The virulence of bacterial pathogens often relies on their secreted effector proteins that interfere with or manipulate the host signal transduction system. Our research on bacterial virulence has demonstrated that bacterial effectors can function to induce novel posttranslational modifications on key host signaling molecules. While continuing to define the functional mechanism of other important effectors, we are also exploring the idea of using activities of bacterial effectors to guide new understanding of eukaryotic signaling mechanisms. Our research on innate immunity focuses on how macrophage cells use the cytosolic inflammasome pathway to sense and counteract bacterial infection and virulence.
Biochemical and Signaling Functions of Bacterial Virulence Effectors and Implications for Eukaryotic Signal Transduction
Gram-negative bacterial pathogens use specialized secretion systems such as type III or IV secretion systems to inject virulence effector proteins into host cells. The effectors usually harbor unique and potent activities that modify key proteins in the host signal transduction system. Mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-ĸB mediate two major signaling pathways that signal transcriptional induction of many pro-inflammatory cytokines upon host sensing of infection. However, suppression of host innate immune signaling is often observed with enteric pathogens such as Shigella, Salmonella, and enteropathogenic Escherichia coli (EPEC), and this effect relies on their conserved type III secretion systems. Using these enteric pathogens as the model, we discovered several novel biochemical strategies utilized by bacterial type III effectors to block MAPK and NF-ĸB signaling.
The OspF family of effectors, conserved in Shigella, Salmonella, and the plant pathogen Pseudomonas syringae, defines a novel phosphothreonine lyase that removes the phosphate from the phosphothreonine residue in MAPKs through a β-elimination mechanism. This irreversible "dephosphorylation" leads to permanent inactivation of host MAPK signaling and inhibited cytokine production in Shigella-infected cells. Very recently, we demonstrated that an EPEC effector, NleE, which is conserved in the related pathogen enterohaemorrhagic E. coli as well as in certain species of Shigella and Salmonella, targets ubiquitin-chain-sensing proteins TAB2/3 in the canonical NF-ĸB pathway. NleE is a novel S-adenosine-l-methionine-dependent methyltransferase that methylates one of the four zinc-coordinating cysteines in the Npl4 zinc finger (NZF) domain of TAB2/3. NleE-catalyzed cysteine methylation diminishes the activity of TAB2/3 NZF domain in binding to Lys63-linked polyubiquitin chains, which is responsible for EPEC suppression of NF-ĸB signaling.
Consistent with the importance of ubiquitin signaling in innate immunity, bacterial pathogens have evolved to modulate the ubiquitin system. We and others showed that Shigella and Salmonella encode a group of structurally distinct ubiquitin ligase effectors that presumably ubiquitinate host molecules for proteasomal degradation. We also discovered that the type III effectors Cif from EPEC and CHBP from Burkholderia pseudomallei can directly modify ubiquitin and ubiquitin-like protein NEDD8 by hydrolyzing Gln-40 in ubiquitin/NEDD8 into glutamate. This deamidation modification inactivates ubiquitin/NEDD8 and leads to dysfunctioning of the ubiquitin system, causing disruption of multiple host cellular processes, including the innate immune response.
These discoveries are not only revealing in understanding the pathogenesis mechanism of the corresponding pathogen, but they also illustrate three new posttranslational modifications: eliminylation, ubiquitin/NEDD8 deamidation, and cysteine methylation. We are currently investigating whether these kinds of modifications also play a role in regulating eukaryotic signal transduction. Therefore, we are developing proteomic approaches to identify possible eukaryotic proteins that undergo eliminylation or cysteine methylation modification. We are also pursuing the idea of using the unique activities of these effectors as a tool to probe host cellular processes and hope to be able to discover new signal transduction pathways based on the sensitivity to effector-mediated new posttranslational modifications.
Small GTPase signaling is another frequent target of bacterial effectors. The Rho-family small GTPase controls actin cytoskeleton dynamics and plays a critical role in mediating bacterial invasion and phogocytosis of the pathogen by host cells, which has been observed with Yersinia and Salmonella species. For intracellular pathogens such as Legionella pneumophila, Shigella, and Salmonella, the function of Rab small GTPase is crucial for trafficking of the bacteria and bacteria-containing vacuoles. Both Rho and Rab GTPases are lipid modified for membrane anchoring, and their activities are regulated by guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP). GEFs catalyze the exchange of GDP for GTP for activation, whereas GAPs negatively regulate the switch by enhancing the intrinsic GTPase activity. Earlier research, including that from our lab, has provided examples of bacterial effectors that either modulate the lipid modification of the GTPases or mimic the activities of host GEFs or GAPs. Using diverse pathogens such as Shigella and Legionella as the model, we are currently working to identify additional biochemical mechanisms used by the secreted bacterial effectors to target the small GTPase switch and/or modulate small GTPase signal transduction pathways.
Inflammasome Pathway in Macrophage Innate Immune Defense Against Bacterial Infection
The innate immune system recognizes a wide range of pathogen-derived products, thereby initiating inflammatory responses by secreting cytokines and chemokines. One of the most important inflammatory cytokines secreted by macrophages is IL-1β, and excessive IL-1β production is associated with autoinflammatory disorders. IL-1β maturation depends on caspase-1, whose activation is mediated by the newly proposed, but poorly understood, large cytoplasmic complex called inflammasome. Inflammasome-mediated caspase-1 activation also triggers macrophage inflammatory death, another factor contributing to restriction of bacterial replication. A class of NOD-like receptor (NLR) proteins, structurally related to the plant-disease-resistant R proteins, is believed to sense microbial infection or endogenous danger signals and mediate assembly of the inflammasome complex. However, the biochemical mechanism underlying inflammasome assembly and activation remains poorly understood.
One of the NLR proteins called NLRC4 is critical for macrophages to sense cytosolic bacterial flagellin and induce caspase-1 activation, but the receptor for flagellin is not known. We recently identified a family of cytosolic BIR-domain NLR proteins called NAIPs that are inflammasome receptors for a class of bacterial products. NAIP5 and NAIP2 in mice directly recognize bacterial flagellin and the type III secretion apparatus rod component, respectively, whereas the sole NAIP family member in humans is the receptor for the needle subunit of the type III secretion system. Engagement of these bacterial ligands by NAIP receptors stimulates a physical association between NAIP and NLRC4 and presumably formation of the large inflammasome complex. These discoveries for the first time demonstrate the NLR proteins (NAIPs) as inflammasome receptors for microbial products and further indicate that certain NLR proteins such as NLRC4 are indeed signaling adaptors in mediating inflammasome assembly and caspase-1 activation. Using biochemical and reconstitution assays that we established with the NLRC4 inflammasome, we are currently trying to understand the detailed molecular and structural mechanisms underlying specific recognition of flagellin and the type III secretion system components by the NAIP receptors as well as the role of other NAIP proteins in sensing bacterial infection.
Genetic studies have demonstrated that two other NLR proteins (NLRP3 and mNalp1b) and a non-NLR protein (AIM2) also mediate inflammasome signaling. The mNalp1b inflammasome responds to the metalloprotease activity of Bacillus anthracis lethal toxin. NLRP3 senses diverse stimuli, including extracellular ATP, and lysosomal damage triggered by insoluble materials such as silica, asbestos, amyloid-β, urate crystals, and aluminum. AIM2 directly recognizes cytosolic dsDNA and activates caspase-1 through the ASC adaptor; however, the functional mechanisms of NLRP3 and mNalp1b are not yet established. We are combining biochemical reconstitution, cell biology, and mouse genetics approaches to investigate the biochemical mechanism of NLRP3 and mNalp1b inflammasome signaling as well as to explore the role of other "orphan" NLR proteins in inflammasome-mediated innate immune surveillance of cytosolic bacterial pathogens.