Pseudomonas aeruginosa ExoT Acts In Vivo as a GTPase-Activating Protein for RhoA, Rac1, and Cdc42

ABSTRACT The Pseudomonas aeruginosa protein ExoT is a bacterial GTPase-activating protein (GAP) that has in vitro activity toward Rho, Rac, and Cdc42 GTPases. Expression of ExoT both inhibits the internalization of strain PA103 by macrophages and epithelial cells and is associated with morphological changes (cell rounding and detachment) of infected cells. We find that expression of ExoT leads to the loss of GTP-bound RhoA, Rac1, and Cdc42 in transfected HeLa cells, demonstrating that ExoT has GAP activity in vivo toward all three GTPases. GAP activity is absolutely dependent on the presence of arginine at position 149 but is not affected by whether ExoT is expressed in the absence or presence of other P. aeruginosa type III secreted proteins. We also demonstrate that expression of ExoT in epithelial cells is sufficient to cause stress fiber disassembly by means of ExoT's GAP activity toward RhoA.

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Correlation of Structural Changes at Different Levels of the Jejunal Villus with Positive and Negative Net Water Transport in Vivo and in Vitro

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y75-063 ◽

1975 ◽

Vol 53 (3) ◽

pp. 439-450 ◽

Cited By ~ 16

Author(s):

T. F. McElligott ◽

I. T. Beck ◽

P. K. Dinda ◽

S. Thompson

Keyword(s):

Epithelial Cells ◽

Water Content ◽

Water Transport ◽

Structural Changes ◽

Morphological Changes ◽

Sugar Transport ◽

Apical Part ◽

Different Levels

Experiments were done for identification and localization of certain structural changes at different levels of jejunal villus of the hamster during positive and negative water transport across the intestine in vivo and in vitro. Positive transport occurred when the mucosal surface of the intestine was bathed (in vitro experiments) or perfused (in vivo experiments) with isotonic Krebs–Ringer bicarbonate solution containing 10 mM glucose, and negative water transport was achieved by rendering this solution hypertonic with 150 mM mannitol. Results indicate that during positive net water transport, the intestine in vivo transported more fluid and exhibited a more conspicuous dilatation of the lateral intercellular spaces (L.I.S.) than did the in vitro preparation. Dilatation of the L.I.S. in both preparations was present only in the apical part of the villus, suggesting that this is the principal site of water absorption. When the mucosal solution was made hypertonic with mannitol, the L.I.S. in the in vivo intestine totally collapsed, whereas in the in vitro intestine these spaces remained open very slightly. These morphological changes correspond well with our finding that in the presence of the hypertonic mucosal solution there was a greater net negative water transport in vivo than in vitro. Incubation of the intestine in the isotonic mucosal solution produced subnuclear swelling of the mid-villus epithelial cells, and this morphological change was associated with an increase in the water content of the tissue. Perfusion of the in vivo intestine with the isotonic solution produced neither the swellings nor the increase in water content of the tissue. In the presence of hypertonic mucosal solution there was a water loss from the tissue both in vivo and in vitro, and these swellings were not observed. These results are discussed in relation to intestinal sugar transport and to the maturity of the epithelial cells, and it is concluded that transport studies on in vitro preparations may provide valid information on a qualitative basis, if not on a strictly quantitative basis.

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Pet secretion, internalization and induction of cell death during infection of epithelial cells by enteroaggregative Escherichia coli

Microbiology ◽

10.1099/mic.0.029116-0 ◽

2009 ◽

Vol 155 (9) ◽

pp. 2895-2906 ◽

Cited By ~ 14

Author(s):

Miguel Betancourt-Sanchez ◽

Fernando Navarro-Garcia

Keyword(s):

Escherichia Coli ◽

Cell Death ◽

Epithelial Cells ◽

Morphological Changes ◽

Eukaryotic Cell ◽

Transcriptional Level ◽

Cell Detachment ◽

Wild Type ◽

Infected Cells

In an in vitro model using HEp-2 cells treated with purified plasmid-encoded toxin (Pet), we have identified morphological changes characterized by cell rounding and detachment after toxin internalization; these changes progress to cell death. However, these effects have not yet been shown to occur during the infection of epithelial cells by enteroaggregative Escherichia coli (EAEC). Here, we show that the secretion of Pet by EAEC is regulated at the transcriptional level, since secretion was inhibited in eukaryotic cell culture medium, although Pet was efficiently secreted in the same medium supplemented with tryptone. Inefficient secretion of Pet by EAEC in DMEM prevented cell detachment, whereas efficient Pet secretion in DMEM/tryptone increased cell detachment in a HEp-2 cell adherence assay. Interestingly, Pet toxin was efficiently delivered to epithelial cells, since it was internalized into epithelial cells infected with EAEC at similar concentrations to those obtained by using 37 μg ml−1 purified Pet protein. Additionally, Pet was not internalized when the epithelial cells were infected with a pet clone, HB101(pCEFN1), unlike the wild-type strain, which has a high adherence capability. There is a correlation between Pet secretion by EAEC, the internalization of Pet into epithelial cells, cell detachment and cell death in EAEC-infected cells. The ratio between live and dead cells decreased in cells treated with wild-type EAEC in comparison with cells treated with an isogenic mutant in the pet gene, whereas the effects were restored by complementing the mutant with the pet gene. All these data indicate that Pet is an important virulence factor in the pathogenesis of EAEC infection.

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Human Tear Fluid Protects against Pseudomonas aeruginosa Keratitis in a Murine Experimental Model

Infection and Immunity ◽

10.1128/iai.01404-06 ◽

2007 ◽

Vol 75 (5) ◽

pp. 2325-2332 ◽

Cited By ~ 34

Author(s):

Mary S. F. Kwong ◽

David J. Evans ◽

Minjian Ni ◽

Brigitte A. Cowell ◽

Suzanne M. J. Fleiszig

Keyword(s):

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Bacterial Colonization ◽

Healing Process ◽

Tear Fluid ◽

Protective Mechanism ◽

Healthy Human ◽

Epithelial Barrier Function

ABSTRACT Pseudomonas aeruginosa keratitis is an acute sight-threatening infection. We previously reported that human tear fluid could protect individual human corneal epithelial cells in vitro against invasion by and cytotoxicity due to clinical and laboratory isolates of P. aeruginosa and that the protective mechanism was independent of bacteriostatic activity. In the present study, we examined the effects of human tear fluid in vivo. Tears were collected from healthy human volunteers and were studied in vivo in mice. The effects on the virulence of both invasive and cytotoxic clinical isolates of P. aeruginosa were examined. Tear fluid was found to reduce the severity of disease when corneas were challenged with cytotoxic bacteria immediately after scratch injury, and it completely protected against susceptibility to infection by a cytotoxic strain in a model in which corneas were infected during the healing process 6 h after scratching. Visible protection correlated with the inhibition of bacterial colonization 1, 4, and 48 h postinoculation. Tear fluid also significantly reduced the severity of infections caused by invasive P. aeruginosa in the 6-h-healing model. This result also coincided with significantly reduced bacterial colonization at 48 h. In vitro, human tear fluid significantly reduced the ability of invasive and cytotoxic bacteria to translocate across corneal epithelia and increased transepithelial resistance with or without bacterial inoculation. These data show that human tear fluid can protect against P. aeruginosa corneal infection in vivo and that the mechanism likely involves enhanced epithelial barrier function in addition to protection of individual epithelial cells against bacterial internalization and cytotoxicity.

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Dimethyl sulfoxide decreases interleukin-8-mediated neutrophil recruitment in the airways

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1996.271.5.l838 ◽

1996 ◽

Vol 271 (5) ◽

pp. L838-L843 ◽

Cited By ~ 1

Author(s):

P. P. Massion ◽

A. Linden ◽

H. Inoue ◽

M. Mathy ◽

K. M. Grattan ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Dimethyl Sulfoxide ◽

Neutrophil Recruitment ◽

Interleukin 8 ◽

Bronchial Epithelial ◽

Anti Inflammatory

In this study, we investigated the role of dimethyl sulfoxide (DMSO) in inhibiting interleukin-8 (IL-8)-mediated neutrophil recruitment induced by Pseudomonas aeruginosa (PA) bacterial supernatant. First, we tested whether DMSO could inhibit IL-8 production induced by PA in human bronchial epithelial (16-HBE) cells in vitro. In these cells, exposure to PA or H2O2 induced IL-8 production dose dependently, an effect that was inhibited by 1% DMSO at both the protein and RNA level. Second, we tested whether DMSO could block the recruitment of neutrophils induced by PA in a bypassed segment of dog trachea in vivo. PA supernatant was placed in the tracheal segment for 6 h in four dogs, and neutrophil recruitment and IL-8 concentrations were measured in the superfusate. DMSO prevented the recruitment of neutrophils and IL-8 production induced by PA time dependently. The results suggest that DMSO may play an anti-inflammatory role in the airway by inhibiting IL-8 production in epithelial cells.

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Effect of Imipenem and Amikacin Combination against Multi-Drug Resistant Pseudomonas aeruginosa

Antibiotics ◽

10.3390/antibiotics10111429 ◽

2021 ◽

Vol 10 (11) ◽

pp. 1429

Author(s):

Sara Mahmoud Farhan ◽

Mohamed Raafat ◽

Mohammed A. S. Abourehab ◽

Rehab Mahmoud Abd El-Baky ◽

Salah Abdalla ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Synergistic Effect ◽

Morphological Changes ◽

P Value ◽

High Morbidity ◽

Drug Resistant ◽

Pcr Techniques ◽

Extensively Drug Resistance

Pseudomonas aeruginosa is an opportunistic nosocomial pathogen associated with high morbidity and mortality rates. Combination of antibiotics has been found to combat multi-drug resistant or extensively drug resistance P. aeruginosa. In this study we investigate the in vitro and in vivo effect of amikacin and imipenem combination against resistant P. aeruginosa. The checkerboard technique and time-killing curve have been performed for in vitro studies showed synergistic effect for combination. A peritonitis mouse model has been used for evaluation of the therapeutic efficacy of this combination which confirmed this synergistic effect. The in vitro and in vivo techniques showed synergistic interaction between tested drugs with fractional inhibitory concentration indices (FICIs) of ≤0.5. Conventional PCR and quantitative real-time PCR techniques were used in molecular detection of bla IMP and aac(6′)-Ib as 35.5% and 42.2% of P. aeruginosa harbored bla IMP and aac(6′)-Ib respectively. Drug combination viewed statistically significant reduction in bacterial counts (p value < 0.5). The lowest bla IMP and aac(6′)-Ib expression was observed after treatment with 0.25 MIC of imipenem + 0.5 MIC of amikacin. Morphological changes in P. aeruginosa isolates were detected by scanning electron microscope (SEM) showing cell shrinkage and disruption in the outer membrane of P. aeruginosa that were more prominent with combination therapy than with monotherapy.

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Changes in toxin production of environmental Pseudomonas aeruginosa isolates exposed to sub-inhibitory concentrations of three common antibiotics

PLoS ONE ◽

10.1371/journal.pone.0248014 ◽

2021 ◽

Vol 16 (3) ◽

pp. e0248014

Author(s):

Biljana Mojsoska ◽

Melanie Ghoul ◽

Gabriel G. Perron ◽

Håvard Jenssen ◽

Fatima AlZahra’a Alatraktchi

Keyword(s):

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Toxin Production ◽

Biologically Relevant ◽

A Value ◽

Severe Infections ◽

Bacterial Virulence Factor ◽

And Diffusion

Pseudomonas aeruginosa is an environmental pathogen that can cause severe infections in immunocompromised patients. P. aeruginosa infections are typically treated with multiple antibiotics including tobramycin, ciprofloxacin, and meropenem. However, antibiotics do not always entirely clear the bacteria from the infection site, where they may remain virulent. This is because the effective antibiotic concentration and diffusion in vitro may differ from the in vivo environment in patients. Therefore, it is important to understand the effect of non-lethal sub-inhibitory antibiotic concentrations on bacterial phenotype. Here, we investigate if sub-inhibitory antimicrobial concentrations cause alterations in bacterial virulence factor production using pyocyanin as a model toxin. We tested this using the aforementioned antibiotics on 10 environmental P. aeruginosa strains. Using on-the-spot electrochemical screening, we were able to directly quantify changes in production of pyocyanin in a measurement time of 17 seconds. Upon selecting 3 representative strains to further test the effects of sub-minimum inhibitory concentration (MICs), we found that pyocyanin production changed significantly when the bacteria were exposed to 10-fold MIC of the 3 antibiotics tested, and this was strain specific. A series of biologically relevant measured pyocyanin concentrations were also used to assess the effects of increased virulence on a culture of epithelial cells. We found a decreased viability of the epithelial cells when incubated with biologically relevant pyocyanin concentrations. This suggests that the antibiotic-induced virulence also is a value worth being enclosed in regular testing of pathogens.

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Effect of Hypoxia on the Pathogenesis of Acinetobacter baumannii and Pseudomonas aeruginosa In Vitro and in Murine Experimental Models of Infection

Infection and Immunity ◽

10.1128/iai.00543-18 ◽

2018 ◽

Vol 86 (10) ◽

Cited By ~ 7

Author(s):

María Luisa Gil-Marqués ◽

María Eugenía Pachón-Ibáñez ◽

Jerónimo Pachón ◽

Younes Smani

Keyword(s):

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Acinetobacter Baumannii ◽

Experimental Models ◽

Host Cells ◽

Content Type ◽

Bactericidal Activities ◽

Bacterial Concentrations

ABSTRACT Hypoxia modulates bacterial virulence and the inflammation response through hypoxia-inducible factor 1α (HIF-1α). Here we study the influence of hypoxia on Acinetobacter baumannii and Pseudomonas aeruginosa infections. In vitro, hypoxia increases the bactericidal activities of epithelial cells against A. baumannii and P. aeruginosa, reducing extracellular bacterial concentrations to 50.5% ± 7.5% and 90.8% ± 13.9%, respectively, at 2 h postinfection. The same phenomenon occurs in macrophages (67.6% ± 18.2% for A. baumannii at 2 h and 50.3% ± 10.9% for P. aeruginosa at 24 h). Hypoxia decreases the adherence of A. baumannii to epithelial cells (42.87% ± 8.16% at 2 h) and macrophages (52.0% ± 18.7% at 24 h), as well as that of P. aeruginosa (24.9% ± 4.5% in epithelial cells and 65.7% ± 5.5% in macrophages at 2 h). Moreover, hypoxia decreases the invasion of epithelial cells (48.6% ± 3.8%) and macrophages (8.7% ± 6.9%) by A. baumannii at 24 h postinfection and by P. aeruginosa at 2 h postinfection (75.0% ± 16.3% and 63.4% ± 5.4%, respectively). In vivo, hypoxia diminishes bacterial loads in fluids and tissues in animal models of infection by both pathogens. In contrast, mouse survival time was shorter under hypoxia (23.92 versus 36.42 h) with A. baumannii infection. No differences in the production of cytokines or HIF-1α were found between hypoxia and normoxia in vitro or in vivo. We conclude that hypoxia increases the bactericidal activities of host cells against both pathogens and reduces the interaction of pathogens with host cells. Moreover, hypoxia accelerates the rate at which animals die despite the lower bacterial concentrations in vivo.

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In Vitro Activity of Rifabutin and Rifampin against Antibiotic-Resistant Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae

mSphere ◽

10.1128/msphere.00920-21 ◽

2021 ◽

Author(s):

Bosul Lee ◽

Jun Yan ◽

Amber Ulhaq ◽

Sarah Miller ◽

Wonjae Seo ◽

...

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

Klebsiella Pneumoniae ◽

General Effect ◽

In Vitro Activity ◽

Antibiotic Resistant ◽

Content Type

Rifabutin has been recently described as a potential adjunctive therapy for antibiotic-resistant A. baumannii infections due to hypersensitivity in iron-depleted media, which may more closely mimic an in vivo environment. Here, we report that this hyperactivity is specific for A. baumannii , rather than being a general effect for other pathogens.

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Substitution of Ser-17 of pp60c-src: biological and biochemical characterization in chicken embryo fibroblasts

Molecular and Cellular Biology ◽

10.1128/mcb.8.4.1826-1830.1988 ◽

1988 ◽

Vol 8 (4) ◽

pp. 1826-1830

Author(s):

Y Hirota ◽

J Kato ◽

T Takeya

Keyword(s):

Chicken Embryo ◽

Biochemical Characterization ◽

Morphological Changes ◽

Rous Sarcoma ◽

Sarcoma Virus ◽

Infected Cells ◽

Embryo Fibroblasts ◽

Chicken Embryo Fibroblasts

pp60c-src is phosphorylated mainly on Ser-17 and Tyr-527 in vivo. In this study, we examined the effect of the phosphorylation of Ser-17 on the properties of pp60c-src by introducing Rous sarcoma virus variants carrying pp60c-src in which Ser-17 had been substituted, into chicken embryo fibroblasts. The Ala-17 substitution in wild-type pp60c-src and pp60c-src carrying Phe-527 caused a two- to threefold elevation in the kinase activity in vitro of these proteins; the former variant resulted in no morphological changes of infected cells, whereas the latter variant transformed chicken embryo fibroblasts. Since the substitution of Tyr-527 per se has been reported to activate pp60c-src, these results suggest that the abolishment of the phosphorylation of Ser-17 does not affect noticeably the properties of pp60c-src in chicken embryo fibroblasts.

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