Photobacterium damselae subsp. damselae Major Virulence Factors Dly, Plasmid-Encoded HlyA, and Chromosome-Encoded HlyA Are Secreted via the Type II Secretion System

ABSTRACT Photobacterium damselae subsp. damselae is a marine bacterium that causes septicemia in marine animals and in humans. Previously, we had determined a major role of pPHDD1 plasmid-encoded Dly (damselysin) and HlyA (HlyA pl ) and the chromosome-encoded HlyA (HlyA ch ) hemolysins in virulence. However, the mechanisms by which these toxins are secreted remain unknown. In this study, we found that a mini-Tn 10 transposon mutant in a plasmidless strain showing an impaired hemolytic phenotype contained an insertion in epsL , a component of a type II secretion system (T2SS). Reconstruction of the mutant by allelic exchange confirmed the specific involvement of epsL in HlyA ch secretion. In…

Recovery of human fibroblasts from attack by the pore-forming alpha-toxin of Staphylococcus aureus.

When applied at low concentrations (10 micrograms/ml), staphylococcal alpha-toxin generates a small channel in keratinocyte and lymphocyte membranes that permits selective transmembrane flux of monovalent ions. Here we show that a moderate concentration (1-50 micrograms/ml) of alpha-toxin similarly produces a small pore in membranes of human fibroblasts. This process leads to rapid leakage of K+ and to a drop in cellular ATP to 10-20% of normal levels in 2 h. In the presence of medium supplemented with serum and at pH 7.4, the cells are able to recover from toxin attack, so that normal levels of K+ and ATP are reached after 6-8 h at 37 degrees C. The repair process is dependent on the prese…

Hypersusceptibility of neutrophil granulocytes towards lethal action of free fatty acids contained in enzyme-modified atherogenic low density lipoprotein.

Abstract Objective The bulk of LDL entrapped in the arterial intima is modified by hydrolytic enzymes, leading to extensive cleavage of cholesterylesters and liberation of fatty acids. The latter induce apoptosis in endothelial cells but are far less cytotoxic towards macrophages. We have compared the cytotoxic effects of enzymatically modified LDL (E-LDL) on macrophages and polymorphonuclear granulocytes (PMN). Methods and results E-LDL displayed toxicity towards PMN at far lower concentrations than towards monocyte-derived macrophages. Native or oxidized LDL had no effect. Free fatty acids contained in E-LDL were the cause of the observed toxicity, which could be mimicked by linoleic acid…

Global Functional Analyses of Cellular Responses to Pore-Forming Toxins

Here we present the first global functional analysis of cellular responses to pore-forming toxins (PFTs). PFTs are uniquely important bacterial virulence factors, comprising the single largest class of bacterial protein toxins and being important for the pathogenesis in humans of many Gram positive and Gram negative bacteria. Their mode of action is deceptively simple, poking holes in the plasma membrane of cells. The scattered studies to date of PFT-host cell interactions indicate a handful of genes are involved in cellular defenses to PFTs. How many genes are involved in cellular defenses against PFTs and how cellular defenses are coordinated are unknown. To address these questions, we pe…

Activation induced by pore-forming bacterial toxins

Phobalysin, a Small β-Pore-Forming Toxin of Photobacterium damselae subsp. damselae

ABSTRACT Photobacterium damselae subsp. damselae , an important pathogen of marine animals, may also cause septicemia or hyperaggressive necrotizing fasciitis in humans. We previously showed that hemolysin genes are critical for virulence of this organism in mice and fish. In the present study, we characterized the hlyA gene product, a putative small β-pore-forming toxin, and termed it phobalysin P (PhlyP), for “photobacterial lysin encoded on a plasmid.” PhlyP formed stable oligomers and small membrane pores, causing efflux of K + , with no significant leakage of lactate dehydrogenase but entry of vital dyes. The latter feature distinguished PhlyP from the related Vibrio cholerae cytolysin…

eIF2α confers cellular tolerance to S. aureus α-toxin

We report on the role of conserved stress-response pathways for cellular tolerance to a pore forming toxin. First, we observed that small molecular weight inhibitors including of eIF2α-phosphatase, jun-N-terminal kinase (JNK), and PI3-kinase sensitized normal mouse embryonal fibroblasts (MEFs) to the small pore forming S. aureus α-toxin. Sensitization depended on expression of mADAM10, the murine ortholog of a proposed high-affinity receptor for α-toxin in human cells. Similarly, eIF2α (S51A/S51A) MEFs, which harbor an Ala knock-in mutation at the regulated Ser51 phosphorylation site of eukaryotic translation initiation factor 2α, were hyper-sensitive to α-toxin. Inhibition of translation w…

A subunit of eukaryotic translation initiation factor 2α-phosphatase (CreP/PPP1R15B) regulates membrane traffic.

The constitutive reverter of eIF2α phosphorylation (CReP)/PPP1r15B targets the catalytic subunit of protein phosphatase 1 (PP1c) to phosphorylated eIF2α (p-eIF2α) to promote its dephosphorylation and translation initiation. Here, we report a novel role and mode of action of CReP. We found that CReP regulates uptake of the pore-forming Staphylococcus aureus α-toxin by epithelial cells. This function was independent of PP1c and translation, although p-eIF2α was involved. The latter accumulated at sites of toxin attack and appeared conjointly with α-toxin in early endosomes. CReP localized to membranes, interacted with phosphomimetic eIF2α, and, upon overexpression, induced and decorated a pop…

GyrA sequence-based typing of Legionella.

Comparative sequence analysis of a 423-bp segment of the gyrA gene including a region homologous to the quinolone resistance-determining region (QRDR) of other species was evaluated as a novel typing method for Legionella strains. The study was performed with 29 reference strains representing 11 different Legionella species, with various serogroups, and with 13 clinical isolates of L. pneumophila. Pulsed-field gel electrophoresis and serotyping were employed for comparison of the clinical isolates. QRDR sequencing proved to be a highly discriminative tool for typing Legionellae, and permitted identification of species, serogroups and even different strains within serogroup 1. None of the is…

Interferons increase cell resistance to Staphylococcal alpha-toxin.

ABSTRACTMany bacterial pathogens, includingStaphylococcus aureus, use a variety of pore-forming toxins as important virulence factors. Staphylococcal alpha-toxin, a prototype β-barrel pore-forming toxin, triggers the release of proinflammatory mediators and induces primarily necrotic death in susceptible cells. However, whether host factors released in response to staphylococcal infections may increase cell resistance to alpha-toxin is not known. Here we show that prior exposure to interferons (IFNs) prevents alpha-toxin-induced membrane permeabilization, the depletion of ATP, and cell death. Moreover, pretreatment with IFN-α decreases alpha-toxin-induced secretion of interleukin 1β (IL-1β)…

A case of endocarditis with vasculitis due to Actinobacillus actinomycetemcomitans: a 16S rDNA signature for distinction from related organisms.

Atherogenic properties of enzymatically degraded LDL: selective induction of MCP-1 and cytotoxic effects on human macrophages.

Abstract —The mechanisms underlying the selective accumulation of macrophages in early atherosclerotic lesions are poorly understood but are likely to be related to specific properties of altered low density lipoprotein (LDL) deposited in the subendothelium. Enzymatic, nonoxidative degradation of LDL converts the lipoprotein to a potentially atherogenic moiety, enzymatically altered LDL (E-LDL), which activates complement and is rapidly taken up by human macrophages via a scavenger receptor–dependent pathway. Immunohistological evidence indicates that E-LDL is present in an extracellular location in the early lesion. We report that E-LDL causes massive release of monocyte chemotactic prote…

Isolation of Abiotrophia adiacens from a brain abscess which developed in a patient after neurosurgery.

ABSTRACT We report the case of a patient who developed a large brain abscess after neurosurgery. Cerebrospinal fluid from the abscess drainage yielded Abiotrophia adiacens -specific PCR products and microorganisms that were identified by conventional microbiological methods and by 16S ribosomal DNA analysis as Abiotrophia adiacens , which was formerly classified as a member of nutritionally variant streptococci.

Caspase-2 is an initiator caspase responsible for pore-forming toxin-mediated apoptosis

Bacterial pathogens modulate host cell apoptosis to establish a successful infection. Pore-forming toxins (PFTs) secreted by pathogenic bacteria are major virulence factors and have been shown to induce various forms of cell death in infected cells. Here we demonstrate that the highly conserved caspase-2 is required for PFT-mediated apoptosis. Despite being the second mammalian caspase to be identified, the role of caspase-2 during apoptosis remains enigmatic. We show that caspase-2 functions as an initiator caspase during Staphylococcus aureus alpha-toxin- and Aeromonas aerolysin-mediated apoptosis in epithelial cells. Downregulation of caspase-2 leads to a strong inhibition of PFT-mediate…

Pore-forming Staphylococcus aureus alpha-toxin triggers epidermal growth factor receptor-dependent proliferation.

Staphylococcal alpha-toxin is an archetypal killer protein that homo-oligomerizes in target cells to create small transmembrane pores. The membrane-perforating beta-barrel motif is a conserved attack element of cytolysins of Gram-positive and Gram-negative bacteria. Following the recognition that nucleated cells can survive membrane permeabilization, a profile of abundant transcripts was obtained in transiently perforated keratinocytes. Several immediate early genes were found to be upregulated, reminiscent of the cellular response to growth factors. Cell cycle analyses revealed doubling of S + G2/M phase cells 26 h post toxin treatment. Determination of cell counts uncovered that after an …

Vital dyes and virtual deaths.

Dear Editor, In 2009 and 2012, Cell Death & Differentiation has published recommendations of the Nomenclature Committee on Cell Death (NCCD) for the Classification of cell death and Molecular definitions of cell death subroutines, respectively. Under the subtitle When is a cell ‘dead'?, and in an accompanying table entitled Molecular or morphological criteria to define dead cells, the 2009 paper featured a set of three criteria; the fulfillment of any single one is considered to indicate cell death.1 The first criterion in this list is Loss of plasma membrane integrity, and parameters commonly used to assess integrity of the plasma membrane were mentioned, namely exclusion from cells of vit…

Embryonic neural cell adhesion molecules on human natural killer cells

The neural cell adhesion molecules (NCAM) are surface glycoproteins that were first described in brain tissue. NCAM mediate adhesion in a variety of cell-cell interactions. In the present study we show that the so-called "embryonic" NCAM, i.e., the highly polysialylated forms of these proteins, are expressed on natural killer cells and some CD3+ cells in man. Homotypic binding of NCAM, believed to be of importance for cell-cell adhesion in neural tissues, appears not to be essential for NK cell-mediated killing. Yet, NCAM might be involved in NK cell migration, homing or related functions.

Staphylococcus aureus α-toxin: small pore, large consequences

Abstract The small β-pore-forming α-toxin, also termed α-hemolysin or Hla is considered to be an important virulence factor of Staphylococcus aureus. Perforation of the plasma membrane (PM) by Hla leads to uncontrolled flux of ions and water. Already a small number of toxin pores seems to be sufficient to induce complex cellular responses, many of which depend on the efflux of potassium. In this article, we discuss the implications of secondary membrane lesions, for example, by endogenous channels, for Hla-mediated toxicity, for calcium-influx and membrane repair. Activation of purinergic receptors has been proposed to be a major contributor to the lytic effects of various pore forming prot…

Differential role of p38 mitogen activated protein kinase for cellular recovery from attack by pore-forming S. aureus alpha-toxin or streptolysin O.

Following the observation that cells are able to recover from membrane lesions incurred by Staphylococcus aureus alpha-toxin and streptolysin O (SLO), we investigated the role of p38 in this process. p38 phosphorylation occurred in response to attack by both toxins, commencing within minutes after toxin treatment and waning after several hours. While SLO reportedly activates p38 via ASK1 and ROS, we show that this pathway does not play a major role for p38 induction in alpha-toxin-treated cells. Strikingly divergent effects of p38 blockade were noted depending on the toxin employed. In the case of alpha-toxin, inhibition of p38 within the time frame of its activation led to disruption of th…

Fatty acids liberated from low-density lipoprotein trigger endothelial apoptosis via mitogen-activated protein kinases.

Enzymatic modification of low-density lipoprotein (LDL) as it probably occurs in the arterial intima drastically increases its cytotoxicity, which could be relevant for the progression of atherosclerotic lesions. LDL was treated with a protease and cholesterylesterase to generate a derivative similar to lesional LDL, with a high content of free cholesterol and fatty acids. Exposure of endothelial cells to the enzymatically modified lipoprotein (E-LDL), but not to native or oxidized LDL, resulted in programmed cell death. Apoptosis was triggered by apoptosis signal-regulating kinase 1 dependent phosphorylation of p38. Depletion and reconstitution experiments identified free fatty acids (FFA)…

Simultaneous identification of campylobacters and prediction of quinolone resistance by comparative sequence analysis.

Comparative sequence analysis of a 30-bp segment in the quinolone resistance-determining region of campylobacters not only allows for the detection of base changes associated with resistance but also is a powerful tool for species identification based on silent mutations.

Staphylococcus aureus α-Toxin’s Close Contacts Ensure the Kill

The membrane pore-forming α-toxin is an important virulence factor of Staphylococcus aureus. Target cells can remove pores from their surface, but recent work shows that α-toxin may undermine this self-defense by clinging to epithelial cell junctions. The findings could lead to the development of novel remedies against S. aureus infections.

A novel SP-1 site in the human interleukin-1β promoter confers preferential transcriptional activity in keratinocytes

To investigate the mechanisms of transcriptional activation of interleukin-1beta (IL-1beta) in non-monocytic cells, we constructed a series of reporter plasmids with the bacterial chloramphenicol acetyltransferase gene linked to various parts of the human IL-1beta promoter and performed transient transfection experiments. We identified a promoter segment that activates transcription most efficiently in keratinocytes. Electrophoretic mobility shift assays (EMSA) with a 43-mer oligonucleotide derived from the functionally identified cis-acting element revealed specific complexes. By competition analysis with transcription factor consensus sequence oligonucleotides and by immunosupershift, tra…

Elimination of a bacterial pore-forming toxin by sequential endocytosis and exocytosis

Staphylococcus aureus alpha-toxin is the archetype of bacterial pore forming toxins and a key virulence factor secreted by the majority of clinical isolates of S. aureus. Toxin monomers bind to target cells and oligomerize to form small beta-barrel pores in the plasma membrane. Many nucleated cells are able to repair a limited number of lesions by unknown, calcium-independent mechanisms. Here we show that cells can internalize alpha-toxin, that uptake is essential for cellular survival, and that pore-complexes are not proteolytically degraded, but returned to the extracellular milieu in the context of exosome-like structures, which we term toxosomes.

Immunohistochemical localization of polysialic acid in tissue sections: differential binding to polynucleotides and DNA of a murine IgG and a human IgM monoclonal antibody.

For immunolocalization of alpha(2-8)-linked polysialic acid, which forms part of the neural cell adhesion molecule (N-CAM), two monoclonal antibodies, MAb735 and IgMNOV, were employed. Both antibodies have previously been shown to bind the extremely low immunogenic capsular polysaccharide of group B meningococci, which also consists of alpha(2-8) polysialic acid, but not to other, even closely related forms of polysialic acid. Despite the identical polysaccharide specificity of these two MAb, we observed marked differences of the staining pattern in tissue sections. We showed that these differences in immunostaining were due to the crossreactivity of IgMNOV with polynucleotides and DNA. MA…

Enzymatically modified nonoxidized low-density lipoprotein induces interleukin-8 in human endothelial cells: role of free fatty acids.

Background— Treatment of low-density lipoprotein (LDL) with a protease and cholesterolesterase transforms the lipoprotein to an entity that resembles lipoprotein particles in atherosclerotic lesions, which have a high content of free cholesterol, reflecting extensive de-esterification in the intima. Because de-esterification would occur beneath the endothelium, we examined the effects of enzymatically modified LDL (E-LDL) on cultured endothelial cells. Methods and Results— Incubation of endothelial cells with E-LDL provoked selective accumulation of interleukin (IL)-8 mRNA and production of the cytokine. Chemical analyses and depletion experiments indicated that the effect was caused by th…

Nuclear receptors modulate the interaction of Sp1 and GC-rich DNA via ternary complex formation

Binding sites for transcription factor Sp1have been implicated in the transcriptional regulation of several genes by hormones or vitamins, and here we show that a GC-rich element contributes to the retinoic acid response of the interleukin 1β promoter. To explain such observations, it has been proposed that nuclear receptors can interact with Sp1 bound to GC-rich DNA. However, evidence supporting this model has remained indirect. So far, nuclear receptors have not been detected in a complex with Sp1 and GC-rich DNA, and the expected ternary complexes in non-denaturing gels were not seen. In search for these missing links we found that nuclear receptors [retinoic acid receptor (RAR), thyroid…

Role of C-reactive protein in atherogenesis: can the apolipoprotein E knockout mouse provide the answer?

Objective—Human C-reactive protein (CRP) was reported to accelerate atherosclerotic lesion development in male but not in female apolipoprotein E (apoE) knockout mice. Here, mice expressing rabbit CRP (rbCRP) were crossbred onto apoE knockout animals, and the effect on atherogenesis was studied.Methods and Results—Hemolytic complement activity could not be detected in apoE knockout mice. Furthermore, in contrast to human complement, neither rabbit nor human CRP complexed to modified low-density lipoprotein–activated murine complement. At 52 weeks, rbCRP levels were similar in male and female transgenic animals. Serum cholesterol levels were equivalent in female animals irrespective of rbCRP…

Subcytocidal attack by staphylococcal alpha-toxin activates NF-kappaB and induces interleukin-8 production.

ABSTRACTFormation of transmembrane pores by staphylococcal alpha-toxin can provoke a spectrum of events depending on target cell species and toxin dose, and in certain cases, repair of the lesions has been observed. Here, we report that transcriptional processes are activated as a response of cells to low toxin doses. Exposure of monocytic (THP-1) or epithelial (ECV304) cells to 40 to 160 ng/ml alpha-toxin provoked a drop in cellular ATP level that was followed by secretion of substantial amounts of interleukin-8 (IL-8). Cells transfected with constructs comprising the proximal IL-8 promoter fused to luciferase or to green fluorescent protein cDNA exhibited enhanced reporter gene expression…

Clathrin- and Caveolin-Independent Entry of Human Papillomavirus Type 16—Involvement of Tetraspanin-Enriched Microdomains (TEMs)

BACKGROUND: Infectious entry of human papillomaviruses into their host cells is an important step in the viral life cycle. For cell binding these viruses use proteoglycans as initial attachment sites. Subsequent transfer to a secondary receptor molecule seems to be involved in virus uptake. Depending on the papillomavirus subtype, it has been reported that entry occurs by clathrin- or caveolin-mediated mechanisms. Regarding human papillomavirus type 16 (HPV16), the primary etiologic agent for development of cervical cancer, clathrin-mediated endocytosis was described as infectious entry pathway. METHODOLOGY/PRINCIPAL FINDINGS: Using immunofluorescence and infection studies we show in contra…

Unsaturated Fatty Acids Drive Disintegrin and Metalloproteinase (ADAM)-dependent Cell Adhesion, Proliferation, and Migration by Modulating Membrane Fluidity*

The disintegrin-metalloproteinases ADAM10 and ADAM17 mediate the release of several cell signaling molecules and cell adhesion molecules such as vascular endothelial cadherin or L-selectin affecting endothelial permeability and leukocyte transmigration. Dysregulation of ADAM activity may contribute to the pathogenesis of vascular diseases, but the mechanisms underlying the control of ADAM functions are still incompletely understood. Atherosclerosis is characterized by lipid plaque formation and local accumulation of unsaturated free fatty acids (FFA). Here, we show that unsaturated FFA increase ADAM-mediated substrate cleavage. We demonstrate that these alterations are not due to genuine ch…

Vascular Leakage in Severe Dengue Virus Infections: A Potential Role for the Nonstructural Viral Protein NS1 and Complement

Background Vascular leakage and shock are the major causes of death in patients with dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Thirty years ago, complement activation was proposed to be a key underlying event, but the cause of complement activation has remained unknown. Methods The major nonstructural dengue virus (DV) protein NS1 was tested for its capacity to activate human complement in its membrane-associated and soluble forms. Plasma samples from 163 patients with DV infection and from 19 patients with other febrile illnesses were prospectively analyzed for viral load and for levels of NS1 and complement-activation products. Blood and pleural fluids from 9 patient…

Possible protective role for C-reactive protein in atherogenesis: complement activation by modified lipoproteins halts before detrimental terminal sequence.

Background—Previous work indicated that enzymatically remodeled LDL (E-LDL) might activate complement in atherosclerotic lesions via a C-reactive protein (CRP)–dependent and CRP-independent pathway. We sought to substantiate this contention and determine whether both pathways drive the sequence to completion.Methods and Results—E-LDL was prepared by sequential treatment of LDL with a protease and cholesteryl esterase. Trypsin, proteinase K, cathepsin H, or plasmin was used with similar results. Functional tests were used to assess total complement hemolytic activity, and immunoassays were used to demonstrate C3 cleavage and to quantify C3a, C4a, C5a, and C5b-9. E-LDL preparations activated …

Enzymatically hydrolyzed low-density lipoprotein modulates inflammatory responses in endothelial cells

SummaryThere is evidence that low-density lipoprotein (LDL) is modified by hydrolytic enzymes,and that the product (E-LDL) induces selective production of interleukin 8 (IL-8) in endothelial cells. Since nuclear factor-kappaB (NF-κB) is a major regulator of IL-8 transcription, we studied its activation in endothelial cells treated with E-LDL. Unexpectedly,the modified lipoprotein not only failed to activate NF-κB, but completely blocked its activation by tumour necrosis factor-alpha (TNF-α) in EA.hy926-cells, as assessed by electrophoretic mobility shift assays and immunofluorescence. Inhibition occurred upstream of NF-κB translocation, as inhibitor of NF-κB- (IκB)-phosphorylation was suppr…

Case of fatal systemic infection with an Aureobacterium sp.: identification of isolate by 16S rRNA gene analysis

The case of a 75-year-old man who succumbed to a disseminated infection most likely caused by a species of the genus Aureobacterium is reported. Identification of the isolate was achieved by comparative 16S rRNA gene analysis. Aureobacteria are commonly found in the environment. However, only recently have they been recognized as a cause of infections including septicemia and soft tissue infections. To our knowledge, this is the first documentation of a fatal infection caused by an Aureobacterium sp.

Dissecting the role of ADAM10 as a mediator of Staphylococcus aureus α-toxin action

Staphylococcus aureus is a leading cause of bacterial infections in humans, including life-threatening diseases such as pneumonia and sepsis. Its small membrane-pore-forming α-toxin is considered an important virulence factor. By destroying cell–cell contacts through cleavage of cadherins, the metalloproteinase ADAM10 (a disintegrin and metalloproteinase 10) critically contributes to α-toxin-dependent pathology of experimental S. aureus infections in mice. Moreover, ADAM10 was proposed to be a receptor for α-toxin. However, it is unclear whether the catalytic activity or specific domains of ADAM10 are involved in mediating binding and/or subsequent cytotoxicity of α-toxin. Also, it is not k…

Molecular basis of early epithelial response to streptococcal exotoxin: role of STIM1 and Orai1 proteins

Streptolysin O (SLO) is a cholesterol-dependent cytolysin (CDC) from Streptococcus pyogenes. SLO induces diverse types of Ca(2+) signalling in host cells which play a key role in membrane repair and cell fate determination. The mechanisms behind SLO-induced Ca(2+) signalling remain poorly understood. Here, we show that in NCI-H441 cells, wild-type SLO as well as non-pore-forming mutant induces long-lasting intracellular Ca(2+) oscillations via IP(3) -mediated depletion of intracellular stores and activation of store-operated Ca(2+) (SOC) entry. SLO-induced activation of SOC entry was confirmed by Ca(2+) add-back experiments, pharmacologically and by overexpression as well as silencing of ST…

Enzymatic modification of low-density lipoprotein in the arterial wall: a new role for plasmin and matrix metalloproteinases in atherogenesis.

Objective— Functionally interactive proteases of the plasminogen/plasmin and the matrix metalloproteinase (MMP) system degrade and reorganize the extracellular matrix of the vessel wall in atherosclerosis. Here we investigated whether such proteases are able to confer atherogenic properties onto low density lipoprotein by nonoxidative modification. Methods and Results— Similar to the recently described enzymatically-modified low-density lipoprotein (E-LDL), native LDL exposed to plasmin or matrix MMP-2 or MMP-9 and cholesterylester-hydrolase (CEH) showed extensive deesterification, with ratios of free cholesterol to total cholesterol rising to 0.8 compared with 0.2 in native LDL. When the …

Resealing of large transmembrane pores produced by streptolysin O in nucleated cells is accompanied by NF‐κB activation and downstream events

Streptolysin O (SLO), archetype of a cholesterol-binding bacterial cytolysin, forms large pores in the plasma membrane of mammalian cells. We have recently reported that when a limited number of pores are generated in a cell, they can be sealed in a Ca++-dependent process. Here, we show that resealing is followed by the release of IL-6 and IL-8 from keratinocytes and from endothelial cells, both relevant targets for SLO attack. Production of cytokines by these cells was preceded by activation of transcription factor nuclear factor kappaB, which thus emerges as a common denominator of stress responses to various pore-forming agents, including alpha-toxin of Staphylococcus aureus and compleme…

Enzymatically modified LDL induces cathepsin H in human monocytes: potential relevance in early atherogenesis.

Objective—Modification with proteases and cholesterylesterase transforms LDL to a moiety that resembles lipoproteins isolated from atherosclerotic lesions and possesses atherogenic properties. To identify changes in monocyte-derived foam cells laden with enzymatically modified LDL (E-LDL), we compared patterns of the most abundant transcripts in these cells after incubation with LDL or E-LDL.Methods and Results—Serial analyses of gene expression (SAGE) libraries were constructed from human monocytes after treatment with LDL or E-LDL. Several tags were differentially expressed in LDL-treated versus E-LDL–treated cells, whereby marked selective induction by E-LDL of cathepsin H was conspicuou…

Helicobacter sp. strain Mainz isolated from an AIDS patient with septic arthritis: case report and nonradioactive analysis of 16S rRNA sequence

A campylobacter-like organism was isolated from an effusion of the left knee joint of an AIDS patient 2 weeks after bacteremia with a morphologically identical organism. Amplified genomic 16S rRNA sequences were analyzed by a nonradioactive blotting technique. The closest match was found with Helicobacter fenelliae (97.7% homology). Sequence data and phenotype suggest that the isolate may represent a so far unrecognized species of the genus Helicobacter.

Pore-forming toxins activate MAPK p38 by causing loss of cellular potassium.

Mitogen activated protein kinase (MAPK) p38 has emerged as a survival protein in cells that are attacked by bacterial toxins forming small membrane pores. Activation of p38 by pore forming toxins (PFT) has been attributed to osmotic stress, but here we show that loss of K+ is likely to be the critical parameter. Several lines of evidence support this conclusion: first, osmoprotection did not prevent p38-phosphorylation in alpha-toxin-loaded cells. Second, treatment of cells with a K+ ionophore, or simple incubation in K+-free medium sufficed to cause robust p38-phosphorylation. Third, media containing high [K+] prevented p38-activation by Staphylococcus aureus alpha-toxin, Vibrio cholerae c…

Potassium regulates IL-1 beta processing via calcium-independent phospholipase A2.

Abstract We report that potassium leakage from cells leads to activation of the Ca2+-independent phospholipase A2 (iPLA2), and the latter plays a pivotal role in regulating the cleavage of pro-IL-1β by the IL-converting enzyme caspase-1 in human monocytes. K+ efflux led to increases of cellular levels of glycerophosphocholine, an unambiguous indicator of phospholipase A2 activation. Both maturation of IL-1β and formation of glycerophosphocholine were blocked by bromoenol lactone, the specific iPLA2 inhibitor. Bromoenol lactone-dependent inhibition of IL-1β processing was not due to perturbation of the export machinery for pro-IL-1β and IL-1β or to caspase-1 suppression. Conspicuously, activ…

Repair of a Bacterial Small β-Barrel Toxin Pore Depends on Channel Width

ABSTRACT Membrane repair emerges as an innate defense protecting target cells against bacterial pore-forming toxins. Here, we report the first paradigm of Ca2+-dependent repair following attack by a small β-pore-forming toxin, namely, plasmid-encoded phobalysin of Photobacterium damselae subsp. damselae. In striking contrast, Vibrio cholerae cytolysin, the closest ortholog of phobalysin, subverted repair. Mutational analysis uncovered a role of channel width in toxicity and repair. Thus, the replacement of serine at phobalysin´s presumed channel narrow point with the bulkier tryptophan, the corresponding residue in Vibrio cholerae cytolysin (W318), modulated Ca2+ influx, lysosomal exocytosi…

Chromosome-Encoded Hemolysin, Phospholipase, and Collagenase in Plasmidless Isolates of Photobacterium damselae subsp. damselae Contribute to Virulence for Fish.

ABSTRACT Photobacterium damselae subsp. damselae is a pathogen of marine animals, including fish of importance in aquaculture. The virulence plasmid pPHDD1, characteristic of highly hemolytic isolates, encodes the hemolysins damselysin (Dly) and phobalysin (PhlyP). Strains lacking pPHDD1 constitute the vast majority of the isolates from fish outbreaks, but genetic studies to identify virulence factors in plasmidless strains are scarce. Here, we show that the chromosome I-encoded hemolysin PhlyC plays roles in virulence and cell toxicity in pPHDD1-negative isolates of this pathogen. By combining the analyses of whole genomes and of gene deletion mutants, we identified two hitherto uncharacte…