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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 calpastatin 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 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. Calpastatin was detected with rabbit polyclonal antibody (red). BPH-1 nuclei and U. parvum (white arrow) were identified with DAPI stain (blue). Western blot analysis for the detection of calpastatin was performed on cytosolic (cyt) and nuclear (nuc) fractions from uninfected cells (BPH), infected (UP), uninfected supernatant treated (BPH S) and infected supernatant treated (UP S) cells. M equals molecular weight marker. GAPDH was used as a loading control and a confirmation that the nuclear fraction was not contaminated with cytosolic proteins. Quantitation of calpastatin in cytosolic fractions was performed by densitometry. The average quantity within each blot was normalized by dividing the average quantity of calpastatin protein band by the average quantity of the GAPDH band within each blot. Values represent the mean ± SD of 2 biological replicates from 2 independent experiments.
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Figure 4: Intracellular distribution and quantification of calpastatin 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 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. Calpastatin was detected with rabbit polyclonal antibody (red). BPH-1 nuclei and U. parvum (white arrow) were identified with DAPI stain (blue). Western blot analysis for the detection of calpastatin was performed on cytosolic (cyt) and nuclear (nuc) fractions from uninfected cells (BPH), infected (UP), uninfected supernatant treated (BPH S) and infected supernatant treated (UP S) cells. M equals molecular weight marker. GAPDH was used as a loading control and a confirmation that the nuclear fraction was not contaminated with cytosolic proteins. Quantitation of calpastatin in cytosolic fractions was performed by densitometry. The average quantity within each blot was normalized by dividing the average quantity of calpastatin protein band by the average quantity of the GAPDH band within each blot. Values represent the mean ± SD of 2 biological replicates from 2 independent experiments.

Mentions: Recent studies have shown that infection of host cells with Mycoplasma hyorhinis caused inhibition of calpain activity through upregulation of its inhibitor, calpastatin [25]. Therefore, we also evaluated the effect of U. parvum infection on the intracellular distribution and the relative concentrations of calpain and calpastatin in BPH-1 cells. We did not detect a difference in the intracellular distribution of calpain among uninfected BPH-1 cells, U. parvum infected cells, and cells incubated with supernatants by confocal microscopy (data not shown). We also did not observe any appreciable differences in the amount of calpain present within the cytosolic and nuclear fractions of these cells by Western blot (data not shown). However, we did observe differences in both the intracellular distribution of calpastatin and its relative concentration among the groups. Specifically, U. parvum infected cells exhibited large aggregates of calpastatin within the nucleus, and these aggregates were more prominent than what was observed in the other groups (Figure 4A). Moreover, Western blot showed that calpastatin was reduced in the cytosolic fraction of U. parvum infected cells (Figure 4B), which was confirmed by densitometry (Figure 4C.)


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 calpastatin 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 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. Calpastatin was detected with rabbit polyclonal antibody (red). BPH-1 nuclei and U. parvum (white arrow) were identified with DAPI stain (blue). Western blot analysis for the detection of calpastatin was performed on cytosolic (cyt) and nuclear (nuc) fractions from uninfected cells (BPH), infected (UP), uninfected supernatant treated (BPH S) and infected supernatant treated (UP S) cells. M equals molecular weight marker. GAPDH was used as a loading control and a confirmation that the nuclear fraction was not contaminated with cytosolic proteins. Quantitation of calpastatin in cytosolic fractions was performed by densitometry. The average quantity within each blot was normalized by dividing the average quantity of calpastatin protein band by the average quantity of the GAPDH band within each blot. Values represent the mean ± SD of 2 biological replicates from 2 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Intracellular distribution and quantification of calpastatin 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 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. Calpastatin was detected with rabbit polyclonal antibody (red). BPH-1 nuclei and U. parvum (white arrow) were identified with DAPI stain (blue). Western blot analysis for the detection of calpastatin was performed on cytosolic (cyt) and nuclear (nuc) fractions from uninfected cells (BPH), infected (UP), uninfected supernatant treated (BPH S) and infected supernatant treated (UP S) cells. M equals molecular weight marker. GAPDH was used as a loading control and a confirmation that the nuclear fraction was not contaminated with cytosolic proteins. Quantitation of calpastatin in cytosolic fractions was performed by densitometry. The average quantity within each blot was normalized by dividing the average quantity of calpastatin protein band by the average quantity of the GAPDH band within each blot. Values represent the mean ± SD of 2 biological replicates from 2 independent experiments.
Mentions: Recent studies have shown that infection of host cells with Mycoplasma hyorhinis caused inhibition of calpain activity through upregulation of its inhibitor, calpastatin [25]. Therefore, we also evaluated the effect of U. parvum infection on the intracellular distribution and the relative concentrations of calpain and calpastatin in BPH-1 cells. We did not detect a difference in the intracellular distribution of calpain among uninfected BPH-1 cells, U. parvum infected cells, and cells incubated with supernatants by confocal microscopy (data not shown). We also did not observe any appreciable differences in the amount of calpain present within the cytosolic and nuclear fractions of these cells by Western blot (data not shown). However, we did observe differences in both the intracellular distribution of calpastatin and its relative concentration among the groups. Specifically, U. parvum infected cells exhibited large aggregates of calpastatin within the nucleus, and these aggregates were more prominent than what was observed in the other groups (Figure 4A). Moreover, Western blot showed that calpastatin was reduced in the cytosolic fraction of U. parvum infected cells (Figure 4B), which was confirmed by densitometry (Figure 4C.)

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