Effect of temperature on Burkholderia pseudomallei growth, proteomic changes, motility and resistance to stress environments.

Burkholderia pseudomallei is a flagellated, gram-negative environmental bacterium that causes melioidosis, a severe infectious disease of humans and animals in tropical areas. We hypothesised that B. pseudomallei may undergo phenotypic adaptation in response to an increase in growth temperature. We analysed the growth curves of B. pseudomallei strain 153 cultured in Luria-Bertani broth at five different temperatures (25 °C-42 °C) and compared the proteomes of bacteria cultured at 37 °C and 42 °C. B. pseudomallei exhibited the highest growth rate at 37 °C with modest reductions at 30 °C, 40 °C and 42 °C but a more marked delay at 25 °C. Proteome analysis revealed 34 differentially expressed protein spots between bacterial cultures at 42 °C versus 37 °C. These were identified as chaperones (7 spots), metabolic enzymes (12 spots), antioxidants (10 spots), motility proteins (2 spots), structural proteins (2 spots) and hypothetical proteins (1 spot). Of the 22 down-regulated proteins at 42 °C, redundancy in motility and antioxidant proteins was observed. qRT-PCR confirmed decreased expression of fliC and katE. Experiments on three B. pseudomallei strains demonstrated that these had the highest motility, greatest resistance to H2O2 and greatest tolerance to salt stress at 37 °C. Our data suggest that temperature affects B. pseudomallei motility and resistance to stress

Alterations in cellular proteome and secretome upon differentiation from monocyte to macrophage by treatment with phorbol myristate acetate: Insights into biological processes

[[sponsorship]]生物化學研究所[[note]]已出版;[SCI];有審查制度;具代表性[[note]]http://gateway.isiknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=Drexel&SrcApp=hagerty_opac&KeyRecord=1874-3919&DestApp=JCR&RQ=IF_CAT_BOXPLOT[[note]]http://gateway.isiknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=RID&SrcApp=RID&DestLinkType=FullRecord&DestApp=ALL_WOS&KeyUT=00027455240002

Alterations in Macrophage Cellular Proteome Induced by Calcium Oxalate Crystals: The Association of HSP90 and F‑Actin Is Important for Phagosome Formation

The presence of macrophages in renal interstitium is the key feature of progressive renal inflammation in kidney stone disease. However, response of macrophages to calcium oxalate monohydrate (COM) crystals, the major crystalline composition of kidney stone, remained unclear. This study aimed to investigate alterations in the cellular proteome of macrophages induced by COM crystals using a proteomics approach. U937-derived macrophages (by phorbol-12-myristate-13-acetate activation) were incubated without or with 100 μg/mL COM crystals for 24 h. Their cellular proteins were resolved by 2-DE (<i>n</i> = 10 gels; 5 were derived from 5 independent cultures in each group) and visualized with Deep Purple fluorescent dye. Spot matching, quantitative intensity analysis, and statistics revealed 18 differentially expressed protein spots, which were successfully identified by Q-TOF MS and MS/MS analyses. The altered levels of α-tubulin, β-actin and ezrin were validated by Western blot analysis. Protein interaction network analysis using STRING software showed that 90 kDa heat shock protein (HSP90) was associated with β-actin and α-tubulin (all these three proteins were increased in the COM-treated macrophages). Multiple immunofluorescence stainings confirmed the associations of HSP90 with filamentous form of actin (F-actin) and α-tubulin. However, only the association between HSP90 and F-actin was found on the phagosome membrane surrounding COM crystal, indicating that the association of HSP90 with F-actin, but not with α-tubulin, is important for phagosome formation. Silencing of HSP90 (siHSP90) reduced expression of cytoskeletal proteins and phagosome marker (Rab5) and successfully diminished COM crystal-induced phagocytosis and migration of macrophages. Our findings enlightened the significant role of these altered proteins, especially HSP90, in enhanced phagocytic activity of the COM-exposed macrophages

Alterations in Macrophage Cellular Proteome Induced by Calcium Oxalate Crystals: The Association of HSP90 and F‑Actin Is Important for Phagosome Formation

The presence of macrophages in renal interstitium is the key feature of progressive renal inflammation in kidney stone disease. However, response of macrophages to calcium oxalate monohydrate (COM) crystals, the major crystalline composition of kidney stone, remained unclear. This study aimed to investigate alterations in the cellular proteome of macrophages induced by COM crystals using a proteomics approach. U937-derived macrophages (by phorbol-12-myristate-13-acetate activation) were incubated without or with 100 μg/mL COM crystals for 24 h. Their cellular proteins were resolved by 2-DE (<i>n</i> = 10 gels; 5 were derived from 5 independent cultures in each group) and visualized with Deep Purple fluorescent dye. Spot matching, quantitative intensity analysis, and statistics revealed 18 differentially expressed protein spots, which were successfully identified by Q-TOF MS and MS/MS analyses. The altered levels of α-tubulin, β-actin and ezrin were validated by Western blot analysis. Protein interaction network analysis using STRING software showed that 90 kDa heat shock protein (HSP90) was associated with β-actin and α-tubulin (all these three proteins were increased in the COM-treated macrophages). Multiple immunofluorescence stainings confirmed the associations of HSP90 with filamentous form of actin (F-actin) and α-tubulin. However, only the association between HSP90 and F-actin was found on the phagosome membrane surrounding COM crystal, indicating that the association of HSP90 with F-actin, but not with α-tubulin, is important for phagosome formation. Silencing of HSP90 (siHSP90) reduced expression of cytoskeletal proteins and phagosome marker (Rab5) and successfully diminished COM crystal-induced phagocytosis and migration of macrophages. Our findings enlightened the significant role of these altered proteins, especially HSP90, in enhanced phagocytic activity of the COM-exposed macrophages

Antibodies to whole cell antigens of <i>B</i>. <i>pseudomallei</i> K96243 wild type and OPS mutant (<i>ΔwbiD</i> K96243).

<p>ELISAs were performed on the plates coated with antigens using serum from melioidosis patients, Thai healthy donors and U.S. healthy donors at dilution of 1:2000. Box plots represent 25^th and 75^th percentile boundaries in the box with the median line within the box; the whiskers indicate the 10^th and 90^th percentiles. The plots show OD 450 nm for each antigen.</p

Receiver Operating Characteristics (ROC) plots of the ELISAs of Hcp1, OPS and combined Hcp1/OPS with different groups of serum.

<p>(A) Serum form melioidosis patients versus Thai healthy donors, (B) Serum form melioidosis patients versus U.S. healthy donors. The serum samples were diluted 1:2000 for all ELISAs.</p

Scatterplots of individual antibody titers to Hcp1 and OPS in serum samples of 103 melioidosis patients who were survived within one year.

<p>(A) Sera from all time points, (B) sera of week 0, (C) sera of week 12 and (D) sera of week 52. For each plot, the relationship between variables as determined by linear regression (dotted line), Spearman correlation (correlation coefficient, rho) and P-value are shown.</p

Sensitivity and specificity of Hcp1-ELISA, OPS-ELISA and Hcp1/OPS-ELISA.

<p>The assay values were calculated from Thai patients who had melioidosis, tuberculosis, scrub typhus, leptospirosis and Thai healthy donors. The cut-off values with Thai healthy donors as controls were used at specificity of 95% for each assay.</p

Antibody titers to Hcp1 antigen (A) and OPS antigen (B) in melioidosis patients who survived or died.

<p>ELISAs were performed on the plates coated with antigens using undiluted sera and two-fold serially diluted sera from patients. Box plots represent 25^th and 75^th percentile boundaries in the box with the median line within the box; the whiskers indicate the 10^th and 90^th percentiles. The plots show antibody titers for each group of patients. The antibody titer was determined by ELISA using cut-off titers at specificity of 95%.</p

Antibody titers to Hcp1 antigen (A) and OPS antigen (B) in melioidosis patients with bacteremia and non-bacteremia patients.

<p>ELISAs were performed on the plates coated with antigens using undiluted sera and two-fold serially diluted sera from patients. Box plots represent 25^th and 75^th percentile boundaries in the box with the median line within the box; the whiskers indicate the 10^th and 90^th percentiles. The plots show antibody titers for each group of patients. The antibody titer was determined by ELISA using cut-off titers at specificity of 95%.</p