenic for humans, animals and plants, the molecular basis for exoprotein recruitment by this secretion machine as well as the underlying targeting motifs remain unknown. To address this question, we used bacterial two-hybrid, surface plasmon resonance, in vivo site-specific photo-cross-linking approaches and functional analyses. We showed that the fibronectin-like Fn3 domain of exoprotein PelI from Dickeya dadantii interacts with four periplasmic domains of the T2SS components GspD and GspC. The interaction between exoprotein and the GspC PDZ domain is positively modulated by the GspD N1 domain, suggesting that exoprotein secretion is driven by a succession of synergistic interactions. We found that an exposed 9-residue-long loop region of PelI interacts with the GspC PDZ domain. This loop acts as a specific secretion signal that controls exoprotein recruitment by the T2SS. Concerted in silico and in vivo approaches reveal the occurrence of equivalent secretion motifs in other exoproteins, suggesting a plausible general mechanism of exoprotein recruitment by the T2SS.

Exoprotein Production Correlates with Morphotype Changes of Nonmotile Shewanella oneidensis Mutants

We report a previously undescribed mechanism for the rugose morphotype in Shewanella oneidensis, a research model for investigating redox transformations of environmental contaminants. Bacteria may form smooth or rugose colonies on agar plates. In general, conversion from the smooth to rugose colony morphotype is attributed to increased production of exopolysaccharide (EPS). In this work, we discovered that aflagellate S. oneidensis mutants grew into rugose colonies, whereas those with nonfunctional flagellar filaments remained smooth. EPS production was not altered in either case, but mutants with the rugose morphotype showed significantly reduced exoprotein secretion. The idea that exoproteins at a reduced level correlate with rugosity gained support from smooth suppressor strains of an aflagellate rugose fliD (encoding the capping protein) mutant, which restored the exoprotein level to the levels of the wild-type and mutant strains with a smooth morphotype. Further analyses revealed that SO1072 (a putative GlcNAc-binding protein) was one of the highly upregulated exoproteins in these suppressor strains. Most intriguingly, this study identified a compensatory mechanism of SO1072 to flagellins possibly mediated by bis-(3'-5')-cyclic dimeric GMP.

Structural and Functional Studies on the Interaction of GspC and GspD in the Type II Secretion System

Type II secretion systems (T2SSs) are critical for secretion of many proteins from Gram-negative bacteria. In the T2SS, the outer membrane secretin GspD forms a multimeric pore for translocation of secreted proteins. GspD and the inner membrane protein GspC interact with each other via periplasmic domains. Three different crystal structures of the homology region domain of GspC (GspCHR) in complex with either two or three domains of the N-terminal region of GspD from enterotoxigenic Escherichia coli show that GspCHR adopts an all-β topology. N-terminal β-strands of GspC and the N0 domain of GspD are major components of the interface between these inner and outer membrane proteins from the T2SS. The biological relevance of the observed GspC–GspD interface is shown by analysis of variant proteins in two-hybrid studies and by the effect of mutations in homologous genes on extracellular secretion and subcellular distribution of GspC in Vibrio cholerae. Substitutions of interface residues of GspD have a dramatic effect on the focal distribution of GspC in V. cholerae. These studies indicate that the GspCHR–GspDN0 interactions observed in the crystal structure are essential for T2SS function. Possible implications of our structures for the stoichiometry of the T2SS and exoprotein secretion are discussed.

Investigations into σ B -Modulated Regulatory Pathways Governing Extracellular Virulence Determinant Production in Staphylococcus aureus

The commonly used Staphylococcus aureus laboratory strain 8325-4 bears a naturally occurring 11-bp deletion in the sigmaB-regulating phosphatase rsbU. We have previously published a report (M. J. Horsburgh, J. L. Aish, I. J. White, L. Shaw, J. K. Lithgow, and S. J. Foster, J. Bacteriol. 184:5457-5467, 2002) on restoring the rsbU deletion, producing a sigmaB-functional 8325-4 derivative, SH1000. SH1000 is pleiotropically altered in phenotype from 8325-4, displaying enhanced pigmentation, increased growth yields, and a marked decrease in secreted exoproteins. This reduction in exoprotein secretion appears to result from a sixfold reduction in agr expression. In this study we have undertaken transposon mutagenesis of SH1000 to identify components involved in the modulation of extracellular proteases and alpha-hemolysin compared to 8325-4. In total, 13 genes were identified displaying increased alpha-hemolysin transcription and extracellular proteolysis. Phenotypic analysis revealed that each mutant also had decreased pigmentation and a general increase in protein secretion. Interestingly this phenotype was not identical in each case but was variable from mutant to mutant. None of the genes identified encoded classic regulatory proteins but were predominantly metabolic enzymes involved in amino acid biosynthesis and transport. Further analysis revealed that all of these mutations were clustered in a 35-kb region of the chromosome. By complementation and genetic manipulation we were able to demonstrate the validity of these mutations. Interestingly transcriptional analysis revealed that rather than being regulated by sigmaB, these genes appeared to have a role in the regulation of sigmaB activity. Thus, we propose that the loss of individual genes in this chromosomal hot spot region results in a destabilization of cellular harmony and disruption of the sigmaB regulatory cascade.

Structure and Function of the XpsE N-Terminal Domain, an Essential Component of the Xanthomonas campestris Type II Secretion System

Secretion of fully folded extracellular proteins across the outer membrane of Gram-negative bacteria is mainly assisted by the ATP-dependent type II secretion system (T2SS). Depending on species, 12-15 proteins are usually required for the function of T2SS by forming a trans-envelope multiprotein secretion complex. Here we report crystal structures of an essential component of the Xanthomonas campestris T2SS, the 21-kDa N-terminal domain of cytosolic secretion ATPase XpsE (XpsEN), in two conformational states. By mediating interaction between XpsE and the cytoplasmic membrane protein XpsL, XpsEN anchors XpsE to the membrane-associated secretion complex to allow the coupling between ATP utilization and exoprotein secretion. The structure of XpsEN observed in crystal form P4(3)2(1)2 is composed of a 90-residue alpha/beta sandwich core domain capped by a 62-residue N-terminal helical region. The core domain exhibits structural similarity with the NifU-like domain, suggesting that XpsE(N) may be involved in the regulation of XpsE ATPase activity. Surprisingly, although a similar core domain structure was observed in crystal form I4(1)22, the N-terminal 36 residues of the helical region undergo a large structural rearrangement. Deletion analysis indicates that these residues are required for exoprotein secretion by mediating the XpsE/XpsL interaction. Site-directed mutagenesis study further suggests the more compact conformation observed in the P4(3)2(1)2 crystal likely represents the XpsL binding-competent state. Based on these findings, we speculate that XpsE might function in T2SS by cycling between two conformational states. As a closely related protein to XpsE, secretion ATPase PilB may function similarly in the type IV pilus assembly.

The Escherichia coli Hemolysin.

The Escherichia coli hemolysin, earlier referred to as the hemolysin, is the best-characterized repeats in toxin (RTX) secreted by a type I exoprotein secretion system. The E. coli hemolysin is a significant virulence factor in murine models of peritonitis and ascending urinary tract infection, which suggests it is likely to be an important cytotoxin in human, extraintestinal E. coli diseases. Among E. coli or Salmonella strains there are no known examples of strict RTX leukotoxins in which lytic activity is limited to white blood cells. The general gene organization of the Vibrio cholerae RTX locus is similar to that seen with either of the E. coli hly and ehx loci with C, B, and D RTX homologs, clearly indicating it is a member of the RTX family. The hemolysin occurs less frequently in cystitis strains and only rarely among normal fecal strains. Among the extraintestinal E. coli isolates, the hlyCABDgenes were among the first virulence factors localized to unique, tRNA-associated segments of E. coli chromosomes. The hemolysin genes were eventually linked to P-type pilin and cytotoxic necrotizing factor-1 genes. Recent progress with its study has slowed down because of the difficulty in deriving the physical structure of the hemolysin protein or other RTX toxins and establishing its precise cytotoxic mechanism and role in pathogenesis of extraintestinal E. coli disease. Genomic sequencing has revealed that there are additional RTX-like genes found among many different pathogens; perhaps new efforts to discover their functions will aid progress in the RTX toxin field.

Membrane association and multimerization of secreton component pulC.

The PulC component of the Klebsiella oxytoca pullulanase secretion machinery (the secreton) was found by subcellular fractionation to be associated with both the cytoplasmic (inner) and outer membranes. Association with the outer membrane was independent of other secreton components, including the outer membrane protein PulD (secretin). The association of PulC with the inner membrane is mediated by the signal anchor sequence located close to its N terminus. These results suggest that PulC forms a bridge between the two membranes that is disrupted when bacteria are broken open for fractionation. Neither the signal anchor sequence nor the cytoplasmic N-terminal region that precedes it was found to be required for PulC function, indicating that PulC does not undergo sequence-specific interactions with other cytoplasmic membrane proteins. Cross-linking of whole cells resulted in the formation of a ca. 110-kDa band that reacted with PulC-specific serum and whose detection depended on the presence of PulD. However, antibodies against PulD failed to react with this band, suggesting that it could be a homo-PulC trimer whose formation requires PulD. The data are discussed in terms of the possible role of PulC in energy transduction for exoprotein secretion.

Protein secretion in Escherichia coli K-12: dead or alive?

Escherichia coli K-12 possesses a large number of chromosomal genes that, in other Gram-negative bacteria, are involved either in exoprotein secretion or in the formation of type IV pili. Some of these E. coli genes have been shown to encode proteins when expressed from heterologous promoters. Furthermore, at least two of these proteins are functional in heterologous complementation tests, but none of the genes examined so far is expressed when E. coli is grown under standard laboratory conditions. We propose that transcription of these genes is turned off during growth in laboratory medium, that their expression is controlled by environmental sensor proteins and that they could play an important role in pathogenicity or in the utilization of large polymers.

Modulation of mannosylphosphodolichol synthase and dolichol kinase activity in Trichoderma, related to protein secretion.

It has been postulated that exoprotein secretion in Trichoderma is related to their O-glycosylation. In the present paper the involvement of phosphodolichol in this process is described and the key role of mannosylphosphodolichol (MPD) synthase in protein O-mannosylation is discussed. The effect of water soluble phospholipid precursors such as choline and Tween 80, known also to increase secretion of cellulases when added to the medium, on MPD-synthase activity is presented. This effect is positive in the Trichoderma reesei QM 9414 (a low producing strain) but has no influence on the enzyme activity from the RUT C-30 strain selected to overproduce secretion of exoproteins and known to contain an increased cellular amount of endoplasmic reticulum. The positive effect of addition of choline and Tween to the medium on the level of dolichol kinase activity is also demonstrated. The influence of cultivation temperature on the activity of the various enzymes involved in dolichol-dependent protein glycosylation i.e. MPD-synthase, dolichyl kinase and MPD/Protein mannosyl transferase was tested. For all enzymes cultivation at 35 degrees C led to the elevated activity, which was most striking for dolichol kinase, whereas for MPD-synthase and MPD/Protein mannosyl transferase the difference was only apparent in the assay when endogenous phosphodolichol was used as a substrate. Furthermore, lipid extract from the membranes cultivated at elevated temperature, when added to the enzyme obtained from Trichoderma grown at 25 degrees C, enhanced the dolichol kinase activity measured in the absence of exogenous dolichol.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of Peroxidase Secretion and Subcellular Organization of Phanerochaete chrysosporium INA-12 in the Presence of Various Soybean Phospholipid Fractions

Stimulation of lignin peroxidase production by exogenous phospholipids depends on the composition of the phospholipid fraction prepared by using the Nattermann process. The fraction composed mainly of negatively charged phospholipids (NAT 89) was the most efficient source for exoprotein secretion by Phanerochaete chrysosporium INA-12. The results of biochemical marker assays and ultrastructural morphology determination by electron microscopy were correlated. Activities of succinate dehydrogenase, a mitochondrial marker, and cytochrome c oxidoreductase, an endoplasmic reticulum (ER) marker, were increased 1.3- and 2.2-fold, respectively, in the presence of NAT 89. Electron microscopy observations suggested that the amount of mitochondria and ER in culture containing phospholipids was increased at the optimum day of lignin peroxidase production. Therefore, phospholipids enhanced energetic metabolism of strain INA-12 and markedly modified fungus physiology. Since ER is involved in enzyme synthesis, we suggest that its increased amount in mycelium cultured with NAT 89 is directly associated with the higher production of lignin peroxidase.

Stimulation of exoprotein secretion by choline and Tween 80 in Trichoderma reesei QM 9414 correlates with increased activities of dolichol phosphate mannose synthase

Summary: Addition of choline (20 mM) or Tween 80 (0.06%) to the culture medium of Trichoderma reesei QM 9414 increased (a) the secretion of protein under both carbon-catabolite-repressed and -derepressed conditions, and (b) cellulase secretion under carbon-catabolite-derepressed conditions. In contrast, no stimulation by choline or Tween 80 was observed with the hypersecretory strain T. reesei RUT C-30. In view of the obligatory role of O-glycosylation in protein secretion by this fungus, an investigation was made into the effects on this process of choline and Tween 80. A membrane preparation was isolated from both strains of T. reesei and used to assay enzymes involved in O-glycosylation. Significant differences were observed with respect to the activity of dolichol phosphate mannose (Dol-P-Man) synthase only. Strain QM 9414, grown on media supplemented with choline or Tween 80 exhibited a two- to threefold higher activity of Dol-P-Man synthase compared to a control lacking these supplements. This stimulatory effect was observed during growth under both carbon-catabolite-repressed and -derepressed conditions. In contrast, strain RUT C-30 exhibited decreased activities of Dol-P-Man synthase when grown in media supplemented with choline. Choline had no effect on Dol-P-Man synthase in vitro, whereas Tween 80 decreased the activity. Thus the effect of Tween 80 or choline on protein secretion by T. reesei may be due to a stimulation of formation and/or activity of Dol-P-Man synthase, thereby elevating the level of O-glycosylation and protein secretion.

The patterns of extracellular protein formation by spontaneously-occurring rifampicin-resistant mutants of Staphylococcus aureus

Spontaneously-occurring rifampicin-resistant mutants of Sataphylococcus aureus were isolated on 4% (w/v) Tryptone Soya Agar containing 4 and 40 times the m.i.c. for rifampicin. A number of colonies were selected at each rifampicin concentration and were grown aerobically in 3% (w/v) Tryptone Soya Broth for 24 h at 37°C. In the case of S. aureus RN4220 all the mutants grew to bacterial densities up to approximately 1.7 times more than parent organism. The corresponding levels of extracellular protein secretion varied over a 5-fold range, all the mutants being less productive than the parent. By contrast, mutants of the wild-type Wood 46 strain achieved bacterial densities of only 45–83% that of the parent whilst exoprotein secretion showed a smaller 1.7-fold variation. However, widely-differing patterns of exoproteins were revealed by SDS-polyacrylamide gel electrophoresis of the parent and mutant organisms of both strains.

Characterization of a Pseudomonas aeruginosa transposon insertion mutant with defective release of exoenzymes.

A Pseudomonas aeruginosa transposon insertion mutant with defective release of several exoenzymes has been characterized. The Tn5-751 insertion mutation was located in the previously described xcp-1 locus at 0 min on the chromosomal map and caused several exoenzymes to remain in cell-bound form. At least one of the exoenzymes, elastase, was accumulated in the periplasmic space. The periplasmic elastase had the same Mr as the extracellular enzyme produced by the wild-type strain. The virulence of the mutant was comparable to that of wild-type strains in experimental burn infection in mice. The presence of an easily selectable antibiotic resistance marker in the xcp-1 locus offers the possibility of cloning the gene(s) involved in exoprotein secretion.

Choline stimulates synthesis of extracellular proteins in Trichoderma reesei QM 9414

A correlation between intracellular phospholipid levels and the rate of exoprotein synthesis was investigated in the filamentous fungus Trichoderma reesei QM 9414 during growth on cellulose. When the incubation temperature was varied between 20 and 37°C, the exoprotein synthesis rate correlated with the total cellular amount of phospholipids, but not with an individual phospholipid component. In contrast, when phospholipid bases were added exogenuously, a significant stimulation of exoprotein synthesis was observed with choline. The addition of the surfactant Tween 80—which also stimulates exoprotein secretion in T. reesei QM 9414—prevented choline stimulation. Optimal stimulation occurred around 20 mM choline. Choline stimulated exoprotein synthesis in general as shown by increased activities of several extracellular enzymes. Mycelia required preincubation for at least 20 h before stimulation of choline could be seen. Mycelia pregrown in the absence or presence of choline were equally effective in formation of β-glucosidase upon induction with methyl-β-d-glucoside, and the addition of choline to the induction medium had no effect. Choline did not alter the osmotic stability of protoplasts of T. reesei. Electron microscopic examinations and analysis of chemical constituents as well as marker enzymes from choline grown and non-choline grown mycelia revealed higher contents of mitochondria and endoplasmic reticula in choline grown mycelia. The possibility is discussed that choline may stimulate exoprotein synthesis by increasing the cellular content of endoplasmic reticula.

The characteristics of extracellular protein secretion by Staphylococcus staphylolyticus.

The differential rate of extracellular protein formation by Staphylococcus staphylolyticus, the lysostaphin-producing organism, was biphasic with a low rate of exoprotein secretion during exponential growth and an increased rate during the post-exponential phase of growth. After 20 h, when no further exoprotein was secreted, exoprotein accounted for 5% of the total protein in the culture. The secretion of three extracellular enzymes was monitored and found to represent a constant proportion of total exoprotein at exoprotein concentrations greater than 0.1 mg ml-1.

Messenger ribonucleic acid content of Bacillus amyloliquefaciens throughout its growth cycle compared with Bacillus subtilis 168.

The messenger RNA contents of Bacillus amyloliquefaciens and B. subtilis 168, grown in a 1% maltose-0.5% casein hydrolysate complex medium, were determined throughout their growth cycles by a hybridization technique. In both cases there was a level equal to about 3% of the total cellular RNA during the exponential phase. In B. subtilis this level was maintained into the stationary phase. By contrast, in B. amyloliquefaciens the proportion of messenger RNA increased after the end of exponential growth levelling off in the stationary phase at a value twice that observed in exponential growth. The total messenger RNA in each organism was resolved into two components, that involved in the formation of cell proteins and that concerned in extracellular protein production, by determining the relative rates of incorporation of l-[14C]valine into the two protein fractions. In both cases the cell protein component was the same and remained a relatively constant proportion of the total cellular material throughout the growth cycles. The exoprotein mRNA paralleled exoprotein secretion in each species, remaining at a constant low level in B. subtilis and undergoing a tenfold increase after the end of exponential growth in B. amyloliquefaciens. Applying a serial hybridization procedure to B. amyloliquefaciens, no evidence was obtained for the accumulation of a specific component of the messenger RNA in the exponential or post-exponential phase of growth, which was not detected by hybridization.