The binding of cholera toxin to the periplasmic vestibule of the type II secretion channel

The type II secretion system (T2SS) is a large macromolecular complex spanning the inner and outer membranes of many Gram-negative bacteria. The T2SS is responsible for the secretion of virulence factors such as cholera toxin (CT) and heat-labile enterotoxin (LT) from Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. CT and LT are closely related AB₅ heterohexamers, composed of one A subunit and a B-pentamer. Both CT and LT are translocated, as folded protein complexes, from the periplasm across the outer membrane through the type II secretion channel, the secretin GspD. We recently published the 19 Å structure of the V. cholerae secretin (VcGspD) in its closed state and showed by SPR measurements that the periplasmic domain of GspD interacts with the B-pentamer complex. Here we extend these studies by characterizing the binding of the cholera toxin B-pentamer to VcGspD using electron microscopy of negatively stained preparations. Our studies indicate that the pentamer is captured within the large periplasmic vestibule of VcGspD. These new results agree well with our previously published studies and are in accord with a piston-driven type II secretion mechanism.Key words: secretin, GspD, electron cryomicroscopy, type II secretion system (T2SS), cholera toxin     

Functional magnetic resonance study of deliberate deception

The goal of the study was analysis of the cerebral mechanisms of deliberate deception. The eventrelated functional magnetic resonance (ER fMRI) imaging technique was used to assess the changes in the functional brain activity by means of recording the blood oxygen level-dependent (BOLD) signal. Twelve right-handed healthy volunteers aged 19–44 years participated in the study. The BOLD images were obtained during three experimental trials: deliberate deception, manipulative honest and control truthful trials (catch trials). The deliberate deception and manipulative honest actions were characterized by a BOLD signal increase in the anterior cingulate (Brodmann’s area (BA) 32), frontal (BAs 9/10, 6), and parietal (BA 40) cortices as compared with a truthful response. Comparison of the ER fMRI data with the results of earlier studies where event-related potentials (ERPs) were recorded under similar conditions indicates the involvement of the brain mechanism of error detection in deliberate deception.     

The vegetative state as a manifestation of a stable pathological state

The results of comprehensive monitoring of the state of 25 patients in the vegetative state (prolonged coma) before and after compensating for the factors of secondary brain damage were analyzed and followed up for no less than six months. The primary results showed that the best recovery of consciousness and cognitive functions was observed if, according to the positron emission tomography data, a diffuse decrease in the glucose metabolism rate (GMR_glu), significantly exceeding the extent of the zones of morphological/anatomical lesions, was present in the brain before treatment, while the minimal improvement was observed if the GMR_glu was sufficiently intact. These and other paradoxical results can be explained if the vegetative state is regarded as a stable pathological state of the brain, which offers new approaches to the treatment of this group of patients.     

Epizootics of "avian" influenza as the manifestation of pandemic

In this review of literature modern notions on the role of birds in the evolution of the pathogenicity signs and immune system of the main and intermediate hosts of influenza viruses, as well as on the mechanisms of overcoming interspecific barriers, are analyzed. The chronology of the spread of "avian" influenza among humans, starting from 1997, the properties of the natural reservoir of this infection, and in particular influenza viruses A, the ways of their variability and evolution are presented. The conclusion has been made that the mixing, joint evolution, recombination and reassortment of viral genomes may be caused by global events in individual geographical regions.     

Characterization of the Bellevalia sarmatica (Georgi) Woronov populations from Volgograd oblast using molecular genetic identification

DNA samples obtained from the populations of the Red Data Book species Bellevalia sarmatica (Georgi) Worovow from Volgograd oblast were examined using RAPD and AFLP analyses. DNA marking revealed considerable differences in the levels of interpopulation and intraspecific polymorphisms the Bellevalia. Furthermore, RAPD analysis, despite of lower levels of interpopulation polymorphism identified, made it possible to obtain clearer data on population subdivision upon statistical treatment of the results. The results of this study can be used in developing conservation strategies for maintenance of the species abundance and genetic diversity, as well as in elaborating the criteria for construction genetic collections.     

Secretins: dynamic channels for protein transport across membranes

Secretins form megadalton bacterial-membrane channels in at least four sophisticated multiprotein systems that are crucial for translocation of proteins and assembled fibers across the outer membrane of many species of bacteria. Secretin subunits contain multiple domains, which interact with numerous other proteins, including pilotins, secretion-system partner proteins, and exoproteins. Our understanding of the structure of secretins is rapidly progressing, and it is now recognized that features common to all secretins include a cylindrical arrangement of 12-15 subunits, a large periplasmic vestibule with a wide opening at one end and a periplasmic gate at the other. Secretins might also play a key role in the biogenesis of their cognate secretion systems.     

Calcium Is Essential for the Major Pseudopilin in the Type 2 Secretion System

The pseudopilus is a key feature of the type 2 secretion system (T2SS) and is made up of multiple pseudopilins that are similar in fold to the type 4 pilins. However, pilins have disulfide bridges, whereas the major pseudopilins of T2SS do not. A key question is therefore how the pseudopilins, and in particular, the most abundant major pseudopilin, GspG, obtain sufficient stability to perform their function. Crystal structures of Vibrio cholerae, Vibrio vulnificus, and enterohemorrhagic Escherichia coli (EHEC) GspG were elucidated, and all show a calcium ion bound at the same site. Conservation of the calcium ligands fully supports the suggestion that calcium ion binding by the major pseudopilin is essential for the T2SS. Functional studies of GspG with mutated calcium ion-coordinating ligands were performed to investigate this hypothesis and show that in vivo protease secretion by the T2SS is severely impaired. Taking all evidence together, this allows the conclusion that, in complete contrast to the situation in the type 4 pili system homologs, in the T2SS, the major protein component of the central pseudopilus is dependent on calcium ions for activity.In Gram-negative bacteria, the type 2 secretion system (T2SS)² is used for the secretion of several important proteins across the outer membrane (1). The T2SS is also called the terminal branch of the general secretory pathway (Gsp) (2) and, in Vibrio species, the extracellular protein secretion (Eps) apparatus (3). This sophisticated multiprotein machinery spans both the inner and the outer membrane of Gram-negative bacteria and contains 11–15 different proteins. The T2SS consists of three major subassemblies (4–9): (i) the outer membrane complex comprising mainly the crucial multisubunit secretin GspD; (ii) the pseudopilus, which consists of one major and several minor pseudopilins; and (iii) an inner membrane platform, containing the cytoplasmic secretion ATPase GspE and the membrane proteins GspL, GspM, GspC, and GspF.The pseudopilus is a key element of the T2SS that forms a helical fiber spanning the periplasm. The fiber is assembled from multiple subunits of the major pseudopilin GspG (4, 5, 10–14). The pseudopilus is thought to form a plug of the secretin pore in the outer membrane and/or to function as a piston during protein secretion. In recent years, studies of the T2SS pseudopilins led to structure determinations of all individual pseudopilins (13, 15–17). The recent structure of the helical ternary complex of GspK-GspI-GspJ suggested that these three minor pseudopilins form the tip of the pseudopilus (17). A crystal structure of GspG from Klebsiella oxytoca was in a previous study combined with electron microscopy data to arrive at a helical arrangement, with no evidence for special features, such as disulfide bridges, other covalent links, or metal-binding sites, for stabilizing this major pseudopilin or the pseudopilus (13).The pseudopilins of the T2SS share a common fold with the type 4 pilins (15–21). Pilins are proteins incorporated into pili, long appendages on the surface of bacteria forming thin, strong fibers with multiple functions (19, 21). Type 4 pilins and pseudopilins contain a prepilin leader sequence that is cleaved off by a prepilin peptidase, yielding mature protein (10, 11, 22). A distinct feature of the type 4 pilins is the occurrence of a disulfide bridge connecting β4 to a Cys in the so-called “D-region” near the C terminus (21). In a recent study (23) on the thin fibers of Gram-positive bacteria, isopeptide units appeared to be essential for providing these filaments sufficient cohesion and stability. A key question was therefore whether the major pseudopilin GspG also requires a special feature to obtain sufficient stability to perform its function.     

Latent sequence periodicity of some oncogenes and DNA-binding protein genes

A method of latent periodicity search is developed. We use mutualinformation to reveal the latent periodicity of mRNA sequences.The latent periodicity of an mRNA sequence is a periodicitywith a low level of similarity between any two periods insidethe mRNA sequence. The mutual information between an artificialnumerical sequence and an mRNA sequence is calculated. The lengthof the artificial sequence period is varied from 2 to 150. Thehigh level of the mutual information between artificial andmRNA sequences allows us to find any type of latent periodicityof mRNA sequence. The latent periodicity of many mRNA codingregions has been found. For example, the retinoblastoma geneof HSRBS clone contains a region with a latent period equalto 45 bases. The A-RAF oncogene of HSARAFIR clone contains aregion with a latent period equal to 84 bases. Integrated sequencesfor the regions with latent periodicity are determined. Thepotential significance of latent periodicity is discussed.     

Take five — Type VII secretion systems of Mycobacteria

Mycobacteria use type VII secretion (T7S) systems to secrete proteins across their complex cell envelope. Pathogenic mycobacteria, such as the notorious pathogen Mycobacterium tuberculosis, have up to five of these secretion systems, named ESX-1 to ESX-5. At least three of these secretion systems are essential for mycobacterial virulence and/or viability. Elucidating T7S is therefore essential to understand the success of M. tuberculosis and other pathogenic mycobacteria as pathogens, and could be instrumental to identify novel targets for drug- and vaccine-development. Recently, significant progress has been achieved in the identification of T7S substrates and a general secretion motif. In addition, a start has been made with unraveling the mechanism of secretion and the structural analysis of the different subunits. This review summarizes these recent findings, which are incorporated in a working model of this complex machinery. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Search of tandem repeats with insertion and deletions in the <Emphasis Type="Italic">A. thaliana</Emphasis> genome

A new mathematical method was used for the first time to search for tandem repeats with insertions and deletions in the full-length sequence of the A. thaliana genome. The method is based on a new algorithm for multiple alignment of sequences of certain periods without using paired comparisons of sequences. We identified 13997 periodic sites 2 to 50 characters long, only approximately 30% of which were known earlier. The possible origin and use of the identified sites with tandem repeats are discussed.