Investigating the effects of host factors (proteins and non-proteins) on mycobacteria

Abstract

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis, is one of the leading causes of death due to a single infectious agent and results in more than 1 million human deaths every year. M.tb infection of the host initiates a local inflammatory response, resulting in the migration of a number of host plasma protein and non-protein factors to the site of infection. In addition, some of these factors are also produced locally at the site of infection. It is envisaged that these host factors are likely to come in direct contact with M.tb and immune cells and may modulate the outcome of the infection. In this study, a number of host factors including transferrin, lactoferrin, fibrinogen, C-reactive protein, alpha-2-macroglobulin (α2M), vitronectin, plasminogen, low-density lipoprotein (LDL), high-density lipoprotein (HDL), serotonin, L-alpha dipalmitoyl phosphatidylcholine (DPPC) and platelet activating factor C-16 (PAF C-16) were screened in vitro for their direct effect on the growth of mycobacteria using M.smegmatis as a model. As a result of this screening, PAF C-16, a phospholipid compound was identified that directly inhibited the growth of M.smegmatis and M.bovis BCG in a dose and time-dependent manner. Use of a range of PAF C-16 structural analogues, including Lyso-PAF, PAF C-18, Hexanolamino PAF, 2-O-methyl PAF & Pyrrolidino PAF, revealed that small modifications in structure did not alter the direct growth inhibition property of PAF C-16 and similar levels of M.smegmatis and M.bovis BCG growth inhibition were observed as compared to PAF C-16. Structural dissection of PAF C-16 suggested that the attachment of carbon tail to the glycerol backbone via ether bond at sn-1 position was important for its direct growth inhibition activity against mycobacteria. Microscopy and flow cytometry with PAF C-16 treated M.smegmatis and M.bovis BCG showed damage to the bacterial cell membrane. The addition of membrane-stabilizing agents, α-tocopherol, tween-80 and tween-20, partially mitigated the growth inhibitory effect of PAF C-16. These results suggested that the growth inhibition activity of PAF C-16 against mycobacteria is most likely due to its detergent-like effect, resulting in damage to the bacterial cell membrane. PAF C-16 and its structural analogues were also investigated for their effect on the growth of intracellular M.smegmatis inside THP1 cells. In vitro, PAF C-16, PAF C-18 and Hexanolamino PAF inhibited the growth of intracellular M.smegmatis, whereas, analogues such as Lyso-PAF and 2-O-methyl PAF failed to show any growth inhibitory effect, suggesting that the presence of acetyl group at sn-2 position was important for growth inhibition of intracellular M.smegmatis. Use of PAF receptor antagonists partially mitigated the inhibitory effect of PAF C-16 on the growth of intracellular M.smegmatis, suggesting this inhibition was through receptor-mediated signalling pathways. Blocking of PAF C-16 signalling pathway components such as phospholipase C and phospholipase A2, resulted in the increased survival of intracellular M.smegmatis. Arachidonic acid, a product of PAF C-16 signalling pathway directly inhibited the growth of M.smegmatis. Furthermore, inhibition of iNOS enzyme and antibody-mediated neutralization of TNF-α partially mitigated the inhibitory effect of PAF C-16 on intracellular M.smegmatis growth, suggesting that the production of NO and TNF-α were also involved in PAF C-16 induced intracellular growth inhibition. Overall, this study has identified PAF C-16, its structural analogues such as Lyso-PAF, PAF C-18, Hexanolamino PAF and other compounds including 1-O-hexadecyl-sn-glycerol, miltefosine and hexadecyl lactate with novel anti-mycobacterial activity. Further investigations are needed to demonstrate their effectiveness against M.tb both in vitro and in animal models to assess their therapeutic potential as anti-TB drugs.