The effect of thiol-active compounds and sterols on the membrane-associated hemolysin of Mycoplasma pulmonis

Jarvill-Taylor, K

doi:10.1016/0378-1097(95)00115-l

Cited by 5 publications

(11 citation statements)

References 3 publications

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“…The determination of the effects of cholesterol or 1,2‐dioleyl‐phosphatidylcholine (DOPC) on haemolytic activity was essentially as described earlier (Jarvill‐Taylor and Minion, 1995): a 1.2% (w/w) cholesterol solution in 99.5% ethanol or a 2.4% (w/w) DOPC solution in 99.5% ethanol was added directly to the assay wells (0.2, 0.5, 1.0, 2.0, 5.0 and 10 μl). Controls consisted of erythrocytes incubated with PBS, ethanol, cholesterol or DOPC respectively.…”

Section: Methodsmentioning

confidence: 99%

Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli

Oscarsson

Mizunoe

et al. 1999

Molecular Microbiology

116

153

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SummaryEscherichia coli K-12 carries a gene for a protein denoted ClyA or SheA that can mediate a cytolytic phenotype. The ClyA protein is not expressed at detectable levels in most strains of E. coli, but overproduction suitable for purification was accomplished by cloning the structural gene in an hns mutant strain. Highly purified ClyA protein was cytotoxic to macrophage cells in culture and caused detachment and lysis of the mammalian cells. Results from osmotic protection assays were consistent with the suggestion that the protein formed pores with a diameter of up to 3 nm. Using Acholeplasma laidlawii cells and multilamellar liposomes, we studied the effect of ClyA on membranes with varying compositions and in the presence of different ions. ClyA induced cytolytic release of the fluorescent tracer from carboxyfluorescein-loaded liposomes, and the release was stimulated if cholesterol was present in the membranes whereas addition of calcium had no effect. Pretreatment of the ClyA protein with cholesterol inhibited the pore formation, suggesting that ClyA could bind to cholesterol. Efficient coprecipitation of ClyA with either cholesterol or 1,2,3-trioctadecanoylglycerol in aqueous solutions showed that ClyA directly interacted with the hydrophobic molecular aggregates. We tested the possible functional importance of selected ClyA protein regions by site-directed mutagenesis. Defined mutants of ClyA were obtained with alterations in postulated transmembrane structures in the central part and in a postulated membrane-targeting domain in the C-terminal part. Our results were consistent with the suggestion that particular amphiphilic segments are required for ClyA activity. We propose that these domains are necessary for ClyA to form pores.

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Section: Methodsmentioning

confidence: 99%

Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli

Oscarsson

Mizunoe

et al. 1999

Molecular Microbiology

116

153

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“…In previous studies of pathogenic mycoplasmas, hemadsorption and cytadherence activities were closely associated with virulence potential (2, 30). For example, hydrogen peroxide-mediated and membrane-associated hemolytic activities have been found in numerous Mycoplasma species (15,24,30), and analysis of mycoplasma genomic sequences revealed the presence of a hemolysin-like gene (VXpSPT7_orf424) in Mycoplasma pneumoniae (13) and a homologous gene (MG146) in Mycoplasma genitalium (10). It is well known that bacterial hemolysins lyse erythrocytes (RBCs) and a variety of other cell types, including mast cells, neutrophils, and polymorphonuclear cells, which enables hemolytic microorganisms to directly damage host tissues as well as induce inflammatory responses (5).…”

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confidence: 99%

Hemolytic and Hemoxidative Activities inMycoplasma penetrans

Kannan

Baseman

2000

Infect Immun

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Mycoplasma penetrans is a newly isolated Mollicute from the urine of patients infected with human immunodeficiency virus that demonstrates the capacity to adhere to and invade human cells. A previous report, based on assays with mouse red blood cells (RBCs), indicated that M. penetrans lacked hemolytic activity. In our studies, we incubated different isolates of M. penetrans with various RBC species and observed hemolytic zones surrounding individual mycoplasma colonies. All M. penetrans strains displayed hemolysis after 2 to 3 days of incubation. Hemolytic activity diffused from single colonies, eventually causing complete lysis. Hemolysis was most pronounced with sheep RBCs, followed by horse, chicken, and human cells. Furthermore, hemolytic activity was demonstrable in both intact mycoplasma cell preparations and spent culture supernatant. However, unlike intact mycoplasmas, the hemolytic activity in the supernatant was dependent on the reducing agent, cysteine. In addition to hemolysis, a brown precipitate was closely associated with mycoplasma colonies, suggesting oxidation of hemoglobin. Absorption spectra indicated that hemoglobin was oxidized to methemoglobin, and the addition of catalase demonstrated H 2 O 2 -mediated hemoxidation. Other experiments suggested that hemoxidation enhanced total hemolysis, providing the first evidence of both hemolytic and hemoxidative activities in M. penetrans.Mycoplasmas, members of the cell wall-less Mollicutes and considered among the smallest self-replicating cells, have been detected in a wide range of hosts, including humans, other vertebrates, invertebrates, and plants (2,19,20,27). In previous studies of pathogenic mycoplasmas, hemadsorption and cytadherence activities were closely associated with virulence potential (2, 30). For example, hydrogen peroxide-mediated and membrane-associated hemolytic activities have been found in numerous Mycoplasma species (15,24,30), and analysis of mycoplasma genomic sequences revealed the presence of a hemolysin-like gene (VXpSPT7_orf424) in Mycoplasma pneumoniae (13) and a homologous gene (MG146) in Mycoplasma genitalium (10). It is well known that bacterial hemolysins lyse erythrocytes (RBCs) and a variety of other cell types, including mast cells, neutrophils, and polymorphonuclear cells, which enables hemolytic microorganisms to directly damage host tissues as well as induce inflammatory responses (5). However, a new species, Mycoplasma penetrans, which was isolated from the urine of patients infected with human immunodeficiency virus (22), was shown to actively invade mammalian cells in culture (21) and cause cytopathology in experimentally infected chicken embryos (12) but lack hemolytic activity (24). In this report we describe hemolytic activity in all isolates of M. penetrans and show that H 2 O 2 -mediated hemoxidative activity contributes to total mycoplasma-mediated hemolysis. MATERIALS AND METHODSBacterial strains and medium. M. penetrans cells (GTU-54 and isolates from France and Texas) (11) were obtained from J. T...

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“…Minion and Jarvill-Taylor established that the membranes of most mycoplasmas contain an activity which is capable of lysing trypsinized mouse red blood cells (138). It was also demonstrated that the hemolysin from M. pulmonis is inhibited by cholesterol and selected cholesterol derivatives (83). The activity was affected by heat treatment, thiol-reducing agents, thiol-activating agents and oxidizing compounds (83,138), thus resembling the gram positive thiol-activated hemolysins (streptolysin, pneumolysin, and listeriolysin).…”

Section: Membrane-associated Activitiesmentioning

confidence: 98%

“…It was also demonstrated that the hemolysin from M. pulmonis is inhibited by cholesterol and selected cholesterol derivatives (83). The activity was affected by heat treatment, thiol-reducing agents, thiol-activating agents and oxidizing compounds (83,138), thus resembling the gram positive thiol-activated hemolysins (streptolysin, pneumolysin, and listeriolysin). The stereo-specificity in sterol recognition, however, was distinctly different than the pattern observed with the gram positive thiol-activated hemolysins (83).…”

Section: Membrane-associated Activitiesmentioning

confidence: 98%

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Characterization of membrane-associated nuclease activity in Mycoplasma pulmonis

Jarvill-Taylor

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Introduction 45 Materials and Methods 47 Organisms and media 47 Isolation of M. pulmonis chromosomal DNA Antisera Genomic libra^ 49 Library screening 49 Recombinant DNA Nuclease detection assays TnlOOO mutagenesis Hybridization smdies DNA sequence analysis Nucleotide sequence accession number Results Characterization of the anti-nuclease antisera Identification and characterization of immunoreactive X phage Cloning and functional analysis of the chromosomal fragment from phage 13-2B DNA sequencing and computer analysis Analysis of mnuA products produced in E. coli Presence of mnuA-like sequences in other mycoplasma species Discussion Acknowledgments CHAPTER 4. MOLECULAR ANALYSIS OF THE uvrB HOMOLOG IN

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The effect of thiol-active compounds and sterols on the membrane-associated hemolysin of Mycoplasma pulmonis

Cited by 5 publications

References 3 publications

Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli

Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli

Hemolytic and Hemoxidative Activities inMycoplasma penetrans

Characterization of membrane-associated nuclease activity in Mycoplasma pulmonis

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