Characterisation of dysregulated proteins in macrophages infected with Mycobacterium smegmatis focusing on matrix metalloproteases and their effectors

Seele, Palesa  Pamela

Characterisation of dysregulated proteins in macrophages infected with Mycobacterium smegmatis focusing on matrix metalloproteases and their effectors

Doctoral Thesis

2019

Abstract

Cavitation is a key facilitator in transmission of Mycobacterium tuberculosis (M. tb). Upregulation of matrix metalloproteases (MMPs) has been documented in patients with tuberculosis (TB), while their tissue inhibitor (TIMPs) levels remained the same. Animals which can develop cavities have well-conserved MMP-1 orthologs suggesting a pivotal role of MMP-1 in cavitation. The migration of immune cells to the site of infection and maturation of the granuloma is associated with MMP-9 expression. Our understanding of the phenotypic changes induced by Mycobacterium smegmatis (M. smeg) in THP-1 macrophages is central to understanding its avirulent nature especially its effects on MMPs. The aim of this study was to evaluate the role of MMP-1 and MMP-9, and their effectors in macrophages infected with M. smeg. Differentiated THP-1 monocytes were incubated in serum-free media with or without bacilli. Thereafter, the secretome and lysate were harvested at different time points. The activity of MMPs was analysed by zymography. The activity of MMP-1 and MMP-9 were specifically determined using an MMP-1 fluorogenic assay and a non-fluorogenic MMP-9 substrate monitored using the HPLC. Discovery proteomics was performed for the 18-hour time point with the use of mass spectrometry. The generated data was used to evaluate dysregulated proteins and those that act as upstream and downstream effectors of MMPs. The phenotypic changes induced by M. smeg were also analysed. In addition to that, the hosts’ response to lipoarabinomannan H37Rv (LAM) treatment was assessed by discovery proteomics and zymography. There was an increase in gelatinase activity of secreted MMP-9 which was maintained between the 1 and 18-hour time points. The fold difference in activity between uninfected and infected declined at 24 hours, and at the 72-hour time point the uninfected was slightly higher versus the infected. The data also suggests a switch in the proteolytic repertoire of the macrophages between the 6- and 18-hour time points to one that potentially generates the same degradation products as the uninfected macrophages. The intracellular gelatinase activity of MMP-9 (82 kDa) was not significantly altered by the M. smeg infection, in fact the activity was slightly higher in the uninfected in the 18 and 24-hour time points. In contrast to MMP-9, MMP-1 was secreted in the later time points and was significantly decreased by the infection. This supports the postulation that upregulation of MMP-1 is specific for M. tb infection. The proteomics data depict significant upregulation of MMP-9 in the lysate and secretome, while TIMP-1 was exclusively expressed and secreted by infected macrophages, validating the non-destructive ECM phenotype induced by M. smeg. The dysregulation of IL-1β and COX pathways were implicated in the overexpression of MMP-9, as well as tRNA aminoacylation in alternative splicing of MMPs. The GO enrichment of exosomes is postulated to play a role in the recycling of MMP-9. Intercellular communication is hypothesised to be delivered to neighbouring cells through exosomes carrying DNA, RNA, proteins and DNA/RNA binding proteins, and via signalling scaffolds formed by the 14-3-3 proteins amongst others. LAM treatment did not induce dysregulation in the activity of expressed and secreted MMP-9, however, TIMP-1 was upregulated explaining the lack of differential gelatinase activity between treated and non-treated macrophages. The C-type mannose receptor 2 (MRC2) and C-type lectin domain family 11 (CLEC11A) were only expressed by the LAM-treated macrophages and may partake in the recognition and uptake of M. tb. Interestingly, the data indicates the presence of chromatin in the secretome which may be responsible for the formation of extracellular traps (NETs) and facilitating the transport of LAM across the glomerular basal membrane (GBM) through exosomes. Inhibition of MMP activity by TIMPs could result in decreased aggregation of NETS (aggNETS) that trap the LAM from being transported by binding to the chromatin. This decreases the concentration of LAM in urine and means MMP inhibitors that chelate the active-site zinc could decrease the sensitivity of urine-LAM detection kits.

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