Transmission of methicillin-resistant Staphylococcus aureus via deceased donor liver transplantation confirmed by whole genome sequencing.

Donor-derived bacterial infection is a recognized complication of solid organ transplantation (SOT). The present report describes the clinical details and successful outcome in a liver transplant recipient despite transmission of methicillin-resistant Staphylococcus aureus (MRSA) from a deceased donor with MRSA endocarditis and bacteremia. We further describe whole genome sequencing (WGS) and complete de novo assembly of the donor and recipient MRSA isolate genomes, which confirms that both isolates are genetically 100% identical. We propose that similar application of WGS techniques to future investigations of donor bacterial transmission would strengthen the definition of proven bacterial transmission in SOT, particularly in the presence of highly clonal bacteria such as MRSA. WGS will further improve our understanding of the epidemiology of bacterial transmission in SOT and the risk of adverse patient outcomes when it occurs.

Type III secretion by Salmonella typhimurium does not require contact with a eukaryotic host.

The type III secretion system encoded by pathogenicity island I in Salmonella typhimurium delivers proteins to the external milieu and into the eukaryotic host cell. The principal factor in induction of the secretion system was found to be a change in the pH of the culture medium from acidic to mildly alkaline. The synthesis of components of the secretion machinery and the production and secretion of substrates occur simultaneously and do not require contact with a eukaryotic host cell. This argues against the concept that type III secretion in S. typhimurium is a process in which the delivery of a presynthesized pool of substrates is triggered by contact with a eukaryotic host cell.

The Salmonella typhimurium InvH protein is an outer membrane lipoprotein required for the proper localization of InvG.

The secretion of pathogenicity factors by Salmonella typhimurium is mediated by a type III secretion system that includes an outer membrane protein of the secretin family. Related secretins are also required for f1 phage assembly and type II secretion. When the C-terminal 43 amino acids of the S. typhimurium secretin InvG are added to f1 pIV, the chimeric f1 pIV-'InvG43 protein becomes dependent on the co-expression of another gene, invH, for function in phage assembly. [3H]-palmitic acid labelling, globomycin sensitivity and density gradient flotation were used to demonstrate that InvH is an outer membrane lipoprotein that is processed by signal peptidase II. A complex between chimeric f1 pIV-'InvG43 and InvH was demonstrated in vivo. InvH was shown to be required for the proper localization of InvG in the outer membrane and for the secretion of the virulence factor SipC. These results suggest that InvH and InvG are part of the functional outer membrane translocation complex in type III secretion systems.

The C-terminal domain of the secretin PulD contains the binding site for its cognate chaperone, PulS, and confers PulS dependence on pIVf1 function.

Related outer membrane proteins, termed secretins, participate in the secretion of macromolecules across the outer membrane of many Gram-negative bacteria. In the pullulanase-secretion system, PulS, an outer membrane-associated lipoprotein, is required both for the integrity and the proper outer membrane localization of the PulD secretin. Here we show that the PulS-binding site is located within the C-terminal 65 residues of PulD. Addition of this domain to the filamentous phage secretin, pIV, or to the unrelated maltose-binding protein rendered both proteins dependent on PulS for stability. A chimeric protein composed of bacteriophage f1 pIV and the C-terminal domain of PuID required properly localized PulS to support phage assembly. An in vivo complex formed between the pIV-PulD65 chimera and PulS was detected by co-immunoprecipitation and by affinity chromatography.

Module swaps between related translocator proteins pIV(f1), pIV(IKe) and PulD: identification of a specificity domain.

In Gram-negative bacteria, type II and type III secretion and filamentous phage assembly systems use related outer membrane proteins for substrate-specific transport across the outer membrane. We show here that the specificity domain of the phage f1 outer membrane protein pIV is contained within the 149 N-terminal amino acid residues. When the pIV(f1) specificity domain is fused to the translocator domain of the related pIV of phage IKe, the chimeric construct supports f1 but not IKe assembly. Functional coupling between the two domains in this chimeric construct is poor and is improved by a single amino acid change in the translocator domain of the pIV(IKe). In native pIV(IKe), two amino acid changes within its specificity domain are both necessary and sufficient to change the specificity from IKe to f1 assembly. Analysis of 39 chimeric constructs between pIV(f1) and the outer membrane protein PulD of the pullulanase secretion system failed to identify a comparable exchangeable specificity domain. These results indicate that the two domains may not function autonomously, and suggest that tertiary and quarternary changes of the entire translocator component rather than of an autonomous functional domain are required for specific translocation across the outer membrane.

Cell wall sorting of lipoproteins in Staphylococcus aureus.

Many surface proteins are thought to be anchored to the cell wall of gram-positive organisms via their C termini, while the N-terminal domains of these molecules are displayed on the bacterial surface. Cell wall anchoring of surface proteins in Staphylococcus aureus requires both an N-terminal leader peptide and a C-terminal cell wall sorting signal. By fusing the cell wall sorting of protein A to the C terminus of staphylococcal beta-lactamase, we demonstrate here that lipoproteins can also be anchored to the cell wall of S. aureus. The topology of cell wall-anchored beta-lactamase is reminiscent of that described for Braun's murein lipoprotein in that the N terminus of the polypeptide chain is membrane anchored whereas the C-terminal end is tethered to the bacterial cell wall.

Inhibition of Rev activity and human immunodeficiency virus type 1 replication by antisense oligodeoxynucleotide phosphorothioate analogs directed against the Rev-responsive element.

The interaction between the Rev protein of human immunodeficiency virus type 1 and its highly structured and conserved RNA target, the Rev-responsive element, is required for virus replication. We demonstrate that antisense oligodeoxynucleotide phosphorothioate analogs directed against the Rev-responsive element effectively inhibit Rev activity, as well as human immunodeficiency virus type 1 replication, and are candidates for antiviral therapy.

Keratinocyte lipid fluidity under the influence of cholesterols, hydrocortisones, "active lipid", tocopherol and retinoic acid--a fluorescence polarization study with regard to physiological and pathophysiological epidermopoiesis and its therapeutic accessibility.

Lipid fluidity of freshly isolated human (H) and guinea pig (GP) keratinocytes (K) was determined as the reciprocal of diphenylhexatriene (DPH) fluorescence polarization (P-value), the temperature being kept at 25 degrees C and cell density standardized to 550,000 per ml (level of statistical significance a less than 0.05). An experimental model involving short-term incubations (2.5 hours, 37 degrees C) of GPK in 1% ethanolic lipid solutions (15 mg lipid agent per ml ethanol) was set up to investigate accumulation a) of cholesterol due to terminal differentiation of keratinocytes and b) of cholesteryl sulfate due to the lack of steroid sulfatase activity in recessive X-linked ichthyosis (RXLI). In comparison to the control including 1% ethanol (P = 0.291 +/- 0.004), significant rigidifying effects were demonstrated for cholesteryl hemisuccinate (0.331 +/- 0.005) and cholesteryl sulfate (0.310 +/- 0.002). Correspondingly, a significant increase of the P-value was also induced by cholesteryl hemisuccinate in HK. Rigidification of GPK by a preincubation with cholesteryl sulfate (P = 0.306 +/- 0.002) could be antagonized by a subsequent short-term incubation with "active lipid (mixture 721)" (0.285 +/- 0.003, a less than 0.05) which may be relevant for future therapeutic strategies in RXLI. Other steran molecules such as hydrocortisone-21-hemisuccinate or hydrocortisone acetate did not affect lipid fluidity. With regard to the therapeutic potency of retinoids in epidermopoietic disorders, incubations of HK with all-trans-retinoic-acid were compared to those with also lipophilic vitamin E, i.e. d-alpha-tocopherol, for 2.5 hours at 37 degrees C using 1% DMSO as a solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

Trans-activating rev protein of the human immunodeficiency virus 1 interacts directly and specifically with its target RNA.

The 20-kDa phosphorylated rev protein from human immunodeficiency virus 1 has been shown to transactivate posttranscriptionally the expression of viral structural proteins by selective stabilization and nuclear export of unspliced and incompletely spliced viral mRNA. We could demonstrate in gel-mobility and immunoprecipitation assays that the recombinant rev protein purified from a baculovirus expression system forms a distinct and specific complex with its target RNA (rev-responsive element), a 234-nucleotide sequence within the envelope coding region of human immunodeficiency virus 1. No complex formation could be observed using RNAs with similar secondary structure nor with other human immunodeficiency virus 1 recombinant proteins. Deletion analysis mapped this specific binding to the first 90 nucleotides of this rev-responsive element, which contains a U2 small nuclear RNA homologous region. We propose that the specific binding of rev to its target RNA sequence plays an essential part in releasing an incompletely spliced viral mRNA containing this target sequence to the cytoplasm.

Susceptibility to HIV-1 infection of a human B-lymphoblastoid cell line, DG75, transfected with subgenomic DNA fragments of Epstein-Barr virus.

We have previously shown that the presence of the EBV genome and active EBV infection in some Burkitt's lymphoma cell lines confer the susceptibility of HIV-1 infection in spite of the absence of surface CD4 receptors in these cells. In the present study we used an EBV genome negative Burkitt's lymphoma B-cell line, DG75, transfected with three different subgenomic fragments of EBV expressing the nuclear antigens (EBNA1 and EBNA2) and the latent membrane protein (LMP), respectively. Immunofluorescence analysis demonstrated CD4 expression in more than 90% of the EBNA1, EBNA2 and LMP transfected cell lines, whereas the antigen could only be detected in less than 4% of the parental DG75 cell line. Unlike the wild type DG75 line, the three transfected cell lines were shown to be susceptible to HIV-1 by both IFA and production of virions. Northern blotting of poly(A) selected mRNA of the four cell lines and hybridization to a human CD4 cDNA (pT4B) demonstrated a 3 kb band in all three EBNA1, EBNA2 and LMP transfected cells as well as in the wild type DG75 cells. Approximate quantification indicates equivalent level of T4 mRNA expression in the transfected cell lines as compared to T-cell lines (Hut-78, H9-9).

Lack of dynamic lipid changes after binding of interleukin 2 in chronic lymphatic leukemia lymphocytes indicates defective transmembrane signalling.

The role of membrane fluidity in the process of signal transduction after binding of Interleukin-2 (Il-2) to its specific cell-surface receptor was investigated in lymphocytes from normal donors and patients with Chronic Lymphocytic Leukemia (CLL). Membrane fluidity was assessed by fluorescence polarization analysis of the apolar fluorophor 1,6-diphenyl-1, 3, 5-hexatrien (DPH) incorporated in the lipid core of the cell membrane. Phytohemagglutinin (PHA) stimulation of lymphocytes for 72h disclosed marked membrane fluidization in normal lymphocytes without affecting lipid phase separation lacking in leukemic cells. Binding of Il-2 induced a significant decrease of the thermotropic transition temperature and overall membrane fluidization within 1h. These effects were not observed in CLL lymphocytes. Results are discussed in terms of defective transmembrane signalling in leukemic cells and pathogenetic implications for uncoupling from proliferation and differentiation signals in the development of leukemia.

Expression of proliferation and differentiation antigens in response to modulation of membrane fluidity in chronic lymphocytic leukemia lymphocytes.

Isolated lymphocytes from patients with chronic lymphocytic leukemia (CLL) and from healthy controls were investigated for cell membrane fluidity and lipid phase separation. Expression of surface immunoglobulins, OKB7 and HLA-DR antigen, transferrin and interleukin-2 receptor was determined in relation to modulation of membrane lipid composition in each individual case. Structural and functional changes of membrane lipids and impaired lipid-protein interactions could be demonstrated in CLL lymphocytes. However, there is no simple correlation of membrane fluidity and receptor expression. This interaction poses a complex physicochemical problem, which must consider the original structural and functional state of the lipid bilayer. Disturded lipid-protein interactions are discussed in terms of defective transmembrane signalling and cell-cell interaction, allowing uncoupling of the leukemic cell from the network of growth and differentiation factors.

Cholesterol modulation of membrane fluidity and ecto-nucleotide triphosphatase activity in human normal and CLL lymphocytes.

Lymphocytes isolated from the peripheral blood of patients with chronic lymphocytic leukemia (CLL) and from healthy donors were studied for membrane fluidity and lipid phase separation. The degree of fluidity of the surface membrane lipids was estimated by fluorescence polarization analysis using the probe 1,6-diphenyl-1,3,5-hexatriene (DPH) embedded in the lipid core of the surface membrane of intact cells. CLL lymphocytes showed a more fluid lipid bilayer in their plasma membrane than normal lymphocytes. Treatment of both groups of lymphocytes with cholesteryl hemisuccinate (CHS) complexed with PVP and BSA resulted in rigidification of cell membranes. A lipid thermotropic transition temperatures was observed at 23.6 + 1.1 degree in normal lymphocytes and 16.3 + 1.0 degree in CLL lymphocytes, which rose to 32.3 + 1.3 degree in normal and was abolished in CLL lymphocytes after treatment with CHS. "Compound lipid fluidizer" reduced the thermotropic transition temperature to 17.5 + 1.0 degree and 15.1 + 0.9 degree in normal and CLL lymphocytes, respectively. A statistically significant increase (p less than 0.01) in the specific activity of the ecto-ATPase was observed in CLL lymphocytes as compared to normal cells. A dramatic increase of the ecto-ATPase activity was also observed when normal lymphocytes were treated with 0.05-0.5 mM CHS. The biological significance of these results is discussed in terms of modifications in the lipid-protein interactions in lymphocyte plasma membrane induced by CHS and the implications in the proliferation of leukemic cells.

Cell membrane fluidity in chronic lymphocytic leukemia (CLL) lymphocytes and its relation to membrane receptor expression.

Cell membrane fluidity (CMF), Ig receptor expression and ecto-ATPase activity were investigated in lymphocytes from patients with chronic lymphocytic leukemia (CLL) and from healthy controls. Significant differences were observed in the parameters of individual CLL cases as compared to normal controls. These findings suggest that malignant transformation of lymphocytes to CLL cells is accompanied by disturbances in cell membrane structure and function, which can be modulated to some extent by exogenous influences on the lipid composition of the cell membrane. The results also suggest, however, that CLL is apparently an inhomogeneous disease with major variations in the investigated parameters among individual cases.