Genome-Wide CRISPR-Cas9 Screen Does Not Identify Host Factors Modulating Streptococcus agalactiae ß-Hemolysin/Cytolysin-Induced Cell Death.

Pore-forming toxins (PFTs) are commonly produced by pathogenic bacteria, and understanding them is key to the development of virulence-targeted therapies. Streptococcus agalactiae, or group B Streptococcus (GBS), produces several factors that enhance its pathogenicity, including the PFT ß-hemolysin/cytolysin (ßhc). Little is understood about the cellular factors involved in ßhc pore formation. We conducted a whole-genome CRISPR-Cas9 forward genetic screen to identify host genes that might contribute to ßhc pore formation and cell death. While the screen identified the established receptor, CD59, in control experiments using the toxin intermedilysin (ILY), no clear candidate genes were identified that were required for ßhc-mediated lethality. Of the top targets from the screen, two genes involved in membrane remodeling and repair represented candidates that might modulate the kinetics of ßhc-induced cell death. Upon attempted validation of the results using monoclonal cell lines with targeted disruption of these genes, no effect on ßhc-mediated cell lysis was observed. The CRISPR-Cas9 screen results are consistent with the hypothesis that ßhc does not require a single nonessential host factor to mediate target cell death. IMPORTANCE CRISPR-Cas9 forward genetic screens have been used to identify host cell targets required by bacterial toxins. They have been used successfully to both verify known targets and elucidate novel host factors required by toxins. Here, we show that this approach fails to identify host factors required for cell death due to ßhc, a toxin required for GBS virulence. These data suggest that ßhc may not require a host cell receptor for toxin function or may require a host receptor that is an essential gene and would not be identified using this screening strategy.

Role of perforin secretion from CD8+ T-cells in neuronal cytotoxicity in multiple sclerosis.

OBJECTIVES: Multiple sclerosis (MS) is the most prevalent autoimmune disease of the central nervous system, and is characterized by inflammation and myelin damage. The immune system initiates the autoimmune response, although the mechanisms of neuronal damage have not been elucidated. The purpose of the present study was to investigate autoreactive CD4+ and CD8+ T lymphocytes, in conjunction with other inflammatory cells and cytokines in active MS lesions. METHODS: EAE animal models was established by plantar injections of MBP (200 µg per rat). Purified CD4+ or CD8+ T-cells were isolated from heparinized peripheral blood (EAE animals and control animals) via negative selection. To examine effects of presence of autoreactive CD4+ and CD8+ T lymphocytes, we carried out ELISA, Western blot analysis and TUNEL. In addition, we examined the direct effects of various factors on neuronal cell death using MTT assay. RESULTS: The data revealed that CD8+ T-cells were more toxic to neurons compared to CD4+ T-cells, in both the MBP and EAE conditions. Bax was greater increased when neurons were co-cultured with CD8+ T-cells in the MBP group. There is a significant increase in IL-17 secretion by CD4+ T-cells in both the MBP group and EAE group. Neuronal viability were affected by Perforin (1.5 µg/mL). CONCLUSION: The present study extends previous research by demonstrating the role of CD8+ T-cells in MS and supports perforin secretion by CD8+ T-cells as a potential therapeutic factor. Furthermore, we determined that CD4+ T-cells can enhance CD8+ T-cell neuronal cytotoxicity via induction of intense inflammation.

Cardiovascular effects of Sp-CTx, a cytolysin from the scorpionfish (Scorpaena plumieri) venom.

Fish venom cytolysins are multifunctional proteins that in addition to their cytolytic/hemolytic effects display neurotoxic, cardiotoxic and inflammatory activities, being described as "protein lethal factors". A pore-forming cytolysin called Sp-CTx (Scorpaena plumieriCytolytic Toxin) has been recently purified from the venom of the scorpionfish Scorpaena plumieri. It is a glycoprotein with dimeric constitution, comprising subunits of approximately 65 kDa. Previous studies have revealed that this toxin has a vasorelaxant activity that appears to involve the L-arginine-nitric oxide synthase pathway; however its cardiovascular effects have not been fully comprehended. The present study examined the cardiovascular effects of Sp-CTx in vivo and in vitro. In anesthetized rats Sp-CTx (70 µg/kg i.v) produced a biphasic response which consisted of an initial systolic and diastolic pressure increase followed by a sustained decrease of these parameters and the heart rate. In isolated rats hearts Sp-CTx (10(-9) to 5 × 10(-6) M) produced concentration-dependent and transient ventricular positive inotropic effect and vasoconstriction response on coronary bed. In papillary muscle, Sp-CTx (10(-7) M) also produced an increase in contractile isometric force, which was attenuated by the catecholamine releasing agent tyramine (100 µM) and the ß-adrenergic antagonist propranolol (10 µM). On isolated ventricular cardiomyocytes Sp-CTx (1 nM) increased the L-type Ca(2+) current density. The results show that Sp-CTx induces disorders in the cardiovascular system through increase of sarcolemmal calcium influx, which in turn is partially caused by the release of endogenous noradrenaline.

Signaling beyond Punching Holes: Modulation of Cellular Responses by Vibrio cholerae Cytolysin.

Pore-forming toxins (PFTs) are a distinct class of membrane-damaging cytolytic proteins that contribute significantly towards the virulence processes employed by various pathogenic bacteria. Vibrio cholerae cytolysin (VCC) is a prominent member of the beta-barrel PFT (beta-PFT) family. It is secreted by most of the pathogenic strains of the intestinal pathogen V. cholerae. Owing to its potent membrane-damaging cell-killing activity, VCC is believed to play critical roles in V. cholerae pathogenesis, particularly in those strains that lack the cholera toxin. Large numbers of studies have explored the mechanistic basis of the cell-killing activity of VCC. Consistent with the beta-PFT mode of action, VCC has been shown to act on the target cells by forming transmembrane oligomeric beta-barrel pores, thereby leading to permeabilization of the target cell membranes. Apart from the pore-formation-induced direct cell-killing action, VCC exhibits the potential to initiate a plethora of signal transduction pathways that may lead to apoptosis, or may act to enhance the cell survival/activation responses, depending on the type of target cells. In this review, we will present a concise view of our current understanding regarding the multiple aspects of these cellular responses, and their underlying signaling mechanisms, evoked by VCC.

Antigen dependently activated cluster of differentiation 8-positive T cells cause perforin-mediated neurotoxicity in experimental stroke.

Neuroinflammation plays a key role in secondary brain damage after stroke. Although deleterious effects of proinflammatory cytokines are well characterized, direct cytotoxic effects of invading immune cells on the ischemic brain and the importance of their antigen-dependent activation are essentially unknown. Here we examined the effects of adaptive and innate immune cells-cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells-that share the direct perforin-mediated cytotoxic pathway on outcome after cerebral ischemia in mice. Although CTLs and NK cells both invaded the ischemic brain, only brain-infiltrating CTLs but not NK cells were more activated than their splenic counterparts. Depletion of CTLs decreased infarct volumes and behavioral deficit in two ischemia models, whereas NK cell depletion had no effect. Correspondingly, adoptive CTL transfer from wild-type into Rag1 knock-out mice increased infarct size. Adoptive CTL transfer from perforin knock-out or interferon-γ knock-out mice into Rag1 knock-out mice revealed that CTL neurotoxicity was mediated by perforin. Accordingly, CTLs isolated from wild-type or interferon-γ knock-out but not from perforin knock-out mice induced neuronal cell death in vitro. CTLs derived from ovalbumin-specific T-cell receptor transgenic mice were not activated and infiltrated less into the ischemic brain compared with wild-type CTLs. Their transfer did not increase the infarct size of Rag1 knock-out mice, indicating antigen-dependent activation as an essential component of CTL neurotoxicity. Our findings underscore the importance of antigen-dependent, direct cytotoxic immune responses in stroke and suggest modulation of CTLs and their effector pathways as a potential new strategy for stroke therapy.

Targeted deletion of the ara operon of Salmonella typhimurium enhances L-arabinose accumulation and drives PBAD-promoted expression of anti-cancer toxins and imaging agents.

Tumor-specific expression of antitumor drugs can be achieved using attenuated Salmonella typhimurium harboring the PBAD promoter, which is induced by L-arabinose. However, L-arabinose does not accumulate because it is metabolized to D-xylulose-5-P by enzymes encoded by the ara operon in Salmonellae. To address this problem, we developed an engineered strain of S. typhimurium in which the ara operon is deleted. Linear DNA transformation was performed using λ red recombinase to exchange the ara operon with linear DNA carrying an antibiotic-resistance gene with homology to regions adjacent to the ara operon. The ara operon-deleted strain and its parental strain were transformed with a plasmid encoding Renilla luciferase variant 8 (RLuc8) or cytolysin A (clyA) under the control of the PBAD promoter. Luciferase assays demonstrated that RLuc8 expression was 49-fold higher in the ara operon-deleted S. typhimurium than in the parental strain after the addition of L-arabinose. In vivo bioluminescence imaging showed that the tumor tissue targeted by the ara operon-deleted Salmonella had a stronger imaging signal (~30-fold) than that targeted by the parental strain. Mice with murine colon cancer (CT26) that had been injected with the ara operon-deleted S. typhimurium expressing clyA showed significant tumor suppression. The present report demonstrates that deletion of the ara operon of S. typhimurium enhances L-arabinose accumulation and thereby drives PBAD-promoted expression of cytotoxic agents and imaging agents. This is a promising approach for tumor therapy and imaging.

Human-specific bacterial pore-forming toxins induce programmed necrosis in erythrocytes.

UNLABELLED: A subgroup of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins (PFTs) has an unusually narrow host range due to a requirement for binding to human CD59 (hCD59), a glycosylphosphatidylinositol (GPI)-linked complement regulatory molecule. hCD59-specific CDCs are produced by several organisms that inhabit human mucosal surfaces and can act as pathogens, including Gardnerella vaginalis and Streptococcus intermedius. The consequences and potential selective advantages of such PFT host limitation have remained unknown. Here, we demonstrate that, in addition to species restriction, PFT ligation of hCD59 triggers a previously unrecognized pathway for programmed necrosis in primary erythrocytes (red blood cells [RBCs]) from humans and transgenic mice expressing hCD59. Because they lack nuclei and mitochondria, RBCs have typically been thought to possess limited capacity to undergo programmed cell death. RBC programmed necrosis shares key molecular factors with nucleated cell necroptosis, including dependence on Fas/FasL signaling and RIP1 phosphorylation, necrosome assembly, and restriction by caspase-8. Death due to programmed necrosis in RBCs is executed by acid sphingomyelinase-dependent ceramide formation, NADPH oxidase- and iron-dependent reactive oxygen species formation, and glycolytic formation of advanced glycation end products. Bacterial PFTs that are hCD59 independent do not induce RBC programmed necrosis. RBC programmed necrosis is biochemically distinct from eryptosis, the only other known programmed cell death pathway in mature RBCs. Importantly, RBC programmed necrosis enhances the growth of PFT-producing pathogens during exposure to primary RBCs, consistent with a role for such signaling in microbial growth and pathogenesis. IMPORTANCE: In this work, we provide the first description of a new form of programmed cell death in erythrocytes (RBCs) that occurs as a consequence of cellular attack by human-specific bacterial toxins. By defining a new RBC death pathway that shares important components with necroptosis, a programmed necrosis module that occurs in nucleated cells, these findings expand our understanding of RBC biology and RBC-pathogen interactions. In addition, our work provides a link between cholesterol-dependent cytolysin (CDC) host restriction and promotion of bacterial growth in the presence of RBCs, which may provide a selective advantage to human-associated bacterial strains that elaborate such toxins and a potential explanation for the narrowing of host range observed in this toxin family.

CD40 signaling in CD8+CD40+ T cells turns on contra-T regulatory cell functions.

CD4(+) regulatory T cells (Treg cells) mediate immunosuppression, whereas CD8(+) T cells confer resistance in many diseases. It is unknown whether CD8(+) T cells confer protection by antagonizing the Treg cells. Using a model of stage-specific immune responses against Leishmania donovani infection in susceptible BALB/c mice, we report that CD3(+)CD8(+)CD40(+) T cells executed CD40-dependent cytotoxicity on CD3(+)CD4(+)CD127(dim)GITR(+)CD25(+) Treg cells during the initial phase of the infection but were later apoptosed by IL-10. CD40 signaled through Ras, PI3K, and protein kinase C, resulting in p38MAPK- or ERK-1/2-independent, but NF-kappaB-dependent, induction of the cytotoxic mediators granzyme and perforin. Adoptive transfer of CD3(+)CD8(+)CD40(+) T cells reduced the L. donovani infection in BALB/c mice. These results identify CD3(+)CD8(+)CD40(+) T cells as the contra-Treg cells and imply a novel immunotherapeutic principle.

Role of calcium/calmodulin signaling pathway in Vibrio vulnificus cytolysin-induced hyperpermeability.

Endothelial hyperpermeability, a hallmark of septicemia, is induced by stress fiber formation, which is primarily regulated by the calcium/calmodulin signaling pathway in endothelial cells. We previously reported that trifluoperazine, a calcium/calmodulin antagonist, blocks Vibrio vulnificus cytolysin (VVC) -induced lethality at in vivo animal model. The object of this study was therefore to examine whether VVC induces stress fiber formation through calcium/calmodulin signaling in endothelial cells. Here, we monitored calcium-influx after treatment of VVC using confocal microscopy in CPAE cells, pulmonary endothelial cell line. Interestingly, we found that VVC-induced dose-dependently increases of [Ca(2+)](i) in CPAE cells. Moreover, VVC-induced stress fiber formation as well as phosphorylation of myosin light chain (MLC) in a dose- and time-dependent manner, which was completely blocked by trifluoperazine. These results suggest that the calcium/calmodulin signaling pathway plays a pivotal role in VVC-induced hyperpermeability.

Vibrio vulnificus cytolysin induces apoptosis in HUVEC, SGC-7901 and SMMC-7721 cells via caspase-9/3-dependent pathway.

Vibrio vulnificus cytolysin (VVC) is known to be a pore-forming toxin which shows cytotoxicity for mammalian cells in culture and induces apoptosis in endothelial cells. In order to determine whether VVC induces apoptosis in vascular endothelial cells and tumor cells, the cytotoxicity induced by recombinant VVC (rVVC) and its potential mechanism in HUVEC, SGC-7901 and SMMC-7721 cells were investigated. Our study demonstrated that rVVC induced the release of intracellular K(+) from all the target cells, yet lactate dehydrogenase was not released by rVVC. It indicates that osmotic lysis might not contribute to the cytolysin-induced cytotoxicity. The study also demonstrated that rVVC induced apoptosis in HUVEC, SGC-7901 and SMMC-7721 cells in time- and dosage-dependent manners, which was associated with the activation of caspase-9 and -3, but not caspase-8. During the apoptotic process of the target cells, rVVC labeled with FITC was monitored to attach initially to the surface of the cells and entered the cytoplasma subsequently. These findings suggest that VVC may be not only a pore-forming toxin, but also a transmembrane toxin with powerful ability to induce apoptosis in human vascular endothelial cells and tumor cells.