Abstract
BackgroundBurkholderia pseudomallei is an intracellular bacteria causing Melioidosis, the disease widely disseminates in Southeast Asia and Northern Australia. B. pseudomallei has ability to invade various types of host cell and to interfere with host defense mechanisms, such as nitric oxide (NO). Due to the cross-talk among alternative killing mechanisms in host immune response against invading microbes, autophagy is the molecular mechanism belonging to intracellular elimination of eukaryotic cells that has been widely discussed. However, bacterial evasion strategy of B. pseudomallei and host-bacterial protein–protein interaction within autophagic machinery remain unknown.MethodsHere, we demonstrated the protein–protein interaction study between different strains of B. pseudomallei, including wild type PP844 and rpoS mutant, with autophagy-related protein LC3 that has been constructed, using the modified immunoaffinity hydrophobic chromatography based-technique. Liquid chromatography tandem-mass spectrometry (LC–MS/MS) analysis was utilized for identifying the eluted proteins obtained from the established column. In addition, the expression level of gene encoding candidate protein was predicted prior to verification using real-time quantitative reverse transcription PCR assay (RT-qPCR).ResultsLC3 recombinant proteins could be entrapped inside the column before encountering their bacterial interacting partners. Based on affinity interaction, the binding capacity of LC3 with antibody displayed over 50% readily for hydrophobically binding with bacterial proteins. Following protein identification, bacterial ATP-binding cassette (ABC) transporter periplasmic substrate-binding protein (BPSL2203) was identified as a candidate LC3-interacting protein, which was found only in B. pseudomallei wild type. Gene expression analysis and bioinformatics of BPSL2203 were validated the proteomic result which are suggesting the role of RpoS-dependent gene regulation.ConclusionsRemarkably, utilization of the modified immunoaffinity hydrophobic chromatography with LC–MS/MS is a convenient and reliable approach to a study in B. pseudomallei-LC3 protein–protein interaction.
Highlights
Burkholderia pseudomallei is an intracellular bacteria causing Melioidosis, the disease widely disseminates in Southeast Asia and Northern Australia
Induction of autophagy in U937 infected with B. pseudomallei Since B. pseudomallei PP844 has been studied about the ability to suppress the expression of inducible nitric oxide synthase (iNOS) rather than rpoS mutant in both phagocytic and non-phagocytic cells [11, 12], the induction of an alternative host killing mechanism as autophagy in elimination of intracellular pathogen, B. pseudomallei has been addressed in murine macrophage [36]
To investigate the role of autophagy induced by B. pseudomallei infected human macrophage U937cell line, the expression level of microtubule-associated protein light chain 3 (LC3), a key autophagy-related protein was determined
Summary
Burkholderia pseudomallei is an intracellular bacteria causing Melioidosis, the disease widely disseminates in Southeast Asia and Northern Australia. After internalization in phagocytic cells, B. pseudomallei is able to escape from intracellular phagocytic and endocytic vacuoles, and followed by inducing actin polymerization to facilitate intracellular bacterial motility [7]. This pathogen can inhibit host innate immune response by interfering the inducible nitric oxide synthase (iNOS) expression in mouse macrophage cell line (RAW 264.7) [8], and can induce the multinucleated giant cells (MNGCs) formation for distributing bacterial infection to the adjacent cells before undergoing apoptotic cell death [9, 10]. RpoS sigma factor, one bacterial virulence factor has been studied the regulation in MNGCs formation and iNOS expression in both phagocytic and non-phagocytic cells [11, 12]
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