Date Published: June 14, 2018
Publisher: Public Library of Science
Author(s): Sirikamon Koosakulnirand, Phornpun Phokrai, Kemajittra Jenjaroen, Rosemary A. Roberts, Pongsak Utaisincharoen, Susanna J. Dunachie, Paul J. Brett, Mary N. Burtnick, Narisara Chantratita, R. Mark Wooten.
Burkholderia pseudomallei is a flagellated Gram-negative bacterium which is the causative agent of melioidosis. The disease poses a major public health problem in tropical regions and diabetes is a major risk factor. The high mortality rate of melioidosis is associated with severe sepsis which involves the overwhelming production of pro-inflammatory cytokines. Bacterial flagellar protein (flagellin) activates Toll-like receptor 5 (TLR5)-mediated innate immune signaling pathways and induces adaptive immune response. However, previous studies of TLR5 signaling in melioidosis have been performed using recombinant flagellin from Salmonella Typhimurium instead of B. pseudomallei. This study aimed to investigate human innate immune response and antibody response against a recombinant B. pseudomallei flagellin (rFliC). We prepared B. pseudomallei rFliC and used it to stimulate HEK-BlueTM-hTLR5 and THP1-DualTM cells to assess TLR5 activation. Subsequently, whole blood stimulation assays with rFliC were performed ex vivo. TLR5-flagellin interactions trigger activation of transcription factor NF-κB in HEK-BlueTM-hTLR5 cells. Pro-inflammatory cytokine (IL-1β, IL-6, and TNF-α) productions from whole blood in response to rFliC differed between fourteen healthy individuals. The levels of these cytokines changed in a dose and time-dependent manner. ELISA was used to determine rFliC-specific antibodies in serum samples from different groups of melioidosis patients and healthy subjects. IgG antibody to rFliC in melioidosis patients with diabetes were higher compared with non-diabetic patients. Our results show that B. pseudomallei flagellin is a potent immune stimulator and that the immune responses to rFliC are different among individuals. This may provide valuable insights toward the potential use of rFliC in vaccine development.
Burkholderia pseudomallei is a flagellated, environmental, Gram-negative bacterium which is the causative agent of melioidosis, a community-acquired infection that is endemic in Southeast Asia and northern Australia. The mortality rates of melioidosis vary between countries ranging from 14% in Australia to 40% in northeast Thailand [1–3]. Melioidosis is commonly associated with host risk factors, the majority of which is diabetes presenting in 23% to 60% of patients and type II diabetes is common. Clinical symptoms in melioidosis patients are varied, but often present as pneumonia, bacteremia, genitourinary infections, skin infections and abscesses in internal organs. In many cases, melioidosis can present as chronic and persistent infections [1, 3]. The high mortality rate of melioidosis is attributed to bacteremia and severe sepsis, which involves the overwhelming production of pro-inflammatory cytokines.
Previous studies have suggested B. pseudomallei flagellin is a potential vaccine candidate [10–13, 24]. The present study confirmed the previous studies that flagellin is a component of B. pseudomallei that activates human innate and adaptive immune responses [8, 25–27]. Our observation that rFliC activated HEK-BlueTM-hTLR5 cells confirmed that B. pseudomallei flagellin has TLR5-dependent immunostimulatory function, which is similar to FLA-ST although some differences between the immunoreactive epitopes between rFliC and FLA-ST has been reported. S. Typhimurium flagellin (FliC) contains dominant epitopes at residues 339–350 (FliC339-350) and 427–441(FliC427-441) within C-terminal region [28, 29] while dominant immunoreactive epitopes of B. pseudomallei flagellin are two peptides at positions F51-69 and F270-288 .