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Ureaplasma parvum infection alters filamin A dynamics in host cells.

Allam AB, Alvarez S, Brown MB, Reyes L - BMC Infect. Dis. (2011)

Bottom Line: In the BPH-1 model, we confirmed that U. parvum perturbed the regulation of filamin A.Specifically, infected BPH-1 cells exhibited a significant increase in filamin A phosphorylated at serine 2152 (P ≤ 0.01), which correlated with impaired proteolysis of the protein and its normal intracellular distribution.Phosphorylation of filamin A occurs in response to various cell signaling cascades that regulate cell motility, differentiation, apoptosis and inflammation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious Disease & Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA.

ABSTRACT

Background: Ureaplasmas are among the most common bacteria isolated from the human urogenital tract. Ureaplasmas can produce asymptomatic infections or disease characterized by an exaggerated inflammatory response. Most investigations have focused on elucidating the pathogenic potential of Ureaplasma species, but little attention has been paid to understanding the mechanisms by which these organisms are capable of establishing asymptomatic infection.

Methods: We employed differential proteome profiling of bladder tissues from rats experimentally infected with U. parvum in order to identify host cell processes perturbed by colonization with the microbe. Tissues were grouped into four categories: sham inoculated controls, animals that spontaneously cleared infection, asymptomatic urinary tract infection (UTI), and complicated UTI. One protein that was perturbed by infection (filamin A) was used to further elucidate the mechanism of U. parvum-induced disruption in human benign prostate cells (BPH-1). BPH-1 cells were evaluated by confocal microscopy, immunoblotting and ELISA.

Results: Bladder tissue from animals actively colonized with U. parvum displayed significant alterations in actin binding proteins (profilin 1, vinculin, α actinin, and filamin A) that regulate both actin polymerization and cell cytoskeletal function pertaining to focal adhesion formation and signal transduction (Fisher's exact test, P < 0.004; ANOVA, P < 0.02). This phenomenon was independent of clinical profile (asymptomatic vs. complicated UTI). We selected filamin A as a target for additional studies. In the BPH-1 model, we confirmed that U. parvum perturbed the regulation of filamin A. Specifically, infected BPH-1 cells exhibited a significant increase in filamin A phosphorylated at serine 2152 (P ≤ 0.01), which correlated with impaired proteolysis of the protein and its normal intracellular distribution.

Conclusion: Filamin A dynamics were perturbed in both models of infection. Phosphorylation of filamin A occurs in response to various cell signaling cascades that regulate cell motility, differentiation, apoptosis and inflammation. Thus, this phenomenon may be a useful molecular marker for identifying the specific host cell pathways that are perturbed during U. parvum infection.

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Intracellular distribution and quantification of phosphorylated filamin A in uninfected, U. parvum infected, and supernatant treated BPH-1 cells. Cells were exposed to sterile 10B broth, 109 CFU of U. parvum, or cell culture supernatant (super) for 72 hours before examination by confocal microscopy (A), or ELISA (B). Phosphorylated filamin A (red) was detected with a rabbit monoclonal antibody (EP2310AY). Intact filamin A (green) was detected with mouse anti-filamin 1. BPH-1 nuclei and U. parvum (white arrow) were identified with DAPI stain (blue). All images were taken at 600× magnification and the scale bar is equal to 10 μm. ELISA for phosphorylated filamin A was performed on whole cell lysates. Absorbance values were divided by the total mg protein determined by BCA assay. Values represent the mean ± SD (n = 5) of phosphorylated filamin A in uninfected (BPH-1) and infected (UP), uninfected supernatant treated (BPH super), and infected supernatant treated (UP super) cells. **P Value Was obtained by Fishers PLSD.
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Figure 3: Intracellular distribution and quantification of phosphorylated filamin A in uninfected, U. parvum infected, and supernatant treated BPH-1 cells. Cells were exposed to sterile 10B broth, 109 CFU of U. parvum, or cell culture supernatant (super) for 72 hours before examination by confocal microscopy (A), or ELISA (B). Phosphorylated filamin A (red) was detected with a rabbit monoclonal antibody (EP2310AY). Intact filamin A (green) was detected with mouse anti-filamin 1. BPH-1 nuclei and U. parvum (white arrow) were identified with DAPI stain (blue). All images were taken at 600× magnification and the scale bar is equal to 10 μm. ELISA for phosphorylated filamin A was performed on whole cell lysates. Absorbance values were divided by the total mg protein determined by BCA assay. Values represent the mean ± SD (n = 5) of phosphorylated filamin A in uninfected (BPH-1) and infected (UP), uninfected supernatant treated (BPH super), and infected supernatant treated (UP super) cells. **P Value Was obtained by Fishers PLSD.

Mentions: In prostate cells, a dominant pathway of filamin A regulation involves cleavage of the protein by calpain [23,24]. Calpain mediated cleavage of filamin A can be reduced by phosphorylation of the protein at serine2152. Therefore, we assessed the degree of filamin A phosphorylation at serine2152 by immunofluorescent microscopy (Figure 3A) and ELISA (Figure 3B). Both detection methods confirmed that only cells infected with U. parvum displayed a significant increase in phosphorylated filamin A.


Ureaplasma parvum infection alters filamin A dynamics in host cells.

Allam AB, Alvarez S, Brown MB, Reyes L - BMC Infect. Dis. (2011)

Intracellular distribution and quantification of phosphorylated filamin A in uninfected, U. parvum infected, and supernatant treated BPH-1 cells. Cells were exposed to sterile 10B broth, 109 CFU of U. parvum, or cell culture supernatant (super) for 72 hours before examination by confocal microscopy (A), or ELISA (B). Phosphorylated filamin A (red) was detected with a rabbit monoclonal antibody (EP2310AY). Intact filamin A (green) was detected with mouse anti-filamin 1. BPH-1 nuclei and U. parvum (white arrow) were identified with DAPI stain (blue). All images were taken at 600× magnification and the scale bar is equal to 10 μm. ELISA for phosphorylated filamin A was performed on whole cell lysates. Absorbance values were divided by the total mg protein determined by BCA assay. Values represent the mean ± SD (n = 5) of phosphorylated filamin A in uninfected (BPH-1) and infected (UP), uninfected supernatant treated (BPH super), and infected supernatant treated (UP super) cells. **P Value Was obtained by Fishers PLSD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3107797&req=5

Figure 3: Intracellular distribution and quantification of phosphorylated filamin A in uninfected, U. parvum infected, and supernatant treated BPH-1 cells. Cells were exposed to sterile 10B broth, 109 CFU of U. parvum, or cell culture supernatant (super) for 72 hours before examination by confocal microscopy (A), or ELISA (B). Phosphorylated filamin A (red) was detected with a rabbit monoclonal antibody (EP2310AY). Intact filamin A (green) was detected with mouse anti-filamin 1. BPH-1 nuclei and U. parvum (white arrow) were identified with DAPI stain (blue). All images were taken at 600× magnification and the scale bar is equal to 10 μm. ELISA for phosphorylated filamin A was performed on whole cell lysates. Absorbance values were divided by the total mg protein determined by BCA assay. Values represent the mean ± SD (n = 5) of phosphorylated filamin A in uninfected (BPH-1) and infected (UP), uninfected supernatant treated (BPH super), and infected supernatant treated (UP super) cells. **P Value Was obtained by Fishers PLSD.
Mentions: In prostate cells, a dominant pathway of filamin A regulation involves cleavage of the protein by calpain [23,24]. Calpain mediated cleavage of filamin A can be reduced by phosphorylation of the protein at serine2152. Therefore, we assessed the degree of filamin A phosphorylation at serine2152 by immunofluorescent microscopy (Figure 3A) and ELISA (Figure 3B). Both detection methods confirmed that only cells infected with U. parvum displayed a significant increase in phosphorylated filamin A.

Bottom Line: In the BPH-1 model, we confirmed that U. parvum perturbed the regulation of filamin A.Specifically, infected BPH-1 cells exhibited a significant increase in filamin A phosphorylated at serine 2152 (P ≤ 0.01), which correlated with impaired proteolysis of the protein and its normal intracellular distribution.Phosphorylation of filamin A occurs in response to various cell signaling cascades that regulate cell motility, differentiation, apoptosis and inflammation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious Disease & Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA.

ABSTRACT

Background: Ureaplasmas are among the most common bacteria isolated from the human urogenital tract. Ureaplasmas can produce asymptomatic infections or disease characterized by an exaggerated inflammatory response. Most investigations have focused on elucidating the pathogenic potential of Ureaplasma species, but little attention has been paid to understanding the mechanisms by which these organisms are capable of establishing asymptomatic infection.

Methods: We employed differential proteome profiling of bladder tissues from rats experimentally infected with U. parvum in order to identify host cell processes perturbed by colonization with the microbe. Tissues were grouped into four categories: sham inoculated controls, animals that spontaneously cleared infection, asymptomatic urinary tract infection (UTI), and complicated UTI. One protein that was perturbed by infection (filamin A) was used to further elucidate the mechanism of U. parvum-induced disruption in human benign prostate cells (BPH-1). BPH-1 cells were evaluated by confocal microscopy, immunoblotting and ELISA.

Results: Bladder tissue from animals actively colonized with U. parvum displayed significant alterations in actin binding proteins (profilin 1, vinculin, α actinin, and filamin A) that regulate both actin polymerization and cell cytoskeletal function pertaining to focal adhesion formation and signal transduction (Fisher's exact test, P < 0.004; ANOVA, P < 0.02). This phenomenon was independent of clinical profile (asymptomatic vs. complicated UTI). We selected filamin A as a target for additional studies. In the BPH-1 model, we confirmed that U. parvum perturbed the regulation of filamin A. Specifically, infected BPH-1 cells exhibited a significant increase in filamin A phosphorylated at serine 2152 (P ≤ 0.01), which correlated with impaired proteolysis of the protein and its normal intracellular distribution.

Conclusion: Filamin A dynamics were perturbed in both models of infection. Phosphorylation of filamin A occurs in response to various cell signaling cascades that regulate cell motility, differentiation, apoptosis and inflammation. Thus, this phenomenon may be a useful molecular marker for identifying the specific host cell pathways that are perturbed during U. parvum infection.

Show MeSH
Related in: MedlinePlus