Tat Mutants Research Articles

Chronic HIV-1 Tat action induces HLA-DR downregulation in B cells: A mechanism for lymphoma immune escape in people living with HIV.

Despite the success of combination antiretroviral therapy, people living with human immunodeficiency virus (HIV) still have an increased risk of Epstein-Barr virus (EBV)-associated B cell malignancies. In the HIV setting, B cell physiology is altered by coexistence with HIV-infected cells and the chronic action of secreted viral proteins, for example, HIV-1 Tat that, once released, efficiently penetrates noninfected cells. We modeled the chronic action of HIV-1 Tat on B cells by ectopically expressing Tat or TatC22G mutant in two lymphoblastoid B cell lines. The RNA-sequencing analysis revealed that Tat deregulated the expression of hundreds of genes in B cells, including the downregulation of a subset of major histocompatibility complex (MHC) class II-related genes. Tat-induced downregulation of HLA-DRB1 and HLA-DRB5 genes led to a decrease in HLA-DR surface expression; this effect was reproduced by coculturing B cells with Tat-expressing T cells. Chronic Tat presence decreased the NF-ᴋB pathway activity in B cells; this downregulated NF-ᴋB-dependent transcriptional targets, including MHC class II genes. Notably, HLA-DRB1 and surface HLA-DR expression was also decreased in B cells from people with HIV. Tat-induced HLA-DR downregulation in B cells impaired EBV-specific CD4+ T cell response, which contributed to the escape from immune surveillance and could eventually promote B cell lymphomagenesis in people with HIV.

GigaAssay – An adaptable high-throughput saturation mutagenesis assay platform

High-throughput assay systems have had a large impact on understanding the mechanisms of basic cell functions. However, high-throughput assays that directly assess molecular functions are limited. Herein, we describe the “GigaAssay”, a modular high-throughput one-pot assay system for measuring molecular functions of thousands of genetic variants at once. In this system, each cell was infected with one virus from a library encoding thousands of Tat mutant proteins, with each viral particle encoding a random unique molecular identifier (UMI). We demonstrate proof of concept by measuring transcription of a GFP reporter in an engineered reporter cell line driven by binding of the HIV Tat transcription factor to the HIV long terminal repeat. Infected cells were flow-sorted into 3 bins based on their GFP fluorescence readout. The transcriptional activity of each Tat mutant was calculated from the ratio of signals from each bin. The use of UMIs in the GigaAssay produced a high average accuracy (95%) and positive predictive value (98%) determined by comparison to literature benchmark data, known C-terminal truncations, and blinded independent mutant tests. Including the substitution tolerance with structure/function analysis shows restricted substitution types spatially concentrated in the Cys-rich region. Tat has abundant intragenic epistasis (10%) when single and double mutants are compared.

The Rcs System Contributes to the Motility Defects of the Twin-Arginine Translocation System Mutant of Extraintestinal Pathogenic Escherichia coli.

Flagellum-mediated bacterial motility is important for bacteria to take up nutrients, adapt to environmental changes, and establish infection. The twin-arginine translocation system (Tat) is an important protein export system, playing a critical role in bacterial physiology and pathogenesis. It has been observed for a long time that the Tat system is critical for bacterial motility. However, the underlying mechanism remains unrevealed. In this study, a comparative transcriptomics analysis was performed with extraintestinal pathogenic Escherichia coli (ExPEC), which identified a considerable number of genes differentially expressed when the Tat system was disrupted. Among them, a large proportion of flagellar biosynthesis genes showed downregulation, indicating that transcription regulation plays an important role in mediating the motility defects. We further identified three Tat substrate proteins, MdoD, AmiA, and AmiC, that were responsible for the nonmotile phenotype. The Rcs system was deleted in the Δtat, the ΔmdoD, and the ΔamiAΔamiC strains, which restored the motility of ΔmdoD and partially restored the motility of Δtat and ΔamiAΔamiC. The flagella were also observed in all of the ΔtatΔrcsDB, ΔmdoDΔrcsDB, and ΔamiAΔamiCΔrcsDB strains, but not in the Δtat, ΔmdoD, and ΔamiAΔamiC strains, by using transmission electron microscopy. Quantitative reverse transcription-PCR data revealed that the regulons of the Rcs system displayed differential expression in the tat mutant, indicating that the Rcs signaling was activated. Our results suggest that the Rcs system plays an important role in mediating the motility defects of the tat mutant of ExPEC. IMPORTANCE The Tat system is an important protein export system critical for bacterial physiology and pathogenesis. It has been observed for a long time that the Tat system is critical for bacterial motility. However, the underlying mechanism remains unrevealed. In this study, we combine transcriptomics analysis and bacterial genetics, which reveal that transcription regulation plays an important role in mediating the motility defects of the tat mutant of extraintestinal pathogenic Escherichia coli. The Tat substrate proteins responsible for the motility defects are identified. We further show that the Rcs system contributes to the motility suppression. We for the first time reveal the link between the Tat system and bacterial motility, which is important for understanding the physiological functions of the Tat system.

Disruption of the Pseudomonas aeruginosa Tat system perturbs PQS-dependent quorum sensing and biofilm maturation through lack of the Rieske cytochrome bc1 sub-unit.

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of PQS-dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA, rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat substrate(s) responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the primary signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc1 complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. This defect was also phenocopied by deletion of cytB or cytC1. Thus, either lack of the Rieske sub-unit or mutation of cytochrome bc1 genes results in the perturbation of PQS-dependent autoinduction resulting in eDNA deficient biofilms, reduced antibiotic tolerance and compromised virulence factor production.

Proteomics Investigation of the Time Course Responses of RAW264.7 Macrophages to Infections With the Wild-Type and Twin-Arginine Translocation Mutant Strains of Brucella melitensis.

Brucella, a notorious intracellular pathogen, causes chronic infections in many mammals, including humans. The twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane; protein substrates translocated by Brucella include ABC transporters, oxidoreductases, and cell envelope biosynthesis proteins. Previously, we showed that a Tat mutant of Brucella melitensis M28 exhibits reduced survival within murine macrophages. In this study, we compared the host responses elicited by wild-type M28 and its Tat-mutant strains ex vivo. We utilized label-free quantitative proteomics to assess proteomic changes in RAW264.7 macrophages after infection with M28 and its Tat mutants. A total of 6085 macrophage proteins were identified with high confidence, and 79, 50, and 99 proteins were differentially produced upon infection with the Tat mutant at 4, 24, and 48 hpi, respectively, relative to the wild-type infection. Gene ontology and KEGG enrichment analysis indicated that immune response-related proteins were enriched among the upregulated proteins. Compared to the wild-type M28 infection, the most upregulated proteins upon Tat-mutant infection included the cytosolic nucleic acid signaling pathway-related proteins IFIH1, DHX58, IFI202, IFI204, and ISG15 and the NF-κB signaling pathway-related proteins PTGS2, CD40, and TRAF1, suggesting that the host increases the production of these proteins in response to Tat mutant infection. Upregulation of some proteins was further verified by a parallel reaction monitoring (PRM) assay. ELISA and qRT-PCR assays indicated that Tat mutant infection significantly induced proinflammatory cytokine (TNF-α and IL-6) and nitric oxide (NO) production. Finally, we showed that the Tat mutant displays higher sensitivity to nitrosative stress than the wild type and that treatment with the NO synthase inhibitor L-NMMA significantly increases the intracellular survival of the Tat mutant, indicating that NO production contributes to restricting Tat mutant survival within macrophages. Collectively, this work improves our understanding of host immune responses to Tat mutants and provides insights into the mechanisms underlying the attenuated virulence of Tat mutants.

Twin-arginine translocation (Tat) mutants in Salmonella enterica serovar Typhimurium have increased susceptibility to cell wall targeting antibiotics.

ABSTRACTThe twin-arginine translocation (Tat) system is a protein secretion system that is conserved in bacteria, archaea and plants. In Gram-negative bacteria, it is required for the export of folded proteins from the cytoplasm to the periplasm. There are 30 experimentally verified Tat substrates in Salmonella, including hydrogenase subunits, enzymes required for anaerobic respiration and enzymes involved in peptidoglycan remodeling during cell division. Multiple studies have demonstrated the susceptibility of tat mutants to antimicrobial compounds such as SDS and bile; however, in this work, we use growth curves and viable plate counts to demonstrate that cell wall targeting antibiotics (penicillins, carbapenems, cephalosporins and fosfomycin) have increased killing against a Δtat strain. Further, we demonstrate that this increased killing is primarily due to defects in translocation of critical Tat substrates: MepK, AmiA, AmiC and SufI. Finally, we show that a ΔhyaAB ΔhybABC ΔhydBC strain has an altered ΔΨ that impacts proper secretion of critical Tat substrates in aerobic growth conditions.

Double trouble: Bacillus depends on a functional Tat machinery to avoid severe oxidative stress and starvation upon entry into a NaCl-depleted environment

The widely conserved twin-arginine translocases (Tat) allow the transport of fully folded cofactor-containing proteins across biological membranes. In doing so, these translocases serve different biological functions ranging from energy conversion to cell division. In the Gram-positive soil bacterium Bacillus subtilis, the Tat machinery is essential for effective growth in media lacking iron or NaCl. It was previously shown that this phenomenon relates to the Tat-dependent export of the heme-containing peroxidase EfeB, which converts Fe2+ to Fe3+ at the expense of hydrogen peroxide. However, the reasons why the majority of tat mutant bacteria perish upon dilution in NaCl-deprived medium and how, after several hours, a sub-population adapts to this condition was unknown. Here we show that, upon growth in the absence of NaCl, the bacteria face two major problems, namely severe oxidative stress at the membrane and starvation leading to death. The tat mutant cells can overcome these challenges if they are fed with arginine, which implies that severe arginine depletion is a major cause of death and resumed arginine synthesis permits their survival. Altogether, our findings show that the Tat system of B. subtilis is needed to preclude severe oxidative stress and starvation upon sudden drops in the environmental Na+ concentration as caused by flooding or rain.

Morphine exposure exacerbates HIV-1 Tat driven changes to neuroinflammatory factors in cultured astrocytes.

Despite antiretroviral therapy human immunodeficiency virus type-1 (HIV-1) infection results in neuroinflammation of the central nervous system that can cause HIV-associated neurocognitive disorders (HAND). The molecular mechanisms involved in the development of HAND are unclear, however, they are likely due to both direct and indirect consequences of HIV-1 infection and inflammation of the central nervous system. Additionally, opioid abuse in infected individuals has the potential to exacerbate HIV-comorbidities, such as HAND. Although restricted for productive HIV replication, astrocytes (comprising 40-70% of all brain cells) likely play a significant role in neuropathogenesis in infected individuals due to the production and response of viral proteins. The HIV-1 protein Tat is critical for viral transcription, causes neuroinflammation, and can be secreted from infected cells to affect uninfected bystander cells. The Wnt/β-catenin signaling cascade plays an integral role in restricting HIV-1 infection in part by negatively regulating HIV-1 Tat function. Conversely, Tat can overcome this negative regulation and inhibit β-catenin signaling by sequestering the critical transcription factor TCF-4 from binding to β-catenin. Here, we aimed to explore how opiate exposure affects Tat-mediated suppression of β-catenin in astrocytes and the downstream modulation of neuroinflammatory genes. We observed that morphine can potentiate Tat suppression of β-catenin activity in human astrocytes. In contrast, Tat mutants deficient in secretion, and lacking neurotoxic effects, do not affect β-catenin activity in the presence or absence of morphine. Finally, morphine treatment of astrocytes was sufficient to reduce the expression of genes involved in neuroinflammation. Examining the molecular mechanisms of how HIV-1 infection and opiate exposure exacerbate neuroinflammation may help us inform or predict disease progression prior to HAND development.

Strong In Vivo Inhibition of HIV-1 Replication by Nullbasic, a Tat Mutant

Nullbasic is a mutant form of the HIV-1 transcriptional activator protein (Tat) that strongly inhibits HIV-1 transcription and replication in lymphocytes in vitro To investigate Nullbasic inhibition in vivo, we employed an NSG mouse model where animals were engrafted with primary human CD4+ cells expressing a Nullbasic-ZsGreen1 (NB-ZSG) fusion protein or ZSG. NB-ZSG and ZSG were delivered by using a retroviral vector where CD4+ cells were transduced either prior to (preinfection) or following (postinfection) HIV-1 infection. The transduced cells were analyzed in vitro up to 10 days postinfection (dpi) and in vivo up to 39 dpi. Compared to ZSG, NB-ZSG strongly inhibited HIV-1 replication both in vitro and in vivo using preinfection treatment. In vitro, HIV-1 mRNA levels in cells were reduced by up to 60-fold. In vivo, HIV-1 RNA was undetectable in plasma samples during the course of the experiment, and HIV-1 mRNA levels in resident CD4+ cells in organ tissue were reduced up to 2,800-fold. Postinfection treatment of HIV-1-infected cells with NB-ZSG attenuated HIV-1 infection for up to 14 days. In vitro, a 25-fold reduction of viral mRNA in cells was observed but diminished to a <2-fold reduction by 10 dpi. In vivo, HIV-1 RNA was undetectable in plasma of NB-ZSG mice at 14 dpi but afterwards was not significantly different between NB-ZSG mice and control mice. However, we observed higher levels of CD4+ cells in NB-ZSG mice than in control mice, suggesting that NB-ZSG imparted a survival advantage to HIV-1-infected animals.IMPORTANCE HIV-1 infection is effectively controlled by antiviral therapy that inhibits virus replication and reduces viral loads below detectable levels in patients. However, therapy interruption leads to viral rebound due to latently infected cells, which serve as a source of continued viral infection. Interest in strategies leading to a functional cure for HIV-1 infection by long-term or permanent viral suppression is growing. Here, we show that a mutant form of the HIV-1 Tat protein, referred to as Nullbasic, inhibits HIV-1 transcription in infected CD4+ cells in vivo Analysis shows that stable expression of Nullbasic in CD4+ cells could lead to durable anti-HIV-1 activity. Nullbasic, as a gene therapy candidate, could be a part of a functional-cure strategy to suppress HIV-1 transcription and replication.

Tat system is required for the virulence of Dickeya zeae on rice plants

The Gram-negative pathogenic bacterium Dickeya zeae causes soft rot in rice, corn and many other plants. The molecular mechanisms underlying D. zeae virulence are not clearly understood. In this study, we characterized the twin-arginine translocation (Tat) system of this pathogen. The mutants of tatA, tatB, tatC and tatE of D. zeae EC1 were generated through the use of the suicide vector pKNG101 and their phenotypic characters were determined. Our results showed that mutations in D. zeae Tat system have no effect on the bacterial growth. △tatA, △tatB and △tatE mutants reduced the motility distinctly, and could not form biofilm, while the △tatC mutant did not show a distinctive decrease on the motility and biofilm formation. Less production of proteases, pectate lyases and cellulases were shown in △tatA, △tatB, △tatC and △tatE mutants as compared with the wild type EC1, whereas the toxin in Tat mutants was reduced, as compared with the wild type. The Tat mutants △tatA, △tatB, △tatC and △tatE could cause soft rot in potato and Chinese cabbage and hypersensitive reactions (HR) in tobacco, that were not different significantly from those of the wild type EC1. However, △tatA, △tatB and △tatE were not significantly effective in the inhibition of rice seed germination compared to the wild type. Furthermore, the disease incidence of the Tat mutants decreased significantly when they were inoculated in rice plants. This reveals that the Tat system has a different role in the virulence of D. zeae in rice, potato and cabbage and it is required for the virulence of D. zeae in rice plants.

HIV-1 Tat phosphorylation on Ser-16 residue modulates HIV-1 transcription

BackgroundHIV-1 transcription activator protein Tat is phosphorylated in vitro by CDK2 and DNA-PK on Ser-16 residue and by PKR on Tat Ser-46 residue. Here we analyzed Tat phosphorylation in cultured cells and its functionality.ResultsMass spectrometry analysis showed primarily Tat Ser-16 phosphorylation in cultured cells. In vitro, CDK2/cyclin E predominantly phosphorylated Tat Ser-16 and PKR—Tat Ser-46. Alanine mutations of either Ser-16 or Ser-46 decreased overall Tat phosphorylation. Phosphorylation of Tat Ser-16 was reduced in cultured cells treated by a small molecule inhibitor of CDK2 and, to a lesser extent, an inhibitor of DNA-PK. Conditional knock-downs of CDK2 and PKR inhibited and induced one round HIV-1 replication respectively. HIV-1 proviral transcription was inhibited by Tat alanine mutants and partially restored by S16E mutation. Pseudotyped HIV-1 with Tat S16E mutation replicated well, and HIV-1 Tat S46E—poorly, but no live viruses were obtained with Tat S16A or Tat S46A mutations. TAR RNA binding was affected by Tat Ser-16 alanine mutation. Binding to cyclin T1 showed decreased binding of all Ser-16 and Ser-46 Tat mutants with S16D and Tat S46D mutationts showing the strongest effect. Molecular modelling and molecular dynamic analysis revealed significant structural changes in Tat/CDK9/cyclin T1 complex with phosphorylated Ser-16 residue, but not with phosphorylated Ser-46 residue.ConclusionPhosphorylation of Tat Ser-16 induces HIV-1 transcription, facilitates binding to TAR RNA and rearranges CDK9/cyclin T1/Tat complex. Thus, phosphorylation of Tat Ser-16 regulates HIV-1 transcription and may serve as target for HIV-1 therapeutics.

Transcriptomic and Phenotypic Analysis Reveals New Functions for the Tat Pathway in Yersinia pseudotuberculosis.

The twin-arginine translocation (Tat) system mediates the secretion of folded proteins that are identified via an N-terminal signal peptide in bacteria, plants, and archaea. Tat systems are associated with virulence in many bacterial pathogens, and our previous studies revealed that Tat-deficient Yersinia pseudotuberculosis was severely attenuated for virulence. Aiming to identify Tat-dependent pathways and phenotypes of relevance for in vivo infection, we analyzed the global transcriptome of parental and ΔtatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26°C and 37°C. The most significant changes in the transcriptome of the ΔtatC mutant were seen at 26°C during stationary-phase growth, and these included the altered expression of genes related to virulence, stress responses, and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat-dependent phenotypes, including decreased YadA expression, impaired growth under iron-limited and high-copper conditions, as well as acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypic defects observed in the Tat-deficient strain were generally more pronounced than those in mutants lacking the Tat substrate predicted to contribute to that specific function. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection. In addition to its established role in mediating the secretion of housekeeping enzymes, the Tat system has been recognized as being involved in infection. In some clinically relevant bacteria, such as Pseudomonas spp., several key virulence determinants can readily be identified among the Tat substrates. In enteropathogens, such as Yersinia spp., there are no obvious virulence determinants among the Tat substrates. Tat mutants show no growth defect in vitro but are highly attenuated in in vivo This makes Tat an attractive target for the development of novel antimicrobials. Therefore, it is important to establish the causes of the attenuation. Here, we show that the attenuation is likely due to synergistic effects of different Tat-dependent phenotypes that each contributes to lowered in vivo fitness.

Exosome-associated release, uptake, and neurotoxicity of HIV-1 Tat protein.

HIV-1 Tat is an indispensible transactivator for HIV gene transcription and replication. It has been shown to exit cells as a free protein and enter neighboring cells or interact with surface receptors of neighboring cells to regulate gene expression and cell function. In this study, we report, for the first time, exosome-associated Tat release and uptake. Using a HIV-1 LTR-driven luciferase reporter-based cell assay and Western blotting or in combination with exosome inhibitor, OptiPrep gradient fractionation, and exosome depletion, we demonstrated significant presence of HIV-1 Tat in exosomes derived from Tat-expressing primary astrocytes, Tat-transfected U373.MG and 293T, and HIV-infected MT4. We further showed that exosome-associated Tat from Tat-expressing astrocytes was capable of causing neurite shortening and neuron death, further supporting that this new form of extracellular Tat is biologically active. Lastly, we constructed a Tat mutant deleted of its basic domain and determined the role of the basic domain in Tat trafficking into exosomes. Basic domain-deleted Tat exhibited no apparent effects on Tat trafficking into exosomes, while maintained its dominant-negative function in Tat-mediated LTR transactivation. Taken together, these results show a significant fraction of Tat is secreted and present in the form of exosomes and may contribute to the stability of extracellular Tat and broaden the spectrum of its target cells.

Cytoplasmic Distribution of HIV-1 Tat Sensitizes Jurkat T cells to Sulphamethoxazole-Hydroxylamine Induced Toxicity

Background: One medication commonly used by HIV-1-infected individuals is the antimicrobial sulphamethoxazole (SMX), which is used in the treatment and prophylaxis of pneumocystis pneumonia. However, SMX is responsible for a very high incidence of hypersensitivity adverse drug reactions (ADRs) in the HIV-1 population. While the pathophysiology of ADRs in general is unknown, sulphamethoxazole-mediated ADRs have been linked to its reactive metabolite sulphamethoxazole-hydroxylamine (SMX-HA). Our previous work has shown that increased expression of the HIV-1 Tat protein in T cells correlated with increased apoptosis after incubation with SMX-HA. In this study we sought to determine the region of the Tat protein responsible for this effect and the mechanism by which Tat contributed to SMX-HA mediated apoptosis. Methods: We established Jurkat T and Cos 7 cell lines that stably expressed full-length Tat (Tat101) and deletion mutants (Tat86, Tat72, Tat48 and TatΔ). These cell lines were then incubated with SMX-HA and assayed for cell viability and production of reactive oxygen species (ROS). We further used confocal microscopy to assess the intracellular distribution of the Tat proteins and to determine if changes in the expression and/or localization of key cytoskeleton proteins contributed to Tat-mediated apoptosis after SMX-HA treatment. Results: Deletion of regions of Tat that lead to increased cytoplasmic accumulation significantly contributed to increased cell death in the presence of SMX-HA. The increased cell death did not require induction of ROS. Quantitative analysis also showed that the Tat-expressing cell lines had significantly lower levels of β-actin and α-tubulin present both before and after treatment with SMX-HA. Increased cytoplasmic localization of Tat correlated with greater disturbances in the distribution of actin filaments. Conclusion: The presence of cytoplasmic Tat in T and epithelial cell lines increases their sensitivity to SMX-HA induced cell death, an effect mediated by the first 48 amino acids of TAT.

Formate dehydrogenase localization and activity are dependent on an intact twin arginine translocation system (Tat) in Campylobacter jejuni 81-176.

The oxidation of formate constitutes a main energy source for Campylobacter jejuni, an important foodborne pathogen. Formate occurs in the hosts' gut as a byproduct of fermentation, which may contribute to C. jejuni's adaptation to that niche. C. jejuni possesses a periplasmic formate dehydrogenase (Fdh) that facilitates the metabolism of formate. In C. jejuni NCTC-11168, a poorly invasive strain, Fdh localizes to the periplasm via the twin arginine translocation (Tat) system, and the fdhA encodes the signature Tat signal motif. However, screening the genome of C. jejuni 81-176, a highly invasive strain, showed that the Tat signal motif was encoded on a separate gene (CJJ81176_1504) directly upstream of the fdhA (CJJ81176_1503). This possibly suggested a difference in formate metabolism between these two strains. Therefore, we investigated whether the Fdh was Tat dependent in C. jejuni 81-176. For this purpose, we measured formate respiration in C. jejuni 81-176 (wild-type), a Tat mutant (ΔtatC-81-176), and a complementation strain (C-ΔtatC-81-176) using BIOLOG-AN plates. We also used a viologen-based assay to specifically measure the Fdh enzymatic activity in whole cells and periplasmic preparations of the wild-type and mutant strains. Collectively, our data indicated that the Fdh activity and localization in C. jejuni 81-176 were dependent on a functional Tat system. The proper maturation and maintenance of Fdh are essential for cognate energy production, which might explain the association of the Fdh with the Tat system across different strains. The latter highlights the potential of the Tat system and Fdh as appealing targets for novel anti-C. jejuni therapeutics.

A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1.

BackgroundPreviously we described a transdominant negative mutant of the HIV-1 Tat protein, termed Nullbasic, that downregulated the steady state levels of unspliced and singly spliced viral mRNA, an activity caused by inhibition of HIV-1 Rev activity. Nullbasic also altered the subcellular localizations of Rev and other cellular proteins, including CRM1, B23 and C23 in a Rev-dependent manner, suggesting that Nullbasic may disrupt Rev function and trafficking by intervening with an unidentified component of the Rev nucleocytoplasmic transport complex.ResultsTo seek a possible mechanism that could explain how Nullbasic inhibits Rev activity, we used a proteomics approach to identify host cellular proteins that interact with Nullbasic. Forty-six Nullbasic-binding proteins were identified by mass spectrometry including the DEAD-box RNA helicase, DDX1. To determine the effect of DDX1 on Nullbasic-mediated Rev activity, we performed cell-based immunoprecipitation assays, Rev reporter assays and bio-layer interferometry (BLI) assays. Interaction between DDX1 and Nullbasic was observed by co-immunoprecipitation of Nullbasic with endogenous DDX1 from cell lysates. BLI assays showed a direct interaction between Nullbasic and DDX1. Nullbasic affected DDX1 subcellular distribution in a Rev-independent manner. Interestingly overexpression of DDX1 in cells not only restored Rev-dependent mRNA export and gene expression in a Rev reporter assay but also partly reversed Nullbasic-induced Rev subcellular mislocalization. Moreover, HIV-1 wild type Tat co-immunoprecipitated with DDX1 and overexpression of Tat could rescue the unspliced viral mRNA levels inhibited by Nullbasic in HIV-1 expressing cells.ConclusionsNullbasic was used to further define the complex mechanisms involved in the Rev-dependent nuclear export of the 9 kb and 4 kb viral RNAs. All together, these data indicate that DDX1 can be sequestered by Nullbasic leading to destabilization of the Rev nucleocytoplasmic transport complex and decreased levels of Rev-dependent viral transcripts. The outcomes support a role for DDX1 in maintenance of a Rev nuclear complex that transports viral RRE-containing mRNA to the cytoplasm. To our knowledge Nullbasic is the first anti-HIV protein that specifically targets the cellular protein DDX1 to block Rev’s activity. Furthermore, our research raises the possibility that wild type Tat may play a previously unrecognized but very important role in Rev function.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0121-9) contains supplementary material, which is available to authorized users.

A Yersinia pestis tat mutant is attenuated in bubonic and small-aerosol pneumonic challenge models of infection but not as attenuated by intranasal challenge.

Bacterial proteins destined for the Tat pathway are folded before crossing the inner membrane and are typically identified by an N-terminal signal peptide containing a twin arginine motif. Translocation by the Tat pathway is dependent on the products of genes which encode proteins possessing the binding site of the signal peptide and mediating the actual translocation event. In the fully virulent CO92 strain of Yersinia pestis, the tatA gene was deleted. The mutant was assayed for loss of virulence through various in vitro and in vivo assays. Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type. Cell morphology of the mutant bacteria was altered and demonstrated a more elongated form. In addition, while cultures of the mutant strain were able to produce a biofilm, we observed a loss of adhesion of the mutant biofilm structure compared to the biofilm produced by the wild-type strain. Immuno-electron microscopy revealed a partial disruption of the F1 antigen on the surface of the mutant. The virulence of the ΔtatA mutant was assessed in various murine models of plague. The mutant was severely attenuated in the bubonic model with full virulence restored by complementation with the native gene. After small-particle aerosol challenge in a pneumonic model of infection, the mutant was also shown to be attenuated. In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains. However, an increased time-to-death and delay in bacterial dissemination was observed in mice infected with the ΔtatA mutant as compared to the parent strain. Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge.

HIV-1 Tat second exon limits the extent of Tat-mediated modulation of interferon-stimulated genes in antigen presenting cells.

BackgroundWe have shown that HIV-1 Tat interaction with MAP2K3, MAP2K6, and IRF7 promoters is key to IFN-stimulated genes (ISG) activation in immature dendritic cells and macrophages.ResultsWe evaluated how Tat alleles and mutants differ in cellular gene modulation of immature dendritic cells and monocyte-derived macrophages and what similarities this modulation has with that induced by interferons. The tested alleles and mutants modulated to different degrees ISG, without concomitant induction of interferons. The first exon TatSF21-72 and the minimal transactivator TatSF21-58, all modulated genes to a significantly greater extent than full-length wild type, two-exon Tat, indicating that Tat second exon is critical in reducing the innate response triggered by HIV-1 in these cells. Mutants with reduced LTR transactivation had a substantially reduced effect on host gene expression modulation than wild type TatSF2. However, the more potent LTR transactivator TatSF2A58T modulated ISG expression to a lower degree compared to TatSF2. A cellular gene modulation similar to that induced by Tat and Tat mutants in immature dendritic cells could be observed in monocyte-derived macrophages, with the most significant pathways affected by Tat being the same in both cell types. Tat expression in cells deleted of the type I IFN locus or receptor resulted in a gene modulation pattern similar to that induced in primary immature dendritic cells and monocyte-derived macrophages, excluding the involvement of type I IFNs in Tat-mediated gene modulation. ISG activation depends on Tat interaction with MAP2K3, MAP2K6, and IRF7 promoters and a single exon Tat protein more strongly modulated the luciferase activity mediated by MAP2K3, MAP2K6, and IRF7 promoter sequences located 5′ of the RNA start site than the wild type two-exon Tat, while a cysteine and lysine Tat mutants, reduced in LTR transactivation, had negligible effects on these promoters. Chemical inhibition of CDK9 or Sp1 decreased Tat activation of MAP2K3-, MAP2K6-, and IRF7-mediated luciferase transcription.ConclusionsTaken together, these data indicate that the second exon of Tat is critical to the containment of the innate response stimulated by Tat in antigen presenting cells and support a role for Tat in stimulating cellular transcription via its interaction with transcription factors present at promoters.

A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1

BackgroundPreviously we described a transdominant negative mutant of the HIV-1 Tat protein, termed Nullbasic, that downregulated the steady state levels of unspliced and singly spliced viral mRNA, an activity caused by inhibition of HIV-1 Rev activity. Nullbasic also altered the subcellular localizations of Rev and other cellular proteins, including CRM1, B23 and C23 in a Rev-dependent manner, suggesting that Nullbasic may disrupt Rev function and trafficking by intervening with an unidentified component of the Rev nucleocytoplasmic transport complex.ResultsTo seek a possible mechanism that could explain how Nullbasic inhibits Rev activity, we used a proteomics approach to identify host cellular proteins that interact with Nullbasic. Forty-six Nullbasic-binding proteins were identified by mass spectrometry including the DEAD-box RNA helicase, DDX1. To determine the effect of DDX1 on Nullbasic-mediated Rev activity, we performed cell-based immunoprecipitation assays, Rev reporter assays and bio-layer interferometry (BLI) assays. Interaction between DDX1 and Nullbasic was observed by co-immunoprecipitation of Nullbasic with endogenous DDX1 from cell lysates. BLI assays showed a direct interaction between Nullbasic and DDX1. Nullbasic affected DDX1 subcellular distribution in a Rev-independent manner. Interestingly overexpression of DDX1 in cells not only restored Rev-dependent mRNA export and gene expression in a Rev reporter assay but also partly reversed Nullbasic-induced Rev subcellular mislocalization. Moreover, HIV-1 wild type Tat co-immunoprecipitated with DDX1 and overexpression of Tat could rescue the unspliced viral mRNA levels inhibited by Nullbasic in HIV-1 expressing cells.ConclusionsNullbasic was used to further define the complex mechanisms involved in the Rev-dependent nuclear export of the 9 kb and 4 kb viral RNAs. All together, these data indicate that DDX1 can be sequestered by Nullbasic leading to destabilization of the Rev nucleocytoplasmic transport complex and decreased levels of Rev-dependent viral transcripts. The outcomes support a role for DDX1 in maintenance of a Rev nuclear complex that transports viral RRE-containing mRNA to the cytoplasm. To our knowledge Nullbasic is the first anti-HIV protein that specifically targets the cellular protein DDX1 to block Rev’s activity. Furthermore, our research raises the possibility that wild type Tat may play a previously unrecognized but very important role in Rev function.

The Twin Arginine Translocation System Is Essential for Aerobic Growth and Full Virulence of Burkholderia thailandensis

The twin arginine translocation (Tat) system in bacteria is responsible for transporting folded proteins across the cytoplasmic membrane, and in some bacteria, Tat-exported substrates have been linked to virulence. We report here that the Tat machinery is present in Burkholderia pseudomallei, B. mallei, and B. thailandensis, and we show that the system is essential for aerobic but not anaerobic growth. Switching off of the Tat system in B. thailandensis grown anaerobically resulted in filamentous bacteria, and bacteria showed increased sensitivity to some β-lactam antibiotics. In Galleria mellonella and zebrafish infection models, the Tat conditional mutant was attenuated. The aerobic growth-restricted phenotype indicates that Tat substrates may play a functional role in oxygen-dependent energy conservation. In other bacteria, aerobic growth restriction in Tat mutants has been attributed to the inability to translocate PetA, the Rieske iron-sulfur protein which forms part of the quinol-cytochrome c oxidoreductase complex. Here, we show that PetA is not responsible for aerobic growth restriction in B. thailandensis. However, we have identified an operon encoding 2 proteins of unknown function (BTH_I2176 and BTH_I2175) that play a role in aerobic growth restriction, and we present evidence that BTH_I2176 is Tat translocated.