Cytokines, growth, and environment factors in bone marrow plasma of acute lymphoblastic leukemia pediatric patients.

Acute lymphoblastic leukemia (ALL) cells depend on the microenvironment of the host in vivo and do not survive in in vitro culture. Conversely, the suppression of non-malignant tissues is one of the leading characteristics of the course of ALL. Both the non-malignant suppression and malignant cell survival may be partly affected by soluble factors within the bone marrow (BM) environment. Here, we aimed to identify proteins in BM plasma of children with ALL that may contribute to ALL aggressiveness and/or the microenvironment-mediated survival of ALL cells. LBMp (leukemic bone marrow plasma) at the time of ALL diagnosis was compared to control plasma of bone marrow (CBMp) or peripheral blood (CPBp) using a cytokine antibody array. The cytokine antibody array enabled simultaneous detection of 79 proteins per sample. Candidate proteins exhibiting significantly different profiles were further analyzed and confirmed by ELISA. mRNA expression of one of the candidate proteins (TIMP1) was studied using quantitative reverse transcriptase polymerase chain reaction (qRTPCR). The cytokine antibody array experiments identified 23 proteins that differed significantly (p<0.05); of these, two proteins (TIMP1 and LIF) withstood the Bonferroni correction. In contrast, little difference was observed between CBMp and CPBp. At the diagnosis of ALL, changes in the soluble microenvironment are detectable in BM plasma. These changes probably participate in the pathogenesis and/or result from the changes in the cell composition.

Morphological and proteomic analysis of early stage air-liquid interface biofilm formation in Mycobacterium smegmatis.

We studied the early stages of pellicle formation by Mycobacterium smegmatis on the surface of a liquid medium [air-liquid interface (A-L)]. Using optical and scanning electron microscopy, we showed the formation of a compact biofilm pellicle from micro-colonies over a period of 8-30 h. The cells in the pellicle changed size and cell division pattern during this period. Based on our findings, we created a model of M. smegmatis A-L early pellicle formation showing the coordinate growth of cells in the micro-colonies and in the homogeneous film between them, where the accessibility to oxygen and nutrients is different. A proteomic approach utilizing high-resolution two-dimensional gel electrophoresis, in combination with mass spectrometry-based protein identification, was used to analyse the protein expression profiles of the different morphological stages of the pellicle. The proteins identified formed four expression groups; the most interesting of these groups contained the proteins with highest expression in the biofilm development phase, when the floating micro-colonies containing long and more robust cells associate into flocs and start to form a compact pellicle. The majority of these proteins, including GroEL1, are involved in cell wall synthesis or modification, mostly through the involvement of mycolic acid biosynthesis, and their expression maxima correlated with the changes in cell size and the rigidity of the bacterial cell wall observed by scanning electron microscopy.

Fitness and proteome changes accompanying the development of erythromycin resistance in a population of Escherichia coli grown in continuous culture.

We studied the impact of a sublethal concentration of erythromycin on the fitness and proteome of a continuously cultivated population of Escherichia coli. The development of resistance to erythromycin in the population was followed over time by the gradient plate method and minimum inhibitory concentration (MIC) measurements. We measured the growth rate, standardized efficiency of synthesis of radiolabeled proteins, and translation accuracy of the system. The proteome changes were followed over time in two parallel experiments that differed in the presence or absence of erythromycin. A comparison of the proteomes at each time point (43, 68, and 103 h) revealed a group of unique proteins differing in expression. From all 35 proteins differing throughout the cultivation, only three were common to more than one time point. In the final population, a significant proportion of upregulated proteins was localized to the outer or inner cytoplasmic membranes or to the periplasmic space. In a population growing for more than 100 generations in the presence of antibiotic, erythromycin-resistant bacterial clones with improved fitness in comparison to early resistant culture predominated. This phenomenon was accompanied by distinct changes in protein expression during a stepwise, population-based development of erythromycin resistance.

Surface hydrophobicity and roughness influences the morphology and biochemistry of streptomycetes during attached growth and differentiation.

Streptomycetes, soil-dwelling mycelial bacteria, can colonise surface of organic soil debris and soil particles. We analysed the effects of two different inert surfaces, glass and zirconia/silica, on the growth and antibiotic production in Streptomyces granaticolor. The surfaces used were in the form of microbeads and were surrounded by liquid growth media. Following the production of the antibiotic granaticin, more biomass was formed as well as a greater amount of antibiotic per milligram of protein on the glass beads than on the zirconia/silica beads. Comparison of young mycelium (6 h) proteomes, obtained from the cultures attached to the glass and zirconia/silica beads, revealed three proteins with altered expression levels (dihydrolipoamide dehydrogenase, amidophosphoribosyltransferase and cystathionine beta-synthase) and one unique protein (glyceraldehyde-3-phosphate dehydrogenase) that was present only in cells grown on glass beads. All of the identified proteins function primarily as cytoplasmic enzymes involved in different parts of metabolism; however, in several microorganisms, they are exposed on the cell surface and have been shown to be involved in adhesion or biofilm formation.

A microsporidian pathogen of the predatory beetle Rhizophagus grandis (Coleoptera: Rhizophagidae).

A new Microsporidium sp. infects Rhizophagus grandis Gyllenhall, a beetle which preys on the bark beetle Dendroctonus micans Kugellan in Turkey. Mature spores are single, uninucleate, oval in shape (3.75 +/- 0.27 microm in length by 2.47 +/- 0.13 microm in width), with a subapically fixed polar filament. The polar filament is anisofilar, coiled in 7-8 normal and 3-4 reduced coils. Other characteristic features of the microsporidium are the four/five nuclear divisions to form 16/32 (commonly 16) spores, subpersistent sporophorous vesicles (pansporoblasts) remaining till formation of the endospore, and the vesicles dissolved with free mature spores. The polaroplast is divided into three zones: an amorphous zone, dense layers, and a lamellartubular area extending to the central part of the spore.

Identification of multiple substrates of the StkP Ser/Thr protein kinase in Streptococcus pneumoniae.

Monitoring the external environment and responding to its changes are essential for the survival of all living organisms. The transmission of extracellular signals in prokaryotes is mediated mainly by two-component systems. In addition, genomic analyses have revealed that many bacteria contain eukaryotic-type Ser/Thr protein kinases. The human pathogen Streptococcus pneumoniae encodes 13 two-component systems and has a single copy of a eukaryotic-like Ser/Thr protein kinase gene designated stkP. Previous studies demonstrated the pleiotropic role of the transmembrane protein kinase StkP in pneumococcal physiology. StkP regulates virulence, competence, and stress resistance and plays a role in the regulation of gene expression. To determine the intracellular signaling pathways controlled by StkP, we used a proteomic approach for identification of its substrates. We detected six proteins phosphorylated on threonine by StkP continuously during growth. We identified three new substrates of StkP: the Mn-dependent inorganic pyrophosphatase PpaC, the hypothetical protein spr0334, and the cell division protein DivIVA. Contrary to the results of a previous study, we did not confirm that the alpha-subunit of RNA polymerase is a target of StkP. We showed that StkP activation and substrate recognition depend on the presence of a peptidoglycan-binding domain comprising four extracellular penicillin-binding protein- and Ser/Thr kinase-associated domain (PASTA domain) repeats. We found that StkP is regulated in a growth-dependent manner and likely senses intracellular peptidoglycan subunits present in the cell division septa. In addition, stkP inactivation results in cell division defects. Thus, the data presented here suggest that StkP plays an important role in the regulation of cell division in pneumococcus.

Budding: a new stage in the development of Chytridiopsis typographi (Zygomycetes: Microsporidia).

Chytridiopsis typographi Weiser, 1954, the microsporidian pathogen of the spruce bark beetle, Ips typographus L. (Coleoptera: Scolytidae), has an early developmental period with plurinucleate mother cells, each of which produces a single bud. The globular bud is connected with the mother cell by a collar and the cellular constituents are pushed to the distant end of the bud. Both the mother cell and the bud continue to develop; the bud then separates from the mother cell and grows to produce a cell of the same type. Both cells then continue sporogonial development and produce sporophorous vesicles with 16-32 spores. The process of a single mother cell producing a single bud that grows to an identical stage is new in the development of C. typographi and has no analogy in other Microsporidia.

Unikaryon phyllotretae sp. n. (Protista, Microspora), a new microsporidian pathogen of Phyllotreta undulata (Coleoptera; Chrysomelidae).

The microsporidium Unikaryon phyllotretae sp. n., a new pathogen of Phyllotreta undulata, is described based on light microscopic and ultrastructural characteristics. Microscopic examination of parasitized individuals revealed two types of spores. The majority of the spores were of the first type, which are oval and measured 2.74+/-0.17 x 1.93+/-0.17 microm when fresh. Fresh spores of the second type (very rare) are elongated and measured 4.39+/-0.18 x 1.61+/-0.20 microm. All life stages have single nuclei. Sporogony ends with uninucleate single sporoblasts and spores. The spores were only observed in Malpighian tubules. The isofilar polar filament of the parasite has six to eight coils, and a well-developed polaroplast was of the lamellated type, with closely packed anterior lamellae and loosely packed posterior lamellae.

Phosphorylation of Type IA restriction-modification complex enzyme EcoKI on the HsdR subunit.

Phosphorylation of Type I restriction-modification (R-M) enzymes EcoKI, EcoAI, and EcoR124I - representatives of IA, IB, and IC families, respectively - was analysed in vivo by immunoblotting of endogenous phosphoproteins isolated from Escherichia coli strains harbouring the corresponding hsd genes, and in vitro by a phosphorylation assay using protein kinase present in subcellular fractions of E. coli. From all three R-M enzymes, the HsdR subunit of EcoKI system was the only subunit that was phosphorylated. Further, evidence is presented that HsdR is phosphorylated in vivo only when coproduced with HsdM and HsdS subunits - as part of assembled EcoKI restriction endonuclease, while the individually produced HsdR subunit is not phosphorylated. In vitro phosphorylation of the HsdR subunit of purified EcoKI endonuclease occurs on Thr, and is strictly dependent on the addition of a catalytic amount of cytoplasmic fraction isolated from E. coli. So far this is the first case of phosphorylation of a Type I R-M enzyme reported.

The iron-regulated transcriptome and proteome of Neisseria meningitidis serogroup C.

Restricting bacterial growth by iron-chelating proteins that reduce iron availability in mucosal secretions and body fluids belongs to basic mechanisms of innate immunity. Most pathogens and commensals thus developed gene regulons responding to iron concentration and encoding iron acquisition systems and genes involved in host colonization and virulence. Here, we analyzed the steady-state composition of the iron-regulated proteome and transcriptome of an invasive serogroup C clinical isolate of Neisseria meningitidis. The proteome of meningococci grown under iron-depleted and iron-replete conditions was analyzed by 2-DE and proteins exhibiting significantly altered expression were identified by MALDI-TOF MS analysis. In parallel, total RNA was isolated from the same cultures and iron-regulated genes were identified using whole-genome DNA microarrays. The proteome and the transcriptome were found to overlap by only 19 iron-regulated genes/proteins, with 111 genes/proteins being significantly up-regulated in iron-replete cultures and 130 genes/proteins being up-regulated during iron starvation, respectively. Comparisons with published transcriptomic data for N. meningitidis serogroup B, moreover, indicate that expression of up to 20% of all meningococcal genes can be subject to regulation in function of iron availability.

Vairimorpha disparis n. comb. (Microsporidia: Burenellidae): a redescription and taxonomic revision of Thelohania disparis Timofejeva 1956, a microsporidian parasite of the gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae).

Investigation of pathogens of populations of the gypsy moth, Lymantria dispar (L.) in Central and Eastern Europe revealed the existence of a microsporidium (Fungi: Microsporidia) of the genus Vairimorpha. The parasite produced three spore morphotypes. Internally infective spores are formed in the gut and adjacent muscle and connective tissue; single diplokaryotic spores and monokaryotic spores grouped by eight in sporophorous vesicles develop in the fat body tissues. The small subunit rDNA gene sequences of various isolates of the Vairimorpha microsporidia, obtained from L. dispar in various habitats in the investigated region, revealed their mutual identity. In phylogenetic analyses, the organism clustered with other L. dispar microsporidia that form only diplokaryotic spores in the sporogony cycle. The octospores of certain microsporidia infecting Lepidoptera that were previously described as Thelohania spp., have recently been shown to be one of the several spore morphotypes produced by species in the genus Vairimorpha. Because the description and drawings of a parasite described as Thelohania disparis by Timofejeva fit the characteristics of Vairimorpha, and all octospore-producing microsporidia collected from L. dispar since 1985 are genetically identical Vairimorpha species, it is believed that the parasite characterized here is identical to T. disparis Timofejeva 1956, and is herein redescribed, characterized, and transferred to the genus Vairimorpha as the new combination Vairimorpha disparis n. comb.

Nosema chrysorrhoeae n. sp. (Microsporidia), isolated from browntail moth (Euproctis chrysorrhoea L.) (Lepidoptera, Lymantriidae) in Bulgaria: characterization and phylogenetic relationships.

A new microsporidian parasite Nosema chrysorrhoeae n. sp., isolated in Bulgaria from the browntail moth (Euproctis chrysorrhoea L.), is described. Its life cycle includes two sequential developmental cycles that are similar to the general developmental cycles of the Nosema-like microsporidia and are indistinguishable from those of two Nosema spp. from Lymantria dispar. The primary cycle takes place in the midgut tissues and produces binucleate primary spores. The secondary developmental cycle takes place exclusively in the silk glands and produces binucleate environmental spores. N. chrysorrhoeae is specific to the browntail moth. Phylogenetic analysis based on the ssu rRNA gene sequence places N. chrysorrhoeae in the Nosema/Vairimorpha clade, with the microsporidia from lymantriid and hymenopteran hosts. Partial sequences of the lsu rRNA gene and ITS of related species Nosema kovacevici (Purrini K., Weiser J., 1975. Natürliche Feinde des Goldafters, Euproctis chrysorrhoea L., im Gebiet von Kosovo, FSR Jugoslawien. Anzeiger fuer Schädlingskunde, Pflanzen-Umweltschutz, 48, 11-12), Nosema serbica Weiser, 1963 and Nosema sp. from Lymantria monacha was obtained and compared with N. chrysorrhoeae. The molecular data indicate the necessity of future taxonomic reevaluation of the genera Nosema and Vairimorpha.

Microsporidia and the Society for Invertebrate Pathology [corrected]: a personal point of view.

In this overview, I trace the history of the study of microsporidia, with special emphasis on the collegial relationships that developed at the international level and were fostered by the establishment of the Society for Insect Pathology, which later became the Society for Invertebrate Pathology. Study of these organisms of invertebrates in the early days seemed to be mere curiosities, but it soon became clear that they were major disease-causing agents in insects, and later even in vertebrates, especially humans with compromised immune systems. Though microsporidia have not proven effective as pesticides, they do play a role in the regulation of insect populations, especially insects such as the gypsy moth, grasshoppers, and occasionally mosquitoes.

DNA isolation from museum and type collection slides of microsporidia.

DNA from 19 species of microsporidia was isolated and amplified from infected host tissue that were originally prepared between the years 1946 and 1996. The smears, on glass microscope slides, were either Giemsa-stained or unstained. Methanol-fixed, Giemsa-stained smears proved to be suitable for DNA isolation; DNA was amplified from only two of 14 unstained slides. The isolated DNA was successfully amplified in PCRs using small subunit and large subunit rDNA primers and sequenced. The high efficiency of DNA isolation demonstrates the usefulness of archival and type collection slides for some molecular biology and molecular taxonomy studies.

Cultivation system using glass beads immersed in liquid medium facilitates studies of Streptomyces differentiation.

A two-phase cultivation system was developed which will enable studies of streptomycete differentiation by molecular biological and global techniques such as transcriptomics and proteomics. The system is based on a solid phase formed by glass beads corresponding to particles in soil, clay, or sand natural habitats of streptomycetes. The beads are immersed in a liquid medium that allows easy modification or replacement of nutrients and growth factors as well as radioactive labeling of proteins. Scanning electron microscopy was used to analyze morphological differentiation of streptomycetes on glass beads and two-dimensional protein electrophoresis to demonstrate the potential of the system for analyses of protein synthesis profiles during the developmental program. This system facilitates studies of differentiation including expression and post-translation modifications of streptomycetes proteins, secondary metabolite biosynthesis, and morphological development.

Molecular basis of intrinsic macrolide resistance in the Mycobacterium tuberculosis complex.

The intrinsic resistance of the Mycobacterium tuberculosis complex (MTC) to most antibiotics, including macrolides, is generally attributed to the low permeability of the mycobacterial cell wall. However, nontuberculous mycobacteria (NTM) are much more sensitive to macrolides than members of the MTC. A search for macrolide resistance determinants within the genome of M. tuberculosis revealed the presence of a sequence encoding a putative rRNA methyltransferase. The deduced protein is similar to Erm methyltransferases, which confer macrolide-lincosamide-streptogramin (MLS) resistance by methylation of 23S rRNA, and was named ErmMT. The corresponding gene, ermMT (erm37), is present in all members of the MTC but is absent in NTM species. Part of ermMT is deleted in some vaccine strains of Mycobacterium bovis BCG, such as the Pasteur strain, which lack the RD2 region. The Pasteur strain was susceptible to MLS antibiotics, whereas MTC species harboring the RD2 region were resistant to them. The expression of ermMT in the macrolide-sensitive Mycobacterium smegmatis and BCG Pasteur conferred MLS resistance. The resistance patterns and ribosomal affinity for erythromycin of Mycobacterium host strains expressing ermMT, srmA (monomethyltransferase from Streptomyces ambofaciens), and ermE (dimethyltransferase from Saccharopolyspora erythraea) were compared, and the ones conferred by ErmMT were similar to those conferred by SrmA, corresponding to the MLS type I phenotype. These results suggest that ermMT plays a major role in the intrinsic macrolide resistance of members of the MTC and could be the first example of a gene conferring resistance by target modification in mycobacteria.

Proteome of Caulobacter crescentus cell cycle publicly accessible on SWICZ server.

Here we present the Swiss-Czech Proteomics Server (SWICZ), which hosts the proteomic database summarizing information about the cell cycle of the aquatic bacterium Caulobacter crescentus. The database provides a searchable tool for easy access of global protein synthesis and protein stability data as examined during the C. crescentus cell cycle. Protein synthesis data collected from five different cell cycle stages were determined for each protein spot as a relative value of the total amount of [(35)S]methionine incorporation. Protein stability of pulse-labeled extracts were measured during a chase period equivalent to one cell cycle unit. Quantitative information for individual proteins together with descriptive data such as protein identities, apparent molecular masses and isoelectric points, were combined with information on protein function, genomic context, and the cell cycle stage, and were then assembled in a relational database with a world wide web interface (http://proteom.biomed.cas.cz), which allows the database records to be searched and displays the recovered information. A total of 1250 protein spots were reproducibly detected on two-dimensional gel electropherograms, 295 of which were identified by mass spectroscopy. The database is accessible either through clickable two-dimensional gel electrophoretic maps or by means of a set of dedicated search engines. Basic characterization of the experimental procedures, data processing, and a comprehensive description of the web site are presented. In its current state, the SWICZ proteome database provides a platform for the incorporation of new data emerging from extended functional studies on the C. crescentus proteome.

Streptomycetes cultured on glass beads: Sample preparation for SEM.

We demonstrate the preparation of samples of streptomycetes (Streptomyces coelicolor, S. aureofaciens) cultured on glass beads (balotina) for scanning electron microscopy. The main trick of the method consists in immobilization of glass beads with low-melting agarose. The samples are then fixed in OsO(4) vapors followed by dehydration in vapors of absolute ethanol. No air-to-liquid transition during the sample preparation occurs. Consequently, whole cell cycle of streptomycetes in the term of mycelial morphology can readily be studied by this method.