Pseudomonas aeruginosa lipopolysaccharides and pathogenesis

Pseudomonas aeruginosa lipopolysaccharides and pathogenesis

REVIE"WS Pseudomonas aeruginosa lipopolysaccharides and pathogenesis Joanna B. Goldberg and Gerald B. Pier P seudomonas aeruginosa disagreement as ...

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REVIE"WS

Pseudomonas aeruginosa lipopolysaccharides and pathogenesis Joanna B. Goldberg and Gerald B. Pier

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seudomonas aeruginosa disagreement as to whether the Pseudomonas aeruginosa is responsible for a con• common antigen is attached to lipopolysaccharide (LPS) plays a key role stellation of pathological the same LPS core as the 0 anti• in pathogenesis. In acute infections, conditions; the lipopolysacchar• gen or to a distinct core and lipid a smooth LPS protects the organism ide (LPS) of this organism can A component. Rivera et al. claim from complement-mediated killing and, be produced in varied forms that differential migration of during chronic lung infections, an altered depending on the type of infec• D-rhamnan-containing LPS and rough LPS helps the organism evade host tion established by the organ• O-antigen-containing LPS in an defense mechanisms. ism. P. aeruginosa LPS protects SDS gel indicates the presence J.B. Goldberg· is in the Dept of Microbiology, the bacterial cell from host de• of two distinct LPS (Refs 4,5). University of Virginia Health Sciences Center, fenses and mediates entry of However, using delipidated 0 Charlottesville, VA 22908, USA; bacteria into eukaryotic cells. side chains that would not ag• G.B. Pier is at the Channing Laboratory, Nontoxic preparations or de• gregate together and mono• Dept of Medicine, Brigham and Women's Hospital, rivatives of LPS components are Harvard Medical School, 181 Longwood Avenue, clonal and polyclonal antibodies Boston, MA 02115-5899, USA. potential vaccine candidates. specific to either the 0 antigens ·tel: +1 804243 2774, fax: +1 804982 1071, Recent advances in our knowl• or D-rhamnan antigen, Hatano e-mail: [email protected] edge of the structure, function et al. showed that these two and genetics of P. aeruginosa structures coprecipitated, indi• LPS synthesis have increased our understanding of cating that P. aeruginosa synthesizes only a single lipid the pathogenic properties of this organism. A/core molecule that can be substituted by 0 antigens andlor the D-rhamnan polymer6 • Structure of P. aeruglnosa LPS P. aeruginosa LPS has a similar structure to that of other LPS expressed on P. aeruglnosa strains from Gram-negative bacteria (Fig. 1). The 0 polysacchar• various sources ide (also known as B band) is the immunodominant P. aeruginosa strains isolated from clinical specimens antigen on P. aeruginosa. Currently, 20 different sero• may be either LPS smooth (expressing many long 0 groups of P. aeruginosa are recognized on the basis of side chains) or LPS rough (expressing few, short or no variation in O-antigen structure; serogroups can be o side chains). Strains recovered from environmental further divided into subgroups based on serological sources and those isolated from acutely infected pa• crossreactivityl. The LPS core of P. aeruginosa also tients usually have an LPS-smooth phenotype that im• has a similar structure to that found in other Gram• parts serum resistance by a mechanism similar to that negative bacteria: it is composed of ketodeoxyoctonate, described for other Gram-negative bacteria 7• When heptose, hexosamine and hexoses. The structure of the tested in animal models of acute infection, LPS-rough LPS core has been determined for four strains of P. isolates are virtually avirulent 8 • The 0 side chains from P. aeruginosa LPS-smooth aeruginosa from different serogroups; all these struc• tures are distinct2 • It is not known whether all strains strains are heterogeneous in length. In addition, over of a particular serogroup have the same LPS core 80% of the LPS produced by P. aeruginosa contains structure. core oligosaccharides that lack covalently attached The endotoxic portion of the P. aeruginosa LPS O-antigen side chains9.1O. Thus, the core structure of (lipid A) is significantly less toxic than that of Escher• P. aeruginosa, unlike that of E. coli and S. typhimurium, ichia coli and Salmonella typhimurium and differs is more exposed. Certain growth conditions have been from enteric lipid A in that the lipid substituents that found to alter the production of 0 antigens and com• acylate the glucosamine disaccharide backbone are mop antigen ll ,12, although the mechanism by which shorter and the phosphate groups are attached in a this variable expression is controlled is unknown. P. aeruginosa strains that cause chronic lung infec• different manner3. Although P. aeruginosa lipid A is less toxic than that of enteric bacteria, it is sufficiently tions in cystic fibrosis (CF) patients are unusual in that toxic in humans to have precluded the widespread use they are often serum sensitive and LPS rough13, which suggests that LPS-mediated serum resistance is not cru• of LPS-based vaccines. P. aeruginosa LPS contains an additional polysac• cial for the survival of P. aeruginosa in the milieu of the CF lung. These strains arise from LPS-smooth isolates charide antigen (also known as A band) common to many serogroups, which is a polymer composed prin• that are found in the environment and are thought to l4 cipally of D-rhamnose monosaccharides. There is some initiate infection in CF patients . The loss of 0 side Copyright e 1996 Elsevier Science Ltd. All rights reserved. 0966 842X196/$15.00

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long-chain fatty acid

/

~

n

o side chain

Core

Lipid A

FIg. 1. Schematic representation of lipopolysaccharide (LPS) from Pseudomonas aeruginosa. P. aeruginosa lipid A inserts into the outer membrane and has three fatty acids on each glucosamine. The lPS core. attached to the lipid A. is composed of ketodeoxyoctonate. heptose. hexosamine and hexoses. The O-polysaccharide side chain (also called 0 antigen). attached to the lPS core. is composed of tri- or tetrasaccharide repeating units. The hexagons represent monosaccharides; the solid circles represent phosphates; and 'n' indicates an O-antigen repeating unit.

chains may allow these bacteria to evade the strong host response against O-~ide-chain antigens 13. \Yhile t~e mechanisms responsIble for the loss of O-slde-cham antigens by CF isolates are not yet known, recent work indicates that different genes of the rfb locus, which en• code enzymes for the synthesis of LPS 0 polysacchar• ides, are mutated in different CF isolates (see below) 15. In addition to the LPS-rough phenotype, P. aerugi• nosa strains isolated from the lungs of CF patients ex• press high levels of an exopc;>lysaccharide call~d algina~e or mucoid exopolysacchande. OverproductIOn of thIS exopolysaccharide is responsible for the characteris• tic mucoid appearance of these strains when grown on solid medium in the laboratory. The genetics and regulation of alginate production has been reviewed recently16-18. P. aeruginosa isolates from CF patients are often nonmotile, lack flagella and express other virulence factors at a lower leveP4. Immune response to P. aeruglnosa LPS and vaccine development

Both animal models and human studies have estab• lished the 0 antigens of P. aeruginosa as the principal targets of protective antibodies 19. These antibodies mediate opsonophagocytic killing of bacteria, which is an in vitro correlate of protective immunity to P. aerugi• nosa. Serum with O-antigen-specific opsonins protects humans during the acute phase of P. aeruginosa bac• teremia, resulting in significantly higher survival rates20 . Thus, there is intense interest in the development of O-antigen-specific vaccines for human immunization. Cryz and colleagues have prepared conjugates of purified 0 antigens and P. aeruginosa exotoxin A and found that they elicited antibodies that mediated op• sonic killing in vitro and passively protected animals against experimental infection21 . This conjugate vac• cine was used to prepare a hyperimmune globulin that has been.evaluated for therapeutic efficacy in intensive• care patients. Unfortunately, the results of this trial

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indicated that the preparation failed to reduce the in• cidence of P. aeruginosa infection22 . Pier and colleagues have found that a proportion of the P. aeruginosa 0 antigens are made as a high• molecular-weight 0 polysaccharide; these antigens can be purified and used as a nontoxic LPS-specific vac• cine23 . Monovalent preparations elicit antibodies in humans24 and protect animals against infection25 . How• ever, subtle structural variations in P. aeruginosa LPS, which give rise to the subgroup determinants, can re• sult in 0 antigens that elicit antibodies that are highly specific to the strain from which the polysaccharide was isolated 26 . Currently, an investigation into the specificity of P. aeruginosa high-molecular-weight O-polysaccharide vaccines from closely related sub• group strains is under way to clarify the manner in which the polysaccharides differ immunochemically. The use of monoclonal antibodies may provide an alternative route to an effective immunotherapeutic reagent for P. aeruginosa infections. Although initial attempts to produce human monoclonal antibodies in high quantities have been disappointing, the avail• ability of new vectors for cloning antibody variable• region genes and the expression of these genes with human constant-region genes may make sufficient material for clinical evaluation available in the near future. Murine and human monoclonal antibodies to P. aeruginosa LPS, produced for laboratory evaluation, demonstrate a high level of protection against experi• mental infection 25 . LPS as a ligand for binding or entry Into eukaryotlc cells

P. aeruginosa is known to adhere to, and interact with, epithelial cells, and recent results indicate a role for P. aeruginosa LPS as a bacterial ligand for interaction with eukaryotic cells. Fletcher et al. 27 showed LPS• mediated adherence of P. aeruginosa to rat corneal epi• thelium and to contact lenses. Moreover, Gupta et al. 28

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(c)

(a)

Fig. 2. Model of uptake of Pseudomonas aeruginosa by respiratory epithelial cells leading to bacterial clearance from the lung. (8) P. aeruginosa with a complete lipopolysaccharide (LPS) core (red) is taken up by cell lines expressing wild-type CFTR (cystic fibrosis transmembrane conductance regulator; blue). (b) P. aeruginosa with a complete LPS core is poorly ingested by cell lines expressing mutant ~F508 CFTR (green) that is not expressed on the cell surface. (c) P. aeruginosa with an incomplete LPS core (yellow) is poorly ingested by cell lines expressing wild-type CFTR. ER, endoplasmic reticulum.

showed that P. aeruginosa pili and LPS both bind to the glycolipid asialo GMt on corneal cells. Other work• ers29, however, have been unable to demonstrate the presence of this glycolipid on corneal cells from rab• bits and humans and therefore question the relevance of these findings. Interestingly, although attempts to inhibit P. aeruginosa corneal wound infections by the addition of exogenous LPS to infectious inocula to block adherence were successful, the change in eye pa• thology was minimal, thus raising questions concern• ing the role of LPS-mediated adherence in the overall pathogenic process (G.B. Pier, unpublished). P. aeruginosa has recently been reported to be in• gested by corneal cells during experimental eye infec• tion 3o,31; this ingestion is mediated by the outer-core portion of the LPS (Ref. 32). Bacterial entry into cor• neal cells may contribute to eye pathology and partially explain why this infection is often difficult to treat and why antibiotic therapy is not always effective. These studies revealed that P. aeruginosa strains with a com• plete outer-core LPS were ingested by rabbit corneal epithelial cells better than LPS mutants with a trun• cated core. Purified LPS, or delipidated oligosacchar• ides containing a terminal-glucose residue on the LPS core, inhibited both bacterial attachment and entry into corneal cells. In contrast to the wounded eye, where the epithelial cells ingesting P. aeruginosa are buried within the cor• nea, a mucosal surface may achieve bacterial clearance by shedding epithelial cells that have ingested bacteria. This process may be compromised in CF, as histologi• cal analyses of affected lung tissues removed at autopsy or for transplantation indicate that P. aeruginosa is not seen within airway cells but as microcolonies en• cased in the extracellular mucus layer in the airway lumen 33 • Others have reported that transformed type II

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pneumocytes expressing wild-type CFfR (CF trans• membrane conductance regulator) can ingest P. aerugi• nosa in vitro 34 • Further support for this hypothesis comes from studies indicating that derivatives of a transformed airway epithelial cell line originating from a CF patient homozygous for the most common mu• tation in CFfR, MS08, were deficient in the uptake of P. aeruginosa compared with the same cell line ex• pressing wild-type CFTR following transfection35 • In contrast to ten other respiratory pathogens tested, P. aeruginosa was the only bacterial species efficiently ingested by airway epithelial cells with wild-type C~; the P. aeruginosa LPS core was found to be the bacten~l ligand for this ingestion35 • In a model of acute P. aerugt• nosa pulmonary infection in neonatal mice 36, anim~ls infected intra nasally with P. aeruginosa mixed wIth complete LPS-core oligosaccharide had significantly more bacteria in their lungs than mice receiving either no inhibitor or incomplete LPS-core oligosaccharide, leading to significantly less clearance and higher levels of bacteria in the lung. Thus, the deficiency in P. aeruginosa uptake by the epithelial cells in the airways of CF patients may underlie their hypersusceptibility to P. aeruginosa infection35 • A model for the contribution of LPS-mediated epithelial entry in normal bacterial clearance and pathogenesis of P. aeruginosa infection in CF is shown in Fig. 2.

GenetiCS of P. aSlUglnosa LPS synthesis The identification and characterization of the genes re• quired for the production of P. aeruginosa 0 antigens (the rfb genes) are at an early stage compared with stud• ies of these genes from E. coli and S. typhimurium 37 • However similar to these enteric bacteria, a clustering of r{b gedes to a particular region of the P. aeruginosa chromosome has been observed. The plasmid, pLPS2,

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contains all the genes necessary for synthesis of the serogroup 011 antigen, as evidenced by expression of this antigen by E. coli and various Salmonella spp. that have been transformed or transduced with this plas• mid 38,39. In some LPS-rough isolates of P. aeruginosa, the presence of pLPS2 led to the expression of a second LPS serogroup antigen, in addition to serogroup 011, which represents restoration of the ability of the re• cipient strain to synthesize its original 0 side chain38 • Subcloning of pLPS2 revealed that different regions in the rfb locus overcome the mutations in various LPS• rough CF isolates, suggesting that different genetic events can result in the emergence of the LPS-rough phenotype 15 • Sequence analysis of these genes suggests that they encode enzymes involved in the synthesis of LPS rather than regulatory proteins (GenBank accession number U44089). Lightfoot and Lam isolated a similar rfb locus from the serogroup 05 strain PAOI. Plasmid pFV100, ob• tained by complementing a transposon insertion mu• tant that was defective in LPS production40, induced expression of P. aeruginosa serogroup 05 LPS upon transformation to E. coli strain HB101 (Ref. 41). Two LPS genes have thus far been identified on pFV100. One gene, rfc, was identified as encoding an O-antigen polymerase42 • Coyne and Goldberg identified the same gene from the cosmid clone pLPS7 (GenBank accession number U26685), also obtained from strain PA01 (Ref. 43). This gene has similarities to other putative rfc genes, including rare codon usage and a highly hydrophobic inferred amino acid sequence. However, as the O-antigen-polymerase genes isolated to date do not show homology to one another, the function of these genes can only be inferred44 • A second LPS gene, rfbA, has also been identifed from pFV100; however, the function of this gene has not been determined 41 • A comparable gene was also found in the rfb cluster of P. aeruginosa serogroup 011 (GenBank accession num• ber U44089); the inferred amino acid sequence is similar to that of the rfbA gene product of serogroup 05 and the rfe gene product of E. coli and other Rfe-like proteins. A gene, gca, involved in the synthesis of the P. aeru• ginosa common antigen has been identified by Lightfoot and Lam 40 • The gca gene product converts GOP-man• nose to GOP-rhamnose and has been suggested to be a GOP-rhamnose synthetase45 • The inferred amino acid sequence showed similarity to YefA (GOP-D-mannose dehydratase from E. coli) and RfbO (dehydratase, oxi• doreductase or epimerase from Vibrio cholerae). The gca gene is conserved across all serogroups of P. aeru• ginosa and within some other Pseudomonas species46 • Studies of the algC gene have linked the synthesis of the polysaccharide alginate and LPS. The algC gene, previously identified in the pathway for alginate biosynthesis, encodes phosphomannomutase (PMM), which interconverts mannose 6-phosphate and man• nose 1-phosphate47 , and phosphoglucomutase (PGM) which interconverts glucose 6-phosphate and glucos~ I-phosphate. The PG~ activity was confirmed by suc• cessful complementatlOn of a pgm mutation from E. coli with the algC gene from P. aeruginosa 48• The algC gene was also found to restore complete LPS synthesis

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Questions for future research - What controls the variable expression of 0 antigens and com• mon antigen? - Can a safe and effective O-antigen-based vaccine against P. aeru• ginosa be developed? - What is the cellular receptor for P. aeruginosa ingestion by epi• thelialcells? -What is the biosynthetic pathway for the production of lipopoly• saccharide (LPS)? - What is the mechanism of conversion from an LPS-smooth to an lPS-rough form in P. aeruginosa infections in the cystic fibrosis lung? -Is there coordinate regulation of LPS and alginate expression?

to an LPS mutant of strain PA01 that lacks glucose residues on its LPS core 49 • Further studies have shown that PGM activity is required for the synthesis of a complete LPS, and PMM activity is required for the biosynthesis of alginate. Ye et al. 50 have confirmed the bifunctional nature of the purified algC gene product from P. aeruginosa and its involvement in the synthesis of both alginate and LPS. Conclusions P. aeruginosa is an opportunistic pathogen that is found ubiquitously in the environment and is capable of in• fecting patients whose health is compromised. The LPS of P. aeruginosa plays an important role in the patho• genesis of this organism. Long 0 polysaccharides of LPS-smooth strains are capable of resisting comple• ment-mediated phagocytosis, while LPS-rough strains evade host defenses owing to the loss of a ligand that is important for host clearance. The ability of P. aeru• ginosa to alter the expression of its LPS allows it to inhabit various ecological niches and cause different types of life-threatening infections. Acknowledgements

We thank the members of both of our laboratories, as well as our nu• merous colleagues and collaborators throughout the years. This work was supported by NIH grants to J.B.G. (AI35674 and AI30050) and to G.B.P. (AI22806 and AI22535) and by Cystic Fibrosis Foundation Grants to J.B.G. and G.B.P. References

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Trends In Microbiology thanks the following people for their help and guidance In 1996 M. Achtman G. Ada A.C. Allison J.W. Almond L.C. Archard R.E. Aurigemma O.G. Baca M.A. Barry C.E. Barry III S. Bhakdi L. Bj6rck G.E. Blair M.S. Blake M.J. Blaser J. Bliska A. Bochkarev J.D. Boeke P. Boistard D. Boucias V.L. Braciale V. Braun N.J. Brewin W.J. Britton A. Bucheton M.J. Buchmeier G.!. Byme R. Calderone N.P. Cianciotto P. Cleary D.L. Clemens D.B. Clewell J. Coffin F.M. Cohan J. Collinge A. Collmer P. Cossart M. Daniels N.J. Deacon

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