A Sugar Rush for Innate Immunity

Cell Host & Microbe

Previews A Sugar Rush for Innate Immunity Pontus Orning,1,2 Trude H. Flo,1 and Egil Lien1,2,* 1Centre of Molecular Inflammation Research, Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, 7491 Trondheim, Norway 2Program in Innate Immunity, Department of Medicine, Division of Infectious Diseases and Immunology, UMass Medical School, Worcester, MA 01605, USA *Correspondence: [email protected] https://doi.org/10.1016/j.chom.2018.09.017

Early detection of microbial patterns is a hallmark of innate immunity and essential for clearance of invading pathogens. A recent Nature publication by Zhou et al. (2018) has uncovered ALPK1 as a pattern recognition receptor for Gram-negative bacteria triggering NF-kB activation and identified the bacterial sugar ADP-Hep as its ligand. Innate immunity plays a crucial role as a first line of defense during microbial infection. A robust and rapid induction of the innate immune response is paramount for this defense. Upon infection, conserved and recognizable pathogenassociated molecular patterns (PAMPs) will allow innate immune cells to mount efficient protective responses to ensure that infection does not progress. These PAMPs are sensed through pattern recognition receptors (PRRs), and the discovery of a range of PRRs, including TLRs, was a major breakthrough in the field of immunity and for understanding how our cells distinguish between self and non-self. Shao and colleagues recently discovered a PRR and its specific carbohydrate PAMP (Zhou et al., 2018); this finding is a significant advance in our understanding of how innate immune cells combat bacterial infections. Their discovery offers insights into how cells can detect foreign metabolites in the defense against pathogens. By using combinatorial, genome-wide screens both from the bacterial side and from the host side, Zhou et al. (2018) were able to pinpoint the receptor and its exact ligand of a previously unknown immune pathway. Through the discovery of CRISPR and advancements in deep sequencing, these approaches hold immense potential for the way we study pathogen-host interactions. Bacterial secretion systems can greatly influence innate immune signaling (Ratner et al., 2017). Zhou et al. (2018) started out by performing a transposon screen in Type III secretion system (T3SS)-effector-protein deficient Yersinia pseudotuberculosis to detect bacterial components that trigger NF-kB activation independently of

T3SS effector manipulation. Out of 21,000 different mutants, they identified a mutation in the hldE gene encoding a protein responsible for synthesizing the sugar ADP-d/l-glycero-b-d-manno-heptose (ADP-Hep). This is an intermediate in the biosynthetic pathway of lipopolysaccharide (LPS), an essential component of the Gram-negative outer membrane— hence, a good candidate for a PAMP. They validated this hit by showing that addition of synthetic ADP-Hep directly to cells was sufficient for the sugar to enter into the cytosol and trigger NF-kB activation. The group then went on to perform a CRISPR screen on the host cells using NF-kB reporters as readout for activation following ADP-Hep stimulation. This resulted in the identification of Alphakinase1 (ALPK1), TRAF-interacting protein with forkhead-associated domain (TIFA), and TNF-receptor-associated factor (TRAF) 6 as key players in inducing NF-kB activation following ADP-Hep stimulation. ADP-Hep triggered association of the ALPK1, TIFA, and TRAF6, indicating that they might be forming a complex required for sensing ADP-Hep and activating NF-kB. By performing mass spectrometry, the authors identified direct binding of ADP-Hep to ALPK1 and elegantly followed up by determining the crystal structure of the ADPHep-bound ALPK1. This revealed that ADP-Hep activated ALPK1 by interrupting certain intra-molecular interactions close to the catalytic cleft of ALPK1. The authors proposed that binding of ADP-Hep caused a conformational change, exposing the catalytic cleft and allowing ALPK1 to phosphorylate and activate TIFA, which led to downstream NF-kB activation.

ADP-Hep dependent NF-kB activation after Y. pseudotuberculosis infection was also dependent on YopB, a bacterial protein which is a component of the T3SSinduced ‘‘pore’’ in the host cell. It is known that other PAMPs such as LPS and flagellin can be secreted through the T3SS needle, thereby activating cytosolic sensors. These findings could suggest that, even though ADP-Hep added directly to cells triggers ALPK1 activation, the major way that this occurs during bacterial infection is by transportation through the T3SS needle. Interestingly, binding and activation of ALPK1 was highly specific to one sugar intermediate of the LPS biosynthesis pathway, ADP-Hep, with neither its precursor nor its downstream product being able to bind and activate ALPK1. However, certain host adenylyltransferases were able to convert the precursor d-glycerob-d-manno-heptose 1,7-bisphosphate (HBP) into ADP-Hep-7-P, also capable of activating ALPK1. It could be that host immune cells can sense a range of bacterial metabolites using the same PRR pathway simply by converting different metabolites into components resembling a specific ligand. This is a fascinating concept. It is also noteworthy that ADP-Hep was capable of triggering ALPK1 by adding it extracellularly to cells, whereas HBP had to be electroporated into the host cell cytosol. This could indicate that there are specific ADP-Hep transporters shuttling the sugar into the host cell to be recognized by ALPK1. The functions of these proposed transporters are unknown, but it is possible that certain immune cells use them to constantly sample the surroundings for foreign metabolites, which could imply the presence of

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invading pathogens. This ALPK1, is it possible that could present a new way imother types of bacterial infecmune cells can recognize tions can lead to metabolic and combat microbial products being recognized by threats. A model of ALPK1 the same pathway? recognition of ADP-Hep and Immune cells have develHBP is shown in Figure 1. oped an intricate arsenal Finally, injection of ADPof PRRs that recognize a Hep into mice triggered plethora of different pathorobust neutrophil influx and gens. Given the widespread inflammation, demonstrating conservation and critical functhe in vivo significance of tion of many bacterial metabothis pathway. This was comlites, it makes sense that host pletely abolished in ALPK1cells would have evolved deficient mice. Mice lacking PRRs capable of detecting ALPK1 also appeared sensisome of these metabolites. tive to Burkholderia cenoceThe work by Zhou et al. (2018) pacia infection with dimindescribes the discovery of one ished cytokine responses such PRR capable of detecting and higher bacterial burden, a bacterial sugar molecule allowing the authors to (Figure 1). The study thereby adds to the numerous examascertain the involvement of ples of cytosolic detection ALPK1 in combating disease. of bacteria, emphasizes the Additionally, other recent importance of distinguishing studies also established the self from non-self, and will contribution of ALPK1 in spur further interest into detecinfection with Shigella flexneri tion of bacterial metabolites and Helicobacter pylori (Miliby cells. Innate immunity, get vojevic et al., 2017; Zimmerready for a new sugar rush! mann et al., 2017). All of these strains are LPS-containing, Figure 1. Bacterial Sugar Molecules Trigger NF-kB and Inflammation Gram-negative bacteria. ACKNOWLEDGMENTS The bacterial sugar ADP-Hep, an intermediate in the LPS biosynthetic Since ADP-Hep is part of pathway, can be transported into host cells by a type III secretion system (T3SS, right) or via an unknown pathway (middle) from T3SS-negative bacThe authors are supported by the LPS synthesis pathway, teria. ADP-Hep can then interact with ALPK1 to trigger TIFA-TRAF6- medifunding from the Research Council ALPK1 might play a role in ated activation of NF-kB and inflammation. The pathway can also be trigof Norway Center of Excellence the defense against other gered by the bacterial sugar HBP; HBP can also be transferred via an Funding Scheme, Project 223255/ unknown mechanism into the host cells, whereupon it can be metabolized by Gram-negatives as well. F50 (to P.O., T.H.F., and E.L.) and host adenylyltransferase enzymes into ADP-Hep 7-P to trigger ALPK1, TIFA, FRIMEDBIO 245663(to T.H.F.); NaIt is becoming increasingly and, subsequently, NF-kB. tional Institutes of Health grant clear that metabolism and AI129527 (to E.L.); the Norwegian certain metabolic compoCancer Society grant B05035/001(to nents are playing a major role in immunity. Based upon these findings, new ques- E.L.); the Liaison Committee between NTNU and Krebs cycle intermediates such as succi- tions can be raised about the pathway. the Central Norway Regional Health Authority grant 90176000 (to T.H.F.); and the Olav Thon Foundanate and itaconate have been shown to What is its importance in phagocytes tion (to T.H.F.). influence immunity in different ways (Mills compared to other potent sensor systems et al., 2016, 2018). The study from Zhou such as TLR4 signaling? The available REFERENCES et al. (2018) highlights another aspect of in vivo data surely suggest that the ALPK1 metabolism in immunity: the recognition can have an independent contribution. Barker, J.R., Koestler, B.J., Carpenter, V.K., Burof bacterial metabolites as PAMPs. It The significance could be tested in different dette, D.L., Waters, C.M., Vance, R.E., and ValdiR.H. (2013). STING-dependent recognition of has been previously proposed that bacte- models, with bacteria expressing LPS via, cyclic di-AMP mediates type I interferon responses rial cyclic di-AMP and other cyclic nucleo- structures with various abilities to trigger during Chlamydia trachomatis infection. MBio 4, tides can be detected by the innate im- TLR4. Furthermore, how is the interplay be- e00018–e13. mune protein STING as a way of tween this pathway and various T3SS Dey, B., Dey, R.J., Cheung, L.S., Pokkali, S., Guo, combating pathogens (Barker et al., effectors? Also, how is ADP-Hep being H., Lee, J.H., and Bishai, W.R. (2015). 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Open Sesame: New Keys to Unlocking the Gate to Norovirus Infection Marit Lingemann1 and Stefan Taube1,* 1Institute of Virology and Cell Biology, University of Luebeck, Luebeck, Germany *Correspondence: [email protected] https://doi.org/10.1016/j.chom.2018.09.018

Three recent papers highlight the impact of an entry receptor on murine norovirus pathogenesis. CD300lf was shown to be the first proteinaceous entry receptor for a norovirus, specialized intestinal epithelial cells constitute the reservoir for persistence, and a new link between the receptor and a lipid biosynthetic enzyme was revealed. Human noroviruses (HuNoVs), singlestranded, positive-sense RNA viruses in the Caliciviridae family, are a major cause of acute gastroenteritis world-wide and a substantial burden to public health. Studying the biology of HuNoV infection has been hampered by a fairly strict species tropism and the lack of robust and physiologically relevant cell culture models. Murine noroviruses (MNVs) have emerged as a model to unravel basic mechanisms of norovirus (NoV) biology and pathogenesis because they recapitulate many aspects of HuNoV infection and can be efficiently cultivated in vitro and studied in their natural small animal host. Identifying murine macrophages and dendritic cells as a primary source for MNV replication was a paradigm shift, as it was assumed that all enteric viruses, including HuNoVs, primarily target epithelial cells. Recent studies are beginning to elucidate the HuNoV cell tropism. Novel platforms to study HuNoV infection in a small animal model (Taube et al., 2013), a B cell model (Jones et al., 2014), and a threedimensional intestinal enteroid model (HIE) (Ettayebi et al., 2016) are being

developed and now reveal a dual tropism for cells of hematopoietic origin, including macrophages and B cells, as well as epithelial enterocytes. Cellular factors that determine the viral tropism are also slowly being discovered. Specifically, secreted and cell-associated carbohydrates, particularly histo-blood group antigens (HBGAs), are critical determinants for HuNoV infection in the human host and also affect HuNoV replication in cell culture (Jones et al., 2014; Ettayebi et al., 2016). However, expression of functional fucosyltransferase 2 (FUT2), the enzyme that transfers fucose to HBGA precursors, is not sufficient to render otherwise permissive cells susceptible, suggesting that the key barrier to HuNoV infection is entry. This suggests that HBGAs alone do not open the gate to HuNoV infection and that a bona fide entry receptor for HuNoV is still missing. Another co-factor is bile acid, which is required for strain-dependent HuNoV replication in the enteroid model (Ettayebi et al., 2016). Following glycan binding, caliciviruses are thought to engage a protein receptor

for viral entry. The first proteinaceous entry receptor was identified for feline calicivirus (FCV), a member of the vesivirus genus. The feline junctional adhesion molecules 1 (fJam1), an immunoglobulin-like protein that localizes to lung epithelial and endothelial cells, determines the cellular tropism of FCV. Binding of FCV to fJam1 induces a conformational change in the viral capsid, inducing flexibility of the protruding domain leading to loss of icosahedral symmetry. Its human counterpart hJam1 was later shown to confer susceptibility to yet another vesivirus, the human-pathogenic Hom-1 strain of the San Miguel sea lion virus (SMSLV) (Sosnovtsev et al., 2017). This indicates a broad role of Jam1 in the ability of vesiviruses to cross the species barrier. Recently, another immunoglobulin-like protein, murine CD300lf, and its paralog CD300ld were identified as functional receptors for MNV entry by means of a CRISPR-Cas9 technology (Orchard et al., 2016; Haga et al., 2016). CD300lf and CD300ld are both type I transmembrane proteins that share their luminal

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