The Pasteurella multocida toxin is encoded within a lysogenic bacteriophage

Toxigenic strains of Pasteurella multocida produce a 146 kDa toxin (PMT) that acts as a potent mitogen. Sequence analysis of the structural gene for PMT, toxA, previously suggested it was horizontally acquired, because it had a low G + C content relative to the P. multocida genome. To address this, the sequence of DNA flanking toxA was determined. The sequence analysis showed the presence of homologues to bacteriophage tail protein genes and a bacteriophage antirepressor, suggesting that the toxin gene resides within a prophage. In addition to phage genes, the toxA flanking DNA contained a homologue of a restriction/modification system that was shown to be functional. The presence of a bacteriophage was demonstrated in spent medium from toxigenic P. multocida isolates. Its production was increased by mitomycin C addition, a treatment that is known to induce the lytic cycle of many temperate bacteriophages. The genomes of bacteriophages from three different toxigenic P. multocida strains had similar but not identical restriction profiles, and were approximately 45-50 kb in length. The prophages from two of these had integrated at the same site in the chromosome, in a tRNA gene. Southern blot analysis confirmed that these bacteriophages contained the toxA gene.     

Construction of derivatives of the diphtheria toxin gene and their expression in Escherichia coli cells

A fragment of diphtheria toxin (tox) gene from beta 45 phage DNA was cloned on pUC19 plasmid in E. coli cells. The fragment is coding for toxA fragment of the toxin and contains the control region of the tox gene. The tox gene promoter is active in E. coli. The toxA protein is found mainly in periplasm of E. coli cells. The protein is enzymatically active in ADP-ribosilation of elongation factor 2 from eucaryotic cells. An in frame toxA-lacZ' fusion was constructed on pUC8 plasmid. The hybrid protein expresses both toxA and lacZ' activities. Two or seven base pairs were deleted from the central part of toxA gene by means of S1 nuclease digestion. Translation of hybrid toxA-lacZ' mRNA should be terminated downward the delections due to the frameshifts caused by them. Nevertheless, a functionally active alpha-peptide of beta-galactosidase is expressed by both the deletion fusions. The existence of another translational start site functioning in E. coli and located inside 3'-end region of toxA mRNA is suggested.     

Determination of the amino acid change responsible for the nontoxic, cross-reactive exotoxin A protein (CRM 66) of Pseudomonas aeruginosa PAO-PR1.

Analysis of purified exotoxin A from parental Pseudomonas aeruginosa PAO1 and mutant strain PAO-PR1, which produces enzymatically inactive exotoxin A (CRM 66), revealed that CRM 66 lost 90% of parental enzymatic activity. Nucleotide sequence analysis of cloned exotoxin A genes showed a single amino acid substitution in CRM 66. Position 426 in the mature protein of parental (PAO1) exotoxin A is histidine, whereas in CRM 66, it is tyrosine.     

Construction and use of a nontoxigenic strain of Pseudomonas aeruginosa for the production of recombinant exotoxin A.

To express recombinant forms of Pseudomonas aeruginosa exotoxin A in high yield, we have developed a nontoxigenic strain of P. aeruginosa derived from the hypertoxigenic strain PA103. The nontoxigenic strain, designated PA103A, was produced by the excision marker rescue technique to replace the toxA structural gene in PA103 with an insertionally inactivated toxA gene. The PA103A strain (ToxA-) was used subsequently as the host strain for the expression and production of several recombinant versions of exotoxin A, and the results were compared with exotoxin A production in other P. aeruginosa and Escherichia coli strains. Use of the PA103A strain transformed with the high-copy-number pRO1614 plasmid bearing various toxA alleles resulted in final purification yields of exotoxin A averaging 23 mg/liter of culture. By comparison, exotoxin A production in other expression systems and host strains yields approximately 1/4 to 1/10 as much toxin.     

Analysis of immunization with DNA encoding Pseudomonas aeruginosa exotoxin A

The promising arena of DNA-based vaccines has led us to investigate possible candidates for immunization against bacterial pathogens. One such target is the opportunistic pathogen Pseudomonas aeruginosa which produces exotoxin A (PE), a well-characterized virulence factor encoded by the toxA gene. In its native protein form, PE is highly cytotoxic for susceptible eukaryotic cells through ADP-ribosylation of elongation factor-2 following internalization and processing of the toxin. To study the biologic and immunological effects of PE following in situ expression, we have constructed eukaryotic plasmid expression vectors containing either the wild-type or a mutated, non-cytotoxic toxA gene. In vitro analysis by transfection of UM449 cells suggests that expression of the wild-type toxA gene is lethal for transfected cells whereas transfection with a mutated toxA gene results in the production of inactive PE which can be readily detected by immunoblot analysis of cell lysates. To investigate the effects resulting from the intracellular expression of potentially cytotoxic gene products in DNA vaccine constructs, we immunized mice with both the wild-type and mutant toxA plasmid constructs and analyzed the resulting humoral and cellular immune responses. Immunization with the mutated toxA gene results in production of neutralizing antibodies against native PE and potentiates a T(H)1-type response, whereas only a minimal humoral response can be detected in mice immunized with wild-type toxA. DNA-based vaccination with the non-cytotoxic toxA(mut) gene confers complete protection against challenge with the wild-type PE. Therefore, genetic immunization with genes encoding potentially cytotoxic gene products raises concern with regard to the selection of feasible gene targets for DNA vaccine development.     

Development of a toxA gene knock-out mutant of Pasteurella multocida and evaluation of its protective effects

Pasteurella multocida is an important veterinary and opportunistic human pathogen. In particular, strains of P. multocida serogroup D cause progressive atrophic rhinitis, and produce a potent, intracellular, mitogenic toxin known as P. multocida toxin (PMT), which is encoded by the toxA gene. To further investigate the toxigenic and pathogenic effects of PMT, a toxA-deleted mutant was developed by homologous gene recombination. When administrated to mice, the toxigenicity of the toxA mutant P. multocida was drastically reduced, suggesting that the PMT contributes the major part of the toxigenicity of P. multocida. Similar results were obtained in a subsequent experiment, while high mortalities were observed when toxA(+) P. multocida bacterial culture or culture lysate were administrated. Mice immunized with toxA(-) P. multocida were not protected (none survived) following challenge with toxA(+) P. multocida or bacterial culture lysate (toxin). These results suggest that the toxigenicity of P. multocida is mainly derived from PMT.     

Primary structure of heat-labile enterotoxin produced by Escherichia coli pathogenic for humans

Heat-labile enterotoxin of Escherichia coli pathogenic for humans (LTh) or for piglets (LTp) and Vibrio cholerae enterotoxin (CT) are structurally and functionally similar toxins. We have determined the complete nucleotide sequence of the toxA gene which encodes the subunit A of LTh (LTh A). The deduced amino acid sequence consists of 258 residues including a signal peptide of 18 residues. According to the previously completed LTh B sequence (103 residues), the predicted holotoxin (1A5B) of LTh comprises 755 residues and has Mr = 87,866. With respect to LTh A and LTh B, secondary structures, local hydrophilicity, and sites for antigenic determinants were predicted. Both codon usage and G + C content of the toxA gene and the LTh B gene (toxB) were markedly different from those observed with several E. coli chromosomal genes. Its relatively low G + C content was rather close to that of the V. cholerae chromosome. Although the toxA gene shares a common ancestor with the LTp A gene (eltA), the two genes are apparently distinguishable from each other in their sequences. Like LTh B reported previously, the predicted sequence of the catalytic fragment LTh A1 also showed more homology to that of CT A1 than did that of LTp A1. In contrast, unique sequences were found in LTh A2.     

Genetic rearrangement associated with in vivo mucoid conversion of Pseudomonas aeruginosa PAO is due to insertion elements.

The conversion of Pseudomonas aeruginosa PAO to the mucoid phenotype has been reported for a chronic pulmonary infection model in rats (D. E. Woods, P. A. Sokol, L. E. Bryan, D. G. Storey, S. J. Mattingly, H. J. Vogel, and H. Ceri, J. Infect. Dis. 163:143-149, 1991). This conversion was associated with a genetic rearrangement upstream of the exotoxin A gene. To characterize the genetic rearrangement, the region upstream of the toxA gene was cloned from PAO, PAO-muc (a mucoid strain), and PAO-rev (a nonmucoid revertant strain). The nucleotide sequence of a 4.8-kb fragment from PAO-muc was determined. A+T-rich regions of approximately 2 kb (IS-PA-4) and 0.4 kb (IS-PA-5) were identified in this fragment. DNA probes constructed internal to these regions hybridized to PAO-muc but not to PAO or PAO-rev, suggesting that PAO-muc contains an insertion element. Sequence analysis of the nonmucoid clones indicated that a 2,561-bp fragment corresponding to IS-PA-4 and a 992-bp fragment corresponding to IS-PA-5 were not present in PAO or PAO-rev. Both nonmucoid clones, however, contained in the same location as IS-PA-4, a 1,313-bp region which was not present in PAO-muc. DNA probes complementary to this sequence, designated IS-PA-6, did not hybridize with PAO-muc, indicating that this sequence had been replaced upon conversion to the mucoid phenotype. Between IS-PA-4 and IS-PA-5 there was a 500-bp sequence which was 94% identical to the 500-bp sequence downstream of IS-PA-6. These insertion elements had some DNA sequence similarity to plasmid and transposon sequences, suggesting that they may be of plasmid origin. IS-PA-4 and IS-PA-5 were shown also to be present in two mucoid isolates from cystic fibrosis patients. The insertions occurred in the same location upstream of the toxA gene, suggesting that this type of genetic recombination may also be associated with mucoid conversion in some P. aeruginosa clinical isolates.     

Isolation of an Hfr donor of Pseudomonas aeruginosa PAO by insertion of the plasmid RP1 into the tryptophan synthase gene

Summary A derivative of the IncP-1 plasmid RP1, temperature-sensitive for maintenance, was inserted into the Pseudomonas aeruginosa chromosome by selection for a plasmid marker (carbenicillin resistance) at nonppermissive temperature. In one strain, PAO 1000, the plasmid was stably integrated in the trpA, B gene cluster mapped at 27 min, as shown by the following evidence. (i) Trp⁺ transductants lost all plasmid markers. (ii) Cleared lysates of PAO 1000 showed no plasmid band typical of the autonomous RP1 in agarose gel electrophoresis. (iii) No transfer of carbenicillin resistance by PAO 1000 was detectable. (iv) PAO 1000 mobilised the chromosome from an origin at, or very near, the plasmid insertion site with high frequency (recovery of proximal markers 10^–3 per donor). Matings on the plate with and without interruption of conjugation showed that chromosome transfer was unidirectional. (v) Recombinants from PAO 1000-mediated crosses did not inherit plasmid markers or the trpA, B mutation. A derivative of PAO 1000 was obtained which had lost the Hfr property and all plasmid markers except carbenicillin resistance. This strain (PAO 1001), when carrying the autonomous RP1 plasmid, was capable of unidirectional chromosome mobilisation like PAO 1000, but with 50-fold lower efficiency. We propose that integration of the temperature-sensitive RP1 plasmid in PAO 1000 occurred via transposition of Tnl, the element specifying carbenicillin resistance.     

Gradient of genomic diversity in the Pseudomonas aeruginosa chromosome

In 545 Pseudomonas aeruginosa strains, mainly collected from patients with cystic fibrosis, Spel-Dral macrorestriction fragment lenght diversity was scanned for using probes of known map position on th P.earuginosa PAO chromosome. Southern analysis of the 60 unrelated clones uncovered a gradient of macrorestriction fragment lenght polymorphisms (RFLPs) from the origin of replication towards the auxotroh-poor region of the P. aeruginosa population in the region encompassed by the rrn operons. The oriC -reactive Spel fragment was conserved in nearly all isolates examined. Few fragment lenght classes were seen for the alga60-, algR- and toxA -encoding Spel fragments. Fragment siz varied within one class by up to 20 kb. Two probes from the auxotroph-poor region detected a broad size range for the Spel fragment, suggestiong extensive genomic deversity in these reions. Subclonalvariation of fragment size was detected at all investigated loci in at least one of the analysed clones, but within one particlular clone, Spel -RFLPs were found at only few loci.     

Development of broad-host-range vectors and gene banks: self-cloning of the Pseudomonas aeruginosa PAO chromosome.

A host-vector system for Pseudomonas aeruginosa PAO was developed. Scattered regions of the strain PAO chromosome were cloned by direct selection for complementation of auxotrophs or from a DNA gene bank which contains over 1,000 independently isolated chromosome-vector recombinant plasmids. The use of partially digested chromosomal DNA facilitated the selection of a variety of strain PAO chromosomal markers. The progenitor of the vector was a small, multicopy plasmid, pRO1600, found in a PAO strain which had acquired RP1 in a mating experiment. The bacterial host range that could be determined by transformation of vectors produced from pRO1600 resembles that for plasmid RP1. Two derivative plasmids were formed: pRO1613, for cloning DNA cleaved with restriction endonuclease PstI, and pRO1614, which was formed by deleting part of pRO1613 and fusion with plasmid pBR322. Plasmid pRO1614 utilizes known cloning sites within the tetracycline resistance region of pBR322.