Cloning and expression of a murein hydrolase lipoprotein from Escherichia coli

On the basis of the published N-terminal amino acid sequence of the soluble lytic transglycosylase 35 (Slt35) of Escherichia coli, an open reading frame (ORF) was cloned from the 60.8 min region of the E. coli chromosome. The nucleotide sequence of the ORF, containing a putative lipoprotein-processing site, was shown by [³H]-palmitate labelling to encode a lipoprotein with an apparent molecular mass of 36 kDa. A larger protein, presumably the prolipoprotein form, accumulated in the presence of globomycin. Over-expression of the gene, designated mltB (for membrane-bound lytic transglycosylase B), caused a 55-fold increase in murein hydrolase activity in the membrane fraction and resulted in rapid cell lysis. After membrane fractionation by sucrose-density-gradient centrifugation, most of the induced enzyme activity was present in the outer and intermediate membrane fractions. Murein hydrolase activity in the soluble fraction of a homogenate of cells induced for MltB increased with time. This release of enzyme activity into the supernatant could be inhibited by the addition of the serine-protease inhibitor phenylmethyl-sulphonyl fluoride. It is concluded that the previously isolated Slt35 protein is a proteolytic degradation product of the murein hydrolase lipoprotein MltB. Surprisingly, a deletion in the mltB gene showed no obvious phenotype.     

Extracellular regulation of fibroblast multiplication: A direct kinetic approach to analysis of role of low molecular weight nutrients and serum growth factors

The principles of enzyme kinetic analysis were applied to quantitate the relationships among serum-derived growth factors, nutrients, and the rate of survival and multiplication of human fibroblasts in culture. The survival or multiplication rate of a population of cells plotted against an increasing concentration of a growth factor or nutrient in the medium exhibited a hyperbolic pattern that is characteristic of a dissociable, saturable interaction between cells and the ligands. Parameters equivalent to the K_m and V_max of enzyme kinetics were assigned to nutrients and growth factors. When all nutrient concentrations were optimized and in steady state, serum factors accelerated the rate of multiplication of a normal cell population. The same set of nutrients that supported a maximal rate of multiplication in the presence of serum factors supported the maintenance of non-proliferating cells in the absence of serum factors. Therefore, under this condition, serum factors are required for cell division and play a purely regulatory iole in multiplication of the cell population. The quantitative requirement for 18 nutrients of 29 that were examined was significantly higher (P < 0.001) for cell multiplication in the presence of serum factors than for cell maintenance in the absence of serum factors. This indicated specific nutrients that may be quantitatively important in cell division processes as well as in cell maintenance. The quantitative requirement for Ca²⁺, Mg²⁺, K⁺, Pi, and 2-oxocarboxylic acid for cell multiplication was modified by serum factors and other purified growth factors. The requirement for over 30 other nutrients could not clearly be related to the level of serum factors in the medium. Serum factors also determined the Ca²⁺, K⁺, and 2-oxocarboxylic acid requirement for maintenance of non-proliferating cells. Therefore, when either Ca²⁺, K⁺, or 2-oxocarboxylic acid concentration was limiting, factors in serum played a role as cell “survival or maintenance” factors in addition to their role in cell division as “growth regulatory” factors. However, with equivalent levels of serum factors in the medium, the requirement for Ca²⁺, K⁺, and 2-oxocarboxylic acids was still much higher for multiplication than for maintenance. Kinetic analysis revealed that the concentrations of individual nutrients modify the quantitative requirement for others for cell multiplication in a specific pattern. Thus, specific quantitative relationships among different nutrients in the medium are important in the control of the multiplication rate of the cell population. When all nutrient concentrations were optimal for multiplication of normal cells, the multiplication response of SV40-virus-transformed cells to serum factors was similar to that of normal cells. When serum factors were held constant, transformed cells required significantly less (P < 0.001) of 12 of the 26 nutrients examined. Therefore, the transformed cells only have a growth advantage when the external concentration of specific nutrients limits the multiplication rate of normal cells. Taken together, the results suggest that the control of cell multiplication is intimately related to external concentrations of nutrients. Specific growth regulatory factors may stimulate cell proliferation by modification of the response of normal cells to nutrients. Transforming agents may confer a selective growth advantage on cells by a constitutive alteration of their response to extracellular nutrients.     

PVv3, a New Shuttle Vector for Gene Expression in Vibrio vulnificus

An efficient electroporation procedure for Vibrio vulnificus was designed using the new cloning vector pVv3 (3,107 bp). Transformation efficiencies up to 2 × 10⁶ transformants per μg DNA were achieved. The vector stably replicated in both V. vulnificus and Escherichia coli and was also successfully introduced into Vibrio parahaemolyticus and Vibrio cholerae. To demonstrate the suitability of the vector for molecular cloning, the green fluorescent protein (GFP) gene and the vvhBA hemolysin operon were inserted into the vector and functionally expressed in Vibrio and E. coli.     

Microbial view of central nervous system autoimmunity

Not much is known about the initial events leading to the development of the central nervous system (CNS)-specific autoimmune disorder Multiple Sclerosis (MS). Environmental factors are suspected to trigger the pathogenic events in people with genetic disease susceptibility. Historically, many infectious microbes were linked to MS, but no infection has ever been demonstrated to be the cause of the disease. Recent emerging evidence from animal models of MS suggests a causal link with resident commensal bacteria. Microbial organisms may trigger the activation of CNS-specific, auto-aggressive lymphocytes either through molecular mimicry or via bystander activation. In addition, several gut microbial metabolites and bacterial products may interact with the immune system to modulate CNS autoimmunity.     

Coherence between Cellular Responses and in Vitro Splicing Inhibition for the Anti-tumor Drug Pladienolide B and Its Analogs

Pladienolide B (PB) is a potent cancer cell growth inhibitor that targets the SF3B1 subunit of the spliceosome. There is considerable interest in the compound as a potential chemotherapeutic, as well as a tool to study SF3B1 function in splicing and cancer development. The molecular structure of PB, a bacterial natural product, contains a 12-member macrolide ring with an extended epoxide-containing side chain. Using a novel concise enantioselective synthesis, we created a series of PB structural analogs and the structurally related compound herboxidiene. We show that two methyl groups in the PB side chain, as well as a feature of the macrolide ring shared with herboxidiene, are required for splicing inhibition in vitro. Unexpectedly, we find that the epoxy group contributes only modestly to PB potency and is not absolutely necessary for activity. The orientations of at least two chiral centers off the macrolide ring have no effect on PB activity. Importantly, the ability of analogs to inhibit splicing in vitro directly correlated with their effects in a series of cellular assays. Those effects likely arise from inhibition of some, but not all, endogenous splicing events in cells, as previously reported for the structurally distinct SF3B1 inhibitor spliceostatin A. Together, our data support the idea that the impact of PB on cells is derived from its ability to impair the function of SF3B1 in splicing and also demonstrate that simplification of the PB scaffold is feasible.     

The Moraxella catarrhalis-induced pro-inflammatory immune response is enhanced by the activation of the epidermal growth factor receptor in human pulmonary epithelial cells

Background

Chronic lower airway inflammation is considered to be a major cause of pathogenesis and disease progression in chronic obstructive pulmonary disease (COPD). Moraxella catarrhalis is a COPD-associated pathogen causing exacerbations and bacterial colonization in the lower airways of patients, which may contribute to chronic inflammation. Increasing evidence suggests that the epidermal growth factor receptor (EGFR) modulates inflammatory processes in the human airways. The goal of this study was to investigate the role of EGFR in the M. catarrhalis-induced pro-inflammatory immune response in airway epithelial cells.

Methods

The effects of inhibition and gene silencing of EGFR on M. catarrhalis-dependent pro-inflammatory cytokine expression in human primary bronchial epithelial cells (NHBEs), as well as the pulmonary epithelial cell lines BEAS-2B and A549 were analyzed. We also assessed the involvement of EGFR-dependent ERK and NF-κB signaling pathways.

Results

The M. catarrhalis-induced pro-inflammatory immune response depends, at least in part, on the phosphorylation and activation of the EGF receptor. Interaction of M. catarrhalis with EGFR increases the secretion of pro-inflammatory cytokines, which is mediated via ERK and NF-κB activation.

Conclusion

The interaction between M. catarrhalis and EGFR increases airway inflammation caused by this pathogen. Our data suggest that the inhibition of EGFR signaling in COPD could be an interesting target for reducing M. catarrhalis-induced airway inflammation.