6 research outputs found
<i>P. aeruginosa</i> T3SS needle tip complex protein PcrV associates with lung injury.
<p><b>A</b>. Injured areas of the lung were prepared for histological hematoxylin/eosin analysis at 48-hours post-inoculation. Animals were inoculated with saline solution (vehicle control), with ~1 x 10<sup>7</sup> CFUs of PA103 (ΔU/ΔT), or with ~1 x 10<sup>7</sup> CFUs of PA103 (ΔPcrV). Lung sections imaged at 10X (top panels) and 20X (bottom panels) revealed that intratracheal instillation of PA103 (ΔU/ΔT) or PA103 (ΔPcrV) caused lung injury. Inoculation with PA103 (ΔU/ΔT) resulted in fluid accumulation around the bronchovascular bundles (vascular cuffs, arrow heads, upper panel) and lesions reminiscent of diffuse alveolar damage (arrow heads, lower panel), characterized by fluid accumulation and inflammatory cell infiltration within the air space. Inoculation with PA103 (ΔPcrV) caused collapsed air spaces, resembling atelectasis with recruitment of inflammatory cells. These histological patterns suggest that the two PA103 mutants are capable to cause lung damage/injury, although the lesions appear distinct from one another. <b>B</b>. Injured areas of the lung were prepared for histological myeloperoxidase (MPO) staining analysis to reveal the extent of neutrophil infiltration. Inoculation with PA103 (ΔU/ΔT) caused significant neutrophil recruitment compared to either vehicle control or to inoculation with PA103 (ΔPcrV). The average numbers of MPO positive cells from at least 5 independent fields of view from three different animals per condition are presented. Error bars are the standard error of the mean. The asterisks indicate a statistically significant difference (<i>P</i> < 0.05) by one-way ANOVA and Newman-Keuls <i>post-hoc</i> test. <b>C</b>. Lungs from vehicle control (saline solution) and <i>P. aeruginosa</i>-inoculated animals were isolated <i>en </i><i>bloc</i> at 48-hours post-inoculation for analysis of hydraulic permeability (K<sub>f</sub>). Inoculation with PA103 (ΔU/ΔT) caused significant permeability increases compared to either vehicle control or to inoculation with PA103 (ΔPcrV). The average K<sub>f</sub> values from 5 different animals per group are shown. Error bars are the standard error of the mean. The asterisks indicate a statistically significant difference (<i>P</i> < 0.05) by one-way ANOVA and Newman-Keuls <i>post-hoc</i> test.</p
<i>P. aeruginosa</i> T3SS needle tip complex protein PcrV associates with increased PMVEC permeability during early phase infection.
<p><b>A</b>. Isolated rat PMVECs were grown on transwells to confluence and permeability measured as a function of FITC-dextran flux across the monolayer. PMVECs were inoculated with medium alone, saline solution (vehicle control), or with either <i>P. aeruginosa</i> mutant at a 40:1 MOI. Flux assays were initiated at 4-hours post-inoculation by the addition of FITC-dextran and flux monitored for 1-hour. Average flux rate data at 5-hours post-inoculation from the SS vehicle control transwells were used to normalized the flux rate from PA103 (ΔU/ΔT) or PA103 (ΔPcrV) (n=3 for each condition). Inoculation with PA103 (ΔU/ΔT) caused significant increases in PMVEC permeability compared to inoculation with PA103 (ΔPcrV). Error bars are the standard error of the mean. The asterisk indicates a significant difference by unpaired <i>t</i>-test (<i>P</i> < 0.05). <b>B</b>. Mock cell culture infection experiments were performed where ~2.5 x 10<sup>7</sup> CFU/mL of each <i>P. aeruginosa</i> mutant strain was inoculated into DMEM culture medium (no PMVECs) and bacterial growth followed over time by direct plating. Error bars are the standard error of the mean of technical replicates from at least 3 independent experiments. There were no observable differences in growth rates between the two <i>P. aeruginosa</i> mutant strains. <b>C</b>. The ability of each of the P. <i>aeruginosa</i> mutant strains to adhere to PMVECs was assessed. Isolated rat PMVECs were grown in culture dishes to confluence and inoculated with either <i>P. aeruginosa</i> mutant at a 40:1 MOI. At one hour post-inoculation, monolayers were washed copiously to remove planktonic bacteria, PMVECs permeabilized, and total associated bacteria determined by direct plating. Error bars are the standard error of the mean of technical replicates from at least 3 independent experiments. There were no observable differences in adherence to PMVECs between the two <i>P. aeruginosa</i> mutant strains.</p
In the Absence of Effector Proteins, the <i>Pseudomonas aeruginosa</i> Type Three Secretion System Needle Tip Complex Contributes to Lung Injury and Systemic Inflammatory Responses
<div><p>Herein we describe a pathogenic role for the <i>Pseudomonas aeruginosa</i> type three secretion system (T3SS) needle tip complex protein, PcrV, in causing lung endothelial injury. We first established a model in which <i>P. aeruginosa</i> wild type strain PA103 caused pneumonia-induced sepsis and distal organ dysfunction. Interestingly, a PA103 derivative strain lacking its two known secreted effectors, ExoU and ExoT [denoted PA103 (ΔU/ΔT)], also caused sepsis and modest distal organ injury whereas an isogenic PA103 strain lacking the T3SS needle tip complex assembly protein [denoted PA103 (ΔPcrV)] did not. PA103 (ΔU/ΔT) infection caused neutrophil influx into the lung parenchyma, lung endothelial injury, and distal organ injury (reminiscent of sepsis). In contrast, PA103 (ΔPcrV) infection caused nominal neutrophil infiltration and lung endothelial injury, but no distal organ injury. We further examined pathogenic mechanisms of the T3SS needle tip complex using cultured rat pulmonary microvascular endothelial cells (PMVECs) and revealed a two-phase, temporal nature of infection. At 5-hours post-inoculation (early phase infection), PA103 (ΔU/ΔT) elicited PMVEC barrier disruption via perturbation of the actin cytoskeleton and did so in a cell death-independent manner. Conversely, PA103 (ΔPcrV) infection did not elicit early phase PMVEC barrier disruption. At 24-hours post-inoculation (late phase infection), PA103 (ΔU/ΔT) induced PMVEC damage and death that displayed an apoptotic component. Although PA103 (ΔPcrV) infection induced late phase PMVEC damage and death, it did so to an attenuated extent. The PA103 (ΔU/ΔT) and PA103 (ΔPcrV) mutants grew at similar rates and were able to adhere equally to PMVECs post-inoculation indicating that the observed differences in damage and barrier disruption are likely attributable to T3SS needle tip complex-mediated pathogenic differences post host cell attachment. Together, these infection data suggest that the T3SS needle tip complex and/or another undefined secreted effector(s) are important determinants of <i>P. aeruginosa</i> pneumonia-induced lung endothelial barrier disruption.</p> </div
<i>P. aeruginosa</i> T3SS needle tip complex protein PcrV elicits increased PMVEC permeability during early phase infection via cytoskeletal perturbation in a host cell death-independent manner.
<p><b>A</b>. Isolated rat PMVECs were grown in culture dishes to confluence and viability assessed by Trypan Blue exclusion assay. PMVECs were inoculated with saline solution (vehicle control), or with either <i>P. aeruginosa</i> mutant at a 40:1 MOI. PMVEC viability was assessed at 5-hours post-inoculation, and data are expressed as percent total viable cells. Neither PA103 mutant caused significant cell death compared to vehicle control-treated PMVECs. The average viable PMVEC numbers from at least 3 independent experiments per condition are presented. Error bars are the standard error of the mean. <b>B</b>. Isolated rat PMVECs were grown in culture dishes to confluence and damage assessed by LDH extracellular release assay. PMVECs were inoculated with saline solution (vehicle control), or with either <i>P. aeruginosa</i> mutant at a 40:1 MOI. PMVEC damage was assessed over a time course post-inoculation. Culture medium was sampled to measure extracellular LDH and data normalized to the total monolayer (intracellular) LDH. While measurable, compared to vehicle control-treated PMVECs, both PA103 mutants caused only negligible rates of LDH release. The average amounts of extracellular LDH from at least 3 independent experiments are presented. <b>C</b>. Isolated rat PMVECs were grown on cover slips to confluence and actin cytoskeleton was visualized by FITC-phalloidin staining. Z-stacks from whole cellular volume images were acquired via confocal fluorescence microscopy. PMVECs were inoculated with saline solution (vehicle control), or with either <i>P. aeruginosa</i> mutant at a 40:1 MOI. Inoculation with PA103 (ΔU/ΔT) mutant caused evident cytoskeletal rearrangement (stress fibers) compared to vehicle control and PA103 (ΔPcrV) inoculation.</p
<i>P. aeruginosa</i> T3SS needle tip complex protein PcrV associates with mortality in a rat model of lung infection.
<p><b>A</b>. Intratracheal instillation of the P. <i>aeruginosa</i> wild type PA103 and mutant PA103 (ΔU/ΔT) strains into rats resulted in dose-dependent mortality compared to the mutant PA103 (ΔPcrV) which caused no mortality at 1-week post-infection. Doses (CFUs/mL) are listed in the figure. Data represent at least n=20 per group. <b>B</b>. One week survival analysis (Kaplan Meier curve) at a sub-LD<sub>50</sub> dose (~1 x 10<sup>7</sup> CFUs). Mortality in the PA103 (ΔU/ΔT) cohort was observed within 48-hours post-infection. Black dotted line represents vehicle control (saline solution), red dotted line represents infection with PA103 (ΔPcrV), and blue dotted line represents Infection with PA103 (ΔU/ΔT). Data represent at least n=20 per group.</p
<i>P. aeruginosa</i> T3SS needle tip complex protein PcrV increases PMVEC permeability associated with cell damage during late phase infection.
<p><b>A</b>. Isolated rat PMVECs were grown on gold electrodes to confluence and resistance across the monolayer determined using an ECIS system. PMVECs were inoculated with medium alone, saline solution (vehicle control), or with either <i>P. aeruginosa</i> mutant at a 40:1 MOI. Inoculation with PA103 (ΔU/ΔT) caused significant increases in PMVEC permeability (i.e., decreased electrical resistance) compared to no treatment (medium alone), vehicle control inoculation, or inoculation with PA103 (ΔPcrV). The average numbers of resistance recordings from at least 6 independent experiments per condition are presented. Error bars are the standard error of the mean. The asterisk indicates that PA103 (ΔU/ΔT) was significantly different compared to all other conditions (<i>P</i> < 0.05) by one-way ANOVA and Newman-Keuls <i>post-hoc</i> test. <b>B</b>. Isolated rat PMVECs were grown in culture dishes to confluence and viability assessed by Trypan Blue exclusion assay. PMVECs were inoculated with saline solution (vehicle control), or with either <i>P. aeruginosa</i> mutant at a 40:1 MOI. PMVEC viability was assessed at 24-hours post-inoculation, and data are expressed as percent total viable cells. Inoculation with PA103 (ΔU/ΔT) significantly increased PMVEC death compared to vehicle control inoculation and PA103 (ΔPcrV). The average viable PMVEC numbers from at least 10 independent experiments per condition are presented. Error bars are the standard error of the mean. The asterisks indicate that PA103 (ΔU/ΔT) was significantly different compared to all other conditions (<i>P</i> < 0.05) by one-way ANOVA and Newman-Keuls <i>post-hoc</i> test. <b>C</b>. Isolated rat PMVECs were grown on glass cover slips to confluence and cell death assessed by TUNEL staining. PMVECs were inoculated with saline solution (vehicle control), or with either <i>P. aeruginosa</i> mutant at a 40:1 MOI. PMVEC death was assessed at 24-hours post-inoculation, and numbers of dead cells are expressed as a percent of the total number of cells. Inoculation with PA103 (ΔU/ΔT) significantly increased PMVEC death compared to vehicle control inoculation and PA103 (ΔPcrV). Error bars are the standard error of the mean from 3 independent experiments. The asterisks indicate that PA103 (ΔU/ΔT) was significantly different compared to all other conditions (<i>P</i> < 0.05) by one-way ANOVA and Newman-Keuls <i>post-hoc</i> test. <b>D</b>. Isolated rat PMVECs were grown in culture dishes to confluence and cellular fitness assessed by WST-1 metabolism assay. PMVECs were inoculated with saline solution (vehicle control), or with either <i>P. aeruginosa</i> mutant at a 40:1 MOI. PMVECs were assessed at 24-hours post-exposure for each condition, and data are expressed as percent of the vehicle control. Infection with PA103 (ΔU/ΔT) significantly decreased PMVEC fitness compared to vehicle control inoculation and PA103 (ΔPcrV). The average amount of WST-1 metabolism from at least 5 independent experiments per condition is presented. Error bars are the standard error of the mean. The asterisks indicate that PA103 (ΔU/ΔT) was significantly different compared to all other conditions (<i>P</i> < 0.05) by one-way ANOVA and Newman-Keuls <i>post-hoc</i> test.</p