A DUF4148 family protein produced inside RAW264.7 cells is a critical Burkholderia pseudomallei virulence factor

Abstract

Burkholderia pseudomallei: is the etiological agent of the disease melioidosis and is a Tier 1 select agent. It survives and replicates inside phagocytic cells by escaping from the endocytic vacuole, replicating in the cytosol, spreading to other cells via actin polymerization and promoting the fusion of infected and uninfected host cells to form multinucleated giant cells. In this study, we utilized a proteomics approach to identify bacterial proteins produced inside RAW264.7 murine macrophages and host proteins produced in response to B. pseudomallei infection. Cells infected with B. pseudomallei strain K96243 were lysed and the lysate proteins digested and analyzed using nanoflow reversed-phase liquid chromatography and tandem mass spectrometry. Approximately 160 bacterial proteins were identified in the infected macrophages, including BimA, TssA, TssB, Hcp1 and TssM. Several previously uncharacterized B. pseudomallei proteins were also identified, including BPSS1996 and BPSL2748. Mutations were constructed in the genes encoding these novel proteins and their relative virulence was assessed in BALB/c mice. The 50% lethal dose for the BPSS1996 mutant was approximately 55-fold higher than that of the wild type, suggesting that BPSS1996 is required for full virulence. Sera from B. pseudomallei-infected animals reacted with BPSS1996 and it was found to localize to the bacterial surface using indirect immunofluorescence. Finally, we identified 274 host proteins that were exclusively present or absent in infected RAW264.7 cells, including chemokines and cytokines involved in controlling the initial stages of infection.

Deciphering the human antibody response against Burkholderia pseudomallei during melioidosis using a comprehensive immunoproteome approach

“…Further examples, pH stress and/or osmolarity stress by NaCl, provoked the secretion of type 2 and 3 secretion system-dependent proteins ( 71 , 77 ) and we found strong antibody response against BPSS1512 (TssM) and BPSS1529 (BipD), proteins secreted by a type 2 or 3 secretion system, respectively. These proteins are important for the survival inside cells because they are involved in the modulation of cell immunity or the phagosomal escape ( 78 – 80 ), which represent conditions of temporarily increased osmolarity and low pH.…”

Glycometabolism change during Burkholderia pseudomallei infection in RAW264.7 cells by proteomic analysis

“…The bioinformatics analysis showed that many proteins involved in glycometabolism are changed when infected with B. pseudomallei HNBP001, which contain proteins involved in glycolysis, carbon metabolism, galactose metabolism, pentose phosphate pathway, and the fructose and mannose metabolism pathway. In early study, Welkos et al 27 have identified 160 B. pseudomallei ’s proteins in the infected RAW 264.7 cells, and 274 host proteins that were exclusively present or absent in infected cells. However, they focused on B. pseudomallei ’s proteins, constructed mutation genes and assessed their relative virulence.…”

Burkholderia pseudomallei Complex Subunit and Glycoconjugate Vaccines and Their Potential to Elicit Cross-Protection to Burkholderia cepacia Complex

“…Alkyl hydroperoxide reductases such as AhpC in B. pseudomallei are frequently upregulated in bacteria during the intracellular phase of infection to survive host-derived hydrogen peroxide [ 224 ]. As a result, natural exposure to such intracellular bacteria is often associated with robust immune responses to these proteins, making them popular candidate antigens for a variety of pathogenic bacteria [ 225 , 226 , 227 ].…”