Yunhao Tan

Comprehensive Identification of Protein Substrates of the Dot/Icm Type IV Transporter of Legionella pneumophila

A large number of proteins transferred by the Legionella pneumophila Dot/Icm system have been identified by various strategies. With no exceptions, these strategies are based on one or more characteristics associated with the tested proteins. Given the high level of diversity exhibited by the identified proteins, it is possible that some substrates have been missed in these screenings. In this study, we took a systematic method to survey the L. pneumophila genome by testing hypothetical orfs larger than 300 base pairs for Dot/Icm-dependent translocation. 798 of the 832 analyzed orfs were successfully fused to the carboxyl end of β-lactamase. The transfer of the fusions into mammalian cells was determined using the β-lactamase reporter substrate CCF4-AM. These efforts led to the identification of 164 proteins positive in translocation. Among these, 70 proteins are novel substrates of the Dot/Icm system. These results brought the total number of experimentally confirmed Dot/Icm substrates to 275. Sequence analysis of the C-termini of these identified proteins revealed that Lpg2844, which contains few features known to be important for Dot/Icm-dependent protein transfer can be translocated at a high efficiency. Thus, our efforts have identified a large number of novel substrates of the Dot/Icm system and have revealed the diverse features recognizable by this protein transporter.

Legionella pneumophila SidD is a deAMPylase that modifies Rab1

Legionella pneumophila actively modulates host vesicle trafficking pathways to facilitate its intracellular replication with effectors translocated by the Dot/Icm type IV secretion system (T4SS). The SidM/DrrA protein functions by locking the small GTPase Rab1 into an active form by its guanine nucleotide exchange factor (GEF) and AMPylation activity. Here we demonstrate that the L. pneumophila protein SidD preferably deAMPylates Rab1. We found that the deAMPylation activity of SidD could suppress the toxicity of SidM to yeast and is required to release Rab1 from bacterial phagosomes efficiently. A molecular mechanism for the temporal control of Rab1 activity in different phases of L. pneumophila infection is thus established. These observations indicate that AMPylation-mediated signal transduction is a reversible process regulated by specific enzymes.

Legionella pneumophila regulates the small GTPase Rab1 activity by reversible phosphorylcholination

Effectors delivered into host cells by the Legionella pneumophila Dot/Icm type IV transporter are essential for the biogenesis of the specialized vacuole that permits its intracellular growth. The biochemical function of most of these effectors is unknown, making it difficult to assign their roles in the establishment of successful infection. We found that several yeast genes involved in membrane trafficking, including the small GTPase Ypt1, strongly suppress the cytotoxicity of Lpg0695(AnkX), a protein known to interfere severely with host vesicle trafficking when overexpressed. Mass spectrometry analysis of Rab1 purified from a yeast strain inducibly expressing AnkX revealed that this small GTPase is modified posttranslationally at Ser76 by a phosphorylcholine moiety. Using cytidine diphosphate-choline as the donor for phosphorylcholine, AnkX catalyzes the transfer of phosphorylcholine to Rab1 in a filamentation-induced by cAMP(Fic) domain-dependent manner. Further, we found that the activity of AnkX is regulated by the Dot/Icm substrate Lpg0696(Lem3), which functions as a dephosphorylcholinase to reverse AnkX-mediated modification on Rab1. Phosphorylcholination interfered with Rab1 activity by making it less accessible to the bacterial GTPase activation protein LepB; this interference can be alleviated fully by Lem3. Our results reveal reversible phosphorylcholination as a mechanism for balanced modulation of host cellular processes by a bacterial pathogen.

Large-scale identification and translocation of type IV secretion substrates by Coxiella burnetii

Coxiella burnetii is an obligate intracellular bacterial pathogen responsible for acute and chronic Q fever. This bacterium harbors a type IV secretion system (T4SS) highly similar to the Dot/Icm of Legionella pneumophila that is believed to be essential for its infectivity. Protein substrates of the Coxiella T4SS are predicted to facilitate the biogenesis of a phagosome permissive for its intracellular growth. However, due to the lack of genetic systems, protein transfer by the C. burnetii Dot/Icm has not been demonstrated. In this study, we report the identification of 32 substrates of the C. burnetii Dot/Icm system using a fluorescence-based β-lactamase (TEM1) translocation assay as well as the calmodulin-dependent adenylate cyclase (CyaA) assay in the surrogate host L. pneumophila. Notably, 26 identified T4SS substrates are hypothetical proteins without predicted function. Candidate secretion substrates were obtained by using (i) a genetic screen to identify C. burnetii proteins interacting with DotF, a component of the T4SS, and (ii) bioinformatic approaches to retrieve candidate genes that harbor characteristics associated with previously reported substrates of the Dot/Icm system from both C. burnetii and L. pneumophila. Moreover, we have developed a shuttle plasmid that allows the expression of recombinant proteins in C. burnetii as TEM fusion products. Using this system, we demonstrated that a Dot/Icm substrate identified with L. pneumophila was also translocated by C. burnetii in a process that requires its C terminus, providing direct genetic evidence of a functional T4SS in C. burnetii.

An endogenous caspase-11 ligand elicits interleukin-1 release from living dendritic cells

Immune activation in context Dendritic cells (DCs) initiate protective immunity upon binding molecules derived from microbes or released from dying cells. Zanoni et al. examined how microbial and endogenous signals interact to shape the course of the ensuing immune response (see the Perspective by Napier and Monack). They found that oxPAPC, an oxidized phospholipid released from dying cells, binds to a protein called caspase-11 in DCs, activating an inflammatory program in these cells. Whereas caspase-11 binding to oxPAPC and bacterial lipopolysaccharide causes DCs to produce the cytokine interleukin-1 (IL-1) and undergo cell death, binding to oxPAPC alone triggers DCs to secrete IL-1 and induce strong adaptive immunity. Thus, context-dependent signals can shape the ensuing immune response. Science, this issue p. 1232; see also p. 1173 Oxidized phospholipids released from dying cells shape dendritic cell immunity. Dendritic cells (DCs) use pattern recognition receptors to detect microorganisms and activate protective immunity. These cells and receptors are thought to operate in an all-or-nothing manner, existing in an immunologically active or inactive state. Here, we report that encounters with microbial products and self-encoded oxidized phospholipids (oxPAPC) induce an enhanced DC activation state, which we call “hyperactive.” Hyperactive DCs induce potent adaptive immune responses and are elicited by caspase-11, an enzyme that binds oxPAPC and bacterial lipopolysaccharide (LPS). oxPAPC and LPS bind caspase-11 via distinct domains and elicit different inflammasome-dependent activities. Both lipids induce caspase-11–dependent interleukin-1 release, but only LPS induces pyroptosis. The cells and receptors of the innate immune system can therefore achieve different activation states, which may permit context-dependent responses to infection.

A Cross-Disciplinary Perspective on the Innate Immune Responses to Bacterial Lipopolysaccharide

The study of innate immunity to bacteria has focused heavily on the mechanisms by which mammalian cells detect lipopolysaccharide (LPS), the conserved surface component of Gram-negative bacteria. While Toll-like receptor 4 (TLR4) is responsible for all the host transcriptional responses to LPS, recent discoveries have revealed the existence of several TLR4-independent responses to LPS. These discoveries not only broaden our view of the means by which mammalian cells interact with bacteria, but they also highlight new selective pressures that may have promoted the evolution of bacterial immune evasion strategies. In this review, we highlight past and recent discoveries on host LPS sensing mechanisms and discuss bacterial countermeasures that promote infection. By looking at both sides of the host-pathogen interaction equation, we hope to provide comprehensive insights into host defense mechanisms and bacterial pathogenesis.

Ubiquitination independent of E1 and E2 enzymes by bacterial effectors.

Signalling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalysed by the E1, E2 and E3 three-enzyme cascade, which links the carboxy terminus of ubiquitin to the ε-amino group of, in most cases, a lysine of the substrate via an isopeptide bond. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents. For example, many bacterial pathogens exploit ubiquitin signalling using virulence factors that function as E3 ligases, deubiquitinases or as enzymes that directly attack ubiquitin. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a permissive niche for its replication in phagocytes. Here we demonstrate that members of the SidE effector family of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum. Moreover, we show that these proteins are capable of catalysing ubiquitination without the need for the E1 and E2 enzymes. A putative mono-ADP-ribosyltransferase motif critical for the ubiquitination activity is also essential for the role of the SidE family in intracellular bacterial replication in a protozoan host. The E1/E2-independent ubiquitination catalysed by these enzymes is energized by nicotinamide adenine dinucleotide, which activates ubiquitin by the formation of ADP-ribosylated ubiquitin. These results establish that ubiquitination can be catalysed by a single enzyme, the activity of which does not require ATP.

Identification of Coxiella burnetii Type IV Secretion Substrates Required for Intracellular Replication and Coxiella-Containing Vacuole Formation

Coxiella burnetii, the etiological agent of acute and chronic Q fever in humans, is a naturally intracellular pathogen that directs the formation of an acidic Coxiella-containing vacuole (CCV) derived from the host lysosomal network. Central to its pathogenesis is a specialized type IVB secretion system (T4SS) that delivers effectors essential for intracellular replication and CCV formation. Using a bioinformatics-guided approach, 234 T4SS candidate substrates were identified. Expression of each candidate as a TEM-1 β-lactamase fusion protein led to the identification of 53 substrates that were translocated in a Dot/Icm-dependent manner. Ectopic expression in HeLa cells revealed that these substrates trafficked to distinct subcellular sites, including the endoplasmic reticulum, mitochondrion, and nucleus. Expression in Saccharomyces cerevisiae identified several substrates that were capable of interfering with yeast growth, suggesting that these substrates target crucial host processes. To determine if any of these T4SS substrates are necessary for intracellular replication, we isolated 20 clonal T4SS substrate mutants using the Himar1 transposon and transposase. Among these, 10 mutants exhibited defects in intracellular growth and CCV formation in HeLa and J774A.1 cells but displayed normal growth in bacteriological medium. Collectively, these results indicate that C. burnetii encodes a large repertoire of T4SS substrates that play integral roles in host cell subversion and CCV formation and suggest less redundancy in effector function than has been found in the comparative Legionella Dot/Icm model.

The Pore-Forming Protein Gasdermin D Regulates Interleukin-1 Secretion from Living Macrophages

Summary The interleukin‐1 (IL‐1) family cytokines are cytosolic proteins that exhibit inflammatory activity upon release into the extracellular space. These factors are released following various cell death processes, with pyroptosis being a common mechanism. Recently, it was recognized that phagocytes can achieve a state of hyperactivation, which is defined by their ability to secrete IL‐1 while retaining viability, yet it is unclear how IL‐1 can be secreted from living cells. Herein, we report that the pyroptosis regulator gasdermin D (GSDMD) was necessary for IL‐1&bgr; secretion from living macrophages that have been exposed to inflammasome activators, such as bacteria and their products or host‐derived oxidized lipids. Cell‐ and liposome‐based assays demonstrated that GSDMD pores were required for IL‐1&bgr; transport across an intact lipid bilayer. These findings identify a non‐pyroptotic function for GSDMD, and raise the possibility that GSDMD pores represent conduits for the secretion of cytosolic cytokines under conditions of cell hyperactivation. Graphical Abstract Figure. No Caption available. HighlightsMultiple microbial and self‐derived stimuli induce IL‐1 release from living macrophagesInflammasomes can be detected within cells that display multiple signs of viabilityLiving macrophages require gasdermin D to induce pore formation and IL‐1 releaseGasdermin D pores facilitate the release of IL‐1 from liposomes and intact cells &NA; Inflammasomes elicit pyroptosis or cell hyperactivation, with the latter defined as living cells that release IL‐1. Evavold et al report that the pore‐forming protein gasdermin D regulates IL‐1 release from hyperactive macrophages. Cell‐ and liposome‐based assays revealed that gasdermin D pores permit IL‐1 passage across intact lipid bilayers.

Mechanisms of Toll-like Receptor 4 Endocytosis Reveal a Common Immune-Evasion Strategy Used by Pathogenic and Commensal Bacteria

Microbe-induced receptor trafficking has emerged as an essential means to promote innate immune signal transduction. Upon detection of bacterial lipopolysaccharides (LPS), CD14 induces an inflammatory endocytosis pathway that delivers Toll-like receptor 4 (TLR4) to endosomes. Although several regulators of CD14-dependent TLR4 endocytosis have been identified, the cargo-selection mechanism during this process remains unknown. We reveal that, in contrast to classic cytosolic interactions that promoted the endocytosis of transmembrane receptors, TLR4 was selected as cargo for inflammatory endocytosis entirely through extracellular interactions. Mechanistically, the extracellular protein MD-2 bound to and dimerized TLR4 in order to promote this endocytic event. Our analysis of LPS variants from human pathogens and gut commensals revealed a common mechanism by which bacteria prevent inflammatory endocytosis. We suggest that evasion of CD14-dependent endocytosis is an attribute that transcends the concept of pathogenesis and might be a fundamental feature of bacteria that inhabit eukaryotic hosts.