Contribution of ExsA–Regulated Factors to Corneal Infection by Cytotoxic and Invasive<i>Pseudomonas aeruginosa</i>in a Murine Scarification Model

Lee, Ellen J; Cowell, Brigitte A.; Evans, David J.; Fleiszig, Suzanne M. J.

doi:10.1167/iovs.02-1302

Cited by 71 publications

(73 citation statements)

References 38 publications

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“…For use in experiments, bacteria were resuspended in tissue culture medium (equal parts DMEM/Hams F12) to an optical density (at 650 nm) of ~0.1 to 0.2 corresponding to a viable count of ~2-3 × 10 8 cfu/mL. Control experiments in this, and previous studies (Vallis et al, 1999;Lee et al, 2003) have confirmed that these plasmid-complemented strains grow equally well in vitro with or without exposure to mammalian cells.…”

Section: Bacteriamentioning

confidence: 93%

“…Viable counts were performed on homogenates using TSA containing carbenicillin 300ug/ml to select plasmid-bearing bacteria. We have previously observed that this plasmid is retained by P. aeruginosa in vivo over 48 h (Lee et al, 2003). In other experiments, mice were sacrificed at 6 h after infection to explore the role of ExoU and its phospholipase activity on early bacterial colonization at a point prior to significant immune cell infiltration.…”

Section: Murine Corneal Infection and P Aeruginosa Colonizationmentioning

confidence: 99%

“…Cytotoxic isolates can also kill epithelial cells on the surface of intact corneas ex vivo (Fleiszig et al, 1998). Using a murine scarification model of keratitis, we previously demonstrated that ExoU also makes a significant contribution to ocular colonization and corneal disease, and that it represses phagocyte infiltration of the central region of infected corneas in vivo (Lee et al, 2003;Zolfaghar et al, 2006). Others have shown that complementation of exoU into an invasive strain of P. aeruginosa enhances its virulence in a murine model of acute pneumonia (Allewelt et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

“…This was tested by comparing corneal disease pathology and ocular colonization by a double effector mutant of the cytotoxic P. aeruginosa strain PA103 (PA103ΔexoUexoT::Tc) complemented with a functional exoU gene on the plasmid pUCP18 (pUCPexoU) with disease caused by the same strain when complemented with exoU containing a mutation in the catalytic aspartate region of the phospholipase domain (pUCPexoUD344A), or a control (pUCP18). Testing in an effector null background was done to remove any potential complicating factors due to ExoT, another type-III secreted effector encoded by strain PA103, that we have previously shown to act redundantly with ExoU in contributing to P. aeruginosa-induced pathology in the cornea (Lee et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

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Mutation of the phospholipase catalytic domain of the Pseudomonas aeruginosa cytotoxin ExoU abolishes colonization promoting activity and reduces corneal disease severity

Tam

Lewis

et al. 2007

Experimental Eye Research

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We have previously shown that ExoU, a type III secreted cytotoxin of Pseudomonas aeruginosa, causes acute cytotoxicity towards corneal epithelial cells in vitro, and contributes to corneal disease pathology and ocular colonization in vivo. Subsequently, we reported that ExoU represses phagocyte infiltration of infected corneas in vivo. ExoU has patatin-like phospholipase activity that is required for cytotoxic activity in vitro (mammalian cell injury and death) and for disease in a murine model of pneumonia. We hypothesized that the phospholipase activity was required for ExoU-mediated corneal disease and ocular colonization. Using the murine scarification model, corneal disease pathology was examined after inoculation with ~10 6 cfu of a P. aeruginosa effector mutant (PA103ΔexoUexoT::Tc) complemented with either exoU (pUCPexoU), phospholipase-inactive exoU (pUCPexoUD344A) or a plasmid control (pUCP18). Eyes were photographed and disease severity scored at 24 and 48 h post-infection. Viable bacteria colonizing infected eyes were quantified at 6 and 48 h. Complementation with exoU caused significantly more pathology (increased disease severity scores) and enabled bacteria to better colonize (bỹ 1000-fold) at 48 h as compared to phospholipase-inactive exoU which did not differ from plasmid control. Surprisingly, exoU did not contribute to early (6 h) colonization. In-vitro assays confirmed that the phospholipase domain of exoU was required for cytotoxicity towards human corneal epithelial cells. Taken together these data show that the phospholipase activity of the P. aeruginosa cytotoxin, ExoU, plays a role in the pathogenesis of corneal infection via mechanism(s) occurring after initial colonization of a susceptible cornea.

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Section: Bacteriamentioning

confidence: 93%

Section: Murine Corneal Infection and P Aeruginosa Colonizationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Mutation of the phospholipase catalytic domain of the Pseudomonas aeruginosa cytotoxin ExoU abolishes colonization promoting activity and reduces corneal disease severity

Tam

Lewis

et al. 2007

Experimental Eye Research

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“…Both T2SS and the T3SS play independent roles in death due to Pseudomonas lung infection and are related to disease severity [18]. T3SS also enhances infection severity in several animal models: acute pneumonia [19,21,52], keratitis [52], bacteremia [22], peritonitis [53], burn infections [20] and gut-derived sepsis in neutropenia [54]. Moreover, T3SS in vitro detection was correlated with increased morbi-mortality and relapse in human P. aeruginosa ventilator-associated pneumonia and blood stream infections [55,56].…”

Section: Discussionmentioning

confidence: 99%

Pseudomonas aeruginosa flagellum is critical for invasion, cutaneous persistence and induction of inflammatory response of skin epidermis

Garcia

Morello

Garnier³

et al. 2018

Virulence

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Pseudomonas aeruginosa, an opportunistic pathogen involved in skin and lung diseases, possesses numerous virulence factors, including type 2 and 3 secretion systems (T2SS and T3SS) and its flagellum, whose functions remain poorly known during cutaneous infection. Using isogenic mutants deleted from genes encoding each or all of these three virulence factors, we investigated their role in induction of inflammatory response and in tissue invasiveness in human primary keratinocytes and reconstructed epidermis. Our results showed that flagellum, but not T2SS and T3SS, is involved in induction of a large panel of cytokine, chemokine, and antimicrobial peptide (AMP) mRNA in the infected keratinocytes. Chemokine secretion and AMP tissular production were also dependent on the presence of the bacterial flagellum. This pro-inflammatory effect was significantly reduced in keratinocytes infected in presence of anti-toll-like receptor 5 (TLR5) neutralizing antibody. Bacterial invasion of human epidermis and persistence in a mouse model of sub-cutaneous infection were dependent on the P. aeruginosa flagellum. We demonstrated that flagellum constitutes the main virulence factor of P. aeruginosa involved not only in early induction of the epidermis inflammatory response but also in bacterial invasion and cutaneous persistence. P. aeruginosa is mainly sensed by TLR5 during the early innate immune response of human primary keratinocytes.

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Association Between Cytotoxic and Invasive Pseudomonas aeruginosa and Clinical Outcomes in Bacterial Keratitis

Borkar¹,

Fleiszig²,

Leong³

et al. 2013

JAMA Ophthalmol

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To determine whether cytotoxic and invasive Pseudomonas aeruginosa strains differentially influence clinical presentation, outcomes, or therapeutic response in bacterial keratitis.Methods: Pseudomonas aeruginosa isolates from the National Eye Institute-funded Steroids for Corneal Ulcers Trial were subtyped as cytotoxic or invasive strains. The main outcome measure compared between the 2 subtypes was change in visual acuity at 3 months using Huber robust regression, adjusting for topical corticosteroid treatment.Results: Of 101 confirmed P aeruginosa isolates from the Steroids for Corneal Ulcers Trial, 74 had a classically cytotoxic or invasive genotype. While corneal ulcers caused by genotypically invasive P aeruginosa strains were associated at presentation with significantly better visual acuity than corneal ulcers caused by genotypically cyto-toxic P aeruginosa strains when adjusting for the effect of ulcer location (P = .008), invasive ulcers had improved significantly less than cytotoxic ulcers at 3 months (0.35; 95% CI, 0.04-0.66 logMAR; P=.03 [3.5-line difference]). Compared with topical moxifloxacin alone, adjunctive treatment with topical corticosteroids was associated with significantly more improvement in visual acuity in the invasive subgroup (P =.04) but was associated with less improvement in visual acuity in the cytotoxic subgroup (P =.07).Conclusions: Rational profiling of differentially expressed virulence determinants (eg, cytotoxicity and invasiveness for P aeruginosa) could be used as a tool for decision making in the management of infections to optimize outcomes.

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Contribution of ExsA–Regulated Factors to Corneal Infection by Cytotoxic and InvasivePseudomonas aeruginosain a Murine Scarification Model

Abstract: ExsA contributed to corneal virulence of only cytotoxic P. aeruginosa, with contributions made by both ExoU and ExoT to bacterial survival and disease severity. This differs from cytotoxic P. aeruginosa virulence in the lung, which is ExoU-dependent.

Cited by 71 publications

References 38 publications

Mutation of the phospholipase catalytic domain of the Pseudomonas aeruginosa cytotoxin ExoU abolishes colonization promoting activity and reduces corneal disease severity

Mutation of the phospholipase catalytic domain of the Pseudomonas aeruginosa cytotoxin ExoU abolishes colonization promoting activity and reduces corneal disease severity

Pseudomonas aeruginosa flagellum is critical for invasion, cutaneous persistence and induction of inflammatory response of skin epidermis

Association Between Cytotoxic and Invasive Pseudomonas aeruginosa and Clinical Outcomes in Bacterial Keratitis

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