Structural Characterization of a Novel Chlamydia pneumoniae Type III Secretion-Associated Protein, Cpn0803

Type III secretion (T3S) is an essential virulence factor used by Gram-negative pathogenic bacteria to deliver effector proteins into the host cell to establish and maintain an intracellular infection. Chlamydia is known to use T3S to facilitate invasion of host cells but many proteins in the system remain uncharacterized. The C. trachomatis protein CT584 has previously been implicated in T3S. Thus, we analyzed the CT584 ortholog in C. pneumoniae (Cpn0803) and found that it associates with known T3S proteins including the needle-filament protein (CdsF), the ATPase (CdsN), and the C-ring protein (CdsQ). Using membrane lipid strips, Cpn0803 interacted with phosphatidic acid and phosphatidylinositol, suggesting that Cpn0803 may associate with host cells. Crystallographic analysis revealed a unique structure of Cpn0803 with a hydrophobic pocket buried within the dimerization interface that may be important for binding small molecules. Also, the binding domains on Cpn0803 for CdsN, CdsQ, and CdsF were identified using Pepscan epitope mapping. Collectively, these data suggest that Cpn0803 plays a role in T3S

Ab Initio Identification of Novel Regulatory Elements in the Genome of Trypanosoma brucei by Bayesian Inference on Sequence Segmentation

Background: The rapid increase in the availability of genome information has created considerable demand for both comparative and ab initio predictive bioinformatic analyses. The biology laid bare in the genomes of many organisms is often novel, presenting new challenges for bioinformatic interrogation. A paradigm for this is the collected genomes of the kinetoplastid parasites, a group which includes Trypanosoma brucei the causative agent of human African trypanosomiasis. These genomes, though outwardly simple in organisation and gene content, have historically challenged many theories for gene expression regulation in eukaryotes. Methodology/Principle Findings: Here we utilise a Bayesian approach to identify local changes in nucleotide composition in the genome of T. brucei. We show that there are several elements which are found at the starts and ends of multicopy gene arrays and that there are compositional elements that are common to all intergenic regions. We also show that there is a composition-inversion element that occurs at the position of the trans-splice site. Conclusions/Significance: The nature of the elements discovered reinforces the hypothesis that context dependant RN

Crystal Structure of an Integron Gene Cassette-Associated Protein from Vibrio cholerae Identifies a Cationic Drug-Binding Module

Background The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes. Methodology/Principal Findings We report the 1.8 A crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators. Conclusions/Significance Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.National Health and Medical Research Council (Australia) (NHMRC grant 488502)National Institutes of Health (U.S.) (Grant GM62414-0 )Ontario. Ministry of Revenue (Challenge Fund

Stochastically Timed Competition Between Division and Differentiation Fates Regulates the Transition From B Lymphoblast to Plasma Cell

In response to external stimuli, naïve B cells proliferate and take on a range of fates important for immunity. How their fate is determined is a topic of much recent research, with candidates including asymmetric cell division, lineage priming, stochastic assignment, and microenvironment instruction. Here we manipulate the generation of plasmablasts from B lymphocytes in vitro by varying CD40 stimulation strength to determine its influence on potential sources of fate control. Using long-term live cell imaging, we directly measure times to differentiate, divide, and die of hundreds of pairs of sibling cells. These data reveal that while the allocation of fates is significantly altered by signal strength, the proportion of siblings identified with asymmetric fates is unchanged. In contrast, we find that plasmablast generation is enhanced by slowing times to divide, which is consistent with a hypothesis of competing timed stochastic fate outcomes. We conclude that this mechanistically simple source of alternative fate regulation is important, and that useful quantitative models of signal integration can be developed based on its principles