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A reverse-phase protein microarray-based screen identifies host signaling dynamics upon Burkholderia spp. infection.

Chiang CY, Uzoma I, Lane DJ, Memišević V, Alem F, Yao K, Kota KP, Bavari S, Wallqvist A, Hakami RM, Panchal RG - Front Microbiol (2015)

Bottom Line: The lack of effective therapeutic treatments poses serious public health threats.Reverse-phase protein microarray technology was previously proven to identify and characterize novel biomarkers and molecular signatures associated with infectious disease and cancer.Modulating the inflammatory response by perturbing their activities may provide therapeutic routes for future treatments.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Translational Sciences Division, United States Army Medical Research Institute of Infectious Diseases, Frederick MD, USA.

ABSTRACT
Burkholderia is a diverse genus of gram-negative bacteria that causes high mortality rate in humans, equines and cattle. The lack of effective therapeutic treatments poses serious public health threats. Developing insights toward host-Burkholderia spp. interaction is critical for understanding the pathogenesis of infection as well as identifying therapeutic targets for drug development. Reverse-phase protein microarray technology was previously proven to identify and characterize novel biomarkers and molecular signatures associated with infectious disease and cancer. In the present study, this technology was utilized to interrogate changes in host protein expression and phosphorylation events in macrophages infected with a collection of geographically diverse strains of Burkholderia spp. The expression or phosphorylation state of 25 proteins was altered during Burkholderia spp. infections of which eight proteins were selected for further characterization by immunoblotting. Increased phosphorylation of AMPK-α1, Src, and GSK3β suggested the importance of their roles in regulating Burkholderia spp. mediated innate immune response. Modulating the inflammatory response by perturbing their activities may provide therapeutic routes for future treatments.

No MeSH data available.


Related in: MedlinePlus

Burkholderia spp. induces NF-κB mediated responses. (A) The phosphorylation state of indicated proteins acquired by RPMA. (B) Samples were prepared as described in Figure 2B. The expression or phosphorylation state of indicated proteins was evaluated by immunoblotting, followed by (C) densitometry quantification. Immunoblots in Figures 2–4 were performed concurrently with the same sample and loading control, GAPDH. The immunoblot data is representative of three independent trials.
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Figure 3: Burkholderia spp. induces NF-κB mediated responses. (A) The phosphorylation state of indicated proteins acquired by RPMA. (B) Samples were prepared as described in Figure 2B. The expression or phosphorylation state of indicated proteins was evaluated by immunoblotting, followed by (C) densitometry quantification. Immunoblots in Figures 2–4 were performed concurrently with the same sample and loading control, GAPDH. The immunoblot data is representative of three independent trials.

Mentions: NF-κB pathway constitutes an important part of the host defense against pathogens (Akira et al., 2006). Several key regulators of NF-κB signaling pathway were also identified by RPMA. These include the phosphorylated forms of Src and GSK3β (Sasaki et al., 2005; Steinbrecher et al., 2005; Han et al., 2010). RPMA detected changes in Src and GSK3β phosphorylation (Figure 3A). The activities of these proteins were later characterized by immunoblotting. Phosphorylated Src, an integrin associated non-receptor tyrosine kinase, accumulated over time throughout the course of the study (Figures 3B,C). Furthermore, the level of phosphorylated GSK3β at serine 9 and 21, conferring the inactive form, increased over time in RAW264.7 macrophages treated with LPS, infected by Bm ATCC23344 and Bp E8. No apparent accumulation of phosphorylated GSK3β was observed in Bm 2002721278 infected RAW264.7 macrophages (Figures 3B,C).


A reverse-phase protein microarray-based screen identifies host signaling dynamics upon Burkholderia spp. infection.

Chiang CY, Uzoma I, Lane DJ, Memišević V, Alem F, Yao K, Kota KP, Bavari S, Wallqvist A, Hakami RM, Panchal RG - Front Microbiol (2015)

Burkholderia spp. induces NF-κB mediated responses. (A) The phosphorylation state of indicated proteins acquired by RPMA. (B) Samples were prepared as described in Figure 2B. The expression or phosphorylation state of indicated proteins was evaluated by immunoblotting, followed by (C) densitometry quantification. Immunoblots in Figures 2–4 were performed concurrently with the same sample and loading control, GAPDH. The immunoblot data is representative of three independent trials.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4515560&req=5

Figure 3: Burkholderia spp. induces NF-κB mediated responses. (A) The phosphorylation state of indicated proteins acquired by RPMA. (B) Samples were prepared as described in Figure 2B. The expression or phosphorylation state of indicated proteins was evaluated by immunoblotting, followed by (C) densitometry quantification. Immunoblots in Figures 2–4 were performed concurrently with the same sample and loading control, GAPDH. The immunoblot data is representative of three independent trials.
Mentions: NF-κB pathway constitutes an important part of the host defense against pathogens (Akira et al., 2006). Several key regulators of NF-κB signaling pathway were also identified by RPMA. These include the phosphorylated forms of Src and GSK3β (Sasaki et al., 2005; Steinbrecher et al., 2005; Han et al., 2010). RPMA detected changes in Src and GSK3β phosphorylation (Figure 3A). The activities of these proteins were later characterized by immunoblotting. Phosphorylated Src, an integrin associated non-receptor tyrosine kinase, accumulated over time throughout the course of the study (Figures 3B,C). Furthermore, the level of phosphorylated GSK3β at serine 9 and 21, conferring the inactive form, increased over time in RAW264.7 macrophages treated with LPS, infected by Bm ATCC23344 and Bp E8. No apparent accumulation of phosphorylated GSK3β was observed in Bm 2002721278 infected RAW264.7 macrophages (Figures 3B,C).

Bottom Line: The lack of effective therapeutic treatments poses serious public health threats.Reverse-phase protein microarray technology was previously proven to identify and characterize novel biomarkers and molecular signatures associated with infectious disease and cancer.Modulating the inflammatory response by perturbing their activities may provide therapeutic routes for future treatments.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Translational Sciences Division, United States Army Medical Research Institute of Infectious Diseases, Frederick MD, USA.

ABSTRACT
Burkholderia is a diverse genus of gram-negative bacteria that causes high mortality rate in humans, equines and cattle. The lack of effective therapeutic treatments poses serious public health threats. Developing insights toward host-Burkholderia spp. interaction is critical for understanding the pathogenesis of infection as well as identifying therapeutic targets for drug development. Reverse-phase protein microarray technology was previously proven to identify and characterize novel biomarkers and molecular signatures associated with infectious disease and cancer. In the present study, this technology was utilized to interrogate changes in host protein expression and phosphorylation events in macrophages infected with a collection of geographically diverse strains of Burkholderia spp. The expression or phosphorylation state of 25 proteins was altered during Burkholderia spp. infections of which eight proteins were selected for further characterization by immunoblotting. Increased phosphorylation of AMPK-α1, Src, and GSK3β suggested the importance of their roles in regulating Burkholderia spp. mediated innate immune response. Modulating the inflammatory response by perturbing their activities may provide therapeutic routes for future treatments.

No MeSH data available.


Related in: MedlinePlus