Microbiology

Polysaccharide Antigens of Pseudomonas Aeruginosa Yuriy A. Knirel

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ABSTRACT The major polysaccharide antigens of P . aeruginosa are the cell-wall lipopolysaccharides many of which have an acidic polysaccharide chain (O-antigen) rich in unusual amino sugars. The o-rhamnose-rich polysaccharide antigen common to many serologically distinct strains is also associated with the lipopolysaccharide. The high-molecular-weight plysaccharides with O-specificity are present in extracellular slime produced by strains isolated from the environmental and from the immunocompromised hosts. The extracellular antigenic polysaccharide of another type (bacterial alginate) is expressed by mucoid strains isolated from patients with cystic fibrosis. Serotype-specific immune responses after infection are directed at the lipopolysaccharides and these heat-stable antigens serve as the basis for differentiation of P. aeruginosa strains. Both the cell-wall antigens including conjugates of the O-polysaccharides with different proteins and the extracellular antigens have been used to prepare specific antibodies tested for protection against infections due to P. aeruginosa.

1. INTRODUCTION Pseudomonas aeruginosa is a widespread gram-negative bacterium which required minimal conditions for growth and is able to infect man as well as warm- and cold-blooded animals. Being almost hannless for the healthy person this opportunistic pathogen produces severe diseases in immunocompromized patients and plays a role in the pathogenesis of cystic fibrosis. The P. aeruginosa cell envelope is typical for gram-negative bacteria. It involves an inner cytoplasmic membrane and a cell wall which comprises a peptidoglycan layer creating the required rigidity and a flexible outer membrane bilayer. The outer monolayer of the outer membrane consists of lipopolysaccharide (LPS) and proteins, its inner monolayer of phospholipids and proteins. LPS is exposed on the cell surface and plays an important role in interaction of the bacterium with other biological systems (mammalian immune system, bacteriophages, etc.). Like many other gram-negative bacteria P. aeruginosa has no discrete polysaccharide capsule covering the cell envelope, but it produces various extracellular compounds of polysaccharidic nature. In strains isolated from the environment and from immunocompromised hosts these are glycolipoprotein (GLP) and acidic or neutral polysaccharides. The main extracellular material of so-called mucoid strains usually isolated 1990

from patients with cystic fibrosis is an alginate-like acidic polysaccharide. In the present review all the aforementioned polysaccharides and glycoconjugates are considered, which are the most important surface antigens of P . aeruginosa. Attention is primarily drawn to those aspects of their studies where various properties of the antigens, their role in vital activity of the bacterium and in pathogenesis of P. aeruginosa infections are correlated with the structures of these biopolymers. Development of experimental vaccines based on P. aeruginosa p l y saccharide antigens for therapy and prophylaxis is also discussed. The section dealing with somatic LPS antigens involves consideration of the general architecture of the macromolecule, its fine chemical structure, correlation of the structure with serological characteristics, and immunological properties of LPS (virulence, immunogenicity, protective capacity). Extracellular high-molecular-weight polysaccharides structurally and serologically related to plysaccharide chains of LPSs as well as P. aeruginosa common polysaccharide antigen associated with LPS are also considered in this chapter. The second main chapter of the review summarizes the data on extracellular polysaccharide antigens: alginate-like polysaccharide of mucoid strains, GLP, and low-molecular-weight neutral polysaccharides. In the 1980s, several reviews covering the structures and properties of P. aeruginosa LPSs and alginate as well as results and prospects of use of experimental vaccines on the basis of P . aeruginosa plysaccharide antigens were published, l - l 0 and additional information is acquired from these reviews.

II. OUTER-MEMBRANE LIPOPOLYSACCHARIDE A. General 1. Characterization LPS of P. aeruginosa possesses the same general molecular architecture as the most thoroughly studied LPSs of enterobacteria. The molecule of complete LPS (S-form) contains a hydrophobic lipid A part to which a polysaccharide chain is attached via a core oligosaccharide.' This structure is characteristic of wild-type smooth strains but not of rough strains. Some rough-type strains, however, may express side chains, although in reduced amount." R-form LPS is characterized b j the absence of any side chain. This form is present in both

Y. A. Knirel earned his Ph.D. at N . D. Zclinsky Instltute of Organic Chemistry, Academy of Sciences U . S . S . R . . Moscow, U.S.S.R. Dr.Knirel is Senior Scientist in the Department of C a r b hydrate Chemistry, Instltute of Organic Chemistry. Academy of Sciences U . S . S . R . , Moscow, U.S.S.R.

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Critical Reviews In smooth and rough strains. Some P. aeruginosa mutants produce an incomplete core which is occasionally but not usually substituted by a side chain.”.l3 Lipid A is based on a phosphorylated disaccharide of glucosamine which carries ester- and amide-bound fatty acids. The core is a rather large oligosaccharide containing higher sugars (heptose and 2-keto-3-deoxyoctonic acid, KDO), ethanolamine, and phosphate in the inner part, proximal to lipid A, whereas six-carbon sugars and alanine are components of the outer part. The polysaccharide chain of S-form LPS represents a heteropolymer built up of oligosaccharide (from dito penta-saccharide) repeating units. Only one such unit is linked to the core in the intermediate SR-form LPS. Structurally distinct polysaccharide chains may be produced by the individual strains. Lipid A is the part of the LPS molecule that by hydrophobic interaction with phospholipid is buried in the outer membrane of cell wall, thus making LPS an integral part of this membrane. It plays a significant role in organization and stability of the outer membrane. Lipid A is responsible for the induction of endotoxin effects by LPS in mammals, and it participates in the initiation of a number of pathophysiological marufestations.2 Due to a high level of phosphorylation, the core oligosaccharide possesses exceptional metal-binding properties. This makes the outer membrane particularly dependent on stabilization by divalent cations acting as bridges between neighboring LPS molecules. Chelation of these cations by EDTA causes disordering of the membrane with the release of LPS.’7~18 The disruption of the outer membrane integrity and its antibiotic permeability caused by polycationic aminoglycoside and peptide antibiotics and polyamines results from binding of these compounds to anionic groups of LPS.’.’’ The reduction in LPS phosphate content due to a mutation, and thus a decrease in the number of membrane stabilizing sites may result in antibiotic supersusceptibility and loss of the outer membrane permeability barrier to hydrophobic antibiotics.” The polysaccharide chain is the peripheral part of the LPS molecule and can extend about 10 to 12 nm away from the cell into the environment.*’ In the majority of P. aeruginosa strains, the polysaccharide chain is acidic, but the level of negative charge does not exceed one acidic function per three sugar residues (see Section LI.D.3.b). This feature seems to be characteristic of LPS from all gram-negative bacteria while no such limitation is observed for extracellular bacterial antigens, for which much a higher concentration of negative charge is common (see, e.g., the description of the alginate-like antigen of P. aeruginosa in Section 1LI.A). The probable reason for the limitation of the level of negative charge in the side chain of LPS is that a higher concentration of the charge would lead to a decrease in mobility of polysaccharide chain and in its protrusion into the external medium as a result of binding to outer membrane via divalent-cation bridges. Both core and polysaccharide chain contain antigenic determinants at which specific immune responses are directed. 274

The specificity of the core may be cryptic in S-form LPS because of the masking of active sites by the side chain attached. The polysaccharide side chain is responsible for the manifesting of 0-specificity and is called 0-antigen. Each serologically distinguishable strain of P. aeruginosa produces a unique 0-antigen having a specific composition and structure. The presence and the amount of 0-antigen as well as the length of 0-antigen chain influences various other P. aeruginosa cell surface phenomena, including antibiotic s~sceptibility.”~’~ bacteriophage rec~gnition,’~.‘~ virulence and sensitivity to bactericidal action of ~ e r u m , ’ ~ .and ~ ’ capacity of LPS to induce protective antibodies.28 Extracellular polysaccharides with the same specificities and structures as 0-antigens have been found in cultural fluid or in the slime of different P . aeruginosa strain^.'^-'' Their production increases after cessation of logarithmic growth of bacteria. It is unclear whether these antigens are produced by bacteria as pure polysaccharides or they are attached to a lipid moiety. In the majority of the tested P . aeruginosa strains of both smooth and rough type, another LPS species is present, whose polysaccharide chain differs serologically and structurally from 0-antigen chain. 16.34-36 In wild type strains, this common polysaccharide antigen is covered by 0-antigen chain and is not exposed on the cell surface, but it is shown on the surface of rough-type strain^.".^'

2. Heterogeneity Heterogeneity of LPS is attributed to the existence in the individual strains of S-form molecules which contain 0-specific polymer chains of different length together with those of SR- and R-form having one or no 0-antigen repeating unit, respectively. Further heterogeneity may arise from the presence of LPS species with structurally different polysaccharide side chains and from substoichiometric modifications in the lipid A-core moiety. Several methods are employed to resolve different LPS populations, with polyacrylamide gel electrophoresis (PAGE) and gel filtration being the most successful. The latter method separates molecules on the basis of their size while the former method on the basis of both size and charge.38 Due to the tendency of LPS to aggregate, the separation is carried out in the presence of a detergent, usually sodium dodecyl sulphate (SDS) or sodium deoxycholate, dissociating the aggregates. LPS electrophoretic run on polyacrylamide gel displays a high degree of heterogeneity (Figure 1). The dense fastest migrating bands near the gel bottom are believed to be lipid A-core and lipid A-core with one repeating unit of side chain attached, i.e., R- and SR-form of LPS. respectively. A progressive, ladder-like pattern of bands is revealed up the gel, which represents molecules of SR-form with increasing length of 0-antigen side chain. The neighboring bands are believed to belong to the chains which differ by one repeating unit. In some cases the bands are resolved as doublets suggesting a

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Microbiology

Fraction number FIGURE 1. Silver-stained SDS-PAGE patterns of LPS in EDTNglycine monovalent extracts from 16 P.acruginosa IATS serotypes. (From Maclntyre, S . , McVeigh. 1..and Owen P . , fnfccr. fmmun.. 51, 675, 1988. With permission.)

substoichiometric modification in the lipid A-core which results in a difference in the charge of the molecules.’s~28 Such a modification is most probably associated with the change in phosphate content. SDS-PAGE allowed also a distinction between LPS species with complete and incomplete core; the latter moves faster on gel. 12.39 Gel chromatography of P. aeruginosa smooth-type LPSs on Sepharose 6B or Sephacryl S-300 afforded two main fractions, the fraction eluted first corresponded to S-form and that eluted second to R- and SR-forms of LPS.2.’8 Chromatography on Sephadex G-200 of LPS preparations from a series of wild-type strains derived from strain PA01 (Figure 2 ) resulted in even more subfractions representing as many as four populations of molecules with 0-antigen polysaccharide chains of different length which are made in large amounts. I s Such a separation monitored by SDS-PAGE also showed the presence in some of the strains tested of another LPS species carrying common antigen polysaccharide chain which was recovered in a minor peak and made up 10 to 15% of the total LPS sample. LPS species containing 0-antigen and common antigen chains afforded in SDS-PAGE two distinct sets of ladder-like bands. The molecules of the latter moved slower than those of the former having the same mol wt evidently due to the different charge of the polysaccharide chains: the 0-antigen of the strains studied is acidic while common antigen is neutral (see Section 1I.D). An R-form LPS fraction contaminated by a considerable amount of short-chain S-form and common antigen containing

FIGURE 2. Fractionation of LPS from P.acruginosa strain 503 on Sephadex G-200. Fraction comspondmg to LPS species carrying common polysaccharide chain is indicated by an asterisk. (From R~vcra,M., Bryan, L. E., Hancock. R . E. W . , and Mdjroarty. E. J . . 1.Bacrcriol., 170. 512, 1988. Wilh permission.)

LPS species has been separated from long-chain S-form LPS fraction by ultracentrifugation at 75,000 x g.36 Relatively little lipid A-core is usually capped with a long side chain. According to different estimations the mol fraction of S-form does not exceed 15% (usually 3 to 1 l % ) , and in the case of rough-type strains it may be as low as 0.2%.’.2.15.41 Larger amounts of a very-long-chain population than of the long-chain and intermediate-chain populations have been found in LPSs from a series of smooth-type strains derived from PAOl strain.I5 As for the number of molecules having substituted lipid A-core (total s- and SR-forms), it amounts to 25% of the total LPS molecules.2.B The capping frequency and the 0-antigen chain length depend markedly on growth conditions. As the temperature of growth increases, the total amount of capped forms of LPS from strain PAOl also increases from 19.3% at 15°C to 37.6% at 45°C whereas the content of S-form decreases and the predominant capped species at the lugher temperature is the SRThe effect of different culture media has also been investigated.2 While the overall capping frequency in strain PAO1, which amounts to approximately 20% changes insignificantly, there is some variation between the relative amounts of S-form vs. SR-form molecules (from 5.3 to 11.9% to 10.9 and 8.0%,respectively). In vivo grown strain P A 0 1 has been shown to produce LPS which displays an altered banding pattern compared to that of the in vitro grown strain, with an

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Critical Reviews In increase in short-chain species and a loss of some long-chain species.a A considerable influence on the observed LPS banding pattern may also be the procedure of LPS isolation (see Section 11. A. 3). The average length of 0-antigen chains in International Antigenic Typing System (IATS) serotype 6 strain is approximately 20 repeating units, as estimated on the basis of the LPS banding pattern in SDS-PAGE.” Taking into account the structure of the corresponding 0-antigen repeating unit (see Section II.D.3.b), this length corresponds to a mol wt of approximately 14,000 Da. The similar rnol wt has been supposed for the 0-antigen chain of strain PA01 . 4 1 The longest O-antigen chain in serotype 6 strain comprises about 50 repeating units,’8 that corresponds to a rnol wt of about 45,000 Da. A relatively small average rnol wt (10,000or 14,000 Da) has been determined for the 0-antigen-core polymer from Fisher immunotype 2 whereas larger values (from 25,000 to 75,000 Da) are characteristic for many other P. aeruginosa strains. 12.29.31-34.44 Extracellular polysaccharides with 0-specificity have much higher rnol wt (100,000to 350,000Da) than outer-membrane 0-antigens, and it has been found that their size is larger when they are produced during stationary phase of growth as compared to the size of polysaccharides isolated from younger ( 18 h) c ~ l t u r e . ’ ~ - ~ ~ Different estimations showed that the common polysaccharide antigen has a mol wt of 12,000 to 27,000Da and is on the average shorter than the 0-antigen in the same strain. 15.16.34.36

3. Isolation LPSs from wild-type P.aeruginosa strains are most widely isolated by the method of Westphal and Jann, which involves extraction of cells with a hot phenovwater mixture followed by separation of the phases and purification of LPS by sedimentation as a pellet using ultracentrifugation or by removal of nucleic acids contaminations using precipitation with Cet a ~ l o n Most . ~ ~ of the LPS preparations are recovered from the aqueous phase. In some cases, however, LPS is isolated from the phenol layer, the aqueous layer being almost free of the antigen, or LPS is partitioned between both I a y e r ~ . ~ , ~ ~Th’ . *IS. ~ ’ phenomenon is evidently due to the presence of sugars with hydrophobic groups, such as acetyl and methyl (C-6) groups of 2-acetamido-2,6-dideoxyhexoses,in the polysaccharide side chain (see Section II.D.3).46 The inconvenience arising from the undefined behavior of LPS may be avoided by excluding the step of separation of the phases and removal of phenol from the combined phenovwater layer by dialysis followed by usual treatment^.^^,^^ It should be noted that LPS preparations obtained in this way usually have a higher content of proteins (3 to 9%) in comparison to that of 0.5 to 2% for preparations recovered from the aqueous layer. Yields of LPS isolated by the Westphal and Jann procedure from the wild-type P. oeru-

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ginosa strains amount to 4 to 9% of the dry cell weight depending on the ~rganism.‘~ In some cases, lower yields (less than 1%) were o b ~ e r v e d ;however, ’ ~ ~ ~ ~ this may be attributed to lower content of LPS in the cell wall of these strains. The phenovwater extraction may also be used to isolate LPS from rough-type strains of P. aeruginosa; however, better results were obtained by applying phenoL/chloroform/petrol ether extraction by the method of Galanos et al.5’ (for example, 1 and 2.2%, respectively, for the rough-type strain AKl 121).so Application of this method to smooth-type strains resulted in very poor yields of LPS.so.52 Extraction of LPS from wild-type strains may be performed by trichloroacetic acid to yield LPS-protein preparations, which contain 80 to 90% of the total amount of LPS, the protein content being estimated at about 12 to 15%.2s Another method of LPS extraction is based on solubilization of the LPS-protein complex by treatment of mechanically disrupted cells with SDSEDTA followed by digestion of nucleic acids and proteins by the appropriate en~yrnes.~) This procedure is applicable to both smooth- and rough-type strains and gave higher yields of rather pure LPS (3.4 to 5.9%. that is up to 80% of the theoretical) than both the Westphal and Jann and Galanos et al. methods applied to the same strains. The authors noted that a potential problem associated with the new procedure is the heating step at alkaline pH, which is necessary for complete removal of pronase-resistant proteins. This treatment could cause partial transesterification or even loss of ester-linked fatty acids in the lipid A moiety; however, the change does not seem more significant than in phenovwater extraction. Such a heating may also result in partial destruction of alkali-labile 0-specific chain (see Section II.A.4.b). The heating at alkaline pH may, however, be avoided without a considerable increase in the content of proteins (up to 5% as compared to 0.5 to 2.5% for the procedure including the heating step). The method proposed by Uchida and Mizushima consists of the recovery of LPS in an insoluble form together with denatured proteins in a hot MgC1,Rriton X-100 solution followed by solubilization with EDTARriton X-100.52This procedure afforded good yields for the majority of the strains tested (2.7 to 5 % that is 40 to 80% of the total amount of LPS) and is less time consuming and less laborious as compared to the method of Darveau and Hancock. Protein contamination is, however, somewhat higher (5 to 15% when the proteolysis step is omitted). Gentle methods of extraction which lead to LPS-protein complex are based on treatment of cells with EDTA in a buffer or with aqueous E D T A l g l y ~ i n e . ~ A ~ .loosely ’ ~ ~ ~ bound LPSprotein complex was extracted from cells of P. aeruginosa Lanyi-Bergan serogroup 01 1 by saline solution; the preparation contained half of the LPS which could be isolated by the phenol/ water method.* LPS (or LPS-protein complex) similar to that extracted from cells is spontaneously released by some

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Microbiology stTdins.17.s7 that seems characteristic to a greater or lesser extent for all P . aeruginosa strains, especially after the exponential growth has ceased. An increased amount of proteins which are often present in LPS preparations isolated by any other than classical Westphal and Jann procedure could be removed by phenouwater treatments5 or, as mentioned earlier by proteolysis, for example, with protease K.1’.52For many purposes (e.g., for SDSPAGE analysis or preparation of 0-antigen polysaccharides), the LPS-protein complex may be used without any punfication. Different extraction procedures can generate LPS preparations which differ in the capping frequency and the length of polysaccharide chain. Thus, the phenoYwater procedure is known to select long-chain populations of LPS; whereas, the method of Darveau and Hancock as well as that of Uchida and Mizushima give preparations more representative as concerned short chain but not very-long-chain p o p u l a t i o n ~ . ~The ~.~~.~~ spectrum and relative proportion of LPS populations with long and very long side chains in preparations obtained by using the EDTNglycine method resemble closely those in LPS isolated by the procedure of Westphal and Jann; whereas, the 10 20 Fraction number number of lipid A-core molecules and short-chain populations is intermediate between that in LPS prepared by phenollwater FIGURE 3. Fractionation of acid-degraded LPS from P. acruginosa Lanyiand SDSiEDTA extractions. LPS preparations obtained by Bergan subgroup 0 4 a , 4 c on Sephadex G-50. (From Kninl. Y .A,, unpublished phenovwater and trichloroacetic acid extractions are almost data.) identical.3 Since LPS species, different in length of the polysaccharide derived from R- and RS-forms, respectively. In some cases, chain, may possess different biological properties, such as serthese species may be separated from one Material ological specificity and protective one should be of the third peak represents low-molecular-weight products of careful in the interpretation of data involving LPS preparations hydrolysis, such as KDO and its phosphorylated derivatives. of undefined heterogeneity, which are obtained by different The degraded polysaccharide from 0-antigen-deficient strains extraction procedures. exhibits only the two last peaks provided the common polysaccharide antigen is absent. 4. Degradation -7 Heterogeneity in a high-molecular-weight polysaccharide a. ACIDIC DEGRADATION fraction may be observed by using for separation Sephadex GThe carbohydrate moiety of LPS is linked to lipid A by the ketosidic linkage of the terminal KDO residue of the core. This 75, G-100,0rG-2OO,’~.@’which reflect the presence of different 0-antigen populations with definite chain length produced in linkage is extremely acid labile and is cleaved upon a very large amount. Like SDS-PAGE of LPS, the elution profile of mild acid hydrolysis (usually with 1 to 2% acetic acid, in some degraded polysaccharide is thus a characteristic pattern of incases with very dilute hydrochloric acid, on heating). Such dividual strain. cleavage gives a soluble carbohydrate material (so called deUsually 0-antigen polysaccharides are stable enough to graded pol ysaccharide) and lipid A (usually with contaminating survive the conditions of mild acid degradation of LPS. Howprotein) separated as an insoluble precipitate. ever, some 0-antigens contain monosaccharides with glycosThe degraded polysaccharide can be fractionated by gel idic linkage as unstable towards cleavage by acids as that of chromatography. When Sephadex G-50 is used, three peaks KDO. An example of this kind is the 0-antigen of P. aeruare usually observed from LPS of wild-type strains (Figure 3). ginosu Lanyi-Bergan serogroup 0 7 which includes a derivative The first peak corresponds to high-molecular weight side-chain of a higher 3-deoxyaldulosonic acid other than KDO (pseupolysaccharide derived from S-form LPS. For the majority of daminic acid, see Section II.D.3). Acid degradation of the the P. aeruginosa strains, it involves about 10 to 30% of the serogroup 0 7 LPS resulted both in liberation of lipid A and starting LPS eight;"^.'^ however, for several strains, its conin depolymerization of the polysaccharide chain, so that all the tribution is much higher (up to 70%).29.55The second peak 0-antigen material was recovered in the fraction of oligosaccorresponds to the core oligosaccharide and the core with one charides smaller than the core oligosaccharide.61The high(sometimes two) 0-antigen repeating units attached which are

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Critical Reviews In molecular-weight fraction of the degraded polysaccharide from this LPS was represented by the common polysaccharide antigen which has been isolated in this way in the yield of 1 to 3%. l 6

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b. ALKALINE DEGRADATION

Mild alkaline degradation (usually with dilute sodium hydroxide solution on heating) removes ester-bound fatty acids without elimination of amide-bound fatty acids and without cleavage of the linkage between the core and lipid A moieties. Due to a decrease of nonpolar interactions, alkali-degraded LPS has practically no tendency to aggregate and may be fractionated by gel chromatography without any detergent to reveal heterogeneity similar to that observed for the corresponding acid-degraded LPS Care should be taken in the cases of LPS containing alkalilabile substitutents, such as 0-acetyl, N-formyl, and N-acetimidoyl groups characteristic of many P . aeruginosa 0-antigens (see Section II.D.3). They may be partly or even completely removed or destroyed during the course of degradation, which may cause significant alterations in 0-antigen properties. Thus, 0-deacetylation has been shown to change 0-specificity of P. aeruginosa Lanyi-Bergan subgroup 02a,2b antigen.62 Alkaline degradation of LPS from P. aeruginosa Homma group G was accompanied by depolymerization of the O-antigen chain due to the presence of derivatives of galactosaminuronamide. l 4 These derivatives readily undergo alkaline (3elimination resulting in release of a monosaccharide attached and formation of oligosaccharide fragments (see Section II.D.3.b). 0-Antigen of P . aeruginosa Fisher immunotype 1, which is structurally related to the Homma group G 0-antigen and contains the same galactosaminuronamide derivatives, is also destroyed with alkali.63 As in the case of serogroup 0 7 LPS with the acid-labile O-antigen,6' degradation of the alkalilabile Homma group G LPS allowed the isolation of the common polysaccharide antigen linked to core-0-deacetylated lipid A.36

B. Serology LPS is a heat-stable surface antigen and due to this fact gram-negative bacteria keep their specific antibody-producing and antibody-binding capacity even after heating up to 130°C. Using preheated cell suspensions for immunization 0-sera can be produced which contain antibodies against 0-antigen and do not contain antibodies against heat-labile surface antigens, such as flagella, pili, or slime antigens. 0-Sera are very useful for serotyping P. aeruginosa strains, which is necessary for understanding the environmental and epidemiological distribution of this serologically heterogeneous organism. Bacterial agglutination with 0-sera is the method of choice for the determination of 0-antigens, both tube and slide agglutination tests being useful. On account of the absence of any antigenic factors which would mask 0-agglutination, such

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as capsule, live and heated suspensions of the overwhelming majority of P. aeruginosa strains give identical patterns of agglutination with 0-sera, the use of live bacteria being justifiable for routine serogrouping. The highest 0-titers are observed, however, with bacteria steamed for 2.5 h and then heated at 130°C for 1 h.M Techniques other than agglutination, such as passive haemagglutination, immunoprecipitation, immunoelectrophoresis are not used routinely, but are applied in many studies with LPS preparation. Thus, crossed immunoelectrophoresis was used for analysis of a P . aeruginosa LPS-based v a ~ c i n e . ~ ' Figure 4 shows that a complex mixture of antigenically distinct LPS constituting the PEV vaccine may be readily resolved by using this method. The SDS-PAGE technique also proves valuable for solving questionable problems of serotyping since strains of different serotypes express a great diversity in the spacing and intensities of bands of S-form LPS (see Figure 1) whereas strains of the same serotype have the same banding pattern.58 SDS-PAGE immunoelectrophoresis and immunoblotting with antibodies specific to 0-antigen may be useful for further confirmation of the results of serotype determination.

FIGURE 4. Analysis by crossed immunoelcctrophorcsis of the polyvalent LPS-based P . ocruginoso vaccine PEV. (From Maclntyrc, S . , McVeigh. T., and Owen, P.,Infect. fmmun.. 51, 675. 1988. With permission.)

Several independent serotyping systems for P . aeruginosa based on 0-antigen specificity have been established and their history has been reviewed. '.@M Use of different classification schemes complicated the comparison of results of studies of P. aeruginosa antigens in various research groups. To avoid these difficulties a survey of different serotyping schemes has been canied out aiming at the establishment of all of the most prominent (major) somatic antigens of P. aeruginosa." As a result a scheme comprising initially 17 groups has been proposed as the IATS. It includes 12 antigens described in the original scheme of H a b ~ , ~together ' with 5 additional antigens from other schemes which were found to be distinct (Table 1).

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Microbiology Table 2 Origin of Lanyi-Bergan Reference Strains and Correspondence of O-groups of Different Classification Schemes

Table 1 Origin of IATS Reference Strains LATS serotype 1

7

3 4

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5

6 7 8 9 10 11 12 13 14

15 16 17

Origin Habs group 1 Habs group 2 Habs group 3 Habs group 4 Habs group 5 Habs group 6 Habs group 7 Habs group 8 Habs group 9 Habs group 10 Habs group 11 Habs group 12 Sandvlk group II Verder-Evans group V Lanyi group 12 Lanyi group 3 Mcitcn group X

Strain ATCC no.

O-Group (subgroup)

ATCC 33348 ATCC 33349 ATCC 33350 ATCC 3335 I ATCC 33352 ATCC 33353 ATCC 33354 ATCC 33355 ATCC 33356 ATCC 33357 ATCC 33358 ATCC 33359 ATCC 33060 ATCC 33361 ATCC 33362 ATCC 33363 ATCC 33364

I 2a,2b Za.Zb.2~ (2Wc 2a. 2d 2a.2d.2~ (2a)JdJf 3a.3b 3a.3b.3~ 3a,3d 4a,4b 4a,4c 6a. . . . 6a,6b 6a.k 6a,6d 7a.7bI7c 7a.7b.7d 7a,7d 9a.9b.9d 9a,9c 9a.9d 10a,10b 10a.lOc 1la.llb

Provisional serotypes 18 19 20 21

Chinese scheme group II

Strain PA-87 Strain PS-113 Vcrder-Evans group I

ATCC 43390 ATCC 43731 ATCC 43732 ATCC 43732

Data from References 66 and 69

Up to four more major antigens have been recently identified in P. aeruginosa, which are candidates for groups 18 to 21 in JATS,68.69and there are reasons to believe that the number of serogroups will increase further. The IATS being a simplified antigenic scheme taking into account only major antigens is useful as a tool for rapid epidemiological tracing and is accepted by many investigators. However, this scheme does not reflect serological relationships between different groups (for example, between groups 2, 5 , 16, and 18; 7 and 8; 13 and 14). These relationships are due to the presence of minor antigens appearing in various combinations with the major antigens. Moreover, some strains having the same major antigen as one of the serotype strain but distinct minor antigens are not included in the IATS. Minor antigens are well demonstrable using a technique with adequately absorbed O-sera, and they are very useful for a finer differentiation of strains which is necessary for many laboratory purposes. The subgroup differentiation based on minor antigens may also give more reliable results in epidemiology. A serotyping scheme taking into account both major and minor somatic antigens has been proposed by Lanyi and by Lanyi and B e ~ g a n . ~ This . ’ ~ scheme was supported later by Homma and supplemented by Akatova and Smirnova (Table 2).’*.’* Strains are united here into serogroups on the basis of the presence of a common group antigen, and subdivision w i b

1la.llc 12 13a, 13b 13a. 13c 14

I5 a

Origin’

IATS

Fisher

Homma

Strain 170014 or Habs group I

1

4

I

Strain 170003 Wokatsch p u p 25 Straio 170002 or Habs group 2 Strain 170005 Strain 170006 Strain 170007 Wokatsch group 14 Strain 170001 or Habs group 3 Wokatsch group 13 Strain 1700021 or Habs p u p 4 Strain 170040 Habs group 6 Swain 170008 Strain 170009 Strain 170010 Strain 170011 Strain 170012 Strain 170013 Strain 170020 Wokatsch group 16 Strain 170019 Habs group 10 Strain 170002 Strain 170015 or Habs p u p 1 1 Strain 170016 Strain 170023 or Habs group 12 Sandvik group II Verder-Evans group V Meitcn group X Strain 170022

16 2 5

20

B

3

A

4

F

6

1

G 7 8 6

C

9

D

21 10

H 5

I1

2

E

12

L

13 14

K

17

N

15

1

Numben of strains according to the Hungarian National Collection of Medical Bacteria.

Data from References 3 and 64

complex serogroups is based on the presence of distinct partial antigens. Group antigens are not always the most prominent ones, and they may be underdeveloped in some strains (in Table 2 this is shown by parentheses, e.g., 0(2a),2c). Nevertheless, these strains have the same antigenic structure as their counterparts with a well-developed group antigen, and their partial antigens have never been found associated with antigens belonging to other groups. Chemical studies also showed that 0-

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Critical Reviews In antigens of these strains are structurally similar to each other the longest-chain LPS species also is much less virulent than and substantiated their positioning in one serogroup (see Secstrain PACl , the 50% lethal dose of PI4 and mutant PAC605 tions II.D.3.b and LI.E.1). strains being not significantly different . 2 7 Comparative study Relationships between reference strains of the Lanyi-Berof a variety of wild-type P. aeruginosa strains also showed gan scheme and other classification schemes including IATS that the virulence decreased as the proportion of LPS species have been established,@ and some of them are given in Table having short-chain antigen increased. 26 Thus the virulence ap2. peared closely associated with both the presence and the chain Serogroup distribution of P. aeruginosa strains isolated length of 0-antigen in LPS. It is unknown, however, whether from a variety of sources has been repeatedly ~ t u d i e d . ~ . ~ ~ - ’the ~ fine structure of 0-antigen influences the virulence of Strains belonging to IATS serogroups 2, 6, and 11 have been bacteria. found to be prevalent whereas some other serogroups, such as In noncapsulated bacteria, such as P . aeruginosa, LPS can serogroups 14 and 15, occur rather rarely. The frequency of serve as the target for both bactericidal and opsonic antibodies. occurrence of different Linyi-Bergan serogroups among P. In many 0-antigen-deficient P. aeruginosa strains, mutations aeruginosa isolates from various clinical sources has been resulted in loss of serum resistance, so that they readily undergo estimated. 64 killing by low concentration of normal human serum mediated A serious problem with serotyping of P. aeruginosa arises by c~mpIement.’~~*’.~’~~’ This, however, does not seem to be from the relatively high frequency of occurrence of nontypable the only reason for decreased virulence. Thus, a serum-senstrains, which are agglutinated with no typing 0-serum or with sitive P. aeruginosa strain PA103 has been shown to be virmore than one serum or are autoagglutinable. Such strains, ~ I e n t , ’and, ~ on the other hand, all natural wild-type P . which are most typical of isolates from patients with cystic aeruginosa strains with different virulence are serum resistant. fibrosis lung infection, appear to be devoid of LPS 0-antigen The absence of 0-antigen chain would be expected to decrease chain or express it in reduced Lack of agglutinthe ability of the mutant strains to evade other host-defense ation may be accounted for by masking of 0-antigen by another mechanisms, particularly phagocytosis. Indeed, the mutant strain surface antigen whereas polyagglutinability is probably due to PAC605 was opsonized by complement and then phagocytized the accessibility of a common antigen related to LPS which to a higher extent than its parent PACl train.'^ It is also becomes more exposed in the absence of 0-antigen. remarkable that the 0-antigen-deficient PAC605 strain could The category of polyagglu~nabilitymay be eliminated by be effectively opsonized by complement both in the presence using monoclonal antibodies (homogenous myeloma immuand in the absence of antibodies whereas opsonization of PAC1 noglobulins with predetermined specficity), which have proven and P14 strains strongly depends on the presence of antibodies to be more sensitive and specific in serotyping than polyclonal to LPS.2’ ones.58.76.77 Thus through the use of the complete panel of the agglutinating 0-antigen-specific monoclonal antibodies, 45% 2. lmmunogenicity of P. aeruginosa isolates nontypable with typing sera normally Bacterial LPSs as well as polysaccharides are well-known used were found to be typable.” to be responsible for humoral immune response. The immunogenecity, i.e., the ability to stimulate the formation of specfic antibodies, depends on the molecular size. The accumulated C. lmmunobiology data permits the conclusion that 45,000 5 5,000 is the mol 1. Virulence wt above which free polysaccharides are immunogenic and Bacterial virulence may be regarded as the capacity to resist below which their immunogenecity falls off rapidly.” the host’s defense mechanism, so that the microorganism can LPS is known as a highly immunogenic antigen, that is multiply and eventually damage or kill the host. LPS is one of the virulence factors of gram-negative bacteria, and its strucprobably due to its tendency to form aggregates with appeared ture plays an important role in manifesting this property. molecular weights in the millions. Acid-degraded LPS, alkaWild-type strains with complete core and 0-antigen chain line-degraded LPS, and extracellular high-molecular-weight are more virulent than mutants lacking the 0-antigen moiety. polysaccharides of P. aeruginosa are not immunogenic at doses Thus, an 0-antigen-deficient mutant P. aeruginosa strain less than 1 kg whereas intact LPS is, and thus the lipid A portion is essential for primary immune response to P. aerwPA220-R2 has been found to be comparatively nonvirulent with a 50% lethal dose for mice more than 1,000-fold higher than ginosa LPS.*’ The immunogenic dosage level may depend on that of its parent wild-type strain PA220.26The same decrease the immunogenicity of LPS from various P. aeruginosa strains as well as on the immunological response of different animals in virulence, as compared to the parent strain, has been oband animal strains. served for strain PAC605, which is an 0-antigen-deficient spontaneous mutant of P. aeruginosa strain PACl .27 Another 0-antigen polysaccharides prepared by acid degradation of wild-type strain P14, which has the same amount of anchor P. aeruginosa LPS usually have a mol wt less than 50,000 and places of LPS on the cell surface as the strain PACl but lacks are not immunogenic; only one case has been documented

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Microbiology when the free O-antigen of Fisher immunotype 1 having mol wt 60,000 and more was immunogenic in mice.63On the contrary, extracellular polysaccharides with O-specificity having a mol wt of more than 100,OOO appeared to be immunogenic.4.30.31.63.74.82 Alkali-treated detoxified LPS is able to elicit specific antibodies; however, it is a worse immunogen in mice than the intact LPS.83Immunogenicity of delipidated O-antigen polysaccharide may be increased by coupling with proteins, and the preparation and properties of such conjugates are discussed in Section II.C.5. As can be expected, immunization with LPSs or polysaccharides of various P. aeruginosa strains in the majority of cases results in serotype-specific immune response. In a few cases, antibodies to other serotypes are made;4,*3the phenomenon has no rational explanation. 3. Protective Capacity

the common core antigen by O-antigen or by structural differences in core regions of different serotype strains. During the last two decades, LPS was tested as a vaccine against infections due to P. aeruginosa. Since in nature infection may be caused by a strain belonging to any immunotype(serotype), it is important that the vaccine should be protective against the whole range. Multivalent vaccines Pseudogen based on LPSs of seven Fisher immunotype strains, and PEV produced by EDTNgIycine extraction of 16 IATS serotype strains have been studied in animal models and in humans and proved to be p r o t e ~ t i v e . ~Whereas . ~ ~ . ~ ~severe adverse reactions, such as malaise, fever, localized induration, pain, were observed after administration of Pseudogen, PEV elicited only few toxic side effects. The reason for this apparent discrepancy most likely relates to differences in the quantities of LPS administrated: one human dose of Pseudogen and PEV contains about 1.750 or 30 k g of LPS, r e ~ p e c t i v e l y . ~ ~

a. LIPOPOLYSACCHARIDES

LPS and LPS-containing P. aeruginosa cell-wall preparations have been shown to elicit both opsonic and complementmediated bactericidal antibodies.85.86Direct administration of purified LPS (active immunization) can protect animals against fatal experimental infections by P . aeruginosa,’8,87-91and the level of protection correlates with anti-LPS antibody titer^.*^,^ High anti-LPS titers have been correlated also with increased rates of survival among patients with P . aeruginosa septicemia. Experiments performed on the subfractionated LPS species have revealed a relationship between O-antigen chain length and protective capacity of LPS. Thus, P. aeruginosa IATS serogroup 6 LPS species with over 18 O-antigen repeating units were 50 to 100 times more protective in mice than those with zero to two repeating units and the most efficient protection afforded by LPS can be accounted for by LPS species possessing 10 or more O-antigen repeating units.” Whereas nonspecific protection induced by LPS has been observed, which seems to be related to the presence of Lipid A m ~ i e t y , LPSs ~ ~ . ~of~wild-type strains act predominantly in a serotype-specific manner,85.w.9e97that is consistent with the important role of specific antibodies directed to O-antigen chain. On the basis of challenge protection in mice, Fisher et al. have defined seven groups of cross-protective homogeneity (immunotypes 1 to 7).98 All these groups are characterized by different major somatic antigens and correspond, thus, to different LATS serogroups and different Lanyi-Bergm subgroups (see Table 2).61.M.93 Immunization with LPS of O-antigen-deficient P. aeruginosa mutant strains has been shown also to protect mice against infection when challenged by the homologous strain, and the protection correlated with a specific immune re~ p o n s e . ~However, ’ LPS of the mutant strains tested were not effective in prevention of the infection caused by various P . aeruginosa wild-type strains, as may be expected on the basis of structural similarity of their core region of LPS. The absence of cross-protection may be accounted for by the masking of

b. HIGH-MOLECULAR-WEIGHT POLYSACCHARIDES

Extracellular high-molecular-weight polysaccharides with 0-specificity induced in human O-antigen-specific antibodies which can opsonize P. aeruginosa for phagocytic killing.84 Unexpectedly, immunization of mice with the polysaccharide of Fisher immunotype 4 strain resulted in protection comparable to a nonimmune control, that is evidently accounted for by the presence in most adults of a high level of naturally occurring antibody to immunotype 4 O - a ~ ~ t i g e n . ~ ~ The antibodies induced by immunization with the polysaccharides produced by various Fisher immunotype strains are protective in animals against both intraperitoneal and burnwound experimental P. aeruginosa i n f e ~ t i o n . ~T’ ,cell-me~~ diated immunity elicited by immunizing mice with Fisher immunotype 1 polysaccharide has been reported, which may also be important for p r o t e c t i ~ n . ~ . ~ . ~ . ~ ~ High-molecular-weight polysaccharides provided less protection from experimental P. aeruginosa infection as compared to L P S S , ~and . ~approximately 1000-fold more polysaccharide was needed to protect mice to an equivalent degree.w However, the polysaccharide vaccine, even containing relatively large amount of the antigen, is safe enough, eliciting only slight pain and tenderness at the infection site. Thus, the polysaccharides of Fisher immunotypes 1 and 2 have been used to immunize humans without any harm at doses of 100 to 250 k g which were enough to elicit both binding and opsonic antibody at the level comparable to the level in patients who survive P. aeruginosa s e p s i ~ . ~ ~ . ~ ’ 4. O-Antigen-Protein Conjugates A useful tool in studies of immunological phenomena related to LPS are O-antigen-protein conjugates. First f. aeruginosa conjugates were prepared by covalent coupling of the detoxified (alkali-treated) LPS of Fisher immunotype 5 to different proteins: tetanus toxoid, P. aeruginosa pili or exotoxin A.83A variety of conjugates has been prepared on the basis of 1990

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Critical Reviews In 0-antigen polysaccharides derived from acid-degraded LPS using different protein carriers, spacers, and coupling procedures.J483.92.101-103These, as well as immunological properties of the conjugates obtained are summarized in Table 3. The conjugates, including those based on the highly toxic (in the free state) toxin A, have been found to be nontoxic in animals and in humans.102.104s10s The majority of them are capable of eliciting antibodies to both 0-antigen and protein moieties, the antibodies directed to 0-antigen being opsonic for the homologous organisms. 102.106Immunization with a multivalent vaccine comprising 0-antigen-toxin A conjugates derived from seven Fisher immunotype strains engendered an antibody response which recognized all LPS serotypes included in the vaccine. 10s.107 Administration of each of the conjugates tested prevented mice from fatal experimental P. aeruginosa infection when challenged by the homologous strain, the degree of protection with the conjugates based on toxin A amounting to 10,OOOfold and higher.Io8In several conjugates of this type, the toxin A-neutralizing capacity correlated well with antitoxin A antibody titers, and the conjugates protected mice also against toxin A intoxication. Io8 It has been found that the reduction of mol wt of Fisher immunotypes 1 and 7 0-antigens from 45, -50,000 to about 10,OOO resulted in significant enhancement of protective capacity without change in immunospecificity." Different length of 0-antigen chain may also be one of the reason for the marked variability in the immune response directed against 0-antigen portion of conjugates produced from LPS of different strains. Ion Another reason for poor immunogenicity of some of the 0antigen-protein conjugates may be associated with destruction of the 0-antigen polysaccharide during the course of acid deg-

radation of LPS, especially in the case of acid-labile 0-antigens (see Section II.A.4.a. and II.D.3. b).

5. Passive Immunization Passive immunization implies transfer of immunity by administration of antiserum containing preformed protective antibodies. This has been demonstrated with all P. aeruginosa antigens discussed above, that is using antibodies induced by administration of L P S S , ~ ~ ~high-molecular~ ~ " ~ ' ~ ~ ' ~ ~ weight p o l y s a ~ c h a r i d e s , ~' I ~and . I 0-antigen-protein conjugates. 101.I04.105.1Oa Antibodies directed against 0-antigen have been shown to provide passive protection in mice from challenge with live organ~sm,%.101,108.1 1 1 - 1 I5 and as little as 1 Fg of passively transferred antibody was found to be able to confer the marked effect. 32.IIZ.I16 Protective properties of the antibodies are associated mainly with the immunoglobulin G class;' however, other classes have also been shown to induce protection. 83.I 13.115. I 17 Comparison of human immunoglobulin G preparations enriched in polyclonal or monoclonal antibodies to Fisher immunotype 1 LPS showed that the polyclonal preparation is a more potent opsonin. On the other hand, the polyclonal preparation demonstrated an absolute requirement for complement while the monoclonal preparation appeared capable of mediating enhanced phagocytic activity without complement.lW As could be expected, agglutinating a n a w e s have far higher protective capacity than nonagglutinating Since protection afforded by anti-LPS is serotype specific, antibodies directed to each of the P. aeruginosa serotypes are to be prepared for potential treatment or prophylaxis. Human antibodies are preferable for practical use as animal antibodies

Table 3 Properties of Pseudomonas aeruginosa 0-Antigen-Protein Conjugates Serotype or immunotype

Protein

Spacer

Coupling procedure

Immunogenicity

+

Fisher 1 Fisher 3 Fisher 1, 2. 7

w

-

BSA, toxin A

(CHA

A B C

Fisher 5

w

(CH,),

D

Pili Toxin A Toxin A

-

D

(CH,), CO(CH,),CO

D A

IATS 1 . 2 4 , 6, 10, 1 1 IATS 6 Meiten I1

BSA

(CHI),

A B

+

N.t.

+ + -

+

+ N.t.

Protective capacity

Ref.

+

101

N.t.

I03 44

N.I. N.t. N.I.

83

+

+

102

+ +

106

92

Nore: . BSA - bovine serum albumin, TT - tetanus toxoid; A - partially peridateoxidized 0-antigen is coupled to tctramethylenediamine or adipic acid dihydrazide-derivatized protein by reductive deamination with sodium cyanoborohydride. B - cahodiimidc is used as a coupling reagent, BSA being methylated before coupling, C - 0-antigen activated by carbodiimide or. in the case of Fisher immunotype 2, by cyanogen bromide is coupled to protein derivatized with hexamethylenediamine, D - detoxified LPS activated by N-hydroxysuccinirnide is coupled to protein. TT and pili being derivatizcd with tetramethylenediamine before coupling, and N.t. - not tested.

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Microbiology represent foreign proteins in man. On the way to prepare such an immunoglobulin preparation, human monoclonal antibodies against O-antigens of P.aeruginosa Homma serogroups A, B, E, G, and I have been produced and found to possess opsonophagocytic killing activity of the corresponding serotype strain. ’ l6 It should be noted, however, that in preparing multivalent monoclonal preparations for passive transfer of immunity it is important to characterize the fine specificity of the antibodies involved. If an antibody is specific to an antigen common to a serogroup, one can expect that it will also protect against all the variant strains which possess this antigen, i.e., which belong to the same serogroup. In contrast, if an antibody turns out to be specific to a partial antigen, it may be not protective against strains of other subgroups in the same serogroup which lack this partial antigen (see Section U.B). Antibodies directed at the core of LPS seem to be of particular interest since the inner part of this region contains structures common to LPS from a variety of strains (see Section ff .D.2). However, mouse monoclonal antibodies directed to the core turned out to be not opsonic and despite such antibodies are believed to act as endotoxin-neutralizing agents; they have not been found to be protective. Monoclonal antibodies, both mouse and human, specific to P. aeruginosa common polysaccharide antigen have also been tested for protective a ~ t i v i t y .They ~ ~ . ~failed ~ to protect mice against challenge with wild-type serotypable strains, e.g., Homma serogroup G strain, that is most likely due to the fact that the common antigen is not exposed on the cell surface of wild-type strains. On the contrary, antibodies promote phagocytosis of nontypable O-antigen-deficient strains and protect mice against the infection caused by such strains.

D. Chemistry 1. Lipid A Lipid A of P. aeruginosa consists of the backbone p 1,6linked disaccharide of glucosamine phosphorylated at positions 1 and 4’. Both amino groups and part of hydroxy groups of the disaccharide are acylated by different fatty acids. ‘.‘la Some of them, such as dodecanoic (C12:O). 3-hydroxydecanoic (3OH-C10:0), 2-hydroxydodecanoic (2-OH-C12:0), and 3-hydroxydodecanoic (3-OH-C12:O) acids, are found in all tested strains. Also often encountered in considerable amount is hexadecanoic acid (C16:O) while other fatty acids occur only as minor components if not contaminants.1*12.17~4331.118 2-OH-C12:0 has been shown to have the L configuration while 3-hydroxy fatty acids are of the D c~nfiguration.”~ 3-OH-C12.0 is the only amide bound acid whereas other acids are ester linked to hydroxy groups of the glucosamine backbone or of 3-hydroxy fatty acids.1.’18.’20 A lipid A precursor of P. aeruginosa strain K799 obtained by inhibition of LPS biosynthesis at the stage of transfer of KDO to lipid A has undergone the most detailed structural

investigation. l2] The major precursor species accumulated has the same composition as lipid A derived from the LPS of this strain, and it contains glucosamine, phosphate, C12:0, 2-OHC12:0, 3-OH-C10:0, and 3-OH-C12.0 in the molar ratios of 22: 1: 1:2:2, respectively. As judged from fast-atom bornbardment mass spectrometry analysis, the species turned out to be heterogeneous. Based on these and the other data,J2.11*-120 it has been concluded that the lipid A precursor represents a mixture of four hexaacyl derivatives, the individual species differing from each other only in the content and distribution of C12:O and 2-OH-C12:0 linked to hydroxy groups of amidebound 3-OH-Cl2:O. Their structures can be represented by the general formula shown in Figure 5 . One of the KDO residues of the core is linked to hydroxy group at position 6’ of the nonreducing glucosamine residue while hydroxy group at position 4 of the reducing glucosamine residue remains unsubstituted.

FIGURE 5. SUUCNES of four lipid A precursor species from P. oeruginosn strain K759. Major precursors: R = CI2:O. R’ = 2-OH-C12:0 or R = 2OHC12:O. R’ = C12:O; minor precursors: R = R’ = C12:O or R = R‘ = 2-OH-C12:0. (After data of References 120 and 121 .)

An alternative distribution of hydroxy fatty acids has been suggested in lipid A of P. aeruginosa strain 1224.Iz2This, however, does not seem reasonable, and the mode of attachment of 3-hydroxy fatty acids is, most likely, the same in lipid A of d P. aeruginosa strains. One can propose that fatty acids, when present with a longer chain, such as C16:O. replace part of C12:O and 2-OH-CI2:O; this suggestion, however, has never been proved. The structure of P. aeruginosa lipid A is very similar to that of rather well studied enterobacterial lipid A , which has the same phosphorylated glucosamine backbone and the same

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Critical Reviews In mode of substitution of amino and hydroxy groups. However, in contrast to lipid A of enteric bacteria, no additional components have been found attached through phosphate groups. Another difference is that 3-hydroxy fatty acids are shorter in lipid A of P . aeruginosa (3-OH-C10:O and 3-OH-C12:0 rather than 3-OH-C14:0), which may result in a higher fluidity of LPS and influence the barrier properties of the lipid domains of outer membrane.* It is noteworthy that the biosynthesis of lipid A in P . aeruginosa proceeds although similar, but not identical to that of enterobacterial lipid A. The major precursor of the latter includes only amide-bound fatty acids while that of the former contains all fatty acids present in lipid A of the mature LPS before addition of KDO.

2. Core Composition of P . aeruginosa core oligosaccharide has been studied in many details, and the results have been reviewed.'.8 The inner core is composed of heptose (L-glyceroD-mnno-heptose, 1) and KDO (3-deoxy-D-manno-octu1osonic acid, 2 ) and is highly phosphorylated (up to 11 to 16 phosphate residues per LPS molecule including phosphate associated with lipid A). These are present as hiphosphate, pyrophosphate monoester, pyrophosphate diester (most probably connecting ethanolamine to the core), and monophosphate.8 The role of phosphorylation for stabilization of the outer membrane is discussed in Section I1.A. 1. C HZOH

CH OH

t-?,,

no-(

FIGURE 7. Structures of LPS cores from P. aeruginosa strains NCTC 1999 (3) and PAClR ( 4 ) . (After data of References 13 and 124.)

2

1 FIGURE 6.

In smooth-type strains and in those of rough-type strains which produce complete core, constitutents of the outer core region are glucose, rhamnose, galactosamine, and alanine. Part of the alanine residues acylate the amino group of galactosamine while the position of the others remains obscure. Some of the alanine and glucose residues may be linked to the inner region of the core. As for fucosamine and quinovosamine, which have been proposed to enter the core of several Fisher immunotype strains,43they originate, most probably, from the single 0-antigen unit of SR-form contaminating the studied core material derived from the acid-degraded LPS. In core-

284

defective mutant strains glucose or/and rhamnose may be absent, and a mutant is known which is devoid of the whole outer core. The core oligosaccharide, to which the common polysaccharide antigen is attached, is similar in composition to that of 0-antigen-containing LPS, but includes additionally xylose and an unidentified 3-O-methyl-6-deo~yhexose.~~ It has been proposed that these two components form a separate oligosaccharide chain comprising seven residues of the 0-methylated sugar and two residues of xylose. Despite the similarity in monosaccharide composition, the complete core from various strains may differ in the structure. This follows from a serological test using two rough corespecific monoclonal antibodies, which reacted with only 6 of 17 IATS serotype strain^.^' Two rather different partial structures 3 and 4 have been proposed for the complete core of smooth P. aeruginosa strain NCTC 1999 and for semirough mutant PAClR derived from P . aeruginosa strain PACI, res p e c t i v e l ~ . ' Both ~ . ~ ~structures ~ shown in Figure 7 lack some details, and some others were assigned tentatively. It has been found that in the core 4, 0-antigen chain is attached at position 3 of the P-glucose r e s i d ~ e . ' ~

In the study of the core from PAClR strain, which was based mainly on methylation and I3C NMR spectroscopy, a series of the core defective mutants derived from this strain were examined, and the structures of their incomplete core have been e~tab1ished.I~ The data obtained, together with the other data,'= permit the conclusion that biosynthesis of the outer core in P. aeruginosa, as in enteric bacteria, proceeds by stepwise transfer of sugar residues, the transfer of rhamnose following the transfer of glucose. Differences in the structures of the core from strain to strain and, probably, from molecule to molecule in the same strain may be due to different content and position of phosphate and ethanolamine in the inner core. As for the structure of the heptose-KDO region of the core, it is unknown whether it varies from strain to strain. On the basis of the possibility of inactivating the heptose-specific coliphage T7, it has been proposed that the inner core of P. aeruginosa is structurally similar to that of enteroba~teria;'~'however, the detailed structure remains obscure.

Volume 17, Issue 4

Microbiology 3. 0-Antigen

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a. COMPOSITION

The composition of P . aeruginosa 0-antigens has been found to differ essentially from that of other bacterial antigens studied. l o First of all these antigens contain few neutral sugars, among which only rhamnose encountered rather frequently. Very typical are monoamino and diamino sugars, many of which carry a carboxyl function. Such monoamino sugars as D-glucosamine and D-gdactosamine which are widespread in namre occur only in a few of P. aeruginosa 0-antigens whereas the far less common 6deoxyhexosamines (D-quinovosamine, D-, and L-fucosamine) are present in most of them. Derivatives of an even rarer diamino sugar bacillosamine 5 to 7 have also been found.i26.i27 Acidic aminosugars are represented by 2-amino-2-deoxyuronic acids (derivatives of D- and L-galacfo isomers 8 to 12), 2,3-diamino-2,3-dideoxyuronicacids (derivatives of Dgluco, ~ - m a n n oand , L-gulo isomer 13 to 17), and 5,7-diamino3,5,7,9-tetradeoxynonulosonicacids (derivatives of L-glyceroL-rnanno isomer or pseudaminic acid 18 to 20 and D-glyceroL-galacro isomer 21). The last two classes of monosaccharides have been found for the first time in nature as constitutents of P . aeruginosa L P S S . The ~ ~ ~higher ulosonic acids are similar in structure to neuraminic acid (5-amino-3,5-dideoxy-~-glycero-D-galacfo-nonulosonicacid), which is a well known component of many biologically important carbohydrates. The new acids differ from neuraminic acid in the presence of an additional aminogroup at C-7,a deoxy unit at C-9 and in the configuration. Most aminogroups of the amino sugars are acetylated, but in several 0-antigens they carry other acyl substituents which occur rather rarely in natural carbohydrates, such as the formyl group (derivatives 10, 12, 19, and 20), the (R)- and (S)-3hydroxybutyryl groups (derivatives 6, 7, 18, and 20), and the acetimidoyl group (derivatives 15, 17, and 22). The last group is of basic character and endows the derivative of L-fucosamine 22 with basic properties and the derivatives of uronic acids 15 and 17 with amphoteric properties. Derivatives of D-gdaCtOSamiIIUrOniC acid have been found in the form of both free acids 9 and 10 and primary amides 11 and 12. Many constitutent monosaccharides (rhamnose, Nacetyl-D-fucosamine, derivatives of D- and L-galactosaminuronic acids and pseudaminic acid) may carry 0-acetyl group. Table 4 shows the distribution of the monosaccharides and nonsugar components in 0-antigens of different Lanyi-Bergan serotype strains.

on

OH

NHR-

8

HNR

HNR

0 COOH I

no

14

R = k

16

R = k

15

R = cH,C(=M1)

17

R =CH,C(=tW)

% -NUAc

AcHH

no

OH

CWH

21

b. STRUCTURES OF REPEATING UNITS 22

Structural elucidation of 0-antigens of f.ueruginosa LanyiBergan serotype strains has been completed quite recently, and the data are summarized.'O The structures of these 0-antigens, together with those of some other strains, are given in Table 5.

FIGURE 8. Monosaccharide constituents of P . uenrginosu 0-antigens. (After data of References 10. 14. 127.)

1990

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Critical Reviews In Table 4 Distribution of Monosaccharides and Non-carbohydrate Components in Pseudomonas Aeruginosa 0-Antigens Lanyi-Bergan serogroup

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Components

Monosacchandes DRib DXyl DGlC LRha ffilcN DGalN D M ~ ~ N DQUd

DFUCN LFucN oBac(ZN4N) DGalNA LG~NA DGlc(2N3N)A oMan(2N3N)A LCuI(2NlN)A

Pse(SN7N) Non(SN7N) N-Substituents Ac

1

10

+ + + +

+

+

+

+

+ +

+

-+

Fm (R)-CH,CH(OH)CH,CO (S)-CH,CH(OH)CH:CO NH = C(CH,)

+

+

t

+

+

+

0-Substituents Ac

+

t

Carboxyl substituents c

NH

Note; Abbreviations: Ac - acetyl, Frn - formyl, Qua- 2-amino-2.6-dideoxyglucosc (quinovosarnine). FucN - 2-amino2.4-diamino-2,4,6-tndeoxyglucosc (bacillosamine), GalNA - galactosami2,6,dideoxyfucose (fucosamine), Bac(2N4N) nuronic acid, Glc(ZN3N)A. Man(ZN3N)A. Gul(2N3N)A - 2,3diamino-2,3dideaxy-gluc-,-man-. -PI-uronic acid. Pse(SN7N) - 5 . 7 - d i ~ 3 . 5 . 7 . 9 - ~ t r a d e o x y - ~ - g l y c e r o - L acid c (pseudaminic acid), Non(5N7N) - 5.7diamine3,5,7.9tetradcox y-D-g~ycero-L-gu~~clo-nonulosonic acid.

-

Data from References 10. 14, 127. and 129

As one can see, most of the 0-antigens are regular polysaccharides built up of linear tri-or tetra-saccharide repeating units. There is only one branched polysaccharide with a pentasaccharide repeating unit (subgroup 0 13a, 13c). The regularity of some of the 0-antigens is masked by several factors. The most common is nonstoichiometric 0acetylation a s , for example, in subgroups 0 3 a , 3 b and 03a,3b,3c, where the residue of rhamnose carries the 0-acetyl group in only 40% of the total repeating units, or in subgroup 013a,13b, where the degree of 0-acetylation of the residues of N-acetylgalactosaminuronic acid is about 60%. In many other 0-antigens containing 0-acetyl groups the degree of 0acetylation amounts to 80 to 90%. Another factor masking the regularity of 0-antigens is nonstoichiometric amidation of carboxyl group of the derivatives

286

'

of galactosaminuronic acid, such as in serogroup 06.1J,130 Depending on the subgroup, the residue of N-formylgalactosaminuronic acid may be amidated in all, in part, or in none of the repeating units. And, finally, the regularity may be masked by partial epimerization at c-5 of one of the derivatives of 2,3-diamino-2,3dideoxyuronic acids in serogroup 02."' Such epimerization implies conversion of the p-D-mUnn0 isomer into the a-L-gdo isomer or vice versa. Thus, 0-antigen of subgroup 0(2a),2d,2f involves in comparable amounts trisaccharide units of two types which differ from one another in the configuration at C-5 of the uronic acid derivative carrying N-acetimidoyl group. 0antigens of subgroups 0(2a),2c and 02a,2d,2e, together with the main repeating units containing 2.3-diacetamido-2,3-dideoxy-a-L-guluronic acid, involves a small proportion (about

Volume 17. Issue 4

Microbiology Table 5 Structures of O-Antigens of P. Aeruginosa Lhnyi-Bergan group (subgroup)

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01

Other goups Fisher 4

Structure of repeating unit +~)-P-D--GIc(ZNAC~NAC)A+ 1+3h-D-FucNAc-( 1-+3)-a-~Qu&Ac-( I+4)-a-D-GalNAc-(

02a.Zb

+ ~ ) - ~ - F M ~ ( Z N A C ~ N ) A -I+~)-P-WMU~(ZNAC~NAC)A+ ( I+~)-~-D-FucNAc-( I + 31 CH,C=NH

02a,2b,Ze

44)-p-~-Ma11(

O(Za).lc'

Fisher 3

I-.

2NAc3N)A-( I+~)-P-D-M~~(ZNAC~NAC)A-( I+~)-P-D-FucNA~-( I+ 31 14 CH,C=NH OAc

-r4)-p-~-Man(ZNAc3N)A-( l-.4)-a-~Gul(ZNAc3NAc)A-( 31 CH,C=NH

I - + 3 ) - @ - ~ F u c N A c i I+

O2a.2d

-4)-p-wMan(ZNAc3N)A-( I + ~ ) - P - D - M ~ ~ ( ~ N A C ~ N A C )I A + -~( ~ - D - F u c N A c - I+~ 31 CH,C=NH

02a.2d.Ze'

I - + ~ ) - ~ - L ~ U ~ ( ~ N A C ~ N A1+3)a-~-FucNAc-t C)A-( I-, -4)-p-~Man(?NAc3N)A-( 31 CH,C=NH

+ ~ ) - ~ - L G u I ( Z N A C ~ N ) A1+4)-p-~Man(ZNAc3NAc)A-( -( 31 CH,C=CH -70%

I +3)-a-~-FucNAc+

I+

and

14 OAc

I+~)-~-D-FucNAc-( I+ +~)-P-D-M~~(ZNAC~N)A-( I+~)-~-D-MwI(ZNAC~NAC)A-( 14 31 CH ,C=CH 30% OAc

-

Fisher 7'

+~)-~-LGIJI(ZNAC~N)A-(I+~)-P-D-MuI(ZNAC~NAC)A+ I+~)-~-D-FucNAc-( I+ 31 CH,C=NH +6)-a-D4lcNAc-(

03a.3b

1+4)-a-~-GalNAcA-( I-r3)-p-~-Bac(2NAc4NH

I+2)-a-~-Rha-( I -+ 13 (S)-CH,CH(OH)CH:CO -40% OAc 41

03a.3b.3~

I+6)-a-~-GlcNAc-( 1+4)-a-~-GalNAcA-( l+3)+-~-Bac(ZNAc4N)-( I+Z)-a-L-Rha-( 13 41 13 OAc (S)-CH,CH(OH)CH?CO -40% OAc

03a.3d

+6)-a-&lcNAc-( Hornma A

l+4)-a-~GalNAcA-(

I-+~)-P-D-B~c(ZNAC~NACHI+3)-a-~-Rha-(

l-r

-+~~-D-G~CNAC-(I~~)-~-LG~~NACA-(I+~)-P-D-B~C(ZN~ N A c H l+Z)-a-L-Rha-(l-r 13 OAc

13

21

(R)-€H,CH(OH)CH,CO

-20% OAc

04a,4b 04a.4~ 06a.. . .

-4)-a-~-GalNAcA-( l+4)-a-~-GalNFmA+ 13 61 OAc NH,

1990

l-+3)-a-D-QuiNAc+

I-+3)-a-~-Rha+

I+

287

Critical Reviews In Table 5 (continued) Structures of 0-Antigens of P. Aeruginosa Linyi-Bergan group (subgroup)

Other groups

Structure of repeating unit

06a.6b

06a, 6c

Homma Gb

+4)-Q-&dNAcA-(

I + 4 j a - ~ - G a N F m A 3 I+3)-u-@uiNAc-(

I + 3 ) - c - ~ - R h d I+

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61

NH? +4)-a-WCalNAcA-( l-+4~-mCialNFmA-t 1+3)-P-~-QuiNAc-t 13 61 61 OAc NH, -20% NH,

06a.M

1+3)-a-~-Rha

Polysaccharide antigens of Pseudomonas aeruginosa.

The major polysaccharide antigens of P. aeruginosa are the cell-wall lipopolysaccharides many of which have an acidic polysaccharide chain (O-antigen)...
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