Activity of cefpirome combined with beta-lactamase inhibitors and affinity for the penicillin-binding proteins of methicillin-resistantStaphylococcus aureus

The susceptibility of 47 clinical isolates of methicillin-resistantStaphylococcus aureus (MRSA) to cefpirome, ceftazidime and methicillin was determined with Isosensitest media, with/without 5 % NaCl and incubation at 30°, 37° and 44°C for 24 and 48 h. At 24 h the MIC50 of cefpirome was 8 mg/l compared to 64 mg/l ceftazidime; at 48 h this increased to 32 mg/l cefpirome. The addition of 10 mg/l clavulanic acid or sulbactam lowered the MIC of cefpirome (at 48 h) by greater than four-fold in 23 % and 11 % of the strains, respectively. Cefpirome had primary affinity for penicillin-binding protein (PBP) 1 and 2 in five MRSA and one methicillin-susceptibleStaphylococcus aureus. PBP 2a was present in all MRSA and was not saturated by 64 mg/l cefpirome. Clavulanic acid at a concentration of 10 mg/l bound to PBP 2 by > 50 % in all strains, and when combined with cefpirome, the density of PBP 2a was also reduced but not completely abolished. The data from this study suggests that the mechanism of synergy of a -lactamase inhibitor plus a cephalosporin for MRSA may be due to an additive effect against PBPs and not just inhibition of a -lactamase. No cefpirome-resistant mutants could be selected from a methicillin-susceptibleStaphylococcus aureus, but mutants were selected from an MRSA (expressing homogeneous methicillin resistance) for which MICs of cefpirome were 8 to 32 mg/l.     

Interaction of human papillomavirus type 16 L2 with cellular proteins: identification of novel nuclear body-associated proteins

Two structural proteins form the Papillomavirus (PV) capsids. While the functions of the major structural protein L1 are well established, the exact functions for the minor structural protein L2 are much less well defined, except for some information on a role in viral entry and maturation of infectious virions. To gain more insight in the function of L2 we used the yeast two hybrid system with the Human Papillomavirus (HPV) 11 L2 and HPV16 L2 as bait proteins to isolate putative cellular interaction partners. We identified four proteins interacting with L2 proteins of at least two different HPV types and this interaction was confirmed in vitro by pull-down assays. Further evidence for this interaction was obtained by in vivo localization studies. Two of the proteins, the previously described PATZ and a novel protein, designated PLINP, were localized in discrete nuclear domains and colocalized with L2. The third protein, designated PMSP, is a newly identified cytoplasmic protein which was recruited to nuclear dots when coexpressed with L2. The fourth protein interacting with HPV16, 11 and 1 L2, the tubular-nephritis antigen related protein (TIN-Ag-RP), shows a cytoplasmic as well as a membrane bound subcellular distribution. Taken together, our data indicate that L2 of HPVs with different phenotypes interacts with several cellular host proteins, recruits one of them to the nucleus, and is complexed with at least three cellular proteins in specific nuclear domains. These findings suggest an HPV type-independent modulatory function of L2 on host-cell functions that involves discrete nuclear domains and alteration of the subcellular distribution of cellular proteins. The interacting cellular proteins identified may play a role in the viral life cycle and establishment of viral persistence.     

Interaction of the replication proteins and the capsid protein of porcine circovirus type 1 and 2 with host proteins

Porcine circovirus type 2 (PCV2) is an important pathogen in swine, whereas porcine circovirus type 1 (PCV1) is apathogenic. To analyze the interactions between PCV and its host, we have used a yeast two-hybrid assay to identify cellular proteins interacting with Cap and Rep proteins of both PCV genotypes. Six cellular proteins were found to interact with Cap (MKRN1, gC1qR, Par-4, NAP1, NPM1 and Hsp40) and three with Rep (ZNF265, TDG and VG5Q). These interactions were confirmed by co-immunoprecipitation. Investigation of the localisation of the proteins by immunofluorescence revealed in some cases only limited spatial overlapping with Cap, while in others a clear co-localisation and prominent protein redistribution was observed. The nine cellular proteins are associated with distinct aspects of viral lifecycle and our data is likely to support future research in the field of PCV2 pathogenesis.     

Overexpression of Id protein inhibits the muscle differentiation program: in vivo association of Id with E2A proteins.

The helix-loop-helix (HLH) protein Id lacks the basic DNA-binding domain common to this class of proteins. In vitro experiments suggested that Id could associate tightly with two other HLH proteins encoded by the E2A gene, E12 and E47 (referred to here collectively as E proteins) and prevent their binding to a sequence present in the muscle creatine kinase (MCK) enhancer either as homo-oligomers or hetero-oligomers with MyoD. In this report we present evidence for the in vivo roles of Id and E proteins: (1) Id and E proteins co-fractionate and co-immunoprecipitate in whole-cell extracts prepared from myoblasts; (2) the loss of Id protein observed during the conversion of proliferating myoblasts into mature myotubes correlates with the formation of MyoD/E hetero-oligomeric complexes in whole-cell extracts (these complexes do not form when purified Id protein is added to the extracts); and (3) stable overexpression of Id mRNA and protein in the C2C12 muscle cell line inhibits differentiation in these cells 16 hr post-induction. The myotubes that do eventually form 48 hr post-induction have no detectable Id protein in the nucleus despite the persistence of exogenous Id mRNA. These data support a model in which Id can inhibit muscle cell differentiation by associating with E proteins and preventing them from forming active hetero-oligomeric complexes with the muscle determination gene products.     

Interaction of fimbriae of Haemophilus influenzae type B with heparin-binding extracellular matrix proteins

The interaction of the fimbriae of Haemophilus influenzae type b (Hib) with two heparin-binding extracellular matrix proteins, human fibronectin (Fn) and heparin-binding growth-associated molecule (HB-GAM) from mouse, were studied. The fimbriated Hib strain 770235 fim+, as well as the recombinant strain E. coli HB101(pMH140), which expressed Hib fimbriae, adhered strongly to Fn and HB-GAM immobilized on glass. Purified Hib fimbriae bound to Fn and HB-GAM, and within the Fn molecule, the binding was localized to the N-terminal 30,000-molecular-weight (30K) and 40K fragments, which contain heparin-binding domains I and II, respectively. Fimbrial binding to Fn, HB-GAM, and the 30K and the 40K fragments was inhibited by high concentrations of heparin. The results show that fimbriae of Hib interact with heparin-binding extracellular matrix proteins. The nonfimbriated Hib strain 770235 fim- exhibited a low level of adherence to Fn but did not react with HB-GAM, indicating that Hib strains also possess a fimbria-independent mechanism to interact with Fn.     

Plasmid regulation and temperature-sensitive behavior of the Yersinia pestis penicillin-binding proteins.

Six major bands corresponding to penicillin-binding proteins (PBPs) with molecular weights ranging from 43,000 to 97,000 were detected in cell envelopes of Yersinia pestis EV76 grown at 28 degrees C. When cells were transferred to 37 degrees C and incubated for extended periods of time, the amounts of all PBPs, except for PBP2, were gradually reduced in cell envelopes of a strain carrying a 75-kb virulence-associated plasmid (as measured by penicillin-binding capacity), whereas in a strain cured of the plasmid, all PBPs were stable. The results indicated that the stability and/or the expression of Y. pestis PBPs is affected by a temperature-inducible pathway associated with the virulence-associated plasmid.     

Interaction of heparinized polymer materials with plasma proteins and platelets

Conclusion A negative correlation was found between adsorption of plasma proteins and surface affinity for AT-III. This correlation suggests that maximal activity of surface-bound HP and, therefore, maximal hemocompatibility of polymer material can be attained by minimization of adsorbed plasma proteins. The adsorbed layer is also enriched with AT-III. In addition, a negative correlation in the AT-III-RNAP pair shows that this condition is also necessary for decreasing risk of thrombosis, because it reduces the number of adhered platelets.Thus, high concentration of AT-III in the adsorbed layer is the specific feature of heparinized biomaterials which exerts a fundamental effect on cellular and molecular mechanisms of the interaction of blood with polymer materials.Scientific-Research Institute for Transplantology and Artificial Organs, Russian Ministry of Health, Moscow. Translated from Meditsinskaya Tekhnika, No. 2, pp. 18–22, March–April, 1994.     

Interaction of macrophage cationic proteins with the outer membrane of Pseudomonas aeruginosa.

The interaction of the polycationic rabbit alveolar macrophage cationic proteins MCP-1 and MCP-2 (or their identical neutrophil equivalents NP-1 and NP-2) with the surface of Pseudomonas aeruginosa was investigated. Both proteins bound avidly to purified lipopolysaccharide, as judged by their ability to competitively displace the probe dansyl polymyxin with 50% inhibition (I50) values of 2 to 3 microM. Similar I50 were measured with dansyl polymyxin as a probe for cell surface binding, suggesting that the initial binding site for MCP-1 and MCP-2 on the surface of cells was lipopolysaccharide. Both MCP-1 and MCP-2 permeabilized outer membranes to the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN). The initial rate of NPN uptake plotted against the concentration of MCP-1 or MCP-2 gave sigmoidal curves, suggesting cooperative permeabilization of the outer membrane. Replotting the data as a Hill plot gave an affinity parameter, S0.5, the concentration of MCP giving a half-maximal increase in the rate of NPN uptake, of 5 and 25 microM for MCP-1 and MCP-2, respectively, and thus subsequent studies concentrated on the more active permeabilizer MCP-1. Permeabilization of outer membranes to NPN was a function of buffer pH, with lower pH considerably favoring the permeabilizing effects of MCP-1. Thin-section electron microscopic visualization of MCP-1-treated cells showed production of extended blebs. Further evidence of an altered cell surface after MCP-1 treatment was obtained by demonstrating that treated unopsonized cells were more efficiently phagocytosed by unelicited rabbit alveolar macrophages. The data overall suggest that macrophage cationic proteins interact with the P. aeruginosa outer membrane in a manner typical of other polycations and suggest that one of their major functions may be to permeabilize the outer membrane.     

Interaction of murine macrophage-membrane proteins with components of the pathogenic fungus Histoplasma capsulatum

The interaction of macrophage-membrane proteins and histoplasmin, a crude antigen of the pathogenic fungus Histoplasma capsulatum, was studied using murine peritoneal macrophages. Membrane proteins were purified via membrane attachment to polycationic beads and solubilized in Tris–HCl/SDS/DTT/glycerol for protein extraction; afterwards they were adsorbed or not with H. capsulatum yeast or lectin binding-enriched by affinity chromatography. Membrane proteins and histoplasmin interactions were detected by ELISA and immunoblotting assays using anti-H. capsulatum human or mouse serum and biotinylated goat anti-human or anti-mouse IgG/streptavidin-peroxidase system to reveal the interaction. Results indicate that macrophage-membrane proteins and histoplasmin components interact in a dose-dependent reaction, and adsorption of macrophage-membrane proteins by yeast cells induces a critical decrease in the interaction. Macrophage-membrane glycoproteins with terminal D -galactosyl residues, purified by chromatography with Abrus precatorius lectin, bound to histoplasmin; and two bands of 68 kD and 180 kD of transferred membrane protein samples interacted with histoplasmin components, as revealed by immunoblot assays. Specificity for β-galactoside residues on the macrophage-membrane was confirmed by galactose inhibition of the interaction between macrophage-membrane proteins and histoplasmin components, in competitive ELISA using sugars, as well as by enzymatic cleavage of the galactoside residues.     

Interaction of human complement proteins with serum-sensitive and serum-resistant strains of Escherichia coli

Exposure of serum-susceptible Escherichia coli strains to lethal concns of lysozyme-free human serum resulted in stable binding of complement components to the outer membrane (OM), but not to the cytoplasmic membrane (CM). The short prekilling phase of the reaction was accompanied by binding of C3b; loss of viability was immediately preceeded by stable deposition onto the OM of the component proteins of the membrane attack complex. During the early stages of the active killing phase, bound monomeric C9 could be resolved into two distinct bands on SDS-polyacrylamide gels. Serum exposure lead to a progressive loss of CM recoverability, which appeared to result from partial degradation of CM phospholipids. In contrast, exposure of a resistant E, coli strain to human serum resulted in little change in the membrane profile and very little stable deposition of terminal complement components onto the OM.     

Interaction of proteins with aggregates of catecholamines and nucleotides: possible biological implications

Aggregates of noradrenaline and adenosine-5-triphosphate (ATP) sediment jointly with gelatine on analytical ultracentrifugation. The sediumentation rate of this misture is higher than that of gelatine or of the aggregates alone. Furthermore, gelatine causes an increase in volume of the bottom phase which separates in aqueous solutions of ATP, noradrenaline and calcium. These effects are not obtained with the globular protein albumin.It is concluded that gelatine reversibly binds noradrenaline-ATP aggregates. Since the chromogranins have similar physicochemical properties to gelatine, an interaction of these proteins with aggregates of ATP and noradrenaline may be anticipated to occur in the adrenal chromaffin granules.     

Interaction of gelatin with stereospecific binding proteins and its enhancement of competitive binding assays

The effect of gelatin (0.5 g/l) on binding curves at high dilution of three classes of stereo-specific binding proteins was studied. These included two antibodies (to oestradiol and aldosterone), six transins (horse and dog transcortins, human thyroxine-binding globulin, human sex steroid-binding globulin, and guinea pig transprogestin), and one receptor (bovine adrenal protein kinase). Gelatin increased the apparent binding of all these proteins, particularly at the highest dilutions and sometimes in a striking manner. While much of this action can be attributed to its decreasing the adhesion of the dilute binding protein to glass, gelatin also increased the apparent uptake of some tracers by certain adsorbents. Similar findings were obtained using human gamma globulin (2 g/l). These effects resulted in increased sensitivity and improved reproducibility in the assays employing them.     

Interaction of outer envelope proteins of Chlamydia psittaci GPIC with the HeLa cell surface.

The chlamydial life cycle involves the intimate interaction of components of the infectious elementary body (EB) surface with receptors on the susceptible eukaryotic cell plasma membrane. We have developed an in vitro ligand binding assay system for the identification and characterization of detergent-extracted EB envelope proteins capable of binding to glutaraldehyde-fixed HeLa cell surfaces. With this assay, the developmentally regulated cysteine-rich envelope protein Omp2 of Chlamydia psittaci strain guinea pig inclusion conjunctivitis was shown to bind specifically to HeLa cells. HeLa cells bound Omp2 selectively over other cell wall-associated proteins, including the major outer membrane protein, and the binding of Omp2 was abolished under conditions which alter its conformation. Furthermore, trypsin treatment, which reduces EB adherence, resulted in the proteolytic removal of a small terminal peptide of Omp2 at the EB surface and inactivated Omp2 in the ligand binding assay, while having a negligible effect on the major outer membrane protein. Collectively, our results suggest that Omp2 possesses the capacity to engage in a specific interaction with the host eukaryotic cell. We speculate that, since Omp2 is present only in the infectious EB form, the observed in vitro interaction may be representative of a determining step of the chlamydial pathogenic process.     

Interaction of human apolipoprotein AI and HIV-1 envelope proteins with the native and recombinant CD4 receptors

The method of enzyme-linked immunosorbent assay (ELISA) was used to show an interaction of soluble recombinant CD4-receptor (rsCD4) with human apolipoprotein A-1. Competitive interactions between envelope proteins VIH-1 (gp120 and gp41), on the one hand, and human apolipoprotein A-1 with CD4 receptor, present in the cellular membranes of line MT4 human lymphocytes, were demonstrated by the method of flow cytofluorimetry. It was suggested that the competitive interactions between the above proteins could manifest in respect to the apolipoprotein A-1 receptor, which affects the involvement of the latter in the regulation of protein biosynthesis and which leads to a decrease in the body weight of HIV-infected patients.     

Contradictory Roles for Antibody and Complement in the Interaction of Brucella abortus with Its Host

Abstract

The ability of serum complement to kill bacteria has been linked to host resistance to Gram-negative bacteria. A mechanism for killing extracellular organisms during early invasion, following release from infected phagocytic cells, or during bacteremia would contribute to a host's ability to resist disease. In fact, the ability of serum complement to kill bacteria has been linked to disease resistance. Brucella abortus are Gram-negative intracellular pathogens. Resistance to these bacteria involves the coordinated activities of the cellular and humoral immune systems. The existence of serum-resistant forms of B. abortus has been established, and it has been shown that these bacteria can resist the killing action of complement even in the presence of specific antibody. Antibody is usually necessary for complement-mediated killing of smooth (virulent) forms of Gram-negative bacteria. An anomolous situation exists with some isolates of smooth B. abortus. Sera containing high titers of specific antibody do not support killing unless they are diluted. In the bovine, this phenomenon is associated with IgGl and IgG2 antibodies. This finding may account for the lack of positive correlation between antibody levels and resistance to disease, which has led, perhaps wrongly, to the idea that antibody and complement are not important in resistance to brucellosis.

Available evidence suggests that antibody may have contradictory roles in the interactions between a host and bacteria. Avirulent (rough) forms of the organism would be rapidly killed by complement shortly after invasion, but serum-resistant smooth forms would survive and invade resident phagocytic cells. During the process of invasion and phagocytosis, the bacteria would initiate an immune response. With time, some B. abortus organisms would be released from infected phagocytic cells. In the early stages of this process, the bacteria would encounter IgM antibody and low concentrations of IgG antibody. These would cause complement-mediated killing, and infection would be restricted to resident phagocytic cells. However, the immune response to B. abortus antigens would be intensified, and IgG antibody levels would increase. High concentrations of antibody do not support complement-mediated killing of extracellular B. abortus, but the bacteria would be opsonized by antibody and complement component fragments. This would lead to increased phagocytosis of extracellular B. abortus as they appear, and concomitant extension of disease. Because die high levels of antibody would block complement-mediated lulling of B. abortus, resistance to disease at this point would be dependent on cell-mediated immunity.