Limits...
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.

Show MeSH

Related in: MedlinePlus

Intracellular distribution intact and cleaved filamin A in BPH-1 cells. Cells were exposed to sterile 10B broth, 109 CFU of U. parvum (UP), or cell culture supernatant (super) for 72 hours before examination by confocal microscopy (A), Western blot (B) and densitometry (C). Confocal images were taken at 600× magnification and the scale bar is equal to 10 μm. Cleaved and intact filamin A (Fil A) were stained with rabbit- anti C terminal filamin A (red). Intact Fil A was stained with mouse anti-filamin 1 (green). BPH-1 nuclei and U. parvum (white arrow) were identified with DAPI stain (blue). Western blot analysis for the detection of cleaved filamin A was performed on cytosolic (cyt) and nuclear (nuc) fractions from uninfected (BPH) and infected (UP) cells. The black arrow is delineating GAPDH, which was used as a loading control and a confirmation that the nuclear fraction was not contaminated with cytosolic proteins. Quantitation of intact filamin A was performed by densitometry of the cytosolic fractions of uninfected and U. parvum infected cells. The average quantity within each blot was normalized by dividing the average quantity of filamin A protein band by the average quantity of the GAPDH band. Values represent the mean ± SD of 3 replicates from 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3107797&req=5

Figure 2: Intracellular distribution intact and cleaved filamin A in BPH-1 cells. Cells were exposed to sterile 10B broth, 109 CFU of U. parvum (UP), or cell culture supernatant (super) for 72 hours before examination by confocal microscopy (A), Western blot (B) and densitometry (C). Confocal images were taken at 600× magnification and the scale bar is equal to 10 μm. Cleaved and intact filamin A (Fil A) were stained with rabbit- anti C terminal filamin A (red). Intact Fil A was stained with mouse anti-filamin 1 (green). BPH-1 nuclei and U. parvum (white arrow) were identified with DAPI stain (blue). Western blot analysis for the detection of cleaved filamin A was performed on cytosolic (cyt) and nuclear (nuc) fractions from uninfected (BPH) and infected (UP) cells. The black arrow is delineating GAPDH, which was used as a loading control and a confirmation that the nuclear fraction was not contaminated with cytosolic proteins. Quantitation of intact filamin A was performed by densitometry of the cytosolic fractions of uninfected and U. parvum infected cells. The average quantity within each blot was normalized by dividing the average quantity of filamin A protein band by the average quantity of the GAPDH band. Values represent the mean ± SD of 3 replicates from 3 independent experiments.

Mentions: We evaluated the intracellular distribution of filamin A in uninfected and infected BPH-1 cells by confocal microscopy and immunoblotting. Uninfected cells showed a punctate pattern of filamin A within the nucleus (see Figure 2a) that was seen only with the antibody that recognized the cleaved form of filamin A. When a monoclonal antibody that recognized only intact filamin A was used, nuclear filamin A appeared to be present in a striated form that resembled actin stress fibers. This was confirmed with co-localization studies of filamin A with polymerized actin as shown in Additional file 3. This intracellular distribution of filamin A was consistent with previous reports in normal prostate cells [23,24]. Interestingly, a significant proportion of U. parvum infected BPH-1 cells exhibited a marked reduction in the punctate nuclear staining of filamin A coupled with a concurrent increased of filamin A in the cytosol (P < 0.0001). Specifically, 68 ± 10% of U. parvum infected cells as compared with 14.4 ± 4% of uninfected cells showed this abnormal phenotype.


Ureaplasma parvum infection alters filamin A dynamics in host cells.

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

Intracellular distribution intact and cleaved filamin A in BPH-1 cells. Cells were exposed to sterile 10B broth, 109 CFU of U. parvum (UP), or cell culture supernatant (super) for 72 hours before examination by confocal microscopy (A), Western blot (B) and densitometry (C). Confocal images were taken at 600× magnification and the scale bar is equal to 10 μm. Cleaved and intact filamin A (Fil A) were stained with rabbit- anti C terminal filamin A (red). Intact Fil A was stained with mouse anti-filamin 1 (green). BPH-1 nuclei and U. parvum (white arrow) were identified with DAPI stain (blue). Western blot analysis for the detection of cleaved filamin A was performed on cytosolic (cyt) and nuclear (nuc) fractions from uninfected (BPH) and infected (UP) cells. The black arrow is delineating GAPDH, which was used as a loading control and a confirmation that the nuclear fraction was not contaminated with cytosolic proteins. Quantitation of intact filamin A was performed by densitometry of the cytosolic fractions of uninfected and U. parvum infected cells. The average quantity within each blot was normalized by dividing the average quantity of filamin A protein band by the average quantity of the GAPDH band. Values represent the mean ± SD of 3 replicates from 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Intracellular distribution intact and cleaved filamin A in BPH-1 cells. Cells were exposed to sterile 10B broth, 109 CFU of U. parvum (UP), or cell culture supernatant (super) for 72 hours before examination by confocal microscopy (A), Western blot (B) and densitometry (C). Confocal images were taken at 600× magnification and the scale bar is equal to 10 μm. Cleaved and intact filamin A (Fil A) were stained with rabbit- anti C terminal filamin A (red). Intact Fil A was stained with mouse anti-filamin 1 (green). BPH-1 nuclei and U. parvum (white arrow) were identified with DAPI stain (blue). Western blot analysis for the detection of cleaved filamin A was performed on cytosolic (cyt) and nuclear (nuc) fractions from uninfected (BPH) and infected (UP) cells. The black arrow is delineating GAPDH, which was used as a loading control and a confirmation that the nuclear fraction was not contaminated with cytosolic proteins. Quantitation of intact filamin A was performed by densitometry of the cytosolic fractions of uninfected and U. parvum infected cells. The average quantity within each blot was normalized by dividing the average quantity of filamin A protein band by the average quantity of the GAPDH band. Values represent the mean ± SD of 3 replicates from 3 independent experiments.
Mentions: We evaluated the intracellular distribution of filamin A in uninfected and infected BPH-1 cells by confocal microscopy and immunoblotting. Uninfected cells showed a punctate pattern of filamin A within the nucleus (see Figure 2a) that was seen only with the antibody that recognized the cleaved form of filamin A. When a monoclonal antibody that recognized only intact filamin A was used, nuclear filamin A appeared to be present in a striated form that resembled actin stress fibers. This was confirmed with co-localization studies of filamin A with polymerized actin as shown in Additional file 3. This intracellular distribution of filamin A was consistent with previous reports in normal prostate cells [23,24]. Interestingly, a significant proportion of U. parvum infected BPH-1 cells exhibited a marked reduction in the punctate nuclear staining of filamin A coupled with a concurrent increased of filamin A in the cytosol (P < 0.0001). Specifically, 68 ± 10% of U. parvum infected cells as compared with 14.4 ± 4% of uninfected cells showed this abnormal phenotype.

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