Role for the Src Family Kinase Fyn in Sphingolipid Acquisition by Chlamydiae

Abstract

The bacterial obligate intracellular pathogen Chlamydia trachomatis replicates within a membrane-bound vacuole termed the inclusion. From within this protective environment, chlamydiae usurp numerous functions of the host cell to promote chlamydial survival and replication. Here we utilized a small interfering RNA (siRNA)-based screening protocol designed to identify host proteins involved in the trafficking of sphingomyelin to the chlamydial inclusion. Twenty-six host proteins whose deficiency significantly decreased sphingomyelin trafficking to the inclusion and 16 proteins whose deficiency significantly increased sphingomyelin trafficking to the inclusion were identified. The reduced sphingomyelin trafficking caused by downregulation of the Src family tyrosine kinase Fyn was confirmed in more-detailed analyses. Fyn silencing did not alter sphingomyelin synthesis or trafficking in the absence of chlamydial infection but reduced the amount of sphingomyelin trafficked to the inclusion in infected cells, as determined by two independent quantitative assays. Additionally, inhibition of Src family kinases resulted in increased cellular retention of sphingomyelin and significantly decreased incorporation into elementary bodies of both C. trachomatis and Chlamydophila caviae.Chlamydiae are Gram-negative obligate intracellular bacteria that cause diseases with significant medical and economic impact. Distinct serologically defined variants of Chlamydia trachomatis are the leading cause of infectious blindness worldwide as well as of sexually transmitted disease in the Western world (52). Chlamydia species share a unique biphasic life cycle, alternating between infectious elementary bodies (EBs) and replicative reticulate bodies (RBs) (39). During its entire intracellular developmental cycle, C. trachomatis resides within a parasitophorous vacuole, termed an inclusion, which is not fusogenic with endocytic compartments but which acquires host-derived lipids, including sphingomyelin, cholesterol, neutral lipids, and phospholipids, via both vesicular and nonvesicular trafficking pathways (17).Chlamydiae accept lipid traffic from a number of cellular sources, including the Golgi apparatus (12,21,22), multivesicular bodies (MVBs) (48), lipid droplets (13, 29), and others (23, 58). Acquisition of sphingomyelin and cholesterol from the Golgi apparatus is initiated very early in the chlamydial developmental cycle, with incorporation of fluorescent sphingolipid analogs into the bacteria by 2 h postinfection (21). Chlamydial protein synthesis is required for the initiation of this interaction, presumably by modification of the inclusion membrane to become fusogenic with vesicular traffic from the Golgi apparatus (21,24,53). Specificity of this pathway is further demonstrated by the preferential acquisition of sphingomyelin from a basolaterally directed pathway that segregates the immediate products of ceramide metabolism, glucosylceramide and sphingomyelin, toward apical and basolateral surfaces, respectively (37). The molecula...

Vesicle-Associated Membrane Protein 4 and Syntaxin 6 Interactions at the Chlamydial Inclusion

“…Because sphingolipid acquisition is a critical step in the development of obligate intracellular chlamydial organisms (18,19), there are multiple compensatory mechanisms, as evident in the role of Src family kinase Fyn (62) and CERT (63,64), an endoplasmic reticulum-associated ceramide transfer protein, in chlamydial sphingomyelin acquisition. Data in this study identify the first SNARE protein involved in sphingomyelin trafficking to the chlamydial inclusion; however, there are other proteins involved in vesicle fusion that have been demonstrated to play a role in chlamydial sphingomyelin acquisition, such as small GTPase Rab proteins, which are involved in vesicle formation and trafficking (65).…”

Lipid acquisition by intracellular Chlamydiae

“…A recent siRNA screen to identify host proteins required for SM acquisition by C. trachomatis has also implicated Src family kinases (SFK), specifically Fyn, in SM acquisition (Mital and Hackstadt, 2011a). Activated Fyn and Src kinases localize in discrete microdomains at the inclusion that are enriched in cholesterol and overlap with a subset of Incs, including IncB, Inc101, Inc222 and Inc850 (Mital et al ., 2010).…”

“…Activated Fyn and Src kinases localize in discrete microdomains at the inclusion that are enriched in cholesterol and overlap with a subset of Incs, including IncB, Inc101, Inc222 and Inc850 (Mital et al ., 2010). Depletion of Fyn decreases C6‐NBD‐sphingolipid retention by both the inclusion and EBs; however, depletion of Fyn alone does not impair production of infectious progeny, indicating that SM trafficking mediated by Fyn signalling functions redundantly with other lipid trafficking pathways (Mital and Hackstadt, 2011a). SFK regulate microtubule‐ and dynein‐dependent trafficking of the inclusion to the microtubule organizing centre (MTOC) (Mital and Hackstadt, 2011b), therefore, it is speculated that Fyn may mediate linkage of the inclusion to the microtubule network and thereby intersect SM containing vesicles trafficking along microtubules.…”

Sphingolipid Trafficking and Purification in Chlamydia trachomatis–Infected Cells

“…Previous studies have demonstrated that in chlamydial infected cells labeled with C 6 -NBD-ceramide, the chlamydial inclusion intercepts Golgi-derived NBD-sphingomyelin (Hackstadt et al, 1996; Hackstadt et al, 1995; Moore et al, 2008; Wolf and Hackstadt, 2001). Most recently, methods developed from these initial studies helped to identify a role in SRC family kinases in the trafficking of sphingomyelin to the chlamydial inclusion (Mital and Hackstadt, 2011). Basic Protocol 1 outlines the steps for labeling cells with C 6 -NBD-ceramide to study lipid transfer and metabolism.…”