Bacterial two-component and hetero-heptameric pore-forming cytolytic toxins: structures, pore-forming mechanism, and organization of the genes

Staphylococcal gamma-hemolysin (Hlg), leukocidin (Luk), and Panton-Valentine leukocidin (PVL) are two-component and hetero-oligomeric pore-forming cytolytic toxins (or cytolysin), that were first identified in bacteria. No information on the existence of hetero-oligomeric pore-forming cytolytic toxins in bacteria except for staphylococcal strains is available so far. Hlg (Hlg1 of 34 kDa/Hlg2 of 32 kDa) effectively lyses erythrocytes from human and other mammalian species. Luk (LukF of 34 kDa/LukS of 33 kDa) is cytolytic toward human and rabbit polymorphonuclear leukocytes and rabbit erythrocytes, and PVL (LukF-PV of 34 kDa/LukS-PV of 33 kDa) reveals cytolytic activity with a high cell specificity to leukocytes. Hlg1 is identical to LukF and that the cell specificities of the cytolysins are determined by Hlg2 and LukS. Based on the primary and 3-dimensional structures of the toxin components, Hlg, Luk, and PVL are thought to form a family of proteins. In the first chapter of this article, we describe the molecular basis of the membrane pore-forming nature of Hlg, Luk, and PVL. We also describe a requirement of the phosphorylation of LukS and LukS-PV by protein kinase for their leukocytolytic activity besides their pore formation on human leukocytes.Recently, the assembly mechanism of the LukF and Hlg2 monomers into pore-forming hetero-oligomers of Hlg on human erythrocyte membranes has been clarified for the first time by our study using a single-molecular fluorescence imaging technique. We estimated 11 sequential equilibrium constants for the assembly pathway which includes the beginning with membrane binding of monomers, proceeds through single pore oligomerization, and culminates in the formation of clusters of the pores. In the second chapter of this article, we refer to an assembly mechanism of LukF and Hlg2 on human erythrocytes as well as the roles of the membranes of the target cells in pore formation by Hlg.The LukF, LukS, and Hlg2 proteins are derived from the Hlg locus (hlg), and have been found in 99% of clinical isolates of Staphylococcus aureus. In contrast, LukF-PV and LukS-PV are derived from the PVL locus (pvl) which is distinct from the hlg locus, and only a small percentage of clinically isolated S. aureus strains carries pvl. Recently, we discovered pvl on the genome of lysogenic bacteriophages, psiPVL, and determined the entire gene of the phage. We also demonstrated the phage conversion of S. aureus leading to the production of PVL through the discovery of a PVL-carrying temperate phage, psiSLT, from a clinical isolate of S. aureus. In the third chapter of this article, we discuss genetic analyses of the Hlg, Luk, and PVL genes. We also discuss the current status of knowledge of the genetic organization of PVL-converting phages in order to achieve an understanding of their molecular evolution.     

Molecular phylogeny of monocotyledons inferred from combined analysis of plastid<Emphasis Type="Italic"> matK</Emphasis> and<Emphasis Type="Italic"> rbcL</Emphasis> gene sequences

Using matK and rbcL sequences (3,269 bp in total) from 113 genera of 45 families, we conducted a combined analysis to contribute to the understanding of major evolutionary relationships in the monocotyledons. Trees resulting from the parsimony analysis are similar to those generated by earlier single or multiple gene analyses, but their strict consensus tree provides much better resolution of relationships among major clades. We find that Acorus (Acorales) is a sister group to the rest of the monocots, which receives 100% bootstrap support. A clade comprising Alismatales is diverged as the next branch, followed successively by Petrosaviaceae, the Dioscoreales–Pandanales clade, Liliales, Asparagales and commelinoids. All of these clades are strongly supported (with more than 90% bootstrap support). The sister-group relationship is also strongly supported between Alismatales and the remaining monocots (except for Acorus) (100%), between Petrosaviaceae and the remaining monocots (except for Acorus and Alismatales) (100%), between the clade comprising Dioscoreales and Pandanales and the clade comprising Liliales, Asparagales and commelinoids (87%), and between Liliales and the Asparagales–commelinoids clade (89%). Only the sister-group relationship between Asparagales and commelinoids is weakly supported (68%). Results also support the inclusion of Petrosaviaceae in its own order Petrosaviales, Nartheciaceae in Dioscoreales and Hanguanaceae in Commelinales.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s10265-003-0133-3     

Specification of amino acid residues essential for the catalytic reaction of cold-active protein-tyrosine phosphatase of a psychrophile, Shewanella sp

Protein-tyrosine phosphatase [EC 3.1.3.48] from a psychrophile, Shewanella sp. shows high activity at low temperatures and has the conserved amino acid sequence of protein-Ser/Thr-phosphatases. Site-directed mutagenesis with the conserved amino acid residues indicated that His148 could be important as a general acid catalyst and Asp115 assists the protonation with His148 of the leaving group of a substrate, and that Asp76 and Asp112 were involved in binding to magnesium ions.     

Current concepts on Escherichia coli K1 translocation of the blood-brain barrier

The mortality and morbidity associated with neonatal gram-negative meningitis have remained significant despite advances in antimicrobial chemotherapy. Escherichia coli K1 is the most common gram-negative organism causing neonatal meningitis. Our incomplete knowledge of the pathogenesis of this disease is one of the main reasons for this high mortality and morbidity. We have previously established both in vitro and in vivo models of the blood-brain barrier (BBB) using human brain microvascular endothelial cells (HBMEC) and hematogenous meningitis in neonatal rats, respectively. With these in vitro and in vivo models, we have shown that successful crossing of the BBB by circulating E. coli requires a high-degree of bacteremia, E. coli binding to and invasion of HBMEC, and E. coli traversal of the BBB as live bacteria. Our previous studies using TnphoA, signature-tagged mutagenesis and differential fluorescence induction identified several E. coli K1 determinants such as OmpA, Ibe proteins, AslA, TraJ and CNF1 contributing to invasion of HBMEC in vitro and traversal of the blood-brain barrier in vivo. We have shown that some of these determinants interact with specific receptors on HBMEC, suggesting E. coli translocation of the BBB is the result of specific pathogen-host cell interactions. Recent studies using functional genomics techniques have identified additional E. coli K1 factors that contribute to the high degree of bacteremia and HBMEC binding/invasion/transcytosis. In this review, we summarize the current knowledge on the mechanisms underlying the successful E. coli translocation of the BBB.     

Crystallization and preliminary X-ray analysis of plant class I chitinase from rice

We report here on crystallization and preliminary X-ray analysis of plant class I chitinase from rice (OsChia1b). Similar single crystals of full-length OsChia1b were obtained under two independent conditions. The crystals grown under these conditions diffracted up to 2.1 and 2.5 angstroms resolution, respectively, at a synchrotron beamline, and were found to belong to the tetragonal space group P4(3)2(1)2.     

Hes genes regulate size, shape and histogenesis of the nervous system by control of the timing of neural stem cell differentiation

Radial glial cells derive from neuroepithelial cells, and both cell types are identified as neural stem cells. Neural stem cells are known to change their competency over time during development: they initially undergo self-renewal only and then give rise to neurons first and glial cells later. Maintenance of neural stem cells until late stages is thus believed to be essential for generation of cells in correct numbers and diverse types, but little is known about how the timing of cell differentiation is regulated and how its deregulation influences brain organogenesis. Here, we report that inactivation of Hes1 and Hes5, known Notch effectors, and additional inactivation of Hes3 extensively accelerate cell differentiation and cause a wide range of defects in brain formation. In Hes-deficient embryos, initially formed neuroepithelial cells are not properly maintained, and radial glial cells are prematurely differentiated into neurons and depleted without generation of late-born cells. Furthermore, loss of radial glia disrupts the inner and outer barriers of the neural tube, disorganizing the histogenesis. In addition, the forebrain lacks the optic vesicles and the ganglionic eminences. Thus, Hes genes are essential for generation of brain structures of appropriate size, shape and cell arrangement by controlling the timing of cell differentiation. Our data also indicate that embryonic neural stem cells change their characters over time in the following order: Hes-independent neuroepithelial cells, transitory Hes-dependent neuroepithelial cells and Hes-dependent radial glial cells.     

Complex spatial distribution and dynamics of an abundant Escherichia coli outer membrane protein, LamB

Advanced techniques for observing protein localization in live bacteria show that the distributions are dynamic. For technical reasons, most such techniques have not been applied to outer membrane proteins in Gram-negative bacteria. We have developed two novel live-cell imaging techniques to observe the surface distribution of LamB, an abundant integral outer membrane protein in Escherichia coli responsible for maltose uptake and for attachment of bacteriophage lambda. Using fluorescently labelled bacteriophage lambda tails, we quantitatively described the spatial distribution and dynamic movement of LamB in the outer membrane. LamB accumulated in spiral patterns. The distribution depended on cell length and changed rapidly. The majority of the protein diffused along spirals extending across the cell body. Tracking single particles, we found that there are two populations of LamB--one shows very restricted diffusion and the other shows greater mobility. The presence of two populations recalls the partitioning of eukaryotic membrane proteins between 'mobile' and 'immobile' populations. In this study, we have demonstrated that LamB moves along the bacterial surface and that these movements are restricted by an underlying dynamic spiral pattern.     

Cross-talk between the estrogen receptor-, protein kinase A-, and mitogen-activated protein kinase-mediated signaling pathways in the regulation of lactotroph proliferation in primary culture

Using pharmacological means we investigated the functional interactions between the estrogen receptor (ER)-, protein kinase A (PKA)-, and mitogen-activating protein kinase (MAPK)-mediated pathways in the regulation of lactotroph proliferation in primary culture. Treatment of lactotrophs for 28 h with the PKA inhibitor H89 or KT5720, an effective inhibitor of forskolin-induced proliferation, inhibited both insulin- and estradiol-induced proliferation. Inhibition of the MAPK activity by PD98059 or U0126 suppressed not only insulin-induced proliferation but also forskolin- and estradiol-induced proliferation. However, treatment for 28 h with the antiestrogens 4-hydroxy tamoxifen and ICI182780 failed to antagonize estradiol-induced lactotroph proliferation but instead enhanced it. Prolonging the antiestrogen treatment time from 28 to 88 h was effective in antagonizing estradiol-induced proliferation with this long-term treatment also inhibiting insulin- and forskolin-induced proliferation. There was no decrease in these mitogen-induced proliferations following treatment with a progesterone antagonist or protein kinase C inhibitor. These results suggest that cross-talk occurs between the ER-, PKA-, and MAPK-mediated signaling pathways in the regulation of lactotroph proliferation, and that antiestrogens stimulate and inhibit estradiol-induced proliferation in a time-dependent manner.