Effects of phthalates on marine organisms: cytotoxicity and genotoxicity of mono-(2-ethylhexyl)-phthalate (MEHP) on European sea bass (Dicentrarchus labrax) embryonic cell line.

INTRODUCTION: Mono-(2-ethylhexyl) phthalate (MEHP) represents a toxicological risk for marine organisms due to its widespread presence in aquatic environments. METHODS: MEHP effects on cell viability, cell death and genotoxicity were investigated on the DLEC cell line, derived from early embryos of the European sea bass Dicentrarchus labrax L. RESULTS: A dose-dependent cytotoxic effect, with no induction of necrotic process, except at its highest concentration, was observed. Moreover, chromosomal instability was detected, both in binucleated and mononucleated cells, coupled with a minor inhibition of cell proliferation, whereas genomic instability was not revealed. To our knowledge, the overall results suggest the first evidence of a possible aneugenic effect of this compound in the DLEC cell line, that is the induction of chromosomal loss events without the induction of primary DNA damage. CONCLUSIONS: MEHP should be considered more harmful than its parent compound DEHP, because it induces genomic instability in the DLEC cell line without triggering cell death.

In vitro comparative cytotoxic assessment of pristine and carboxylic functionalized multiwalled carbon nanotubes on LN18 cells.

Multiwalled carbon nanotubes (MWCNTs) have been used in biomedical applications due to their ability to enter the cells. Carboxylic functionalization of MWCNT (MWCNT-COOH) is used to mitigate the toxicity of MWCNTs. Our study focuses on comparing the toxicity of MWCNT and MWCNT-COOH on the neuronal cells, LN18. Concentrations of 5, 10, 20, and 40 µg ml-1 were used for the study, and cytotoxicity was determined at 0, 1, 3, 6, 12, 24, and 48 h of incubation. Cell viability was assessed by Trypan Blue, MTT, and Live dead cell assays, and the oxidative stress produced was determined by reactive oxygen species (ROS) and Lipid peroxidation assays. MWCNT-COOH showed higher cell viability than MWCNT for 20 and 40 µg ml-1 at 24 and 48 h. This was also visually observed in the live dead cell imaging. However, at 48 h, the morphology of the cells appeared more stretched for all the concentrations of MWCNT and MWCNT-COOH in comparison to the control. A significant amount of ROS production can also be observed at the same concentration and time. Viability and oxidative stress results together revealed that MWCNT-COOH is less toxic when compared to MWCNT at longer incubation periods and higher concentrations. However, otherwise, the effect of both are comparable. A concentration of 5-10 µg ml-1 is ideal while using MWCNT and MWCNT-COOH as the toxicity is negligible. These findings can further be extended to various functionalizations of MWCNT for wider applications.

Diselenide-crosslinked carboxymethyl chitosan nanoparticles for doxorubicin delivery: Preparation and in vivo evaluation.

In this paper, we report a simple approach to fabricate diselenide-crosslinked carboxymethyl chitosan nanoparticles (DSe-CMC NPs) for doxorubicin (DOX) delivery, with disulfide analogs (DS-CMC NPs) as control. DS-CMC NPs and DSe-CMC NPs featured a spherical morphology and narrow size distribution with the average size about 200 nm. Carboxymethyl chitosan (CMC) as the starting material not only improved the biocompatibility of the nanocarriers but also enhanced physiological stability. Due to electrostatic interactions between DOX and CMC, the nanoparticles had high drug encapsulation efficiency (∼25 %). The nanoparticles disintegration and drug release were accelerated by the cleavage of diselenide bonds through oxidation by H2O2 or reduction by GSH. In vitro cell experiments revealed that DOX-loaded DSe-CMC NPs possessed the highest drug accumulation and cytotoxicity in tumor cells. Moreover, DOX-loaded DSe-CMC NPs performed the enhanced growth inhibition in vivo than that of DS-CMC NPs. Thus, the diselenide-crosslinked nanoparticles possess great potentials for DOX delivery.

Cytotoxicity of Venoms and Cytotoxins from Asiatic Cobras (Naja kaouthia, Naja sumatrana, Naja atra) and Neutralization by Antivenoms from Thailand, Vietnam, and Taiwan.

Envenoming by cobras (Naja spp.) often results in extensive local tissue necrosis when optimal treatment with antivenom is not available. This study investigated the cytotoxicity of venoms and purified cytotoxins from the Monocled Cobra (Naja kaouthia), Taiwan Cobra (Naja atra), and Equatorial Spitting Cobra (Naja sumatrana) in a mouse fibroblast cell line, followed by neutralization of the cytotoxicity by three regional antivenoms: the Thai Naja kaouthia monovalent antivenom (NkMAV), Vietnamese snake antivenom (SAV) and Taiwanese Neuro bivalent antivenom (NBAV). The cytotoxins of N. atra (NA-CTX) and N. sumatrana (NS-CTX) were identified as P-type cytotoxins, whereas that of N. kaouthia (NK-CTX) is S-type. All venoms and purified cytotoxins demonstrated varying concentration-dependent cytotoxicity in the following trend: highest for N. atra, followed by N. sumatrana and N. kaouthia. The antivenoms moderately neutralized the cytotoxicity of N. kaouthia venom but were weak against N. atra and N. sumatrana venom cytotoxicity. The neutralization potencies of the antivenoms against the cytotoxins were varied and generally low across NA-CTX, NS-CTX, and NK-CTX, possibly attributed to limited antigenicity of CTXs and/or different formulation of antivenom products. The study underscores the need for antivenom improvement and/or new therapies in treating local tissue toxicity caused by cobra envenomings.

1-[4-(2-Dimethylaminoethoxy)phenylcarbonyl]-3,5-Bis(3,4,5-Trimethoxybenzylidene)- 4-Piperidone Hydrochloride and Related Compounds: Potent Cytotoxins Demonstrate Greater Toxicity to Neoplasms than Non- Malignant Cells.

BACKGROUND: The incidence of cancer has been increasing worldwide. Unfortunately, the drugs used in cancer chemotherapy are toxic to both neoplasms and normal tissues, while many available medications have low potencies. Conjugated α,ß-unsaturated ketones differ structurally from contemporary anticancer medications , some of which have noteworthy antineoplastic properties. OBJECTIVES: This study aimed to design and synthesize highly potent cytotoxins with far greater toxicity to neoplasms than to non-malignant cells. METHODS: A series of N-acyl-3,5-bis(benzylidene)-4-piperidone hydrochlorides 4a-n were prepared and evaluated against Ca9-22, HSC-2, HSC-3, and HSC-4 squamous cell carcinomas as well as against HGF, HPLF, and HPC non-malignant cells. QSAR and western blot analyses were performed. RESULTS: The majority of compounds display submicromolar CC50 values towards the neoplasms; the figures for some of the compounds are below 10-7 M. In general, 4a-n have much lower CC50 values than those of melphalan, 5-fluorouracil, and methotrexate, while some compounds are equitoxic with doxorubicin. The compounds are far less toxic to the non-malignant cells, giving rise to substantial selectivity index (SI) figures. A QSAR study revealed that both potency and the SI data were controlled to a large extent by the electronic properties of the substituents in the arylidene aryl rings. Two representative compounds, 4f and 4g, caused apoptosis in HSC-2 cells. CONCLUSION: The compounds in series 4 are potent cytotoxins displaying tumor-selective toxicity. In particular, 4g with an average CC50 value of 0.04 µM towards four malignant cell lines and a selectivity index of 46.3 is clearly a lead molecule that should be further evaluated.

Novel Aspects of the SubA Subunit of the Subtilase Cytotoxin.

The subtilase cytotoxin (SubAB) belongs to the family of AB5 toxins and is produced together with Shiga toxin (Stx) by certain Stx-producing E. coli strains (STEC). For most AB-type toxins, it is assumed that cytotoxic effects can only be induced by a complete holotoxin complex consisting of SubA and SubB. However, it has been shown for SubAB that the enzymatically active subunit SubA, without its transport and binding domain SubB, induces cell death in different eukaryotic cell lines. Interestingly, the molecular structure of SubA resembles that of the SubAB complex. SubA alone is capable of binding to cells and then being taken up autonomously. Once inside the host cell, SubA is transported, similar to the SubAB holotoxin, via a retrograde transport into the endoplasmatic reticulum (ER). In the ER, it exhibits its enzymatic activity by cleaving the chaperone BiP/GRP78 and thereby triggering cell death. Therefore, the existence of toxic single SubA subunits that have not found a B-pentamer for holotoxin assembly might improve the pathogenic potential of subtilase-producing strains. Moreover, from a pharmacological aspect, SubA might be an interesting molecule for the targeted transport of therapeutic molecules into the ER, in order to investigate and specifically modulate processes in the context of ER stress-associated diseases. Since recent studies on bacterial AB5 toxins contributed mainly to the understanding of the biology of AB-type holotoxins, this mini-review specifically focus on that recently observed single A-effect of the subtilase cytotoxin and addresses whether a fundamental shift of the traditional AB5 paradigm might be required.

Cytolysin A (ClyA): A Bacterial Virulence Factor with Potential Applications in Nanopore Technology, Vaccine Development, and Tumor Therapy.

Cytolysin A (ClyA) is a pore-forming toxin that is produced by some bacteria from the Enterobacteriaceae family. This review provides an overview of the current state of knowledge regarding ClyA, including the prevalence of the encoding gene and its transcriptional regulation, the secretion pathway used by the protein, and the mechanism of protein assembly, and highlights potential applications of ClyA in biotechnology. ClyA expression is regulated at the transcriptional level, primarily in response to environmental stressors, and ClyA can exist stably both as a soluble monomer and as an oligomeric membrane complex. At high concentrations, ClyA induces cytolysis, whereas at low concentrations ClyA can affect intracellular signaling. ClyA is secreted in outer membrane vesicles (OMVs), which has important implications for biotechnology applications. For example, the native pore-forming ability of ClyA suggests that it could be used as a component of nanopore-based technologies, such as sequencing platforms. ClyA has also been exploited in vaccine development owing to its ability to present antigens on the OMV surface and provoke a robust immune response. In addition, ClyA alone or OMVs carrying ClyA fusion proteins have been investigated for their potential use as anti-tumor agents.

Engineered Liposomes Protect Immortalized Immune Cells from Cytolysins Secreted by Group A and Group G Streptococci.

The increasing antibiotic resistance of bacterial pathogens fosters the development of alternative, non-antibiotic treatments. Antivirulence therapy, which is neither bacteriostatic nor bactericidal, acts by depriving bacterial pathogens of their virulence factors. To establish a successful infection, many bacterial pathogens secrete exotoxins/cytolysins that perforate the host cell plasma membrane. Recently developed liposomal nanotraps, mimicking the outer layer of the targeted cell membranes, serve as decoys for exotoxins, thus diverting them from attacking host cells. In this study, we develop a liposomal nanotrap formulation that is capable of protecting immortalized immune cells from the whole palette of cytolysins secreted by Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis-important human pathogens that can cause life-threatening bacteremia. We show that the mixture of cholesterol-containing liposomes with liposomes composed exclusively of phospholipids is protective against the combined action of all streptococcal exotoxins. Our findings pave the way for further development of liposomal antivirulence therapy in order to provide more efficient treatment of bacterial infections, including those caused by antibiotic resistant pathogens.

Activation of multiple proteolysis systems contributes to acute cadmium cytotoxicity.

Cadmium exhibits both toxic and carcinogenic effects, and its cytotoxicity is linked to various cellular pathways, such as oxidative stress, ubiquitin-proteasome, and p53-mediated response pathways. The molecular mechanism(s) underlying cadmium cytotoxicity appears to be complex, but remains largely unclear. Here, we examined the effects of cadmium on the protein catabolism using two surrogate markers, DNA topoisomerases I and II alpha and its contribution to cytotoxicity. We have found that cadmium exposure induced time- and concentration-dependent decreases in the protein level of surrogate markers and therefore suggest that cadmium may be involved in proteolysis system activation. A pharmacological study further revealed the novel role(s) of these proteolytic activities and reactive oxygen species (ROS) in the cadmium-induced acute toxicity: (i) Proteasome inhibition only partially relieved the cadmium-induced proteolysis of topoisomerases; (ii) Moreover, we report for the first time that the activation of metalloproteases, serine proteases, and cysteine proteases contributes to the acute cadmium cytotoxicity; (iii) Consistent with the notion that both ROS generation and proteolysis system activation contribute to the cadmium-induced proteolysis and cytotoxicity, the scavenger N-acetylcysteine and aforementioned protease inhibition not only reduced the cadmium-induced topoisomerase degradation but also alleviated the cadmium-induced cell killing. Taken together, acute cadmium exposure may activate multiple proteolytic systems and ROS formation, subsequently leading to intracellular damage and cytotoxicity. Thus, our results provide a novel insight into potential action mechanism(s) by which cadmium exerts its cytotoxic effect and suggest potential strategies to prevent cadmium-associated acute toxicity.

Antimicrobial, Insecticidal and Cytotoxic Activity of Linear Venom Peptides from the Pseudoscorpion Chelifer cancroides.

Linear cationic venom peptides are antimicrobial peptides (AMPs) that exert their effects by damaging cell membranes. These peptides can be highly specific, and for some, a significant therapeutic value was proposed, in particular for treatment of bacterial infections. A prolific source of novel AMPs are arthropod venoms, especially those of hitherto neglected groups such as pseudoscorpions. In this study, we describe for the first time pharmacological effects of AMPs discovered in pseudoscorpion venom. We examined the antimicrobial, cytotoxic, and insecticidal activity of full-length Checacin1, a major component of the Chelifer cancroides venom, and three truncated forms of this peptide. The antimicrobial tests revealed a potent inhibitory activity of Checacin1 against several bacteria and fungi, including methicillin resistant Staphylococcus aureus (MRSA) and even Gram-negative pathogens. All peptides reduced survival rates of aphids, with Checacin1 and the C-terminally truncated Checacin11-21 exhibiting effects comparable to Spinosad, a commercially used pesticide. Cytotoxic effects on mammalian cells were observed mainly for the full-length Checacin1. All tested peptides might be potential candidates for developing lead structures for aphid pest treatment. However, as these peptides were not yet tested on other insects, aphid specificity has not been proven. The N- and C-terminal fragments of Checacin1 are less potent against aphids but exhibit no cytotoxicity on mammalian cells at the tested concentration of 100 µM.

Membrane-Disrupting Activity of Cobra Cytotoxins Is Determined by Configuration of the N-Terminal Loop.

In aqueous solutions, cobra cytotoxins (CTX), three-finger folded proteins, exhibit conformational equilibrium between conformers with either cis or trans peptide bonds in the N-terminal loop (loop-I). The equilibrium is shifted to the cis form in toxins with a pair of adjacent Pro residues in this loop. It is known that CTX with a single Pro residue in loop-I and a cis peptide bond do not interact with lipid membranes. Thus, if a cis peptide bond is present in loop-I, as in a Pro-Pro containing CTX, this should weaken its lipid interactions and likely cytotoxic activities. To test this, we have isolated seven CTX from Naja naja and N. haje cobra venoms. Antibacterial and cytotoxic activities of these CTX, as well as their capability to induce calcein leakage from phospholipid liposomes, were evaluated. We have found that CTX with a Pro-Pro peptide bond indeed exhibit attenuated membrane-perturbing activity in model membranes and lower cytotoxic/antibacterial activity compared to their counterparts with a single Pro residue in loop-I.

The hepatocyte export carrier inhibition assay improves the separation of hepatotoxic from non-hepatotoxic compounds.

An in vitro/in silico method that determines the risk of human drug induced liver injury in relation to oral doses and blood concentrations of drugs was recently introduced. This method utilizes information on the maximal blood concentration (Cmax) for a specific dose of a test compound, which can be estimated using physiologically-based pharmacokinetic modelling, and a cytotoxicity test in cultured human hepatocytes. In the present study, we analyzed if the addition of an assay that measures the inhibition of bile acid export carriers, like BSEP and/or MRP2, to the existing method improves the differentiation of hepatotoxic and non-hepatotoxic compounds. Therefore, an export assay for 5-chloromethylfluorescein diacetate (CMFDA) was established. We tested 36 compounds in a concentration-dependent manner for which the risk of hepatotoxicity for specific oral doses and the capacity to inhibit hepatocyte export carriers are known. Compared to the CTB cytotoxicity test, substantially lower EC10 values were obtained using the CMFDA assay for several known BSEP and/or MRP2 inhibitors. To quantify if the addition of the CMFDA assay to our test system improves the overall separation of hepatotoxic from non-hepatotoxic compounds, the toxicity separation index (TSI) was calculated. We obtained a better TSI using the lower alert concentration from either the CMFDA or the CTB test (TSI: 0.886) compared to considering the CTB test alone (TSI: 0.775). In conclusion, the data show that integration of the CMFDA assay with an in vitro test battery improves the differentiation of hepatotoxic and non-hepatotoxic compounds in a set of compounds that includes bile acid export carrier inhibitors.

New quinones, a sesquiterpene and phenol compounds with cytotoxic activity from the aerial parts of Morinda umbellata L.

Eight previously undescribed compounds, two quinones (1-2), one sesquiterpene (3), and five phenol compounds (4-8), including three enantiomers (6a, 7a, and 8a), along with three corresponding known enantiomers (6b-8b) were isolated from the aerial parts of Morinda umbellata L. Their structures were elucidated by 1D and 2D NMR spectroscopy, X-ray diffraction, and experimental and calculated ECD spectra, respectively. Compound 5 was found to have weak cytotoxity, which inhibited the growth of seven human cancer cell lines (A2780, HeLa, MCF-7, BGC-823, H7420, Ketr3 and SW 1990) with IC50 values from 13.3 to 15.1 µM.

Cytotoxic new caged-polyprenylated xanthonoids from Garcinia oligantha.

Caged-polyprenylated xanthonoids represent a rare class of natural products. This type of compounds is mainly isolated from Genus Garcinia. Phytochemical studies on the leaves and twigs of Garcinia oligantha led to the isolation of four new caged-polyprenylated xanthonoids, oliganthone CF (1-4), and two new simple xanthones (5-6), oliganthaxanthone D and oliganthaxanthone E. Eight known other polyprenylated xanthones (7-14) including five caged-polyprenylated xanthonoids (7-11) were also isolated. Their structures were elucidated based on the analyses of extensive spectroscopic data. All the isolated compounds except for 5, 6 and 14 showed cell viability reducing effect against human lung cancer A549 cells. Compounds 1-3 were proved to be potential apoptosis inducing agents.

Inhibition of Methamphetamine-Induced Cytotoxicity in the U87-Cell Line by Atorvastatin-Conjugated Carbon Nanotubes.

In biological systems, carbon nanotubes can enhance the biological effects of drugs and reduce their side effects. Methamphetamine (METH) is a stimulant drug that induces cell death in various cell types, primarily neural cells. On the other hand, specific doses of atorvastatin (ATO) can stimulate cell growth and inhibit cell death in different cell lines. This study aimed to investigate the improvement effect of ATO@single-walled carbon nanotube (SWCNT) on METH-induced cell cytotoxicity in the U87 glioblastoma cell line. In this study, cells were cultured in 10 mM of METH during the cell treatment with 0-10 nM of ATO and ATO@SWCNT. The conjugated drugs to SWCNT as Van der Waals were detected using field emission scanning electron microscopy, Fourier transform-infrared spectroscopy, and other analyses. Then, the in vitro proliferating of ATO@SWCNT was explored against glioblastoma cells compared to pure ATO. This examine was performed using methyl thiazole tetrazolium approach, terminal deoxynucleotidyl transferase deoxy uridine-triphosphate nick end labeling assay, caspase-3 method, lactate dehydrogenase assay, and RH-123 assay with 10 mM METH. The results obtained from transmission electron microscopy analysis showed the average size of 50 nm for ATO@SWCNT. This study indicated that U87 cells, which were exposed to METH and suffered cell death, were severely reduced in the presence of ATO, especially ATO@SWCNT (for its anti-apoptotic effect), but they survived. This study suggests that ATO, which was primarily used to reduce blood lipids, can significantly reduce brain cell death. The findings of this study indicate that by using SWCNT, more drugs can reach the target cells. This method reduces the total amount of required medication and shows a more beneficial therapeutic effect.

Fish Cytolysins in All Their Complexity.

The majority of the effects observed upon envenomation by scorpaenoid fish species can be reproduced by the cytolysins present in their venoms. Fish cytolysins are multifunctional proteins that elicit lethal, cytolytic, cardiovascular, inflammatory, nociceptive, and neuromuscular activities, representing a novel class of protein toxins. These large proteins (MW 150-320 kDa) are composed by two different subunits, termed α and ß, with about 700 amino acid residues each, being usually active in oligomeric form. There is a high degree of similarity between the primary sequences of cytolysins from different fish species. This suggests these molecules share similar mechanisms of action, which, at least regarding the cytolytic activity, has been proved to involve pore formation. Although the remaining components of fish venoms have interesting biological activities, fish cytolysins stand out because of their multifunctional nature and their ability to reproduce the main events of envenomation on their own. Considerable knowledge about fish cytolysins has been accumulated over the years, although there remains much to be unveiled. In this review, we compiled and compared the current information on the biochemical aspects and pharmacological activities of fish cytolysins, going over their structures, activities, mechanisms of action, and perspectives for the future.

Cytotoxicity of an Innovative Pressurised Cyclic Solid-Liquid (PCSL) Extract from Artemisia annua.

Therapeutic treatments with Artemisia annua have a long-established tradition in various diseases due to its antibacterial, antioxidant, antiviral, anti-malaria and anti-cancer effects. However, in relation to the latter, virtually all reports focused on toxic effects of A. annua extracts were obtained mostly through conventional maceration methods. In the present study, an innovative extraction procedure from A. annua, based on pressurised cyclic solid-liquid (PCSL) extraction, resulted in the production of a new phytocomplex with enhanced anti-cancer properties. This extraction procedure generated a pressure gradient due to compressions and following decompressions, allowing to directly perform the extraction without any maceration. The toxic effects of A. annua PCSL extract were tested on different cells, including three cancer cell lines. The results of this study clearly indicate that the exposure of human, murine and canine cancer cells to serial dilutions of PCSL extract resulted in higher toxicity and stronger propensity to induce apoptosis than that detected by subjecting the same cells to Artemisia extracts obtained through canonical extraction by maceration. Collected data suggest that PCSL extract of A. annua could be a promising and economic new therapeutic tool to treat human and animal tumours.

Cytotoxic Lactalbumin-Oleic Acid Complexes in the Human Milk Diet of Preterm Infants.

Frozen storage is necessary to preserve expressed human milk for critically ill and very preterm infants. Milk pasteurization is essential for donor milk given to this special population. Due to these storage and processing conditions, subtle changes occur in milk nutrients. These changes may have clinical implications. Potentially, bioactive complexes of unknown significance could be found in human milk given to preterm infants. One such complex, a cytotoxic α-lactalbumin-oleic acid complex named "HAMLET," (Human Alpha-Lactalbumin Made Lethal to Tumor cells) is a folding variant of alpha-lactalbumin that is bound to oleic acid. This complex, isolated from human milk casein, has specific toxicity to both carcinogenic cell lines and immature non-transformed cells. Both HAMLET and free oleic acid trigger similar apoptotic mechanisms in tissue and stimulate inflammation via the NF-κB and MAPK p38 signaling pathways. This protein-lipid complex could potentially trigger various inflammatory pathways with unknown consequences, especially in immature intestinal tissues. The very preterm population is dependent on human milk as a medicinal and broadly bioactive nutriment. Therefore, HAMLET's possible presence and bioactive role in milk should be addressed in neonatal research. Through a pediatric lens, HAMLET's discovery, formation and bioactive benefits will be reviewed.

Synthesis and cytotoxicity evaluation of alpha beta unsaturated carboxylic acids of thiophene analogs by using polymer supported microwave-assisted method.

α- ß unsaturated carboxylic acids containing a heterocyclic moiety is one of the most potent class of bioactive compounds whose speedy generation through novel synthetic techniques has become an enigma for the synthetic chemists. This research project demonstrates a novel method for the synthesis of these compounds using polymer-supported microwave-assisted methodology carried out through one-pot multicomponent reaction. Both soluble and insoluble polymers have been used and their results are comprehensively analyzed. Moreover, the compounds are characterized through spectral analysis like FTIR, GC-MASS, 1HNMR Spectroscopy. The cytotoxicity of synthesized compounds is evaluated through MTT assay using HEPG 2 cells.

The cell envelope of Staphylococcus aureus selectively controls the sorting of virulence factors.

Staphylococcus aureus bi-component pore-forming leukocidins are secreted toxins that directly target and lyse immune cells. Intriguingly, one of the leukocidins, Leukocidin AB (LukAB), is found associated with the bacterial cell envelope in addition to secreted into the extracellular milieu. Here, we report that retention of LukAB on the bacterial cells provides S. aureus with a pre-synthesized active toxin that kills immune cells. On the bacteria, LukAB is distributed as discrete foci in two distinct compartments: membrane-proximal and surface-exposed. Through genetic screens, we show that a membrane lipid, lysyl-phosphatidylglycerol (LPG), and lipoteichoic acid (LTA) contribute to LukAB deposition and release. Furthermore, by studying non-covalently surface-bound proteins we discovered that the sorting of additional exoproteins, such as IsaB, Hel, ScaH, and Geh, are also controlled by LPG and LTA. Collectively, our study reveals a multistep secretion system that controls exoprotein storage and protein translocation across the S. aureus cell wall.