A novel bacteriophage Tail-Associated Muralytic Enzyme (TAME) from Phage K and its development into a potent antistaphylococcal protein

<p>Abstract</p> <p>Background</p> <p><it>Staphylococcus aureus </it>is a major cause of nosocomial and community-acquired infections. However, the rapid emergence of antibiotic resistance limits the choice of therapeutic options for treating infections caused by this organism. Muralytic enzymes from bacteriophages have recently gained attention for their potential as antibacterial agents against antibiotic-resistant gram-positive organisms. Phage K is a polyvalent virulent phage of the <it>Myoviridae </it>family that is active against many <it>Staphylococcus </it>species.</p> <p>Results</p> <p>We identified a phage K gene, designated <it>orf</it>56, as encoding the phage tail-associated muralytic enzyme (TAME). The gene product (ORF56) contains a C-terminal domain corresponding to cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), which demonstrated muralytic activity on a staphylococcal cell wall substrate and was lethal to <it>S. aureus </it>cells. We constructed N-terminal truncated forms of ORF56 and arrived at a 16-kDa protein (Lys16) that retained antistaphylococcal activity. We then generated a chimeric gene construct encoding Lys16 and a staphylococcal cell wall-binding SH3b domain. This chimeric protein (P128) showed potent antistaphylococcal activity on global clinical isolates of <it>S. aureus </it>including methicillin-resistant strains. In addition, P128 was effective in decolonizing rat nares of <it>S. aureus </it>USA300 in an experimental model.</p> <p>Conclusions</p> <p>We identified a phage K gene that encodes a protein associated with the phage tail structure. The muralytic activity of the phage K TAME was localized to the C-terminal CHAP domain. This potent antistaphylococcal TAME was combined with an efficient <it>Staphylococcus</it>-specific cell-wall targeting domain SH3b, resulting in the chimeric protein P128. This protein shows bactericidal activity against globally prevalent antibiotic resistant clinical isolates of <it>S. aureus </it>and against the genus <it>Staphylococcus </it>in general. <it>In vivo</it>, P128 was efficacious against methicillin-resistant <it>S. aureus </it>in a rat nasal colonization model.</p

Identification of Distinct Unmutated Chronic Lymphocytic Leukemia Subsets in Mice Based on Their T Cell Dependency

Chronic lymphocytic leukemia (CLL) can be divided into prognostically distinct subsets with stereotyped or non-stereotyped, mutated or unmutated B cell receptors (BCRs). Individual subsets vary in antigen specificity and origin, but the impact of antigenic pressure on the CLL BCR repertoire remains unknown. Here, we employed IgH.TEμ mice that spontaneously develop CLL, expressing mostly unmutated BCRs of which ~35% harbor VH11-2/Vκ14-126 and recognize phosphatidylcholine. Proportions of VH11/Vκ14-expressing CLL were increased in the absence of functional germinal centers in IgH.TEμ mice deficient for CD40L or activation-induced cytidine deaminase. Conversely, in vivo T cell-dependent immunization decreased the proportions of VH11/Vκ14-expressing CLL. Furthermore, CLL onset was accelerated by enhanced BCR signaling in Siglec-G−/− mice or in mice expressing constitutively active Bruton's tyrosine kinase. Transcriptional profiling revealed that VH11 and non-VH11 CLL differed in the upregulation of specific pathways implicated in cell signaling and metabolism. Interestingly, principal component analyses using the 148 differentially expressed genes revealed that VH11 and non-VH11 CLL clustered with BCR-stimulated and anti-CD40-stimulated B cells, respectively. We identified an expression signature consisting of 13 genes that were differentially expressed in a larger panel of T cell-dependent non-VH11 CLL compared with T cell-independent VH11/Vκ14 or mutated IgH.TEμ CLL. Parallel differences in the expression of these 13 signature genes were observed between heterogeneous and stereotypic human unmutated CLL. Our findings provide evidence for two distinct unmutated CLL subsets with a specific transcriptional signature: one is T cell-independent and B-1 cell-derived while the other arises upon antigen stimulation in the context of T-cell help

Asian Physics and Software Meeting

TV meeting ID: #936 (KEK MCU

TNF-α-induced protein 3 levels in lung dendritic cells instruct TH2 or TH17 cell differentiation in eosinophilic or neutrophilic asthma

Background: It is currently unknown why allergen exposure or environmental triggers in patients with mild-to-moderate asthma result in T(H)2-mediated eosinophilic inflammation, whereas patients with severe asthma often present with T(H)17-mediated neutrophilic inflammation. The activation state of dendritic cells (DCs) is crucial for both T(H)2 and T(H)17 cell differentiation and is mediated through nuclear factor kappa B activation. Ablation of TNF-alpha-induced protein 3 (TNFAIP3), one of the crucial negative regulators of nuclear factor kappa B activation in myeloid cells and DCs, was shown to control DC activation. Objective: In this study we investigated the precise role of TNFAIP3 in myeloid cells for the development of T(H)2- and T(H)17-cell mediated asthma. Methods: We exposed mice with conditional deletion of the Tnfaip3 gene in either myeloid cells (by using the lysozyme M [LysM] promotor) or specifically in DCs (by using the Cd11c promotor) to acute and chronic house dust mite (HDM)-driven asthma models. Results: We demonstrated that reduced Tnfaip3 gene expression in DCs in either Tnfaip3(CD11c) or Tnfaip3(LysM) mice dose-dependently controlled development of T(H)17-mediated neutrophilic severe asthma in both acute and chronic HDM-driven models, whereas wild-type mice had a purely T(H)2-mediated eosinophilic inflammation. TNFAIP3-deficient DCs induced HDM-specific T(H)17 cell differentiation through increased expression of the T(H)17-instructing cytokines IL-1 beta, IL-6, and IL-23, whereas HDM-specific T(H)2 cell differentiation was hampered by increased IL-12 and IL-6 production. Conclusions: These data show that the extent of TNFAIP3 expression in DCs controls T(H)2/T(H)17 cell differentiation. This implies that reducing DC activation could be a new pharmacologic intervention to treat patients with severe asthma who present with T(H)17-mediated neutrophilic inflammation

Cell lines generated from a chronic lymphocytic leukemia mouse model exhibit constitutive Btk and Akt signaling

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of mature CD5(+) B cells in blood. Spontaneous apoptosis of CLL cells in vitro has hampered in-depth investigation of CLL pathogenesis. Here we describe the generation of three monoclonal mouse cell lines, EMC2, EMC4 and EMC6, from the IgH. TE mu CLL mouse model based on sporadic expression of SV40 large T antigen. The cell lines exhibit a stable CD5(+) CD43(+) IgM(+) CD19(+) CLL phenotype in culture and can be adoptively transferred into Rag1(-/-) mice. RNA-seq analysis revealed only minor differences between the cell lines and their primary tumors and suggested that NF-kappa B and mTOR signaling pathways were involved in cell line outgrowth. In vitro survival and proliferation was dependent on constitutive phosphorylation of Bruton's tyrosine kinase (Btk) at Y551/Y223, and Akt(S473). Treatment of the cell lines with small molecule inhibitors specific for Btk (ibrutinib) or PI3K (idelalisib), which is upstream of Akt, resulted in reduced viability, proliferation and fibronectin-dependent cell adhesion. Treatment of cell line-engrafted Rag1(-/-) mice with ibrutinib was associated with transient lymphocytosis, reduced splenomegaly and increased overall survival. Thus, by generating stable cell lines we established a novel platform for in vitro and in vivo investigation of CLL signal transduction and treatment modalitie

Overexpression of SH2-containing inositol phosphatase contributes to chronic lymphocytic leukemia survival

Balanced activity of kinases and phosphatases downstream of the BCR is essential for B cell differentiation and function and is disturbed in chronic lymphocytic leukemia (CLL). In this study, we employed IgH.TEμ mice, which spontaneously develop CLL, and stable EMC CLL cell lines derived from these mice to explore the role of phosphatases in CLL. Genome-wide expression profiling comparing IgH.TEμ CLL cells with wild-type splenic B cells identified 96 differentially expressed phosphatase genes, including SH2-containing inositol phosphatase (Ship2). We found that B cell-specific deletion of Ship2, but not of its close homolog Ship1, significantly reduced CLL formation in IgH.TEμ mice. Treatment of EMC cell lines with Ship1/2 small molecule inhibitors resulted in the induction of caspase-dependent apoptosis. Using flow cytometry and Western blot analysis, we observed that blocking Ship1/2 abrogated EMC cell survival by exerting dual effects on the BCR signaling cascade. On one hand, specific Ship1 inhibition enhanced calcium signaling and thereby abrogated an anergic response to BCR stimulation in CLL cells. On the other hand, concomitant Ship1/Ship2 inhibition or specific Ship2 inhibition reduced constitutive activation of the mTORC1/ribosomal protein S6 pathway and downregulated constitutive expression of the antiapoptotic protein Mcl-1, in both EMC cell lines and primary IgH.TEμ CLL cells. Importantly, also in human CLL, we found overexpression of many phosphatases including SHIP2. Inhibition of SHIP1/SHIP2 reduced cellular survival and S6 phosphorylation and enhanced basal calcium levels in human CLL cells. Taken together, we provide evidence that SHIP2 contributes to CLL pathogenesis in mouse and human CLL