Conjugal transfer of a chromosomal gene determining production of enterotoxin in vibrio cholerae.

Matings between strains of Vibrio cholerae differing in toxinogenicity, nutritional requirements, and antibiotic susceptibilities were performed in order to determine the location of the gene tox that controls production of cholera enterotoxin. Segregation analysis shows that tox is linked to a gene required for histidine biosynthesis. Our data indicate that the tox gene is located on the bacterial chromosome and not on a plasmid in the strains of V. cholerae studied.

Studies on toxinogenesis in Vibrio cholerae. III. Characterization of nontoxinogenic mutants in vitro and in experimental animals.

Spontaneous and chemically induced mutants with reduced ability to produce cholera enterotoxin (choleragen) as an extracellular protein were isolated from Vibrio cholerae strains 569B Inaba, a classical cholera vibrio, and 3083-2 Ogawa, an El Tor vibrio. By qualitative and quantitative immunological assay in vitro such mutants could be separated into different classes characterized either by production of no detectable choleragen (tox minus), or of small quantities of extracellular choleragen, or of large quantities of cell-associated choleragen but little extracellular choleragen. Analysis of proteins in concentrated culture supernates by electrophoresis in polyacrylamide gels showed that cultures from tox minus strains lacked proteins with electrophoretic mobilities corresponding with choleragen or the spontaneously formed toxoid (choleragenoid). Infant rabbits infected with the tox minus strains remained asymptomatic or developed milder symptoms than rabbits infected with the tox+ parental strains. When symptoms of cholera developed after inoculation with tox minus mutants, detectable numbers of tox+ revertants could be isolated from the intestines of the infected animals. Two tox minus strains, designated M13 and M27, caused no sumptoms and showed no evidence of reversion to tox+ during single passage in infant rabbits, and mutant M13 also remained avirulent and stably tox minus during six cycles of serial passage in infant rabbits. Strains M13 and M27 were also noncholeragenic in acult rabbit ileal loops. Quantitative cultures of the intestines from infected infant rabbits demonstrated that the avirulent mutant M13 can multiply in vivo and can persist in the intestinal tract for at least 48 h.

Pathologic mechanisms of multifocal choroiditis with retinal detachment after carotid injection of Streptococcus mutans and other bacteria in dogs.

Multifocal choroiditis with overlying retinal detachment occurs after carotid injection of certain bacteria in dogs. The ocular lesions occur mainly in the tapetal area of the retina, correlate with microabscesses in the inner choroid and subretinal space, and occasionally occur in the inner retina and anterior uveal tract. The major pathophysiologic factor involved in the dog model of septic choroiditis appears to be embolization of the choriocapillairies by "live" bacteria, which clump and adhere well to tissues. In the dosages used, antibiotics did not prevent or alter the severity of the fundus lesions.

Alveolar epithelial injury and pleural empyema in acute P. aeruginosa pneumonia in anesthetized rabbits.

We developed an experimental model of acute Pseudomonas aeruginosa pneumonia in anesthetized ventilated rabbits to determine whether bacterial-induced injury to the alveolar epithelium would occur and the effect of the injury on the pleural space. Dose-response studies established that 10(9) colony-forming units of P. aeruginosa (wild-type strain, PAO-1) were required to injure the epithelial barrier and to cause pleural empyema with exudative pleural effusions that contained both the instilled alveolar protein tracer and P. aeruginosa. We explored the mechanisms of P. aeruginosa-induced lung and pleural injury by using three isogenic bacterial strains to compare several extracellular virulence products. PAO-S21, which carries an insertion mutation in a regulatory gene that prevents the production of exoenzyme S, resulted in no lung or pleural injury. PAO-R1, which carries a deletion in a regulatory gene that controls the production of elastase and alkaline protease, caused the same degree of lung and pleural injury as PAO-1 did. Instillation of PLC-SRN, which has both structural genes encoding phospholipase C activity deleted, resulted in a moderate reduction in alveolar epithelial injury. Although other products may be involved, exoenzyme S and phospholipase C are important in mediating injury to the alveolar epithelial barrier in acute P. aeruginosa pneumonia in rabbits.

Comparative usefulness of ribotyping, exotoxin A genotyping, and SalI restriction fragment length polymorphism analysis for Pseudomonas aeruginosa lineage assessment.

Ribotyping, exotoxin A genotyping (EAGP), and restriction fragment length polymorphism (RFLP) analysis of total DNA with SalI (SalI RFLP) were compared for intraspecies discrimination of 93 Pseudomonas aeruginosa isolates. Type-ability of all methods was 100% and the results of typing with each method remained unchanged during laboratory manipulation. Clonal groups defined with each molecular method were largely coincident and, in those cases where inconsistencies were detected, isolates were analyzed by transverse alternating field gel electrophoresis (TAFE) and arbitrarily primed polymerase chain reaction (AP-PCR). SalI RFLP analysis was highly discriminative so as to distinguish unrelated isolates of close lineage. However, it was not a good method to identify isolates of unrelated lineage because SalI RFLP appeared to be subjected to convergent evolution. The index of discrimination suggested by Hunter and Gaston was determined to assess the discriminatory power of the molecular methods utilized either alone or in several combinations. Combined use of ribotyping and SalI RFLP analysis reached the highest index of discrimination (0.982) and proved to be a very valuable tool for epidemiological differentiation of P. aeruginosa isolates.

Role of haemolytic and non-haemolytic phospholipase C from Pseudomonas aeruginosa in interleukin-8 release from human monocytes.

A massive accumulation of neutrophils, mainly due to enhanced interleukin-8 (IL-8) levels, is believed to contribute to the deleterious effects of Pseudomonas aeruginosa lung infection, e.g., in cystic fibrosis (CF). Antibodies to phospholipase C, an exoenzyme of P. aeruginosa, are detected early and at high levels in CF patients. However, P. aeruginosa produces at least two types of phospholipase C (PLC), one haemolytic (PLC-H) and the other non-haemolytic (PLC-N), both with mol.wts of c. 77 kDa. Experiments were performed to evaluate the potential contribution of P. aeruginosa PLC to neutrophil accumulation during infection. Therefore, P. aeruginosa PLC-H and PLC-N were compared with regard to IL-8 generation from human monocytes. Purified PLC-H as well as culture supernates (mol.wt > 50 kDa) of a P. aeruginosa strain capable of producing both PLC-H and PLC-N, and mutant strains deficient in the production of one or other phospholipase, or both, were examined. Purified PLC-H (only at low concentrations up to 1 unit/4 x 10(5) monocytes), induced a dose-dependent increase in IL-8 release and IL-8-specific mRNA expression over that of unstimulated cells (at 4-, 12- and 24-h incubation times). Higher concentrations of PLC-H led to a decrease in IL-8 release and IL-8-specific mRNA expression. These findings were confirmed by the results obtained with the supernates of cultures of mutant strains of P. aeruginosa PAO1 that produced either a PLC-H or PLC-N or neither. Stimulation and inhibition of IL-8 release and mRNA expression were associated with a culture supernate fraction of mol. wt > 50 kDa and containing PLC-H. These results contribute to the understanding of the role of both P. aeruginosa PLC in IL-8 generation during their interaction with human monocytes.

Pseudomonas aeruginosa: biology, mechanisms of virulence, epidemiology.

Pseudomonas aeruginosa is a gram-negative pathogen, versatile and opportunistic in terms of its genetics, metabolic potential, and mechanisms of virulence. This versatility enables it to respond to variable and frequently adverse environmental conditions. Considered by many to be an aerobic organism, it is capable of growing anaerobically if certain substrates are available, for example, nitrates or arginine. Diversity of mechanisms of genetic exchange, including transformation, transduction, and conjugation, help P. aeruginosa adapt to changing conditions by acquiring new genetic information. Genetic manipulations have been exploited in recent years to study the basic biology of this bacterial species and the roles of its numerous virulence factors. Recently, transposon mutagenesis techniques and recombinant DNA methods (cloning) have been used to study some of the virulence factors of P. aeruginosa. The pathogenesis of P. aeruginosa infections is multifactorial, as manifested by the numerous toxins, or virulence factors, it produces and the variety of diseases it causes. P. aeruginosa is invasive and toxigenic. Infections appear to occur in stages: bacterial adherence, colonization, invasion and dissemination, and systemic or toxemic disease. Virulence factors can contribute to one or several stages of pathogenesis. Surface factors, including pili, lipopolysaccharide, and polysaccharide slime (alginate), probably contribute to the first two stages. Polysaccharide slime and lipopolysaccharide may also contribute to other processes later in the course of infection. Toxins, including exotoxin A and phospholipase C (hemolysin), and proteases of P. aeruginosa may contribute to tissue damage and dissemination. They may also aid in the procurement of nutrients required by the bacteria in the early stages of infection. The significance of the different virulence factors probably depends on the infection. Alginate production and phospholipase C are likely to have special significance in respiratory infections, particularly in cystic fibrosis.

Analysis of transcription of the exotoxin A gene of Pseudomonas aeruginosa.

Analysis of RNA isolated from Pseudomonas aeruginosa PA103 and PAKS grown under Fe2+-limiting (0.08 microgram/ml) and Fe2+-sufficient (10 micrograms/ml) conditions demonstrated that exotoxin A (ETA) expression is regulated by Fe2+ at the level of transcription. S1 nuclease mapping revealed two 5' termini of the tox transcript, 89 base pairs (bp) (S1A) and 62 bp (S1B) 5' to the ETA initiation codon. There appeared to be no consensus promoter sequence for either tox transcript. An 8-bp direct repeat was found 5' to the start of transcript S1A. Transcript S1B mapped 8 bp upstream of a dodecamer sequence conserved between the ETA and phospholipase C genes of P. aeruginosa. Multicopy plasmids in which the expression of ETA is directed from the Escherichia coli trp promoter (ptrpETA-RSF1010) or the tox promoter (pCMtox) were constructed and mobilized into a Tox-P. aeruginosa strain, WR5. WR5 synthesized and secreted high levels of ETA when it was expressed from the E. coli trp promoter; however, the synthesis of ETA from its own promoter in this strain was very low. These and other data suggest that the expression of ETA is under a positive control mechanism. A fusion of the ETA promoter fragment to lacZ was constructed. Use of this fusion plasmid revealed that this DNA fragment directed the synthesis of beta-galactosidase in E. coli at very low levels and that the synthesis of beta-galactosidase from this fusion in E. coli was not regulated by Fe2+.

Identification of a new phospholipase C activity by analysis of an insertional mutation in the hemolytic phospholipase C structural gene of Pseudomonas aeruginosa.

The phospholipase C (PLC) gene of Pseudomonas aeruginosa encodes a heat-labile secreted hemolysin which is part of a Pi-regulated operon. The structural gene for PLC, plcS, was mutated in vitro by insertion of a tetracycline resistance gene cartridge. Gene replacement techniques were used to introduce the mutated plcS gene into the P. aeruginosa chromosome in place of the wild-type gene. The precise replacement of wild-type sequences by mutant sequences was confirmed by Southern hybridization. The mutant strain, designated PLC S, is nonhemolytic and lacks a 78-kilodalton protein corresponding to the size of the wild-type PLC. However, there is an additional phospholipase activity present in PLC S capable of hydrolyzing p-nitrophenylphosphorylcholine, a synthetic PLC substrate, and phosphatidylcholine. This enzymatic activity is not a result of a truncated product produced from the mutated plcS gene. The phospholipase activity of PLC S was identified as a nonhemolytic PLC.

Nucleotide sequence and expression of a phosphate-regulated gene encoding a secreted hemolysin of Pseudomonas aeruginosa.

A 3.3-kilobase-pair fragment of Pseudomonas aeruginosa DNA containing the phospholipase C (heat-labile hemolysin) gene was sequenced, and the location of the gene was determined. The gene product contains at its NH2 terminus a 38-amino acid sequence which structurally resembles the signal peptides of other secreted proteins but is unusually long and positively charged (6+). The location of the translation start codon was determined by constructing a series of plasmids in which the promoter of a transcription vector was ligated to Pseudomonas DNA containing deletions at the 5' end of the gene. The plasmids were used to transform Escherichia coli, and the resulting clones were assayed for hemolysin activity. In addition, sizes of truncated proteins produced by mutants with translation terminators introduced at specific sites were analyzed in E. coli maxicells. The gene is transcribed, starting just upstream of the hemolysin gene, as an mRNA of approximately 2,800 bases. Analysis of the nucleotide sequence, analysis of mutants in maxicells, and transcriptional studies indicate that the hemolysin is part of an operon composed of two genes. Phosphate regulation of the operon is at the transcriptional level. The location of the 5' end of the transcript was determined by S1 mapping.

Osmoprotectant-dependent expression of plcH, encoding the hemolytic phospholipase C, is subject to novel catabolite repression control in Pseudomonas aeruginosa PAO1.

Expression of the hemolytic phospholipase C (PlcH) of Pseudomonas aeruginosa is induced under phosphate starvation conditions or in the presence of the osmoprotectants choline and glycine betaine. Because choline and glycine betaine may serve as carbon and energy sources in addition to conferring osmoprotection to P. aeruginosa, it seemed possible that induction of plcH is subject to catabolite repression control (CRC) by tricarboxylic cycle intermediates such as succinate. Total phospholipase (PLC) activity in osmoprotectant-induced cultures of P. aeruginosa PAO1 supplemented with 20 mM succinate was three- to fourfold lower than the levels in cultures supplemented with the non-catabolite-repressive substrate lactate. Analyses of osmoprotectant-dependent plcH expression in a derivative of strain PAO1 containing a plcH::lacZ operon fusion showed that (i) succinate prevented induction of plcH expression by osmoprotectants; and (ii) addition of succinate reduced or shut down further expression of plcH in osmoprotectant-induced bacteria, while cultures supplemented with lactate had little or no change in plcH expression. RNase protection analysis confirmed that repression of plcH occurs at the transcriptional level. However, a P. aeruginosa mutant decoupled in CRC exhibited a phenotype similar to that of the wild-type strain (PAO1) with respect to succinate-dependent repression of plcH expression. Osmoprotectant-induced total PLC activities, levels of expression of plcH measured with the same plcH::lacZ fusion, and levels of plcH transcription in a CRC-deficient strain reflected those seen in strain PAO1. This indicates that CRC of plcH functions by a distinct mechanism which differs from that regulating the glucose or mannitol catabolic pathway. A strain carrying a mutation in vfr, which encodes the Escherichia coli Crp homolog in P. aeruginosa, still exhibited a wild-type phenotype with respect to osmoprotectant-dependent expression and CRC of plcH. These data indicate that there is a novel CRC system that regulates the expression of plcH in P. aeruginosa.

Septic choroiditis with serous retinal detachment in Streptococcus mutans-injected dogs.

This report describes a dog model of multifocal choroiditis with serous retinal detachment after carotid injection of Streptococcus mutans. The fundus lesions occurred mainly in the tapetal area of the retina, and, on histopathologic examination, microabscesses in the choroid and subretinal space were observed.

Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa: purification and preliminary characterization.

Phospholipase C (heat-labile hemolysin) was purified from Pseudomonas aeruginosa culture supernatants to near homogeneity by ammonium sulfate precipitation followed by a novel application of DEAE-Sephacel chromatography. Enzymatic activity remained associated with DEAE-Sephacel even in the presence of 1 M NaCl, but was eluted with a linear gradient of 0 to 5% tetradecyltrimethylammonium bromide. Elution from DEAE-Sephacel was also obtained with 2% lysophosphatidylcholine, and to a lesser extent with 2% phosphorylcholine, but not at all with choline. The enzyme was highly active toward phospholipids possessing substituted ammonium groups (e.g., phosphatidycholine, lysophosphatidylcholine, and sphingomyelin); however, it had little if any activity toward phospholipids lacking substituted ammonium groups (e.g., phosphatidylethanolamine, phosphatidylserine, and phosphaditylglycerol). Collectively, these data suggest that phospholipase C from P. aeruginosa exhibits high affinity for substituted ammonium groups, but requires an additional hydrophobic moiety for optimum binding. The specific activity of the purified enzyme preparation increased 1,900-fold compared with that of culture supernatants. The molecular weight of the phospholipase C was estimated to be 78,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephacryl S-200 column chromatography and was 76,000 by high-performance size exclusion chromatography. The isoelectric point was 5.5. Amino acid analysis showed that phospholipase C was rich in glycine, serine, threonine, aspartyl, glutamyl, and aromatic amino acids, but was cystine free.

Isolation and genetic characterization of toxin-deficient mutants of Pseudomonas aeruginosa PAO.

Two independently derived, exotoxin A-deficient (Tox- phenotype), nitroso-guanidine-induced mutants of Pseudomonas aeruginosa PAO1 were isolated by using sensitive immunological assays. One mutant, designated PAOT10, was detected as a colony which failed to produce a halo of immunoprecipitation in an antiserum-agar assay. The other mutant (PAOT20) was independently isolated and was detected by a negative reaction in a staphylococcal coagglutination assay with protein A-containing staphylococci and affinity-purified antibodies. Both mutants produced parental levels of extracellular protein. However, whereas the qualitative and quantitative compositions of proteins produced by PAOT20 were indistinguishable from those of the parental strain as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and measurement of extracellular protease, there were marked differences between PAOT10 and the parental strain. The mutation in PAOT10 (tox-1) as mapped by linkage analysis was located between trp-6 and proA. In contrast, linkage analysis and cotransduction placed the mutation in PAOT20 (tox-2), very near trp-6. Data are presented which suggest that tox-1 and tox-2 are regulatory loci.

Gene repression by the ferric uptake regulator in Pseudomonas aeruginosa: cycle selection of iron-regulated genes.

The expression of at least 24 distinct genes of Pseudomonas aeruginosa PAO1 is under direct control of the "ferric uptake regulator" (Fur). Novel targets of the Fur protein were isolated in a powerful SELEX (systematic evolution of ligands by exponential enrichment)-like cycle selection consisting of in vitro DNA-Fur interaction, binding to anti-Fur antibody, purification on protein G, and PCR amplification. DNA fragments obtained after at least three exponential enrichment cycles were cloned and subjected to DNA mobility-shift assays and DNase I footprint analyses to verify the specific interaction with the Fur protein in vitro. Iron-dependent expression of the corresponding genes in vivo was monitored by RNase protection analysis. In total, 20 different DNA fragments were identified which represent actual Pseudomonas iron-regulated genes (PIGs). While four PIGs are identical to already known genes (pfeR, pvdS, tonB, and fumC, respectively), 16 PIGs represent previously unknown genes. Homology studies of the putative proteins encoded by the PIGs allowed us to speculate about their possible function. Two PIG products were highly similar to siderophore receptors from various species, and three PIG products were significantly homologous to alternative sigma factors. Furthermore, homologs of the Escherichia coli ORF1-tolQ, nuoA, stringent starvation protein Ssp, and of a two-component regulatory system similar to the Pseudomonas syringae LemA sensor kinase were identified. The putative gene products of seven additional PIGs did not show significant homologies to any known proteins. The PIGs were mapped on the P.aeruginosa chromosome. Their possible role in iron metabolism and virulence of P. aeruginosa is discussed.

The response of Pseudomonas aeruginosa to iron: genetics, biochemistry and virulence.

During the past decade significant progress has been made towards identifying some of the schemes that Pseudomonas aeruginosa uses to obtain iron and towards cataloguing and characterizing many of the genes and gene products that are likely to play a role in these processes. This review will largely recount what we have learned in the past few years about how P. aeruginosa regulates its acquisition, intake and, to some extent, trafficking of iron, and the role of iron acquisition systems in the virulence of this remarkable opportunistic pathogen. More specifically, the genetics, biochemistry and biology of an essential regulator (Ferric uptake regulator - Fur) and a Fur-regulated alternative sigma factor (PvdS), which are central to these processes, will be discussed. These regulatory proteins directly or indirectly regulate a substantial number of other genes encoding proteins with remarkably diverse functions. These genes include: (i) other regulatory genes, (ii) genes involved in basic metabolic processes (e.g. Krebs cycle), (iii) genes required to survive oxidative stress (e.g. superoxide dismutase), (iv) genes necessary for scavenging iron (e.g. siderophores and their cognate receptors) or genes that contribute to the virulence (e.g. exotoxin A) of this opportunistic pathogen. Despite this recent expansion of knowledge about the response of P. aeruginosa to iron, many significant biological issues surrounding iron acquisition still need to be addressed. Virtually nothing is known about which of the distinct iron acquisition mechanisms P. aeruginosa brings to bear on these questions outside the laboratory, whether it be in soil, in a pipeline, on plants or in the lungs of cystic fibrosis patients.

Possible insertion sequences in a mosaic genome organization upstream of the exotoxin A gene in Pseudomonas aeruginosa.

Nucleotide sequence and Southern hybridization data revealed a mosaic genome organization in a region that extends several thousand base pairs upstream of the exotoxin A (toxA) gene in Pseudomonas aeruginosa. An interstrain comparison of DNA in this region showed a pattern of alternating segments of homologous and nonhomologous sequences. Two nonhomologous elements, approximately 1 kilobase pair upstream of the gene in strains PA103 and Ps388, were characterized in more detail. The sequence elements, denoted IS-PA-1 and IS-PA-2 for the different strains, are about 1,000 and 785 base pairs long, respectively, and have 5-base-pair direct repeats at their boundaries, consistent with their being DNA insertion sequences. The distribution of these elements in 34 different strains was determined. IS-PA-1 was found in a single copy upstream of toxA in half of the strains and was found in two copies in four of the strains. Some strains contained neither element, and one strain carried both. The genome of another strain, WR5, which lacks toxA, was shown to contain a 350-base-pair region that was highly homologous to DNA sequences located just upstream of toxA in other strains. The WR5 genome lacked several kilobase pairs of DNA that was found both upstream and downstream of this homologous region in the other strains.

Role of the ferric uptake regulator of Pseudomonas aeruginosa in the regulation of siderophores and exotoxin A expression: purification and activity on iron-regulated promoters.

The cloned Pseudomonas aeruginosa fur (ferric uptake regulator) gene was overexpressed in P. aeruginosa by using a T7 expression system, and the Fur protein (PA-Fur) was purified by using a combination of ion-exchange chromatography and metal affinity chromatography. The DNA binding activity of the PA-Fur protein was confirmed by gel mobility shift assays and DNase I footprints of the synthetic DNA fragment GATAAT GATAATCATTATC, representing a perfect "Fur box". In addition, it was shown that PA-Fur is capable of binding to promoter and operator determinants of the tightly iron-regulated Escherichia coli fepA-fes enterobactin gene system. The activity of PA-Fur on the promoters of iron-regulated genes involved in the production of two siderophores, pyochelin and pyoverdin, and in the expression of exotoxin A was investigated. Data indicating that the promoters of the pchR gene, encoding a transcriptional activator for pyochelin synthesis, and of the pvdS gene, encoding a positive regulator for pyoverdin production, are specifically recognized by Fur-Fe(II) are presented, suggesting that PA-Fur represses expression of pchR and pvdS during growth in an iron-replete environment. However, neither the promoter region of the gene encoding exotoxin A (toxA) nor the promoters of the regAB operon, required for toxA expression, interacted with high concentrations of purified PA-Fur. These data indicate that iron regulation of exotoxin A production involves additional factors which may ultimately be under the control of PA-Fur.

Regulation of exotoxin A synthesis in Pseudomonas aeruginosa: characterization of toxA-lacZ fusions in wild-type and mutant strains.

A mobilizable plasmid which carries the promoter for the exotoxin A (ETA) structural gene fused to lacZ was integrated into the chromosome of wild-type and mutant strains of Pseudomonas aeruginosa at the toxA locus by homologous recombination. beta-galactosidase synthesis in the strains (cointegrates) carrying the toxA-lacZ fusions was regulated like ETA synthesis is in P. aeruginosa. Two multicopy plasmids carrying a positive regulatory gene designated toxR were constructed which are identical except with respect to the orientation of toxR to the lacZ promoter on the plasmid. These plasmids were then introduced into P. aeruginosa cointegrate strains. When toxR was using its own promoter, synthesis of beta-galactosidase in the cointegrate strains was increased but the pattern of iron regulation was not altered. In contrast, when the lacZ promoter was directing synthesis of the toxR product in the cointegrate strains, iron regulation of beta-galactosidase and ETA synthesis were abolished.