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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

A schematic diagram of the experimental design and overview of the results. (A) Lysates harvested from Burkholderia spp. infected RAW264.7 macrophages were arrayed on nitrocellulose coated slides. Each slide was incubated with one primary antibody with a total of 114 antibodies in the library. The primary antibody was detected by biotin-labeled secondary antibody, which, in turn, was recognized by IRDye680 fluorophore conjugated streptavidin. Signals were acquired, quantified, and analyzed according to materials and methods section. (B) RAW264.7 macrophages were infected at multiplicity of infection (MOI) of 10 with indicated Burkholderia spp. for 0.5, 1, 4, and 8 h. Lysates were harvested and subjected to reverse-phase protein microarray (RPMA) studies. A heat map of fold changes over untreated samples is depicted. Experiments were performed on two independent days and data from these repeat studies are shown.
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Figure 1: A schematic diagram of the experimental design and overview of the results. (A) Lysates harvested from Burkholderia spp. infected RAW264.7 macrophages were arrayed on nitrocellulose coated slides. Each slide was incubated with one primary antibody with a total of 114 antibodies in the library. The primary antibody was detected by biotin-labeled secondary antibody, which, in turn, was recognized by IRDye680 fluorophore conjugated streptavidin. Signals were acquired, quantified, and analyzed according to materials and methods section. (B) RAW264.7 macrophages were infected at multiplicity of infection (MOI) of 10 with indicated Burkholderia spp. for 0.5, 1, 4, and 8 h. Lysates were harvested and subjected to reverse-phase protein microarray (RPMA) studies. A heat map of fold changes over untreated samples is depicted. Experiments were performed on two independent days and data from these repeat studies are shown.

Mentions: To gain insight into host proteins that are differentially expressed or post-translationally modified following Burkholderia spp. infection, a high throughput RPMA-based screening platform was utilized. RAW264.7 macrophage were adopted due to its ability to phagocytose Burkholderia spp., which in turn exploits the host’s cellular systems by promoting host actin polymerization on the bacterial surface and inducing multinucleated giant cells (MNGCs) formation (Galyov et al., 2010; Pegoraro et al., 2014). Host responses to a collection of nine Burkholderia spp. originating from various geographical locations isolated from human, animals, or environmental sources were investigated. The ancestry for the majority of the strains is known along with their virulence profiles and genome sequences. RAW264.7 macrophage lysates harvested at various time points post Burkholderia spp. infection were individually arrayed onto nitrocellulose coated slides and probed with a total of 114 well characterized antibodies (Figure 1A). Statistical analysis revealed 25 candidates whose change in expression was twofold or higher in both replicates in any strain, at any time point and at MOI of 10 (Figure 1B; Supplementary Figure S1). At MOI of 1, very few robust changes were observed in host signaling molecules. Hence, subsequent studies were performed using MOI of 10. RPMA data analysis did not identify any proteins whose expression was considerably down regulated compared to uninfected and untreated control. The selected candidates were further characterized by performing traditional immunoblots using cell lysates collected from RAW264.7 macrophage treated with LPS, or infected with Bm ATCC23344, and Bp E8. Although not included in the initial RPMA, Bm 2002721278, an avirulent strain (personal communication), was included for purposes of comparative analyses of host signaling dynamics between virulent and avirulent Bm.


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)

A schematic diagram of the experimental design and overview of the results. (A) Lysates harvested from Burkholderia spp. infected RAW264.7 macrophages were arrayed on nitrocellulose coated slides. Each slide was incubated with one primary antibody with a total of 114 antibodies in the library. The primary antibody was detected by biotin-labeled secondary antibody, which, in turn, was recognized by IRDye680 fluorophore conjugated streptavidin. Signals were acquired, quantified, and analyzed according to materials and methods section. (B) RAW264.7 macrophages were infected at multiplicity of infection (MOI) of 10 with indicated Burkholderia spp. for 0.5, 1, 4, and 8 h. Lysates were harvested and subjected to reverse-phase protein microarray (RPMA) studies. A heat map of fold changes over untreated samples is depicted. Experiments were performed on two independent days and data from these repeat studies are shown.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: A schematic diagram of the experimental design and overview of the results. (A) Lysates harvested from Burkholderia spp. infected RAW264.7 macrophages were arrayed on nitrocellulose coated slides. Each slide was incubated with one primary antibody with a total of 114 antibodies in the library. The primary antibody was detected by biotin-labeled secondary antibody, which, in turn, was recognized by IRDye680 fluorophore conjugated streptavidin. Signals were acquired, quantified, and analyzed according to materials and methods section. (B) RAW264.7 macrophages were infected at multiplicity of infection (MOI) of 10 with indicated Burkholderia spp. for 0.5, 1, 4, and 8 h. Lysates were harvested and subjected to reverse-phase protein microarray (RPMA) studies. A heat map of fold changes over untreated samples is depicted. Experiments were performed on two independent days and data from these repeat studies are shown.
Mentions: To gain insight into host proteins that are differentially expressed or post-translationally modified following Burkholderia spp. infection, a high throughput RPMA-based screening platform was utilized. RAW264.7 macrophage were adopted due to its ability to phagocytose Burkholderia spp., which in turn exploits the host’s cellular systems by promoting host actin polymerization on the bacterial surface and inducing multinucleated giant cells (MNGCs) formation (Galyov et al., 2010; Pegoraro et al., 2014). Host responses to a collection of nine Burkholderia spp. originating from various geographical locations isolated from human, animals, or environmental sources were investigated. The ancestry for the majority of the strains is known along with their virulence profiles and genome sequences. RAW264.7 macrophage lysates harvested at various time points post Burkholderia spp. infection were individually arrayed onto nitrocellulose coated slides and probed with a total of 114 well characterized antibodies (Figure 1A). Statistical analysis revealed 25 candidates whose change in expression was twofold or higher in both replicates in any strain, at any time point and at MOI of 10 (Figure 1B; Supplementary Figure S1). At MOI of 1, very few robust changes were observed in host signaling molecules. Hence, subsequent studies were performed using MOI of 10. RPMA data analysis did not identify any proteins whose expression was considerably down regulated compared to uninfected and untreated control. The selected candidates were further characterized by performing traditional immunoblots using cell lysates collected from RAW264.7 macrophage treated with LPS, or infected with Bm ATCC23344, and Bp E8. Although not included in the initial RPMA, Bm 2002721278, an avirulent strain (personal communication), was included for purposes of comparative analyses of host signaling dynamics between virulent and avirulent Bm.

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