Enteric pathogens deploy cell cycle inhibiting factors to block the bactericidal activity of Perforin-2

Abstract

Perforin-2 (MPEG1) is an effector of the innate immune system that limits the proliferation and spread of medically relevant Gram-negative, -positive, and acid fast bacteria. We show here that a cullin-RING E3 ubiquitin ligase (CRL) complex containing cullin-1 and βTrCP monoubiquitylates Perforin-2 in response to pathogen associated molecular patterns such as LPS. Ubiquitylation triggers a rapid redistribution of Perforin-2 and is essential for its bactericidal activity. Enteric pathogens such as Yersinia pseudotuberculosis and enteropathogenic Escherichia coli disarm host cells by injecting cell cycle inhibiting factors (Cifs) into mammalian cells to deamidate the ubiquitin-like protein NEDD8. Because CRL activity is dependent upon NEDD8, Cif blocks ubiquitin dependent trafficking of Perforin-2 and thus, its bactericidal activity. Collectively, these studies further underscore the biological significance of Perforin-2 and elucidate critical molecular events that culminate in Perforin-2-dependent killing of both intracellular and extracellular, cell-adherent bacteria.DOI: http://dx.doi.org/10.7554/eLife.06505.001

Killing of Microbes and Cancer by the Immune System with Three Mammalian Pore-Forming Killer Proteins

“…He subsequently employed electron microscopy and analytical ultracentrifugation, among other techniques, to determine the reason for this unexpected property (4, 15). With the electron microscope, he observed structures (1) that were nearly identical to the images of the entire C5b–9 membrane attack complex (MAC) that had been previously reported (4, 16). Yet, Eckhard’s samples were comprised solely of C9 .…”

“…The three pore formers are complement component C9 secreted into blood and interstitial fluid, Perforin-1 expressed in cytotoxic T cells and natural killer (NK) cells, and Perforin-2 expressed by all phagocytic and all non-phagocytic cells examined to date. C9, Perforin-1, and Perforin-2 share the conserved membrane attack complex perforin (MACPF) domain, which functions in these proteins as a pore-forming killer domain (Figure 1) (1–3). These MACPF domains consist of 316–372 amino acids and although they share low overall homology (<50%) they contain the signature motifs that define them as members of the MACPF family (see Prosite entries PS00279 and PDOC00251 at ).…”

“…Abbreviations: TSP1, thrombospondin type-1 repeat; LDL, low-density lipoprotein receptor class A repeat; EGF, epidermal growth factor-like domain; C2, calcium-dependent phospholipid-binding domain. This figure was adapted from McCormack et al (1). …”

“…With the necessary cell line and reagents in place, the group was able to use electron microscopy to image pore-like structures in the membranes of transfected cells and in the membranes of bacteria exposed to cells expressing MPEG1 (14). Moreover, Eckhard and collaborators found ample evidence that the pore-forming protein was a killer, bactericidal protein (1, 14, 55, 56). These observations supported Eckhard’s hypothesis that MPEG1 kills its targets by forming clusters of pores on target membranes, similar to C9 and Perforin-1.…”

MPEG1/perforin-2 mutations in human pulmonary nontuberculous mycobacterial infections

“…In the resting state, perforin-2 resides in the endoplasmic reticulum, Golgi, and early endosomal membranes (2). Upon infection, perforin-2 is monoubiquitinated in response to LPS and IFNγ, and redistributes within the cell to the endosomal/phagosomal bodies that contain phagocytosed bacteria (1,2,4). Following this relocalization, perforin-2 polymerizes and likely refolds to form large clusters of pores that render attacked organisms susceptible to the effects of lysozyme, reactive oxygen species, and nitric oxide, contributing to effective microbial elimination (1,2).…”