Pseudomonas aeruginosa utilizes the type III secreted toxin ExoS to avoid acidified compartments within epithelial cells.

Invasive Pseudomonas aeruginosa (PA) can enter epithelial cells wherein they mediate formation of plasma membrane bleb-niches for intracellular compartmentalization. This phenotype, and capacity for intracellular replication, requires the ADP-ribosyltransferase (ADPr) activity of ExoS, a PA type III secretion system (T3SS) effector protein. Thus, PA T3SS mutants lack these capacities and instead traffic to perinuclear vacuoles. Here, we tested the hypothesis that the T3SS, via the ADPr activity of ExoS, allows PA to evade acidic vacuoles that otherwise suppress its intracellular viability. The acidification state of bacteria-occupied vacuoles within infected corneal epithelial cells was studied using LysoTracker to visualize acidic, lysosomal vacuoles. Steady state analysis showed that within cells wild-type PAO1 localized to both membrane bleb-niches and vacuoles, while both exsA (transcriptional activator) and popB (effector translocation) T3SS mutants were only found in vacuoles. The acidification state of occupied vacuoles suggested a relationship with ExoS expression, i.e. vacuoles occupied by the exsA mutant (unable to express ExoS) were more often acidified than either popB mutant or wild-type PAO1 occupied vacuoles (p < 0.001). An exoS-gfp reporter construct pJNE05 confirmed that high exoS transcriptional output coincided with low occupation of acidified vacuoles, and vice versa, for both popB mutants and wild-type bacteria. Complementation of a triple effector null mutant of PAO1 with exoS (pUCPexoS) reduced the number of acidified bacteria-occupied vacuoles per cell; pUCPexoSE381D which lacks ADPr activity did not. The H(+)-ATPase inhibitor bafilomycin rescued intracellular replication to wild-type levels for exsA mutants, showing its viability is suppressed by vacuolar acidification. Taken together, the data show that the mechanism by which ExoS ADPr activity allows intracellular replication by PA involves suppression of vacuolar acidification. They also show that variability in ExoS expression by wild-type PA inside cells can differentially influence the fate of individual intracellular bacteria, even within the same cell.

Surfactant protein D contributes to ocular defense against Pseudomonas aeruginosa in a murine model of dry eye disease.

Dry eye disease can cause ocular surface inflammation that disrupts the corneal epithelial barrier. While dry eye patients are known to have an increased risk of corneal infection, it is not known whether there is a direct causal relationship between these two conditions. Here, we tested the hypothesis that experimentally-induced dry eye (EDE) increases susceptibility to corneal infection using a mouse model. In doing so, we also examined the role of surfactant protein D (SP-D), which we have previously shown is involved in corneal defense against infection. Scopolamine injections and fan-driven air were used to cause EDE in C57BL/6 or Black Swiss mice (wild-type and SP-D gene-knockout). Controls received PBS injections and were housed normally. After 5 or 10 days, otherwise uninjured corneas were inoculated with 10(9) cfu of Pseudomonas aeruginosa strain PAO1. Anesthesia was maintained for 3 h post-inoculation. Viable bacteria were quantified in ocular surface washes and corneal homogenates 6 h post-inoculation. SP-D was measured by Western immunoblot, and corneal pathology assessed from 6 h to 4 days. EDE mice showed reduced tear volumes after 5 and 10 days (each by ∼75%, p<0.001) and showed fluorescein staining (i.e. epithelial disruption). Surprisingly, there was no significant difference in corneal pathology between EDE mice and controls (∼10-14% incidence). Before bacterial inoculation, EDE mice showed elevated SP-D in ocular washes. After inoculation, fewer bacteria were recovered from ocular washes of EDE mice (<2% of controls, p = 0.0004). Furthermore, SP-D knockout mice showed a significant increase in P. aeruginosa corneal colonization under EDE conditions. Taken together, these data suggest that SP-D contributes to corneal defense against P. aeruginosa colonization and infection in EDE despite the loss of barrier function to fluorescein.

Spontaneous bacterial keratitis in CD36 knockout mice.

PURPOSE: CD36 is a Class B scavenger receptor that is constitutively expressed in the corneal epithelium and has been implicated in many homeostatic functions, including the homeostasis of the epidermal barrier. The aim of this study is to determine (1) whether CD36 is required for the maintenance of the corneal epithelial barrier to infection, and (2) whether CD36-deficient mice present with an increased susceptibility to bacterial keratitis. METHODS: The corneas of CD36(-/-), TSP1(-/-), TLR2(-/-), and C57BL/6 WT mice were screened via slit lamp microscopy or ex vivo analysis. The epithelial tight junctions and mucin layer were assessed via LC-biotin and Rose Bengal staining, respectively. Bacterial quantification was performed on corneal buttons and GFP-expressing Staphylococcus aureus was used to study bacterial binding. RESULTS: CD36(-/-) mice develop spontaneous corneal defects that increased in frequency and severity with age. The mild corneal defects were characterized by a disruption in epithelial tight junctions and the mucin layer, an infiltrate of macrophages, and increased bacterial binding. Bacterial quantification revealed high levels of Staphylococcus xylosus in the corneas of CD36(-/-) mice with severe defects, but not in wild-type controls. CONCLUSIONS: CD36(-/-) mice develop spontaneous bacterial keratitis independent of TLR2 and TSP1. The authors conclude that CD36 is a critical component of the corneal epithelial barrier, and in the absence of CD36 the barrier breaks down, allowing bacteria to bind to the corneal epithelium and resulting in spontaneous keratitis. This is the first report of spontaneous bacterial keratitis in mice.

Role of defensins in corneal epithelial barrier function against Pseudomonas aeruginosa traversal.

Studies have shown that epithelium-expressed antimicrobial peptides (AMPs), e.g., ß-defensins, play a role in clearing bacteria from mouse corneas already infected with Pseudomonas aeruginosa. Less is known about the role of AMPs in allowing the cornea to resist infection when healthy. We previously reported that contact lens exposure, a major cause of P. aeruginosa keratitis, can inhibit the upregulation of human ß-defensin 2 (hBD-2) by corneal epithelial cells in response to P. aeruginosa antigens in vitro. Here, we studied the role of AMPs in maintaining the corneal epithelial barrier to P. aeruginosa penetration using both in vitro (human) and in vivo (mouse) experiments. Results showed that preexposing human corneal epithelial multilayers to bacterial antigens in a culture supernatant (known to upregulate AMP expression) reduced epithelial susceptibility to P. aeruginosa traversal up to 6-fold (P < 0.001). Accordingly, small interfering RNA (siRNA) knockdown of any one of four AMPs expressed by human epithelia promoted P. aeruginosa traversal by more than 3-fold (P < 0.001). The combination knockdown of AMPs further enhanced susceptibility to bacterial traversal by ∼8-fold (P < 0.001). In vivo experiments showed that the loss of murine ß-defensin 3 (mBD-3), a murine ortholog of hBD-2, enhanced corneal susceptibility to P. aeruginosa. The uninjured ocular surface of mBD-3(-/-) mice showed a reduced capacity to clear P. aeruginosa, and their corneal epithelia were more susceptible to bacterial colonization, even when inoculated ex vivo to exclude tear fluid effects. Together, these in vitro and in vivo data show functional roles for AMPs in normal corneal epithelial cell barrier function against P. aeruginosa.

Response of corneal epithelial cells to Staphylococcus aureus.

Staphylococcus aureus is a leading cause of invasive infection. It also infects wet mucosal tissues including the cornea and conjunctiva. Conflicting evidence exists on the expression of Toll-like receptors by human corneal epithelial cells. It was therefore of interest to determine how epithelial cells from this immune privileged tissue respond to S. aureus. Further, it was of interest to determine whether cytolytic toxins, with the potential to cause ion flux or potentially permit effector molecule movement across the target cell membrane, alter the response. Microarrays were used to globally assess the response of human corneal epithelial cells to S. aureus. A large increase in abundance of transcripts encoding the antimicrobial dendritic cell chemokine, CCL20, was observed. CCL20 release into the medium was detected, and this response was found to be largely TLR2 and NOD2 independent. Corneal epithelial cells also respond to S. aureus by increasing the intracellular abundance of mRNA for inflammatory mediators, transcription factors, and genes related to MAP kinase pathways, in ways similar to other cell types. The corneal epithelial cell response was surprisingly unaffected by toxin exposure. Toxin exposure did, however, induce a stress response. Although model toxigenic and non-toxigenic strains of S. aureus were employed in the present study, the results obtained were strikingly similar to those reported for stimulation of vaginal epithelial cells by clinical toxic shock toxin expressing isolates, demonstrating that the initial epithelial cellular responses to S. aureus are largely independent of strain as well as epithelial cell tissue source.

Cell surface O-glycans limit Staphylococcus aureus adherence to corneal epithelial cells.

The mucin-rich environment of the intact corneal epithelium is thought to contribute to the prevention of Staphylococcus aureus infection. This study examined whether O-glycans, which constitute the majority of the mucin mass of epithelial cell glycocalyces, prevented bacterial adhesion and growth. Abrogation of mucin O glycosylation using the chemical primer benzyl-alpha-GalNAc resulted in increased adherence of parental strain RN6390 to apical human corneal-limbal epithelial (HCLE) cells and to biotinylated cell surface protein in static and liquid phase adhesion assays, consistent with a role of mucin O-glycans in preventing bacterial adhesion. Comparable results were found with ALC135, an isogenic mutant strain defective in the accessory gene regulators agr and sar, indicating that the agr- and/or sar-regulated virulence factors did not play a major role in mediating adhesion to the corneal cell surface after mucin O-glycan truncation. In exoglycosidase digestion studies, treatment with sialidase from Arthrobacter ureafaciens--which hydrolyzed mucin-associated O-acetyl sialic acid--but not from Clostridium perfringens resulted in an increase in RN6390 and ALC135 adhesion. Abrogation of mucin O glycosylation in HCLE cell cultures did not affect bacterial growth. Overall, these data indicate that mucin O-glycans contribute to the prevention of bacterial adherence to the apical surface of corneal epithelial cells and suggest that alteration of cell surface glycosylation from disease or trauma, including that stemming from contact lens wear, could contribute to a higher risk of infection.

alphaB-crystallin protects retinal tissue during Staphylococcus aureus-induced endophthalmitis.

Bacterial infections of the eye highlight a dilemma that is central to all immune-privileged sites. On the one hand, immune privilege limits inflammation to prevent bystander destruction of normal tissue and loss of vision. On the other hand, bacterial infections require a robust inflammatory response for rapid clearance of the pathogen. We demonstrate that the retina handles this dilemma, in part, by activation of a protective heat shock protein. During Staphylococcus aureus-induced endophthalmitis, the small heat shock protein alphaB-crystallin is upregulated in the retina and prevents apoptosis during immune clearance of the bacteria. In the absence of alphaB-crystallin, mice display increased retinal apoptosis and retinal damage. We found that S. aureus produces a protease capable of cleaving alphaB-crystallin to a form that coincides with increased retinal apoptosis and tissue destruction. We conclude that alphaB-crystallin is important in protecting sensitive retinal tissue during destructive inflammation that occurs during bacterial endophthalmitis.

Functions of MUC16 in corneal epithelial cells.

PURPOSE: The membrane-associated mucin MUC16, a heavily O-glycosylated transmembrane protein, is expressed by the ocular surface epithelia and localized on the tips of the surface microplicae. Although its functions in the ocular surface glycocalyx are unknown, it is thought that MUC16 provides a disadhesive barrier to the epithelial membrane. Two other membrane-associated mucins expressed by ocular surface epithelia, MUC1 and MUC4, are multifunctional and have signaling capabilities through their cytoplasmic tails and EGF-like domains, respectively. The MUC16 cytoplasmic tail has not been characterized, but, because it contains a polybasic amino acid sequence, it potentially interacts with the actin cytoskeleton through ezrin/radixin/moesin (ERM) actin-binding proteins. METHODS: The interaction of MUC16 with the actin cytoskeleton through ERMs was investigated using cytoplasmic tail peptides and ERM pull-down experiments. MUC16 functions were determined using RNA interference in immortalized human corneal-limbal epithelial (HCLE) cells. The effect of MUC16 knockdown on microplicae structure in HCLE cells was determined using scanning and immunoelectron microscopy. HCLE cells were incubated with rose bengal dye to measure the role of MUC16 in ocular surface barrier function. Binding of fluorescently labeled Staphylococcus aureus to HCLE cells was measured to determine the role of MUC16 in the protection of pathogen adherence on the ocular surface epithelium. RESULTS: MUC16 cytoplasmic tail peptides bound the N-terminus of ERMs, with no detectable binding of MUC1 and MUC4 peptides. No effect on surface membrane projections could be detected in HCLE cells after MUC16 suppression; however, HCLE cells incubated with rose bengal showed that exclusion of the dye was significantly reduced in cells with MUC16 suppression. In addition, S. aureus binding to HCLE cells was significantly increased with MUC16 suppression. CONCLUSIONS: These results suggest that MUC16 is a multifunctional molecule linked to the actin cytoskeleton. The expression of MUC16 in the ocular surface glycocalyx helps provide a disadhesive protective barrier for the epithelial surface.

Autotransporter genes pic and tsh are associated with Escherichia coli strains that cause acute pyelonephritis and are expressed during urinary tract infection.

We have identified two chromosomal open reading frames in uropathogenic Escherichia coli (UPEC) strain CFT073 which are highly homologous to serine protease autotransporters Pic and Tsh. Both cloned determinants were correlated with the presence of 105- to 110-kDa proteins in the culture supernatants. Furthermore, in cellular fractionation experiments, 30-kDa polypeptides were identified in the outer membrane; we speculated that these proteins are the beta-barrel portions of the autotransporter homologues. Furthermore, Pic-containing culture supernatants have serine protease activity. In reverse transcription-PCR analyses, the expression of the pic and tsh genes in E. coli CFT073 was higher in broth cultures grown at 37 degrees C than at 25 degrees C. Moreover, pic and tsh were expressed by bacteria isolated from urine of transurethrally infected mice. The tsh determinant was identified in 63% of our clinical UPEC strain isolates (n = 87) and in 33% of fecal strains (n = 27), whereas pic was present in 31% of the pyelonephritis (n = 67) and 7% of the fecal strains. There was no significant correlation between cystitis strains (n = 20) and the pic determinant.

Urease of enterohemorrhagic Escherichia coli: evidence for regulation by fur and a trans-acting factor.

Recent genomic analyses of Escherichia coli O157:H7 strain EDL933 revealed two loci encoding urease gene homologues (ureDABCEFG), which are absent in nonpathogenic E. coli strain K-12. This report demonstrates that the cloned EDL933 ure gene cluster is capable of synthesizing urease in an E. coli DH5alpha background. However, when the gene fragment is transformed back into the native EDL933 background, the enzymatic activity of the cloned determinants is undetectable. We speculate that an unidentified trans-acting factor in enterohemorrhagic E. coli (EHEC) is responsible for this regulation of ure expression. In addition, Fur-like recognition sites are present in three independent O157:H7 isolates upstream of ureD and ureA. Enzymatic assays confirmed a difference in urease expression of cloned EHEC ure clusters in E. coli MC3100Deltafur. Likewise, interruption of fur in O157:H7 isolate IN1 significantly diminished urease activity. We propose that, similar to the function of Fur in regulating the acid response of Salmonella enterica serovar Typhimurium, it modulates urease expression in EHEC, perhaps contributing to the acid tolerance of the organism.