Peptidyl-Prolyl Isomerase ppiB Is Essential for Proteome Homeostasis and Virulence in Burkholderia pseudomallei

Abstract

Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to Southeast Asia and northern Australia. Mortality rates in these areas are high even with antimicrobial treatment, and there are few options for effective therapy. Therefore, there is a need to identify antibacterial targets for the development of novel treatments. Cyclophilins are a family of highly conserved enzymes important in multiple cellular processes. Cyclophilins catalyze the cis-trans isomerization of xaa-proline bonds, a rate-limiting step in protein folding which has been shown to be important for bacterial virulence. B. pseudomallei carries a putative cyclophilin B gene, ppiB, the role of which was investigated. A B. pseudomallei ΔppiB (BpsΔppiB) mutant strain demonstrates impaired biofilm formation and reduced motility. Macrophage invasion and survival assays showed that although the BpsΔppiB strain retained the ability to infect macrophages, it had reduced survival and lacked the ability to spread cell to cell, indicating ppiB is essential for B. pseudomallei virulence. This is reflected in the BALB/c mouse infection model, demonstrating the requirement of ppiB for in vivo disease dissemination and progression. Proteomic analysis demonstrates that the loss of PpiB leads to pleiotropic effects, supporting the role of PpiB in maintaining proteome homeostasis. The loss of PpiB leads to decreased abundance of multiple virulence determinants, including flagellar machinery and alterations in type VI secretion system proteins. In addition, the loss of ppiB leads to increased sensitivity toward multiple antibiotics, including meropenem and doxycycline, highlighting ppiB inhibition as a promising antivirulence target to both treat B. pseudomallei infections and increase antibiotic efficacy.

What Are We Missing by Using Hydrophilic Enrichment? Improving Bacterial Glycoproteome Coverage Using Total Proteome and FAIMS Analyses

“…Bacterial strains and growth conditions: [63]. B. cenocepacia J2315; B. dolosa AU0158 and B. ubonensis MSMB22 strains were grown overnight on LB agar at 37°C as previously described [31].…”

Open Database Searching Enables the Identification and Comparison of Bacterial Glycoproteomes without Defining Glycan Compositions Prior to Searching

“…fetus NCTC 10842 was grown on Brain-Heart Infusion medium (Hardy Diagnostics) with 5% defibrinated horse blood (Hemostat, Dixon, CA) under microaerobic conditions (10% CO 2 , 5% O 2 , 85% N 2 ) at 37 °C as previously reported ( 25 ). Burkholderia pseudomallei K96243 was grown as previously reported ( 54 ) in Luria Bertani (LB) broth. All other bacterial strains were grown overnight on LB agar at 37 °C as previously described ( 37 ).…”

“…(25). Burkholderia pseudomallei K96243 was grown as previously reported (54) in Luria Bertani (LB) broth. All other bacterial strains were grown overnight on LB agar at 37°C as previously described (37).…”

Open database searching enables the identification and comparison of bacterial glycoproteomes without defining glycan compositions prior to searching

“…fetus NCTC 10842 was grown on Brain-Heart Infusion medium (Hardy Diagnostics) with 5% defibrinated horse blood (Hemostat, Dixon, CA) under microaerobic conditions (10% CO 2 , 5% O 2 , 85% N 2 ) at 37 °C as previously reported [25]. Burkholderia pseudomallei K96243 was grown as previously reported [51] in Luria Bertani (LB) broth. All other bacterial strains were grown overnight LB agar at 37 °C as previously described [37].…”