Life at the Front: Dissecting Bacterial-Host Interactions at the Ocular Surface

Laboratory Science JAMES V. JESTER, PHD, SECTION EDITOR

Life at the Front: Dissecting Bacterial-Host Interactions at the Ocular Surface DAVID J. EVANS, PHD,1,4 NANCY A. MCNAMARA, OD, PHD,3AND SUZANNE M. J. FLEISZIG, OD, PHD1, 2

ABSTRACT The ocular surface usually looks quiet, presenting a general impression of biological inactivity. Yet, the ability of the cornea to maintain health while continually exposed to environmental insults, and in the relative absence of immune strategies afforded by other body sites, reflects its complexity. Because it is critical for transparency and, therefore, our survival, the fine structure of the cornea has likely provided the driving force for the evolution of what appears to be a truly remarkable system. While several molecules are now known to participate, we are only beginning to obtain the knowledge to fully explain the mechanisms involved in corneal resistance to infection. Full explanation will require a better understanding of the interplay between microbes and various components of the ocular surface, and of the critical factors determining health as the usual outcome. To understand infectious disease, we need to consider how the scenario changes in conditions associated with susceptibility. What we learn in

Accepted for publication April 2007. From the 1School of Optometry, University of California, Berkeley, 2Graduate Programs in Vision Science, Microbiology, Infectious Disease and Immunity, University of California, Berkeley, 3Francis I. Proctor Foundation for Research in Ophthalmology and Department of Anatomy, University of California, San Francisco, and 4College of Pharmacy, Touro University-California, Vallejo, California, USA. Supported by grants from National Eye Institute (EY11221 to SMJF, EY016203 to NAM), the American Optometric Foundation, and the Cystic Fibrosis Foundation, and from industry, including Alcon, Allergan, Bausch and Lomb, and CibaVision.

the process could yield a wealth of potential therapies for a wide variety of diseases of the eye and of other sites. KEY WORDS bacteria, contact lenses, cornea, epithelium, innate defense, keratitis, pathogenesis, P. aeruginosa, Pseudomonas, tears, type III secretion system

I. INTRODUCTION acterial keratitis is an acute infectious disease of the cornea that can result in vision loss. The healthy cornea is highly resistant, but contact lens wear, ocular injury, or surgery can predispose to bacterial keratitis (Figure 1).1-3 A large range of Gram-positive and Gram-negative pathogens have been reported to be involved, but the most common bacterial species isolated are Pseudomonas aeruginosa, Serratia marcescens (Gramnegative) and Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pneumoniae (Gram-positive).1,4-6 Treatment of bacterial keratitis is frequently complicated by increasing bacterial resistance to antibiotics7,8 and by the often rapid progression of pathology. Thus, improved therapeutic approaches or, ideally, methods to prevent disease onset are needed. An improved understanding of ocular surface defenses against infection, of how and why they become compromised, and of the cellular and molecular mechanisms involved in disease pathogenesis

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Drs. Evans and Fleiszig are involved in a pending US patent application that describes the use of Collectins (eg, SP-D) in the management of bacterial ocular infections. Drs. McNamara and Fleiszig have been awarded a patent that describes using the up-regulation of human beta-defensins for the management of bacterial infections. Dr. Fleiszig is a paid consultant for Allergan, Inc. Single copy reprint requests to Suzanne M.J. Fleiszig, OD, PhD (address below). Corresponding author: Suzanne M. J. Fleiszig, School of Optometry, University of California, Berkeley, CA 94720-2020. (510) 643-0990. E-mail: fl[email protected]

©2007 Ethis Communications, Inc. The Ocular Surface ISSN: 1542-0124. Evans DJ, McNamara NA, Fleiszig SMJ. Life at the front: dissecting bacterial-host interactions at the ocular surface. 2007;5(3):213-227.

Figure 1. Pseudomonas aeruginosa keratitis.

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OUTLINE I. Introduction II. P. aeruginosa virulence mechanisms directed against corneal epithelial cells A. The ExsA-regulated Type III secretion system of P. aeruginosa B. P. aeruginosa invasion of epithelial cells C. Other virulence factors, and regulation of P. aeruginosa virulence III. The ocular surface fights back A. Tear fluid biochemical protection B. Mechanical protection by blinking and tear flow C. Corneal epithelial cell surface factors D. The closed-eye environment IV. Bacteria hijacking, exploiting, or avoiding host defenses V. Development of therapeutic and/or preventive methods VI. Conclusion

would bring us closer to that goal. Bacterial keratitis develops when bacterial strategies that cause disease (virulence mechanisms) enable bacteria to evade ocular surface defenses against infection (innate immunity) or when those defenses are otherwise compromised. Understanding why this occurs requires an understanding of how bacteria interact with ocular surface cells in the context of the in vivo environment at the front of the eye. Bacteria and eukaryotic cells have a long history of co-evolution, with the eukaryotic cell, ie, a cell containing a nucleus, evolving after bacteria. Eukaryotic cells often depend upon bacteria for their normal function, but they recognize that bacteria are also potentially dangerous. Thus, epithelial cells, which line most of the tissue surfaces, have developed their own local immune system to allow them to distinguish pathogenic microbes and protect themselves directly and appropriately. Such local defense is beneficial, as fully activated immune responses involving immune cells can cause collateral damage. However, bacteria have responded to the evolution of these systems. Considering the rapid regeneration rate of bacteria compared to eukaryotes, which has often favored the evolutionary development of bacteria, it is not surprising that many bacterial virulence strategies function by manipulating molecules that are critical for eukaryotic cell function. Many species of bacteria are able to respond to the inner workings of the eukaryotic cell. Because it is becoming widely recognized that bacteria sometimes “know” more about eukaryotic cells than people do, bacteria are now commonly used as research tools by eukaryotic cell biologists.9 We are just beginning to understand how the defense system of human corneal epithelial cells functions, eg, the roles of receptors that recognize bacterial components, secreted antimicrobial peptides, surfactant proteins, mucins, and numerous pro-inflammatory cytokines released 214

in response to bacterial antigens.10-17 Similarly, we are just starting to understand bacterial countermeasures. Because bacteria can sometimes be useful to eukaryotes (and vice versa), constitutive expression and secretion of defense molecules and virulence factors can be counterproductive. Thus, both bacteria and eukaryotic cells tend to regulate factors involved in their interactions with each other. In eukaryotes, this includes the expression of receptors for specific microbial factors that allow selective upregulation of appropriate innate defenses. Toll-like receptors (TLRs), which are expressed on or within numerous eukaryotic cell types, form part of that system. TLRs detect conserved bacterial antigens or pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS) or lipoteichoic acid (LTA).18-27 In their fight for survival (to utilize eukaryotic cells for food and shelter or to defend themselves against being phagocytosed or otherwise killed), bacteria have developed multiple mechanisms to regulate the expression of virulence factors to counter the effects of these initial eukaryotic defenses.25,28-30 Thus developed the back-andforth nature of bacterial-host interactions and their regulation throughout evolution, making these relationships both complex and intriguing. While cell biologists and immunologists are utilizing pathogens for their research, investigators studying microbial pathogenesis are focusing more on the host cell systems they infect. This is leading us to think in new ways about problems in medicine and biology. Bacteria are even being considered for their potential as drug delivery vehicles and for the manipulation of eukaryotic cell function to treat conditions that are not related to infectious disease. For the most part, bacteria and eukaryotic cells have adapted to survive in numerous documented commensal and symbiotic relationships. A notable example is that of commensal microbial (mostly bacterial) flora that colonize human skin and mucosal surfaces (gastrointestinal and reproductive tracts, nasopharynx), and which are thought to protect us against infection and disease, synthesize vitamins, and contribute to overall tissue health. In those instances, colonizing bacteria and host responses could be thought of as being in a state of balanced coexistence. Disturbances of this balance can result in human disease, such as occurs with immunocompromise through infection (eg, HIV), through local or systemic drug treatment (immunosuppressive agents or cancer chemotherapy), or through genetic defects that directly or indirectly affect host defense function (eg, cystic fibrosis).31-36 The risk of infectious keratitis during corneal disease or after surgery or injury is likely attributable to obvious disturbance to the normal structure of the tissue. Other types of immunosuppression may be more subtle and difficult to identify, which appears to be the case for bacterial keratitis in the setting of contact lens wear. Despite significant research effort spanning three decades, no single obvious factor has been shown to be responsible for the increased susceptibility to infection that occurs during contact lens wear. One possibility is that the risk is due to subtle changes

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BACTERIAL-HOST INTERACTIONS AT THE OCULAR SURFACE / Evans, et al Association & invasion in interaction between bacBacterial exotoxins with intracellular survival: terial pathogens and the & lytic enzymes, Involves host CFTR, lipid rafts, tyrosine P. aeruginosa [e.g. proteases] ocular surface, as a result kinases, Ca2+, MEK-ERK, cytoskeleton. Bacterial LPS, flagellin, pilin. of changes to one or more [Invasive strains]. components involved in TTSS-ExsA regulated cytotoxicity & other anti-host effects: ExoU, ExoS, that interaction or its reguExoT, ExoY, & Needle Apparatus. lation, occasionally causing Requires host cell contact. [ExoU only in cytotoxic strains]. failure of the defense system of what is a relatively TEAR FILM immune-privileged tissue. While much effort has CORNEAL Cell death Cell death been devoted to underCell death EPITHELIUM standing the immune responses to infection in Cell death the disease state, less effort has been directed toward studying the defenses involved in maintaining health. It is, however, alTranslocated bacteria; most certain that different Intracellular or paracellular pathways factors are involved and Figure 2. Schematic diagram of P. aeruginosa virulence mechanisms at the ocular surface. Both epithelial that the virulence factors cell invasion by P. aeruginosa (invasive isolates) or ExsA-regulated cytotoxicity toward corneal epithelial cells (cytotoxic isolates) contribute to P. aeruginosa virulence. Invasion, cytotoxicity, and other virulence used by bacteria to initimechanisms are each likely to enhance bacterial translocation of the epithelial barrier. ate disease are different from those participating various bacterial pathogens often use common strategies in pathology at later stages. The cellular and molecular in interacting with eukaryotic hosts, the understanding of mechanisms involved in the development of pathology and P. aeruginosa pathogenesis and ocular surface defenses that disease resolution have been well reviewed elsewhere.15,37 Therefore, this review will focus on the initial interactions resist it applies to other types of bacteria that infect the eye between bacteria and the ocular surface, ie, interactions that and to infections of other tissues. are likely to determine whether health will be maintained II. P. AERUGINOSA VIRULENCE MECHANISMS or pathology will be initiated. DIRECTED AGAINST CORNEAL EPITHELIAL CELLS The cornea is continuously exposed to microorganisms Epithelial cells are the first cell type that bacteria and other forms of insult from airborne particles/droplets, encounter at the ocular surface. Thus, our research has from fingers, from the surrounding eyelids/skin, or from centered mostly upon the interplay between P. aeruginosa various aqueous media. Although certain features of the and the epithelial cells of the cornea. Surprisingly, we ocular surface favor microbial growth (optimal temperature, have found remarkable diversity among clinical isolates pH, nutrient availability, osmolarity, etc.), a combination of P. aeruginosa in this regard. Most significantly, we have of epithelial and tear-fluid-derived factors at the ocular found that some strains invade corneal epithelial cells surface make the ocular surface a difficult location for (invasive strains), whereas other strains quickly kill these bacteria or other microbial pathogens to survive and grow. cells (cytotoxic strains).40-42 It has since been shown by us Nevertheless, some bacteria (true or opportunistic pathoand others that these virulence strategies can also target gens) have established virulence strategies to counter the other mammalian cell types43,44 and that each contributes protective factors. to the ability of P. aeruginosa to initiate disease by assisting The focus of our research has been P. aeruginosa, one bacteria in colonizing tissue surfaces, penetrating epithelial of the leading causes of contact-lens-related keratitis1,38,39 and the innate defenses of the ocular surface against this cell layers and other tissue barriers, avoiding epithelial and pathogen. In this review, we will first explore the virulence immune cell-associated antimicrobials, and destroying or strategies of P. aeruginosa at the corneal epithelium (Section otherwise avoiding the effects of infiltrating phagocytes, II), then the role of the corneal epithelium and tear film in such as polymorphonuclear (PMN) neutrophils and defense against P. aeruginosa (Section III). This is followed macrophages.45-48 Our comparisons of corneal isolates in terms of their interactions with corneal epithelial cells and by discussion of how and why the cornea sometimes subsequent studies, in which we explored the mechanisms becomes susceptible to infection, ie, what changes to the for differences in phenotype, have led to the discovery ocular surface or bacteria must occur to tip the outcome of various previously unknown virulence strategies used from health to disease? The answer to that challenging by P. aeruginosa.41 These are summarized in Figure 2 and question would certainly accelerate efforts toward preventdiscussed further below. ing infection of the eye and possibly other sites. Because

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BACTERIAL-HOST INTERACTIONS AT THE OCULAR SURFACE / Evans, et al A. The ExsA-regulated Type III Secretion System of P. aeruginosa

The most notable difference between cytotoxic and invasive strains is that only cytotoxic strains encode a powerful cytotoxin, ExoU, which causes acute cell death of corneal and other epithelial cells within a few hours of bacterial contact.41,43,49 The cytotoxic effects of ExoU are attributed to its patatin-like phospholipase activity.50,51 Isolates that encode ExoU can even kill epithelial cells in an ExoU-dependent manner when they are on the surface of intact corneas ex vivo.52 Invasive isolates do not encode ExoU, which explains why they are not cytotoxic.43,49 Although ExoU-bearing isolates (cytotoxic strains) are more prevalent in ocular infections than in infections at other sites,53 ExoU secreting strains have been found in other types of P. aeruginosa infection,54 and its expression tends to be associated with more severe disease and poor clinical prognosis.55 Our in vivo studies have shown that ExoU is a key mediator of P. aeruginosa colonization and virulence during corneal infection with a cytotoxic strain46 and that this is associated with less phagocyte infiltration of the center of the cornea.48 The expression of ExoU is controlled by the transcriptional activator ExsA,56,57 and it is a component of a Type III secretion system (TTSS). TTSSs provide a mechanism for bacteria to deliver effector proteins (toxins) directly into the cytoplasm of eukaryotic cells upon contact, and they are now known to be common among Gram-negative bacterial pathogens.28,58,59 Knowledge that P. aeruginosa possesses a TTSS system and knowledge of the details of its involvement in pathogenesis have contributed significantly to our understanding of how P. aeruginosa causes disease in the cornea, as well as at other sites where this bacterium causes infection associated with significant morbidity and mortality, eg, respiratory tract, urinary tract, burn wounds, and blood.31,36,54,55,60,61 ExoU is one of four known effector proteins that can be injected into host cells by the P. aeruginosa ExsA-regulated TTSS; the others are ExoT, ExoS, and ExoY. ExoT and ExoS have dual enzymatic activities, N-terminal GTPase activating protein (GAP) activity toward RhoGTPases and C-terminal ADP-ribosyltransferase (ADPRT) activity, each of which interferes with signal transduction and cytoskeleton function in mammalian cells.62 ExoY is an adenylyl cyclase,63 which can also disrupt the actin cytoskeleton.64 ExoT and ExoS can each inhibit P. aeruginosa invasion of various mammalian cell types, including corneal epithelial cells, through mechanisms involving their GAP activity and actin cytoskeleton disruption.65-67 Although ExoS is normally encoded only by invasive strains, it can substitute for ExoT to inhibit P. aeruginosa invasion of a cytotoxic strain.65 ExoT can also inhibit wound healing via its ADPRT activity.68 ExoS has even greater ADPRT activity than ExoT,69 and this activity causes multiple cytotoxic effects (including apoptotic cell death) in various mammalian cell types by targeting (ADP-ribosylation of) the Ras family of signal transduction proteins and other cytosolic 216

targets.70-74 Although much less is known about ExoY, we have found that it also possesses some invasion inhibitory activity for corneal epithelial cells.64 Interestingly, even in the absence of known effectors, the TTSS can be cytotoxic to mammalian cells. This is thought to involve the needle apparatus used for injecting host cells and oncotic cell death following membrane pore formation.75,76 Together, these components of the TTSS play various roles in P. aeruginosa virulence in animal models of corneal infection46,48 and of acute pneumonia.77-80 Roles include a contribution to more efficient penetration of healing corneal epithelia.81 Cytotoxic strains encode ExoT and, therefore, do not invade epithelial cells efficiently.82 As expected, loss of the ExsA-regulated TTSS (or mutation of the gene encoding ExoT) in a cytotoxic strain enables its internalization by epithelial cells.83 Yet, most invasive P. aeruginosa strains (which efficiently invade mammalian cells) encode ExoT. They also encode ExoS and ExoY,43,54 which also have antiphagocytic activities in the background of a cytotoxic strain.64,65,82 This paradox is partially explained by our findings that 1) an association exists between the expression of elastase and integrity of TTSS effectors; 2) elastase activity is strongly correlated with invasion ability for P. aeruginosa; and 3) the TTSS effector ExoS is quickly degraded by elastase in vitro.84 Thus, elastase expression either directly or indirectly enables invasion, despite the fact that these strains encode TTSS effectors capable of inhibiting invasion. Elastase production is itself regulated by several factors responsive to environmental circumstances, including the density of the bacterial population (quorum sensing). In the context of bacterial virulence in vivo, this is one of many elaborate and efficient mechanisms (others are discussed later in this review) that can regulate the expression of anti-invasive effectors when needed, such as for avoiding phagocytosis by professional phagocytes. (Professional phagocytes are polymorphonuclear leukocytes and the monocyte-macrophage lineage cells, whose major function is phagocytosis.) In other circumstances, the ability to become intracellular can be an advantage, eg, when bacteria need to traverse cell layers or avoid extracellular antimicrobial factors. The multiple biochemical activities of TTSS effectors and their varied biological effects on different types of eukaryotic cells, combined with a complex system of regulation,58,85 make their in vivo roles complex, dependent upon the circumstances of the model, and often difficult to predict (as is often the case for bacterial virulence factors). For example, ExoT, which is encoded by almost all cytotoxic and invasive isolates,43,54 appears to be able to substitute for ExoU in conferring corneal virulence (bacterial colonization and disease severity), but only for cytotoxic strains.46,48 While the lack of a role for ExoT for invasive strains is predicted by in vitro experiments using cultured cells, it is surprising that there is redundancy between ExoT and ExoU for cytotoxic strains, considering that they have no common known enzymatic activities.50,62 The mechanism of ExoT contributions to P. aeruginosa corneal virulence for

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cytototoxic strains is not yet well known. Unlike ExoU, ExoT does not seem to repress phagocyte infiltration.48 The role of ExoT in corneal virulence might involve an inhibition of phagocytosis by professional phagocytes or the inhibition of wound healing, since both have been found to occur in vitro.65,68 TTSS effectors, and the secretion system itself, have been found to contribute to P. aeruginosa virulence in other experimental models of infection, eg, acute pneumonia and systemic spread, albeit to varying degrees and sometimes with a surprising lack of correlation between in vitro biochemical and biological effects and in vivo virulence.78-80,86 The “functional redundancy” between ExoU and ExoT that is seen during corneal infection is not mirrored in the lung infection models, where ExoT functions only to promote bacterial dissemination to other sites.65,79 B. P. aeruginosa Invasion of Epithelial Cells

Although P. aeruginosa was long thought to be an extracellular pathogen, our research has shown that it can indeed invade corneal and other epithelial cell types in vivo and in vitro.40,43-45,87,88 Our subsequent discovery that the TTSS modulates invasion likely explains why the invasive capacity of P. aeruginosa had not previously been recognized. Indeed, much of the research with this pathogen has been done using strain 19660, which is a cytotoxic strain. Our data show that all P. aeruginosa strains have the capacity to invade. Invasive strains invade consistently, while cytotoxic strains invade only if TTSS activity is downregulated. P. aeruginosa invasion of epithelial cells involves the outer-core region of LPS,89 but other surface antigens, including flagellin90 and pilin,91 can also mediate this process. The role of LPS in invasion includes interaction with the cystic fibrosis transmembrane-conductance regulator (CFTR) on host epithelial cells.92 CFTR is a multifunctional ATP-binding cassette (ABC)protein that is genetically defective in patients with cystic fibrosis (CF), resulting in abnormal fluid and electrolyte secretion at airway surface epithelium.93 CF patients are highly susceptible to P. aeruginosa infection of the airways. It is thought that defective mucociliary clearance contributes to chronic P. aeruginosa colonization. However, CFTR defects have also been linked to the pathogenesis of P. aeruginosa airway infections through defects in epithelial invasion and loss of CFTR function as a pattern recognition receptor for P. aeruginosa antigens, resulting in defective epithelial innate defense.88,94,95 Subsequent studies have shown that lipid (ceramide)rich membrane rafts that contain CFTR are involved in the internalization of P. aeruginosa, and are needed for an effective epithelial-derived innate defense response.96,97 Recent studies have shown that internalization of P. aeruginosa by rabbit and human corneal epithelial cells also involves lipidrafts98 and that contact lenses enhance both raft formation and bacterial internalization,99 although in the latter study, the role of CFTR was less clear. P. aeruginosa adherence and invasion of surface epithelial cells of the cornea is

likely to be a defense mechanism if those cells are exfoliated (sloughed) from the ocular surface.40 Conversely, the entrapment of adherent and invaded bacteria on or within epithelial cells at the ocular surface through reduced surface exfoliation (eg, during contact lens wear)100 could switch an ocular defense into a pathogenic mechanism. Other epithelial surface molecules have also been implicated in P. aeruginosa invasion, including asialoGM1,101 which is known to bind both flagellin and pilin antigens, albeit primarily in the activation of innate defense responses via the transcription factor NF-KB.102-104 Interestingly, asialo-GM1 and other asialoglycolipids are also concentrated within lipid-rich membrane rafts, in which their association with TLRs enhances activation of epithelial innate defenses in response to P. aeruginosa and other bacterial antigens.105 Invasion of epithelial cells by P. aeruginosa involves an endocytic process and requires the actin cytoskeleton.40 Associated signal transduction events involved include Src-family tyrosine kinase activity, intracellular calciumcalmodulin, and MEK-ERK.44,83,106,107 More recent studies have shown that phosphoinositol (PI)-3 kinase and protein kinase B/Akt are also involved in P. aeruginosa internalization.108 Invasion (and cytotoxicity) of P. aeruginosa are enhanced by exposing epithelial basolateral cell surfaces (tight junction disruption) or through other mechanisms of altering cell polarity.87,109 There is also evidence of an additional pathway(s) for P. aeruginosa invasion of epithelial cells that are independent of cytoskeleton inhibition, but dependent upon epithelial cell polarity.110 Considerably more work is needed to understand the relationships between expression of epithelial surface receptors for invasion and cellular signaling events that mediate the process. Moreover, little is known of the intracellular fate of internalized bacteria, other than the observation that wild-type strains can replicate extensively within the cell, but without intact LPS core, invaded bacteria show reduced intracellular viability.111 C. Other Virulence Factors and Regulation of P. aeruginosa Virulence

Other virulence factors and mechanisms also contribute to pathogenesis of P. aeruginosa corneal infections apart from the TTSS and cell invasion. For example, studies have shown important roles for P. aeruginosa proteases (eg, elastase, alkaline protease, protease IV) in corneal disease through both direct action on tissues and indirectly via activation of host proteases.112-118 Exotoxin A, one of the earliest characterized P. aeruginosa virulence factors, plays a role in corneal disease persistence.119 Pilus-mediated twitching (surface-associated) motility also contributes to P. aeruginosa virulence in the cornea.91 The expression of P. aeruginosa virulence mechanisms is regulated on multiple levels.58,86,120-122 Two-component (sensor-regulator) systems are integral to this process by allowing bacteria to sense environmental signals and alter gene expression to allow adaptation to changing environ-

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ments. The P. aeruginosa genome contains about 64 twocomponent sensor-regulator systems,123,124 many of which have not been characterized. One of the best-characterized systems, GacS-GacA, is a global regulator of P. aeruginosa virulence. GacS-GacA influences expression of the quorumsensing systems (eg, LasR-LasI), which control expression of proteases, rhamnolipids, pyocyanin, and numerous other virulence genes.125-127 We more recently described another gene, retS (regulator of exopolysaccharide and type III secretion), which forms a hybrid two-component regulatory protein involved in both positively and negatively regulating ~400 P. aeruginosa genes, including the ExsA-regulated TTSS,85,128,129 pilin biosynthesis, type II secretion, and the negative regulation of exopolysaccharide biosynthesis.85 These systems can each influence corneal epithelial virulence mechanisms of P. aeruginosa in vitro,128 as well as virulence in corneal48,130 and other animal models of infection.85,131 These regulatory systems also positively and negatively influence each other to “drive” gene expression toward establishing acute or chronic infection phenotypes.58,85,121,132 For example, we tested the corneal virulence of P. aeruginosa mutated in the retS gene. In vitro, retS mutants show loss of the ExsA-regulated TTSS, and a reduction of other virulence mechanisms, including twitching motility, adherence and invasion of corneal epithelial cells, and intracellular survival after invasion, as compared to wild-type bacteria and exsA mutants.128 In vivo, however, although retS mutants showed reduced early virulence and colonization at 24 h and 48 h post-infection (similar to an exsA mutant),48,128 they showed enhanced corneal virulence and colonization at later stages of disease (> 48 h up to 7 days), even when compared to wild-type bacteria.48 This very concise review provides some insight into how complex the virulence strategies of a single bacterial species can be, even when considering its impact upon a single cell type. Many other Gram-negative bacterial pathogens, eg, Yersinia spp., Salmonella spp., and Shigella spp., express multiple virulence gene regulons encoding factors involved in internalization, cytotoxicity, or other virulence strategies toward mammalian cells, depending on circumstances.59,133-135 Much of what we have learned about P. aeruginosa at the ocular surface is likely to apply to infections with other pathogens, but there are also likely to be differences. While it is important to mull over the multifactorial and cooperative nature of virulence mechanisms, it is also important to ponder the influence of the ocular surface environment, which includes not only epithelial cells, but also tear fluid, immune cells, eyelids and ambient environmental conditions. Perhaps, even the substratum on which the epithelial cells reside and other tissues around the eye should be considered. Of specific interest in this regard is that P. aeruginosa (and other pathogens) possess virulence factors capable of killing and invading corneal epithelial cells grown in a tissue culture dish — the same as any other type of epithelial cell. However, when on the 218

ocular surface in vivo, the same cells become exquisitely resistant to infection with P. aeruginosa and almost all other types of pathogenic microbes. The mechanism(s) by which the ocular surface achieves this resistant state in the virtual absence of factors utilized by other tissues for defense purposes is a mystery that needs to be solved. III. THE OCULAR SURFACE FIGHTS BACK A. Tear Fluid Biochemical Protection

The human tear film contains a number of proteinaceous and nonproteinaceous components, several of which are known to act as antimicrobial agents. The presence of antimicrobial agents in the tear film was first reported in 1922 by Fleming and Allison, who described the bactericidal effects of lysozyme on Gram-positive bacteria.136 Other tear factors with bactericidal activity include secretory phospholipase A2 (sPLA2),137 beta-lysin,138 defensin antimicrobial peptides,139,140 and complement proteins. Other tear factors inhibit bacterial growth (eg, tear lipocalin and lactoferrin141,142), or prevent bacterial adherence to the ocular surface, eg, secretory IgA140,143,144 or secreted mucins.145 This latter group is believed to decrease bacterial colonization of the cornea by binding pathogenic organisms and facilitating their clearance through normal tear exchange and blinking. For example, secreted mucins mix with the aqueous portion of the tear film and have been shown to bind P. aeruginosa and inhibit its adherence to the cornea.145 Similar effects have been shown for secretory IgA.144 Although several components of human tears are believed to provide strong bactericidal activity, the major effector for Gram-positive bacteria was recently identified as sPLA2. However, sPLA2 exhibits little efficacy against Gramnegative bacteria when tested in the ionic environment of tears.137 Indeed, at least 50% of P. aeruginosa corneal isolates can grow readily in human tear fluid that has been removed from the eye.146 This is interesting, given that the ocular surface is often exposed to Gram-negative organisms, and yet they are rarely found at the ocular surface. To understand how tear fluid might protect the eye, we explored the effect of tears on P. aeruginosa interactions with corneal epithelial cells. We found that diluted tear fluid collected from human volunteers protected corneal epithelial cells in culture against P. aeruginosa cytotoxicity and invasion (cytotoxic or invasive strains, respectively).146 Tear cytoprotective activity was not dependent upon the bacteriostatic activity of tears. Heat-stable components of the tear fluid appear to play a significant role, since boiling human tears did not interfere with their ability to protect against several different strains. Thus, defensins and PLA2 (both heat-stable) could be involved. In a separate study, we found that protection of corneal epithelial cells against P. aeruginosa invasion was mediated by multiple fractions of human tears separated by high-performance liquid chromatography (HPLC), with fractions containing sIgA and lysozyme having the greatest activity.147 We have since shown that surfactant protein-D (SP-D) is present in tears and that it is involved in tear fluid protec-

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Y

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Bacterial flagellin tion against invasive strains Bacterial LPS Mechanical action of blink 13 of P. aeruginosa. This mulP. aeruginosa tifunctional protein has been shown to have broadspectrum bacteriostatic and bactericidal activity, to bind TEAR FILM and aggregate pathogens, prevent adherence, to opsonize for phagocytosis, and to modulate excesSignal Signal Corneal transduction transduction sive inflammatory and imEpithelium [Multilayered: mune responses.148,149 The Innate defense only surface Innate defense roles of SP-D, and other gene transcription: gene transcription: cells shown] mucins, cytokines, mucins, cytokines, tear components that have defensins, SP-D, LL-37. defensins, SP-D etc. both antimicrobial and immunomodulatory activCORNEAL ity, eg, galectins,150 offer STROMA SP-D exciting new avenues of Y Antibody (eg, IgG, IgA) + complement investigation. Toll-Like Receptor TLR (eg, TLR2, TLR4, TLR5, TLR9) Extracellular & Intracellular Studies of secreted anOther receptors binding bacterial ligands, eg, asialoGM1, CFTR (both in lipid rafts), NOD (intracellular) timicrobial components have shown that they can Figure 3. Schematic diagram of host defenses against P. aeruginosa at the ocular surface. A multitude be dependent upon one of tear and corneal epithelial-derived factors are thought to play a role, including antimicrobial defensins, surfactant protein-D, complement, antibodies, cytokines, and both secreted and membrane-associated another for their activimucins. Upregulation of these defenses involves recognition of bacterial antigens, eg, LPS and flagellin, ties. For example, enteric by sentinel receptors of the corneal epithelium, eg, TLRs. bacteria are not susceptible to being killed by either without any biochemical actions of tear components. It has lactoferrin or secretory IgA alone, but are susceptible to not yet been established how much tear flow/tear exchange being killed by their combination.151 IgG and complement may act in concert, as in other parts of the body, to cause is required to compromise this important defense, but this bacterial lysis or to aid in phagocytosis.152 Finally, a delicate is important in the context of contact lens wear, which rebalance between tear film proteases and anti-proteases, and duces tear exchange at the ocular surface.158 Some of our unpublished work investigating the effect of shear stress on other tear components with opposing effects, is necessary P. aeruginosa cytotoxicity toward corneal epithelial cells in to maintain homeostasis of the ocular surface.153 It is likely that the list of components known to particivitro has indicated that significant reductions in bacterialpate in tear protection will continue to grow. New sophistiinduced epithelial cell death can be achieved by increasing cation in the area of proteomics has revealed the presence of fluid movement over the cell surface. Interestingly, with 90 proteins in meibomian gland secretions alone,154 and at cultured cells, no single shear stress value can eliminate least 60 tear proteins have been identified in pooled human bacterial-induced cytotoxicity before the appearance of tear samples with use of a combination of reverse-phase mechanically-induced damage by the fluid itself. The HPLC and analysis by nanoLC-nano-ESI-MS/MS.155 A simidata do suggest, however, that a correct combination of lar approach, using LC-MALDI MS, successfully identified increased tear flow under a contact lens with sufficient, but a total of 54 proteins in human tears using less than 5 μl of not excessive, lens movement could reduce the impact of sample.156 Membrane arrays provide a means to characterthis virulence mechanism. ize expression of specific proteins and have been used to It is possible that physical forces could play other roles identify multiple chemokines, cytokines, and growth factors in defense, such as regulating homeostasis at the ocular in tears.157 Given cooperative relationships between tear surface. In vitro and in vivo studies have shown that contact film factors, subtractive methods may be necessary to study lenses can affect corneal epithelial cell proliferation and the relative roles of specific tear components in modulation desquamation, the expression of surface receptors, and of bacterial pathogenesis. susceptibility to P. aeruginosa virulence mechanisms, such as adherence and internalization.98-100,159,160 While some of B. Mechanical Protection by Blinking and Tear Flow these effects have been related to oxygen transmissibility of Physical/mechanical effects induced by blinking and the lens, it is also probable that lens mechanostimulatory tear flow are likely to play major roles in defense of both or stagnation effects are involved, since infections are still the open and the closed eye. Intuitively, we know that prevalent despite introduction of silicone hydrogel lenses, mechanical flow of tear fluid facilitated by the blink of which allow physiological levels of oxygen to reach the the eyelid serves to wash away potential pathogens, even ocular surface.3

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Corneal epithelial cells also play a role in defense at the ocular surface. We have found that polarized corneal epithelial cells (cells with intact tight junctions and normal cell sorting) are significantly less susceptible to P. aeruginosa infection than cells that have lost their polarity by disruption of tight junctions or of normal cell sorting.87,109 Proteins expressed on the basolateral surface, such as E-cadherin, can act as bacterial receptors,161 whereas proteins and glycoproteins unique to the apical surface appear to provide a physical barrier to pathogens.162 Polarized expression of apical and basolateral membrane proteins occurs both in vivo and in vitro when mucosal epithelial cells are grown at an air-liquid interface.163-166 Membrane-bound mucins on the surface of cells are thought to provide a selective physical barrier between the epithelium and the tear film by resisting bacterial binding.17,147,167 A combination of previous immunocytochemical and bacterial binding studies show that highly glycosylated mucins of the most acidic species (sialylated and sulfated) fail to bind P. aeruginosa.168 This is supported by studies showing that partial removal of carbohydrate residues present in human tears and/or the selective removal of sialic acid residues increased P. aeruginosa binding to tear fluid factors. Binding is also increased when negative charges present in the tear film are neutralized, using the positively charged molecule, protamine sulfate. Thus, negatively charged (acidic) mucin species may resist binding of negatively charged bacteria by electronegative forces.147,169 Given that cell surface phospholipids can function as receptors for pathogenic bacteria,170 it is perhaps not surprising that membranebound mucins would function to resist bacterial binding as a defensive strategy to prevent initial colonization. This also suggests that disease processes leading to loss of membrane-bound ocular surface mucins (eg, injury, dry eye) could favor colonization, particularly when tear clearance is impaired during eye closure and contact lens wear. The corneal epithelium possesses several other defense mechanisms. For example, factors that are directly antimicrobial are expressed and secreted by corneal epithelial cells. They include the defensin and cathelicidin (LL-37) cationic antimicrobial peptides.10-12,171,172 Defensins exhibit direct bactericidal activity against bacteria,173,174 and some are constitutively expressed (eg, human B-defensins 1 and 3).11,175 Several years ago, we found that human B-defensin 2 can be upregulated in response to bacteria or purified antigens,12 and others have since confirmed this for other bacteria, eg, Staphylococcus aureus.176 LL-37 also shows broad-spectrum antibacterial activity (including P. aeruginosa) and antiviral activity, while also influencing epithelial cytokine expression and cell migration.172,177 SP-D is another tear factor also expressed by corneal epithelial cells that has multiple defense-related attributes.13 Under some circumstances, effective defense of the ocular surface may require recruitment of other cellular components of immunity.15,37 Consequently, epithelial cells have the capacity to secrete pro-inflammatory and 220

anti-inflammatory cytokines in response to the presence of various microbial antigens, including components of P. aeruginosa.14,22,178-182 In addition, dendritic cells within the corneal epithelium are also likely to serve as an interface between innate and adaptive immunity.183,184 The release of antimicrobial peptides, cytokines, and other innate defense molecules by mucosal epithelial cells within minutes of exposure to pathogens is mediated by interaction of PAMPS with receptors such as the TLRs.185 Antigens, such as LPS, LTA, or flagellin, bind to TLRs (eg, TLR2, TLR4, TLR5) and/or associated surface molecules (eg, asialo-GM1) on epithelial cells to activate intracellular signaling pathways, NFKB and other transcription factors, and the expression of innate defense genes.20,22,104,176,186 Binding of intracellular antigens by TLRs (eg, DNA by TLR9) serves a similar function.187 Signaling via TLRs has been conserved as a defensive strategy of mucosal surfaces against microbial pathogens and plays a complex role in ocular surface defense.19,20,188 Further details of this role have been recently reviewed.189-191 Based upon their role in other tissues, other receptors are also likely to play a role in expression of corneal innate defenses, including the CFTR and NOD2.97,192 Together, these epithelial warning systems function as a first line of defense against bacterial attack. It is likely that further studies will reveal other defense systems and their activation mechanisms and elucidate the processes involved in cooperation between systems and their regulation. D. The Closed-Eye Environment

Profound changes to tear fluid biochemistry occur during sleep. There is a shift from an inducible, lacrimal secretion containing lysozyme, lactoferrin, and tear-specific lipocalin, to a constitutive secretion composed largely of sIgA.153 There is also an accumulation of heavily sialylated glycoprotein, activated complement, serum proteins and the recruitment and activation of massive numbers of PMN neutrophils. Thus, the closed-eye tears are rich in innate defense molecules, reactive complement products, and PMN-derived proteases, which are postulated to provide an active immune and phagocyte-mediated response that protects the cornea from potential pathogens trapped in the stagnant closed-eye tear film. However, one might postulate that active phagocytosis of bacteria by PMNs in the closed-eye state would result in the release of lysosomal enzymes into the tear fluid, with subsequent damage to ocular surface cells. This autologous cell damage appears to be avoided in closed-eye tear fluid by the accumulation of several modulators of complement activation, which shift activation toward opsonization of entrapped microorganisms and the build-up of a wide array of protease inhibitors (see review153). It is interesting to note that PMN recruitment during overnight eye closure is inhibited by the presence of a contact lens.193,194 This suggests that the lens may prevent or delay PMN recruitment to the cornea by either limiting their access or by modifying their chemotactic signal. Further studies of the closed-eye environment during

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sleep will allow us to more fully understand the ramifications of its shift to a more immunogenic state157 and may ultimately provide answers to the long-standing concerns regarding infectious keratitis that accompanies extended wear of contact lenses. It is clear that in the ongoing battle between the host immune system and microbial pathogens, both epithelial cells and tear fluid take a very active role in fighting back (Figure 3). The full contribution of each of the various defenses in protecting the cornea against infection is still not fully understood. It is quite likely that their roles will turn out to be synergistic. It is also probable that there are defense factors with entirely different mechanisms of action yet to be discovered, since our current knowledge falls short of fully explaining why the healthy cornea resists disease caused by almost any pathogen, while retaining its normal homeostasis.

SP-D.197-199 P. aeruginosa can also directly interfere with cytokine signaling through direct proteolytic digestion, eg, of IL-6 and IL-8,200 or inhibition of pro-inflammatory cytokine expression by activating regulatory enzymes, eg, TNF-alpha converting enzyme (TACE).201 It is intriguing that some receptors and signaling events involved in host innate defenses, including asialo-GM1, CFTR, MEK-ERK, NFKB, etc, are also required for bacterial invasion of epithelial cells, a recognized pathogenic strategy. Conversely, bacterial factors known to be major contributors to virulence (such as LPS and flagellin) are often the same bacterial factors that are recognized by host cells to activate their defense systems. These may represent further examples of P. aeruginosa taking advantage of defense strategies for the purpose of virulence and vice versa.

IV. BACTERIA HIJACKING, EXPLOITING, OR AVOIDING HOST DEFENSES

Topical antibiotic therapies have a vital place in the effective treatment of keratitis involving P. aeruginosa and other bacterial pathogens.5,38,202 However, the often quoted “prevention is better than cure” (Desiderius Erasmus) may be especially applicable to P. aeruginosa keratitis, a disease that, once initiated, can quickly cause irreversible damage to the cornea and vision loss, in spite of appropriate therapeutic intervention.1,39 This clinical observation is supported in experimental models of keratitis, in which P. aeruginosa infected corneas show a delayed response to conventional therapies once the disease is set in motion.203 In addition, the continued threat of emerging bacterial resistance to currently useful antibiotics also supports preventive approaches.8,202 Improved oxygen transmissibility of contact lenses has reduced, but not eradicated, the incidence of bacterial keratitis.3 While ocular surface biochemistry is critical in maintaining the health of the cornea and other ocular surface tissues, contact lenses cover the cornea and stagnate tear exchange. Improving tear exchange under a contact lens might be one approach to reducing the incidence of contact lens-related infection. Changes to lens surfaces that prevent adhesion and entrapment of bacterial pathogens might also be useful in this respect. Another approach would be to replace/restore/or protect essential endogenous defense factors to prevent infection from occurring in the first place in susceptible populations. In this respect, SPD, defensins, and other endogenous molecules that have both immunomodulatory and antimicrobial properties are worthy of consideration for therapeutic potential. In the meantime, there is a need for novel therapeutics that limit corneal damage from overactive inflammatory and immune responses and/or enhance activities of innate defenses.

Bacterial pathogens are notorious for finding ways to avoid detection and clearance by host defenses or to exploit them. In essence, these mechanisms define them as pathogens in the first place. P. aeruginosa possesses several “anti-host” strategies. The cytotoxic and anti-phagocytic activities of the ExsA-regulated TSSS and its known effectors are good examples. In addition to epithelial cells, macrophages and PMNs are also susceptible to these toxins or to the TTSS injection apparatus itself.65,76 These actions are likely to prevent bacterial clearance by professional phagocytes, as they attempt to infiltrate tissues that the bacteria have colonized. Indeed, we have found that in the cornea, ExoU can repress phagocyte infiltration during infection.48 The mechanism for this effect is likely to include direct killing of phagocytes, or inhibition of chemotaxis through toxin effects on phagocytes, epithelial cells, and their cytokine signals. Other effectors of the TTSS might also have effects on phagocytes, as they can inhibit phagocytosis by macrophages in vitro.65,67 Other bacteria can use different strategies to avoid being killed by PMNs or macrophages, some of which involve type III secretion. They include survival and replication inside a phagocyte by escaping the phagosome before it merges with the lysosome, preventing phagosome-lysosome fusion, or resisting toxic compounds released into the phagolysosome after fusion. Specific examples of the latter include 1) acquired resistance to killing by defensins, 2) production of enzymes or cell surface polysaccharides that detoxify reactive oxygen species, 3) cell surface proteins that reduce the strength of oxidative burst, and 4) cell walls that are refractory to destruction by lysosomal proteases and lysozyme.135,195 Bacterial TTSSs can also interfere with TLR-mediated defense responses, including the suppression of TLR-signaling.3 Other examples of P. aeruginosa interference with host defense include the enzymatic (protease) degradation of defense molecules, including immunoglobulins196 and

V. DEVELOPMENT OF THERAPEUTIC AND/OR PREVENTIVE METHODS

VI. CONCLUSION This review provides insights into some of the complexities involved in studying host-pathogen interactions. Even in an in vitro situation where a single bacterium interacts with a single host cell, there are two completely different

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types of living organisms to consider, each capable of modifying its gene expression in response to its environment. Thus, the conditions of an experiment, including ingredients of the tissue culture medium, can markedly influence the outcome. While it is generally important to be reductionist to determine the involvement of specific molecules in a biological process, it is also critical to consider how that involvement is modified in the presence of molecules or other cells that are present during disease and over time. Pathogens generally express more than one molecule recognized by host cells, and the host response to one can be confounded by its response to another. Moreover, live pathogens often possess the capacity to modify or compromise host cell function in ways that cannot necessarily be predicted from experiments in which cells are challenged with a single bacterial ligand. Pathogen effects on cells are varied and can range from subtle modification of cytokine responses to toxin-induced killing. If pathogens are phagocytosed by a host cell, they can act intracellularly to affect function as they strive to modify their own trafficking or to otherwise avoid being killed. Pathogens, as living organisms, regulate the expression of their virulence factors and their pattern recognition molecules in response to the prevailing environmental conditions. Cross-talk between pathogens and host cells is common, and this can sometimes involve multiple sequential back-and-forth responses. A classic example is the way in which Salmonella or Francisella change the structure of their LPS in response to host contact, which then modifies host responses to them.134,204 Prevailing conditions often vary at different locations within an infected host, and over time, depending on the path taken by the invading pathogens (in particular, intracellular versus extracellular localization). To further confound this, both bacterial and host cell responses can actually cause alterations to the environment that influence the pathogen or host. During infectious disease in vivo, the state of host cells and infecting bacteria is generally not as well synchronized as can be achieved in in vitro experiments. Considering all of this complexity, it is not at all surprising that in vivo experiments do not always show what in vitro experiments predict, and that host cell responses to bacterial ligands are modified when living bacteria are utilized. For example, TLR-4 mutation has opposite effects if disease is induced with live P. aeruginosa bacteria (more severe disease) as compared to disease induced with LPS derived from the same bacterium (less severe disease).188 Although studies using single bacterial ligands are, of course, important if we are to eventually understand host cell responses to them, research using living bacteria is also required, just as in vitro cell culture experiments need to be validated in vivo in a mammalian host. A multipronged and multidisciplinary approach to studying infectious disease pathogenesis, and the mechanisms used by the ocular surface to resist it, is the most likely to lead us toward making sense out of the complexity of bacterial keratitis and solving this important problem. 222

ACKNOWLEDGEMENTS The authors thank the National Eye Institute (EY11221 to SMJF, EY016203 to NAM), the American Optometric Foundation and the Cystic Fibrosis Foundation for research grant support. In addition, the authors thank several companies that have provided generous and sometimes unrestricted support to support our research into the pathogenesis of infectious keratitis. They include Alcon, Allergan, Bausch and Lomb, and CibaVision.

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