Role of type IV pili in virulence of Pseudomonas syringae pv. tabaci 6605: correlation of motility, multidrug resistance, and HR-inducing activity on a nonhost plant

To investigate the role of type IV pili in the virulence of phytopathogenic bacteria, four mutant strains for pilus biogenesis-related genes were generated in Pseudomonas syringae pv. tabaci 6605. PilA encodes the pilin protein as a major subunit of type IV pili, and the pilO product is reported to be required for pilus assembly. The fimU and fimT genes are predicted to produce minor pilins. Western blot analysis revealed that pilA, pilO, and fimU mutants but not the fimT mutant failed to construct type IV pili. Although the swimming motility of all mutant strains was not impaired in liquid medium, they showed remarkably reduced motilities on semisolid agar medium, suggesting that type IV pili are required for surface motilities. Virulence toward host tobacco plants and hypersensitive response-inducing ability in nonhost Arabidopsis leaves of pilA, pilO, and fimU mutant strains were reduced. These results might be a consequence of reduced expression of type III secretion system-related genes in the mutant strains. Further, all mutant strains showed enhanced expression of resistance-nodulation-division family members mexA, mexB, and oprM, and higher tolerance to antimicrobial compounds. These results indicate that type IV pili are an important virulence factor of this pathogen.     

Characterization of a complex chemosensory signal transduction system which controls twitching motility in Pseudomonas aeruginosa

Virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of a wide range of virulence factors including type IV pili which are required for colonization of host tissues and are associated with a form of surface translocation termed twitching motility. Twitching motility in P. aeruginosa is controlled by a complex signal transduction pathway which shares many modules in common with chemosensory systems controlling flagella rotation in bacteria and which is composed, in part, of the previously described proteins PilG, PilH, PilI, PilJ and PilK. Here we describe another three components of this pathway: ChpA, ChpB and ChpC, as well as two downstream genes, ChpD and ChpE, which may also be involved. The central component of the pathway, ChpA, possesses nine potential sites of phosphorylation: six histidine-containing phosphotransfer (HPt) domains, two novel serine- and threonine-containing phosphotransfer (SPt, TPt) domains and a CheY-like receiver domain at its C-terminus, and as such represents one of the most complex signalling proteins yet described in nature. We show that the Chp chemosensory system controls twitching motility and type IV pili biogenesis through control of pili assembly and/or retraction as well as expression of the pilin subunit gene pilA. The Chp system is also required for full virulence in a mouse model of acute pneumonia.     

Two newly identified SipA domains (F1, F2) steer effector protein localization and contribute to Salmonella host cell manipulation

Salmonella Typhimurium causes bacterial enterocolitis. The type III secretion system (TTSS)-1 is a key virulence determinant of S. Typhimurium mediating host cell invasion and acute enterocolitis. The TTSS-1 effector protein SipA is transported into host cells, accumulates in characteristic foci at the bacteria-host cell interface, manipulates signalling and affects virulence. Two functional domains of SipA have previously been characterized: The N-terminal SipA region (amino acids 1-105) mediates TTSS-1 transport and the C-terminal SipA 'actin-binding' domain (ABD; amino acids 446-685) manipulates F-actin assembly. Little is known about the central region of SipA. In a deletion analysis we found that the central SipA region harbours two distinct functional domains, F1 and F2. They are involved in SipA focus formation and host manipulation. The F1 domain (amino acids 170-271) drives SipA focus formation and domain F2 (amino acids 280-394) enhances this process by mediating SipA-SipA interactions. SipA variants lacking the F1-, the F2- or the actin binding domain were attenuated in virulence assays, namely host cell invasion and/or virulence in a mouse model for enterocolitis. Our results show that the newly identified SipA domains have distinct functions. Nevertheless, cooperation between the SipA domains F1, F2 and ABD is required to promote Salmonella virulence.     

Cryo-EM structure of an early precursor of large ribosomal subunit reveals a half-assembled intermediate

Assembly of eukaryotic ribosome is a complicated and dynamic process that involves a series of intermediates.It is unknown how the highly intertwined structure of 60S large ribosomal subunits is established.Here,we report the structure of an early nucleolar pre-60S ribosome determined by cryo-electron microscopy at 3.7 A resolution,revealing a half-assembled subunit.DomainsⅠ,ⅡandⅣof 25S/5.8S rRNA pack tightly into a native-like substructure,but domains Ⅲ,ⅣandⅤare not assembled.The structure contains 12 assembly factors and 19 ribosomal proteins,many of which are required for early processing of large subunit rRNA.The Brx1-Ebp2 complex would interfere with the assembly of domains Ⅳ and Ⅴ.Rpf1,Mak16,Nsa1 and Rrp1 form a cluster that consolidates the joining of domainsⅠandⅡ.Our structure reveals a key intermediate on the path to establishing the global architecture of 60S subunits.     

Voltage sensors in domains III and IV, but not I and II, are immobilized by Na+ channel fast inactivation

Using site-directed fluorescent labeling, we examined conformational changes in the S4 segment of each domain of the human skeletal muscle sodium channel (hSkM1). The fluorescence signals from S4 segments in domains I and II follow activation and are unaffected as fast inactivation settles. In contrast, the fluorescence signals from S4 segments in domains III and IV show kinetic components during activation and deactivation that correlate with fast inactivation and charge immobilization. These results indicate that in hSkM1, the S4 segments in domains III and IV are responsible for voltage-sensitive conformational changes linked to fast inactivation and are immobilized by fast inactivation, while the S4 segments in domains I and II are unaffected by fast inactivation.     

Molecular dissection of the ST8Sia IV polysialyltransferase. Distinct domains are required for neural cell adhesion molecule recognition and polysialylation

Polysialic acid, a homopolymer of alpha2,8-linked sialic acid expressed on the neural cell adhesion molecule (NCAM), is thought to play critical roles in neural development. Two highly homologous polysialyltransferases, ST8Sia II and ST8Sia IV, which belong to the sialyltransferase gene family, synthesize polysialic acid on NCAM. By contrast, ST8Sia III, which is moderately homologous to ST8Sia II and ST8Sia IV, adds oligosialic acid to itself but very inefficiently to NCAM. Here, we report domains of polysialyltransferases required for NCAM recognition and polysialylation by generating chimeric enzymes between ST8Sia IV and ST8Sia III or ST8Sia II. We first determined the catalytic domain of ST8Sia IV by deletion mutants. To identify domains responsible for NCAM polysialylation, different segments of the ST8Sia IV catalytic domain, identified by the deletion experiments, were replaced with corresponding segments of ST8Sia II and ST8Sia III. We found that larger polysialic acid was formed on the enzymes themselves (autopolysialylation) when chimeric enzymes contained the carboxyl-terminal region of ST8Sia IV. However, chimeric enzymes that contain only the carboxyl-terminal segment of ST8Sia IV and the amino-terminal segment of ST8Sia III showed very weak activity toward NCAM, even though they had strong activity in polysialylating themselves. In fact, chimeric enzymes containing the amino-terminal portion of ST8Sia IV fused to downstream sequences of ST8Sia III inhibited NCAM polysialylation in vitro, although they did not polysialylate NCAM. These results suggest that in polysialyltransferases the NCAM recognition domain is distinct from the polysialylation domain and that some chimeric enzymes may act as a dominant negative enzyme for NCAM polysialylation.     

Arrangement of the respiratory chain complexes in Saccharomyces cerevisiae supercomplex III2IV2 revealed by single particle cryo-electron microscopy

Here we present for the first time a three-dimensional cryo-EM map of the Saccharomyces cerevisiae respiratory supercomplex composed of dimeric complex III flanked on each side by one monomeric complex IV. A precise fit of the existing atomic x-ray structures of complex III from yeast and complex IV from bovine heart into the cryo-EM map resulted in a pseudo-atomic model of the three-dimensional structure for the supercomplex. The distance between cytochrome c binding sites of complexes III and IV is about 6 nm, which supports proposed channeling of cytochrome c between the individual complexes. The opposing surfaces of complexes III and IV differ considerably from those reported for the bovine heart supercomplex as determined by cryo-EM. A closer association between the individual complex domains at the aqueous membrane interface and larger spaces between the membrane-embedded domains where lipid molecules may reside are also demonstrated. The supercomplex contains about 50 molecules of cardiolipin (CL) with a fatty acid composition identical to that of the inner membrane CL pool, consistent with CL-dependent stabilization of the supercomplex.     

Herpesvirus mRNAs are sorted for export via Crm1-dependent and -independent pathways

Cellular pre-mRNA splicing is inhibited by ICP27, a herpes simplex virus regulatory protein, resulting in the shutoff of host protein synthesis. Here we reveal that ICP27 also mediates the export of some virus RNAs via a Crm1-dependent pathway and present evidence that independent domains are required for these functions. Sorting of some viral mRNAs for nuclear export requires Crm1, while other virus mRNAs are exported via another pathway.     

Recognition and delivery of effector proteins into eukaryotic cells by bacterial secretion systems

The direct transport of virulence proteins from bacterium to host has emerged as a common strategy employed by Gram-negative pathogens to establish infections. Specialized secretion systems function to facilitate this process. The delivery of 'effector' proteins by these secretion systems is currently confined to two functionally similar but mechanistically distinct pathways, termed type III and type IV secretion. The type III secretion pathway is ancestrally related to the multiprotein complexes that assemble flagella, whereas the type IV mechanism probably emerged from the protein complexes that support conjugal transfer of DNA. Although both pathways serve to transport proteins from the bacterium to host, the recognition of the effector protein substrates and the secretion information contained in these proteins appear highly distinct. Here, we review the mechanisms involved in the selection of substrates by each of these transport systems and secretion signal information required for substrate transport.     

Proteomic patterns of cervical cancer cell lines, a network perspective

Background

Systemic bacterial infections are highly regulated and complex processes that are orchestrated by numerous virulence factors. Genes that are coordinately controlled by the set of regulators required for systemic infection are potentially required for pathogenicity.

Results

In this study we present a systems biology approach in which sample-matched multi-omic measurements of fourteen virulence-essential regulator mutants were coupled with computational network analysis to efficiently identify Salmonella virulence factors. Immunoblot experiments verified network-predicted virulence factors and a subset was determined to be secreted into the host cytoplasm, suggesting that they are virulence factors directly interacting with host cellular components. Two of these, SrfN and PagK2, were required for full mouse virulence and were shown to be translocated independent of either of the type III secretion systems in Salmonella or the type III injectisome-related flagellar mechanism.

Conclusions

Integrating multi-omic datasets from Salmonella mutants lacking virulence regulators not only identified novel virulence factors but also defined a new class of translocated effectors involved in pathogenesis. The success of this strategy at discovery of known and novel virulence factors suggests that the approach may have applicability for other bacterial pathogens.     

Mutational analysis of the herpes simplex virus virion host shutoff protein: evidence that vhs functions in the absence of other viral proteins.

Herpes simplex virus (HSV) virions contain one or more factors that trigger rapid shutoff of host protein synthesis and accelerated decay of cellular and viral mRNAs in infected cells. HSV isolates bearing mutations at the virion host shutoff (vhs) locus (gene UL41) are defective for both processes, indicating that the vhs protein is required; however, it is not clear whether the role of vhs in shutoff is direct or indirect and if other virion components are also necessary. We therefore used a transient-cotransfection assay to determine if the vhs protein displays activity in the absence of other viral gene products. We found that a vhs expression vector strongly suppressed expression of a cotransfected lacZ reporter gene and that this effect was eliminated by the vhs1 point mutation that abolishes virion-induced host shutoff during HSV infection. Further evidence for the biological relevance of the transfection assay came from the demonstration that five vhs in-frame linker insertion mutations yielded concordant results when assayed in cotransfected cells and following transfer into the viral genome: three mutations eliminated activity in both assays, while two had no effect. On the basis of these results, we conclude that the vhs protein can trigger host shutoff in the absence of other HSV proteins. The cotransfection assay was used to rapidly assess the activities of a panel of linker insertion mutants spanning the vhs polypeptide. All mutations that mapped to regions conserved among the vhs homologs of alphaherpesvirus inactivated function; in contrast, four of five mutations that mapped to regions that are absent from several vhs homologs had no effect. These results further support the biological relevance of the transfection assay and begin to delineate functional domains of the vhs polypeptide.     

A second type of rat phosphoinositide-specific phospholipase C containing a src-related sequence not essential for phosphoinositide-hydrolyzing activity

A fourth type of rat phosphoinositide-specific phospholipase C (PLC IV) has been cloned for cDNA and sequenced. PLC IV is distinct from the other three types of rat PLC (PLC I, II, and III) with respect to primary structure and tissue distribution of its mRNAs. PLC IV contains two homologous regions included commonly in PLC I, II, and III and is most similar to PLC II (identity: 50.2%). PLC IV, in common with PLC II, has a sequence homologous to the N-terminal regulatory domains of nonreceptor tyrosine kinases of the src-family of oncogenes. Using an Escherichia coli expression system, we succeeded in producing active PLC IV in E. coli crude extracts. Various truncation experiments of the PLC IV cDNA revealed that the src-related domain is not necessary for catalytic activity while both domains homologous among PLC I-IV are essential. PLC IV is expressed in various rat tissues and abundant in spleen, suggesting that PLC IV plays a fundamental role in cellular functions such as growth and secretion.     

On the presence and organization of open reading frames of the nonmotile pathogen Brucella abortus similar to class II, III, and IV flagellar genes and to LcrD virulence superfamily

Brucellae are pathogenic, nonmotile bacteria that are facultative intracellular parasites. Little is known about the genetics of these bacteria. Open reading frames from Brucella abortus with similarity to the flagellin, M-ring, and hook of related bacteria were discovered. The open reading frames encode proteins of three of the four flagellum gene classes, namely II, III, and IV. A homolog of the LcrD virulence superfamily was also found. This superfamily is involved in type III protein secretion. B. abortus has the potential for motility and type III secretion.     

Structure of a mutant EF-G reveals domain III and possibly the fusidic acid binding site

The crystal structure of Thermus thermophilus elongation factor G (EF-G) carrying the point mutation His573Ala was determined at a resolution of 2.8 A. The mutant has a more closed structure than that previously reported for wild-type EF-G. This is obtained by a 10 degrees rigid rotation of domains III, IV and V with regard to domains I and II. This rotation results in a displacement of the tip of domain IV by approximately 9 A. The structure of domain III is now fully visible and reveals the double split beta-alpha-beta motif also observed for EF-G domain V and for several ribosomal proteins. A large number of fusidic acid resistant mutations found in domain III have now been possible to locate. Possible locations for the effector loop and a possible binding site for fusidic acid are discussed in relation to some of the fusidic acid resistant mutations.     

The multiple RNA-binding domains of the mRNA poly(A)-binding protein have different RNA-binding activities.

The poly(A)-binding protein (PABP) is the major mRNA-binding protein in eukaryotes, and it is essential for viability of the yeast Saccharomyces cerevisiae. The amino acid sequence of the protein indicates that it consists of four ribonucleoprotein consensus sequence-containing RNA-binding domains (RBDs I, II, III, and IV) and a proline-rich auxiliary domain at the carboxyl terminus. We produced different parts of the S. cerevisiae PABP and studied their binding to poly(A) and other ribohomopolymers in vitro. We found that none of the individual RBDs of the protein bind poly(A) specifically or efficiently. Contiguous two-domain combinations were required for efficient RNA binding, and each pairwise combination (I/II, II/III, and III/IV) had a distinct RNA-binding activity. Specific poly(A)-binding activity was found only in the two amino-terminal RBDs (I/II) which, interestingly, are dispensable for viability of yeast cells, whereas the activity that is sufficient to rescue lethality of a PABP-deleted strain is in the carboxyl-terminal RBDs (III/IV). We conclude that the PABP is a multifunctional RNA-binding protein that has at least two distinct and separable activities: RBDs I/II, which most likely function in binding the PABP to mRNA through the poly(A) tail, and RBDs III/IV, which may function through binding either to a different part of the same mRNA molecule or to other RNA(s).     

Roles of molecular regions in determining differences between voltage dependence of activation of CaV3.1 and CaV1.2 calcium channels

Voltage-dependent calcium channels are classified into low voltage-activated and high voltage-activated channels. We have investigated the molecular basis for this difference in voltage dependence of activation by constructing chimeras between a low voltage-activated channel (Ca(V)3.1) and a high voltage-activated channel (Ca(V)1.2), focusing on steady-state activation properties. Wild type and chimeras were expressed in oocytes, and two-electrode voltage clamp recordings were made of calcium channel currents. Replacement of domains I, III, or IV of the Ca 3.1 channel with the corresponding domain of Ca(V)1.2 led (V)to high voltage-activated channels; for these constructs the current/voltage (I/V) curves were similar to those for Ca(V)1.2 wild type. However, replacement of domain II gave only a small shift to the right of the I/V curve and modulation of the activation kinetics but did not lead to a high voltage-activating channel with an I/V curve like Ca 1.2. We also investigated the role of the voltage sensor (V)S4 by replacing the S4 segment of Ca(V)3.1 with that of Ca 1.2. For domain I, there was no shift in the I/V curve (V)as compared with Ca(V)3.1, and there were relatively small shifts to the right for domains III and IV. Taken together, these results suggest that domains I, III, and IV (rather than domain II) are apparently critical for channel opening and, therefore, contribute strongly to the difference in voltage dependence of activation between Ca 3.1 and Ca(V)1.2. However, the S4 segments in domains I, (V)III, and IV did not account for this difference in voltage dependence.