AUTHOR=Riaz Muhammad Suleman , Kaur Anuvinder , Shwayat Suha Nadim , Behboudi Shahriar , Kishore Uday , Pathan Ansar Ahmed 

TITLE=Dissecting the Mechanism of Intracellular Mycobacterium smegmatis Growth Inhibition by Platelet Activating Factor C-16

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.01046

DOI=10.3389/fmicb.2020.01046

ISSN=1664-302X

ABSTRACT=<p><italic>Mycobacterium tuberculosis</italic> (<italic>M.tb</italic>) infection results in approximately 1.3 million human deaths each year. <italic>M.tb</italic> resides primarily inside macrophages, and maintains persistent infection. In response to infection and inflammation, platelet activating factor C-16 (PAF C-16), a phospholipid compound, is released by various cells including neutophils and monocytes. We have recently shown that PAF C-16 can directly inhibit the growth of two representative non-pathogenic mycobacteria, <italic>Mycobacterium bovis BCG</italic> and <italic>Mycobacterium smegmatis</italic> (<italic>M. smegmatis</italic>), by damaging the bacterial cell membrane. Here, we have examined the effect of PAF C-16 on <italic>M. smegmatis</italic> residing within macrophages, and identified mechanisms involved in their growth inhibitory function. Our results demonstrated that exogenous PAF C-16 inhibited the growth of <italic>M. smegmatis</italic> inside phagocytic cells of monocytic cell line, THP-1; this effect was partially blocked by PAF receptor antagonists, suggesting the involvement of PAF receptor-mediated signaling pathways. Arachidonic acid, a downstream metabolite of PAF C-16 signaling pathway, directly inhibited the growth of <italic>M. smegmatis in vitro</italic>. Moreover, the inhibition of phospholipase C and phospholipase A<sub>2</sub> activities, involved in PAF C-16 signaling pathway, increased survival of intracellular <italic>M. smegmatis</italic>. Interestingly, we also observed that inhibition of inducible nitric oxide synthase (iNOS) enzyme and antibody-mediated neutralization of TNF-α partially mitigated the intracellular growth inhibitory effect of PAF C-16. Use of a number of PAF C-16 structural analogs, including Lyso-PAF, 2-O-methyl PAF, PAF C-18 and Hexanolamino PAF, revealed that the presence of acetyl group (CH<sub>3</sub>CO) at <italic>sn</italic>-2 position of the glycerol backbone of PAF is important for the intracellular growth inhibition activity against <italic>M. smegmatis</italic>. Taken together, these results suggest that exogenous PAF C-16 treatment inhibits intracellular <italic>M. smegmatis</italic> growth, at least partially, in a nitric oxide and TNF-α dependent manner.</p>