The dependence of the antibacterial effect of the polycationic peptide warnerin on the energy state of target cells

The bactericidal effect of the polycationic peptide warnerin, produced by Staphylococcus warneri IEGM KL-1, was found to depend on the energy state of susceptible Staphylococcus epidermidis cells. The pre-treatment of these cells with compounds that diminish the proton-motive force of plasma membranes enhanced cell tolerance to warnerin. The components deltapsi and deltapH of the membrane proton potential influenced the antibacterial activity of warnerin in different ways. In particular, the antibacterial activity of warnerin decreased when the electric component of the proton-motive force of target membranes declined.     

Mip protein of Legionella pneumophila exhibits peptidyl-prolyl-cis/trans isomerase (PPIase) activity

Legionella pneumophila is an intracellular parasite which is able to survive and multiply in human monocytes and alveolar macrophages. The Mip (macrophage infectivity potentiator) protein has been shown to be an essential virulence factor. A search of translated nucleic acid data bases has shown that the Mip protein from strain Wadsworth possesses regions homologous to those found in the FK506-binding proteins (FKBPs) of several different eukaryotic organisms. FKBPs are able to bind to the immunosuppressant macrolide FK506 and possess peptidyl-prolyl cis/trans isomerase (PPIase) activity. The gene coding for the Mip protein was cloned from the chromosome of L. pneumophila strain Philadelphia I and sequenced. It was synthesized in Escherichia coli K-12 and after purification it exhibited PPIase activity catalysing the slow cis/trans isomerization of prolyl peptide bonds in oligopeptides. Mip is inhibited by FK506 and fully resistant to cyclosporin A, as was also found for the recently characterized FKBP-type PPIases of eukaryotes. However, the N-terminal extension of Mip and/or the substitutions of the variable amino acids in the C-terminal FKBP core leads to variations, when compared with eukaryotic FKBPs, in substrate specificity with the oligopeptide substrates of type Suc-Ala-Xaa-Pro-Phe-4-nitroanilide. Nevertheless, the Legionella Mip factor represents a bacterial gene product which shares some characteristics normally found in eukaryotic proteins. In view of the activity of PPIases in protein-folding reactions, such prokaryotic FKBP analogues may represent a new class of bacterial pathogenicity factors.     

Rapid method for enumeration of viable Legionella pneumophila and other Legionella spp. in water

A sensitive and specific method has been developed to enumerate viable L. pneumophila and other Legionella spp. in water by epifluorescence microscopy in a short period of time (a few hours). This method allows the quantification of L. pneumophila or other Legionella spp. as well as the discrimination between viable and nonviable Legionella. It simultaneously combines the specific detection of Legionella cells using antibodies and a bacterial viability marker (ChemChrome V6), the enumeration being achieved by epifluorescence microscopy. The performance of this immunological double-staining (IDS) method was investigated in 38 natural filterable water samples from different aquatic sources, and the viable Legionella counts were compared with those obtained by the standard culture method. The recovery rate of the IDS method is similar to, or higher than, that of the conventional culture method. Under our experimental conditions, the limit of detection of the IDS method was <176 Legionella cells per liter. The examination of several samples in duplicates for the presence of L. pneumophila and other Legionella spp. indicated that the IDS method exhibits an excellent intralaboratory reproducibility, better than that of the standard culture method. This immunological approach allows rapid measurements in emergency situations, such as monitoring the efficacy of disinfection shock treatments. Although its field of application is as yet limited to filterable waters, the double-staining method may be an interesting alternative (not equivalent) to the conventional standard culture methods for enumerating viable Legionella when rapid detection is required.     

Immunogenicity and adjuvanticity of lipopolysaccharide from Legionella pneumophila

Lipopolysaccharide isolated from Legionella pneumophila was found to be a potent antigen and inducer of antibody with strong adjuvant activity for related and unrelated antigens such as sheep erythrocytes by in vivo and in vitro systems. The LPS was also a potent stimulator of blastogenic responses by spleen cells from normal mice as well as from mice immunized with inactivated whole cells of Legionella. It strongly stimulated production of interferon and interleukin 1. These results indicate that the LPS of Legionella may be an important immune regulator in the host response.     

Influence of alpha-helices on the emulsifying properties of proteins

A peptide derived from apomyoglobin by cyanogen bromide cleavage was found to be an active emulsifier. This molecule, peptide 1-55, has two potential amphipathic alpha-helices and a hydrophilic C-terminal domain. The importance of each of these domains to the emulsifying properties of this molecule was investigated by testing the products of gene constructs based on the sequence of peptide 1-55, but lacking one of the three domains. The emulsifying activity of the peptides lacking either of the alpha-helices was correlated with the hydrophobic moments of their respective helices. The hydrophobic moment is a measure of the amphipathicity of alpha-helices; a hydrophobic moment analysis of other emulsifying peptides supports the hypothesis that a high hydrophobic moment contributes to good emulsifying properties in a molecule which contains alpha-helices.     

Design of a partial peptide mimetic of anginex with antiangiogenic and anticancer activity

Based on structure-activity relationships of the angiostatic beta-sheet-forming peptide anginex, we have designed a mimetic, 6DBF7, which inhibits angiogenesis and tumor growth in mice. 6DBF7 is composed of a beta-sheet-inducing dibenzofuran (DBF)-turn mimetic and two short key amino acid sequences from anginex. This novel antiangiogenic molecule is more effective in vivo than parent anginex. In a mouse xenograft model for ovarian carcinoma, 6DBF7 is observed to reduce tumor growth by up to 80%. It is suggested that the activity is based on antiangiogenesis, because in vitro tube formation is inhibited, and because treatment of tumor-bearing mice led to a significant reduction in microvessel density within the tumor. This partial peptide mimetic is the first endothelial cell-specific molecule designed as a substitute for an angiostatic inhibitory peptide.     

Molecular Properties and Extracellular Processing of the Lipase of Staphylococcus warneri M

Staphylococcus warneri M exhibited extracellular lipase activity. By zymogram analysis of extracellular proteins, multiple bands were detected and the profiles changed depending on the bacterial growth phase. N-terminal amino acid sequences of three bands (N1-N3) were determined. From the genome library of S. warneri M whole DNA, the gene-directing lipase activity (named gehC(WM)) was cloned and characterized. The gehC(WM )gene encoded a protein (GehC(WM)), whose calculated molecular mass was 83.4 kDa, and the sequence was similar to the other staphylococcal lipases. Though two lipases have been known from S. warneri 863, GehC(WM) differs from both of them, indicating that this enzyme is the third extracellular lipase of the S. warneri strain. The N-terminal sequences of the N1-N3 polypeptides completely coincided with the deduced amino acid sequences in GehC(WM). GehC(WM) was predicted to be a prepro-protein. In vitro processing and protein sequencing suggested that pro-GehC(WM) is possibly processed by extracellular glutamyl endopeptidase, PROM. Inductively coupled plasma-atomic emission spectrometer analysis showed that purified his-tagged mature GehC(WM) possessed zinc ion. A gehC(WM) knockout mutant was constructed by insertion of an erythromycin resistance gene into the gehC(WM). Zymogram and immunoblot analyses of the gehC(WM )mutant indicated that GehC(WM) was a major extracellular lipase of S. warneri M.     

Effects of Disinfection on Legionella spp., Eukarya, and Biofilms in a Hot Water System

Legionella species are frequently detected in hot water systems, attached to the surface as a biofilm. In this work, the dynamics of Legionella spp. and diverse bacteria and eukarya associated together in the biofilm, coming from a pilot scale 1 system simulating a real hot water system, were investigated throughout 6 months after two successive heat shock treatments followed by three successive chemical treatments. Community structure was assessed by a fingerprint technique, single-strand conformation polymorphism (SSCP). In addition, the diversity and dynamics of Legionella and eukarya were investigated by small-subunit (SSU) ribosomal cloning and sequencing. Our results showed that pathogenic Legionella species remained after the heat shock and chemical treatments (Legionella pneumophila and Legionella anisa, respectively). The biofilm was not removed, and the bacterial community structure was transitorily affected by the treatments. Moreover, several amoebae had been detected in the biofilm before treatments (Thecamoebae sp., Vannella sp., and Hartmanella vermiformis) and after the first heat shock treatment, but only H. vermiformis remained. However, another protozoan affiliated with Alveolata, which is known as a host cell for Legionella, dominated the eukaryal species after the second heat shock and chemical treatment tests. Therefore, effective Legionella disinfection may be dependent on the elimination of these important microbial components. We suggest that eradicating Legionella in hot water networks requires better study of bacterial and eukaryal species associated with Legionella in biofilms.     

Virulence factors of the family Legionellaceae.

Whereas bacteria in the genus Legionella have emerged as relatively frequent causes of pneumonia, the mechanisms underlying their pathogenicity are obscure. The legionellae are facultative intracellular pathogens which multiply within the phagosome of mononuclear phagocytes and are not killed efficiently by polymorphonuclear leukocytes. The functional defects that might permit the intracellular survival of the legionellae have remained an enigma until recently. Phagosome-lysosome fusion is inhibited by a single strain (Philadelphia 1) of Legionella pneumophila serogroup 1, but not by other strains of L. pneumophila or other species. It has been found that following the ingestion of Legionella organisms, the subsequent activation of neutrophils and monocytes in response to both soluble and particulate stimuli is profoundly impaired and the bactericidal activity of these cells is attenuated, suggesting that Legionella bacterial cell-associated factors have an inhibitory effect on phagocyte activation. Two factors elaborated by the legionellae which inhibit phagocyte activation have been described. First, the Legionella (cyto)toxin blocks neutrophil oxidative metabolism in response to various agonists by an unknown mechanism. Second, L. micdadei bacterial cells contain a phosphatase which blocks superoxide anion production by stimulated neutrophils. The Legionella phosphatase disrupts the formation of critical intracellular second messengers in neutrophils. In addition to the toxin and phosphatase, several other moieties that may serve as virulence factors by promoting cell invasion or intracellular survival and multiplication are elaborated by the legionellae. Molecular biological studies show that a cell surface protein named Mip is necessary for the efficient invasion of monocytes. A possible role for a Legionella phospholipase C as a virulence factor is still largely theoretical. L. micdadei contains an unusual protein kinase which catalyzes the phosphorylation of eukaryotic substrates, including phosphatidylinositol and tubulin. Since the phosphorylation of either phosphatidylinositol or tubulin might compromise phagocyte activation and bactericidal functions, this enzyme may well be a virulence factor. Administration of the L. pneumophila exoprotease induces lesions resembling those of Legionella pneumonia and kills guinea pigs, suggesting that this protein plays a role in the pathogenesis of legionellosis. However, recent work with a genetically engineered strain has convincingly shown that the protease is not necessary for intracellular survival or virulence. As might be expected with a complex process like intracellular parasitism, it appears that the capability of Legionella strains to invade and multiply in host phagocytes is multifactorial and that no single moiety which is responsible for the virulence phenotype will be found.     

Modification of swine pepsin and pepsinogen by p-nitrophenyldiazonium chloride

It was found that at pH 5.2 and 40-fold excess of p-nitrophenyldiazonium chloride the inhibitor incorporation into the porcine pepsin molecule involves 1.9 residues, one residue being bound to tyrosine 189. Besides, tyrosines 44, 113, 154 and 174 enter the reaction. Modified pepsin retains 25% of the native enzyme activity. In the pepsinogen molecule the degree of tyrosine 189 modification diminishes 5 times; of 1.5 inhibitor molecules incorporated into the protein 0.78 residues are bound to tyrosine 113. The potential proteolytic activity of modified pepsinogen towards haemoglobin cleavage makes up to 60% of the original one. It is concluded that the activation peptide in the pepsinogen molecule masks the substrate binding site bearing tyrosine 189, thus preventing its modification with p-nitrophenyldiazonium chloride. The activation peptide in the pepsinogen molecule is presumably located in the vicinity of the wide loop bend carrying tyrosine residue 113, which may be the reason for the decreased pKa value of this residue and of its increased reactivity in the azocoupling reaction.     

Peptide mimics of the Bowman-Birk inhibitor reactive site loop

Bowman-Birk Inhibitors (BBIs) are small highly cross-linked proteins that typically display an almost symmetrical "double-headed" structure. Each "head" contains an independent proteinase binding domain. The realization that one BBI molecule could form a 1:1:1 complex with two enzymes led early workers to dissect this activity. Now, after three decades of research, it has been possible to isolate the antiproteinase activity as small ( approximately 11 residues), cyclic, synthetic peptides, which display most of the functional aspects of the protein. More recently, it has been found that these peptide fragments are not just a synthetic curiosity-a natural 14-residue cyclic peptide (SFTI-1), which too encapsulates the BBI inhibitory motif, is found to occur in sunflowers. This article reviews the properties of BBI-based peptides (including SFTI-1) and discusses the features that are important for inhibitory activity.     

A yeast genetic system for the identification and characterization of substrate proteins transferred into host cells by the Legionella pneumophila Dot/Icm system

The Dot/Icm system is a type IVb secretion system used by Legionella pneumophila to modulate vesicular transport in both protozoan and mammalian host cells. It has been shown that proteins and processes that are highly conserved in all eukaryotic cells are targets for some of the proteins injected by the Dot/Icm system. For example, the Legionella protein RalF was shown previously to be a Dot/Icm substrate that functions as a guanine nucleotide exchange factor (GEF) for the Arf family of eukaryotic small GTP-binding proteins. Here we show that ectopic production of the RalF protein in Saccharomyces cerevisiae interferes with yeast growth. Inhibition of yeast growth was found to be dependent on the ability of RalF to function as an Arf-GEF in vivo. The possibility that other Dot/Icm substrate proteins would have the capacity to interfere with yeast growth was used as a rationale to screen plasmid libraries containing random fragments of Legionella chromosomal DNA positioned downstream of a galactose-inducible promoter. This screen identified Legionella proteins that conferred a conditional growth defect when overproduced by yeast cultured in the presence of galactose. Most of the Legionella proteins identified were determined to be substrates of the Dot/Icm system. This screen led to the identification of a new Dot/Icm substrate protein that was called YlfA, for yeast lethal factor A. A paralogue of YlfA was identified on an unlinked region of the Legionella chromosome and this protein was also translocated by the Dot/Icm system. It was determined that a hydrophobic region near the N-terminus of the YlfA protein and an adjacent region predicted to form a coiled-coil domain were necessary for a biological activity that interfered with yeast growth. The YlfA protein did not decorate the Legionella-containing vacuole during the first 7 h of infection but could be observed on the endoplasmic reticulum (ER)-derived replicative vacuole and on punctate structures throughout the host cell at later stages. Ectopic production of YlfA in mammalian cells revealed that the N-terminal hydrophobic domain in YlfA was able to localize the protein to early secretory organelles, including endoplasmic reticulum. These studies show that yeast genetics can be exploited to identify and characterize proteins that are injected into host cells by bacterial pathogens that utilize type IV secretion systems for pathogenesis.     

Studies on the Mechanism of Activation of Pepsinogen

The mechanism of activation of pepsinogen was studied. It was found that no peptide bond cleavage occurred in the molecule of denatured pepsinogen at pH 2. It was inferred from this that a specific secondary and tertiary structure is formed in the molecule of pepsinogen in acid and that it might be necessary for the hydrolysis of the peptide bond. From the circular dichroism studies on pepsinogen and pepsin, it was found that there is a conformational change in the molecule of pepsinogen at pH 4.3~4.5 and that this change is followed by a gradual formation of pepsin.     

Longitudinal evaluation of the efficacy of heat treatment procedures against Legionella spp. in hospital water systems by using a flow cytometric assay

Legionella spp. are frequently isolated in hospital water systems. Heat shock (30 min at 70°C) is recommended by the World Health Organization to control its multiplication. The aim of the study was to evaluate retrospectively the efficacy of heat treatments by using a flow cytometry assay (FCA) able to identify viable but nonculturable (VBNC) cells. The study included Legionella strains (L. pneumophila [3 clusters] and L. anisa [1 cluster]) isolated from four hot water circuits of different hospital buildings in Saint-Etienne, France, during a 20-year prospective surveillance. The strains recovered from the different circuits were not epidemiologically related, but the strains isolated within a same circuit over time exhibited an identical genotypic profile. After an in vitro treatment of 30 min at 70°C, the mean percentage of viable cells and VBNC cells varied from 4.6% to 71.7%. The in vitro differences in heat sensitivity were in agreement with the observed efficacy of preventive and corrective heating measures used to control water contamination. These results suggest that Legionella strains can become heat resistant after heating treatments for a long time and that flow cytometry could be helpful to check the efficacy of heat treatments on Legionella spp. and to optimize the decontamination processes applied to water systems for the control of Legionella proliferation.     

Antiplasmin activity of a peptide that binds to the receptor-binding site of angiogenin.

It has been suggested that angiogenin binds to an actin-like molecule present on the surface of endothelial cells. Actin inhibits plasmin activity, but the angiogenin-actin complex is not active. In this report, we found that plasmin inhibits the interaction between angiogenin and actin suggesting a possibility that both angiogenin and plasmin may bind to a similar site on actin. Here we report that chANG, an antiangiogenin peptide that binds to the actin-binding site of angiogenin, inhibits the proteolytic activity of plasmin without any apparent effect on the activities of plasminogen activators and matrix metalloproteases. Its antiplasmin activity is comparable with that of actin. chANG inhibits plasmin activity via its binding to plasmin kringle domains while scrambled chANG does not bind to plasmin. chANG also inhibits the invasion of angiogenin-secreting human fibrosarcoma and colorectal carcinoma cells without effecting migration. Furthermore, chANG blocks angiogenesis induced by fibrosarcoma cells and metastasis of colorectal carcinoma cells to the liver. Therefore, the 11-amino acid peptide chANG has both antiangiogenin and antiplasmin activity, and could be useful in the development of anticancer agents.     

Enhanced growth of Legionella pneumophila in tetrahydrocannabinol-treated macrophages.

Legionella pneumophila is an opportunistic intracellular pathogen that infects macrophages, both in vivo and in vitro. Tetrahydrocannabinol is a major psychoactive component of marijuana and can affect the functional activity of macrophages. In the present study, it was found that the treatment of macrophage cultures from permissive A/J mice with THC enhanced the growth of Legionella in these cells. Legionella grew much better in macrophages treated with low doses of THC, which caused no alteration in the number or viability of macrophages, as compared with growth in untreated cells. Furthermore, lipopolysaccharide-treated A/J mouse macrophages restricted the growth of Legionella, but this growth restriction was overcome by the addition of THC to LPS-treated macrophage cultures after infection. Thus, it is apparent that THC has the ability to enhance the growth of the intracellular opportunistic pathogen Legionella that grows in A/J mouse macrophages.     

Pituitary lactogenic hormone. XXXVII. Synthesis and properties of the carboxyl terminal cyclic undecapeptide.

The carboxyl terminal cyclic undecapeptide of the ovine prolactin molecule: Leu-Asn-Cys-Arg-Ile-Ile-Tyr-Asn-Asn-Asn-Cys has been synthesized by the solid-phase method. The circular dichroism of the synthetic peptide has been investigated. It was also found that the cyclic undecapeptide does not have pigeon crop-sac stimulating activity, or immunological activity when tested by complement fixation experiments.     

Aspartic proteinases: Fourier transform infrared spectroscopic studies of a model of the active side.

We synthesized and studied by Fourier transform infrared spectroscopy nine monosalts of diamides as models for the active side of aspartic proteinases. One compound, the monosalt of meta-aminobenzoic acid diamide of fumaric acid (m-FUM), shows the same biological activity as pepsin with regard to the splitting of peptide bonds of the Pro-Thi-Glu-Phe-Phe(4-NO2)-Arg-Leu heptapeptide. The monosalt of m-FUM forms with oxindole a complex in which the carboxylic acid group of the monosalt of m-FUM is strongly hydrogen bonded with the O atom of the peptide bond of oxindole. When one water molecule is added to this complex, the strong field of the carboxylate group destabilizes an O-H bond of the water molecule. The distorted water molecule attacks the carbon atom of the peptide group, and the water proton transfers to the peptide N atom. Simultaneously, the C-N bond of the amide group is broken. Hence it is demonstrated that the catalytic mechanism of aspartic acid proteinases is a base catalysis. The results show that for this catalytic mechanism there are sufficient carboxylic and carboxylate groups, as well as a water molecule in the correct arrangement. It was also demonstrated with other monosalts of dicarboxylic acids that well-defined steric conditions of the carboxylic acid and the carboxylate group must be fulfilled to show hydrolytic activity with regard to oxindole molecules.