Sphingomyelin Biosynthesis Is Essential for Phagocytic Signaling during Mycobacterium tuberculosis Host Cell Entry

Abstract

Phagocytosis by alveolar macrophages is the obligate first step in Mycobacterium tuberculosis (Mtb) infection, yet the mechanism underlying this process is incompletely understood. Here, we show that Mtb invasion relies on an intact sphingolipid biosynthetic pathway. Inhibition or knockout of early sphingolipid biosynthetic enzymes greatly reduces Mtb uptake across multiple phagocytic cell types without affecting other forms of endocytosis. While the phagocytic receptor dectin-1 undergoes normal clustering at the pathogen contact sites, sphingolipid biosynthetic mutant cells fail to segregate the regulatory phosphatase CD45 from the clustered receptors. Blocking sphingolipid production also impairs downstream activation of Rho GTPases, actin dynamics, and phosphoinositide turnover at the nascent phagocytic cup. Moreover, we found that production of sphingomyelin, not glycosphingolipids, is essential for Mtb uptake. Collectively, our data support a critical role of sphingomyelin biosynthesis in an early stage of Mtb infection and provide novel insights into the mechanism underlying phagocytic entry of this pathogen. IMPORTANCE Mycobacterium tuberculosis (Mtb) invades alveolar macrophages through phagocytosis to establish infection and cause disease. The molecular mechanisms underlying Mtb entry are still poorly understood. Here, we report that an intact sphingolipid biosynthetic pathway is essential for the uptake of Mtb by phagocytes. Disrupting sphingolipid production affects the segregation of the regulatory phosphatase CD45 from the nascent phagosome, a critical step in the progression of phagocytosis. We also show that blocking sphingolipid biosynthesis impairs activation of small GTPases and phosphoinositide turnover at the host-pathogen contact sites. Moreover, production of sphingomyelin, not glycosphingolipids, is critical for the phagocytic uptake of Mtb. These data demonstrate a vital role for sphingomyelin biosynthesis in an early step of Mtb infection, defining a potential target for antimycobacterial therapeutics.

The Squeaky Yeast Gets Greased: The Roles of Host Lipids in the Clearance of Pathogenic Fungi

“…Similarly, we and others demonstrate that sphingolipid biosynthesis is essential for phagocytic uptake and clearance of C. albicans and other microbial pathogens [38][39][40][41]. In particular, we demonstrated evidence that sphingolipid biosynthesis is essential for formation of the phagocytic synapse upon contact with Zymosan A, a model particle for fungal cells composed of β-1,3-glucans.…”

“…In particular, we demonstrated evidence that sphingolipid biosynthesis is essential for formation of the phagocytic synapse upon contact with Zymosan A, a model particle for fungal cells composed of β-1,3-glucans. In sphingolipid-deficient cells, we observe that the phagocytic receptor Dectin-1 and the anti-phagocytic phosphatase CD45 remain colocalized at the contact site [39]. Thus, membrane order and composition at the phagosomal synapse have significant influences on the clearance of fungal pathogens.…”

Sphingolipid metabolism during Toll‐like receptor 4 (TLR4)‐mediated macrophage activation

“…Mononuclear phagocytes rely on membrane remodelling during phagocytosis and endo‐lysosomal trafficking for eliminating pathogens and process/present antigens. Phagocytosis of pathogens, lysosomal stability, vesicular fusion, receptor‐mediated chemotaxis, autophagy and antigen presentation are all dependent on sphingolipid metabolism (Denard et al, 2019; Jongsma et al, 2021; Kirkegaard et al, 2010; Niekamp et al, 2021; Sims et al, 2010; Utermohlen et al, 2008). Notably, sphingolipids also regulate the TLR4‐mediated innate immune response in macrophages (Koberlin et al, 2015).…”

Multiplicity of Glycosphingolipid-Enriched Microdomain-Driven Immune Signaling

“…Recently, genome-wide CRISPR screening showed that the enzymes ceramide synthase, fatty acid elongase and sphingomyelin synthase 1 (SMS1) are involved in phagocytosis [173]. In addition, sphingomyelin biosynthesis was reported to be critical for the uptake of M. tuberculosis by human macrophages [174]. Quantitative lipidomic analysis demonstrated that ceramide synthase 2 (CerS2) is enriched in early phagosomes and that the amounts of C24 ceramides are increased in late phagosomes [175].…”