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Glycerophosphodiester phosphodiesterase (GlpQ) metabolizes glycerophosphorylcholine from the lung epithelium to produce free choline, which is transformed into phosphorylcholine and presented on the surfaces of many respiratory pathogens. Two orthologs of glpQ genes are found in Streptococcus pneumoniae: glpQ, with a membrane motif, is widespread in pneumococci, whereas glpQ2, which shares high similarity with glpQ in Haemophilus influenzae and Mycoplasma pneumoniae, is present only in S. pneumoniae serotype 3, 6B, 19A, and 19F strains. Recently, serotype 19A has emerged as an epidemiological etiology associated with invasive pneumococcal diseases. Thus, we investigated the pathophysiological role of glpQ2 in a serotype 19A sequence type 320 (19AST320) strain, which was the prevalent sequence type in 19A associated with severe pneumonia and invasive pneumococcal disease in pediatric patients. Mutations in glpQ2 reduced phosphorylcholine expression and the anchorage of choline-binding proteins to the pneumococcal surface during the exponential phase, where the mutants exhibited reduced autolysis and lower natural transformation abilities than the parent strain. The deletion of glpQ2 also decreased the adherence and cytotoxicity to human lung epithelial cell lines, whereas these functions were indistinguishable from those of the wild type in complementation strains. In a murine respiratory tract infection model, glpQ2 was important for nasopharynx and lung colonization. Furthermore, infection with a glpQ2 mutant decreased the severity of pneumonia compared with the parent strain, and glpQ2 gene complementation restored the inflammation level. Therefore, glpQ2 enhances surface phosphorylcholine expression in S. pneumoniae 19AST320 during the exponential phase, which contributes to the severity of pneumonia by promoting adherence and host cell cytotoxicity., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Autolysis of lactic acid bacteria (LAB) plays a vital role in dairy processing. During cheese making, autolysis of LAB affects cheese flavor development through release of intracellular enzymes and restricts the proliferation of cells in yogurt fermentation and probiotics production. In order to explore the mechanism of autolysis, the gene for the autolytic enzymes of L. bulgaricus, N-acetylmuramidase (mur), was cloned and sequenced (GenBank accession number: KF157911). Mur gene overexpression and gene knockout vectors were constructed based on pMG76e and pUC19 vectors. Recombinant plasmids were transformed into L. bulgaricus ljj-6 by electroporation, then three engineered strains with pMG76e-mur vector and fifteen engineered strains with pUC19-mur::EryBII were screened. The autolysis of the mur knockout strain was significantly lower and autolysis of the mur overexpressed strain was significantly higher compared with that of the wild type strain ljj-6. This result suggested that the mur gene played an important role in autolysis of L. bulgaricus. On the other hand, autolytic activity in a low degree was still observed in the mur knockout strain, which implied that other enzymes but autolysin encoded by mur were also involved in autolysis of L. bulgaricus.

Cell autolysis plays important physiological roles in the life cycle of clostridial cells. Understanding the genetic basis of the autolysis phenomenon of pathogenic Clostridium or solvent producing Clostridium cells might provide new insights into this important species. Genes that might be involved in autolysis of Clostridium acetobutylicum, a model clostridial species, were investigated in this study. Twelve putative autolysin genes were predicted in C. acetobutylicum DSM 1731 genome through bioinformatics analysis. Of these 12 genes, gene SMB_G3117 was selected for testing the in tracellular autolysin activity, growth profile, viable cell numbers, and cellular morphology. We found that overexpression of SMB_G3117 gene led to earlier ceased growth, significantly increased number of dead cells, and clear electrolucent cavities, while disruption of SMB_G3117 gene exhibited remarkably reduced intracellular autolysin activity. These results indicate that SMB_G3117 is a novel gene involved in cellular autolysis of C. acetobutylicum.

YvrGHb is a novel two-component system in Bacillus subtilis. The yvrG and yvrHb genes are considered to encode sensor kinase and response regulator respectively. We found that the YvrGHb system positively regulates the 7 transcriptional units (wprA, wapA-yxxG, dltABCDE, sunA, sunT-bdbA-yolJ-bdbB, yvrI-yvrHa, and sigX-rsiX), and negatively regulates the lytABC operon. wprA, wapA, lytB, and lytC encode the main cell surface proteins of B. subtilis. Furthermore, SigX [extracytoplasmic function sigma factor (ECF sigma factor)] relating to the cell surface homeostatic functions, was regulated by this system. The yvrGHb null mutant showed the unusual autolysis and higher susceptibility to the four kinds of antibiotics (aztreonam, cefepime, bacitracin, and fosfomycin). These results indicate that the YvrGHb system is important to maintain the overall state of the cell surface through transcriptional regulation.

Background and Objectives: CD20 has been proposed as a novel suicide gene system for the treatment of graft-versus-host disease (GVHD), a fatal complication of allogeneic bone marrow transplantation: indeed expression of the human non-immunogenic exogenous CD20 protein allows positive immunoselection of transduced cells as well as their killing in vitro with rituximab. Lentiviral vectors are promising tools in the field of gene therapy. We therefore searched for a lentivector giving good efficiency of transduction of human T lymphocytes activated by the sole addition of interleukin (IL)-2 and high expression levels of the CD20 transgene., Design and Methods: The T cell line CEM and peripheral T lymphocytes activated by phytohemagglutinin (PHA) and/or IL-2 were transduced with two different vectors carrying the CD20 transgene driven by either the phosphoglycerate kinase (PGK) or elongation factor 1alpha (EF1alpha) promoter, and using different multiplicities of infection (MOIs)., Results: Both the PGK- and EF1alpha-CD20 vectors allowed efficient transduction of the CEM cell line and PHA-activated T cells, reaching 99 and 90% in the different targets, respectively. However EF1alpha-CD20 led to much higher expression levels of the transgene (mean fluorescence intensity 588-618 compared to 53 for PGK-CD20). Furthermore lymphocytes activated with IL-2 alone could be efficiently transduced with EF1alpha-CD20, reaching 10-25% positivity for CD20 (mean fluorescence intensity 409-424), allowing adequate immunoselection and strong complement-mediated lysis., Interpretation and Conclusions: EF1alpha-CD20 may represent a good candidate vector for gene therapy with the CD20 suicide system in the setting of allogeneic bone marrow transplants.

Since the identification in 1996 of a "gain of function" missense mutation, R122H, in the cationic trypsinogen gene (PRSS1) as a cause of hereditary pancreatitis, continued screening of this gene in both hereditary and sporadic pancreatitis has found more disease-associated missense mutations than expected. In addition, functional analysis has yielded interesting findings regarding their underlying mechanisms resulting in a gain of trypsin. A critical review of these data, in the context of the complicated biogenesis and complex autoactivation and autolysis of trypsin(ogen), highlights that PRSS1 mutations cause the disease by various mechanisms depending on which biochemical process they affect. The discovery of these mutations also modifies the classical perception of the disease and, more importantly, reveals fascinating new aspects of the molecular evolution and normal physiology of trypsinogen. First, activation peptide of trypsinogen is under strong selection pressure to minimize autoactivation in higher vertebrates. Second, the R122 primary autolysis site has further evolved in mammalian trypsinogens. Third, evolutionary divergence from threonine to asparagine at residue 29 in human cationic trypsinogen provides additional advantage. Accordingly, we tentatively assign, in human cationic trypsinogen, the strongly selected activation peptide as the first-line and the R122 autolysis site as the second-line of the built-in defensive mechanisms against premature trypsin activation within the pancreas, respectively, and the positively selected asparagine at residue 29 as an "amplifier" to the R122 "fail-safe" mechanism.

Hyperpolymorphic short tandem repetitive DNA sequences, STRs or microsatellites, have become widely used in human identification, particularly in criminal cases and in mass disasters. In such cases the substrates for the analyses may be decomposed biological material, a fact that has to be taken into account when choosing the appropriate casework methods. In this paper we report the evaluation of five different DNA extraction methods, namely the phenol-chloroform, the silica based, the InstaGene Matrix (BioTest), the glass fiber filter, and the Chelex based methods. The substrates for the analyses are decomposed human liver tissue specimens from forensic autopsy cases. Extracted DNA was quantified and DNA profiled by a set of seven STRs. We have compared laboratory time consumption and costs of the five methods, showing that the Chelex method is the more rapid and less expensive of the methods, the phenol-chlorophorm and silica extractions being the most time consuming and resource demanding ones. A full profile was obtained by the silica method in nine out of ten cases and this method failed to give a reliable type in four out of 70 STR analyses. The phenol-chlorophorm and the glass fiber filter methods failed in 16 analyses, the InstaGene Matrix (BioTest) in 25 and the Chelex extracts in 56 of the 70 STR analyses. By multiple logistic regression we show that the difference between the silica procedure and the other methods are statistically significant. In our hands, the silica gel extraction procedure is an obvious choice when the biological material available is decomposed human tissue--even if this procedure is one of the more laborious ones.

In order to study subunit interactions in calpain, the effects of small subunit truncations on m-calpain activity and heterodimer formation have been measured. It has been shown previously that active calpain is formed by co-expression of the large subunit (80 kDa) of rat m-calpain with a delta 86 form (21 kDa) of the small subunit. cDNA for the full-length 270 amino acid (28.5 kDa) rat calpain small subunit has now been cloned, both with and without an N-terminal histidine tag (NHis10). The full-length small subunit constructs yielded active calpains on co-expression with the large subunit, and the small subunit was autolysed to 20 kDa on exposure of these calpains to Ca2+. A series of deletion mutants of the small subunit, NHis10-delta 86, -delta 99, -delta 107, and -delta 116, gave active heterodimeric calpains with unchanged specific activities, although in decreasing yield, and with a progressive decrease in stability. NHis10-delta 125 formed a heterodimer which was inactive and unstable. Removal of 25 C-terminal residues from delta 86, leaving residues 87-245, abolished both activity and heterodimer formation. The results show that: (a) generation of active m-calpain in Escherichia coli requires heterodimer formation; (b) small subunit residues between 94 and 116 contribute to the stability of the active heterodimer but do not directly affect the catalytic mechanism; (c) residues in the region 245-270 are essential for subunit binding. Finally, it was shown that an inactive mutant Cys103-->Ser-80k/delta 86 calpain, used in order to preclude autolysis, did not dissociate in the presence of Ca2+, a result which does not support the proposal that Ca(2+)-induced dissociation is involved in calpain activation.

In order to carry out a systematic search for mutants affected in cell integrity, the diploid strain Saccharomyces cerevisiae D1 was subjected to mutagenesis with ethyl methane sulphonate (EMS), and mutant clones were screened for thermosensitive autolytic phenotypes. The screening was based on examination of cell populations, from individual mutant clones, stained with propidium iodide to establish the proportion of cells lysing under non-permissive conditions by means of flow cytometry. Osmotic remediation of the autolytic phenotype in the presence of 1 M sorbitol was also checked. Out of 13,300 clones surviving mutagenesis, 34 were confirmed to be thermosensitive autolytic and 7 of them showed some osmotic complementation with regard to growth and cell lysis. The osmotic remediation in the other strains was negligible or affected only one of the two parameters. The expression of the mutant phenotype in the strains isolated led to a sporulation defect (40% of the strains) and significant alterations in morphology, such as cells in chains (35%), altered buds (25%) that eventually might elongate, round unbudded and highly vacuolated cells (12%) and large-sized cells (12%). These observations show that alterations in functions related to cell integrity can be correlated with an altered morphology. Genetic analysis of the mutant strains that could sporulate showed that in many instances the mutant phenotype was the result of more than one mutation, the mutations being individually recessive. However, at least one mutant strain, 933, carried a single mendelian mutation that was dominant in the diploid but haploid segregants were non-viable. Dominance of this mutation was also confirmed in tetraploids obtained by means of protoplast fusion.

The autolytic activities, including unstimulated, Triton X-100-stimulated, and daptomycin-induced, of various sets of methicillin-resistant and related methicillin-susceptible strains were compared. Faster rates of autolysis were noted in two heterogeneous methicillin-resistant transductants than in their methicillin-susceptible parental recipients, in a heterogeneous resistant strain than in a susceptible derivative created by chemical mutagenesis, and in a homogeneous resistant strain than in a derivative that had decreased methicillin resistance and was created by transposon Tn551 mutagenesis. These results suggest that the presence of the methicillin resistance region, mec, either directly or indirectly through an interaction with other host genes, confers a faster rate of autolysis on strains. Various auxilliary genes are known to affect methicillin resistance expression, and one of these genes, femA, was necessary for the expression of this faster rate of autolysis. These differences in autolytic activities were not observed in isolated crude cell walls retaining autolytic activities, suggesting different modes of regulation of autolysins in intact cells and isolated walls. In contrast, one homogeneous, highly resistant strain, DU4916, had a lower autolytic activity than did derived heterogeneous resistant and susceptible strains created by chemical mutagenesis and a strain that had decreased resistance and was created by transposon mutagenesis. Our observations suggest that methicillin resistance expression is associated with an enhanced rate of autolysis, in heterogeneous resistant strains at least.