Rv3091, An Extracellular Patatin-Like Phospholipase in Mycobacterium tuberculosis, Prolongs Intracellular Survival of Recombinant Mycolicibacterium smegmatis by Mediating Phagosomal Escape.

Ziyin Cui,Guanghui Dang,Ningning Song,Hongxiu Liu,Xinxin Zang,Siguo Liu,Yingying Cui,Zhongxing Wang,Zhe Li

doi:10.3389/fmicb.2020.532371

Abstract

Patatin-like phospholipases (PLPs) are important virulence factors of many pathogens. However, there are no prevailing studies regarding PLPs as a virulence factor of Mycobacterium tuberculosis (Mtb). Analysis of Rv3091, a putative protein of Mtb, shows that it belongs to the PLPs family. Here, we cloned and expressed the rv3091 gene in Mycobacterium smegmatis and, subsequently, conducted protein purification and characterization. We show that it possesses phospholipase A1, phospholipase A2, and lipase activity. We confirm the putative active site residues, namely, Ser214 and Asp407, using site directed mutagenesis. The Rv3091 is an extracellular protein that alters the colony morphology of M. smegmatis. The presence of Rv3091 enhances the intracellular survival capability of M. smegmatis in murine peritoneal macrophages. Additionally, it promotes M. smegmatis phagosomal escape from macrophages. Moreover, Rv3091 significantly increased the survival of M. smegmatis and aggravated lesions in C57BL/6 J murine lungs in vivo. Taken together, our results indicate that Rv3091 as an extracellular PLP that is critical to the pathogenicity of mycobacterium as it allows mycobacterium to utilize phospholipids for its growth and provides resistance to phagosome killing, resulting in its enhanced intracellular survival.

Highlights

Tuberculosis (TB) is a severe consumptive disease that is caused by infection with Mycobacterium tuberculosis (Mtb)
We study here the rv3091 gene of Mtb H37Rv, which has been previously described as a putative patatin-like protein superfamily gene in National Center for Biotechnology Information (NCBI; Wijeyesakere et al, 2014; Madeira et al, 2016)
The proteins that share consensus motifs are thought to be essential for patatin-like family of phospholipases (Vanrheenen et al, 2006; Wilson and Knoll, 2018), which include four conserved sequence blocks with four conserved residues, Gly186-Ser214-Gly377-Asp407, in blocks I, II, III, and IV (Figure 1A)

Summary

Introduction

Tuberculosis (TB) is a severe consumptive disease that is caused by infection with Mycobacterium tuberculosis (Mtb). Phospholipase Rv3091 Prolongs Intracellular Survival and treatment programs over an extended period of time, the disease still has not been completely eradicated (Colditz et al, 1994). The main reason is that Mtb is an intracellular pathogen with a complex structure, a unique secretory system, and various immune evasion mechanisms (Gupta et al, 2012; Stoop et al, 2012; Kallenius et al, 2016). Mtb mainly occurs in macrophages, where they survive and replicate without being killed (Dey and Bishai, 2014). Its intracellular survival plays an essential role in the pathogenesis of Mtb; the underlying mechanisms and specific factors remain unclear (Li et al, 2019)

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Conclusion