Strong Hemolytic Activity Research Articles

Discovery, development and optimisation of a novel frog antimicrobial peptide with combined mode of action against drug-resistant bacteria

Antimicrobial peptides (AMP) have emerged as promising candidates for addressing the clinical challenges posed by the rapid evolution of antibiotic-resistant microorganisms. Brevinins, a representative frog-derived AMP family, exhibited broad-spectrum antimicrobial activities, attacking great attentions in previous studies. However, their strong haemolytic activity and cytotoxicity, greatly limit their further development. In this work, we identified and characterised a novel brevinin-1 peptide, brevinin-1pl, from the skin secretions of the northern leopard frog, Rana pipiens. Like many brevinins, brevinin-1pl also displayed strong haemolytic activity, resulting in a lower therapeutic index. We employed several bioinformatics tools to analyse the structure and potential membrane interactions of brevinin-1pl, leading to a series of modifications. Among these analogues, des-Ala16-[Lys4]brevinin-1pl exhibited great enhanced therapeutic efficacy in both in vitro and in vivo tests, particularly against some antibiotics-resistant Escherichia coli strains. Mechanistic studies suggest that des-Ala16-[Lys4]brevinin-1pl may exert bactericidal effects through multiple mechanisms, including membrane disruption and DNA binding. Consequently, des-Ala16-[Lys4]brevinin-1pl holds promise as a candidate for the treatment of drug-resistant Escherichia coli infections.

Characterization of the Hemolytic Activity of Mastoparan Family Peptides from Wasp Venoms.

Biologically active peptides have attracted increasing attention in research on the development of new drugs. Mastoparans, a group of wasp venom linear cationic α-helical peptides, have a variety of biological effects, including mast cell degranulation, activation of protein G, and antimicrobial and anticancer activities. However, the potential hemolytic activity of cationic α-helical peptides greatly limits the clinical applications of mastoparans. Here, we systematically and comprehensively studied the hemolytic activity of mastoparans based on our wasp venom mastoparan family peptide library. The results showed that among 55 mastoparans, 18 had strong hemolytic activity (EC50 ≤ 100 μM), 14 had modest hemolytic activity (100 μM < EC50 ≤ 400 μM) and 23 had little hemolytic activity (EC50 > 400 μM), suggesting functional variation in the molecular diversity of mastoparan family peptides from wasp venom. Based on these data, structure-function relationships were further explored, and, hydrophobicity, but not net charge and amphiphilicity, was found to play a critical role in the hemolytic activity of mastoparans. Combining the reported antimicrobial activity with the present hemolytic activity data, we found that four mastoparan peptides, Parapolybia-MP, Mastoparan-like peptide 12b, Dominulin A and Dominulin B, have promise for applications because of their high antimicrobial activity (MIC ≤ 10 μM) and low hemolytic activity (EC50 ≥ 400 μM). Our research not only identified new leads for the antimicrobial application of mastoparans but also provided a large chemical space to support the molecular design and optimization of mastoparan family peptides with low hemolytic activity regardless of net charge or amphiphilicity.

TurboID screening of ApxI toxin interactants identifies host proteins involved in Actinobacillus pleuropneumoniae-induced apoptosis of immortalized porcine alveolar macrophages

Actinobacillus pleuropneumoniae (APP) is a gram-negative pathogenic bacterium responsible for porcine contagious pleuropneumonia (PCP), which can cause porcine necrotizing and hemorrhagic pleuropneumonia. Actinobacillus pleuropneumoniae-RTX-toxin (Apx) is an APP virulence factor. APP secretes a total of four Apx toxins, among which, ApxI demonstrates strong hemolytic activity and cytotoxicity, causing lysis of porcine erythrocytes and apoptosis of porcine alveolar macrophages. However, the protein interaction network between this toxin and host cells is still poorly understood. TurboID mediates the biotinylation of endogenous proteins, thereby targeting specific proteins and local proteomes through gene fusion. We applied the TurboID enzyme-catalyzed proximity tagging method to identify and study host proteins in immortalized porcine alveolar macrophage (iPAM) cells that interact with the exotoxin ApxI of APP. His-tagged TurboID-ApxIA and TurboID recombinant proteins were expressed and purified. By mass spectrometry, 318 unique interacting proteins were identified in the TurboID ApxIA-treated group. Among them, only one membrane protein, caveolin-1 (CAV1), was identified. A co-immunoprecipitation assay confirmed that CAV1 can interact with ApxIA. In addition, overexpression and RNA interference experiments revealed that CAV1 was involved in ApxI toxin-induced apoptosis of iPAM cells. This study provided first-hand information about the proteome of iPAM cells interacting with the ApxI toxin of APP through the TurboID proximity labeling system, and identified a new host membrane protein involved in this interaction. These results lay a theoretical foundation for the clinical treatment of PCP.

Prevalence of Streptococci spp. and Unexpected Non-Streptococci Strains Associated with Bovine Mastitis Infection in Dairy Cattle in Region IV-A, Philippines

Bovine mastitis is an inflammatory response of the udder tissue in the mammary gland caused by microbial infections. Streptococcus spp. is among the most prevalent mastitis-inducing etiological agents. Thus, this study intended to isolate and evaluate the prevalence of Streptococci in dairy cattle infected with clinical mastitis in Region IV-A, Philippines. Edward Agar medium with 6% defibrinated sheep blood was employed as a selective medium. The bacterial isolates were phenotypically and genotypically characterized. Remarkably, out of 98 isolates, only 26.5% belonged to the genus Streptococcus despite the use of a Streptococci-specific medium. Five Streptococci species and 22 non-Streptococci species were identified. The most prevalent species were S. uberis (prevalence rate: 11.2%). The antimicrobial resistance profiling also revealed that S. agalactiae exhibited resistance to all antimicrobials used, while S. bovis showed hyper-resistance to five out of seven antibiotics. Surprisingly, most of the non-streptococcal isolates exhibited hyper-resistance to multiple antibiotics. For instance, Klebsiella pneumoniae isolates showed high resistance against all antimicrobials. Proteus and Providencia isolates exhibited resistance against six out of seven antibiotics. Strong hemolytic activity was also observed in Bacillus subtilis. The detection of diverse species of microorganisms causing mastitis is significant to the dairy industry as distinct pathogens may entail different risks and necessitate specific treatments, primarily in terms of the antimicrobials that will be utilized to cure the infection. Application of inappropriate antibiotics might unduly expose the udder microbial flora to antimicrobials, increasing the establishment of multidrug-resistant bacteria, which is a severe hazard to animal and human health.

Genomic Epidemiology and Phenotypic Characterization of Staphylococcus aureus from a Tertiary Hospital in Tianjin Municipality, Northern China.

Staphylococcus aureus remains a dangerous pathogen and poses a great threat to public health worldwide. The prevalence of the S. aureus clonotype is temporally and geographically variable. The genomic and phenotypic characteristics of S. aureus isolates in Tianjin, which is among the four big municipalities in China, are unclear. In the present study, 201 nonduplicate S. aureus isolates, including 70 methicillin-resistant S. aureus (MRSA) and 131 methicillin-susceptible S. aureus (MSSA), were collected from 2015 to 2021 in a tertiary hospital in Tianjin. Whole-genome sequencing of S. aureus isolates was carried out to investigate bacterial molecular characteristics, genomic phylogeny, antimicrobial resistance (AMR) gene carriage, and virulence factor gene distribution. The antibiotic resistance profiles, hemolytic activities, and biofilm formation abilities of the S. aureus isolates were also determined. In total, 31 distinct sequence types (STs) and 68 spa types were identified. ST59 (15.9%, 32/201) was the predominant clonotype, followed by ST398 (14.9%, 30/201) and several other major STs (ST1, ST5, ST6, ST22, ST25, ST188, and the newly emerging ST5527). ST59 and ST5527 mainly included MRSA isolates, while ST398 and the other major STs mainly included MSSA isolates. The unique characteristics of the S. aureus isolates belonging to the major STs were determined. ST59 isolates exhibited strong hemolytic activity, and ST398 strains had high biofilm formation capacity, while ST5527 isolates presented the greatest AMR. The genomic epidemiology and phenotypic characteristics of S. aureus isolates determined in this study will help in disease control in nosocomial environments. IMPORTANCE Staphylococcus aureus is an important bacterium pathogen in tertiary hospitals, which provide rich medical resources. Tianjin is one of the four municipalities in China with a population of more than 13 million. However, the epidemiology and molecular characteristics of S. aureus isolates in Tianjin are unknown. In this study, the genomic and phenotypic analyses were performed to investigate 201 S. aureus isolates collected from a tertiary hospital in Tianjin over a time span of 6 years. The refined analysis of predominant clones ST59, ST398, the newly emerging clone ST5527, as well as other major clones, will undoubtedly aid in the control and prevention of infections caused by S. aureus in tertiary hospitals.

Prevalence and Virulence Factors of Vaginal Candidiasis Among Females Collected in Tertiary Care Hospital, Khyber Pakhtunkhwa

Background: The genus Candida includes about 200 different species, but only a few species are human opportunistic pathogens. Candida albicans is a commensal and opportunistic pathogenic agent that causes infection in immune compromised individuals. This work was conducted to study the detection of virulence factors of Candida spp. in clinical female samples from tertiary care hospitals of Kohat. Methods: This cross-sectional study was conducted during April to July 2017. Patients data and samples were collected from Liaqat Memorial Hospital (LMH), Fauji Foundation Hospital, and Family Health Hospital, Kohat. Morphological identification was done by using different media i.e SDA and PDA. Further identification was done through gram staining and germ tube method. Results: A total of one hundred and sixty (n=160) clinical samples were collected from outdoor patients, in which only 50 were Candida positive. Among all the 50 isolates of 13(26%) Candida spp. were germ tube positive. Further identification were done through Chrome Agar in which 17(34%) isolates were C. albicans, followed by 19(38%) were C. tropicalis, and 4(8%) isolates were C. glabrata. Detection of virulence factors is done by different methods in which 30(60%) isolates showed phospholipase activity in which 12(24%) of Candida spp. were large positive. In haemolytic activity showed 28(56%) strong haemolytic activity, 5(10%) were weak positive. Candida positive patients high 36 (72%) in 21 to 40 years. Conclusion: In the present study, the results concluded that patient infected with Candida are highly virulent as about more than 60% of Phospholipase, Biofilm and Haemolytic positive. Candida species have high prevalence rate among 21 t0 40 years from the rest of very late and early age. Finding novel antibacterial chemicals is highly advised in view of the study's findings. Additionally, it is crucial to assess the candida resistant patterns at the genomic and proteomic levels in order to identify the genes causing the patterns of antibiotic resistance. Keyword: Candida, Virulence Factors, Germ Tube, Chrome Agar, opportunistic pathogens, Haemolytic positive, Gram staining, Germ tube method.

ISOLATION AND CHARACTERIZATION OF BACTERIAL PATHOGENIC Bacillus sp. FROM THE Lethrinus lentjanin COASTAL AREA OF INDIA

India stands at the sixth-largest position in the sector of marine and freshwater fisheries and the second-largest in the production of aquaculture. In view of the economic benefits, this highlights the importance of controlling infectious fish diseases that pose threat to the production of aquaculture. It was hypothesized lesions found on the dorsal scales in the Lenthrinus lentjan, could be the reason for the massive morbidity and mortality. Live surface floating fish samples with pathological signs were collected from offshore areas of the Pallam fishing village, located in the Kanyakumari District of Tamil Nadu, India in August 2013. Detailed histological studies confirmed the presence of similar bacterial infections in the tissues adjacent to the lesion sites. The pathogen was isolated and it was subjected to a series of biochemical and 16S RNA sequencing and it was identified the causative bacterial pathogen as Bacillus sp. The pathogen possessed sub-terminal endospore-forming nature and also it has strong hemolytic activities. The organism was named as Bacillus sp. MSU1400. Furtherly, the study was complemented with the Pasteur method, performed on white carp fishes, in vitro, for the confirmation of the pathogen. The pathogen was sensitive to ampicillin, cephalexin, and gentamycin but it was resistant to tetracycline.

Enzymatic and antifungal susceptibility profiles of Candida glabrata isolates from paediatric patients and their genetic diversity based on microsatellite length polymorphism.

This study aimed to detect different genotypes of Candida glabrata isolates in paediatric patients with and without neutropenia utilizing microsatellite length polymorphism (MLP) and its correlation with drug resistance and enzymatic activity were assessed. Samples from neutropenic and non-neutropenic patients were collected from November 2020 to November 2021. Thirty-six C. glabrata strains were isolated and identified using classical and molecular methods. Then, C. glabrata isolates were genotyped by the MLP technique, and their antifungal susceptibility was performed based on the CLSI M27 guideline. Eighteen different multi-loci genotypes (G1-G18) were detected based on MLP analysis. Analysis of molecular variance revealed high genetic variation within populations (94%) and low genetic differentiation amongst populations (6%). Also, 40% (n = 4) of isolates from neutropenic patients were non-wild-type for posaconazole, and 30% (n = 3) were resistant to caspofungin. Very strong hemolytic and proteinase activity were seen in 97·2 and 86·1% of isolates. Candida glabrata strains from neutropenic patients were genetically divergent from other populations. The minimum spanning tree shows that observed genotypes were mainly related to previously reported genotypes from Iran, Spain, and China.

Analysis and Characterization of Chitinase in Bacillus salmalaya Strain 139SI

Introduction: Chitinases are enzymes that hydrolyze the internal β-1,4 glycosidic linkages of chitin, a significant structural component of arthropod exoskeletons and fungus cell walls. Objective: Main objective of current study was to determine the protein and to check the antimicrobial, antifungal activity of chitinase in Bacillus salmalaya strain 139SI. Methodology: Purified and estimation enzymes were quantified by the method of Lowry method. Antimicrobial action of chitinase was done using standard Disc Diffusion method while the hyphal extension inhibition assay was used to test the antifungal activity of pure chitinase strains 139SI, 140SI, and 141SI. Results: In this study, Bacillus salmalaya 139SIexhibited strong hemolytic activity and their protein concentration was measured as 56.43 mg/mL. In addition, strain 139SI had strong antifungal activity against phytopathogenic fungus including Fusarium sp., R. solani and Phytophthora sp. Strain 139SI had the capability in degrading the peptidoglycan component of cell walls of gram-negative bacteria such as Escherichia coli but not against the gram-positive, Staphylococcus aureus. Chitinase activity was observed when 200μl crude extract of 139SI able to degrade 0.09 g chitin of shrimp shell by breaking down shrimp shell structure and bonds of chitin effectively as early as 2 days or up to 7 days. Conclusion: Hence, based on the results, B. salmalaya 139SI has potential to be a novel biofunctional chitinase that could use as a biological agent in degrading the chitin component of fungal cell walls and shells waste of many kind of insect and crustaceans for solving future problem in the agricultural sector and fishery industry.

Chitinase Activity by Chitin Degrading Strain (Bacillus Salmalaya) in Shrimp Waste

Introduction: Chitin degradation by chitinase enzyme can be used on a large scale for bioremediation of seafood waste and is environmentally friendly. Objective: The main aim of this study was to screen the potential of the strain as a chitin degrading agent. Methods: In this present study, the cell-free supernatant of Bacillus salmalaya was determined for its protein concentration. Bacillus salmalaya139SI was isolated from agricultural soil and it was identified by staining technique and colony morphology. The production of chitinase by Bacillus salmalaya139SI was optimized under different concentrations of substrate, pH and temperature. Results: Strain 139SI exhibited strong hemolytic activity and the crude protein concentration of Bacillus salmalaya was 84.09mg/ mL with OD value 0.462. Strain 139SI were also screened on colloidal chitin agar medium supplemented with mineral salt. Chikinase production was determined by clear zones of hydrolysis produced after 7 days of incubation at 37°C. The maximum chitinase production was observed in Brain Heart Infusion broth supplemented with 1.0% colloidal chitin at pH 7 and temperature 35°C after four days of incubation. Chitinase activity was observed when high concentrations of crude extract of 139SI able to degrade shrimp shell by showing the degradation zone at day 4. Conclusion: From the results, we concluded that the Bacillus salmalayahas potential to be a biofunctional chitinase that could degrade complex polysaccharides present in the organic wastes and applicable in cleaning the environment.

Monodesmosidic oleanene-type saponins from kidney vetch (Anthyllis vulneraria L.) with hemolytic activity

• Two saponins (anthylloside A and B) were isolated from Anthyllis vulneraria L. • First detection of saikosaponins in the legume (Fabaceae) family. • Saponin structures were assigned by 1D- and 2D-NMR and HR-ESI-MS experiments. • Anthylloside A exhibits strong hemolytic activity in a blood agar assay. Two undescribed monodesmosidic oleanene-type saponins, namely 3 β - O -{[ β -D-glucopyranosyl-(1→4)]- α -L-arabinopyranosyl}-saikogenin G ( 1 ) and 3 β -O-{[ β -D-glucopyranosyl-(1→4)]- α -L-arabinopyranosyl}-16-deoxysaikogenin F ( 2 ), named anthylloside A and B, have been isolated from an acetone-water extract from aerial parts of kidney vetch ( A. vulneraria L.). Isolation of 1 and 2 was achieved by solvent partition with EtOAc and subsequent repeated chromatographic purification. The structures of the isolated compounds were elucidated by spectrometric (HR-MS) and spectroscopic methods ( 1 H- and 13 C-NMR, UV, IR and CD), as well as GC–MS analysis of samples after 1 N HCl hydrolysis and subsequent derivatization. The configuration and conformation of both 1 and 2 were assigned by means of comprehensive 2D-NMR analyses including 1 H- 1 H−COSY, ROESY, decoupled g HSQC, g HMBC and selective 1D-TOCSY experiments. Moreover, 1 and 2 possess significant hemolytic activity, which was assessed in a blood agar assay and compared to that of three reference saponins.

Potent Activity of Hybrid Arthropod Antimicrobial Peptides Linked by Glycine Spacers.

Arthropod antimicrobial peptides (AMPs) offer a promising source of new leads to address the declining number of novel antibiotics and the increasing prevalence of multidrug-resistant bacterial pathogens. AMPs with potent activity against Gram-negative bacteria and distinct modes of action have been identified in insects and scorpions, allowing the discovery of AMP combinations with additive and/or synergistic effects. Here, we tested the synergistic activity of two AMPs, from the dung beetle Copris tripartitus (CopA3) and the scorpion Heterometrus petersii (Hp1090), against two strains of Escherichia coli. We also tested the antibacterial activity of two hybrid peptides generated by joining CopA3 and Hp1090 with linkers comprising two (InSco2) or six (InSco6) glycine residues. We found that CopA3 and Hp1090 acted synergistically against both bacterial strains, and the hybrid peptide InSco2 showed more potent bactericidal activity than the parental AMPs or InSco6. Molecular dynamics simulations revealed that the short linker stabilizes an N-terminal 310-helix in the hybrid peptide InSco2. This secondary structure forms from a coil region that interacts with phosphatidylethanolamine in the membrane bilayer model. The highest concentration of the hybrid peptides used in this study was associated with stronger hemolytic activity than equivalent concentrations of the parental AMPs. As observed for CopA3, the increasing concentration of InSco2 was also cytotoxic to BHK-21 cells. We conclude that AMP hybrids linked by glycine spacers display potent antibacterial activity and that the cytotoxic activity can be modulated by adjusting the nature of the linker peptide, thus offering a strategy to produce hybrid peptides as safe replacements or adjuncts for conventional antibiotic therapy.

Fine-Tuning of Alkaline Residues on the Hydrophilic Face Provides a Non-toxic Cationic α-Helical Antimicrobial Peptide Against Antibiotic-Resistant ESKAPE Pathogens.

Antibiotic-resistant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) has become a serious threat to public health worldwide. Cationic α-helical antimicrobial peptides (CαAMPs) have attracted much attention as promising solutions in post-antibiotic era. However, strong hemolytic activity and in vivo inefficacy have hindered their pharmaceutical development. Here, we attempt to address these obstacles by investigating BmKn2 and BmKn2-7, two scorpion-derived CαAMPs with the same hydrophobic face and a distinct hydrophilic face. Through structural comparison, mutant design and functional analyses, we found that while keeping the hydrophobic face unchanged, increasing the number of alkaline residues (i.e., Lys + Arg residues) on the hydrophilic face of BmKn2 reduces the hemolytic activity and broadens the antimicrobial spectrum. Strikingly, when keeping the total number of alkaline residues constant, increasing the number of Lys residues on the hydrophilic face of BmKn2-7 significantly reduces the hemolytic activity but does not influence the antimicrobial activity. BmKn2-7K, a mutant of BmKn2-7 in which all of the Arg residues on the hydrophilic face were replaced with Lys, showed the lowest hemolytic activity and potent antimicrobial activity against antibiotic-resistant ESKAPE pathogens. Moreover, in vivo experiments indicate that BmKn2-7K displays potent antimicrobial efficacy against both the penicillin-resistant S. aureus and the carbapenem- and multidrug-resistant A. baumannii, and is non-toxic at the antimicrobial dosages. Taken together, our work highlights the significant functional disparity of Lys vs Arg in the scorpion-derived antimicrobial peptide BmKn2-7, and provides a promising lead molecule for drug development against ESKAPE pathogens.

Toxicity of Jegosaponins A and B from Styrax japonica Siebold et al. Zuccarini in Prostate Cancer Cells and Zebrafish Embryos Resulting from Increased Membrane Permeability.

(1) Background: Screening of medicinal herbs is one of the most powerful approaches to identifying novel therapeutic molecules against many human diseases. To avoid potential harmful effects during medicinal use, toxicity testing is necessary in the early stages of drug discovery. The objective of this study was to identify the cytotoxic mechanisms of jegosaponin A and B from Styrax japonica Siebold et al. Zuccarini; (2) Methods: We screened Japanese medicinal herb extracts using PC-3 prostate cancer cells and found that a methanol extract isolated from the unripe fruit of Styrax japonica Siebold et al. Zuccarini (SJSZ) had an inhibitory effect on cell viability. We further performed fractionation assays with PC-3 cells and identified the bioactive compounds using LC/MS and NMR analysis. We clarified the toxic mechanisms of these compounds using PC-3 cells and zebrafish embryos; (3) Results: We identified two active molecules, jegosaponin A and jegosaponin B, in the inhibitory fractions of the methanol extract. These jegosaponins are toxic to zebrafish embryos during the early developmental stage. Jegosaponin A and B showed strong haemolytic activity in sheep defibrinated blood (EC50 = 2.1 μM, and 20.2 μM, respectively) and increased the cell membrane permeability in PC-3 cells and zebrafish embryos, which were identified using a membrane non-permeable DRAQ7, a fluorescent nucleus staining dye; (4) We identified the cytotoxic compounds jegosaponin A and B from SJSZ, which we showed to exhibit cell membrane disruptive properties using cell- and zebrafish-based testing.

Genetic Basis Underlying the Hyperhemolytic Phenotype of Streptococcus agalactiae Strain CNCTC10/84.

Streptococcus agalactiae (group B streptococcus [GBS]) is a major cause of infections in newborns, pregnant women, and immunocompromised patients. GBS strain CNCTC10/84 is a clinical isolate that has high virulence in animal models of infection and has been used extensively to study GBS pathogenesis. Two unusual features of this strain are hyperhemolytic activity and hypo-CAMP factor activity. These two phenotypes are typical of GBS strains that are functionally deficient in the CovR-CovS two-component regulatory system. A previous whole-genome sequencing study found that strain CNCTC10/84 has intact covR and covS regulatory genes. We investigated CovR-CovS regulation in CNCTC10/84 and discovered that a single-nucleotide insertion in a homopolymeric tract in the covR promoter region underlies the strong hemolytic activity and weak CAMP activity of this strain. Using isogenic mutant strains, we demonstrate that this single-nucleotide insertion confers significantly decreased expression of covR and covS and altered expression of CovR-CovS-regulated genes, including that of genes encoding β-hemolysin and CAMP factor. This single-nucleotide insertion also confers significantly increased GBS survival in human whole blood ex vivoIMPORTANCE Group B streptococcus (GBS) is the leading cause of neonatal sepsis, pneumonia, and meningitis. GBS strain CNCTC10/84 is a highly virulent blood isolate that has been used extensively to study GBS pathogenesis for over 20 years. Strain CNCTC10/84 has an unusually strong hemolytic activity, but the genetic basis is unknown. In this study, we discovered that a single-nucleotide insertion in an intergenic homopolymeric tract is responsible for the elevated hemolytic activity of CNCTC10/84.

SpyCatcher-SpyTagged ApxIA Toxoid and the Immune-Modulating Yeast Ghost Shells.

Actinobacillus pleuropneumoniaesecretes the hemolytic cytotoxins ApxI, ApxII, ApxIII, and ApxIV, which cause pleurop- neumonia in swine. Of these, ApxI is the most toxic. ApxIA, a repeats-in-toxin toxin-like protein, has strong hemolytic and cytotoxic activities. This study aimed to develop an immune modulator ApxIA toxoid, with a Spytag/Spycatcher pair (SC::ST pair), in yeast ghost shells (YGSs). These YGSs were utilized as ApxIA toxoid delivery platforms for -glucan components that can be recognized by the innate immune system. The SC::ST pair was used to conjugate the ApxIA toxoid to YGSs. The YGS-SC::ST-ToxApxIA was successfully phagocytosed by RAW 264.7 macrophages cells, without any toxicity. Further investigation revealed that YGS-SC::ST-ToxApxIA led to defective immune responses, in addition to increased levels of cytokines IL-1β, TNF-α, and IL-10. A membrane proteomic analysis, to determine preferential major histocompatibility complex binding of ApxIA-derived peptides, was performed and four ApxIA peptides were successfully identified by liquid chromatography with tandem mass spectrometry analysis. The identified peptides may serve as poten- tial vaccine candidates in immunobiology studies of A. pleuropneumoniae. Our results indicate that YGS-SC::ST-ToxApxIA can prevent A. pleuropneumoniae pleuropneumonia (APP) by inducing both humoral and cellular immune responses.

Discovery of gramicidin A analogues with altered activities by multidimensional screening of a one-bead-one-compound library

Gramicidin A (1) is a peptide antibiotic that disrupts the transmembrane ion concentration gradient by forming an ion channel in a lipid bilayer. Although long used clinically, it is limited to topical application because of its strong hemolytic activity and mammalian cytotoxicity, likely arising from the common ion transport mechanism. Here we report an integrated high-throughput strategy for discovering analogues of 1 with altered biological activity profiles. The 4096 analogue structures are designed to maintain the charge-neutral, hydrophobic, and channel forming properties of 1. Synthesis of the analogues, tandem mass spectrometry sequencing, and 3 microscale screenings enable us to identify 10 representative analogues. Re-synthesis and detailed functional evaluations find that all 10 analogues share a similar ion channel function, but have different cytotoxic, hemolytic, and antibacterial activities. Our large-scale structure-activity relationship studies reveal the feasibility of developing analogues of 1 that selectively induce toxicity toward target organisms.

Factors associated with the isolation of Candida Spp. from the oral microbiota of people living with HIV

This study aimed to analyze the association between the isolation of Candida species in the oral cavity and the clinical and virulence factors of people living with Human immunodeficiency virus (HIV). This cross-sectional study, comprising 106 people living with HIV who were evaluated in an HIV-AIDS outpatient clinic in Fortaleza, Northeast of Brazil, data collection were conducted during August and November 2018. A saliva sample was also collected to isolate Candida. The virulence factors of extracellular and hemolytic phospholipase activities in isolated Candida strains were analyzed. The prevalence of oral Candida was 40.6%, and the presence of oral Candida was associated with smoking habits (p = .005), daily smoking (p = .026), low weight (p = .030), history of hospitalization (p = .014), TCD4 lymphocyte count &lt; 200 cells/mm3 (p = .018), and detectable viral load (p = .005). The multivariate analysis revealed that the variables smoking habits (p = .008), viral load (p = .011), and body mass index (p = .011) were associated with the presence of oral Candida. Strong hemolytic and phospholipase activities were, respectively, identified in 74.4% and 72% of the isolated strains, mainly Candida albicans. As a main conclusion, the isolation of Candida from the oral cavity in people living with HIV/AIDS, has a strong association with the use of tobacco, even under anappropriate treatment.

Improved Cell Selectivity of Symmetric α‐Helical Peptides Derived From Trp‐Rich Antimicrobial Peptides

Although Trp‐rich antimicrobial peptides (TRAMPs) such as tritrpticin and indolicidin exert potent antimicrobial activity, they have several drawbacks in therapeutic application, such as low cell selectivity and strong hemolytic activity. Here, we designed α‐helical TRAMPs with symmetric amino acid sequences based on pseudo‐symmetric tritrpticin and indolicidin. Compared with the parental pseudo‐symmetric TRAMPs, the symmetric α‐helical TRAMPs were highly effective against Gram‐positive bacteria, including clinically isolated methicillin‐resistant Staphylococcus aureus and vancomycin‐resistant Enterococcus faecium. The symmetric α‐helical TRAMPs exhibited negligible or very low toxicity against mammalian cells, including human erythrocytes. The symmetric peptides caused a rapid collapse of bacterial membrane potential and destabilized negatively charged lipid membranes, indicating that they act on the bacterial cytoplasmic membrane as a primary mode of action. Taken together, these findings imply that symmetric TRAMPs with an α‐helical structure offer significant potential for development as novel therapeutics to overcome the problem of antibiotic resistance in Gram‐positive pathogens.

A preliminary study on the allelopathy and toxicity of the dinoflagellate Karlodinium veneficum

The dinoflagellate Karlodinium veneficum has worldwide distribution and is associated with harmful algal blooms through the production of karlotoxins. We investigated the allelopathy and toxicity to explore the potential ecological implications. Prorocentrum donghaiense was inhibited significantly when grown either in co-cultures or in culture filtrate of K. veneficum. In addition, the effect of the co-occurring microalga species (P. donghaiense) on the hemolytic activity of K. veneficum was also evaluated. P. donghaiense did not inhibit the growth of K. veneficum but increased the hemolytic activity. The culture of K. veneficum was loaded onto an RP-C18 column and eluted with different percentages of aqueous methanol solution. 80% methanol fraction not only inhibited the growth of P. donghaiense by allelopathy but also exhibited strong hemolytic activity, indicating that the allelochemicals and toxins of K. veneficum might be the same components. Furthermore, KmTx 3 (C68H124O24) was identified using HPLC-HRMS from this fraction.