Amphipathic dendritic poly-peptides carrier to deliver antisense oligonucleotides against multi-drug resistant bacteria in vitro and in vivo.

BACKGROUND: Outbreaks of infection due to multidrug-resistant (MDR) bacteria, especially Gram-negative bacteria, have become a global health issue in both hospitals and communities. Antisense oligonucleotides (ASOs) based therapeutics hold a great promise for treating infections caused by MDR bacteria. However, ASOs therapeutics are strangled because of its low cell penetration efficiency caused by the high molecular weight and hydrophilicity. RESULTS: Here, we designed a series of dendritic poly-peptides (DPP1 to DPP12) to encapsulate ASOs to form DSPE-mPEG2000 decorated ASOs/DPP nanoparticles (DP-AD1 to DP-AD12) and observed that amphipathic DP-AD2, 3, 7 or 8 with a positive charge ≥ 8 showed great efficiency to deliver ASOs into bacteria, but only the two histidine residues contained DP-AD7 and DP-AD8 significantly inhibited the bacterial growth and the targeted gene expression of tested bacteria in vitro. DP-AD7anti-acpP remarkably increased the survival rate of septic mice infected by ESBLs-E. coli, exhibiting strong antibacterial effects in vivo. CONCLUSIONS: For the first time, we designed DPP as a potent carrier to deliver ASOs for combating MDR bacteria and demonstrated the essential features, namely, amphipathicity, 8-10 positive charges, and 2 histidine residues, that are required for efficient DPP based delivery, and provide a novel approach for the development and research of the antisense antibacterial strategy.

Restoration of the Antibiotic Susceptibility of Methicillin-Resistant Staphylococcus aureus and Extended-Spectrum ß-Lactamases Escherichia coli Through Combination with Chelerythrine.

Multidrug resistance poses a severe threat to public health and urgently requires new solutions. The natural product chelerythrine (CHE) is a benzophenanthridine alkaloid with antimicrobial potential. In this study, CHE was effective against seven gram-positive bacterial strains, and the minimum inhibitory concentrations (MICs) ranged from 2 to 4 µg/mL. By contrast, CHE showed inferior antibacterial activities against 11 gram-negative strains, and the MICs varied from 16 to 256 µg/mL. We also determined the synergistic/additive effects of combining CHE with nine currently used antibiotics. CHE restored the antibacterial efficacy of the antibiotics against methicillin-resistant Staphylococcus aureus and extended-spectrum ß-lactamases producing Escherichia coli. This study suggests that the combination of CHE with conventional antibiotics may be a promising strategy to combat infections caused by multidrug-resistant organisms.

Exendin-4 induces a novel extended effect of ischemic tolerance via crosstalk with IGF-1R.

Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonist exendin-4 (Ex-4), a drug that has been used in the clinical treatment of type 2 diabetes mellitus, also confers a neuroprotective effect against stroke. Although GLP-1 analogs were reported to induce sustained insulin secretion and glucose tolerance improved after cessation of treatment, no study has revealed whether Ex-4 exerts sustained neuroprotection against stroke and the underlying mechanism after treatment cessation. In this study, mice were pretreated with Ex-4 for 7 days, and middle cerebral artery occlusion (MCAO) was performed on different days after cessation of Ex-4 treatment. Ex-4 ameliorated neurological dysfunction and reduced the infarct volume induced by MCAO. These protective effects lasted for 6 days after the cessation of Ex-4 treatment and were associated with sustained upregulation of PI3K, AKT, mTOR, and HIF-1α levels, as well as HIF-1α downstream genes. Knockdown of GLP-1R or HIF-1α in the brain by short hairpin RNA abolished Ex-4 treatment-mediated neuroprotection. In normal mice, Ex-4 treatment led to instant upregulation of p-PI3K, p-AKT, p-mTOR, and HIF-1α expression levels, which quickly returned to normal after cessation of Ex-4 treatment, while the expression levels of insulin growth factor-1 receptor (IGF-1R) remained high for 6 days after Ex-4 cessation. Additionally, Ex-4 did not directly induce IGF-1 production, which was only induced by MCAO. Ex-4 induces extended cerebral ischemic tolerance. This neuroprotective effect is associated with activation of GLP-1R and upregulation of IGF-1R in the brain, and the latter then activates the PI3K/AKT/mTOR/HIF-1 signaling pathway via binding to IGF-1 secreted from the ischemic brain.

Loop-armed DNA tetrahedron nanoparticles for delivering antisense oligos into bacteria.

BACKGROUND: Antisense oligonucleotides (ASOs) based technology is considered a potential strategy against antibiotic-resistant bacteria; however, a major obstacle to the application of ASOs is how to deliver them into bacteria effectively. DNA tetrahedra (Td) is an emerging carrier for delivering ASOs into eukaryotes, but there is limited information about Td used for bacteria. In this research, we investigated the uptake features of Td and the impact of linkage modes between ASOs and Td on gene-inhibition efficiency in bacteria. RESULTS: Td was more likely to adhere to bacterial membranes, with moderate ability to penetrate into the bacteria. Strikingly, Td could penetrate into bacteria more effectively with the help of Lipofectamine 2000 (LP2000) at a 0.125 µL/µg ratio to Td, but the same concentration of LP2000 had no apparent effect on linear DNA. Furthermore, linkage modes between ASOs and Td influenced gene-knockdown efficiency. Looped structure of ASOs linked to one side of the Td exhibited better gene-knockdown efficiency than the overhung structure. CONCLUSIONS: This study established an effective antisense delivery system based on loop-armed Td, which opens opportunities for developing antisense antibiotics.

A new coumarin compound DCH combats methicillin-resistant Staphylococcus aureus biofilm by targeting arginine repressor.

Staphylococcus aureus infection is difficult to eradicate because of biofilm formation and antibiotic resistance. The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infection necessitates the development of a new agent against bacterial biofilms. We report a new coumarin compound, termed DCH, that effectively combats MRSA in vitro and in vivo and exhibits potent antibiofilm activity without detectable resistance. Cellular proteome analysis suggests that the molecular mechanism of action of DCH involves the arginine catabolic pathway. Using molecular docking and binding affinity assays of DCH, and comparison of the properties of wild-type and ArgR-deficient MRSA strains, we demonstrate that the arginine repressor ArgR, an essential regulator of the arginine catabolic pathway, is the target of DCH. These findings indicate that DCH is a promising lead compound and validate bacterial ArgR as a potential target in the development of new drugs against MRSA biofilms.

PAMP protects intestine from Enterohemorrhagic Escherichia coli infection through destroying cell membrane and inhibiting inflammatory response.

Proadrenomedullin N-terminal 20 peptide (PAMP) is elevated in sepsis, but the function and possible mechanism of PAMP in bacterial infection is elusive. This study is aim to evaluate the role of PAMP in the interaction between the Enterohemorrhagic E. coli (EHEC) and the host barrier. Our results showed that PAMP alleviated the EHEC-induced disruption of goblet cells and mucosal damage in the intestine, increased the expression of occludin in the colon of EHEC-infected mice, and reduced the proinflammatory cytokines level in serum significantly compared with the control group. Meanwhile, lipopolysaccharide (LPS) stimulation could dose-dependently induce the expression of preproADM, the precursor of PAMP, in human intestinal epithelial cell (HIEC) and human umbilical vein endothelial cell (HUVEC). In addition, PAMP inhibited the growth of EHEC O157:H7 and destroyed the inner and outer membrane. At low concentration, PAMP attenuated the EHEC virulence genes including hlyA and eaeA, which was also confirmed from reduced hemolysis to red cells and adhesion to HIEC. These results indicated that EHEC infection would modulate the expression of PAMP in intestinal epithelium or vascular endothelium, and in turn exerted a protective effect in EHEC induced infection by rupturing the bacterial cell membrane and attenuating the bacterial virulence.

Outer membrane protein A (OmpA) as a potential therapeutic target for Acinetobacter baumannii infection.

Acinetobacter baumannii (A. baumannii) is an important opportunistic pathogen causing serious nosocomial infections, which is considered as the most threatening Gram-negative bacteria (GNB). Outer membrane protein A (OmpA), a major component of outer membrane proteins (OMPs) in GNB, is a key virulence factor which mediates bacterial biofilm formation, eukaryotic cell infection, antibiotic resistance and immunomodulation. The characteristics of OmpA in Escherichia coli (E. coli) have been extensively studied since 1974, but only in recent years researchers started to clarify the functions of OmpA in A. baumannii. In this review, we summarized the structure and functions of OmpA in A. baumannii (AbOmpA), collected novel therapeutic strategies against it for treating A. baumannii infection, and emphasized the feasibility of using AbOmpA as a potential therapeutic target.

Contemporaneous Measurement of Outer and Inner Membrane Permeability in Gram-negative Bacteria.

The emergence and rapid spread of multidrug resistance in bacteria have led to the urgent need for novel antibacterial agents. Membrane permeabilization is the mechanism for many antibacterial molecules that are being developed against gram-negative bacteria. Thus, to determine the efficacy of a potential antibacterial molecule, it is important to assess the change in bacterial membrane permeability after treatment. This study describes the protocol for the assays of outer and inner membrane permeability using the fluorescent probes N-phenyl-1-naphthylamine and propidium iodide. Compared with other experiments, such as electron microscopy and the assay of minimal bactericidal concentration, this methodology provides a simpler, faster, and cost-effective way of estimating the membrane-permeabilizing effect and bactericidal efficacy of antibacterial molecules. This study presents an optimized protocol with respect to the classical protocols by incubating bacteria with antibacterial molecules in the culture condition identical to that of antibacterial assays and then detecting the signal of the fluorescent probe in the buffer without broth and antibacterial molecules. This protocol avoids the effect of nutrient deficiency on the physiological status of bacteria and the interference of antibacterial molecules towards the fluorescent probe. Thus, this method can effectively and precisely evaluate the membrane permeability and match the results obtained from other antibacterial assays, such as minimum inhibitory concentration and time-kill curve assays.

Regulation of cytokinesis: FtsZ and its accessory proteins.

Bacterial cell division is a highly controlled process regulated accurately by a diverse array of proteins spatially and temporally working together. Among these proteins, FtsZ is recognized as a cytoskeleton protein because it can assemble into a ring-like structure called Z-ring at midcell. Z-ring recruits downstream proteins, thus forming a multiprotein complex termed the divisome. When the Z-ring scaffold is established and the divisome matures, peptidoglycan (PG) biosynthesis and chromosome segregation are triggered. In this review, we focus on multiple interactions between FtsZ and its accessory proteins in bacterial cell cytokinesis, including FtsZ localization, Z-ring formation and stabilization, PG biosynthesis, and chromosome segregation. Understanding the interactions among these proteins may help discover superior targets on treating bacterial infectious diseases.

In vitro and in vivo anti-biofilm activity of pyran derivative against Staphylococcus aureus and Pseudomonas aeruginosa.

BACKGROUND: The development of bacterial biofilm can cause severe chronic infections and antibiotic resistance. Therefore, it poses a significant threat to public health. Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) are two major pathogens that can cause biofilm-associated infections, which leads to the urgent necessity of developing new agents with biofilm-forming inhibitory ability. METHODS: A series of pyran derivatives were synthesized and characterized, and their in vitro anti-biofilm activity against S. aureus and P. aeruginosa were measured by minimal biofilm inhibitory concentration assay and FITC dye staining. The in vivo antibiofilm therapeutical effects were evaluated in S. aureus induced tissue cage infection mice model and P. aeruginosa induced urinary tract catheter infection rat model. RESULTS: Several pyran derivatives showed the in vitro anti-biofilm activity against S. aureus and P. aeruginosa, and the activity of these compounds was not mediated through the accessory gene regulator (agr) quorum sensing system of S. aureus. One of these pyran derivatives, namely 2-amino-4-(2,6-dichlorophenyl)-3-cyano-5-oxo-4H,5H-pyrano[3,2c]chromene, exhibited significant inhibitory biofilm-formation activity in S. aureus tissue cage infection mice model and in the P. aeruginosa-infected urinary tract catheters of experimental rats. CONCLUSIONS: The data indicated that this pyran derivative is a possible lead compound that can be used for the development of novel anti-biofilm agents against S. aureus and P. aeruginosa infection.

The antimicrobial peptide thanatin disrupts the bacterial outer membrane and inactivates the NDM-1 metallo-ß-lactamase.

New Delhi metallo-ß-lactamase-1 (NDM-1) is the most prevalent type of metallo-ß-lactamase and hydrolyzes almost all clinically used ß-lactam antibiotics. Here we show that the antimicrobial peptide thanatin disrupts the outer membrane of NDM-1-producing bacteria by competitively displacing divalent cations on the outer membrane and inducing the release of lipopolysaccharides. In addition, thanatin inhibits the enzymatic activity of NDM-1 by displacing zinc ions from the active site, and reverses carbapenem resistance in NDM-1-producing bacteria in vitro and in vivo. Thus, thanatin's dual mechanism of action may be useful for combating infections caused by NDM-1-producing pathogens.

Influence of Magnesium Ions on the Preparation and Storage of DNA Tetrahedrons in Micromolar Ranges.

The DNA tetrahedron (Td), as one of the novel DNA-based nanoscale biomaterials, has been extensively studied because of its excellent biocompatibility and increased possibilities for decorating precisely. Although the use of Td in laboratories is well established, knowledge surrounding the factors influencing its preparation and storage is lacking. In this research, we investigated the role of the magnesium ions, which greatly affect the structure and stability of DNA. We assembled 1, 2, 5, 10 and 20 µM Td in buffers containing different Mg2+ concentrations, demonstrating that 2 and 5 mM Mg2+ is optimal in these conditions, and that yields decrease dramatically once the DNA concentration reaches 20 µM or the Mg2+ concentration is lower than 0.5 mM. We also verified that the Td structure is retained better through freeze-thawing than lyophilization. Furthermore, a lower initial Mg2+ (≤2 mM) benefited the maintenance of Td structure in the process of lyophilization. Hence, our research sheds light on the influence of Mg2+ in the process of preparing and storing Td, and also provides some enlightenment on improving yields of other DNA nanostructures.

Efficient Delivery of Antisense Oligonucleotides by an Amphipathic Cell-Penetrating Peptide in Acinetobacter baumannii.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (A. baumannii) was on the top of the list of the most threatening bacteria published by the WHO in 2017. Antisense oligonucleotides (ASOs) based therapy is a promising strategy for combating Multi-Drug Resistant (MDR) bacteria because of its high specificity, easy design and lower induction of resistance, but poor cellular uptake by bacteria has restricted the further utilization of this therapy. METHODS: Here, we used CADY, a secondary amphipathic peptide of 20 residues that could successfully carry siRNA into mammalian cells, to prepare CADY/ASOs nanoparticles (CADY-NPs) targeting acpP (encoding acyl carrier protein), and evaluated the uptake features, the inhibitory effects of CADY-NPs on gene expression and the growth of MDR-A. baumannii. RESULTS: We found that CADY-NPs could be quickly internalized by drug-sensitive and MDR-A. baumannii in an energy independent manner, which could be restrained by chlorpromazine (an inhibitor of clathrin mediated endocytosis) significantly. In addition, CADY-NPs targeting acpP concentrationdependently retarded the growth of MDR-A. baumannii, which was associated with the decreased expression of targeted genes in A. baumannii. CONCLUSION: In conclusion, our research is the first to demonstrate that CADY can deliver ASOs into bacteria and provide a novel strategy for the treatment of MDR-A. baumannii.

Neuroprotection of hydroxysafflor yellow A in experimental cerebral ischemia/reperfusion injury via metabolic inhibition of phenylalanine and mitochondrial biogenesis.

Stroke is the second most frequent cause of mortality, resulting in a huge societal burden worldwide. Timely reperfusion is the most effective therapy; however, it is difficult to prevent ischemia/reperfusion (I/R) injury. In traditional Chinese medicine, hydroxysafflor yellow A (HSYA) has been widely used for the treatment of cerebrovascular disease and as a protective therapy against I/R injury. Evidence has demonstrated that HSYA could reduce the levels of reactive oxygen species and suppress cellular apoptosis; however, whether HSYA alters the metabolic profile as its underlying mechanism for neuroprotection remains unknown. In the present study, using a metabolomic screening, phenylalanine was identified to significantly increase in an experimental model of mouse cerebral I/R injury. Notably, western blotting and qPCR analysis were conducted to test the expression level of apoptosis­associated factors, and HSYA was identified to be able to protect neuronal cells by reducing phenylalanine level associated with I/R injury. Additionally, these findings were confirmed in primary mouse neurons and PC12 cells exposed to oxygen and glucose deprivation/reoxygenation (OGD/R) stress. Of note, HSYA was observed to regulate the mRNA expression of key metabolic enzymes, phenylalanine hydroxylase, tyrosine aminotransferase and aspartate aminotransferase, which are responsible for phenylalanine metabolism. Furthermore, by performing mitochondrial labeling and JC­1 fluorescence assay, HSYA was identified to promote mitochondrial function and biogenesis suppressed by OGD/R. The findings of the present study demonstrated that I/R injury could increase the levels of phenylalanine, and HSYA may inhibit phenylalanine synthesis to enhance mitochondrial function and biogenesis for neuroprotection. The present study proposed a novel metabolite biomarker for cerebral I/R injury and the evaluated the efficacy of HSYA as a potential therapeutic treatment I/R injury.

Inhibiting Jumoji domain containing protein 3 (JMJD3) prevent neuronal apoptosis from stroke.

Control of p53 by histone methylation is closely related in the neuronal apoptosis following ischemic stroke. In mammalian cells, demethylation of methylated lysine residue of histones is catalyzed by Jumonji domain-containing proteins (JMJD) family. Among them, JMJD3 is reported to be a hypoxic target gene and expressed in all cell types of brain including neurons. However, the role of JMJD3 on process of neuronal apoptosis after ischemic stroke is still largely unknown. PCR, immunostaining and Western blotting results indicated that JMJD3 expression was upregulated in cultured neurons upon oxygen-glucose deprivation (OGD) injury. Jmjd3-/- neurons exhibited inhibited cell apoptosis and tolerance to the OGD injury. Chromatin immunoprecipitation and promoter reporter assays showed that the underlying mechanism was through transcriptional activation of p53, thus altering the downstream Bax and Caspase-3 genes. Silencing Jmjd3 improved neurological deficit and reduced infarct volume following ischemic injury in vivo. The present study suggested that JMJD3 was a critical promoter of neuronal apoptosis by regulating the expression of Bax and Caspase-3, and inhibition of JMJD3 might provide a new therapeutic intervention for treating cerebral ischemia.

Inhibiting PSMα-induced neutrophil necroptosis protects mice with MRSA pneumonia by blocking the agr system.

Given its high resistance, enhanced virulence, and high transmissibility, community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) pneumonia is highly associated with high morbidity and mortality. Anti-virulence therapy is a promising strategy that bypasses the evolutionary pressure on the bacterium to develop resistance. RNAIII-inhibiting peptide (RIP), as an accessory gene regulator (agr)-specific inhibitor, significantly restricts the virulence of S. aureus and protects infected mice from death by blocking the agr quorum sensing system. The protective effects of RIP on the neutropenic mice completely disappeared in a neutrophil-deleted mouse infection model, but not in the macrophage-deleted mice. This result confirmed that the in vivo antibacterial activity of RIP is highly associated with neutrophil function. Phenol-soluble modulins (PSMs), as major leukocyte lysis toxins of CA-MRSA, are directly regulated by the agr system. In this experiment, PSMα1, 2, and 3 significantly induced neutrophil necroptosis by activating mixed lineage kinase-like protein (MLKL) phosphorylation and increasing lactate dehydrogenase release. The S. aureus supernatants harvested from the agr or psmα mutant strains both decreased the phosphorylation level of MLKL and cell lysis. PSMα1-mediated neutrophil lysis was significantly inhibited by necrosulfonamide, necrostatin-1, TNFα antibody, and WRW4. These results showed PSMα1 induced necroptosis depends on formylpeptide receptor 2 (FPR2)-mediated autocrine TNFα. Moreover, the neutrophil necroptosis induced by S. aureus was significantly suppressed and pneumonia was effectively prevented by the blockage of agrA and psmα expression levels. These findings indicate that PSMα-induced necroptosis is a major cause of lung pathology in S. aureus pneumonia and suggest that interfering with the agr quorum sensing signaling pathway is a potential therapeutic strategy.

Advances in the delivery of antisense oligonucleotides for combating bacterial infectious diseases.

Discovery and development of new antibacterial drugs against multidrug resistant bacterial strains have become more and more urgent. Antisense oligonucleotides (ASOs) show immense potential to control the spread of resistant microbes due to its high specificity of action, little risk to human gene expression, and easy design and synthesis to target any possible gene. However, efficient delivery of ASOs to their action sites with enough concentration remains a major obstacle, which greatly hampers their clinical application. In this study, we reviewed current progress on delivery strategies of ASOs into bacteria, focused on various non-virus gene vectors, including cell penetrating peptides, lipid nanoparticles, bolaamphiphile-based nanoparticles, DNA nanostructures and Vitamin B12. The current review provided comprehensive understanding and novel perspective for the future application of ASOs in combating bacterial infections.

Exendin-4 Induces Bone Marrow Stromal Cells Migration Through Bone Marrow-Derived Macrophages Polarization via PKA-STAT3 Signaling Pathway.

BACKGROUND/AIMS: The synthesis and degradation processes involved in bone remodeling are critically regulated by osteoblasts and osteoclasts. The GLP-1 receptor agonist Exendin-4 is beneficial for osteoblast differentiation and increases the number of osteoblasts. METHODS: We constructed an ovariectomized model to evaluate the impact of Exendin-4 on bone formation in osteoporosis. A macrophage-depleted model was also created to investigate the effect of macrophages on bone formation. Thirty-two female WT C57BL/6 mice (aged 3 months) were randomly assigned to a normal control group and four ovariectomized (OVX) subgroups: OVX + vehicle group, OVX + Exendin-4 (4.2 µg/kg/day) group, OVX + chloride phosphate liposome group and OVX + chloride phosphate liposome + Exendin-4 group. RESULTS: In this study, we found that Exendin-4 not only increased the number of osteoblasts and decreased the number of osteoclasts, but also increased the number of bone marrow stromal cells (BMSCs) at the bone surface. Moreover, we found that OVX mice treated with Exendin-4 increased TGF-ß1 levels at the bone surface compared with that in OVX mice. Besides, Exendin-4 promoted the polarization of bone marrow-derived macrophages into M2 subtype and increased TGF-ß1 secretion by the M2 subtype. Finally, we found that Exendin-4 induced macrophage polarization via the cAMP-PKA-STAT3 signaling pathway. CONCLUSION: Exendin-4 promotes bone marrow-derived macrophage polarization to the M2 subtype and induces BMSC migration to the bone surface via PKA-STAT3 signaling.

Development of DNA tetrahedron-based drug delivery system.

Nanocarriers of drugs have attracted significant attention to tackle the problems of drug resistance or nucleic acid drug delivery, which can optimize pharmaceutical parameters and enhance the cellular uptake efficiency. Nowadays, DNA nanostructure presents an opportunity in the field of nanomaterial due to its precise control in shape and size, excellent biocompatibility, as well as multiple sites for targeting decoration. DNA tetrahedron, which is stable and easily synthesized, is used for various applications, including nuclear magnetic resonance imaging, molecular diagnosis, targeting drug delivery, and so on. In this review, we will discuss the applications of DNA tetrahedron about drug delivery, intracellular routes and its fates. Also challenges and possible solutions for developing DNA tetrahedron-based drug delivery system are detailed.

Phenol-Soluble Modulin Toxins of Staphylococcus haemolyticus.

Coagulase-negative staphylococci (CoNS) are important nosocomial pathogens and the leading cause of sepsis. The second most frequently implicated species, after Staphylococcus epidermidis, is Staphylococcus haemolyticus. However, we have a significant lack of knowledge about what causes virulence of S. haemolyticus, as virulence factors of this pathogen have remained virtually unexplored. In contrast to the aggressive pathogen Staphylococcus aureus, toxin production has traditionally not been associated with CoNS. Recent findings have suggested that phenol-soluble modulins (PSMs), amphipathic peptide toxins with broad cytolytic activity, are widespread in staphylococci, but there has been no systematic assessment of PSM production in CoNS other than S. epidermidis. Here, we identified, purified, and characterized PSMs of S. haemolyticus. We found three PSMs of the ß-type, which correspond to peptides that before were described to have anti-gonococcal activity. We also detected an α-type PSM that has not previously been described. Furthermore, we confirmed that S. haemolyticus does not produce a δ-toxin, as results from genome sequencing had indicated. All four S. haemolyticus PSMs had strong pro-inflammatory activity, promoting neutrophil chemotaxis. Notably, we identified in particular the novel α-type PSM, S. haemolyticus PSMα, as a potent hemolysin and leukocidin. For the first time, our study describes toxins of this important staphylococcal pathogen with the potential to have a significant impact on virulence during blood infection and sepsis.