1. Top of page
  2. Summary
  3. Cystic fibrosis and Pseudomonas aeruginosa
  4. Alginate biosynthesis
  5. Regulation of alginate biosynthesis
  6. Alginate and biofilm development
  7. Potential therapies, future perspectives and conclusions
  8. Acknowledgements
  9. References

Decades of research have been dedicated to the study of the opportunistic pathogen Pseudomonas aeruginosa, a Gram-negative, environmental bacterium that secretes the exopolysaccharide alginate during chronic lung infection of cystic fibrosis (CF) patients. Although P. aeruginosa utilizes a variety of factors to establish a successful infection in the lungs of CF patients, alginate has stood out as one of the best-studied prognostic indicators of chronic lung infection. While the genetics, biosynthesis and regulation of alginate are well understood, questions still remain concerning its role in biofilm development and its potential as a therapeutic target. The purpose of this review is to provide a brief summary of alginate biosynthesis and regulation, and to highlight recent discoveries in the areas of alginate production, biofilm formation and vaccine design. This information is placed in context with a proposed P. aeruginosa infectious pathway, highlighting avenues for the use of existing therapies as well as the potential for novel agents to reduce or eliminate chronic infections in CF patients.

Cystic fibrosis and Pseudomonas aeruginosa

  1. Top of page
  2. Summary
  3. Cystic fibrosis and Pseudomonas aeruginosa
  4. Alginate biosynthesis
  5. Regulation of alginate biosynthesis
  6. Alginate and biofilm development
  7. Potential therapies, future perspectives and conclusions
  8. Acknowledgements
  9. References

Cystic fibrosis (CF) is a multisystem disorder caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) (Doull, 2001; Boucher, 2004). Mutations in this cyclic AMP-regulated chloride ion channel lead to either mislocalization of CFTR or loss of function, resulting in defective chloride ion transport across epithelial cell surfaces and the build-up of a dehydrated mucus layer in the lung (Boucher, 2004). The mucociliary escalator is largely impaired due to the dense nature of thickened mucosal secretions, which traps microorganisms such as Pseudomonas aeruginosa and other opportunistic pathogens and allows these organisms to establish successful lung infections (Deretic et al., 1995; Pier, 1998). In spite of rigorous antibiotic treatment regimens, persistent microbial infections and the inflammatory responses that they engender gradually lead to progressive pulmonary disease and eventually respiratory failure and death of the CF patient (Govan and Deretic, 1996; Doull, 2001; Boucher, 2004).

Although multiple microbial species can successfully colonize the CF lung, robust infections by P. aeruginosa eventually dominate the microbial population and contribute significantly to disease. How P. aeruginosa adapts to the inflammatory lung environment, establishes a chronic infection and becomes the predominant pathogen in CF has been the subject of intense investigation. Mutations in CFTR increase susceptibility of the host to microbial lung infections, although the precise mechanism by which this occurs is still under debate (Govan and Deretic, 1996; Lyczak et al., 2002; Boucher, 2004). Expression of several virulence factors, including type IV pili, flagella, exotoxins, proteases, lipopolysaccharide (LPS) and type III secretion proteins, enhances the long-term association between P. aeruginosa and the CF lung (Feltman et al., 2001; Darling et al., 2004). In addition to these factors, the conversion of P. aeruginosa microcolonies from a non-mucoid to a mucoid phenotype marks the transition to a more persistent state, characterized by antibiotic resistance and accelerated pulmonary decline (Pedersen et al., 1992; Govan and Deretic, 1996; Lyczak et al., 2002). Ineffective clearance by both innate and acquired immune responses leads to chronic infection, establishing mucoid P. aeruginosa as the major pathogen of CF airway disease (Lyczak et al., 2002).

Colonization of the CF lung by P. aeruginosa occurs early in life and leads to a significant decline in pulmonary function (Govan and Deretic, 1996; Lyczak et al., 2002; Boucher, 2004). One longitudinal study reported that 97.5% of children with CF had evidence of P. aeruginosa infection by age 3 (Burns et al., 2001), and the majority of the P. aeruginosa strains collected from the upper and lower airways of these CF patients displayed non-mucoid and antibiotic susceptible phenotypes when grown under free-swimming (planktonic) conditions. As lung disease progresses, there is a change in the phenotype and growth patterns of the P. aeruginosa colonizing strains recovered from CF sputum. These bacteria often have mucoid colony morphologies (Fig. 1) and adopt a biofilm mode of growth in vivo (Lam et al., 1980; Deretic et al., 1995; Singh et al., 2000). Mucoidy is a descriptive term for the overproduction of the exopolysaccharide alginate, which is a negatively charged, linear copolymer of partially O-acetylated β-1,4-linked d-mannuronic acid and its C5 epimer, α-l-guluronic acid (Linker and Jones, 1966). Biofilms, which are communities of bacteria attached to a surface and encased in a biopolymeric matrix, form within the airway and protect the bacteria from host immune factors and anti-microbial agents (Mah and O’Toole, 2001; Dunne, 2002; Parsek and Singh, 2003). Together, alginate production and biofilm formation by mucoid strains of P. aeruginosa contribute significantly to the resistance of these organisms to treatment regimens and host defences, resulting in a poor prognosis for the CF patient (Govan and Deretic, 1996; Pier, 1998).


Figure 1. Alginate production by P. aeruginosa. Strains were streaked on Luria–Bertani (LB) agar without NaCl and incubated at 37°C for 12 h and at 25°C for 24 h to enhance pigmentation. A. Non-mucoid wild-type PAO1 strain. B. Mucoid CF isolate FRD1 (mucA22).

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This review will focus on alginate biosynthesis, regulation and its role in biofilm development, as well as potential therapeutic options for managing P. aeruginosa infections in CF. Together, the expression and regulation of alginate biosynthesis by P. aeruginosa during chronic CF lung infections illustrate the difficulty and the challenge of designing effective strategies for treatment of these infections. Current strategies focus on alginate as a viable target for the prevention of chronic infection by mucoid P. aeruginosa strains, including both planktonic and biofilm-encased cells, but alternative approaches for preventing early colonization or managing the early infections should also be considered. Emphasis must be placed on treatment strategies before the in vivo switch to mucoidy.

Alginate biosynthesis

  1. Top of page
  2. Summary
  3. Cystic fibrosis and Pseudomonas aeruginosa
  4. Alginate biosynthesis
  5. Regulation of alginate biosynthesis
  6. Alginate and biofilm development
  7. Potential therapies, future perspectives and conclusions
  8. Acknowledgements
  9. References

The alginate biosynthetic pathway has been under scrutiny for a number of years, and several excellent reviews have been published (Govan and Deretic, 1996; Rehm and Valla, 1997; Gacesa, 1998) (see Table 1 and Fig. 2). In brief, the algA, algC and algD genes encode the enzymes required for synthesis of the alginate precursor guanosine diphosphate (GDP)-mannuronic acid (Fig. 2). Once GDP-mannuronic acid is synthesized, the precursor is polymerized and transported across the inner membrane by a hypothesized combination of the alg8 and alg44 gene products (Maharaj et al., 1993; Monday and Schiller, 1996). After polymerization, some of the mannuronate residues are epimerized to guluronate residues by a C-5-epimerase (AlgG). A scaffold model has been proposed where AlgG interacts in the periplasm directly with AlgK in order to protect the growing alginate polymer from degradation by alginate lyase (AlgL) (Jain and Ohman, 1998). Recent evidence suggests that AlgX may be part of this periplasmic scaffold (Robles-Price et al., 2004). The role of alginate lyase is not clear, although there is evidence that alginate is rapidly degraded and varies in size in mucoid strains that hyperexpress AlgL, lending support to the hypothesis that alginate lyase might provide short oligomers that could prime the polymerization reaction (Boyd and Chakrabarty, 1994; Monday and Schiller, 1996). After epimerization, some of the mannuronate residues are acetylated at the O2 and/or O3 positions by the algF, algJ and algI gene products, which might form a complex in the membrane that serves as a reaction centre for O-acetylation (Franklin and Ohman, 2002). The donor of the acetyl group to AlgI is currently unknown. After O-acetylation, the copolymer is transported out of the cell through the outer membrane protein AlgE (Rehm and Valla, 1997).

Table 1.  Elements involved in alginate biosynthesis and regulation.
Gene or proteinAdditional name(s)Structure and/or homologyFunctionReference(s)
   Alginate biosynthesis 
algA  Phosphomannose isomerase and GDP- mannose pyrophosphorylase activitiesShinabarger et al. (1991)
algC Heart-shaped protein; four domains; active site contains bound Mg2+ ionPhosphomannomutase and phosphoglucomutase activities; involved in LPS biosynthesisZielinski et al. (1991); Regni et al. (2004)
algD Tetrameric; active site contains NAD(H) cofactorGDP-mannose dehydrogenaseTatnell et al. (1994); Snook et al. (2003)
algE Hypothesized β-barrelOuter membrane, alginate-specific ion channel responsible for alginate exportGrabert et al. (1990); Chu et al. (1991)
algF  Periplasmic protein involved in alginate O-acetylationFranklin and Ohman (1993); Franklin and Ohman (2002)
algG  Periplasmic-associated, C5-mannuronan epimerase; protects alginate polymer from lyase activityFranklin et al. (1994); Jain et al. (2003)
algI Hypothesized seven transmembrane helicesInner membrane protein involved in alginate O-acetylationFranklin and Ohman (2002)
algJ Member of type IA membrane proteinsMembrane-associated, periplasmic protein involved in alginate O-acetylationFranklin and Ohman (2002)
algK  Periplasmic protein required for proper polymer formationAarons et al. (1997); Jain and Ohman (1998)
algL Polysaccharide lyase (PL-5) familyPeriplasmic alginate lyaseSchiller et al. (1993); Monday and Schiller (1996)
algX  Periplasmic protein required for proper polymer formationMonday and Schiller (1996); Robles-Price et al. (2004)
alg8 Homologous to glycosyl transferase NodCMembrane protein; hypothesized polymeraseMaharaj et al. (1993); Mejia-Ruiz et al. (1997)
alg44  Membrane protein required for alginate productionMaharaj et al. (1993); Mejia-Ruiz et al. (1997)
algB NtrC subfamily of two-component signal transduction systemsPositive activator of algD transcriptionWozniak and Ohman (1991); Goldberg and Dahnke (1992)
algH  Regulates production of alginate by unknown mechanismSchlictman et al. (1995)
algPHp1, algR3Hypothesized coiled-coilHistone-like protein; activates algD transcriptionKato et al. (1990); Konyecsni and Deretic (1990)
algRalgR1LytTR DNA-binding domainPositively activates algC and algD transcriptionDeretic et al. (1989); Nikolskaya and Galperin (2002)
algTalgU, σ22Homologous to E. coliσEAlternative sigma factor responsible for transcription of algD, algR, algT, algZ and rpoHMartin et al. (1993a); Devries and Ohman (1994); Hershberger et al. (1995); Wozniak et al. (2003b)
algQalgR2 Positive activator of algD transcription; repressor of quorum-sensing regulatory genes lasR and rhlRKato et al. (1989); Konyecsni and Deretic (1990)
fimSalgZ Hypothesized cognate sensor kinase for AlgR; positive activator of type IV-mediated twitching motility; negative regulator of alginate productionWhitchurch et al. (1996); Yu et al. (1997)
algZ Homologous to ribbon– helix–helix (RHH) family of DNA-binding proteinsActivator of algD transcriptionBaynham et al. (1999)
CRP  Protein from E. coli binds to algD in vitroDevault et al. (1991)
CysB  Activator of algD transcriptionDelic-Attree et al. (1997)
IHFSubunits encoded by himA and himDHeterodimer composed of α- and β-subunitsHistone-like protein; required for algD transcriptionToussaint et al. (1993); Wozniak (1994)
kinB Homologous to PhoR from B. subtilisInner membrane protein; cognate sensor kinase for AlgBMa et al. (1997)
mucA Homologous to RseA in E. coliInner membrane protein; anti-sigma factor that negatively regulates alginate production by sequestration of AlgT (AlgU)Martin et al. (1993b); Mathee et al. (1997)
mucBalgNHomologous to RseB in E. coliPeriplasmic protein that negatively regulates AlgT; inactivation leads to alginate productionGoldberg et al. (1993); Martin et al. (1993c)
mucCalgM Hypothesized inner membrane protein; might act synergistically with MucA and MucB to negatively regulate AlgT (AlgU)Boucher et al. (1997b); Mathee et al. (1997)
mucDalgYHomologous to serine protease DegP (HtrA)Negatively regulates AlgT by removing activating factors (i.e. denatured proteins)Boucher et al. (1996)
rpoNntrA, σ54 Negative regulator of algD transcription in mucA22 background; responsible for algD transcription in muc23 background; responsible for algC transcriptionBoucher et al. (2000)

Figure 2. Model for the biosynthesis and assembly of alginate in P. aeruginosa. The illustration shown is a summary of recently published data concerning the localization of alginate biosynthetic enzymes within the cell (Franklin and Ohman, 2002; Gimmestad et al., 2003; Robles-Price et al., 2004). Letters A–X and numbers 8 and 44 indicate alginate biosynthetic enzymes (A for AlgA, etc.). I.M. and O.M. represent inner and outer membrane respectively. The letters G and M are for guluronate or mannuronate respectively. ‘Ac’ stands for an acetyl group. Portions of this figure have been reproduced with permission from the following sources: Franklin and Ohman (2002); Gimmestad et al. (2003); copyright American Society for Microbiology.

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Recent structural studies have shed light on the enzymatic complexity behind the synthesis of GDP-mannuronic acid. The algC gene encodes an enzyme with phosphomannomutase (PMM) and phosphoglucomutase (PGM) activities, and PMM/PGM catalyses the reversible conversion of mannose 6-phosphate to mannose 1-phosphate during alginate biosynthesis (Fig. 2). Interestingly, algC is also required for LPS synthesis (Zielinski et al., 1991). Crystallization of PMM/PGM reveals a heart-shaped protein consisting of four domains and a bound Mg2+ ion that is required for activity. PMM/PGM demonstrates preference for both glucose- and mannose-based substrates, which gives it versatility for utilization in both the alginate and LPS biosynthetic pathways (Regni et al., 2004).

The algD gene encodes GDP-mannose dehydrogenase (GMD), which acts as a rate-limiting enzyme in mucoid strains by catalysing the conversion of GDP-mannose to GDP-mannuronic acid, thereby committing the cell to alginate production (Govan and Deretic, 1996; Fig. 2). The GMD crystal structure reveals a tetrameric arrangement with a large binding pocket that contains the active sites for both the cofactor NAD(H) and GDP-mannose (Snook et al., 2003). With detailed structural knowledge now available for both PMM/PGM and GMD, these enzymes are ideal candidates for targeted drug therapies (see below).

Although the steps for the synthesis of alginate precursors, mediated by AlgA, AlgC and AlgD, are well understood, there are gaps in the knowledge of how the polymerization, modification and export of alginate are co-ordinated. In particular the specific role(s) of the putative inner membrane proteins Alg8 and Alg44 and their communication with the periplasmic scaffolding apparatus consisting of AlgG, AlgK and AlgX requires further elucidation. Because of their subcellular localization, these enzymes may provide more logical candidates for the rational delivery of effective inhibitors.

Regulation of alginate biosynthesis

  1. Top of page
  2. Summary
  3. Cystic fibrosis and Pseudomonas aeruginosa
  4. Alginate biosynthesis
  5. Regulation of alginate biosynthesis
  6. Alginate and biofilm development
  7. Potential therapies, future perspectives and conclusions
  8. Acknowledgements
  9. References

Historically, studies concerning the regulation of alginate biosynthesis have centred largely on transcriptional control. Transcription of alginate genes is complex and involves a number of proteins, whose production and/or activity are highly regulated. Expression from the alginate biosynthetic operon (algD-algA PA3540-PA3551) is under the control of the algD promoter. A key element in algD gene regulation is the alternative sigma factor AlgT, also known as AlgU or σ22 (Table 1), which induces the expression of algD and increases the expression of regulatory proteins that enhance algD transcription (Govan and Deretic, 1996). The mucA and mucB gene products inhibit AlgT, as inactivation of mucA or mucB leads to deregulation of AlgT and conversion to mucoidy (see below). Expression of algC is independent of other alginate genes yet algC is under the control of the response regulator AlgR, which binds three regions positioned upstream and downstream of the transcription start site (Fujiwara et al., 1993). Control of algC transcription by AlgR is independent of the orientation or localization of the AlgR binding sites, which resembles eukaryotic enhancer elements.

Many P. aeruginosa CF isolates display a hypermutable phenotype, and expression of alginate is largely due to the acquisition of stable mutations within at least two regions of the chromosome (Govan and Deretic, 1996; Oliver et al., 2000). Collectively referred to as the muc loci, muc-2, muc22 and muc23 are three classes of muc mutations that directly lead to alginate production (Govan and Deretic, 1996). The muc-2 and muc-22 mutations inactivate the mucA gene product (Govan and Deretic, 1996). In one study, over 80% of mucoid P. aeruginosa isolates collected from CF patients harboured mutations in mucA (Boucher et al., 1997a), suggesting that this region is a ‘hot-spot’ for mutagenesis. MucA is an inner membrane anti-sigma factor whose amino-terminus lies in the cytosol and is responsible for direct contact with AlgT, and whose carboxy-terminus is in the periplasm and contacts MucB (Mathee et al., 1997; Rowen and Deretic, 2000). Preliminary evidence suggests that AlgT peripherally associates with the inner membrane, either in association with RNA polymerase and the anti-sigma factor MucA or independent of both proteins (Rowen and Deretic, 2000).

AlgT might not be the only sigma factor that is responsible for algD transcription. Under certain conditions, transcription of algD requires RpoN (σ54). RpoN-dependent expression of algD is only observed in a muc23 background, where mutations are found in currently uncharacterized gene(s) that map to a chromosomal locus distinct from mucA (Boucher et al., 2000). The majority of research in alginate expression has been performed in strains harbouring mucA mutations, but little is understood as to the clinical contribution of the muc23 mutation during mucoid conversion in the CF lung.

Activation from a distance is a strikingly unique feature of the algD promoter. Besides the control exerted by AlgT, MucA and MucB, the response regulators AlgR and AlgB, the DNA-binding protein AlgZ, the histone-like proteins IHF and AlgP (Hp1), and AlgQ also exert control of algD transcription (Table 1). AlgR binds three regions within the algD promoter, the furthest of which is localized to a region spanning −479 to −457 bp upstream of the transcription start site (Mohr et al., 1992). The binding site for the ribbon–helix–helix activator AlgZ is also located a considerable distance (282 bp) away from the start of transcription (Baynham and Wozniak, 1996). The mechanism whereby multiple regulatory proteins bind at significant distances and still promote algD transcription has yet to be delineated. Both IHF and AlgP play a role in alginate production and might contribute to DNA looping (Govan and Deretic, 1996). Future in vitro transcription studies are needed to further delineate the mechanism by which multiple regulatory factors interact to activate algD transcription, and utilization of advanced techniques such as atomic force microscopy might provide the visual evidence to substantiate a DNA-looping model of transcriptional regulation.

Alginate and biofilm development

  1. Top of page
  2. Summary
  3. Cystic fibrosis and Pseudomonas aeruginosa
  4. Alginate biosynthesis
  5. Regulation of alginate biosynthesis
  6. Alginate and biofilm development
  7. Potential therapies, future perspectives and conclusions
  8. Acknowledgements
  9. References

The involvement of alginate production in the formation of P. aeruginosa biofilms has come under recent scrutiny. Doggett et al. (1966) first documented mucoid P. aeruginosa in CF patients. Later, matrix-encased P. aeruginosa microcolonies were observed in post-mortem-sectioned alveoli from CF patients (Lam et al., 1980; Baltimore et al., 1989). These studies and more recent information (Singh et al., 2000; Hoiby et al., 2001) clearly indicate that P. aeruginosa forms biofilms in vivo. The steps of biofilm formation include initial attachment to a surface such as mucin-covered epithelial cells by free-swimming or planktonic bacteria, microcolony formation, development of a mature biofilm and release of planktonic organisms to begin the cycle anew (O’Toole et al., 2000; Sauer et al., 2002) (Fig. 3). Secretion of an exopolymeric substance (EPS) occurs after initial attachment and continues throughout biofilm formation. However, questions remain as to precisely when alginate is expressed in relation to the production of other polysaccharides or matrix material in the course of CF lung infection. Prior work revealed the presence of antibodies to P. aeruginosa alginate in CF patients, although no mucoid cultures were detectable in sputum samples (Pedersen et al., 1990a). Moreover, using alginate-specific antibodies, others have shown that alginate is expressed by non-mucoid strains under limiting oxygen conditions in vitro, and within 1 h after infection of mice (Worlitzsch et al., 2002; Pier et al., 2004). Although these studies suggest alginate expression occurs in non-mucoid strains (i.e. in the absence of muc mutations), this must be reconciled with data from several microarray experiments, which failed to demonstrate that alginate biosynthetic genes are expressed when grown under conditions similar to those described above (Whiteley et al., 2001; Wagner et al., 2003; Frisk et al., 2004; Wolfgang et al., 2004).


Figure 3. Proposed P. aeruginosa virulence strategies during the infectious process and potential therapeutic strategies. In the CF lung, mutations in CFTR lead to aberrant protein function and the build-up of a dehydrated mucous layer that traps microorganisms such as P. aeruginosa and allows for persistent microbial colonization. Patients are initially colonized with motile (planktonic), non-mucoid P. aeruginosa strains, which attach to a surface such as mucin-covered epithelial cells (step 1). Individual bacteria, which express type IV fimbriae and secrete homoserine lactone molecules in a form of cell-to-cell communication (quorum sensing), aggregate and form microcolonies (step 2). Secretion of toxins, proteases and pyocyanin might also significantly contribute to tissue damage. Over time, microcolonies develop into a mature biofilm characterized by secretion of an EPS, loss of flagella and type IV fimbriae, and the formation of three-dimensional structures encasing both aerobically and anaerobically respiring colonies (step 3). ‘EPS′ refers to extracellular polymeric substance such as exopolysaccharides that have been shown to be important for biofilm development, persistence, or architecture (alginate, pel and psl-encoded exopolysaccharides) as well as DNA. Inflammatory cells are recruited to the site of infection, where they release reactive oxygen species (ROS) and cause extensive tissue damage. This applies a selective pressure to the colonizing P. aeruginosa strains, leading to mucA mutations, deregulation of AlgT and subsequent stable mucoid conversion (step 4). Therapies that could potentially target each step are indicated below.

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Recent studies suggest that alginate expression is not required for in vitro biofilm formation by non-mucoid P. aeruginosa strains (Hentzer et al., 2001; Nivens et al., 2001; Wozniak et al., 2003a; Stapper et al., 2004). Moreover, several independent groups have reported the involvement of alternative polysaccharide-encoding genes in biofilm formation for non-mucoid P. aeruginosa strains PAO1 and PA14. These gene clusters, designated psl (polysaccharide synthesis locus) and pel (pellicle formation), are required for biofilm development in P. aeruginosa and encode either a mannose- or a glucose-rich expolysaccharide respectively (Friedman and Kolter, 2004a,b; Jackson et al., 2004; Matsukawa and Greenberg, 2004; Parsek and Fuqua, 2004). In addition to exopolysaccharides, the biofilm matrix contains a significant amount of nucleic acid (Whitchurch et al., 2002; Matsukawa and Greenberg, 2004). There remain significant gaps in our understanding of how P. aeruginosa survives the inflammatory environment of the lung before stable mucoid conversion (Fig. 3, steps 1–3). One must consider that during initial colonization, biofilm formation in the CF lung probably precludes the switch to mucoidy. An intriguing idea is that these polysaccharides and nucleic acids contribute to the matrix EPS of biofilms formed by early colonizing non-mucoid P. aeruginosa strains, before the conversion to alginate-producing variants (Fig. 3, step 3).

Although alginate does not appear to be required for biofilm formation by non-mucoid strains in vitro, alginate production (especially in its O-acetylated form) appears to contribute significantly to the biofilm architecture. An alginate overproducing P. aeruginosa strain forms highly structured biofilms on an abiotic surface as compared with its isogenic, non-mucoid strain. After a delay in biofilm initiation, the mucoid strain produced biofilms with large microcolonies separated by water channels, whereas the non-mucoid strain rapidly attached and initiated growth but did not exhibit the extensive architecture of the mucoid strain (Hentzer et al., 2001; Nivens et al., 2001; Stapper et al., 2004).

In addition, alginate production is not necessarily a reliable indicator of the antibiotic resistance capacity of a CF-colonizing strain. A study using both mucoid and non-mucoid clinical isolates from adult patients found that isolates grown as biofilms were less susceptible to antibiotics than their planktonically grown counterparts, independent of the mucoid status (Aaron et al., 2002). It appears that the mode of growth is more important than alginate production in terms of predicting the ability of these organisms to resist the action of antibiotics commonly used to treat CF lung infections. In view of this, what selective advantage would alginate production provide to biofilm-forming strains colonizing the CF lung? Infection of the CF lung by microorganisms causes inflammatory cells to be recruited to the site of infection, where they release reactive oxygen species (ROS) and cause extensive tissue damage (Govan and Deretic, 1996; Pier, 1998). Alginate appears to protect P. aeruginosa from the consequences of this inflammation as it scavenges free radicals released by activated macrophages in vitro (Simpson et al., 1989). Alginate also appears to provide protection from phagocytic clearance and defensins, most probably because it provides a physical and chemical barrier to the bacterium (Govan and Deretic, 1996; Pier, 1998). Alginate in sputa from CF patients can prevent neutrophil chemotaxis and complement activation but appears to enhance neutrophilic oxidative burst (Pedersen et al., 1990b). Although antibodies to alginate are found in the sera of chronically infected CF patients, these antibodies fail to mediate opsonic killing of P. aeruginosa in vitro (Pier, 1998). Probably most or all of these factors discussed above contribute to the ability of mucoid P. aeruginosa to persist and establish chronic infections in the CF lung.

Although available knowledge concerning how alginate contributes to persistence is considerable, there are still numerous questions that remain unanswered (Fig. 3). Clinical evidence points to initial colonization of the CF lung by non-mucoid strains of P. aeruginosa (Fig. 3, step 1), and there is evidence from histological examination of late-stage CF lungs that matrix-encased P. aeruginosa are found in the bronchioles (Lam et al., 1980; Baltimore et al., 1989; Singh et al., 2000; Hoiby et al., 2001) (Fig. 3, step 4). It is not clear whether mucoid P. aeruginosa strains persist in the CF lung because of alginate expression alone, or whether other factors contribute (Fig. 3, steps 2–4). Data suggest that conversion to mucoidy involves a fundamental change in the physiological and virulence traits of P. aeruginosa. Recent microarray data from AlgT+ mucoid P. aeruginosa strains demonstrated induction of lasB (elastase), aprA (alkaline metalloproteinase) and the hcnABC (hydrogen cyanide production) genes, as well as several lipoproteins (Firoved et al., 2002; 2004; Firoved and Deretic, 2003). These gene products could provide the bacteria with additional tools to combat the host immune response and promote persistence. Studies in P. fluorescens have shown that AlgT is required for tolerance to desiccation and hyperosmolarity, which could also provide a selective growth advantage in such environmental conditions as the CF lung (Schnider-Keel et al., 2001). AlgT has also been shown to exert negative regulation on flagella-mediated motility, and AlgT+ strains are attenuated in a wounded alfalfa seedling model and in both neutropenic and burned mouse models of infection (Yu et al., 1996; Garrett and Wozniak, 1999; Silo-Suh et al., 2002). Together these studies suggest that alginate production might allow the bacteria to survive and persist better than their non-mucoid counterparts, which are more virulent but also better recognized by immune defences. These ‘persistors’ might then grow and divide, establishing a chronic infection that is difficult to eradicate. In this way, the balance is tipped in favour of chronic colonization by mucoid strains of P. aeruginosa in the CF lung.

Potential therapies, future perspectives and conclusions

  1. Top of page
  2. Summary
  3. Cystic fibrosis and Pseudomonas aeruginosa
  4. Alginate biosynthesis
  5. Regulation of alginate biosynthesis
  6. Alginate and biofilm development
  7. Potential therapies, future perspectives and conclusions
  8. Acknowledgements
  9. References

Cystic fibrosis research has come a long way in a very short period. Data from the US indicate that the median age of survival for CF patients has risen from 14 years in 1969 to 32 years in 2000 (Cystic Fibrosis Foundation, 2001). Recent developments in the understanding of airway surface physiology as well as innate immune defences in the airway will facilitate future progress. A recently generated transgenic CFTR mouse model that demonstrates progressive lung infection (Coleman et al., 2003) could assist with the evaluation of novel therapies to prevent colonization, infection and chronic growth of P. aeruginosa. Despite these advances, the vast majority of CF patients die from chronic lung infections caused by P. aeruginosa. While many have studied why P. aeruginosa is uniquely suited to establishing successful infections in the CF lung, the greater challenge lies in devising therapeutic approaches that would address expression of a substantial palette of virulence factors as well as phenotypic diversity among colonizing and established strains. In this regard, a model for the initiation of chronic P. aeruginosa infections in CF and for the formation of biofilms, along with therapies that have the potential to intercede at these steps, is proposed in Fig. 3.

It is believed that the initially colonizing strains in CF are from an environmental reservoir. These strains are predominantly non-mucoid and motile (Fig. 3, step 1). Early aggressive antibiotic treatment of the initial colonizing planktonic population might prevent or at least delay chronic pulmonary infection (Frederiksen et al., 1997). This treatment regimen has been widely adopted by European CF centres and has proven to be successful (Ratjen, 2001). Questions still remain as to whether such therapy should be performed routinely or only during pulmonary exacerbation, and whether the regimen could potentially lead to the emergence of resistant strains. There is no global consensus as to what constitutes the optimal CF treatment regimen after early colonization by non-mucoid strains of P. aeruginosa, and there is a great need for randomized, multicentre trials that compare the efficacies of prevailing treatment strategies so that a more generalized regimen can be proposed (Rosenfeld et al., 2003).

In addition, agents aimed at preventing initial adherence and colonization should be considered (Fig. 3, step 1). These might be based on vaccination strategies targeting the primary adhesins or pharmacological inhibitors of these organelles or their receptors. In this regard, early in vitro animal studies as well as phase I trials with a flagellin-based vaccine have shown promise (Doring and Dorner, 1997), yet no long-term protection studies have been reported. As the role of other surface components such as outer membrane proteins, pili, non-pilus adhesins and polysaccharides are being discerned, these factors might also be examined for their potential as vaccine candidates (Hertle et al., 2001; Price et al., 2001). A pilus-exotoxin A vaccine has been shown to generate neutralizing antibodies in vaccinated rabbits (Hertle et al., 2001), and a trial found that immunizing non-colonized CF patients with an exotoxin A conjugate vaccine significantly lowered P. aeruginosa infections rates as compared with non-vaccinated controls (Cryz et al., 1997). Data also suggest that the pel and psl-encoded exopolysaccharides contribute substantially to cell surface interactions (Friedman and Kolter, 2004b; Jackson et al., 2004; Matsukawa and Greenberg, 2004), and this might be a viable target to reduce or prevent initial colonization.

After attachment, P. aeruginosa develops microcolonies on both biotic and abiotic surfaces (O’Toole et al., 2000; Sauer et al., 2002; Jackson et al., 2003) (Fig. 3, step 2). Prior studies suggest that both type IV fimbriae-mediated twitching motility and quorum sensing play important roles in these processes (Davies et al., 1998; O’Toole and Kolter, 1998), although these observations might be highly dependent on the experimental conditions (Klausen et al., 2003). Inhibitors of quorum sensing and twitching motility have been described (Hentzer et al., 2003; Wozniak and Keyser, 2004), and preliminary in vivo studies suggest that these might have therapeutic value either alone or in combination with conventional antibiotics (Yanagihara et al., 2002; Smith and Iglewski, 2003; Wu et al., 2004). Additional vaccine candidates at this stage of the infection include factors that contribute to tissue damage and cell death such as the type III secretion system or other toxins and adhesins (Sawa et al., 1999; Hertle et al., 2001).

During biofilm formation under laboratory conditions using model P. aeruginosa strains such as PAO1 and PA14, microcolonies mature into complex three-dimensional structures that are entrapped in an EPS matrix (O’Toole et al., 2000; Sauer et al., 2002) (Fig. 3, step 3). The formation of these structures appears highly variable, depending on the strain and growth conditions (Klausen et al., 2003). Several groups including ours have shown that alginate does not appear to be a constituent of these biofilms (Hentzer et al., 2001; Nivens et al., 2001; Wozniak et al., 2003a; Stapper et al., 2004). Despite this, these biofilms display a highly resistant phenotype: physical agents and antibiotics fail to eliminate these organisms, so therapeutic options become limited. In the CF lung, biofilm development becomes more complex. Recent data using alginate-specific antibodies have shown that alginate may be expressed by non-mucoid strains in the mouse lung within 1 h post infection (Pier et al., 2004). Analyses of biofilm microenvironments reveal heterogeneous populations, including regions displaying metabolic and oxygen gradients (Xu et al., 1998). Moreover, P. aeruginosa appears to be localized within hypoxic zones in the lumen of the CF airway (Worlitzsch et al., 2002). This has led to a renewed interest in understanding the physiology and genetics of P. aeruginosa anaerobic metabolism and their effect on exopolysaccharide expression. This might lead to the development of new drugs to combat anaerobic subpopulations, which might enhance the clearance of persister cells that are normally maintained within biofilms despite aggressive conventional antibiotic regimens (Hassett et al., 2002).

As discussed above, there appears to be a strong selective pressure that eventually leads to the appearance of stable alginate-producing variants (Fig. 3, step 4). One such selective factor is likely to be the vigorous inflammatory response, as mucoid conversion has been reproduced under laboratory conditions using sublethal concentrations of hydrogen peroxide or activated neutrophils (Mathee et al., 1999). Agents aimed at reducing the inflammatory response might therefore contribute to preventing the associated damage as well as reducing the frequency of mucoid conversion. This has been proposed as one mechanism to explain the therapeutic value of long-term, low-dose macrolide antibiotics in CF and other chronic airway diseases (Schultz, 2004). Other viable candidates for therapeutics include inhibitors of the enzymes required for alginate synthesis. Initial studies with macrolide antibiotics have indicated that inhibition of GMD activity is possible (Mitsuya et al., 2000) and molecules that inhibit the activity of PMM/PGM are currently under development (Regni et al., 2004). It has also been proposed that AlgL may be used in combination with DNases to reduce the viscosity of CF sputum and provide better antibiotic penetration into alginate-encased cells (Schiller et al., 1993).

Alginate has been the focus of substantial vaccine development in recent years because of its contribution to the establishment of chronic infection in the CF lung (Fig. 3, step 1). The hypothesis underlying current vaccine trials is that aiding the body's ability to eliminate mucoid isolates will not only contribute to a reduction in bacterial numbers, but will also prevent the establishment of chronic infection. The safety and efficacy of alginate as a vaccine candidate has been tested in humans (Pier et al., 1994). Delivery of alginate conjugated to a carrier protein has been shown to elicit opsonic antibodies to alginate in mice (Theilacker et al., 2003). These results appear promising as a large proportion of oposonic antibodies generated by the vaccine are directed towards O-acetate residues, which are hypothesized to contribute to resistance of mucoid P. aeruginosa by preventing complement activation and are also important for attachment and surface-associated growth in biofilms (Nivens et al., 2001; Pier et al., 2001).

So is alginate still relevant in a discussion of P. aeruginosa infection of the CF lung? Without a doubt, the answer is yes. As the infection model becomes more complex, however, the role of alginate biosynthesis and regulation in chronic CF lung infection must be placed in context with new and emerging data that reveal how the establishment of biofilms by non-mucoid strains can equally contribute to a successful and robust infection. As the importance of both topics becomes balanced in the scientific community, the spectrum of therapies will grow to encompass all stages of P. aeruginosa infection of the CF lung, providing clinicians with more choices on how to treat an aggressive and highly versatile pathogen. Further understanding of alginate biosynthesis, regulation and contributions to biofilm formation will continue to shape the direction of therapeutic design with the long-term goal of preventing chronic lung infection by mucoid strains of P. aeruginosa.


  1. Top of page
  2. Summary
  3. Cystic fibrosis and Pseudomonas aeruginosa
  4. Alginate biosynthesis
  5. Regulation of alginate biosynthesis
  6. Alginate and biofilm development
  7. Potential therapies, future perspectives and conclusions
  8. Acknowledgements
  9. References

This work was supported by an American Heart Association Pre-doctoral Fellowship 0215191 U (D.M.R.) and Public Health Service Grants AI-35177 and HL-58334 (D.J.W.). B.E. deFluiter assisted with photography.


  1. Top of page
  2. Summary
  3. Cystic fibrosis and Pseudomonas aeruginosa
  4. Alginate biosynthesis
  5. Regulation of alginate biosynthesis
  6. Alginate and biofilm development
  7. Potential therapies, future perspectives and conclusions
  8. Acknowledgements
  9. References
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