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PubMed1999

Evidence for the presence in Burkholderia pseudomallei of a type III secretion system-associated gene cluster.

Burkholderia pseudomallei, the causative agent of melioidosis, contains a cluster of putative genes homologous to those encoding HpaP, HrcQ, HrcR, HrcS and HrpV in the plant pathogen Ralstonia solanacearum. In R. solanacearum, these genes form part of a type III secretion-associated pathogenicity island. The order of the genes in B. pseudomallei is directly equivalent to that found in R. solanacearum. The B. pseudomallei proteins share 49.5% (HpaP), 52.6% (HrcQ), 80.0% (HrcR), 72.1% (HrcS) and 46.7% (HrpV) similarity, respectively, with their equivalent R. solanacearum proteins. The presence of type III secretion-associated genes in B. pseudomallei pathogens suggests a possible role for type III secretion systems in the pathogenicity of this organism.

Amino Acid Sequence↗

PubMed2006

Helicobacter pylori induces gastric epithelial cell invasion in a c-Met and type IV secretion system-dependent manner.

Helicobacter pylori interacts with gastric epithelial cells, activating signaling pathways important for carcinogenesis. In this study we examined the role of H. pylori on cell invasion and the molecular mechanisms underlying this process. The relevance of H. pylori cag pathogenicity island-encoded type IV secretion system (T4SS), CagA, and VacA for cell invasion was also investigated. We found that H. pylori induces AGS cell invasion in collagen type I and in Matrigel invasion assays. H. pylori-induced cell invasion requires the direct contact between bacteria and cancer cells. H. pylori-mediated cell invasion was dependent on the activation of the c-Met receptor and on increased MMP-2 and MMP-9 activity. The abrogation of the c-Met receptor using the specific NK4 inhibitor or the silencing of c-Met expression with small interference RNA suppressed both cell invasion and MMP activity. Studies with different H. pylori strains revealed that cell invasion, c-Met tyrosine phosphorylation, and increased MMP-2 and MMP-9 activity were all dependent on the presence of a functional bacterial T4SS, but not on VacA cytotoxicity. Our findings demonstrate that H. pylori strains with a functional T4SS stimulate gastric epithelial cell invasion through a c-Met-dependent signaling pathway that comprises an increase in MMP-2 and MMP-9 activity.

Antigens, Bacterial↗

PubMed1984

Ionic dependence of secretory and electrical activity evoked by elevated K+ in a peptidergic neurosecretory system.

Secretion of the octapeptide erythrophore- (red pigment-) concentrating hormone (ECH, RPCH) and extracellularly monitored electrical activity were followed simultaneously from individual, isolated sinus glands (neurohaemal organs), of the crab Cardisoma carnifex. Following introduction of saline having elevated [K], 100-196 mmol l-1 (5-11 X normal), secretion (bioassayed from 1-min fractions during continuous perfusion) increases from barely detectable (less than 1 fmol min-1) to a peak, average 31 fmol min-1, within 5 min, and immediately subsides. Additional responses are obtainable following a period, greater than 30 min, of normal saline perfusion. Secretory responses to K are Ca-dependent. If Ca is restored (in high K) following perfusion in 0-Ca, high K, only a small secretory response is observed. Addition of Mn (10 mmol l-1, normal Ca) reduces secretion to one-tenth. Increased net uptake of 45Ca of 2.5- to 6-fold is observed in individual sinus glands exposed to 10 X K compared to paired, unstimulated organs. The pattern and Ca-dependence of secretory responses to K are unaffected, but the amount of secretion is augmented in Na-deficient or TTX-containing salines. Intracellular recording confirms that brief (10-40 s) bouts of intense firing recorded extracellularly upon commencing a high K perfusion include repetitive firing by terminals, superimposed on rapid depolarization. Firing ceases as the membrane potential reaches a depolarized value (-18 to -15 mV for [K] 100-176 mmol l-1), which is then maintained until restoration of normal saline, when slow repolarization ensues. In 0-Ca, spontaneous impulse firing is increased, resting potential depolarized by 5 to 15 mV, but the bout of impulse firing and the maintained depolarization in response to K are similar. Thus, mechanisms of secretion of a crustacean peptide neurohormone appear closely similar to those of other systems characterized: responsiveness to elevated K, dependence on Ca, depolarization-, but not secretion-dependent inactivation, and lack of dependence on Na inward current. Intracellular recording here permits direct observation of electrical responses of terminals.

Animals↗

PubMed2002

Shigella Spa32 is an essential secretory protein for functional type III secretion machinery and uniformity of its needle length.

The Shigella type III secretion machinery is responsible for delivering to host cells the set of effectors required for invasion. The type III secretion complex comprises a needle composed of MxiH and MxiI and a basal body made up of MxiD, MxiG, and MxiJ. In S. flexneri, the needle length has a narrow range, with a mean of approximately 45 nm, suggesting that it is strictly regulated. Here we show that Spa32, encoded by one of the spa genes, is an essential protein translocated via the type III secretion system and is involved in the control of needle length as well as type III secretion activity. When the spa32 gene was mutated, the type III secretion complexes possessed needles of various lengths, ranging from 40 to 1,150 nm. Upon introduction of a cloned spa32 into the spa32 mutant, the bacteria produced needles of wild-type length. The spa32 mutant overexpressing MxiH produced extremely long (>5 microm) needles. Spa32 was secreted into the medium via the type III secretion system, but secretion did not depend on activation of the system. The spa32 mutant and the mutant overexpressing MxiH did not secrete effectors such as Ipa proteins into the medium or invade HeLa cells. Upon introduction of Salmonella invJ, encoding InvJ, which has 15.4% amino acid identity with Spa32, into the spa32 mutant, the bacteria produced type III needles of wild-type length and efficiently entered HeLa cells. These findings suggest that Spa32 is an essential secreted protein for a functional type III secretion system in Shigella spp. and is involved in the control of needle length. Furthermore, its function is interchangeable with that of Salmonella InvJ.

Amino Acid Sequence↗

PubMed2005

Analysis of ATPases of putative secretion operons in the thermoacidophilic archaeon Sulfolobus solfataricus.

Gram-negative bacteria use a wide variety of complex mechanisms to secrete proteins across their membranes or to assemble secreted proteins into surface structures. As most archaea only possess a cytoplasmic membrane surrounded by a membrane-anchored S-layer, the organization of such complexes might be significantly different from that in Gram-negative bacteria. Five proteins of Sulfolobus solfataricus, SSO0120, SSO0572, SSO2316, SSO2387 and SSO2680, which are homologous to secretion ATPases of bacterial type II, type IV secretion systems and the type IV pili assembly machinery, were identified. The operon structures of these putative secretion systems encoding gene clusters and the expression patterns of the ATPases under different growth conditions were determined, and it was established that all five putative ATPases do show a divalent cation-dependent ATPase activity at high temperature. These results show that the archaeal secretion systems are related to the bacterial secretion systems and might be powered in a similar way.

Adenosine Triphosphatases↗

PubMed2003

Contribution of ExsA-regulated factors to corneal infection by cytotoxic and invasive Pseudomonas aeruginosa in a murine scarification model.

PURPOSE: The exoenzyme S regulatory protein ExsA regulates a type III secretion system in Pseudomonas aeruginosa. In vitro, cytotoxic strains use this system to secrete exotoxin (Exo)U and ExoT causing cytotoxicity and inhibiting their phagocytosis by epithelial cells. Invasive P. aeruginosa secrete ExoT and ExoS, but exsA mutation has little impact on their short-term interactions with epithelia. In the present study, the contribution of these ExsA-regulated proteins toward corneal infections in vivo was investigated. METHODS: After anesthesia, the left cornea of C57BL/6 mice was scratch injured and then inoculated with cytotoxic (PA103) or invasive (PAK) P. aeruginosa or with isogenic mutants in exsA-related genes. Inocula of 10(3) to 10(6) bacteria/5 micro L were used, and at least five animals were assigned to each experimental group. Corneal disease was quantified at regular intervals for 14 days in masked fashion with two different scoring systems. RESULTS: For the cytotoxic strain, mutation of either exoU or exoT alone had little effect on virulence, whereas simultaneous mutation of both exoT and exoU or of exsA resulted in a significantly reduced capacity to cause corneal disease. Complementation of the double exoUexoT mutant with exoU alone restored bacterial colonization levels (>3-log increase) and disease severity to wild-type levels. Complementation with exoT alone increased colonization ( approximately 3-log increase) and increased virulence to almost the same levels as wild-type or exoU-complemented infections. Virulence of the invasive strain was not reduced by mutation of exsA or of genes encoding the ExsA-regulated secreted proteins. CONCLUSIONS: ExsA contributed to corneal virulence of only cytotoxic P. aeruginosa, with contributions made by both ExoU and ExoT to bacterial survival and disease severity. This differs from cytotoxic P. aeruginosa virulence in the lung, which is ExoU-dependent.

ADP Ribose Transferases↗

PubMed2006

DNA adenine methyltransferase influences the virulence of Aeromonas hydrophila.

Among the various virulence factors produced by Aeromonas hydrophila, a type II secretion system (T2SS)-secreted cytotoxic enterotoxin (Act) and the T3SS are crucial in the pathogenesis of Aeromonas-associated infections. Our laboratory molecularly characterized both Act and the T3SS from a diarrheal isolate, SSU of A. hydrophila, and defined the role of some regulatory genes in modulating the biological effects of Act. In this study, we cloned, sequenced, and expressed the DNA adenine methyltransferase gene of A. hydrophila SSU (dam(AhSSU)) in a T7 promoter-based vector system using Escherichia coli ER2566 as a host strain, which could alter the virulence potential of A. hydrophila. Recombinant Dam, designated as M.AhySSUDam, was produced as a histidine-tagged fusion protein and purified from an E. coli cell lysate using nickel affinity chromatography. The purified Dam had methyltransferase activity, based on its ability to transfer a methyl group from S-adenosyl-l-methionine to N(6)-methyladenine-free lambda DNA and to protect methylated lambda DNA from digestion with DpnII but not against the DpnI restriction enzyme. The dam gene was essential for the viability of the bacterium, and overproduction of Dam in A. hydrophila SSU, using an arabinose-inducible, P(BAD) promoter-based system, reduced the virulence of this pathogen. Specifically, overproduction of M.AhySSUDam decreased the motility of the bacterium by 58%. Likewise, the T3SS-associated cytotoxicity, as measured by the release of lactate dehydrogenase enzyme in murine macrophages infected with the Dam-overproducing strain, was diminished by 55% compared to that of a control A. hydrophila SSU strain harboring the pBAD vector alone. On the contrary, cytotoxic and hemolytic activities associated with Act as well as the protease activity in the culture supernatant of a Dam-overproducing strain were increased by 10-, 3-, and 2.4-fold, respectively, compared to those of the control A. hydrophila SSU strain. The Dam-overproducing strain was not lethal to mice (100% survival) when given by the intraperitoneal route at a dose twice that of the 50% lethal dose, which within 2 to 3 days killed 100% of the animals inoculated with the A. hydrophila control strain. Taken together, our data indicated alteration of A. hydrophila virulence by overproduction of Dam.

Aeromonas hydrophila↗

PubMed1999

Enteropathogenic Escherichia coli: a pathogen that inserts its own receptor into host cells.

Enteropathogenic Escherichia coli (EPEC) is a major cause of infant diarrhea, killing hundreds of thousands of children per year worldwide. Intimate attachment to the host cell leading to the formation of actin-rich pedestals beneath the adhering bacteria is an essential feature of EPEC pathogenesis. EPEC attaches to host cells via the outer membrane adhesin, intimin. It was recently shown that EPEC inserts its own receptor for intimate adherence, Tir (translocated intimin receptor) into the host cell membrane. The focus of this review is on the discovery and characterization of this novel receptor, and our current understanding of its role in pedestal formation. Gram-negative bacterial secretion systems, including type III secretion systems, are reviewed and discussed in the context of Tir delivery into the host cell membrane. The relationship and relevance of in vitro models compared to the actual in vivo situation is essential to understanding disease. We have critically reviewed the use of animal models in studying EPEC infection. Elucidating the function of Tir will contribute to our understanding of how EPEC mediates disease.

Adhesins, Bacterial↗

PubMed2004

A therapeutic strategy against the shared virulence mechanism utilized by both Yersinia pestis and Pseudomonas aeruginosa.

Yersinia pestis, which causes pneumonic plague in healthy individuals, has the potential to be used for biologic warfare. Pseudomonas aeruginosis is an opportunistic pathogen that causes severe pneumonia in immunocompromised patients. There is evidence that these two pathogens use a highly homologous virulence mechanism, the type III secretion system. The type III secretion systems of both Yersinia and P. aeruginosa possess a protein named V-antigen which can be used as a target for immunotherapy against bioterrorism and opportunistic infections. This article summarizes the recent progress of V-antigen studies in Yersinia and P. aeruginosa.

Amino Acid Sequence↗

PubMed2005

Profiling the secretomes of plant pathogenic Proteobacteria.

Secreted proteins are central to the success of plant pathogenic bacteria. They are used by plant pathogens to adhere to and degrade plant cell walls, to suppress plant defence responses, and to deliver bacterial DNA and proteins into the cytoplasm of plant cells. However, experimental investigations into the identity and role of secreted proteins in plant pathogenesis have been hindered by the fact that many of these proteins are only expressed or secreted in planta, that knockout mutations of individual proteins frequently have little or no obvious phenotype, and that some obligate and fastidious plant pathogens remain recalcitrant to genetic manipulation. The availability of genome sequence data for a large number of agriculturally and scientifically important plant pathogens enables us to predict and compare the complete secretomes of these bacteria. In this paper we outline strategies that are currently being used to identify secretion systems and secreted proteins in Proteobacterial plant pathogens and discuss the implications of these analyses for future investigations into the molecular mechanisms of plant pathogenesis.

Bacterial Proteins↗

PubMed2000

An Agrobacterium catalase is a virulence factor involved in tumorigenesis.

Most plant pathogenic bacteria adopt the type III secretion systems to secrete virulence factors and/or avirulence gene products, which trigger the plant hypersensitive response (HR) and the oxidative burst with hydrogen peroxide (H2O2) as the main component. However, the soil-borne plant pathogen Agrobacterium tumefaciens uses the type IV secretion pathway to deliver its oncogenic T-DNA that causes crown gall tumours on many plant species. A. tumefaciens does not elicit a typical HR on those plants. Here, we report that inactivation of one of A. tumefaciens catalases (which converts H2O2 to H2O and O2) by a transposon insertion highly attenuated the bacterial ability to cause tumours on plants and to tolerate H2O2 toxicity, but not the bacterial viability in the absence of exogenous H2O2. This provides the first genetic evidence that the Agrobacterium-plant interaction involves a plant defence response, such as H2O2 production, and that catalase is a virulence factor for a plant pathogen.

Amino Acid Sequence↗

PubMed1999

Membrane topology of the lactococcal bacteriocin ATP-binding cassette transporter protein LcnC. Involvement of LcnC in lactococcin a maturation.

Many non-lantibiotic bacteriocins of lactic acid bacteria are produced as precursors with N-terminal leader peptides different from those present in preproteins exported by the general sec-dependent (type II) secretion pathway. These bacteriocins utilize a dedicated (type I) secretion system for externalization. The secretion apparatus for the lactococcins A, B, and M/N (LcnA, B, and M/N) from Lactococcus lactis is composed of the two membrane proteins LcnC and LcnD. LcnC belongs to the ATP-binding cassette transporters, whereas LcnD is a protein with similarities to other accessory proteins of type I secretion systems. This paper shows that the N-terminal part of LcnC is involved in the processing of the precursor of LcnA. By making translational fusions of LcnC to the reporter proteins beta-galactosidase (LacZ) and alkaline phosphatase (PhoA*), it was shown that both the N- and C-terminal parts of LcnC are located in the cytoplasm. As the N terminus of LcnC is required for LcnA maturation and is localized in the cytoplasm, we conclude that the processing of the bacteriocin LcnA to its mature form takes place at the cytosolic side of the cytoplasmic membrane.

ATP-Binding Cassette Transporters↗

PubMed2002

Regulation of mRNA expression in macrophages after Yersinia enterocolitica infection. Role of different Yop effectors.

The Yop virulon, which comprises a complete type III secretion system and secreted proteins, allows bacteria from the genus Yersinia to resist the nonspecific immune response of the host. This virulon, which is encoded by a plasmid called pYV in Yersinia enterocolitica, enables extracellular bacteria to inject six Yop effectors (YopE, -H, -T, -O, -P, -M) into the host cell. To investigate the role of YopP, YopM, and the other pYV-encoded factors on the expression of the host cell genes, we characterized the transcriptome alterations in infected mouse macrophages using the microarray technique. PU5-1.8 macrophages were infected either with an avirulent (pYV(-)), a wild type (pYV(+)), or two knockout (yopP(-) and yopM(-)) mutants of Y. enterocolitica. Expression alterations in response to Y. enterocolitica infection were monitored for 6657 genes. Among those, 857 genes were affected, 339 of which were specifically regulated by the action of the Yop virulon. Further analysis of those 339 genes allowed identification of specific targets of YopP, YopM, or the other pYV-encoded factors. According to these results, the main action of the Yop virulon is to counteract the host cell pro-inflammatory response to the infection. YopP participates to this inhibition, whereas another pYV-encoded factor appears to also be involved in this down-regulation. Besides, YopM was found to induce the regulation of genes involved in cell cycle and cell growth, revealing for the first time an in vitro effect for YopM. In addition to YopM, other pYV factors distinct from YopP affected the expression of genes involved in cycling. In conclusion, these results provide new insight into the mechanisms of Yersinia pathogenicity by identifying the changes in host genes expression after infection and highlight the concerted actions of the different Yop effectors.

Adhesins, Bacterial↗

PubMed2004

TtsI, a key regulator of Rhizobium species NGR234 is required for type III-dependent protein secretion and synthesis of rhamnose-rich polysaccharides.

Formation of nitrogen-fixing nodules on legume roots by Rhizobium sp. NGR234 requires an array of bacterial factors, including nodulation outer proteins (Nops) secreted through a type III secretion system (TTSS). Secretion of Nops is abolished upon inactivation of ttsI (formerly y4xI), a protein with characteristics of two-component response regulators that was predicted to activate transcription of TTSS-related genes. During the symbiotic interaction, the phenotype of NGR omega ttsI differs from that of a mutant with a nonfunctional secretion machine, however. This indicated that TtsI regulates the synthesis of other symbiotic factors as well. Conserved sequences, called tts boxes, proposed to act as binding sites for TtsI, were identified not only within the TTSS cluster but also in the promoter regions of i) genes predicted to encode homologs of virulence factors secreted by pathogenic bacteria, ii) loci involved in the synthesis of a rhamnose-rich component (rhamnan) of the lipopolysaccharides (LPS), and iii) open reading frames that play roles in plasmid partitioning. Transcription studies showed that TtsI and tts boxes are required for the activation of TTSS-related genes and those involved in rhamnose synthesis. Furthermore, extraction of polysaccharides revealed that inactivation of ttsI abolishes the synthesis of the rhamnan component of the LPS. The phenotypes of mutants impaired in TTSS-dependent protein secretion, rhamnan synthesis, or in both functions were compared to assess the roles of some of the TtsI-controlled factors during symbiosis.

Bacterial Outer Membrane Proteins↗

PubMed2004

Lipopolysaccharide O antigen status of Yersinia enterocolitica O:8 is essential for virulence and absence of O antigen affects the expression of other Yersinia virulence factors.

Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacteria. Although much attention has been given to the biological effects of its lipid A portion, a great body of evidence indicates that its O chain polysaccharide (O antigen) portion plays an important role in the bacterium-host interplay. In this work we have studied in-depth the role of the O antigen in Yersinia enterocolitica serotype O:8 pathogenesis. We made a detailed virulence analysis of three mutants having different O antigen phenotypes: (i) LPS with no O antigen (rough mutant); (ii) LPS with one O unit (semirough mutant) and (iii) LPS with random distribution of O antigen chain lengths. We demonstrated that these LPS O antigen mutants were attenuated in virulence regardless of the infection route used. Co-infection experiments revealed that the rough and semirough mutants were severely impaired in their ability to colonize the Peyer's patches and in contrast to the wild-type strain they did not colonize spleen and liver. The mutant with random distribution of O antigen chain lengths, however, survived better but started to be cleared from mouse organs after 8 days. As an explanation to this attenuation we present here evidence that other Yersinia virulence factors depend on the presence of O antigen for their proper function and/or expression. We demonstrated that in the rough mutant: (i) the YadA function but not its expression was altered; (ii) Ail was not expressed and (iii) inv expression was downregulated. On the other hand, expression of flhDC, the flagellar master regulatory operon, was upregulated in this mutant with a concomitant increase in the production of flagellins. Finally, expression of yplA, encoding for the Yersinia phospholipase A, was also upregulated accompanied by an increased flagellar type III secretion system mediated secretion of YplA to culture medium. Together these findings suggest that the absence of O antigen in the outer membrane of Yersinia either directly or indirectly, for example through a cellular or membrane stress, could act as a regulatory signal.

Adhesins, Bacterial↗

PubMed2005

The StcE protease contributes to intimate adherence of enterohemorrhagic Escherichia coli O157:H7 to host cells.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a diarrheal pathogen that causes attaching and effacing (A/E) lesions on intestinal epithelial cells. Strains of the O157 serogroup carry the large virulence plasmid pO157, which encodes the etp type II secretion system that secretes the genetically linked zinc metalloprotease StcE. The Ler regulator controls expression of many genes involved in A/E lesion formation, as well as StcE, suggesting StcE may be important at a similar time during colonization. Our laboratory has previously demonstrated that StcE cleaves C1-esterase inhibitor, a regulator of multiple inflammation pathways. Here we report two new substrates for StcE, mucin 7 and glycoprotein 340, and that purified StcE reduces the viscosity of human saliva. We tested the hypothesis that StcE contributes to intimate adherence of EHEC to host cells by cleavage of glycoproteins from the cell surface. The fluorescent actin stain (FAS) test was used to observe the intimate adherence represented by fluorescently stained bacteria colocalized with regions of bundled actin formed on HEp-2 cells. An E. coli O157:H7 strain with a stcE gene deletion was not affected in its ability to generally adhere to HEp-2 cells, but it did score threefold lower on the FAS test than wild-type or complemented strains. Addition of exogenous recombinant StcE increased intimate adherence of the mutant to wild-type levels. Thus, StcE may help block host clearance of E. coli O157:H7 by destruction of some classes of glycoproteins, and it contributes to intimate adherence of E. coli O157:H7 to the HEp-2 cell surface.

Bacterial Adhesion↗

PubMed1998

YscB of Yersinia pestis functions as a specific chaperone for YopN.

Following contact with a eucaryotic cell, Yersinia species pathogenic for humans (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica) export and translocate a distinct set of virulence proteins (YopE, YopH, YopJ, YopM, and YpkA) from the bacterium into the eucaryotic cell. During in vitro growth at 37 degrees C in the presence of calcium, Yop secretion is blocked; however, in the absence of calcium, Yop secretion is triggered. Yop secretion occurs via a plasmid-encoded type III, or "contact-dependent," secretion system. The secreted YopN (also known as LcrE), TyeA, and LcrG proteins are necessary to prevent Yop secretion in the presence of calcium and prior to contact with a eucaryotic cell. In this paper we characterize the role of the yscB gene product in the regulation of Yop secretion in Y. pestis. A yscB deletion mutant secreted YopM and V antigen both in the presence and in the absence of calcium; however, the export of YopN was specifically reduced in this strain. Complementation with a functional copy of yscB in trans completely restored the wild-type secretion phenotype for YopM, YopN, and V antigen. The YscB amino acid sequence showed significant similarities to those of SycE and SycH, the specific Yop chaperones for YopE and YopH, respectively. Protein cross-linking and immunoprecipitation studies demonstrated a specific interaction between YscB and YopN. In-frame deletions in yopN eliminating the coding region for amino acids 51 to 85 or 6 to 100 prevented the interaction of YopN with YscB. Taken together, these results indicate that YscB functions as a specific chaperone for YopN in Y. pestis.

Amino Acid Sequence↗

PubMed2004

Temporal expression of pertussis toxin and Ptl secretion proteins by Bordetella pertussis.

Pertussis toxin is an AB(5) toxin comprised of protein subunits S1 through S5. The individual subunits are secreted by a Sec-dependent mechanism into the periplasm, where the toxin is assembled. The Ptl type IV secretion system mediates secretion of assembled toxin past the outer membrane. In this study, we examined the time course of protein expression, toxin assembly, and secretion as a function of the bacterial growth cycle. Logarithmic growth was observed after a 1-h lag phase. Secreted toxin was first observed at 3 h. Secretion continued throughout the logarithmic growth phase and decreased as the culture entered the stationary phase after about 24 h. On a per cell basis, toxin secretion occurred at a constant rate of 3 molecules/min/cell from 2 to 18 h. More of toxin subunits S1, S2, and S3 were produced than were secreted, resulting in periplasmic accumulation. Periplasmic S1, S2, and S3 were found to be soluble in the periplasm, as well as membrane associated. About one-half of the periplasmic S1, S2 and S3 subunits were incorporated into holotoxin. Secretion component PtlF was present at a low level at time zero, and the level increased between 2 and 24 h from 30 to 1,000 molecules per cell; however, the initial level of PtlF, 30 molecules per cell, supported maximal secretion. The accumulation of both periplasmic toxin and secretion components suggests that translation rates exceed the rate of secretion and that secretion, not toxin and Ptl complex assembly, is rate limiting.

Animals↗