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An immune response with a sweet tooth

A previously unknown pathway that enables mammalian cells to recognize infection and trigger an immune response requires a kinase enzyme in the host cell to bind a sugar molecule produced by infecting bacteria.
John-Demian Sauer is in the Department of Medical Microbiology and Immunology, University of Wisconsin–Madison, Madison, Wisconsin 53706, USA.
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Bacterial infections are a major cause of disease and death worldwide. The innate branch of the mammalian immune system, which recognizes and reacts to general characteristics of pathogenic organisms, has a key protective role. Writing in Nature, Zhou et al.1 describe a mechanism by which the innate immune system is activated in response to bacterial sugar molecules. This finding broadens our understanding of the types of molecule that can be recognized as hallmarks of bacterial infection and the host proteins that can recognize such molecules.

A key advance in our understanding of how the innate immune system functions was the identification of proteins called pattern-recognition receptors (PRRs), which recognize ‘non-self’ molecules termed pathogen-associated molecular patterns (PAMPs). Beginning with the Toll and Toll-like receptor PRRs24 in the late 1990s, the identification of PRRs and the PAMPs that they recognize has proceeded at a breathtaking pace.

A key function of PRRs is to help drive the expression of secreted proteins called cytokines, which alert the immune system to the presence of infection. The transcription factor NF-κB is a central regulator of cytokine expression. Zhou and colleagues studied human cells grown in vitro to try to identify pathways that activate NF-κB in response to infection by the bacterium Yersinia pseudotuberculosis. This bacterium has a needle-like, multiprotein structure called a type III secretion system (T3SS), which is required for the direct transfer of bacterial proteins into host cells. T3SSs are evolutionarily conserved in many pathogenic bacteria.

Zhou et al. took an unbiased approach and screened a collection of Y. pseudotuberculosis genetic mutants to identify bacterial genes that are linked to NF-κB activation in response to infection. This led the authors to focus on the enzyme HldE, which catalyses steps in the biosynthetic pathway that generates lipopolysaccharide (LPS) molecules. LPS is an essential component of the cell surface of a subset of bacterial pathogens called Gram-negative bacteria.

Using genetically mutated bacteria and purified sugar molecules, the authors sought to pinpoint the molecules in the LPS biosynthetic pathway that stimulate NF-κB activation. They found that the presence of bacterial sugars, including ADP-β-d-manno-heptose (ADP-Hep) and d-glycero-β-d-manno-heptose 1,7-bisphosphate (HBP), in the host-cell cytoplasm triggered NF-κB activation. This is consistent with a study5 of Neisseria meningitidis bacteria that demonstrated that HBP can trigger NF-κB responses in host cells. Crucially, Zhou et al. showed that ADP-Hep is 100 times more potent than is HBP at activating NF-κB. They found that addition of ADP-Hep to the extracellular environment of host cells can activate NF-κB, suggesting that dedicated host-cell transporter proteins deliver ADP-Hep to the host’s cytoplasm.

No PRR was known to recognize ADP-Hep. To search for one, the authors used a gene-editing approach to conduct a screen in which they generated random mutations in host cells and tested whether the mutations affected ADP-Hep recognition. They uncovered two candidate genes that respectively encode the kinase enzyme ALPK1 and the protein TIFA, and showed that these are required for NF-κB activation in response to ADP-Hep in host cells (Fig. 1). A previous study had revealed5 that TIFA is required for recognition of HBP from N. meningitidis. ALPK1 and TIFA signalling has also been linked to HBP-dependent host activation of NF-κB in response to infection by the bacteria Shigella flexneri6 and Helicobacter pylori7. Using biochemical approaches, Zhou and colleagues demonstrated that ADP-Hep binds directly to the amino terminus of ALPK1. The authors solved the X-ray crystal structure of ALPK1 in a complex with ADP-Hep, and validated their structural model by testing the effect of mutations in ALPK1 that were predicted to impair its binding to ADP-Hep.

Figure 1 | Bacterial sugars trigger a host immune response. a, Zhou et al.1 demonstrate that in bacteria such as Yersinia pseudotuberculosis, which has a multiprotein complex called a type III secretion system (T3SS), and in other bacterial species lacking a T3SS, the sugar molecule ADP-β-d-manno-heptose (ADP-Hep) can enter a host cell, by an unknown route (possibly through a transporter protein), and can trigger a signalling pathway that drives inflammation. When ADP-Hep enters the host cell, it binds to ALPK1, which activates the protein TIFA by adding a phosphate group (P) to it. The downstream signalling pathway, not all the steps of which are shown, leads to activation of the protein NF-κB, which drives the expression of cytokine proteins that stimulate an immune response to the infection. b, The authors also report that if the bacterially produced sugar d-glycero-β-d-manno-heptose 1,7-bisphosphate (HBP) enters the host cell (by a route that remains to be determined), it can be converted by host enzymes of the NMNAT family into the molecule ADP-heptose 7-P. This binds to ALPK1 and activates the same pathway as that activated by ADP-Hep.

Zhou et al. also generated ALPK1-deficient mice. The NF-κB-dependent production of cytokines was significantly reduced in these animals after challenge with either ADP-Hep or the pathogenic bacterium Burkholderia cenocepacia, compared with results seen in animals that were not deficient in ALPK1. Moreover, the number of bacteria in the lungs of mice infected with B. cenocepacia was higher in ALPK1-deficient animals than in wild-type mice.

Perhaps Zhou and colleagues’ most striking finding is that mammalian adenylyltransferase enzymes, specifically those of the NMNAT family, catalyse a reaction that converts HBP into a molecule called ADP-heptose 7-P, which can act as a ligand by binding to ALPK1. Previous work5 had suggested that HBP is a PAMP that can directly activate NF-κB. Although HBP can be defined as a PAMP, given that it is a bacterially derived molecule that triggers a host response, Zhou and colleagues’ data indicate that HBP must be converted to ADP-heptose 7-P by host enzymes to trigger this response. The authors report slight differences in the way in which ADP-Hep and ADP-heptose 7-P bind to ALPK1, and use these differences to demonstrate why ADP-Hep and not HBP or ADP-heptose 7-P is the relevant ligand for ALPK1-mediated NF-κB activation, at least in Y. pseudotuberculosis infection.

Zhou and colleagues’ findings have important implications. Evidence that ADP-Hep is a PAMP adds to a growing awareness that bacterial metabolites can act as PAMPs. Given that ADP-Hep is needed to synthesize an essential component of the outer membrane of most Gram-negative bacteria, this makes it an ideal PAMP. However, it is not known how this molecule, which is normally found inside the bacterium, reaches the cytoplasm of the host cell. In Y. pseudotuberculosis, this process requires the T3SS, although it is unclear whether ADP-Hep is actively transported or accidentally leaks through the T3SS, or whether it enters by the pores that the T3SS generates in the host-cell membrane.

The authors report that bacterial species that lack a T3SS can still trigger the ALPK1 pathway in an ADP-Hep-dependent manner, consistent with the ability of purified ADP-Hep to activate the pathway by an extracellular route. This suggests that a dedicated transport system might exist that allows the host cell to sample its extracellular surroundings for the presence of this PAMP, similar to the way in which certain extracellular PAMPs are transported to the cytoplasm for recognition by host proteins8.

Why does bacterial ADP-Hep exposure occur if it activates the innate immune system? Perhaps its release is needed to fulfil some as yet unknown function. Pathogens often evolve mechanisms to evade or thwart an immune-system response. If pathogens have evolved strategies to avoid triggering an ADP-Hep-mediated immune response, understanding such strategies might suggest new therapeutic approaches to fight bacterial infections.

The authors’ observation that host enzymes can convert bacterial metabolites that have poor immune-activating characteristics into potent PAMPs offers a new perspective on the evolutionary battle between pathogens and their hosts. Although Zhou et al. show that ADP-Hep is the relevant immune-triggering ligand for Y. pseudotuberculosis infections, it remains to be seen whether HBP is converted into ADP-heptose 7-P during other bacterial infections. This issue is particularly relevant for pathogens (for example, Shigella) that invade the host-cell cytoplasm and that might shed PAMPs such as HBP directly into the cytoplasm. Zhou and colleagues’ work also offers a fresh perspective on the types of molecule that can act as PAMPs or their PRRs, and where and how researchers should be searching for such molecules.

Nature 561, 37-38 (2018)

doi: 10.1038/d41586-018-05854-y

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