Bacterial Hemolysins Research Articles

Citral and triclosan synergistically silence quorum sensing and potentiate antivirulence response in Pseudomonas aeruginosa.

Among the ESKAPE pathogens, Pseudomonas aeruginosa is an extensively notorious superbug that causes difficult-to-treat infections. Since quorum sensing (QS) directly promotes pseudomonal virulence, targeting QS circuits is a promising approach for disarming phenotypic virulence. Hence, this study scrutinizes the anti-QS, antivirulence, and anti-biofilm potential of citral (CiT; phytochemical) and triclosan (TcN; disinfectant), alone and in combination, against P. aeruginosa PAO1/PA14. The findings confirmed synergism between CiT and TcN and revealed their quorum quenching (QQ) potential. At sub-inhibitory levels, CiT-TcN combination significantly impeded pyocyanin, total bacterial protease, hemolysin, and pyochelin production alongside inhibiting biofilm formation in P. aeruginosa. Moreover, the QQ and antivirulence potential of CiT and TcN was positively correlated by molecular docking studies that predicted strong associations of the drugs with QS receptors of P. aeruginosa. Collectively, the study identifies CiT-TcN as an effective drug combination that harbors QQ, antivirulence, and anti-biofilm prospects against P. aeruginosa.

Attenuation of a DNA cruciform by a conserved regulator directs T3SS1 mediated virulence in Vibrio parahaemolyticus.

Pathogenic Vibrio species account for 3-5 million annual life-threatening human infections. Virulence is driven by bacterial hemolysin and toxin gene expression often positively regulated by the winged helix-turn-helix (wHTH) HlyU transcriptional regulator family and silenced by histone-like nucleoid structural protein (H-NS). In the case of Vibrio parahaemolyticus, HlyU is required for virulence gene expression associated with type 3 Secretion System-1 (T3SS1) although its mechanism of action is not understood. Here, we provide evidence for DNA cruciform attenuation mediated by HlyU binding to support concomitant virulence gene expression. Genetic and biochemical experiments revealed that upon HlyU mediated DNA cruciform attenuation, an intergenic cryptic promoter became accessible allowing for exsA mRNA expression and initiation of an ExsA autoactivation feedback loop at a separate ExsA-dependent promoter. Using a heterologous E. coli expression system, we reconstituted the dual promoter elements which revealed that HlyU binding and DNA cruciform attenuation were strictly required to initiate the ExsA autoactivation loop. The data indicate that HlyU acts to attenuate a transcriptional repressive DNA cruciform to support T3SS1 virulence gene expression and reveals a non-canonical extricating gene regulation mechanism in pathogenic Vibrio species.

Результаты выявления поливирулентных штаммов бактерий Lactobacillus spp. при ассоциативном взаимодействии с простейшими Blastocystis hominis in vivo

The identification of polyvirulent strains of Lactobacillus spp. bacteria with associative interaction with protozoa Blastocystis hominis in vivo was carried out. Lactobacillus spp. and B. hominis strains were obtained from the feces of 396 patients undergoing examination for intestinal dysbiosis. Identification was carried out using microscopic, bacteriological and parasitological methods. 112 reference strains of lactobacilli NK1 and K3SH24 deposited at the Institute of Genetics and Breeding of Industrial Microorganisms were used as control. The degree of virulence of the simplest blastocysts was determined by intraperitoneal administration to white mice (weighing 17.4+1.5 g) 0.5 ml of a culture suspension of the studied microorganisms grown on Suresh medium. Primers to several genes determining the ability to form type 1 fimbriae, type S and P fimbriae, bacterial adhesin intimin and hemolysin were used in the investigation. Testing of 396 Lactobacillus spp. strains isolated from microsymbiocenoses with Blastocystis hominis of varying degrees of virulence showed that the dynamics of detection of pathogenicity genes in lactobacilli increased with an increase in the degree of virulence of protozoa. Most often, in the general pool of lactobacillus strains, the desired amplicons were detected using primers to the fimA gene (up to 67.9%). During associative interaction with moderately and highly virulent blastocysts, an increase in the heterogeneity of the lactobacillus population was revealed, manifested by an increase in the frequency of detection of all studied genetic determinants of pathogenicity, compared with Lactobacillus spp. strains isolated from associations with avirulent B. hominis and in the control group.

Virulence and Antimicrobial Resistance in Canine Staphylococcus spp. Isolates.

Dogs are reservoirs of different Staphylococcus species, but at the same time, they could develop several clinical forms caused by these bacteria. The aim of the present investigation was to characterize 50 clinical Staphylococcus isolates cultured from sick dogs. Bacterial species determination, hemolysins, protease, lipase, gelatinase, slime, and biofilm production, presence of virulence genes (lukS/F-PV, eta, etb, tsst, icaA, and icaD), methicillin resistance, and antimicrobial resistance were investigated. Most isolates (52%) were Staphylococcus pseudointermedius, but 20% and 8% belonged to Staphylococcus xylosus and Staphylococcus chromogenes, respectively. Gelatinase, biofilm, and slime production were very common characters among the investigated strains with 80%, 86%, and 76% positive isolates, respectively. Virulence genes were detected in a very small number of the tested strains. A percentage of 14% of isolates were mecA-positive and phenotypically-resistant to methicillin. Multi-drug resistance was detected in 76% of tested staphylococci; in particular, high levels of resistance were detected for ampicillin, amoxicillin, clindamycin, and erythromycin. In conclusion, although staphylococci are considered to be opportunistic bacteria, the obtained data showed that dogs may be infected by Staphylococcus strains with important virulence characteristics and a high antimicrobial resistance.

Assessment of genetically modified soybean SYHT0H2 for food and feed uses, import and processing, under Regulation (EC) No 1829/2003 (application EFSA-GMO-DE-2012-111).

The scope of application EFSA‐GMO‐DE‐2012‐111 is for food and feed uses, import and processing of genetically modified (GM) soybean SYHT0H2 in the European Union. Soybean SYHT0H2 was developed to confer tolerance to the herbicidal active substances mesotrione and other p‐hydroxyphenylpyruvate dioxygenase (HPPD)‐inhibiting herbicides and glufosinate ammonium. The molecular characterisation data and bioinformatic analyses do not identify issues except for sequence similarity of AvHPPD‐03 to bacterial haemolysins that was considered in food/feed safety assessment. The outcome of the comparative analysis (agronomic/phenotypic and compositional characteristics) did not need further assessment except for the changes in seed levels of α‐tocopherol and γ‐tocopherol that were assessed for food and feed relevance. The GMO Panel does not identify toxicological and allergenicity concerns for the AvHPPD‐03 and PAT proteins expressed in soybean SYHT0H2 and finds no evidence that the genetic modification would change the overall allergenicity of soybean SYHT0H2. The nutritional impact of food/feed from soybean SYHT0H2 is expected to be the same as that of food/feed from the conventional counterpart and commercial non‐GM soybean reference varieties. The GMO Panel concludes that soybean SYHT0H2 is as safe as and nutritionally equivalent to the conventional counterpart and the tested non‐GM soybean reference varieties, and no post‐market monitoring of food/feed is considered necessary. In the case of accidental release of viable soybean SYHT0H2 grains into the environment, soybean SYHT0H2 would not raise environmental safety concerns. The post‐market environmental monitoring plan and reporting intervals are in line with the intended uses of soybean SYHT0H2. In conclusion, the GMO Panel considers that soybean SYHT0H2, as described in this application, is as safe as its conventional counterpart and the tested non‐GM soybean reference varieties with respect to potential effects on human and animal health and the environment.

When might transferrin, hemopexin or haptoglobin administration be of benefit following the transfusion of red blood cells?

After transfusion, a percentage of red blood cells undergo hemolysis within macrophages. Intravascular exposures to hemin and hemoglobin (Hb) can occur after storage bag hemolysis, some transfusion reactions, during use of medical assist devices and in response to bacterial hemolysins. Proteins that regulate iron, hemin and Hb either become saturated after iron excess (transferrin, Tf) or depleted after hemin (hemopexin, Hpx) and Hb (haptoglobin, Hp) excess. Protein saturation or stoichiometric imbalance created by transfusion increases exposure to non-Tf bound iron, hemin and Hb. Tf, Hpx and Hp are being developed for hematological disorders where iron, hemin and Hb contribute to pathophysiology. However, complexed to their ligands, each represents a potential iron source for pathogens, which may complicate the use of these proteins. Erythrophagocytosis by macrophages and processes of cell death that lead to reactive iron exposure are increasingly described. In addition, the effects of transfusion introduced circulatory hemin and Hb are described in the literature, particularly following large volume transfusion, infection and during concomitant medical device use. Supplementation with Tf, Hpx and Hp suggests therapeutic potential in conditions of extravascular/intravascular hemolysis. However, their administration following transfusion may require careful assessment of concomitant disease.

Action of antibiotic oxacillin on invitro growth of methicillin-resistant Staphylococcus aureus (MRSA) previously treated with homeopathic medicines.

Resistance to antibiotics is a major public health concern worldwide. New treatment options are needed and homeopathy is one such option. We sought to assess the effect of the homeopathic medicine Belladonna (Bell) and a nosode (biotherapy) prepared from a multi-drug resistant bacterial species, methicillin-resistant Staphylococcus aureus (MRSA), on the same bacterium. Bell and MRSA nosode were prepared in 6cH and 30cH potencies in 30% alcohol and sterile water, according to the Brazilian Homeopathic Pharmacopeia and tested on MRSA National Collection of Type Cultures (NCTC) 10442. We assessed invitro bacterial growth, deoxyribonuclease (DNAase) and hemolysin activity, and invitro bacterial growth in combination with oxacillin (minimum inhibitory concentration - MIC). All values were compared to control: 30% alcohol and water. Invitro growth of MRSA was statistically significantly inhibited in the presence of Bell and nosode 6cH and 30cH compared to controls (p<0.0001); and with combination of Bell or nosode 6cH and 30cH and oxacillin (p<0.001). Bell 30cH and nosode 6cH and 30cH significantly decreased bacterial DNAse production (p<0.001) and reduced red blood cell lysis. Cultures of MRSA treated with Belladonna or MRSA nosode exhibited reduced growth invitro, reduced enzymatic activity and became more vulnerable to the action of the antibiotic oxacillin. Further studies are needed on the biomolecular basis of these effects.

Coexpression of human perforin improves yeast-mediated delivery of DNA and mRNA to mammalian antigen-presenting cells.

Previous studies underlined the capacity of recombinant yeast as efficient vehicle for the targeted delivery of functional nucleic acids as well as proteinaceous antigens to mammalian antigen-presenting cells (APCs). To improve this yeast-mediated cargo transport into APCs, we investigated the impact of coexpression of the human membrane-perturbing protein perforin in comparison with bacterial listeriolysin O (LLO) on the yeast-based delivery of DNA, mRNA and proteins to mammalian APCs. In contrast to LLO, a cholesterol-dependent pore-forming toxin of Listeria, intracellular expression of human perforin in Saccharomyces cerevisiae had no impact on yeast cell viability, while its coexpression significantly increased translocation of ovalbumin and subsequent activation of ovalbumin-specific T lymphocytes. Likewise, perforin improved the expression of the model antigen enhanced green fluorescent protein after yeast-mediated DNA and mRNA delivery, whereas LLO was only able to enhance DNA delivery. Taken together, our data show that human perforin, besides bacterial hemolysins, represents a promising means to improve the yeast-mediated delivery of functional nucleic acids and proteins to mammalian APCs.

Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans.

Pathogenic members of the genus Leptospira are the causative agents of leptospirosis, a neglected disease of public and veterinary health concern. Leptospirosis is a systemic disease that in its severest forms leads to renal insufficiency, hepatic dysfunction, and pulmonary failure. Many strains of Leptospira produce hemolytic and sphingomyelinase activities, and a number of candidate leptospiral hemolysins have been identified based on sequence similarity to well-characterized bacterial hemolysins. Five of the putative hemolysins are sphingomyelinase paralogs. Although recombinant forms of the sphingomyelinase Sph2 and other hemolysins lyse erythrocytes, none have been demonstrated to contribute to the hemolytic activity secreted by leptospiral cells. In this study, we examined the regulation of sph2 and its relationship to hemolytic and sphingomyelinase activities produced by several L. interrogans strains cultivated under the osmotic conditions found in the mammalian host. The sph2 gene was poorly expressed when the Fiocruz L1-130 (serovar Copenhageni), 56601 (sv. Lai), and L495 (sv. Manilae) strains were cultivated in the standard culture medium EMJH. Raising EMJH osmolarity to physiological levels with sodium chloride enhanced Sph2 production in all three strains. In addition, the Pomona subtype kennewicki strain LC82-25 produced substantially greater amounts of Sph2 during standard EMJH growth than the other strains, and sph2 expression increased further by addition of salt. When 10% rat serum was present in EMJH along with the sodium chloride supplement, Sph2 production increased further in all strains. Osmotic regulation and differences in basal Sph2 production in the Manilae L495 and Pomona strains correlated with the levels of secreted hemolysin and sphingomyelinase activities. Finally, a transposon insertion in sph2 dramatically reduced hemolytic and sphingomyelinase activities during incubation of L. interrogans at physiologic osmolarity. Complementation of the mutation with the sph2 gene partially restored production of hemolytic and sphingomyelinase activities. These results indicate that the sph2 gene product contributes to the hemolytic and sphingomyelinase activities secreted by L. interrogans and most likely dominates those functions under the culture condition tested.

Diverse cellular and organismal functions of the lysosomal thiol reductase GILT.

Gamma-interferon-inducible lysosomal thiol reductase (GILT) is the only enzyme known to catalyze disulfide bond reduction in the endocytic pathway. GILT facilitates the presentation of a subset of epitopes from disulfide bond-containing antigens. Enhanced presentation of MHC class II-restricted epitopes alters central tolerance and modulates CD4+ T cell-mediated autoimmunity. Improved cross-presentation of viral epitopes results in improved cross-priming of viral-specific CD8+ T cells. GILT regulates the cellular redox state. In GILT-/- cells, there is a shift from the reduced to the oxidized form of glutathione, resulting in mitochondrial autophagy, decreased superoxide dismutase 2, and elevated superoxide levels. GILT expression diminishes cellular activation, including decreased phosphorylated ERK1/2, and decreases cellular proliferation. GILT enhances the activity of bacterial hemolysins, such as listeriolysin O, and increases bacterial replication and infection. GILT expression in cancer cells is associated with improved patient survival. These diverse roles of GILT are discussed.

Measurement of the red blood cell lysis by bacterial hemolysin

Measurement of the red blood cell lysis by bacterial hemolysin

Erythrocyte lysis and Xenopus laevis oocyte rupture by recombinant Plasmodium falciparum hemolysin III.

Malaria kills more than 1 million people per year worldwide, with severe malaria anemia accounting for the majority of the deaths. Malaria anemia is multifactorial in etiology, including infected erythrocyte destruction and decrease in erythrocyte production, as well as destruction or clearance of noninfected erythrocytes. We identified a panspecies Plasmodium hemolysin type III related to bacterial hemolysins. The identification of a hemolysin III homologue in Plasmodium suggests a potential role in host erythrocyte lysis. Here, we report the first characterization of Plasmodium falciparum hemolysin III, showing that the soluble recombinant P. falciparum hemolysin III is a pore-forming protein capable of lysing human erythrocytes in a dose-, time-, and temperature-dependent fashion. The recombinant P. falciparum hemolysin III-induced hemolysis was partially inhibited by glibenclamide, a known channel antagonist. Studies with polyethylene glycol molecules of different molecular weights indicated a pore size of approximately 3.2 nm. Heterologous expression of recombinant P. falciparum hemolysin III in Xenopus oocytes demonstrated early hypotonic lysis similar to that of the pore-forming aquaporin control. Live fluorescence microscopy localized transfected recombinant green fluorescent protein (GFP)-tagged P. falciparum hemolysin III to the essential digestive vacuole of the P. falciparum parasite. These transfected trophozoites also possessed a swollen digestive vacuole phenotype. Native Plasmodium hemolysin III in the digestive vacuole may contribute to lysis of the parasitophorous vacuole membrane derived from the host erythrocyte. After merozoite egress from infected erythrocytes, remnant P. falciparum hemolysin III released from digestive vacuoles could potentially contribute to lysis of uninfected erythrocytes to contribute to severe life-threatening anemia.

Transfusion of older stored blood worsens outcomes in canines depending on the presence and severity of pneumonia

In experimental pneumonia we found that transfused older blood increased mortality and lung injury that was associated with increased in vivo hemolysis and elevated plasma cell-free hemoglobin (CFH), non-transferrin-bound iron (NTBI), and plasma labile iron (PLI) levels. In this study, we additionally analyze identically treated animals that received lower or higher bacterial doses. Two-year-old purpose-bred beagles (n = 48) challenged intrabronchially with Staphylococcus aureus (0 [n = 8], 1.0 × 10(9) [n = 8], 1.25 × 10(9) [n = 24], and ≥1.5 × 10(9) [n = 8] colony-forming units/kg) were exchange transfused with either 7- or 42-day-old canine universal donor blood (80 mL/kg in four divided doses). The greater increases in CFH with older blood over days after exchange proved relatively independent of bacterial dose. The lesser increases in CFH observed with fresher blood were bacterial dose dependent potentially related to bacterial hemolysins. Without bacterial challenge, levels of CFH, NTBI, and PLI were significantly higher with older versus fresher blood transfusion but there was no significant measurable injury. With higher-dose bacterial challenge, the elevated NTBI and PLI levels declined more rapidly and to a greater extent after transfusion with older versus fresher blood, and older blood was associated with significantly worse shock, lung injury, and mortality. The augmented in vivo hemolysis of transfused older red blood cells (RBCs) appears to result in excess plasma CFH and iron release, which requires the presence of established infection to worsen outcome. These data suggest that transfused older RBCs increase the risks from infection in septic subjects.

Ion Channel Proteins that Spontaneously Insert into Lipid Bilayer Membranes: An Impedance Spectroscopy Study Employing Tethered Membranes

The Chloride Intracellular Ion Channel (CLIC) protein family, the Annexins, the bacterial toxins Hemolysin (AH), Streptolysin, Perfringolysin Listeriolysin, Pneumonlysin, Ivanolysin and Colicins all possess unusual mechanisms for inserting into cellular and/or host membranes. They are representatives of proteins that spontaneously insert into membranes, by-passing the route for the incorporation of most integral membrane proteins. For example, the soluble form of the 240-amino acid polypeptide CLIC1 is known to exist in at least two conformations due to a large rearrangement of its amino terminus under the influence of oxidation. Oxidation promotes its binding to and insertion into lipid bilayers, forming a chloride selective ion channel. Spontaneous membrane insertion also occurs for the 293-amino acid polypeptide, AH which is secreted as a water-soluble monomer, and when in contact with a membrane inserts and forms heptameric pores. The 36-amino acid peptide (pHLIP) which is a truncated sequence of the C-helix of the integral membrane protein bacteriorhodopsin also inserts into lipid bilayers at low pH. In the case of the 316-amino acid polypeptide Annexin B12, reversible insertion into membranes can also occur at acidic pH. In the present study, we report the functional ion channel activity of a number of spontaneously inserting proteins into Tethered Membranes via a novel impedance spectroscopy assay. We report on conditions that promote insertion and determine the ease of reversible dissociation of protein from the membrane. Also reported will be the kinetics of both the insertion and elimination of the protein based on the functional conductance changes of the membrane.

CREST - a large and diverse superfamily of putative transmembrane hydrolases

BackgroundA number of membrane-spanning proteins possess enzymatic activity and catalyze important reactions involving proteins, lipids or other substrates located within or near lipid bilayers. Alkaline ceramidases are seven-transmembrane proteins that hydrolyze the amide bond in ceramide to form sphingosine. Recently, a group of putative transmembrane receptors called progestin and adipoQ receptors (PAQRs) were found to be distantly related to alkaline ceramidases, raising the possibility that they may also function as membrane enzymes.ResultsUsing sensitive similarity search methods, we identified statistically significant sequence similarities among several transmembrane protein families including alkaline ceramidases and PAQRs. They were unified into a large and diverse superfamily of putative membrane-bound hydrolases called CREST (alkaline ceramidase, PAQR receptor, Per1, SID-1 and TMEM8). The CREST superfamily embraces a plethora of cellular functions and biochemical activities, including putative lipid-modifying enzymes such as ceramidases and the Per1 family of putative phospholipases involved in lipid remodeling of GPI-anchored proteins, putative hormone receptors, bacterial hemolysins, the TMEM8 family of putative tumor suppressors, and the SID-1 family of putative double-stranded RNA transporters involved in RNA interference. Extensive similarity searches and clustering analysis also revealed several groups of proteins with unknown function in the CREST superfamily. Members of the CREST superfamily share seven predicted core transmembrane segments with several conserved sequence motifs.ConclusionsUniversal conservation of a set of histidine and aspartate residues across all groups in the CREST superfamily, coupled with independent discoveries of hydrolase activities in alkaline ceramidases and the Per1 family as well as results from previous mutational studies of Per1, suggests that the majority of CREST members are metal-dependent hydrolases.ReviewersThis article was reviewed by Kira S. Markarova, Igor B. Zhulin and Rob Knight.

Streptococcus pyogenes Ser/Thr Kinase-regulated Cell Wall Hydrolase Is a Cell Division Plane-recognizing and Chain-forming Virulence Factor

Cell division and cell wall synthesis are closely linked complex phenomena and play a crucial role in the maintenance and regulation of bacterial virulence. Eukaryotic-type Ser/Thr kinases reported in prokaryotes, including that in group A Streptococcus (GAS) (Streptococcus pyogenes Ser/Thr kinase (SP-STK)), regulate cell division, growth, and virulence. The mechanism of this regulation is, however, unknown. In this study, we demonstrated that SP-STK-controlled cell division is mediated under the positive regulation of secretory protein that possesses a cysteine and histidine-dependent aminohydrolases/peptidases (CHAP) domain with functionally active cell wall hydrolase activity (henceforth named as CdhA (CHAP-domain-containing and chain-forming cell wall hydrolase). Deletion of the CdhA-encoding gene resulted in severe cell division and growth defects in GAS mutants. The mutant expressing the truncated CdhA (devoid of the CHAP domain), although displayed no such defects, it became attenuated for virulence in mice and highly susceptible to cell wall-acting antibiotics, as observed for the mutant lacking CdhA. When CdhA was overexpressed in the wild-type GAS as well as in heterologous strains, Escherichia coli and Staphylococcus aureus, we observed a distinct increase in bacterial chain length. Our data reveal that CdhA is a multifunctional protein with a major function of the N-terminal region as a cell division plane-recognizing domain and that of the C-terminal CHAP domain as a virulence-regulating domain. CdhA is thus an important therapeutic target.

Schizolysin, a hemolysin from the split gill mushroom Schizophyllum commune

Abstract A monomeric hemolysin with a molecular mass of 29 kDa was isolated from fresh fruiting bodies of the split gill mushroom Schizophyllum commune. The hemolysin was purified by successive adsorption on DEAE-cellulose, carboxymethyl-cellulose and Q-Sepharose and finally gel filtration on Superdex 75. This demonstrated the N-terminal sequence ATNYNKCPGA, different from those of previously reported fungal and bacterial hemolysins. The hemolysin was stable up to 40 degrees C. Only partial activity remained at 50 and 60 degrees C. Activity was indiscernible at 70 degrees C. A pH of 6.0 was optimal for activity. The hemolytic activity was most potently inhibited by dithiothreitol, sucrose and raffinose, followed by cellobiose, maltose, rhamnose, inulin, lactose, fructose and inositol. The metal ions Cu(2+), Mg(2+), Zn(2+), Al(3+) and Fe(3+) significantly, and Pb(2+) to a lesser extent, curtailed the activity of the hemolysin. The hemolysin inhibited HIV-1 reverse transcriptase with an IC(50) of 1.8 microM.

Delivery of foreign antigens by engineered outer membrane vesicle vaccines

As new disease threats arise and existing pathogens grow resistant to conventional interventions, attention increasingly focuses on the development of vaccines to induce protective immune responses. Given their admirable safety records, protein subunit vaccines are attractive for widespread immunization, but their disadvantages include poor immunogenicity and expensive manufacture. We show here that engineered Escherichia coli outer membrane vesicles (OMVs) are an easily purified vaccine-delivery system capable of greatly enhancing the immunogenicity of a low-immunogenicity protein antigen without added adjuvants. Using green-fluorescent protein (GFP) as the model subunit antigen, genetic fusion of GFP with the bacterial hemolysin ClyA resulted in a chimeric protein that elicited strong anti-GFP antibody titers in immunized mice, whereas immunization with GFP alone did not elicit such titers. Harnessing the specific secretion of ClyA to OMVs, the ClyA-GFP fusion was found localized in OMVs, resulting in engineered recombinant OMVs. The anti-GFP humoral response in mice immunized with the engineered OMV formulations was indistinguishable from the response to the purified ClyA-GFP fusion protein alone and equal to purified proteins absorbed to aluminum hydroxide, a standard adjuvant. In a major improvement over current practice, engineered OMVs containing ClyA-GFP were easily isolated by ultracentrifugation, effectively eliminating the need for laborious antigen purification from cell-culture expression systems. With the diverse collection of heterologous proteins that can be functionally localized with OMVs when fused with ClyA, this work signals the possibility of OMVs as a robust and tunable technology platform for a new generation of prophylactic and therapeutic vaccines.

Nanopores as Biosensors: DNA Sequencing and Chiral Discrimination

Biosensors are stochastic sensors inspired by biology. They are potentially important for many applications in bionanotechnology, from DNA sequencing to single-molecule detection and even chiral discrimination. While the macromolecular properties of the individual components i.e. sensor and analyte are well-characterised, the intricacies of their interaction are less well understood. For full exploitation of biomolecules as stochastic sensors, detailed knowledge of their interactions with other biological and chemical species is desirable. Thus, we have performed a series of molecular dynamics simulations of the bacterial toxin, alpha hemolysin (aHL) and derivative model pores to address issues such as the mechanism of DNA transport through the pore, and the molecular basis of chiral discrimination when the protein is fitted with a molecular adapter (in this case the cyclic molecule, beta cyclodextrin (bCD)).We study the orientational discrimination of the DNA molecule by restraining the DNA at one end, inside the protein barrel, and applying an electric field. Simulations of the wildtype protein and mutants give good agreement with published experimental data and allow us to explore the molecular basis of discrimination.Our simulations of a model pore (the aHL barrel with only selected sidechains included), allow us to probe the mechanism of DNA threading into the pore once it has already entered the vestibule of the protein. Our results indicate that only key sidechains are required for the interaction with the DNA molecule, and thus have important implications for the future design of engineered protein pores.Our third set of simulations explores the ability of pores fitted with bCD to discriminate between the enantiomers of ibuprofen. We have used simplified models of pores with full atomistic representation of the bCD and ibuprofen molecules to capture the subtleties of their interaction under an applied external field.

Classical Homeopathy Approach in the Treatment of Methicillin-Resistant Staphylococcus aureus

Resistance to antibiotics is a major public health concern worldwide. New treatment options are needed and homeopathy is one such option. We sought to assess the effect of the homeopathic medicine Belladonna (Bell) and a nosode (biotherapy) prepared from a multi-drug resistant bacterial species, methicillin-resistant Staphylococcus aureus (MRSA), on the same bacterium.Bell and MRSA nosode were prepared in 6cH and 30cH potencies in 30% alcohol and sterile water, according to the Brazilian Homeopathic Pharmacopeia and tested on MRSA National Collection of Type Cultures (NCTC) 10442. We assessed in vitro bacterial growth, deoxyribonuclease (DNAase) and hemolysin activity, and in vitro bacterial growth in combination with oxacillin (minimum inhibitory concentration – MIC). All values were compared to control: 30% alcohol and water.In vitro growth of MRSA was statistically significantly inhibited in the presence of Bell and nosode 6cH and 30cH compared to controls (p < 0.0001); and with combination of Bell or nosode 6cH and 30cH and oxacillin (p < 0.001). Bell 30cH and nosode 6cH and 30cH significantly decreased bacterial DNAse production (p < 0.001) and reduced red blood cell lysis.Cultures of MRSA treated with Belladonna or MRSA nosode exhibited reduced growth in vitro, reduced enzymatic activity and became more vulnerable to the action of the antibiotic oxacillin. Further studies are needed on the biomolecular basis of these effects.