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.

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.

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 ␤-barrel portions of the autotransporter homologues. Furthermore, Piccontaining 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°C than at 25°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.Uropathogenic Escherichia coli (UPEC) encodes a variety of genetic determinants that are associated with virulence. Guyer et al. have previously identified a novel secreted autotransporter toxin (Sat) in culture supernatants of strain CFT073 that is responsible for vacuolating cultured bladder and kidney epithelial cells (10, 11). Sat shares a high degree of homology with an expanding class of serine protease autotransporters in E. coli (SPATEs). In this study, we describe two additional SPATE proteins in CFT073 that are homologous to Pic and Tsh.The Pic protein was originally identified in culture supernatants of enteroaggregative E. coli (EAEC) (12). Its genetic determinant overlaps with shetAB, which encodes the Shet1 enterotoxin (2, 12). This toxin is responsible for fluid accumulation in rabbit ileal loops (7). Like other SPATE proteins, Pic has been shown to have serine protease activity capable of acting on mucin (12).Tsh was first reported as a temperature-sensitive hemagglutinating factor in avian pathogenic E. coli (APEC) (18). Its identical homologue, Hbp, was described as a heme-binding protein with a serine protease activity targeted at hemoglobin (16). Researchers have found that Hbp expression enhances abscess formation in mice during mixed infections with Hbpexpressing E. coli and Bacteroides fragilis (15).We hypothesized that the CFT073 genes homologous to pic and tsh encode SPATE proteins which are secreted and proteolytically active. Moreover, we speculated that these determinants are more prevalent among UPEC isolates than fecal strains.Homology of CFT073 pic and tsh determinants. We identified the pic and tsh determinants while annotating the genomic sequence of E. coli CFT073 (gbAE014075) (22). The Pic homologue is well conserved (96% amino acid identity) among UPEC strain CFT073, EAEC strain 042, and Shigella flexneri 2a. This homology is evenly di...

Studies have shown that epithelium-expressed antimicrobial peptides (AMPs), e.g., ␤-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 ␤-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 ϳ8-fold (P < 0.001). In vivo experiments showed that the loss of murine ␤-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.

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 ␣B-crystallin is upregulated in the retina and prevents apoptosis during immune clearance of the bacteria. In the absence of ␣B-crystallin, mice display increased retinal apoptosis and retinal damage. We found that S. aureus produces a protease capable of cleaving ␣B-crystallin to a form that coincides with increased retinal apoptosis and tissue destruction. We conclude that ␣B-crystallin is important in protecting sensitive retinal tissue during destructive inflammation that occurs during bacterial endophthalmitis.

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 DH5␣ 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 MC3100⌬fur. 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.

Proteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and carbon dioxide. The apourease is comprised of three structural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individual UreABC heterotrimers (UreABC) 3 . To become catalytically active, apourease acquires divalent nickel ions through a poorly understood process involving four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains unclear specifically how these polypeptides associate with the apourease or each other. To identify interactions among P. mirabilis accessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed. A complex containing accessory protein UreD and structural protein UreC was isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and UreG and structural protein UreA. In a yeast two-hybrid screen, UreD was found to directly interact in vivo with coaccessory protein UreF. Unique homomultimeric interactions of UreD and UreF were also detected in vivo. To substantiate the study of urease proteins with a yeast two-hybrid assay, previously described UreE dimers and homomultimeric UreA interactions among apourease trimers were confirmed in vivo. Similarly, a known structural interaction involving UreA and UreC was also verified. This report suggests that in vivo, P. mirabilis UreD may be important for recruitment of UreF to the apourease and that crucial homomultimeric associations occur among these accessory proteins.

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-␣-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.