Zhao-Qing Luo

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.

Quorum-sensing signal binding results in dimerization of TraR and its release from membranes into the cytoplasm

Promoter binding by TraR and LuxR, the activators of two bacterial quorum‐sensing systems, requires their cognate acyl‐homoserine lactone (acyl‐HSL) signals, but the role the signal plays in activating these transcription factors is not known. Soluble active TraR, when purified from cells grown with the acyl‐HSL, contained bound signal and was solely in dimer form. However, genetic and cross‐linking studies showed that TraR is almost exclusively in monomer form in cells grown without signal. Adding signal resulted in dimerization of the protein in a concentration‐dependent manner. In the absence of signal, monomer TraR localized to the inner membrane while growth with the acyl‐HSL resulted in the appearance of dimer TraR in the cytoplasmic compartment. Affinity chromatography indicated that the N‐terminus of TraR from cells grown without signal is hidden. Analysis of heterodimers formed between TraR and its deletion mutants localized the dimerization domain to a region between residues 49 and 156. We conclude that binding signal drives dimerization of TraR and its release from membranes into the cytoplasm.

Legionella pneumophila inhibits macrophage apoptosis by targeting pro-death members of the Bcl2 protein family

To establish a vacuole that supports bacterial replication, Legionella pneumophila translocates a large number of bacterial proteins into host cells via the Dot/Icm type IV secretion system. Functions of most of these translocated proteins are unknown, but recent investigations suggest their roles in modulating diverse host processes such as vesicle trafficking, autophagy, ubiquitination, and apoptosis. Cells infected by L. pneumophila exhibited resistance to apoptotic stimuli, but the bacterial protein directly involved in this process remained elusive. We show here that SidF, one substrate of the Dot/Icm transporter, is involved in the inhibition of infected cells from undergoing apoptosis to allow maximal bacterial multiplication. Permissive macrophages harboring a replicating sidF mutant are more apoptotic and more sensitive to staurosporine-induced cell death. Furthermore, cells expressing SidF are resistant to apoptosis stimuli. SidF contributes to apoptosis resistance in L. pneumophila-infected cells by specifically interacting with and neutralizing the effects of BNIP3 and Bcl-rambo, two proapoptotic members of Bcl2 protein family. Thus, inhibiting the functions of host pro-death proteins by translocated effectors constitutes a mechanism for L. pneumophila to protect host cells from apoptosis.

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.

Construction of a Derivative of Agrobacterium tumefaciens C58 That Does Not Mutate to Tetracycline Resistance

Agrobacterium tumefaciens C58 mutates to tetracycline resistance at high frequency, complicating the use of many broad-host-range cloning and binary vectors that code for resistance to this antibiotic as the selection marker. Such mutations are associated with a resistant gene unit, tetC58, that is present in the genome of this strain. By deleting the tetC58 locus, we constructed NTL4, a derivative of C58 that no longer mutates to tetracycline resistance. The deletion had no detectable effect on genetic or physiological traits of NTL4 or on the ability of this strain to transform plants.

The E Block motif is associated with Legionella pneumophila translocated substrates

Legionella pneumophila promotes intracellular growth by moving bacterial proteins across membranes via the Icm/Dot system. A strategy was devised to identify large numbers of Icm/Dot translocated proteins, and the resulting pool was used to identify common motifs that operate as recognition signals. The 3′ end of the sidC gene, which encodes a known translocated substrate, was replaced with DNA encoding 200 codons from the 3′ end of 442 potential substrate‐encoding genes. The resulting hybrid proteins were then tested in a high throughput assay, in which translocated SidC antigen was detected by indirect immunofluorescence. Among translocated substrates, regions of 6–8 residues called E Blocks were identified that were rich in glutamates. Analysis of SidM/DrrA revealed that loss of three Glu residues, arrayed in a triangle on an α‐helical surface, totally eliminated translocation of a reporter protein. Based on this result, a second strategy was employed to identify Icm/Dot substrates having carboxyl terminal glutamates. From the fusion assay and the bioinformatic queries, carboxyl terminal sequences from 49 previously unidentified proteins were shown to promote translocation into target cells. These studies indicate that by analysing subsets of translocated substrates, patterns can be found that allow predictions of important motifs recognized by Icm/Dot.

The Legionella pneumophila Effector SidJ Is Required for Efficient Recruitment of Endoplasmic Reticulum Proteins to the Bacterial Phagosome

ABSTRACT The virulence of Legionella pneumophila is dependent on the Dot/Icm type IV protein secretion system, which translocates effectors into infected cells. A large number of such translocated proteins have been identified, but few of these proteins are necessary for intracellular replication of the pathogen, making it difficult to correlate these genes with specific cell-biological events associated with L. pneumophila infection. We report here the identification and characterization of a family of two substrates, SidJ and SdjA, with distinctive phenotypes. In contrast to many Dot/Icm substrates, whose expression levels are elevated when bacteria are grown to postexponential phase, SidJ is produced at a constant rate during the entire bacterial growth cycle. Mutation in sidJ causes a significant growth defect in both macrophage and amoeba hosts, but an sdjA mutant is detectably defective only in protozoan hosts. However, in the amoeba host a mutant lacking both sidJ and sdjA does not display a more severe growth defect than the sidJ mutant. Despite its significant intracellular growth defect, the sidJ mutant is still able to effectively evade fusion with lysosomes. Importantly, recruitment of endoplasmic reticulum (ER) proteins by vacuoles containing the sidJ mutant was considerably delayed in both mammalian and amoeba cells. Our results suggest that SidJ modulates host cellular pathways, contributing to the trafficking or retention of ER-derived vesicles to L. pneumophila vacuoles.

Targeting eEF1A by a Legionella pneumophila effector leads to inhibition of protein synthesis and induction of host stress response

The Legionella pneumophila Dot/Icm type IV secretion system is essential for the biogenesis of a phagosome that supports bacterial multiplication, most likely via the functions of its protein substrates. Recent studies indicate that fundamental cellular processes, such as vesicle trafficking, stress response, autophagy and cell death, are modulated by these effectors. However, how each translocated protein contributes to the modulation of these pathways is largely unknown. In a screen to search substrates of the Dot/Icm transporter that can cause host cell death, we identified a gene whose product is lethal to yeast and mammalian cells. We demonstrate that this protein, called SidI, is a substrate of the Dot/Icm type IV protein transporter that targets the host protein translation process. Our results indicate that SidI specifically interacts with eEF1A and eEF1Bγ, two components of the eukaryotic protein translation elongation machinery and such interactions leads to inhibition of host protein synthesis. Furthermore, we have isolated two SidI substitution mutants that retain the target binding activity but have lost toxicity to eukaryotic cells, suggesting potential biochemical effect of SidI on eEF1A and eEF1Bγ. We also show that infection by L. pneumophila leads to eEF1A‐mediated activation of the heat shock regulatory protein HSF1 in a virulence‐dependent manner and deletion of sidI affects such activation. Moreover, similar response occurred in cells transiently transfected to express SidI. Thus, inhibition of host protein synthesis by specific effectors contributes to the induction of stress response in L. pneumophila‐infected cells.