Antigenicity and immunogenicity of the polyagglutinable antigen of Pseudomonas aeruginosa

Abstract Pseudomonas aeruginosa strains isolated from cystic fibrosis patients agglutinate in antisera against anti-polyagglutinable antigen (PA). Anti-PA antibodies were formed in rabbits when immunization was carried out with bacteria possessing core-bound PA, independently of whether the strains were of S or R phenotype. For bacterial agglutination with anti-PA antibodies two prerequisites are essential: the bacterial cell must be of R phenotype and must possess the core-linked PA. In contrast, the PA in the isolated LPS's can be demonstrated in passive haemagglutination for both (S or R) phenotypes, provided the PA is core-linked. Two PA forms have been recognized, one found only in P. aeruginosa species, both in free and bound form. The other one is shared by all members of Pseudomonas genus but is present only in a free, unbound form.     

Structural relationship between the polyagglutinable antigen and the core polysaccharide of Pseudomonas aeruginosa

It has been observed that each strain of the Pseudomonas aeruginosa species harbours the so-called polyagglutinable antigen (PA). Some strains may produce it in a form which is linked to the core moiety of lipopolysaccharide (LPS) and this type of PA can thus be detected by passive haemagglutination using the isolated LPS as coating antigen. Other strains synthesize PA exclusively in a free form, which is also coextractable with LPS, its presence can, however, be demonstrated by the haemagglutination inhibition test. From a polyagglutinable strain of P. aeruginosa an R-type LPS was isolated having the core-linked PA. This LPS preparation was highly immunogenic with regard to its PA moiety. The core-bound PA seems to exert an immunosuppression on the core region, hence, the polyagglutinable strains isolated from cystic fibrosis patients only engender anti-PA antibodies, whereas antibodies against both, side chain and core region of LPS, are not engendered. The mucoid exopolysaccharide also contains the PA which could possibly play an important role in the patient by protecting P. aeruginosa cells against anti-PA antibodies.     

Structural relationship between the polyagglutinable antigen and the core polysaccharide of Pseudomonas aeruginosa

Abstract It has been observed that each strain of the Pseudomonas aeruginosa species harbours the so-called polyagglutinable antigen (PA). Some strains may produce it in a form which is linked to the core moiety of lipopolysaccharide (LPS) and this type of PA can thus be detected by passive haemagglutination using the isolated LPS as coating antigen. Other strains synthesize PA exclusively in a free form, which is also coextractable with LPS, its presence can, however, be demonstrated by the haemagglutination inhibition test. From a polyagglutinable strain of P. aeruginosa an R-type LPS was isolated having the core-linked PA. This LPS preparation was highly immunogenic with regard to its PA moiety. The core-bound PA seems to exert an immunosuppression on the core region, hence, the polyagglutinable strains isolated from cystic fibrosis patients only engender anti-PA antibodies, whereas antibodies against both, side chain and core region of LPS, are not engendered. The mucoid exopolysacharides also contains the PA which could possibly play an important role in the patient by protecting P. aeruginosa cells against anti-PA antibodies.     

Detection of elastase production in Escherichia coli with the elastase structural gene from several non-elastase-producing strains of Pseudomonas aeruginosa.

The elastase structural gene from Pseudomonas aeruginosa IFO 3455 has been cloned and sequenced. Using this gene as a probe, we cloned the DNA fragments (pEL3080R, pEL10, and pEL103R) of the elastase gene from non-elastase-producing strains (P. aeruginosa IFO 3080, N-10, and PA103 respectively). These three Pseudomonas strains showed no detectable levels of elastase antigenicity by Western blotting (immunoblotting) or by elastase activity. When elastase structural genes about 8 kb in length were cloned into pUC18, an Escherichia coli expression vector, we were able to detect both elastase antigenicity and elastolytic activity in two bacterial clones (E. coli pEL10 and E. coli pEL103R). However, neither elastolytic activity nor elastase antigenicity was detected in the E. coli pEL3080R clone, although elastase mRNA was observed. The partial restriction map determined with several restriction enzymes of these three structural genes corresponded to that of P. aeruginosa IFO 3455. We sequenced the three DNA segments of the elastase gene from non-elastase-producing strains and compared the sequences with those from the elastase-producing P. aeruginosa strains IFO 3455 and PAO1. In P. aeruginosa N-10 and PA103, the sequences were almost identical to those from elastase-producing strains, except for several nucleotide differences. These minor differences may reflect a microheterogeneity of the elastase gene. These results suggest that two of the non-elastase-producing strains have the normal elastase structural gene and that elastase production is repressed by regulation of this gene expression in P. aeruginosa. Possible reasons for the lack of expression in these two strains are offered in this paper. In P. aeruginosa IFO 3080, the sequence had a 1-base deletion in the coding region, which should have caused a frameshift variation in the amino acid sequence. At present, we have no explanation for the abnormal posttransciptional behavior of this strain.     

Monoclonal antibodies to the surface antigens of Pseudomonas aeruginosa

Abstract A panel of 48 monoclonal antibodies was prepared against 8 O-serotype strains of Pseudomonas aeruginosa , and 43 of the antibodies reacted specifically with whole cells of the vaccine strain in an enzyme-linked immunosorbent assay (ELISA). 4 antibodies showed varying degrees of reactivity for more than one of the serotype strains, and one antibody bound to all of the serotype strains as well as strains of Pseudomonas putida and Pseudomonas fluorescens . The epitopes recognised by these antibodies were characterised by immunoblotting and the serotype-specific antibodies reacted only with lipopolysaccharide (LPS) of the vaccine strain. The antibodies that bound to more than one serotype strain were specific for outer-membrane proteins common to the serotype strains. The antibody that cross-reacted with all strains of P. aeruginosa apparently recognised an antigen associated with the core or lipid A components of LPS.     

An intragenic deletion in pilQ leads to nonpiliation of a Pseudomonas aeruginosa strain isolated from cystic fibrosis lung

Deficient motility is one of the characteristic hallmarks observed in Pseudomonas aeruginosa strains that chronically colonize the lungs of cystic fibrosis (CF) patients. Pseudomonas aeruginosa TB is a nonpiliated CF isolate known to be defective in twitching motility. Complementation confirmed a direct link of this phenotype to an intragenic out-of-frame deletion in pilQ (PA5040). Sequence alignment of pilQ derived from TB vs. PAO1 suggests that close direct repeats framing the deletion site may have triggered this mutation. This type of mutation could play a role in the emergence of pathoadaptive mutations of P. aeruginosa in the CF lung habitat.     

铜绿假单胞菌PAO1中c-di-GMP代谢相关基因PA0575对表型的影响

【背景】铜绿假单胞菌PAO1中存在与环鸟苷二磷酸(cyclic-di-guanosine monophosphate,c-di-GMP)代谢相关基因PA0575。【目的】探讨铜绿假单胞菌PAO1中环鸟苷二磷酸代谢相关基因PA0575对运动能力及生物膜的影响。【方法】通过PCR对菌株遗传背景进行确认;利用刚果红结合实验及电转PcdrA-gfp质粒间接测量胞内c-di-GMP水平;利用泳动性(swimming)、蜂群泳动(swarming)、蹭行运动(twiching)和生物膜定量实验对细菌进行表型分析,并在运动培养基中添加抗生素研究其对运动能力的影响;针对PA0575基因进行融合蛋白表达载体的构建,并对蛋白进行原核诱导表达。【结果】3株突变体菌株的转座子插入突变位点不一致,胞内c-di-GMP水平检测结果显示,PA0575-1菌株的c-di-GMP含量高于野生型PAO1菌株(P0.05),PA0575-2、PA0575-3菌株胞内c-di-GMP水平与野生型PAO1菌株无差异(P0.05)。运动能力检测实验中,与野生型PAO1菌株相比,PA0575-1菌株泳动性增强(P0.05);PA0575-2、PA0575-3菌株的泳动性、蜂群运动均增强(P0.05);该基因不同位点的突变均导致氯霉素对菌株的运动能力产生抑制作用。生物膜定量结果显示,与野生型PAO1菌株相比,细菌培养18 h后PA0575-1的生物膜含量降低(P0.05),PA0575-2、PA0575-3菌株的生物膜含量升高。最后成功构建了PA0575基因不同结构域的8个表达载体,并获得了异源表达蛋白。【结论】PA0575基因降低铜绿假单胞菌胞内c-di-GMP的水平,影响表型的同时也抑制了氯霉素抗性基因的表达。以上研究为PA0575基因对表型的影响奠定了基础。     

Identification of two metabolites induced by a butyrolactone autoregulator IM-2 in Streptomyces sp. FRI-5

Abstract In order to determine whether non-elastase-producing strains of Pseudomonas aeruginosa such as N-10, PA103 and IFO3080 can express foreign elastase genes, we introduced elastase genes from P. aeruginosa IFO3455 (elastase-producing) as well as from PA103 and N-10 into non-elastase-producing P. aeruginosa strains. Results suggested that gene expression, secretion, and precursor processing systems of elastase were essentially normal in P. aeruginosa N-10 and IFO3080. Our studies using various elastase genes showed that both the elastase structural gene and 5'-upstream regions of P. aeruginosa PA103 were also normal. This was confirmed by the finding that P. aeruginosa N-10 and IFO3080 which carry the PA103 elastase gene produced elastase. Several deleted or chimeric genes were constructed using the 5'-upstream regions of elastase genes from P. aeruginosa N-10 or PA103 and studies of expression revealed that two individual DNA bases seem to be important in suppressing P. aeruginosa N-10 elastase gene expression. Possible reasons for the lack of elastase in these non-elastase-producing strains are discussed.     

Involvement of DNA helicases in chromate resistance by Pseudomonas aeruginosa PAO1

Chromate-hypersensitive mutants of the Pseudomonas aeruginosa PAO1 strain were isolated using transposon insertion mutagenesis. Comparison of the nucleotide sequences of the regions interrupted within the PAO1 genome showed that mutant strains GGP-64 and AJ-22 were affected in open reading frames PA0967 and PA5345, which correspond to the ruvB and recG genes, respectively. These genes encode helicases RuvB and RecG involved in DNA replication, recombination and repair. The chromate resistance phenotype in mutants GGP-64 and AJ-22 was restored by cosmids bearing wild type ruvB or recG genes, respectively. Also, both mutant strains showed an increased susceptibility to the toxic oxyanions tellurite and selenite as well as to mitomycin C, but not to arsenite, paraquat and hydrogen peroxide. It was concluded that P. aeruginosa RuvB and RecG helicases are involved in repairing DNA damage caused by chromate or its derivatives.     

Characterization of two surface-localized antigenic sites on porin protein F of Pseudomonas aeruginosa

A rapid colony immunoblot screening procedure was used to demonstrate the surface localization of porin protein F on bacterial colonies of Pseudomonas aeruginosa. By this method, we demonstrated that protein F was accessible to four different specific monoclonal antibodies in a wide variety of both mucoid and nonmucoid P. aeruginosa strains. Controls were performed to demonstrate that, using this procedure, only surface-exposed epitopes bound monoclonal antibodies and that nonspecific binding of monoclonal antibodies either to cells lacking protein F or to mucoid exopolysaccharide did not occur. Monoclonal antibodies MA4-4, MA2-10, and MA4-10, specific for protein F, also interacted with colonies of Pseudomonas putida and Pseudomonas syringae, whereas the protein F specific monoclonal antibody MA5-8 interacted only with P. aeruginosa strains. Using the above-named monoclonal antibodies, we investigated the antigenic structure of protein F. Monoclonal antibodies MA4-4, MA2-10, and MA4-10 bound to 29-31 kilodalton proteolytic fragments produced after papain or trypsin digestion of purified protein F or of protein F in outer membranes or intact cells. Antibody MA5-8 did not interact with proteolytically digested protein F but did interact with two of the six fragments produced after partial cyanogen bromide cleavage of protein F. Antibodies MA4-4, MA2-10, and MA4-10 did not interact with protein F after reduction of its internal disulphide bonds with 2-mercaptoethanol; in contrast, the reactivity of MA5-8 was unaffected. This data suggests that there are at least two distinct highly conserved surface epitopes on porin protein F.     

Production of cytotoxin by clinical strains of Pseudomonas aeruginosa

Presence of cytotoxin was studied in extracts of 57 strains of Pseudomonas aeruginosa (46 bacteremia, 4 environmental, and 7 Fisher immunotype), 10 Pseudomonas species, and 7 nonpseudomonas isolates. Cytotoxin was identified by Western immunoblot in extracts of all P. aeruginosa isolates. None of the Pseudomonas species or nonpseudomonas isolates were shown to produce this protein. No immunologic cross-reactivity was observed between cytotoxin antibody and P. aeruginosa alkaline protease, toxin A, or elastase. In partially purified extracts of two bacteremia strains and PA 158 (parent strain for cytotoxin production), detection of cytotoxin by Western immunoblot was correlated with biological activity, as measured by the cell swelling assay. Cytotoxin appears to be produced by all strains of P. aeruginosa and biological activity can be demonstrated in extracts of the strains tested. This biological activity is neutralized by specific antibody. Because of its known marked cytotoxic effect on most eukaryotic cells, P. aeruginosa cytotoxin might be an important factor in the pathogenesis of P. aeruginosa infections.     

Quorum sensing is not required for twitching motility in Pseudomonas aeruginosa

It has been reported that mutations in the quorum-sensing genes lasI and rhlI in Pseudomonas aeruginosa result in, among many other things, loss of twitching motility (A. Glessner, R. S. Smith, B. H. Iglewski, and J. B. Robinson, J. Bacteriol. 181:1623-1629, 1999). We constructed knockouts of lasI and rhlI and the corresponding regulatory genes lasR and rhlR and found no effect on twitching motility. However, twitching-defective variants accumulated during culturing of lasI and rhlI mutants. Further analysis showed that the stable twitching-defective variants of lasI and rhlI mutants had arisen as a consequence of secondary mutations in vfr and algR, respectively, both of which encode key regulators affecting a variety of phenotypes, including twitching motility. In addition, when grown in shaking broth culture, lasI and rhlI mutants, but not the wild-type parent, also accumulated unstable variants that lacked both twitching motility and swimming motility and appeared to be identical in phenotype to the S1 and S2 variants that were recently reported to occur at high frequencies in P. aeruginosa strains grown as a biofilm or in static broth culture (E. Deziel, Y. Comeau, and R. Villemur, J. Bacteriol. 183:1195-1204, 2001). These results indicate that mutations in one regulatory system may create distortions that select during subsequent culturing for compensatory mutations in other regulatory genes within the cellular network. This problem may have compromised some past studies of regulatory hierarchies controlled by quorum sensing and of bacterial regulatory systems in general.     

Plasmid control of the Pseudomonas aeruginosa and Pseudomonas putida phenotypes and of linalool and p-cymene oxidation.

Two Pseudomonas strains (PpG777 and PaG158) were derived from the parent isolate Pseudomonas incognita (putida). Strain PpG777 resembles the parental culture in growth on linalool as a source of carbon and slight growth on p-cymene, whereas PaG158 grows well on p-cymene, but not on linalool or other terpenes tested, and has a P. aeruginosa phenotype. Curing studies indicate that linalool metabolism is controlled by an extrachromosomal element whose loss forms a stable strain PaG158 with the p-cymene growth and P. aeruginosa phenotype characters. The plasmid can be transferred by PpG777 to both P. putida and P. aeruginosa strains. Surprisingly, the latter assume the P. putida phenotype. We conclude that the genetic potential to oxidize p-cymene is inherent in PpG777 but expression is repressed. Similarly, this observation implies that support of linalool oxidation effectively conceals the P. aeruginosa character.     

Variable composition of heme oxygenases with different regiospecificities in Pseudomonas species

Heme oxygenases (HO) degrade heme yielding iron, carbon monoxide and one of four possible biliverdin (BV) isomers. Pseudomonas aeruginosa PAO1 is thus far the only organism to contain two HOs with different regiospecificities: BphO and PigA. While BphO cleaves heme to exclusively yield BV IXα, PigA produces the BV isomers IXβ and IXδ. We bioinformatically identified putative HOs in diverse Pseudomonas strains, tested their enzymatic functionality and determined their regiospecificity. Surprisingly, even high amino acid sequence identities to the P. aeruginosa HOs were not sufficient to correctly predict the HO regiospecificity in all cases. Based on our results, Pseudomonas strains differ in their HO composition containing either BphO or PigA or both HO types. Concomitantly with the existence of bphO is the occurrence of at least one gene encoding a bacterial phytochrome implying that only BV IXα is the sufficient phytochrome chromophore. In contrast, pigA genes are organized in gene clusters associated with iron utilization implying a role of PigA in iron acquisition. However, at least in strains containing no PigA this function maybe fulfilled by BphO. Only a combination of homology searches and analyses of genetic environments is appropriate for a reliable prediction of the regiospecificity of Pseudomonas HOs.     

Elucidating the molecular mechanisms of bacterial virulence using non-mammalian hosts

Several strains of the human opportunistic pathogen Pseudomonas aeruginosa infect plants, nematodes and insects. Our laboratory has developed a multihost pathogenesis system based on the P. aeruginosa clinical isolate PA14, in which non-mammalian hosts are used to screen directly for virulence-attenuated mutants. The majority of PA14 mutants isolated using non-mammalian hosts also displayed reduced virulence in a burned mouse model. Surprisingly, only a few host-specific virulence factors were identified, and many of the P. aeruginosa mutants were attenuated in virulence in all the hosts. These studies illustrate the extensive conservation in the virulence mechanisms used by P. aeruginosa to infect evolutionarily diverged hosts, and validate the multihost method of screening for virulence factors relevant to mammalian pathogenesis. Through the use of genetically tractable hosts, the multihost pathogenesis model also provides tools for elucidating host responses and dissecting the fundamental molecular interactions that underlie bacterial pathogenesis.     

Antagonistic action of Pseudomonas aeruginosa against Staphylococci, coliform bacilli and various types of Proteus

Antagonistic activity of 2 fresh isolates and 3 collection strains of Pseudomonas aeruginosa against 177 microbial strains was determined with the method of late antagonism. Among the microbial strains there were 56 staphylococcal strains isolated from patents and carriers. 38 nontypable colon bacilli isolated from healthy persons, 59 enteropathogenic colon bacilli of various serogroups, 12 strains of Proteus and 12 colon bacilli, carriers of multiple drug resistance factors (R factors). All the cultures were sensitive to the antagonistic action of 5 or at least 3 strains of Pseudomonas used in the study. The most active antagonists were the fresh isolates of Pseudomonas as compared to the collection strains. Among the staphylococci S. aureus proved to be the most resistant to the antagonistic action of Pseudomonas as compared to S. epidermidis, the same as the strains isolated from carriers as compared to the strains isolated from patients. As for the enteric bacilli the most resistant were the strains of Proteus. Acquiring of transmissive R factors by the colon bacilli markedly increased their sensitivity to the antagonistic action of Pseudomonas.     

Cloning and sequencing of the Pseudomonas aeruginosa 1244 pilin structural gene

The pilin structural gene of Pseudomonas aeruginosa 1244 was cloned in both cosmids and lambda. Expression of the cloned gene was detected in P. aeruginosa strains PAO2003, PA103, and 653A by an immunoblot reaction utilizing monoclonal antibodies. Western blot analysis showed that pilin expressed from the cloned gene was slightly larger than native 1244 pilin when produced in strains PAO2003 and 653A, but distinctly smaller in PA103. Bacteriophages specific for the 1244 pilus did not lyse strain PAO2003 containing the cloned 1244 pilin gene, indicating that functional 1244 pili were not assembled in this recombinant strain. Nucleotide sequencing revealed a coding region which when translated would produce a 15,615 dalton peptide. The amino-terminal region of this peptide is identical with published pilin sequences. While the rest of the peptides are generally dissimilar, common residues are seen within potentially antigenic regions.