A role for tetraspanin proteins in regulating fusion induced by Burkholderia thailandensis

Abstract

Burkholderia pseudomallei is the causative agent of melioidosis, a disease with high morbidity that is endemic in South East Asia and northern Australia. An unusual feature of the bacterium is its ability to induce multinucleated giant cell formation (MNGC), which appears to be related to bacterial pathogenicity. The mechanism of MNGC formation is not fully understood, but host cell factors as well as known bacterial virulence determinants are likely to contribute. Since members of the tetraspanin family of membrane proteins are involved in various types of cell:cell fusion, their role in MNGC formation induced by Burkholderia thailandensis, a mildly pathogenic species closely related to B. pseudomallei, was investigated. The effect of antibodies to tetraspanins CD9, CD81, and CD63 in MNGC formation induced by B. thailandensis in infected mouse J774.2 and RAW macrophage cell lines was assessed along with that of recombinant proteins corresponding to the large extracellular domain (EC2) of the tetraspanins. B. thailandensis-induced fusion was also examined in macrophages derived from CD9 null and corresponding WT mice, and in J774.2 macrophages over-expressing CD9. Antibodies to CD9 and CD81 promoted MNGC formation induced by B. thailandensis, whereas EC2 proteins of CD9, CD81, and CD63 inhibited MNGC formation. Enhanced MNGC formation was observed in CD9 null macrophages, whereas a decrease in MNGC formation was associated with overexpression of CD9. Overall our findings show that tetraspanins are involved in MNGC formation induced by B. thailandensis and by implication, B. pseudomallei, with CD9 and CD81 acting as negative regulators of this process.

Tetraspanins are involved in Burkholderia pseudomallei-induced cell-to-cell fusion of phagocytic and non-phagocytic cells

“…The inhibition of B. pseudomallei- induced MNGC formation by CD9 and CD81 GST-EC2 proteins is consistent with their effect on this process in B. thailandensis infected mouse macrophages 16 . However, our previous report demonstrated that in this system, anti-CD9 and anti-CD81 MAbs enhanced MNGC formation, while here, the same antibodies inhibited B. pseudomallei- induced MNGC formation (Figs.…”

“…We have also recently demonstrated that reagents targeting tetraspanins CD9, CD63, and CD81 could affect B. thailandensis -induced MNGC in mouse macrophage cell lines. In brief, recombinant proteins corresponding to the EC2s of these tetraspanins inhibited MNGC formation while antibodies to CD9 and CD81 enhanced MNGC formation, and a macrophage cell line generated from a CD9KO mouse showed enhanced MNGC formation 16 . This led us to hypothesize that tetraspanins might play a role in B. pseudomallei infection and bacterial spreading between host cells during melioidosis.…”

“…In order to investigate the role of tetraspanins in B. pseudomallei infection, we used monoclonal antibodies (MAbs) specific to the large extracellular EC2 domain of tetraspanins and recombinant EC2 proteins of CD9, CD63, and CD81 to pretreat a human epithelial cell line A549 and a mouse macrophage cell line J774A.1 before infection. Mouse macrophage cell lines have been widely used as the in vitro model for B. pseudomallei and B. thailandensis infection and MNGC formation 16 – 21 . The mouse tetraspanin CD9 is about 90% homology to Homo sapiens CD9 and has been used to represent mammalian cells in sperm-egg fusion assay 22 , 23 .…”

The roles of tetraspanins in bacterial infections

“…In addition, a significant increase in the bacterially induced cell‐to‐cell fusion enhancing MNGC formation was apparent in CD9 null mouse macrophages, whereas the MNGC formation decreased in cells overexpressing CD9. These findings indicate that CD9 and CD81 act as negative regulators by preventing cell‐to‐cell fusion process and therefore the formation of MNGC structures (Figure 2A) (Elgawidi et al, 2020). Future studies are now required to understand how (a) B. thailandensis can regulate the process of MNGC formation by affecting the function and/or expression of Tspans, (b) to discriminate the direct contribution of B. thailandensis infection in MNGC formation from that of host Tspans and (c) to explain the observations that CD9 deletion and CD9 or CD81 inhibition by neutralising antibodies increases rather than decreases the fusion between cells.…”

Recent advancements in the understanding of tetraspanin functions

“…In this study, comparative analyses using CD9 low-or high-expressing cells suggest that a specific tetraspanin expression optimum promotes the entry process of the pathogen. The inhibitory role of CD9 and CD81 during pathogen infection is also shown by Elgawidi and colleagues [13]. Burkholderia thailandensis is able to induce the formation of multinucleated giant cells where these tetrapanins are involved.…”