Jan Šmarda

S-layers on cell walls of cyanobacteria

S-layers are surface layers of bacterial cell walls. They are formed by two-dimensional, monomolecular crystalline arrays of identical units of protein or glycoprotein macromolecules (subunits). In general, each S-layer exhibits one of four possible 2-D lattice types: oblique (p1 or p2 symmetry), triangle (p3 symmetry), square (p4 symmetry) or hexagonal (p6 symmetry). The S-layer protein compasses up to 15% of the total protein of the bacterial cell and thus represents its major protein. Since 1972, S-layers have also been found in cyanobacteria. So far, they have been observed in 60 strains (isolates) of 23 species, belonging to 12 genera of unicellular Chroococcales and in just five strains or isolates (four species, four genera-only with p1 and p4 lattice symmetry) of filamentous Oscillatoriales; in further families of filamentous cyanobacteria (Nostocales, Stigonematales) they have not been detected, although filamentous cyanobacteria have been frequently studied in the electron microscope. In Chroococcales, relatively large cells of planktonic genera harbouring gas vesicles, S-layers are often present, while picoplanktonic species without gas vesicles usually do not have them. The p6 lattice symmetry appears to be the most common in cyanobacteria, having been found in 41 out of the 60 S-layers observed. All cells of a given strain, all strains capable of forming S-layers and all S-layer forming species of a given genus (as far as it is known) form S-layers of the same lattice type. Hence, the ability to form an S-layer appears to be useful as a supportive morphological marker for species classification. In 41 S-layer formers, the center-to-center spacing of their lattice unit arrays has been measured; the lattice constants range from 5 to 22nm, measured directly on surface of fixed cells. Coarse S-layers of p6 symmetry are the most frequent (with spacing of 15.0-22.0nm); p1 and p2 S-layers are the finest ones (with spacing of 5.0-10.0nm). Medium-spaced lattices (11.0-14.0nm) may be both of the p4 or p6 symmetry types. When measured on isolated S-layers, the spacings show a 10-60% higher value. All the hexagonal unit lattices have the same molecular architecture. Each S-layer unit resembles a truncated cone with an axial pore and with six protein subunits symmetrically placed around its opening. Adjoining units are interspaced by relatively fine channels. The fine detail of every S-layer of every individual strain is unique. Only the S-layer protein subunits of Synechococcus sp. strain GL24 have been analysed by electrophoresis. When incorporated into the S-layer units they confer a net neutral charge to the cell surface. This cyanobacterium induces mineralization of fine-grain gypsum and calcite in a saturated lake fresh water solution. This process is involved in the formation of stromatolites.

Colicins-exocellular lethal proteins of Escherichia coli.

Colicins are toxic exoproteins produced by bacteria of colicinogenic strains ofEscherichia coli and some related species ofEnterobacteriaceae, during the growth of their cultures. They inhibit sensitive bacteria of the same family. About 35%E. coli strains appearing in human intestinal tract are colicinogenic. Synthesis of colicins is coded by genes located on Col plasmids. Until now more than 34 types of colicins have been described, 21 of them in greater detail,viz. colicins A, B, D, E1–E9, Ia, Ib, JS, K, M, N, U, 5, 10. In general, their interaction with sensitive bacteria includes three steps: (1) binding of the colicin molecule to a specific receptor in the bacterial outer membrane; (2) its translocation through the cell envelope; and (3) its lethal interaction with the specific molecular target in the cell. The classification of colicins is based on differences in the molecular events of these three steps.

Exoproducts of the Escherichia coli strain H22 inhibiting some enteric pathogens both in vitro and in vivo

Aims:  The antagonistic activity of the Escherichia coli strain H22 against enteric bacteria was studied both in vitro and in vivo.

Bacteriocin synthesis in uropathogenic and commensal Escherichia coli: colicin E1 is a potential virulence factor

BackgroundBacteriocin production is an important characteristic of E. coli strains of human origin. To date, 26 colicin and 9 microcin types have been analyzed on a molecular level allowing molecular detection of the corresponding genes. The production incidence of 29 bacteriocin types and E. coli phylogroups were tested in a set of 361 E. coli strains isolated from human urinary tract infections (UTI) and in 411 control strains isolated from feces of patients without bacterial gut infection.ResultsProduction of 17 and 20 individual bacteriocin types was found in the UTI and control strains, respectively. Microcin H47 encoding determinants were found more often among UTI strains compared to controls (37.9% and 27.0% respectively, p = 0.02) and strains producing microcin H47 belonged predominantly to phylogroup B2 when compared to other bacteriocin producers (67.4% and 36.7%, respectively; p < 0.0001). Producers of 3 or more identified bacteriocin types were more common in the UTI group (20.0% compared to 12.4% in controls, p = 0.03). In the UTI strains, there was a markedly higher number of those producing colicin E1 compared to controls (22.1% to 10.2%, respectively, p = 0.0008). Moreover, colicin E1 production was more common in the UTI bacteriocinogenic strains with multi-producer capabilities. As shown by Southern blotting, pColE1 DNA was not recognized by the ColIa probe and vice versa suggesting that pColE1 was independently associated with pColIa in UTI strains.ConclusionE. coli strains isolated from human urinary tract infections showed increased incidence of microcin H47 and colicin E1 production, respectively. Moreover, colicin E1 itself appears to be a potentially important virulence factor of certain uropathogenic E. coli strains.

c-Myb regulates matrix metalloproteinases 1/9, and cathepsin D: implications for matrix-dependent breast cancer cell invasion and metastasis

BackgroundThe c-Myb transcription factor is essential for the maintenance of stem-progenitor cells in bone marrow, colon epithelia, and neurogenic niches. c-Myb malfunction contributes to several types of malignancies including breast cancer. However, the function of c-Myb in the metastatic spread of breast tumors remains unexplored. In this study, we report a novel role of c-Myb in the control of specific proteases that regulate the matrix-dependent invasion of breast cancer cells.ResultsEctopically expressed c-Myb enhanced migration and ability of human MDA-MB-231 and mouse 4T1 mammary cancer cells to invade Matrigel but not the collagen I matrix in vitro. c-Myb strongly increased the expression/activity of cathepsin D and matrix metalloproteinase (MMP) 9 and significantly downregulated MMP1. The gene coding for cathepsin D was suggested as the c-Myb-responsive gene and downstream effector of the migration-promoting function of c-Myb. Finally, we demonstrated that c-Myb delayed the growth of mammary tumors in BALB/c mice and affected the metastatic potential of breast cancer cells in an organ-specific manner.ConclusionsThis study identified c-Myb as a matrix-dependent regulator of invasive behavior of breast cancer cells.

Inhibition of topoisomerase IIα: novel function of wedelolactone.

The naturally occurring coumestan wedelolactone has been previously shown to reduce growth of various cancer cells. So far, the growth-suppressing effect of wedelolactone has been attributed to the inhibition of the NFκB transcription factor and/or androgen receptors. We found that wedelolactone suppressed growth and induced apoptosis of androgen receptor-negative MDA-MB-231 breast cancer cells at concentrations that did not inhibit the NFκB activity. The cells responded to wedelolactone by the S and G2/M phase cell cycle arrest and induction of the DNA damage signaling. Wedelolactone interacted with dsDNA and inhibited the activity of DNA topoisomerase IIα. We conclude that wedelolactone can act as growth suppressor independently of NFκB and androgen receptors.

Human tumor cells are selectively inhibited by colicins

The activityin vitro of four types of colicins (A, E1, E3, U) against one human standard fibroblast line and against 11 human tumor-cell lines carrying defined mutations of thep53 gene was quantified by MTT (tetrazolium bromide) assay. Flow cytometry showed that the pore-forming colicins A, E1 and U affected the cell cycle of 5 of these cell lines. Colicins E3 and U did not show any distinct inhibitory effects on the cell lines, while colicins E1 and especially A inhibited the growth of all of them (with one exception concerning colicin E1). Colicin E1 inhibited the growth of the tumor lines by 17–40% and standard fibroblasts MRC5 by 11%. Colicin A exhibited a differentiated 16–56% inhibition, the growth of standard fibroblasts being inhibited by 36%. In three of the lines, colicins A and E1 increased the number of cells in the G1 phase (by 12–58%) and in apoptosis (by 7–58%). These results correlated with the data from sensitivity assays. Hence, the inhibitory effect of colicins on eukaryotic cells is cell-selective, colicin-specific and can be considered to be cytotoxic.

Incidence of colicinogenic strains among human Escherichia coli

During the years 1993–1999, altogether 1,043 Escherichia coli strains from colons of different persons were screened for colicinogeny using a most susceptible procedure and indicator system. In control persons (with healthy colons), 41.37% producers of colicins were found.

Oxynema, a new genus separated from the genus Phormidium (Cyanophyta)

Abstract Taxonomic revision of the traditional polyphyletic cyanobacterial genus Phormidium is based on molecular sequencing combined with the definition of distinct, autapomorphic features. Several genera, clearly separated from each other (with genetic similarity lower than 95%), were already defined and separated from this widely conceived generic unit (Phormidesmis, Wilmottia and others). All of these new generic taxa are characterized by morphological markers also. We have studied another group of species (mainly the strains from salterns in Thailand), which was classified earlier in the genus Phormidium, but it represents an isolated cluster according to 16S rRNA gene sequencing and is characterized by specific uniform and morphological features. Because this whole group represents a phylogenetically and morphologically distinctly separated cluster (see Phormidium group I sensu Komárek & Anagnostidis 2005), we describe it as a special taxon Oxynema genus novum, in agreement with the Botanical Nomenclatoric Code (ICBN; McNeill & al. ed., 2007). The genetically most related clusters always have genetic similarity less than 93% and differ by distinct autapomorphic features. The filaments of members of Oxynema are cylindrical, narrowed and bent at the ends, commonly attenuated to a terminal elongated, more or less sharply pointed cells without calyptra. Thylakoids in cells are distinctly radially arranged, similarly as in the genera Microcoleus and “Phormidium autumnale”-type. The ecology of all members, which belong potentially to these types, is also similar: all species from this cluster were recorded from halophilic habitats, less frequently from thermal springs and soil biotopes with higher salt contents.

Growth/differentiation factor-15 inhibits differentiation into osteoclasts--a novel factor involved in control of osteoclast differentiation.

Survival and capability of cancer cells to form metastases fundamentally depend on interactions with their microenvironment. Secondary tumors originating from prostate carcinomas affect remodeling of bone tissue and can induce both osteolytic and osteocondensing lesions. However, particular molecular mechanisms responsible for selective homing and activity of cancer cells in bone microenvironment have not been clarified yet. Growth/differentiation factor-15 (GDF-15), a distant member of the TGF-beta protein family, has recently been associated with many human cancers, including prostate. We show that both pure GDF-15 and the GDF-15-containing growth medium of 1,25(OH)(2)-vitamin D(3)-treated prostate adenocarcinoma LNCaP cells suppress formation of mature osteoclasts differentiated from RAW264.7 macrophages and bone-marrow precursors by M-CSF/RANKL in a dose-dependent manner. GDF-15 inhibits expression of c-Fos and activity of NFkappaB by delayed degradation of IkappaB. Moreover, GDF-15 inhibits expression of carbonic anhydrase II and cathepsin K, key osteoclast enzymes, and induces changes in SMAD and p38 signaling. The lack of functional osteoclasts can contribute to accumulation of bone matrix by reduction of bone resorption. These results unveil new role of GDF-15 in modulation of osteoclast differentiation and possibly in therapy of bone metastases.