1pseudomonas infection

Review

Establishment of Pseudomonas aeruginosainfection: lessons from a versatile opportunist

Jeffrey B. Lyczaka,b, Carolyn L. Cannonb,c, Gerald B. Piera,b,*

a The Channing Laboratory, Brigham and Women's Hospital, 181 Longwood Avenue, Boston, MA 02115, USAb Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, USAc Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA

ABSTRACT – Pseudomonas aeruginosa is an ubiquitous pathogen capable of infecting virtually alltissues. A large variety of virulence factors contribute to its importance in burn wounds, lung infectionand eye infection. Prominent factors include pili, flagella, lipopolysaccharide, proteases, quorumsensing, exotoxin A and exoenzymes secreted by the type III secretion system. © 2000 Éditionsscientifiques et médicales Elsevier SAS

Pseudomonas aeruginosa / virulence / toxins

1. Introduction

Pseudomonas aeruginosa is a common environmentalGram-negative bacillus which acts as an opportunisticpathogen under several circumstances. The ubiquitousoccurrence of P. aeruginosa in the environment [1, 2] isdue to several factors, including its abilities to colonizemultiple environmental niches and to utilize many envi-ronmental compounds as energy sources [3]. P. aerugi-nosa was likely first reported in human infections in 1862by Luke, who observed rod-shaped particles in blue-greenpus of some infections. Similar coloration had been previ-ously observed by Sedillot on surgical dressings, and isnow known to be caused by the pigment pyocyaninproduced by P. aeruginosa. The microorganism was firstisolated from infections in 1882 by Gessard, who called itBacillus pyocyaneus. Given the widespread occurrence ofP. aeruginosa in the environment, it is noteworthy thathuman disease attributable to it is quite rare in otherwisehealthy individuals.

Nearly all clinical cases of P. aeruginosa infection canbe associated with the compromise of host defense. Whilemany cases of P. aeruginosa infection can be attributed togeneral immunosuppression such as in AIDS patients [4,5] and in neutropenic patients undergoing chemotherapy[6], such scenarios predispose the host to a variety ofbacterial and fungal infections, and therefore do not yieldinformation which is specific to the pathogenesis of P.aeruginosa. In this respect, three of the more informative

human diseases caused by P. aeruginosa are: 1) bacter-emia in severe burn victims; 2) chronic lung infection incystic fibrosis patients; and 3) acute ulcerative keratitis inusers of extended-wear soft contact lenses. Observationsand experimental evaluation of various bacterial virulencefactors have shed a great deal of light on how P. aeruginosais able to cause disease in a wide variety of organs,secondary to disruption of normal physiologic function.Such insights provide an understanding at the molecularand cellular level of how and why P. aeruginosa hasbecome such an important pathogen in human infection.

2. P. aeruginosa bacteremia in severeburn victims

Perhaps the most obvious example of infection follow-ing the compromise of a host defense, bacterial infectionfollowing severe thermal injury can be most simplisticallyattributed to extensive breaches in the skin barrier. The factthat P. aeruginosa occurs so commonly in the environmentmakes it extremely likely that an individual suffering severeburns will be challenged with this microorganism beforethe burns can heal. Exacerbating this situation, hospitalsoften harbor multidrug-resistant P. aeruginosa that canserve as the source of infection. P. aeruginosa has beenfound to contaminate the floors, bed rails, and sinks ofhospitals, and has also been cultured from the hands ofnurses [7]. Besides transmission through fomites and vec-tors, bacterial flora can be carried into a hospital by thepatient and can be an important source of infection for thesame individual after injury [8]. Concerns about multidrug-resistant strains are highlighted in the report of Hsueh et al.[9] who traced the spread of a single multidrug-resistant

* Correspondence and reprints

* Address for correspondence: Channing Laboratory, 181 Longwood Avenue,Boston, MA 02115, USA

Microbes and Infection, 2, 2000, 1051−1060© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved

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strain of P. aeruginosa over a period of several years, andconcluded that the strain was carried by some patientsasymptomatically through several rounds of antibiotictreatment which were administered to treat Pseudomonasand non-Pseudomonas infections. Highly problematic inthis situation is both the spread of P. aeruginosa from onepatient to another and the persistence of this strain inpatients throughout several courses of antibiotic treat-ment.

3. P. aeruginosa virulence factors inburn infections

Numerous P. aeruginosa virulence factors contribute tothe pathogenesis of burn wound infection. Rahme et al.highlighted the occurrence of virulence factors of P. aerugi-nosa contributing to pathogenesis in both burn woundinfection of rodents and leaf destruction in the plant Ara-dopsis thaliana [10]. They identified the toxA, plcS, andgacA genes as contributing to virulence in both plant andanimal infection models. An important role has also beenestablished for P. aeruginosa pili and flagella. Experimentscomparing infection of burn wounds by pilus- andflagellum-deficient P. aeruginosa strains clearly demon-strate that bacteria deficient in either of these structureshave reduced virulence, both in their ability to persist atthe wound site, and in their ability to disseminate through-out the host organism [11]. Dissemination of P. aeruginosathroughout the host is also partially dependent upon pro-duction of bacterial elastase [12] and other proteases.Proteases probably promote infection and disseminationby P. aeruginosa by several mechanisms (figure 1). Elastasehas been shown to degrade collagen and noncollagenhost proteins, and to disrupt the integrity of the hostbasement membrane [13]. Thus, elastase promotes dis-semination by destroying host physical barriers which

would normally inhibit the spread of infection. Proteasescan have adverse effects on several aspects of the innateand acquired host immune response. For example, elastaseinhibits monocyte chemotaxis, [14] which could adverselyaffect early clearance of P. aeruginosa from wound sites byphagocytosis, as well as subsequent presentation of bac-terial antigens to the host immune system. A recent report[15] demonstrated that a P. aeruginosa strain with a dis-ruption in the lasR regulatory gene (producing a straindefective in the synthesis of multiple virulence factors,including two different elastases, LasA and the LasBelastase, along with exotoxin A, and alkaline protease) isincapable of disseminating to distal host sites from acolonized burn wound. Interestingly, the defect in viru-lence of this strain was not due to loss or reduction in theproduction of either elastase, as specific disruptions ineither the lasA and lasB genes did not compromise viru-lence in a burn wound infection model [16]. The lasR geneencodes a protein critical for initiation of the quorumsensing response involved in virulence factor productionand biofilm formation, indicating that other factors con-trolled by lasR are critical determinants of P. aeruginosapathogenesis in burn wound infection [16]. Other P. aerugi-nosa virulence factors reported to be involved in patho-genesis of burn wound infection include phospholipase C[17], the ferripyochelin-binding protein [18], lipopolysac-charide (LPS) [19], and exoproducts secreted by the typeIII secretion apparatus [20].

While the loss of the skin's barrier function is certainlyan important factor in burn wound infection, its compro-mise fails to explain the relatively narrow range of bacte-rial pathogens which are typically cultured from infectedburn wounds [21]. It is therefore likely that additional hostdefense mechanisms specific to some pathogens are morecompromised in severe burns. Felts has recently reported areduction in infection following local application of poly-clonal human antibody to burn sites [22], suggesting that

Figure 1. Schematic representation of the bacterial factors promoting infection of burn wounds by P. aeruginosa. Pseudomonal proteasesact both to destroy host physical barriers and to compromise host immune effectors.

Review Lyczak et al.

1052 Microbes and Infection2000, 1051-1060

in the untreated burn wound, immunoglobulin exists atsubprotective levels. The possibility of a local deficiencyof antibody-mediated immunity in burn wounds is furthersupported by an earlier report [23] that Fc receptor expres-sion by polymorphonuclear leukocytes (PMNs) decreasesby the fifth day post-injury in burn victims. Complementhas also been shown to be depleted in burn wounds [24],probably due to local consumption of complement com-ponents. Local deficiencies in protective antibody, comple-ment proteins, and PMN Fc receptors may explain thedefects in random migration and chemotaxis of PMNsobserved at burn wound sites (see figure 1; [14]). Collec-tively, these data suggest that the ability to colonize a burnwound depends upon the concerted impairment of severalhost immune mechanisms, and that the importance of P.aeruginosa in such infections is due to its ability to takeadvantage of the host immune compromise and secrete avariety of important virulence factors.

4. P. aeruginosa lung infection innosocomial settings and cystic fibrosispatients

P. aeruginosa infection in the hospital manifests prima-rily as acute lung infection in patients in intensive careunits. Models of pseudomonal pneumonia have high-lighted the importance of classic P. aeruginosa virulencefactors such as proteases, flagella, pili and LPS O sidechains. In addition, elegant studies from Frank and col-leagues [25] have recently documented the presence of atype III secretion system in P. aeruginosa that appears toplay a major role in virulence of this organism [26]. Thetype III secretion system delivers important virulence fac-tors, such as ExoS, ExoT and ExoU into mammalian cells.Intracellular delivery of the ExoU virulence factor pre-sents, in vitro, as a cytotoxicity reaction, first described byFleiszig et al. [27]. In vivo studies have shown that immu-nization against one component of the type III secretionsystem, the P. aeruginosa V antigen, protects animalsagainst acute P. aeruginosa pneumonia [28].

In contrast to the acute nature of nosocomialpseudomonal pneumonia, the P. aeruginosa lung infectionseen in cystic fibrosis (CF) patients is indolent in nature.Nonetheless, this chronic bacterial lung infection cur-rently accounts for the majority of the morbidity andmortality seen in the disease. CF is an autosomal recessivedisorder resulting from mutation of the cyclic AMP (cAMP)-regulated chloride ion channel protein known as the cys-tic fibrosis transmembrane conductance regulator (CFTR).This single genetic defect has pleiotropic effects on thedevelopment and function of several tissues and organs,including the pancreas, sweat glands, vas deferens, andintestines. When CF was first identified as a disease, mostaffected individuals died within the first few years of life,owing to insufficient absorption of nutrients secondary topancreatic dysfunction. As treatment for CF patientsimproved over the years, particularly after the introductionof improved nutritional regiments [29], patients beganliving much longer [30]. As a consequence of this increased

survival, chronic bacterial lung infection emerged as theprimary cause of mortality in CF patients.

Although Staphylococcus aureus is often cited as anearly pathogen for CF patients, in fact, there is no crediblestudy that documents direct lung pathology resulting fromthe presence of S. aureus in throat cultures of CF patients[31]. Nonetheless, most clinicians empirically treat CFpatients with antistaphylococcal antibiotics, although arecent review of the field [32] concluded that “It remainsto be determined whether the use of 'prophylactic' versus'intermittent' antistaphylococcal therapy in cystic fibrosisis associated with improved lung function and/or chestradiographic scores, an increase in bacterial resistance, orearlier acquisition of Pseudomonas aeruginosa”. In con-trast, it is abundantly clear that P. aeruginosa is the mostprevalent and problematic pulmonary pathogen in CFpatients. One of the most striking features of infection ofthe CF lung by P. aeruginosa is that the establishment ofchronic infection correlates with the transition by themicrobe to a mucoid phenotype [33] resulting from bac-terial production of a polysaccharide known both as algi-nate and mucoid exopolysaccharide (MEP). This materialplays important roles in bacterial adherence to host cellsand in evasion of the host immune response [34]. MEPproduction by P. aeruginosa was first noted by Doggett andcoworkers [35] in sputum cultures from CF patients. It wascharacterized as an acetylated random polymer of man-nuronic acid and guluronic acid by Linker and Jones [36],with the degree of acetylation varying widely betweenMEP purified from different P. aeruginosa isolates [37]. Inthe lungs of CF patients, Lam and colleagues [38] describedP. aeruginosa growing in a fibrous, polyanionic matrixsurrounding the bacterial cells. In vitro, the mucoid phe-notype is frequently unstable, with a large percentage ofisolates reverting to a nonmucoid phenotype during cul-ture [37]. The instability of the mucoid phenotype in vitrosuggests that mucoidy confers upon P. aeruginosa an invivo growth advantage, perhaps by allowing evasion ofthe host's immune system, or by allowing bacterial growthwhen iron [39], phosphate [40], or nutrients [40] arelimiting.

The genetic regulation of MEP production has been thefocus of intense investigation in recent years. That MEPproduction is controlled by a chromosomal genetic locuswas first demonstrated in the early 1980s [41]. Initialattempts at cloning the genetic loci controlling MEP syn-thesis were frustrated by the inherent instability of themucoid phenotype. The creation of the mucoid-lockmutant strain 8830 allowed plasmid-complementationstudies in which libraries obtained from P. aeruginosastrain 8830 were transformed into mucoid-deficientmutants [42]. These experiments resulted in the cloning ofa minimally essential genetic element of 6.2 kb whichcontained the genes necessary for MEP synthesis. Themucoid phenotype of P. aeruginosa is controlled by tran-scriptional regulation of the GDP-mannose dehydroge-nase encoded by the algD gene, the first gene of thealginate biosynthetic operon [43]. Work conducted overthe previous decade has demonstrated that the algD geneis actually under control of a complex, multi-tier regula-tory mechanism (see figure 2) involving both constitutive

Bacterial and host factors in P. aeruginosa infection Review


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[44] and inducible [45] gene products. The inducible armof algD regulation relies in large part on the alternativesigma factor (σ22) encoded by the algU gene (ref [46]; alsocalled 'algT'), which positively regulates both its owntranscription [47] as well as that of the transcription factorsAlgB and AlgR [45, 47]. Meanwhile, the AlgU sigma factoris under negative control by the anti-sigma factors MucAand MucB [48]. A very recent report demonstrates that themechanism of induction of MEP synthesis by hydrogenperoxide is through mutation of the mucA gene [49], thusabrogating the inhibition of σ22 by MucA. Two genesregulated by AlgU encode the transcription factors AlgBand AlgR [45], which bind to response elements in thealgD promoter. Although these two transcription factorsare phosphorylated during conversion to a mucoid phe-notype [50, 51], more recent data [52] demonstrates thattranscriptional activation of algD by AlgB and AlgR is notaffected by these phosphorylation events. Thus, phospho-rylation of AlgB and AlgR may play important roles inother aspects of alginate regulation, perhaps in the inter-action of these transcription factors with other regulatoryproteins such as AlgZ [53]. Immediately downstream ofalgB is kinB, which encodes the histidine protein kinase,KinB [51]. This kinase likely acts as an environmentalsensor for AlgB. The role that the environment of the CFlung plays in triggering algD transcription, perhaps throughKinB, is unclear.

Elucidation of host factors which predispose individu-als with CF to pulmonary P. aeruginosa infection has beenhampered by the multitude of symptoms observed in CFpatients, and by the plethora of clinical and environmentalfactors which can potentially influence the establishmentof this infection. Since CF is caused by mutation of theCFTR gene, it seems logical to assume that this geneticdefect is directly related to the establishment of P. aerugi-nosa infection, yet the link between the CFTR gene prod-uct and chronic respiratory infection with P. aeruginosaremains to be fully defined. CFTR is expressed primarily inepithelial tissues and serves multiple roles including thecoordination of fluid secretion. Defective epithelial salttransport and fluid secretion underlie many of the clinicalmanifestations of CF, and are the basis of the currentstandard of diagnosis of CF, the sweat electrolyte test. Thefunctional consequences of this defect in salt transportvaries from one tissue/organ to the next. In the pancreas,decreased salt transport leads to thickening of secretionswith subsequent plugging of the pancreatic duct, leadingto eventual autodigestion of the exocrine tissue [54, 55]. Inthe airways, decreased salt transport (see [56] for discus-sion) and concomitant thickening of secretions may nega-tively impact the efficiency of the mucociliary escalator[57]. Additionally, it has been suggested [58] that theabnormal salt concentrations in the airway secretions ofCF patients negatively affects the function of antibacterialpeptides such as defensins, although others have chal-lenged this hypothesis [59]. Both inefficient mucociliaryclearance and/or hyperosmolarity of airway surface liquidmay lead to increased susceptibility of the CF patient toinfection, although neither mechanism explains the spe-cific hypersusceptibility to infection with P. aeruginosa.

Recent evidence suggests that the CFTR protein, inaddition to its role in salt transport, may influence P.aeruginosa lung infection directly through its role as anepithelial cell receptor for this microorganism [60, 61, 62].This binding interaction occurs between the first extracel-lular loop of CFTR (predicted to be in amino acids 108–117of the mature protein) and the complete outer portion ofthe core polysaccharide of the P. aeruginosa LPS; competi-tive inhibitors for either of these moieties block the bind-ing and internalization of P. aeruginosa by airway epithe-lial cells. Epithelial cells expressing mutant CFTR (notably,the ∆F508 trafficking mutant allele of CFTR, whichaccounts for 70% of the CFTR alleles carried by CF patients)internalize P. aeruginosa far less efficiently than do epithe-lial cells expressing normal CFTR protein. Thus, it waspostulated [60–62] that internalization of P. aeruginosa byairway epithelium, perhaps followed by desquamation ofbacteria-laden epithelial cells, constitutes a host defensemechanism which in the normal, healthy lung serves tominimize the bacterial load in the airway. It was furtherproposed that in the CF lung, this mechanism fails tofunction properly, resulting in abnormally high bacterialcarriage which promotes the establishment of chronicbacterial infection.

Figure 2. Control of the mucoid phenotype in P. aeruginosa isattained through a multi-tier regulation of the GDP-mannosedehydrogenase encoded by the algD gene. The regulation utilizesboth constitutive and inducible response elements. The algDpromoter is magnified relative to the other promoters in thefigure (with '-500' and '-200' indictating nucleotide positionswithin the algD promoter) to illustrate that the various transcrip-tion factors utilize different response elements in the algD pro-moter. See text for details.



Not all of the putative host factors which predispose toP. aeruginosa lung infection can be directly linked to thegenetic defect in the CFTR gene product. Chronic infec-tion by P. aeruginosa can be established at any time, butfor some CF patients this may not be until their mid-teens[33]. It has been suggested [63] that early infection withother pathogens primes the airway for later infection by P.aeruginosa. One mechanism by which this might occur isby repeated or chronic inflammation in the airways. Thepresence of bacteria and markers of inflammation in theairways of CF patients begins at an early age. A study byKirchner and colleagues [64] showed increased amountsof DNA and PMNs in bronchoalveolar lavage fluidobtained from infants and young children with CF com-pared with non-CF controls. The DNA which accumulatesis likely derived from dead PMNs, and may increase theviscosity of airway secretions. Birrer and coworkersshowed that 20 of 27 CF patients, including two of fourinfants, had active neutrophil elastase in the bronchoal-veolar lavage fluid, accompanied by a reduction in theamount of secretory leukoprotease inhibitor [65]. Similarresults were reported by Konstan and colleagues [66] andby Khan and coworkers [67]. Researchers have beguninvestigating the possibility that the increase in inflamma-tory mediators seen in the lungs of CF patients derives froman intrinsic property of the epithelium itself, perhaps anexaggerated inflammatory response to bacterial patho-gens. Bonfield and colleagues [68] isolated bronchialepithelial cells from healthy control subjects and patientswith CF and measured the amount of secreted anti-inflammatory cytokine interleukin-10 (IL-10), as well asproinflammatory cytokines IL-8 and IL-6. Cells from nor-mal patients secreted IL-10, but no detectable IL-6 or IL-8,whereas cells from CF patients did not secrete IL-10, butproduced both IL-6 and IL-8. Increased levels of proin-flammatory cytokines in the airway epithelium of CFpatients has also been observed by others [67].

There is a consensus among CF investigators that in theCF airway, the normal host defense against environmentalpathogens is in some way altered, contributing to theabnormal persistence in the airways of P. aeruginosa.Additionally, the inflammatory response to such infectionis somehow dysregulated in the CF airway such thatintense inflammation exists even in the absence of frankinfection. Such inflammation may predispose the CF air-way to the establishment of chronic P. aeruginosa infec-tion, perhaps by damaging local epithelium through theelaboration of high levels of oxidative and non-oxidativeinflammatory mediators, resulting in the compromise ofthe epithelial barrier or in the accumulation of senescenthost cells which impair normal clearance mechanisms.The increased persistence of P. aeruginosa resulting fromthese factors may create the opportunity for the bacteriumto undergo additional phenotypic changes (for example,the conversion to a mucoid phenotype) which, in turn,allows for a more effective evasion of the host response.

5. Ulcerative keratitis of the corneaUlcerative keratitis (UK) is a rapidly progressing inflam-

matory response to bacterial infection of the cornea, and

has been called the most destructive bacterial disease ofthe human cornea [69]. Historically, this infection wasusually associated with injury or trauma to the cornea.However, in 1984, cases of UK were reported amongindividuals who had suffered no acute injury, but whowere users of extended-wear soft contact lenses [70, 71],suggesting that use of such lenses can somehow predis-pose an individual to UK. Despite the description of suchsusceptibility factors, however, the reason for their corre-lation with corneal Pseudomonas infection is still notunderstood. Models which have been proposed rangefrom simple mechanical explanations (such as abrasion ofthe cornea by the contact lens, trapping of the microor-ganisms at the corneal surface, or adherence of the micro-organism to the lens) to elaborate subversion of cornealhost defenses.

The cornea exists in direct contact with the environ-ment, and therefore relies on several mechanisms to pre-vent bacterial infection. Among these are the continuousproduction of tears, which together with eyelid blinking,serves to physically remove bacteria from the eye. Tearsare also important in that they contain secretory immuno-globulin A, amylase and lysozyme, which have beenimplicated in protection of the eye from bacterial infection[72, 73]. The innermost portion of the tear layer is com-prised of mucus which binds strongly to P. aeruginosa,thus inhibiting bacterial adherence to the corneal epithe-lium [74]. It has been reported by Versura and coworkersthat the ocular mucus of contact lens wearers has signifi-cantly reduced levels of sialic acid, N-acetylglucosamine,N-acetylgalactosamine, galactose-N-acetylgalactosamine,and mannose [75]. These same workers suggested thatsuch alterations in mucus composition may affect subse-quent deposition of substances or microorganisms ontothe corneal surface, a supposition supported by the laterfinding that mucus fractions comprising different carbohy-drates bind to P. aeruginosa with different avidities [76].Together, these results suggest that one factor contributingto pseudomonal UK is the loss of adherence of ocularmucus to the bacterium, perhaps impairing a crucial bac-terial removal mechanism.

Phagocytosis of bacteria by PMNs has also been sug-gested to be an important arm of host defense in the cornea[77, 78], although the relationship between PMN activityand host protection is complicated by the large amount ofcollateral damage that PMNs can inflict on nearby hosttissues [79]. Thus, whether the neutrophil plays a role asfriend or foe in the pathogenesis of contact lens-associatedpseudomonal UK remains unclear.

P. aeruginosa elaborates a multitude of factors whichaffect the initiation and the course of corneal infection.Among these are: glycocalyx, endotoxin, exotoxin, pro-tease, flagella and pili (see figure 3). Glycocalyx, whichincludes MEP among its components, appears to functionin several ways during the establishment of P. aeruginosainfection. First, it mediates adhesion of bacteria, both toother bacteria and to host cells and tissue [80], resulting inlarge aggregates of bacteria which are firmly attached tosubstratum. The second function of glycocalyx (perhapsrelated to the first) is the resistance of phagocytosis. Thisresistance may result from the large size of the bacterial



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aggregates which form as a result of bacteria-bacteriaadhesion, or from the firm adhesion of these aggregates tohost tissue. Alternatively, the glycocalyx may serve toblock the interaction of Fc receptors on host immune cellswith antibody bound to bacterial surface antigens [81].

The role of endotoxin, or LPS, in pseudomonal cornealdisease is twofold. As is observed for many endotoxins,administration of purified Pseudomonas LPS into cornealstroma results in PMN chemotaxis and activation [82].This event plays an important role in subsequent hosttissue damage due to oxidative and non-oxidative bacte-riocidal agents, such as protease and reactive oxygenspecies released by these cells. In addition to its role ininitiation of inflammation, however, the LPS of P. aerugi-nosa serves an important role as a ligand for the host cellreceptor CFTR (cystic fibrosis transmembrane conduc-tance regulator; see section on CF lung infection above).Contrary to what is observed in lung infection, in thecornea, it appears that the interaction of P. aeruginosa withthe CFTR protein actually promotes infection [83]. Thereason that Pseudomonas-CFTR binding has two drasti-cally different outcomes in these two host tissues mayrelated to be the architecture of the host tissues, and theCFTR expression pattern in each tissue (see figure 4). Thus,in the airway, CFTR is expressed on an epithelium which isone cell layer thick. Binding of the pseudomonas to thisepithelium results in internalization of the bacterium fol-lowed by desquamation of the epithelial cell. This presum-ably leads to clearance of the internalized bacteria. Thesituation in the cornea differs in that the epithelial cellswhich express the highest amounts of CFTR protein are thebasal epithelial cells (the more superficial epithelial cellsexpress undetectable quantities of CFTR protein; ref [83]).Thus, the corneal epithelial cells which express the most

CFTR, and are possibly most likely to internalizePseudomonas, are buried beneath several epithelial celllayers. Rather than serving to remove Pseudomonas, asoccurs in the airway, internalization of Pseudomonas bycorneal epithelium creates a reservoir of intracellular bac-teria which are capable of replicating within the cornealepithelial cells [84]. Therefore, the contribution of cornealinjury to Pseudomonas infection may be that the formerpermits access of the bacteria to the basal epithelial cellswhich express the CFTR receptor on their membrane. Theimportance of the CFTR-bacterial interaction in cornealinfection by Pseudomonas is further supported by thefinding that transgenic CF mice with the murine ∆F508Cftr allele are highly resistant to P. aeruginosa UK [83].

Pseudomonas produces several exotoxin proteins whichare thought to be prime determinants of pseudomonalvirulence. Exotoxin A has been studied extensively and itsbiochemical activity as been determined to be ADP-ribosylation of protein elongation factor-2 [85, 86], result-ing in a cessation of protein synthesis within the target cell(this enzymatic activity is identical to that of diphtheriatoxin). Early studies on the contribution of exotoxin A toPseudomonas pathogenesis were carried out using puri-fied exotoxin [87], and showed that administration ofpurified toxin resulted in rapid (< 24 h) destruction ofcorneal epithelial cells with subsequent chemotaxis ofPMNs to the site and eventual corneal ulceration. How-ever, it has been difficult to show that exotoxin A plays anecessary role in UK, since exotoxin A-deficient strainscan be as virulent as wild-type strains in some animalmodels of infection [88].

P. aeruginosa proteases are also thought to be crucialfor many facets of Pseudomonas pathogenesis in the eye[89]. The precise role of pseudomonal proteases in thedisease process has been difficult to ascertain with cer-tainty due to the important contribution made by host-derived proteases, which act both to clear infection [78,90] and to exacerbate local host tissue damage [79, 91,92]. There was no effect on virulence in a mouse model ofUK when the lasB gene encoding elastase was inactivated,and the apparent role for the lasA gene product, achievedby a reduction in virulence of a lasA mutant, could not beattributed to this gene by complementation studies orproduction of a defined mutation in the lasA gene [93].Another protease, termed protease IV, has been reported tobe a major virulence factor in a mouse model of P. aerugi-nosa UK [94, 95], but studies of mutant strains comple-mented back to the wild-type have not confirmed thisfinding. Together, these results are supportive of a role ofbacterial proteases in UK infection by P. aeruginosa.

The flagella and pili of Pseudomonas play an importantrole in corneal infection as adhesins. It has been demon-strated that both of these bacterial structures bind specifi-cally to the host cell glycosphingolipid asialo-GM1 [96,97], and that this binding event is essential for epithelialcell invasion and cytotoxicity [98]. While asialo-GM1expression in human cornea has been questioned [99],work by other groups has shown that when mouse corneais scarified and then protease treated, asialo-GM1 expres-sion increases [100]. If true, this observation offers a

Figure 3. A synopsis of bacterial factors (top right) and hostfactors (bottom right) influencing the establishment of UK of thecornea. PMN-secreted mediators (proteases and reactive oxygenspecies (ROS)) probably contribute both to host protection and topathogenesis (see text for explanation)



further explanation for the coincidence of Pseudomonasinfection and corneal injury.

6. Conclusions

P. aeruginosa infections typify those of a pathogen withmany potential virulence factors that allow it to colonizeand infect essentially any mammalian tissue. The organ-ism possesses a multitude of factors that promote adher-ence to host cells and mucins, damage host tissue, elicitinflammation and disrupt defense mechanisms. In spite ofthe ubiquitous nature of this microorganism, and the fre-quency with which it is encountered, most human hostscounteract the infectious process effectively via the innateimmune system. However, compromises to this system,such as in the genetic disease of CF or the use of extended-wear contact lenses, dramatically increase host suscepti-bility to P. aeruginosa infection. At the molecular level thishypersusceptibility has been attributed to the use of CFTRas a receptor for P. aeruginosa, wherein binding andinternalization of the pathogen via CFTR results in clear-ance from a mucosal surface, while entry of the pathogeninto subsurface epithelial cells, such as in the scratch-injured eye, allows the organism to escape host defensesand cause disease. A more detailed molecular and cellularunderstanding of the bacterial and host factors is crucial toan overall comprehension of the pathogenic process ofpseudomonas, and will be of increasing importance to thedevelopment of preventative strategies to be sought for thismajor human pathogen.

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Figure 4. Model to explain the differential effect of the CFTR-Pseudomonas interaction on the outcome of infection. Left: In the airway,CFTR (green color) is expressed by the epithelium, and serves as a receptor promoting internalization of the bacteria (yellow) by epithelialcells. Internalization triggers desquamation of the bacteria-laden epithelial cell, and clearance of Pseudomonas ensues. The deficiency offunctional CFTR protein in CF patients impairs this clearance mechanism. Right: In the cornea, CFTR protein is expressed primary in basalepithelial cells (green cells), with little or no expression in the superficial, squamous epithelium (pink cells). Corneal injury may serve tobreach the tear and mucus layer, and to remove superficial epithelium, thus exposing CFTR-expressing basal cells to the bacteria (yellow).CFTR binding leads to internalization except, in contrast to the airway epithelium, the bacteria-laden epithelial cells are buried beneathseveral epithelial cell layers. CFTR-expressing epithelial cells may therefore become a reservoir for pseudomonas.



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