Abstract
Mycobacterium tuberculosis (M.tb) infection results in approximately 1.3 million human deaths each year. M.tb 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, Mycobacterium bovis BCG and Mycobacterium smegmatis (M. smegmatis), by damaging the bacterial cell membrane. Here, we have examined the effect of PAF C-16 on M. smegmatis residing within macrophages, and identified mechanisms involved in their growth inhibitory function. Our results demonstrated that exogenous PAF C-16 inhibited the growth of M. smegmatis 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 M. smegmatis in vitro. Moreover, the inhibition of phospholipase C and phospholipase A2 activities, involved in PAF C-16 signaling pathway, increased survival of intracellular M. smegmatis. 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 (CH3CO) at sn-2 position of the glycerol backbone of PAF is important for the intracellular growth inhibition activity against M. smegmatis. Taken together, these results suggest that exogenous PAF C-16 treatment inhibits intracellular M. smegmatis growth, at least partially, in a nitric oxide and TNF-α dependent manner.
Highlights
Mycobacterium tuberculosis (M.tb) belongs to the acid-fast group of bacteria and causes an infectious disease in humans, known as Tuberculosis (TB), which mostly affects the respiratory system
Intracellular growth inhibition assays were performed by treating M. smegmatis infected THP-1 cells with Platelet activating factor (PAF) C-16 at concentrations ranging between 0.001–10 μg/ml (1.90 nM– 19.09 μM) for 24 h and the resulting growth inhibitory effect was determined by counting the number of surviving colony forming units (CFUs) on LB agar plates
Prior treatment of M. smegmatis infected THP-1 cells with ABT-491 or WEB-2086 (Test-1 and Test-2, Figure 4) at a concentration of 2 μg/ml partially mitigated the inhibitory effect of PAF C-16 on the growth of intracellular M. smegmatis, as indicated by an increase in the of number CFUs (20–24.5%) compared to 1 μg/ml PAF C-16 treated only condition (n = 4, independent experiments) (Figures 4A,B)
Summary
Mycobacterium tuberculosis (M.tb) belongs to the acid-fast group of bacteria and causes an infectious disease in humans, known as Tuberculosis (TB), which mostly affects the respiratory system. Over the course of time, this latent M.tb infection can reactivate into active disease, and provides a vast reservoir for the spread of M.tb infection New challenges such as HIV-1 and M.tb co-infections in TB patients, multi-drug resistant (MDR), and extensively drug resistant (XDR) strains of M.tb have emerged recently. Mycobacterium tuberculosis infection of the host evokes localized inflammation in the lungs, resulting in the migration of different immune cells and the leakage of plasma proteins and non-proteinaceous factors at the site of infection due to changes in vascular permeability (Sherwood and Toliver-Kinsky, 2004; Amaral et al, 2016) Phospholipids, such as PAF C-16 and proteins such as C1q, are synthesized by the host’s immune cells, which are present at the site of infection (Camussi et al, 1987; Kaul and Loos, 1995). The underlying mechanisms of PAF C-16 induced growth inhibition of intracellular M. smegmatis were investigated
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