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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Immunol. Author manuscript; available in PMC Feb 15, 2013.
Published in final edited form as:
PMCID: PMC3273577

ExoS and ExoT ADP-ribosyltransferase activities mediate Pseudomonas aeruginosa keratitis by promoting neutrophil apoptosis and bacterial survival


P. aeruginosa is a leading cause of blinding corneal ulcers worldwide. To determine the role of type III secretion in the pathogenesis of P. aeruginosa keratitis, corneas of C57BL/6 mice were infected with P. aeruginosa strain PAO1 or PAK, which express ExoS, ExoT and ExoY, but not ExoU. PAO1 and PAK infected corneas developed severe disease with pronounced opacification and rapid bacterial growth. In contrast, corneas infected with ΔpscD or ΔpscJ mutants that cannot assemble a Type III secretion system, or with mutants lacking the translocator proteins, do not develop clinical disease, and are rapidly killed by infiltrating neutrophils. Further, survival of PAO1 and PAK strains in the cornea and development of corneal disease was impaired in ΔexoS, ΔexoT and ΔexoST mutants of both strains, but not in a ΔexoY mutant. ΔexoST mutants were also rapidly killed in neutrophils in vitro and were impaired in their ability to promote neutrophil apoptosis in vivo compared with PAO1. Point mutations in the ADP ribosyltransferase (ADPR) regions of ExoS or ExoT also impaired pro-apoptotic activity in infected neutrophils, and exoST(ADPR-) mutants replicated the ΔexoST phenotype in vitro and in vivo, whereas mutations in rho-GAP showed the same phenotype as PAO1. Together, these findings demonstrate that the pathogenesis of P. aeruginosa keratitis in ExoS and ExoT producing strains is almost entirely due to their ADPR activities, which subvert the host response by targeting the anti-bacterial activity of infiltrating neutrophils.

Keywords: Type III secretion, Pseudomonas, neutrophil, keratitis

Corneal infection with P. aeruginosa is a major cause of visual impairment and blindness worldwide, occurring either as a result of trauma or in association with contact lens wear (1-6). Results of our studies and others using murine model of P. aeruginosa keratitis show that initial activation of the host response in the cornea is dependent on TLR4 and TLR5 expression on resident stromal macrophages, which signal through TIRAP/MyD88 and TRIF to produce IL-1α, IL-1β and the neutrophil chemokines CXCL1 and CXCL2 in the corneal stroma (7-10). IL-1α and IL-1β exacerbate this response by activating IL-1R1/MyD88 responses, leading to neutrophil infiltration to the corneal stroma, bacterial killing and tissue damage, which is manifest as corneal opacification (9). More recently, we demonstrated that production of IFN-γ during P. aeruginosa keratitis stimulates expression of the TLR4 co-receptor, MD-2 on corneal epithelial cells, conferring LPS responsiveness and thereby contributing to the inflammatory response in the cornea (11). Overall, these studies showed a role for TLRs and innate immunity in the pathogenesis of P. aeruginosa keratitis, which is similar, though not identical to the role for neutrophils and TLRs in P. aeruginosa induced lung disease (12-15).

Like many Gram-negative pathogens, P. aeruginosa uses a type III secretion system to deliver effector proteins into targeted host cells. P. aeruginosa type III effector proteins are thought to prevent wound healing and phagocytosis, as well as promote systemic spread of the organism (16). The Type III Secretion System of Gram-negative bacteria is a complex, needle-like organelle designed to inject bacterial toxins and other proteins directly into host cells (reviewed in (16, 17)). It is comprised of a basal body, which spans the bacterial cell envelope, and is connected to a needle that protrudes from the surface of the bacterium. Injection of effector proteins into targeted host-cells is contact dependent and requires the action of the two translocator-proteins, PopB and PopD. These translocator proteins form a pore in the host-cell membrane through which the effector proteins are injected (16, 17).

Four effector proteins (exoenzymes) have been described in P. aeruginosa: ExoS, ExoT, ExoU and ExoY. ExoU has potent phospholipase activity and causes rapid lysis of host cells (18) and ExoY is an adenylate cyclase (16). ExoS and ExoT are closely related and have GTPase activating protein (GAP) and ADP ribosyltransferase (ADPRT) activities (19), The Rho-GAP domains of these two proteins which target a similar subset of GTPases, including Rho, Rac1 and CDC42 (20, 21). Together Rho-GAP and ADPRT activities alter host cell cytoskeletal function, resulting in impaired cell migration and adhesion, in addition to blocking phagocytosis, disrupting epithelial cell barriers and preventing wound healing (16, 19, 22).

Almost every strain of P. aeruginosa contains exoT and about 86% of strains harbor the gene for ExoY; however, for reasons that are not yet clear, most P. aeruginosa isolates express either ExoS or ExoU, but not both effectors (23). ExoS- and ExoU-expressing isolates are recovered from infected corneas at similar frequencies (24). In corneal infections, type III secretion appears to be important for ExoU-producing, cytotoxic variants of P. aeruginosa (25), whereas ExoS-producing strains did not depend on type III secretion to cause disease (26-28). This observation was curious in that lack of ExoT, which is produced by both ExoU and ExoS producing strains of P. aeruginosa, only had a virulence phenotype in the ExoU producing, but not the ExoS producing strain (28). These data also contrast with observations made in the lung model, where type III secretion is an important virulence factor for both ExoS- and ExoU-producing strains of P. aeruginosa (29, 30). Neutrophils are the predominant infiltrating cell-type in lung infections, and it has been proposed that killing of infiltrating neutrophils creates an immunocompromised milieu in the lung in which P. aeruginosa can thrive (31). We and others reported that infection with either ExoU or ExoS expressing strains cause keratitis in murine models, and that bacterial clearance and the severity of infection is dependent on the innate immune response, including neutrophil infiltration to the corneal stroma and bacterial survival (7-9, 11, 26). The obvious importance of neutrophils in clearing P. aeruginosa infections, both in the lung and in the eye, and the identification of neutrophils as the primary target of type III secretion in the lung model of infection, prompted us to revisit the role of type III secretion in eye infections, particularly for ExoS-producing strains of P. aeruginosa.

In the current study, we use strains PAO1 and PAK, which express ExoS, ExoT and ExoY, but not ExoU (32), and demonstrate that type III secretion is required for corneal disease elicited by these two strains of P. aeruginosa. Moreover, we identified the ADP ribosyltransferase activities of ExoS and ExoT as the essential mediators promoting neutrophil apoptosis and bacterial survival in neutrophils in vitro and in vivo.


Generation of Type III secretion mutants

All strains and plasmids used in this study are listed in Table 1. Chromosomal mutants were all derived from the same parental PAO1 strain or PAK strain, as indicated in Table 1, and were generated by allelic exchange. PAO1 strains vary widely in their expression of virulence genes. This difference has, in part, been linked to nfxC mutants that overexpress mexEF-OprN. (33) The strain used in this study is chloramphenicol sensitive, and therefore likely does not overexpress the mexEF-OprN efflux pump. Notably, many PAO1 strains express the type III secretion system poorly (34); however the strain used here expresses the type III secretion genes well (35), All primers used for plasmid construction are listed in Table 2. Flanks specifying the appropriate mutation were amplified using chromosomal DNA as template (unless specified otherwise), joined by splicing by overlap extension (SOE) PCR and cloned into the appropriate plasmid (either the allelic exchange vector pEXG2 or the shuttle vector pPSV35) using the indicated restriction enzymes. Primers specifying the exoT R149K and E383D/E385D mutations were derived from a previous study by the Barbieri lab (36). We reported that the ΔexoST, ΔexoTY, ΔexoSY and Δ3TOX mutant strains are secretion competent and that they export the expected effector proteins (also, all four strains still export PopN) (35). The singe deletion mutants are precursors to these double and triple null mutants.

Table 1
Strains and plasmids
Table 2

Mouse strains

C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME), TLR4−/− and MyD88−/− mice were obtained with permission from Dr. S. Akira, (Research Institute for Microbial Diseases, Osaka University, Japan), and TLR5−/− mice were obtained from Dr Richard Flavell from the Howard Hughes Medical Institute, Yale University, New Haven, CT. TLR4/5−/− mice were generated at CWRU animal facility, and Mafia mice were generated Dr Sandra Burnett, University of Utah, Provo, UT, and are now available from the Jackson Laboratory. All animals were housed under specific pathogen-free conditions in microisolator cages and maintained according to institutional guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

In vivo model of corneal infection

Mice were anesthetized by intraperitoneal injection of 0.4 ml 2,2,2-tribromoethanol (1.2%). Central corneas were scarified with three parallel 1 mm in length abrasions using a 26 gauge needle. A 2.5 μL aliquot containing approximately 1 × 105 PAO1 were applied to the scarified cornea as described in our previous study (9). Sterile PBS was applied to the abraded cornea as a trauma control. A sterile trephine (Miltex, Tuttlingen, Germany), was used to generate a 2 mm diameter punch of silicone hydrogel contact lens (Night and Day™, CIBA Vision, Duluth, GA) which was placed over the central cornea to maintain placement of the bacterial suspension. The contact lenses were removed after 2 h and mice were allowed to recover from the anesthesia.

Quantification of corneal opacification

Image analysis was used to generate an objective measure of corneal opacification based on our studies on fungal keratitis (37). Mouse corneas were illuminated using a gooseneck fiber optic light source, and constant light levels were maintained during image acquisition. Twenty-four bit color images were captured with a SPOT RTKE camera (Diagnostic Instruments, Sterling Hts. MI) connected to a Leica MZF III stereo microscope, and all images were captured using the same exposure time. Image analysis was performed using Metamorph Imaging software (Molecular Devices, Downington PA) and described in detail in Supplemental Figure 1. Briefly, a circular region of constant area was centered on the images of corneas of infected mice, and Metamorph software was used to generate the percent corneal opacity and the total corneal opacity. A threshold value was set from naïve C57BL/6 eyes or eyes with no apparent disease, and corneal opacity was based on values above the threshold, with the more opaque regions displaying a greater integrated pixel intensity value. Areas of glare were demarcated and then set to zero, thus effectively eliminating the iris from the subsequent analysis process.

Mafia mouse model for depletion of macrophages and dendritic cells

Mafia mice express eGFP and a membrane-bound suicide protein comprising the human low-affinity nerve growth factor receptor, the FK506 binding protein (FKBP) and a cytoplasmic domain of Fas (38, 39). AP20187 is a covalently linked dimerizer (Ariad Pharmaceuticals, Cambridge, MA) that crosslinks the FKBP region of the suicide protein and induces caspase 8 – dependent apoptosis as described (38, 40). These mice are on a C57BL/6 background and have a normal phenotype in the absence of the dimerizer.

Bacterial quantification

Whole eyes were homogenized under sterile conditions using the Mixer Mill MM300 (Retsch, Inc., Newtown, PA) at 33 Hz for 4 min. Serial log dilutions were performed and plated onto Brain Heart Infusion Agar (Becton, Dickson and Company, Sparks, MD). Plates were incubated at 37°C for 18 h and the number of CFU was determined by direct counting.

Histology and Immunohistochemistry

Eyes were enucleated and placed in 10% Formalin/PBS for 24 hours, embedded in paraffin, and 5 μm sections were stained by H&E. Neutrophils in the corneal stroma were detected after incubation with rat anti-mouse neutrophil antibody (NIMP-R14; Abcam, Cambridge, MA, 1:100 in 1% FCS-PBS 2 h at room temperature). Sections were washed and incubated with FITC conjugated rabbit anti-rat antibody (Vector Laboratories, Burlingame, CA) diluted 1:200 in 1% FCS-PBS for 45 minutes. Neutrophils in the corneal stroma were enumerated by fluorescence microscopy as described (9).

In vitro neutrophil survival assay

To obtain murine neutrophils, mice were injected with 1 ml 9% casein, 16 h and 3 h prior to peritoneal lavage, and cells were layered onto a sterile 90% Percoll gradient (GE Healthcare, Piscataway, NJ, USA). The neutrophil population was recovered from the second layer on the gradient as determined by cytology and routinely yielded 95-100% pure neutrophils, as described in our previous studies (41). Viability was 95% as determined by Trypan blue exclusion. Neutrophils (5 × 105 cells / ml were incubated in DMEM at 37°C with 5 × 106 bacteria for 30 min, then incubated 30 min with 200 μg/ml Gentamicin (Sigma) to kill extracellular bacteria. Cultures were then washed x2 with PBS, and cells were immediately lysed using 0.1% TritonX-100, and CFU was determined at this time. Remaining cultures were incubated an additional 90 min (total incubation time = 2½ h), washed, lysed and CFU were quantified.

Analysis of total and percent AnnexinV positive corneal neutrophils by flow cytometry

Corneas were excised 24h after infection and residual iris material was removed. Individual corneas were minced using a scalpel, and incubated 1.5h at 37°C in 100 μl collagenase type I (82 units per cornea, Sigma) as described (9). The resulting cell suspensions were filtered, washed with 3 ml fluorescence-activated cell sorter (FACS) buffer (1×PBS w/o Ca2+ or Mg2+, 1% fetal bovine serum, 0.1% sodium azide). Cells were incubated with 20μg Fc blocking antibody (anti-mouse CD16/32; eBioscience) 10 min, on ice and stained with the neutrophil marker NIMPR14 which had been tagged with Alexafluor 488 (eBiosciences) at 0.5μg/106 cells. Cell profiles were acquired on an Accuri C6 flow cytometer (Accuri Cytometers, Inc. Ann Arbor, MI), and were gated based on isotype marker

To determine the percent apoptotic neutrophils, 30,000 corneal cells were incubated with NIMP-R14+ and anti-mouse Annexin V-APC conjugated antibody (eBioscience). Neutrophils were gated and Annexin V expression was analyzed. Histograms showing the percentage annexin positive or negative NIMPR14+ neutrophils were generated using C-Flow software (Accuri).

Statistical Analysis

Student’s t test or ANOVA with Tukey’s multiple comparison test was performed using GraphPad Prism (San Diego, CA). Statistical significance was defined as a P value of < 0.05.


Type III Secretion System is essential for development of P. aeruginosa keratitis

As a first step in dissecting the role of the Type III secretion in the development of P. aeruginosa keratitis, mice were infected with the PAO1 parent strain of P. aeruginosa, the ΔpscD mutant, which does not assemble a Type III secretion apparatus, and the ΔpscD mutant that was complemented with a plasmid expressing PscD (ΔpscD/pscD).

We found that infection with PAO1 caused increasing corneal opacification over time, with severe disease apparent at 72h (Figure 1A-C). In contrast, infection with the ΔpscD mutant induced much less corneal disease. Complementation with a plasmid expressing PscD (ΔpscD/pscD) completely restored the capacity of the ΔpscD mutant to cause clinical disease. To determine the role of Type III secretion on bacterial survival in the cornea, eyes were homogenized and CFU were quantified. Figure 1D shows that at 72h, CFU in PAO1 infected corneas were significantly elevated compared to the inoculum. In contrast, CFU in mice infected with the ΔpscD strain were lower than the inoculum, indicating that these bacteria were being killed. Infection with the complemented ΔpscD/pscD strain restored the wild-type phenotype, thereby demonstrating an essential role for Type III secretion in bacterial survival in the cornea. Repeat experiments showed that the role of Type III secretion on bacterial survival was highly reproducible (Table 3).

Figure 1
The role of Type III secretion in P. aeruginosa keratitis
Table 3
Role of Type III secretion in bacterial survival in corneas of C57BL/6 mice. (CFU is mean colony forming units; pi: post infection)

Histological sections showed a prominent cellular infiltrate in PAO1 infected corneas after 24h (Figure 1E), whereas infection with the ΔpscD strain induced less of an infiltrate. The ΔpscD/pscD strain in which the Type III secretion apparatus was restored showed the wild-type phenotype, and immunostaining of these sections using the neutrophil specific NIMPR-14 antibody revealed a prominent neutrophil infiltrate in the cornea (Figure 1F). Given that neutrophils were recruited to the avascular cornea, we assayed production of the neutrophil chemokines CXCL1/KC and CXCL2/MIP-2 at 3h post infection, which is prior to neutrophil infiltration, or 24h following infection when neutrophils are present. Our previous studies showed that CXCL1 was produced by corneal fibroblasts and macrophages at early time points, whereas CXCL2 was produced by neutrophils and macrophages after 24h (42). Corneas were dissected, homogenized, and chemokine production was measured by ELISA. Figure 1G shows that CXCL1 was elevated at 3h post-infection corneas infected with either PAO1 or ΔpscD mutant, reflecting the early response to the same PAO1 and ΔpscD inoculum. In contrast, CXCL2 production at 24h post infection was significantly lower in the ΔpscD strain compared with PAO1 infected corneas, which is consistent with the lower neutrophil infiltrate and CFU in the absence of Type III secretion.

ΔpscD mutants replicate in MyD88−/− corneas

To determine if the decreased ΔpscD mutant numbers in the cornea are due to an inability to replicate in this tissue or to resist killing by infiltrating phagocytic cells, we infected MyD88−/− corneas. As MyD88−/− mice have impaired neutrophil recruitment to the corneal stroma in response to an ExoU expressing strain (9), we used these mice for infection with the ΔpscD mutant strain, and examined corneal opacification, histopathology and bacterial survival as described above. Figure 2A-C shows significantly less corneal opacity (area and intensity) in PAO1 infected MyD88−/− compared with C57BL/6 mice. In contrast, although the percent area of opacity was significantly less in MyD88−/− than C57BL/6 corneas mice, ΔpscD mutants induced only mild corneal opacification in both mouse strains, with no significant difference in average corneal opacity. Histological analysis of ΔpscD mutant infected MyD88−/− mice shows that in marked contrast to infected C57BL/6 corneas, there was no cellular infiltrate in MyD88−/− corneas (Figure 2D); further, at high magnification, numerous bacteria can be detected in the corneal stroma of MyD88−/− mice (inset). Quantification of bacteria in MyD88−/− and C57BL/6 corneas (Figure 2E) shows significantly higher CFU in MyD88−/− corneas infected with either PAO1 or ΔpscD mutants.

Figure 2
The role of Type III secretion in MyD88−/− mice

These findings demonstrate that Type III secretion is not required for P. aeruginosa survival and growth in the corneal stroma in the absence of infiltrating cells, and is therefore consistent with a role for Type III secretion in inhibiting bacterial killing by infiltrating cells.

PopBD, ExoS and ExoT, but not ExoY are required for development of P. aeruginosa keratitis

The translocation pore can elicit killing of macrophage in vitro even in the absence of effector proteins (30), and can similarly mediate killing of neutrophils in a pore-formation-dependent manner (43), To identify the relative contribution of the individual effector proteins to pathogenesis, as well as the translocation pore itself, we infected corneas of wild-type C57BL/6 mice with mutant strains in which these T3SS components had been inactivated.

To assess the contribution of the translocon, corneas of C57BL/6 mice were abraded and infected with PAO1 or mutant bacteria lacking pscD or the pore-forming translocator proteins (ΔpopBD). Clinical disease and CFU were examined as described above. We found that as with the ΔpscD strain, corneas infected with ΔpopBD mutants had less opacification and lower CFUs than PAO1 (Figure 3A), indicating that the translocation apparatus is essential for development of PAO1 induced keratitis.

Figure 3
The role of popBD, ExoS, ExoT, and ExoY after infection with bacterial strains PAO1 or PAK

To determine the relative contribution of the effector proteins in corneal disease, we infected wild-type mice with a strain lacking the genes for all three effector proteins: exoS exoT and exoY (Δ3TOX) or with a ΔexoY mutant (Figure 3B). Removal of all three effector proteins resulted in a virulence phenotype that was indistinguishable from that of the ΔpscD or ΔpopBD mutant bacteria, suggesting that survival of PAO1 in the cornea is mediated by delivery of effector proteins, rather than translocon-mediated killing of neutrophils. In contrast, we found that deletion of ΔexoY mutants were not significantly different from PAO1 in survival or in inducing corneal opacification, indicating no apparent role for ExoY in P. aeruginosa keratitis.

We further dissected the role of individual effector proteins by examining the virulence phenotype of strains lacking exoS and/or exoT. Figure 3C shows significantly lower CFU when mice were infected with the ΔexoS, ΔexoT or ΔexoST strains compared with PAO1. Corneal opacification was also lower in ΔexoS, ΔexoT and ΔexoST infected mice as shown in representative images below. Further, deletion of exoS or exoT individually resulted in an intermediate defect in colonization relative to wild-type bacteria and a strain lacking both exoS and exoT, suggesting that these two effectors contribute in a non-redundant manner to disease. However, while infection with the strain lacking both exoS and exoT consistently resulted in fewer CFU recovered from the double deletion mutant when compared to the individual ΔexoS and ΔexoT mutant strains, this difference was not statistically significant.

Taken together, these findings indicate that ExoS and ExoT are the primary effectors responsible for the survival of PAO1 in infected corneas, and that ExoS and ExoT have non-redundant activities that promote bacterial survival and growth in the cornea. The observation that ΔexoST double mutant has the same phenotype as the strain lacking all three effectors is further evidence that there is no role for ExoY in corneal disease.

Given the discrepancy between our data and the published lack of type III secretion-related phenotype in strain PAK (28), we also examined the role of type III secretion in PAK-mediated corneal infection. C57BL/6 corneas were abraded and infected with parent stain PAK, the Type III secretion mutant ΔpscJ (equivalent to strain ΔpscD in the PAO1 background), and with ΔexoS, ΔexoT or ΔexoST mutants (Figure 3D). Consistent with our findings in strain PAO1, corneal opacification and CFU were significantly lower in all four mutants compared with PAK, and as with PAO1, the ΔexoST double mutant had lower CFU and opacification scores than single mutants.

Together, these findings demonstrate that strains PAO1 and PAK rely on an intact type III secretion system, and ExoS and ExoT in particular in P. aeruginosa keratitis.

ΔexoST mutants survive and replicate in mice with impaired neutrophil and macrophage infiltration

As ΔexoS and ΔexoT mutants are rapidly killed in C57BL/6 corneas, we next examined if mutants lacking ExoS and ExoT can replicate in the cornea in the absence of infiltrating neutrophils and macrophages, which would indicate a role for these exotoxins in preventing bacterial killing by these cells. To this end, we used three transgenic mouse strains that have impaired neutrophil and macrophage recruitment to the cornea in P. aeruginosa keratitis (9, 39). MyD88−/− and TLR4/5−/− mice were infected with ΔexoST, and CFU was quantified after 48h. We found that in contrast to C57BL/6 mice, CFU of ΔexoST mutants increased in MyD88−/− and TLR4/5−/− corneas compared with (Figure 4).

Figure 4
Survival of ΔexoST mutants in mice with impaired neutrophil and macrophage recruitment to the cornea

As a third approach to examine the ability of ΔexoST to grow in the absence of infiltrating cells, we used transgenic Mafia mice that are on a C57BL/6 background and express fas under control of the c-fms promoter. In these mice, macrophages and dendritic cells expressing fas can also be selectively depleted by injection of the AP20187 dimerizer that induces fas mediated apoptosis (38). Our previous studies using these mice showed that macrophages are the predominant resident cells in the corneal stroma and have an essential role in CXC chemokine production and recruitment of neutrophils (9, 39). Mafia mice were either treated with the dimerizer or left untreated prior to corneal infection with ΔexoST bacteria. After 24h, eyes were homogenized and CFU were assayed as before. Figure 4 shows that as with C57BL/6 mice, ΔexoST were decreased in Mafia mice not given the dimerizer; however, we found a 3-log increase in CFU in infected AP20187-treated Mafia mice, consistent with an essential role for infiltrating cells.

Taken together, these findings indicate that ExoS and ExoT prevent bacterial killing by macrophages and neutrophils that are recruited to the cornea following infection.

ADP ribosyltransferase, but not rho-GAP activities of ExoS and ExoT are essential for P. aeruginosa intracellular survival in neutrophils

ADPR and rho-GAP point mutations in ExoS and ExoT were generated as described in the Methods. Total cell lysates and culture supernatants were processed for Western blot analysis, and polyclonal rabbit serum was used to detect ExoS, ExoT and the cell-associated protein RAN polymerase A (RpoA, fractionation control). Figure 5A shows that whereas ExoS and ExoT proteins were absent in the ΔexoS and ΔexoT null mutants, strains with point mutations had intact ExoS and ExoT proteins. ExoS and ExoT were also secreted into the culture supernatant (Figure 5B).

Figure 5
Survival of ExoS and ExoT ADP ribosyltransferase and rho-GAP mutants in neutrophils and macrophages in vitro

To determine if ExoS and ExoT mediate resistance to neutrophil killing, peritoneal neutrophils and macrophages were incubated with PAO1 or ΔpscD, ΔexoS, ΔexoT, or ΔexoST mutant strains for 30 min to allow for bacterial attachment and phagocytosis. Cells were then incubated for 30 min in the presence of gentamicin to kill extracellular bacteria, the cultures were washed to remove the antibiotic, and incubated another 1.5h when cultures were washed and lysed, and bacterial growth/survival was assessed by CFU analysis.

The number of intracellular bacteria after 30min in gentamicin was similar for all strains (1 × 105 CFU/ml, data not shown). PAO1 survived the additional hour after the initial time point, with 1 × 105 CFU recovered. Figure 5C shows that CFU from neutrophils infected with ΔpscD, ΔexoS, ΔexoT, or ΔexoST strains were significantly lower than PAO1, indicating bacterial killing. In contrast, there was no difference in bacterial survival in macrophages between PAO1 and any of the null mutants (Figure 5D), indicating that there is no apparent role for Type III secretion in P. aeruginosa survival in macrophages.

ExoS and ExoT each have N-terminal rho-GAP and C-terminal ADP ribosyltransferase activities. To determine if rho-GAP or ADPR activities contribute to intracellular survival in neutrophils, P. aeruginosa mutants were generated in which either the rho-GAP activity (G-) or the ADPR activity (A−) were inactivated through point mutations introduced into the chromosomal copy of the corresponding effector gene. Survival of intracellular PAO1 or mutant strains in peritoneal neutrophils was examined. At time = 0 after the initial gentamicin treatment, there was no difference in CFU among the strains (data not shown); however, after 90 min, CFU from neutrophils infected with either the exoS(A−) mutant or the exoS(G/A-) double mutant strains were significantly lower than PAO1 and similar to the ΔexoS strain, whereas CFU from neutrophils infected with the exoS(G-) mutant were not significantly different from PAO1 (Figure 5E), indicating that the ADPR activity of ExoS is essential for P. aeruginosa survival in neutrophils.

Similarly, exoT(A−) and exoT(G/A-) mutants had significantly lower CFU compared with PAO1 and the exoT(G-) mutant, demonstrating that the ADPR activity of ExoT is also essential for survival in neutrophils, whereas the rho-GAP mutant was not significantly different from the PAO1 parent strain (Figure 5F). To determine if there is a synergistic effect between ExoS and ExoT ADPR activities, neutrophils were infected with the exoS(A−) mutant, exoT(A−) mutant, or the exoS(A−)/exoT(A−) double mutants. We found that CFU recovered after infection with the double mutant was not significantly different from the single mutants (Figure 5G), indicating that both ExoS and ExoT ADPR activities are required equally for bacterial survival in neutrophils. Further, while the CFU recovered from neutrophils infected with the ΔexoST double deletion mutant were consistently lower than those recovered from neutrophils infected with the double ADPR-mutant, the difference was not statistically significant, suggesting that while the rho-GAP activities may have a minor role in survival in neutrophils, the bulk of the phenotype relies on the ADPR activities of ExoS and ExoT.

Finally, to determine if the ExoS and ExoT Rho-GAP activities are redundant, neutrophils were infected with exoS(G-), exoT(G-) or exoS(G-) / exoT(G-) double mutants, and CFU was assessed. However, as shown in Figure 5H, there was no significant difference between the double mutants and PAO1, indicating that there is no role for Rho-GAP activity in bacterial survival in neutrophils. Together, these findings demonstrate that survival in neutrophils is dependent on the ADPR activity of ExoS and ExoT.

ADP ribosyltransferase, but not rho-GAP activities of ExoS and ExoT is essential for neutrophil recruitment to the corneal stroma and neutrophil apoptosis

Given that ExoS and ExoT ADP ribosyltransferase activity appears to mediate bacterial survival in neutrophils, and that ExoS and ExoT cause apoptosis in epithelial cells (HeLa cell line) (44, 45), we examined if ExoS and ExoT induce neutrophil apoptosis during corneal infection.

In the first set of experiments, C57BL/6 corneas were infected with PAO1, ΔexoS, ΔexoT, or ΔexoST strains, and after 24h corneas were digested with collagenase, and the total number of neutrophils per cornea and the percent apoptotic neutrophils was assessed by flow cytometry using NIMPR14. As shown in Figure 6A (upper panel), the total number of neutrophils from corneas infected with ΔexoS, ΔexoT, or ΔexoST was significantly lower than corneas infected with PAO1, which is likely due to decreased bacterial survival in neutrophils as shown in Figure 5.

Figure 6
The role of ExoS and ExoT ADPR and rho-GAP activities in neutrophil apoptosis

To identify apoptotic neutrophils in the cornea, infiltrating cells were incubated with NIMPR14 together with an antibody to Annexin V. NIMPR14+ cells were gated and the percent Annexin V positive neutrophils each bacterial strain was examined. We found > 60% annexin positive neutrophils after PAO1 infection, whereas corneas infected with ΔexoS, ΔexoT, or ΔexoST had a significantly lower percentage of Annexin V positive neutrophils (Figure 6A, central panel), which is consistent with a role for ExoS and ExoT in neutrophil apoptosis. Representative flow cytometry scans are shown in the lower panels.

To determine the relative contribution of rho-GAP (G) and ADPR (A) activities of ExoS and ExoT on neutrophil infiltration to the corneal stroma and apoptosis, C57BL/6 corneas were infected with ExoS rho-GAP or ADPR mutants, ExoT rho-GAP or ADPR mutants or ExoST ADPR mutants. As shown in Figure 6B, there was no significant difference in either neutrophil recruitment to the corneal stroma (upper panel), or in the percent annexin positive neutrophils between PAO1 and exoS(G-) mutants (center and lower panels). In marked contrast, the number of infiltrating neutrophils and the number of annexin-positive neutrophils in exoS(A−) infected corneas were significantly less than in PAO1 infected corneas. Further, there was no significant difference in apoptosis among exoS(A−), exoS(G/A-) and ΔexoS mutants, indicating that the ADPR activity accounts for the pro-apoptotic effect of ExoS with no apparent role for rho-GAP activity.

Similarly, the exoT(G-) mutant strain was not significantly different from PAO1 in either neutrophil infiltration or the percent annexin positive neutrophils in the cornea, whereas the exoT(A−) mutant induced significantly less neutrophil recruitment to the cornea and less apoptosis of these neutrophils (Figure 6C). Also, the exoT(G/A-) double mutant had the same phenotype as the exoT(A−) and the ΔexoT strain, demonstrating that, as with ExoS, the ADPR activity accounts for the pro-apoptotic activity of ExoT. To ascertain if there is synergistic activity between the ADPR activities of ExoS and ExoT, corneas were infected with the exoS(A−) mutant, the exoT(A−) mutant, the exoS(A−)/exoT(A−) double mutant or the ΔexoST mutant, and the percent annexin positive cells was examined as before. As shown in Figure 6D, the percent annexin positive cells in the exoS(A−)/exoT(A−) double mutant and the ΔexoST mutant was significantly lower than with either of the single ADPR- mutants, which is consistent with a synergistic role for ADPR activity of ExoS and ExoT in neutrophil apoptosis.

Together, these data reveal an essential role for ADPR but not rho-GAP activity of ExoS and ExoT in promoting neutrophil apoptosis in corneal infections. The increased neutrophil numbers in the presence of ADPR activity is likely due to increased survival of bacteria, which induces further neutrophil recruitment.

ADPR activity of ExoS and ExoT mediates corneal opacification and bacterial survival in the cornea

Given the predominant role for ADPR on neutrophil apoptosis and bacterial survival, we next examined the role of ADPR in P. aeruginosa keratitis. Corneas of C57BL/6 mice were abraded and infected with PAO1 or exoS(A−), exoT(A−), exoS(A−) / exoT(A−) or ΔexoST mutants, and corneal opacification and bacterial recovery were examined after 48h. As shown in Figure 7, each of the ADPR mutants caused less corneal opacification than PAO1. Further, there was significantly less CFU recovered from ADPR mutants than from PAO1 infected corneas. While there appeared to be a reduction in recovered CFU when comparing the ΔexoST and exoST(A−) mutants, this difference was not statistically significant.

Figure 7
The role of ExoS and ExoT ADP ribosyltransferase in corneal disease and bacterial survival

These results demonstrate that ExoS and ExoT ADPR activities are essential for bacterial survival in the cornea and development of corneal disease. As with the neutrophils survival assays, the role of the RhoGAP activities of ExoS and ExoT appear to only play a minor role in survival in the cornea since the difference in survival between the double deletion mutant and the double ADPR-mutant strains was not statistically significant.


The corneal surface is well equipped to prevent bacterial adhesion and infection given that tears contain β-defensins and other anti-bacterial agents, and mucins and surfactants on the external layer of the corneal epithelium inhibit attachment to the corneal epithelial cells (46-48). In addition, corneal epithelial cells form tight junctions, which are an effective barrier to bacterial invasion. Infection of the underlying corneal stroma, which is associated with disease, therefore requires a breach of the epithelial barrier either by trauma (as used in most mouse models) or is associated with contact lens wear and P. aeruginosa biofilm formation (49, 50).

Overall, the results generated in the current study increase our understanding of the pathogenesis of this disease by demonstrating that the type III secretion system in ExoS/T expressing strains subverts the host response in the corneal stroma. Specifically, the two type III secretion-delivered effector proteins ExoS and ExoT, are essential for establishing a productive infection in a mouse model of keratitis. Using mutant mice defective in macrophage and neutrophil functions, we also showed that the type III secretion system, and ExoS and ExoT in particular, are only required for pathogenesis in the presence of a cellular infiltrate, as ΔexoS and ΔexoT mutants were able to replicate in macrophage depleted Mafia mice (which also have impaired neutrophil recruitment), and in MyD88−/− and TLR4/5−/− corneas. Moreover, we found that ExoS and ExoT expressing bacteria cause neutrophil apoptosis, and that ExoS and ExoT are essential for survival in neutrophils in vitro. Together, these observations support the conclusion that the principal role of type III secretion is to subvert the host response by targeting the anti-bacterial activity of infiltrating neutrophils.

The role of ExoS and ExoT in P. aeruginosa keratitis is almost entirely due to the ADPR activities, which appear to have non-redundant roles in bacterial survival in neutrophils, and in induction of neutrophil apoptosis. Both ExoS and ExoT ADPR activities were required for survival in isolated peritoneal neutrophils, which would explain the non-redundant requirement for ExoS and ExoT in our initial infection experiments. In marked contrast to ADPR, inactivation of the Rho-GAP activities of ExoS and ExoT did not significantly affect the ability of P. aeruginosa to survive in neutrophils. However, while not statistically significant, we consistently saw a reduction of recovered CFU in vivo when comparing the ΔexoST double null mutant with either ΔexoS and ΔexoT (Figure 3), or with the strain in which both ADPR activities had been inactivated (Figure 7), suggesting that the Rho-GAP activities of these two enzymes may have a minor function in promoting survival in the cornea.

In corneal and lung epithelial cells, Fleiszig and co-workers showed that P. aeruginosa is sequestered in membrane blebs, formation of which depends on the ADPR activity of ExoS (51, 52). While we focused on the activity of ExoS in promoting survival in neutrophils, this sequestration in non-phagocytic cells could also serve to evade killing by neutrophils and is consistent with the importance of the ADPR activity of ExoS for establishing a productive infection. Our studies also show that corneal epithelial cells can be activated by P. aeruginosa through TLR4/MD-2 to produce CXC1 and CXCL2, which work together with CXCL5 produced by corneal fibroblasts to recruit neutrophils to the corneal stroma (11, 42).

Both ExoS and ExoT have been implicated in induction of apoptosis in vitro (45, 53, 54), although there is some controversy with regard to the role of ExoT in eliciting apoptosis, since it actually prevented cell-death in infected CHO cells (55). Further, differences in cell-type are likely involved also as ExoT-mediated apoptosis was detected in HeLa cells. In the current study, we demonstrate that ExoS and ExoT promote apoptosis in infiltrating neutrophils in vivo. Consistent with prior reports showing ExoS/T dependent apoptosis of HeLa cells (45, 54), induction of apoptosis relied on the ADPR activity of both effectors. It is tempting to speculate that apoptosis of infiltrating phagocytes contributes to persistence of P. aeruginosa in the eye. However, induction of apoptosis is likely not the only factor influencing survival in neutrophils, as these effectors may also interfere with an activity that is directly involved in clearing phagocytosed bacteria, such as fusion with neutrophil granules or generation of an oxidative burst. Even if induction of apoptosis does not influence the immediate survival of P. aeruginosa in neutrophils, it may still contribute to persistence in the tissue at the population level, by killing the primary cell-type responsible for the clearance of these bacteria. The ADPR activities of ExoS and ExoT may therefore promote keratitis by inhibiting the anti-bacterial effects of neutrophils in the corneal stroma, by promoting evasion of neutrophils through sequestration within external corneal epithelial cells (52, 56), and by promoting neutrophil apoptosis.

Although Fleiszig and co-workers showed a clear role for ExoS in P. aeruginosa survival in epithelial cells (52), they reported that the PAK ΔexoS mutant could still cause corneal disease in a mouse keratitis model (28). It is likely that the primary reason for the difference between those findings and results of the current study, which clearly demonstrate that delivery of ExoS and ExoT is essential in the invasive PAO1 and PAK strains of P. aeruginosa (in the absence of ExoU), is due to the higher inoculum used in that study, which likely masked the effect of ExoS (Fleiszig, personal communication).

One emerging area of consensus is that the relative importance of the virulence factors expressed by a given Pseudomonas isolate depends on the site of infection. For example, ExoS and ExoT have non-redundant functions in our model of keratitis, whereas deletion of exoT alone has no phenotype in lung or burn-wound infections (57-59). It is possible that ExoS activity is either redundant with that of ExoT, or has a distinct virulence phenotype depending on the strain used (30, 58-60). In contrast to the keratitis model using the PA103 strain of P. aeruginosa (28), ExoU is the dominant effector in an acute lung model of infection, as deletion of exoU results in a significant reduction in virulence whereas deletion of exoT results in a phenotype only in the context of the exoU mutant strain (60). Effectors therefore have niche-specific roles, even though clearance of P. aeruginosa relies on neutrophils in both the lungs and the cornea (9, 29, 31, 61). The molecular basis for these differences awaits further studies directly comparing infection in these tissues.

In conclusion, given that the pathogenesis of P. aeruginosa keratitis is a consequence of the host response and bacterial virulence factors, our experiments demonstrate a clear role for the type III secretion and ExoS and ExoT in subverting the host response to promote bacterial survival and development of corneal disease. Although the molecular basis for these observations have yet to be determined, we conclude that the ADPR activities of ExoS and ExoT mediate survival of P. aeruginosa in neutrophils and also promote apoptotic cell-death, both of which likely contribute to corneal disease.

Supplementary Material


The authors wish to thank Charles Stopford and Raju Alluri for construction of P. aeruginosa strains, Cathy Doller for outstanding technical assistance, and Scott Howell and Sixto M. Leal, Jr. for discussion on quantification of corneal images.


This work was supported by National Institutes of Health Grants R01 EY14362 (E.P.), P30 EY11373 (E.P.), and by an American Cancer Society Research Scholar Grant RSG-09-198-01-MPC (A.R.). Additional support for this work was provided by the Research to Prevent Blindness Foundation and the Ohio Lions Eye Research Foundation.


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