Pseudomonas aeruginosa EftM Is a Thermoregulated Methyltransferase

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that trimethylates elongation factor-thermo-unstable (EF-Tu) on lysine 5. Lysine 5 methylation occurs in a temperature-dependent manner and is generally only seen when P. aeruginosa is grown at temperatures close to ambient (25 °C) but not at higher temperatures (37 °C). We have previously identified the gene, eftM (for EF-Tu-modifying enzyme), responsible for this modification and shown its activity to be associated with increased bacterial adhesion to and invasion of respiratory epithelial cells. Bioinformatic analyses predicted EftM to be a Class I S-adenosyl-l-methionine (SAM)-dependent methyltransferase. An in vitro methyltransferase assay was employed to show that, in the presence of SAM, EftM directly trimethylates EF-Tu. A natural variant of EftM, with a glycine to arginine substitution at position 50 in the predicted SAM-binding domain, lacks both SAM binding and enzyme activity. Mass spectrometry analysis of the in vitro methyltransferase reaction products revealed that EftM exclusively methylates at lysine 5 of EF-Tu in a distributive manner. Consistent with the in vivo temperature dependence of methylation of EF-Tu, preincubation of EftM at 37 °C abolished methyltransferase activity, whereas this activity was retained when EftM was preincubated at 25 °C. Irreversible protein unfolding at 37 °C was observed, and we propose that this instability is the molecular basis for the temperature dependence of EftM activity. Collectively, our results show that EftM is a thermolabile, SAM-dependent methyltransferase that directly trimethylates lysine 5 of EF-Tu in P. aeruginosa.     

Pseudomonas aeruginosa D-arabinofuranose biosynthetic pathway and its role in type IV pilus assembly

Pseudomonas aeruginosa strains PA7 and Pa5196 glycosylate their type IVa pilins with α1,5-linked D-arabinofuranose (d-Araf), a rare sugar configuration identical to that found in cell wall polymers of the Corynebacterineae. Despite this chemical identity, the pathway for biosynthesis of α1,5-D-Araf in Gram-negative bacteria is unknown. Bioinformatics analyses pointed to a cluster of seven P. aeruginosa genes, including homologues of the Mycobacterium tuberculosis genes Rv3806c, Rv3790, and Rv3791, required for synthesis of a polyprenyl-linked d-ribose precursor and its epimerization to D-Araf. Pa5196 mutants lacking the orthologues of those genes had non-arabinosylated pilins, poor twitching motility, and significantly fewer surface pili than the wild type even in a retraction-deficient (pilT) background. The Pa5196 pilus system assembled heterologous non-glycosylated pilins efficiently, demonstrating that it does not require post-translationally modified subunits. Together the data suggest that pilins of group IV strains need to be glycosylated for productive subunit-subunit interactions. A recombinant P. aeruginosa PAO1 strain co-expressing the genes for d-Araf biosynthesis, the pilin modification enzyme TfpW, and the acceptor PilA(IV) produced arabinosylated pili, confirming that the Pa5196 genes identified are both necessary and sufficient. A P. aeruginosa epimerase knock-out could be complemented with the corresponding Mycobacterium smegmatis gene, demonstrating conservation between the systems of the Corynebacterineae and Pseudomonas. This work describes a novel Gram-negative pathway for biosynthesis of d-Araf, a key therapeutic target in Corynebacterineae.     

The Intermediate Filament Vimentin Mediates MicroRNA miR-378 Function in Cellular Self-renewal by Regulating the Expression of the Sox2 Transcription Factor

MicroRNAs are short noncoding RNAs that are implicated in cell self- renewal and cancer development. We show that miR-378 is up-regulated in human cancers and found that tumor cells transfected with miR-378 acquired properties of tumor stem cells, including cell self-renewal. Overexpression of miR-378 enhanced cell survival and colony formation. Isolated from a single-cell colony, the miR-378-expressing cells formed tumors in nude mice at low cell densities. These cells expressed higher levels of miR-378 and formed more and larger spheres and colonies. We found that the miR-378-expressing cells contained a large number of side population cells and could undergo differentiation. Cells transfected with miR-378 expressed increased levels of Sox2. Expression of miR-378 and Sox2 was found correlated significantly in cancer cell lines and in cancer patient specimens. We also observed opposite levels of vimentin in the cancer cell lines and human breast carcinoma specimens. We further demonstrated that vimentin is a target of miR-378, and ectopic transfection of vimentin inhibited Sox2 expression, resulting in decreased cell survival. Silencing vimentin promoted Sox2 expression and cell survival. Our study demonstrates that miR-378 is a regulator of stem cell marker Sox2 by targeting vimentin, which may serve as a new tool in studying the role of stem cells in tumorigenesis.     

Epidermal Growth Factor Receptor (EGFR) Signaling Requires a Specific Endoplasmic Reticulum Thioredoxin for the Post-translational Control of Receptor Presentation to the Cell Surface

The epidermal growth factor receptor (EGFR) is a well characterized receptor-tyrosine kinase that functions in development and serves a vital role in many human cancers. Understanding EGFR regulatory mechanisms, and hence approaches for clinical intervention, has focused on ligand-receptor interactions and tyrosine kinase activity. Here, we show using the NCI-H460 lung and A431 epidermoid human cancer cell lines that EGFR binding to anterior gradient homolog 2 (AGR2) in the endoplasmic reticulum is required for receptor delivery to the plasma membrane and thus EGFR signaling. Reduced AGR2 protein levels or mutation of an essential cysteine in the active site result in decreased cell surface EGFR and a concomitant decrease in signaling as reflected by AREG, EGR1, and FOS expression. Similar to previously described EGFR nulls, an AGR2 null also resulted in embryonic lethality. Consistent with its role in regulating EGFR-mediated signaling, AGR2 expression is also enhanced in many human cancers and promotes the transformed phenotype. Furthermore, EGFR-mediated signaling in NCI-H460 cells, which are resistant to the tyrosine kinase inhibitor AG1478, is also disrupted with reduced AGR2 expression. The results provide insights into why cancer prognosis or response to therapy often does not correlate with EGFR protein or RNA levels because they do not reflect delivery to the cell surface where signaling is initiated. AGR2, therefore, represents a novel post-translational regulator of EGFR-mediated signaling and a promising target for treating human cancers.     

Dimeric c-di-GMP Is Required for Post-translational Regulation of Alginate Production in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. These results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.     

Paraoxonase 2 Serves a Proapopotic Function in Mouse and Human Cells in Response to the Pseudomonas aeruginosa Quorum-sensing Molecule N-(3-Oxododecanoyl)-homoserine Lactone

Pseudomonas aeruginosa use quorum-sensing molecules, including N-(3-oxododecanoyl)-homoserine lactone (C12), for intercellular communication. C12 activated apoptosis in mouse embryo fibroblasts (MEF) from both wild type (WT) and Bax/Bak double knock-out mice (WT MEF and DKO MEF that were responsive to C12, DKO_R MEF): nuclei fragmented; mitochondrial membrane potential (Δψ_mito) depolarized; Ca²⁺ was released from the endoplasmic reticulum (ER), increasing cytosolic [Ca²⁺] (Ca_cyto); and caspase 3/7 was activated. DKO_R MEF had been isolated from a nonclonal pool of DKO MEF that were non-responsive to C12 (DKO_NR MEF). RNAseq analysis, quantitative PCR, and Western blots showed that WT and DKO_R MEF both expressed genes associated with cancer, including paraoxonase 2 (PON2), whereas DKO_NR MEF expressed little PON2. Adenovirus-mediated expression of human PON2 in DKO_NR MEF rendered them responsive to C12: Δψ_mito depolarized, Ca_cyto increased, and caspase 3/7 activated. Human embryonic kidney 293T (HEK293T) cells expressed low levels of endogenous PON2, and these cells were also less responsive to C12. Overexpression of PON2, but not PON2-H114Q (no lactonase activity) in HEK293T cells caused them to become sensitive to C12. Because [C12] may reach high levels in biofilms in lungs of cystic fibrosis (CF) patients, PON2 lactonase activity may control Δψ_mito, Ca²⁺ release from the ER, and apoptosis in CF airway epithelia. Coupled with previous data, these results also indicate that PON2 uses its lactonase activity to prevent Bax- and Bak-dependent apoptosis in response to common proapoptotic drugs like doxorubicin and staurosporine, but activates Bax- and Bak-independent apoptosis in response to C12.     

Caenorhabditis elegans Recognizes a Bacterial Quorum-sensing Signal Molecule through the AWCON Neuron

In a process known as quorum sensing, bacteria use chemicals called autoinducers for cell-cell communication. Population-wide detection of autoinducers enables bacteria to orchestrate collective behaviors. In the animal kingdom detection of chemicals is vital for success in locating food, finding hosts, and avoiding predators. This behavior, termed chemotaxis, is especially well studied in the nematode Caenorhabditis elegans. Here we demonstrate that the Vibrio cholerae autoinducer (S)-3-hydroxytridecan-4-one, termed CAI-1, influences chemotaxis in C. elegans. C. elegans prefers V. cholerae that produces CAI-1 over a V. cholerae mutant defective for CAI-1 production. The position of the CAI-1 ketone moiety is the key feature driving CAI-1-directed nematode behavior. CAI-1 is detected by the C. elegans amphid sensory neuron AWC^ON. Laser ablation of the AWC^ON cell, but not other amphid sensory neurons, abolished chemoattraction to CAI-1. These analyses define the structural features of a bacterial-produced signal and the nematode chemosensory neuron that permit cross-kingdom interaction.     

Pseudomonas aeruginosa Minor Pilins Prime Type IVa Pilus Assembly and Promote Surface Display of the PilY1 Adhesin

Type IV pili (T4P) contain hundreds of major subunits, but minor subunits are also required for assembly and function. Here we show that Pseudomonas aeruginosa minor pilins prime pilus assembly and traffic the pilus-associated adhesin and anti-retraction protein, PilY1, to the cell surface. PilV, PilW, and PilX require PilY1 for inclusion in surface pili and vice versa, suggestive of complex formation. PilE requires PilVWXY1 for inclusion, suggesting that it binds a novel interface created by two or more components. FimU is incorporated independently of the others and is proposed to couple the putative minor pilin-PilY1 complex to the major subunit. The production of small amounts of T4P by a mutant lacking the minor pilin operon was traced to expression of minor pseudopilins from the P. aeruginosa type II secretion (T2S) system, showing that under retraction-deficient conditions, T2S minor subunits can prime T4P assembly. Deletion of all minor subunits abrogated pilus assembly. In a strain lacking the minor pseudopilins, PilVWXY1 and either FimU or PilE comprised the minimal set of components required for pilus assembly. Supporting functional conservation of T2S and T4P minor components, our 1.4 Å crystal structure of FimU revealed striking architectural similarity to its T2S ortholog GspH, despite minimal sequence identity. We propose that PilVWXY1 form a priming complex for assembly and that PilE and FimU together stably couple the complex to the major subunit. Trafficking of the anti-retraction factor PilY1 to the cell surface allows for production of pili of sufficient length to support adherence and motility.     

16S rDNA用作荧光定量PCR靶基因快速检测铜绿假单胞菌

对20余种细菌16SrDNAs进行多序列比对与进化树分析,设计铜绿假单胞菌(Pseudomonasaeruginosa,PA)荧光定量PCR(fluorescencequantitativePCR,FQ-PCR)特异性引物。提取PA基因组DNA,以特异性引物扩增16SrDNA靶片段,并构建重组质粒pMDT-Pfr。将梯度稀释的pMDT-Pfr质粒作为模板,用于建立定量标准曲线。以SYBRGreenI荧光染料建立20μL反应体系,对不同浓度的PADNA样品进行FQ-PCR检测。同时,以金黄色葡萄球菌、伤寒杆菌、福氏志贺菌、变形杆菌、表皮葡萄球菌、大肠杆菌和结核杆菌的基因组DNA作阴性对照,验证FQ-PCR方法检测PA的特异性。结果显示,设计的FQ-PCR引物的靶向序列,仅对PA16SrDNA有高度同源性;FQ-PCR方法检测PA,其灵敏度达3.6pg/μL的基因组DNA或(2.1×103±3.1×102)拷贝/μL的16SrDNA基因,并且具有很强的特异性;从细菌DNA提取到FQ-PCR检测,可在2h左右完成PA鉴定。较传统的培养鉴定法而言,以16SrDNA作为FQ-PCR靶基因快速检测PA,具有很好的研究价值与应用前景。     

A Physiologically Required G Protein-coupled Receptor (GPCR)-Regulator of G Protein Signaling (RGS) Interaction That Compartmentalizes RGS Activity

G protein-coupled receptors (GPCRs) can interact with regulator of G protein signaling (RGS) proteins. However, the effects of such interactions on signal transduction and their physiological relevance have been largely undetermined. Ligand-bound GPCRs initiate by promoting exchange of GDP for GTP on the Gα subunit of heterotrimeric G proteins. Signaling is terminated by hydrolysis of GTP to GDP through intrinsic GTPase activity of the Gα subunit, a reaction catalyzed by RGS proteins. Using yeast as a tool to study GPCR signaling in isolation, we define an interaction between the cognate GPCR (Mam2) and RGS (Rgs1), mapping the interaction domains. This reaction tethers Rgs1 at the plasma membrane and is essential for physiological signaling response. In vivo quantitative data inform the development of a kinetic model of the GTPase cycle, which extends previous attempts by including GPCR-RGS interactions. In vivo and in silico data confirm that GPCR-RGS interactions can impose an additional layer of regulation through mediating RGS subcellular localization to compartmentalize RGS activity within a cell, thus highlighting their importance as potential targets to modulate GPCR signaling pathways.     

Identification and Functional Characterization of the Phosphorylation Sites of the Neuropeptide FF2 Receptor

The neuropeptide FF₂ (NPFF₂) receptor belongs to the rhodopsin family of G protein-coupled receptors and mediates the effects of several related RFamide neuropeptides. One of the main pharmacological interests of this system resides in its ability to regulate endogenous opioid systems, making it a potential target to reduce the negative effects of chronic opioid use. Phosphorylation of intracellular residues is the most extensively studied post-translational modification regulating G protein-coupled receptor activity. However, until now, no information concerning NPFF₂ receptor phosphorylation is available. In this study, we combined mass spectrometric analysis and site-directed mutagenesis to analyze for the first time the phosphorylation pattern of the NPFF₂ receptor and the role of the various phosphorylation sites in receptor signaling, desensitization, and trafficking in a SH-SY5Y model cell line. We identified the major, likely GRK-dependent, phosphorylation cluster responsible for acute desensitization, ⁴¹²TNST⁴¹⁵ at the end of the C terminus of the receptor, and additional sites involved in desensitization (³⁷²TS³⁷³) and internalization (Ser³⁹⁵). We thus demonstrate the key role played by phosphorylation in the regulation of NPFF₂ receptor activity and trafficking. Our data also provide additional evidence supporting the concept that desensitization and internalization are partially independent processes relying on distinct phosphorylation patterns.     

Impact of the exopolysaccharides Pel and Psl on the initial adhesion of Pseudomonas aeruginosa to sand

In this study, the impact of the exopolysaccharides Pel and Psl on the cell surface electron donor-electron acceptor (acid-base) properties and adhesion to quartz sand was investigated by using Pseudomonas aeruginosa PAO1 and its isogenic EPS-mutant strains Δpel, Δpsl and Δpel/Δpsl. The microbial adhesion to hydrocarbon (MATH) test and titration results showed that both Pel and Psl contribute to the surface hydrophobicity of the cell. The results of contact angle measurement, however, showed no correlation with the cell surface hydrophobicity measured by the MATH test and the titration method. Packed-bed column experiments indicated that the exopolysaccharides Pel and Psl are involved in the initial cell attachment to the sand surface and the extent of their impact is dependent on the ionic strength (IS) of the solution. Overall, the Δpel/Δpsl double mutant had the lowest adhesion coefficient to sand compared with the wild-type PAO1, the Δpel mutant and the Δpsl mutant. It is hypothesized that in addition to bacterial surface hydrophobicity and DLVO forces, other factors, eg steric repulsion caused by extracellular macromolecules, and cell surface appendages (flagella and pili) also contribute significantly to the interaction between the cell surface and a sand grain.     

Aberrant Overexpression of the Rgl2 Ral Small GTPase-specific Guanine Nucleotide Exchange Factor Promotes Pancreatic Cancer Growth through Ral-dependent and Ral-independent Mechanisms

Our recent studies established essential and distinct roles for RalA and RalB small GTPase activation in K-Ras mutant pancreatic ductal adenocarcinoma (PDAC) cell line tumorigencity, invasion, and metastasis. However, the mechanism of Ral GTPase activation in PDAC has not been determined. There are four highly related mammalian RalGEFs (RalGDS, Rgl1, Rgl2, and Rgl3) that can serve as Ras effectors. Whether or not they share distinct or overlapping functions in K-Ras-mediated growth transformation has not been explored. We found that plasma membrane targeting to mimic persistent Ras activation enhanced the growth-transforming activities of RalGEFs. Unexpectedly, transforming activity did not correlate directly with total cell steady-state levels of Ral activation. Next, we observed elevated Rgl2 expression in PDAC tumor tissue and cell lines. Expression of dominant negative Ral, which blocks RalGEF function, as well as interfering RNA suppression of Rgl2, reduced PDAC cell line steady-state Ral activity, growth in soft agar, and Matrigel invasion. Surprisingly, the effect of Rgl2 on anchorage-independent growth could not be rescued by constitutively activated RalA, suggesting a novel Ral-independent function for Rgl2 in transformation. Finally, we determined that Rgl2 and RalB both localized to the leading edge, and this localization of RalB was dependent on endogenous Rgl2 expression. In summary, our observations support nonredundant roles for RalGEFs in Ras-mediated oncogenesis and a key role for Rgl2 in Ral activation and Ral-independent PDAC growth.     

Regulated Localization Is Sufficient for Hormonal Control of Regulator of G Protein Signaling Homology Rho Guanine Nucleotide Exchange Factors (RH-RhoGEFs)

The regulator of G protein signaling homology (RH) Rho guanine nucleotide exchange factors (RhoGEFs) (p115RhoGEF, leukemia-associated RhoGEF, and PDZ-RhoGEF) contain an RH domain and are specific GEFs for the monomeric GTPase RhoA. The RH domains interact specifically with the α subunits of G₁₂ heterotrimeric GTPases. Activated Gα₁₃ modestly stimulates the exchange activity of both p115RhoGEF and leukemia-associated RhoGEF but not PDZ-RhoGEF. Because all three RH-RhoGEFs can localize to the plasma membrane upon expression of activated Gα₁₃, cellular localization of these RhoGEFs has been proposed as a mechanism for controlling their activity. We use a small molecule-regulated heterodimerization system to rapidly control the localization of RH-RhoGEFs. Acute localization of the proteins to the plasma membrane activates RhoA within minutes and to levels that are comparable with activation of RhoA by hormonal stimulation of G protein-coupled receptors. The catalytic activity of membrane-localized RhoGEFs is not dependent on activated Gα₁₃. We further show that the conserved RH domains can rewire two different RacGEFs to activate Rac1 in response to a traditional activator of RhoA. Thus, RH domains act as independent detectors for activated Gα₁₃ and are sufficient to modulate the activity of RhoGEFs by hormones via mediating their localization to substrate, membrane-associated RhoA.     

Structural and Functional Studies of the Pseudomonas aeruginosa Minor Pilin,PilE

Many bacterial pathogens, including Pseudomonas aeruginosa, use type IVa pili (T4aP) for attachment and twitching motility. T4aP are composed primarily of major pilin subunits, which are repeatedly assembled and disassembled to mediate function. A group of pilin-like proteins, the minor pilins FimU and PilVWXE, prime pilus assembly and are incorporated into the pilus. We showed previously that minor pilin PilE depends on the putative priming subcomplex PilVWX and the non-pilin protein PilY1 for incorporation into pili, and that with FimU, PilE may couple the priming subcomplex to the major pilin PilA, allowing for efficient pilus assembly. Here we provide further support for this model, showing interaction of PilE with other minor pilins and the major pilin. A 1.25 Å crystal structure of PilE_Δ1–28 shows a typical type IV pilin fold, demonstrating how it may be incorporated into the pilus. Despite limited sequence identity, PilE is structurally similar to Neisseria meningitidis minor pilins PilX_Nm and PilV_Nm, recently suggested via characterization of mCherry fusions to modulate pilus assembly from within the periplasm. A P. aeruginosa PilE-mCherry fusion failed to complement twitching motility or piliation of a pilE mutant. However, in a retraction-deficient strain where surface piliation depends solely on PilE, the fusion construct restored some surface piliation. PilE-mCherry was present in sheared surface fractions, suggesting that it was incorporated into pili. Together, these data provide evidence that PilE, the sole P. aeruginosa equivalent of PilX_Nm and PilV_Nm, likely connects a priming subcomplex to the major pilin, promoting efficient assembly of T4aP.     

Critical Role for an Acidic Amino Acid Region in Platelet Signaling by the HemITAM (Hemi-immunoreceptor Tyrosine-based Activation Motif) Containing Receptor CLEC-2 (C-type Lectin Receptor-2)

CLEC-2 is a member of new family of C-type lectin receptors characterized by a cytosolic YXXL downstream of three acidic amino acids in a sequence known as a hemITAM (hemi-immunoreceptor tyrosine-based activation motif). Dimerization of two phosphorylated CLEC-2 molecules leads to recruitment of the tyrosine kinase Syk via its tandem SH2 domains and initiation of a downstream signaling cascade. Using Syk-deficient and Zap-70-deficient cell lines we show that hemITAM signaling is restricted to Syk and that the upstream triacidic amino acid sequence is required for signaling. Using surface plasmon resonance and phosphorylation studies, we demonstrate that the triacidic amino acids are required for phosphorylation of the YXXL. These results further emphasize the distinct nature of the proximal events in signaling by hemITAM relative to ITAM receptors.     

Compartmentalization of Distinct cAMP Signaling Pathways in Mammalian Sperm

Fertilization competence is acquired in the female tract in a process known as capacitation. Capacitation is needed for the activation of motility (e.g. hyperactivation) and to prepare the sperm for an exocytotic process known as acrosome reaction. Although the HCO₃⁻-dependent soluble adenylyl cyclase Adcy10 plays a role in motility, less is known about the source of cAMP in the sperm head. Transmembrane adenylyl cyclases (tmACs) are another possible source of cAMP. These enzymes are regulated by stimulatory heterotrimeric G_s proteins; however, the presence of G_s or tmACs in mammalian sperm has been controversial. In this study, we used Western blotting and cholera toxin-dependent ADP-ribosylation to show the G_s presence in the sperm head. Also, we showed that forskolin, a tmAC-specific activator, induces cAMP accumulation in sperm from both WT and Adcy10-null mice. This increase is blocked by the tmAC inhibitor SQ22536 but not by the Adcy10 inhibitor KH7. Although G_s immunoreactivity and tmAC activity are detected in the sperm head, PKA is only found in the tail, where Adcy10 was previously shown to reside. Consistent with an acrosomal localization, G_s reactivity is lost in acrosome-reacted sperm, and forskolin is able to increase intracellular Ca²⁺ and induce the acrosome reaction. Altogether, these data suggest that cAMP pathways are compartmentalized in sperm, with G_s and tmAC in the head and Adcy10 and PKA in the flagellum.