Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher's patients, which reaches the order of $ 84 million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr−) cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (~64 and 59 kDa) and secreted (63–69 kDa) form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditional methods of screening high-producing recombinant cells may represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources.
Snake bite is a neglected public health problem in communities in rural areas of several countries. Bothrops jararaca causes many snake bites in Brazil and previous studies have demonstrated that the pharmacological activities displayed by its venom undergo a significant ontogenetic shift. Similarly, the venom proteome of B. jararaca exhibits a considerable variation upon neonate to adult transition, which is associated with changes in diet from ectothermic prey in early life to endothermic prey in adulthood. Moreover, it has been shown that the Brazilian commercial antibothropic antivenom, which is produced by immunization with adult venom, is less effective in neutralizing newborn venom effects. On the other hand, venom gland transcripts of newborn snakes are poorly known since all transcriptomic studies have been carried out using mRNA from adult specimens.
Here we analyzed venom gland cDNA libraries of newborn and adult B. jararaca in order to evaluate whether the variability demonstrated for its venom proteome and pharmacological activities was correlated with differences in the structure of toxin transcripts. The analysis revealed that the variability in B. jararaca venom gland transcriptomes is quantitative, as illustrated by the very high content of metalloproteinases in the newborn venom glands. Moreover, the variability is also characterized by the structural diversity of SVMP precursors found in newborn and adult transcriptomes. In the adult transcriptome, however, the content of metalloproteinase precursors considerably diminishes and the number of transcripts of serine proteinases, C-type lectins and bradykinin-potentiating peptides increase. Moreover, the comparison of the content of ESTs encoding toxins in adult male and female venom glands showed some gender-related differences.
We demonstrate a substantial shift in toxin transcripts upon snake development and a marked decrease in the metalloproteinase P-III/P-I class ratio which are correlated with changes in the venom proteome complexity and pharmacological activities.
Bothrops jararaca is one of the most abundant venomous snake species in Brazil. It is primarily a nocturnal and generalist animal, however, it exhibits a notable ontogenetic shift in diet, feeding mainly on arthropods, lizards, and amphibians (ectothermic prey) through its juvenile phase and on small mammals (endothermic animals) during adult life. Due to its broad geographical distribution, this species is responsible for the majority of the accidents by Bothrops genus in Brazil. Studies on envenomation cases with newborn and adult B. jararaca snakes have shown distinct patterns, mainly related to blood coagulation disorders, which seems to be prominent in accidents with newborn specimens. Moreover, it has been demonstrated that the Brazilian commercial antibothropic antivenom, which is produced by immunization with adult venom, is less effective in neutralizing newborn venom effects. In this study we analyzed the venom gland transcriptome of newborn snake specimens and compared the content of toxin transcripts with that of adult specimens. We demonstrate that upon B. jararaca development, its repertoire of mRNAs encoding toxins changes both qualitatively and quantitatively and these alterations are associated with the venom proteome profiles and pharmacological activities displayed by newborn and adult specimens.
Glyphosate is a herbicide that is liable to be used in the extensive cultivation of glyphosate-tolerant cultivars. The potential accumulation of the relative effect of glyphosate on the rhizobacterial communities of glyphosate-tolerant maize has been monitored over a period of three years.
The composition of rhizobacterial communities is known to vary with soil texture, hence, the analyses have been performed in two agricultural fields with a different soil texture. The accumulative effects of glyphosate have been monitored by means of high throughput DNA pyrosequencing of the bacterial DNA coding for the 16S rRNA hypervariable V6 region from rhizobacterial communities. The relative composition of the rhizobacterial communities does vary in each field over the three-year period. The overall distribution of the bacterial phyla seems to change from one year to the next similarly in the untreated and glyphosate-treated soils in both fields. The two methods used to estimate bacterial diversity offered consistent results and are equally suitable for diversity assessment.
The glyphosate treatment during the three-year period of seasonal cultivation in two different fields did not seem to significantly change the maize rhizobacterial communities when compared to those of the untreated soil. This may be particularly relevant with respect to a potential authorisation to cultivate glyphosate-tolerant maize in the European Union.
Porcine small intestinal submucosa (SIS) of Cook Biotech is licensed and widely used for tissue remodeling in humans. SIS was shown to be highly effective as an adjuvant in model studies with prostate and ovarian cancer vaccines. However, SIS adjuvanticity relative to alum, another important human-licensed adjuvant, has not yet been delineated in terms of activation of innate immunity via inflammasomes and boosting of antibody responses to soluble proteins and hapten-protein conjugates. We used ovalbumin, and a hapten-protein conjugate, phthalate-keyhole limpet hemocyanin. The evaluation of SIS was conducted in BALB/c and C57BL/6 mice using both intraperitoneal and subcutaneous routes. Inflammatory responses were studied by microarray profiling of chemokines and cytokines and by qPCR of inflammasomes-related genes. Results showed that SIS affected cytokine and chemokines microenvironments such as up-regulation of IL-4 and CD30-ligand and activation of chemotactic factors LIX and KC (neutrophil chemotactic factors), MCP-1 (monocytes chemotactic factors), MIP 1-α (macrophage chemotactic factor) and lymphotactin, as well as, growth factors like M-CSF. SIS also promoted gene expression of Nod-like receptors (NLR) and associated downstream effectors. However, in contrast to alum, SIS had no effects on pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) or NLRP3, but it appeared to promote both Th1 and Th2 responses under different conditions. Lastly, it was as effective as alum in engendering a lasting and specific antibody response, primarily of IgG1 type.
In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the Pm promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/Psal system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i) the nasF transcriptional attenuator, which reduces basal expression levels, (ii) a strong ribosome binding site, and (iii) the Type III Secretion System (TTSS) signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies.
Eight DNA extraction products or methods (Applied Biosystems PrepFiler Forensic DNA Extraction Kit; Bio-Rad Instagene Only, Bio-Rad Instagene & Spin Column Purification; EpiCentre MasterPure DNA & RNA Kit; FujiFilm QuickGene Mini80; Idaho Technologies 1-2-3 Q-Flow Kit; MoBio UltraClean Microbial DNA Isolation Kit; Sigma Extract-N-Amp Plant and Seed Kit) were adapted to facilitate extraction of DNA under BSL3 containment conditions. DNA was extracted from 12 common interferents or sample types, spiked with spores of Bacillus atropheaus. Resulting extracts were tested by real-time PCR. No one method was the best, in terms of DNA extraction, across all sample types. Statistical analysis indicated that the PrepFiler method was the best method from six dry powders (baking, biological washing, milk, plain flour, filler and talcum) and one solid (Underarm deodorant), the UltraClean method was the best from four liquids (aftershave, cola, nutrient broth, vinegar), and the MasterPure method was the best from the swab sample type. The best overall method, in terms of DNA extraction, across all sample types evaluated was the UltraClean method.
Invasion of the erythrocyte by the merozoites of the malaria parasite is a
complex process involving a range of receptor-ligand interactions. Two protein
families termed Erythrocyte Binding Like (EBL) proteins and Reticulocyte Binding
Protein Homologues (RH) play an important role in host cell recognition by the
merozoite. In the rodent malaria parasite, Plasmodium yoelii,
the 235 kDa rhoptry proteins (Py235) are coded for by a multigene family and are
members of the RH. In P. yoelii Py235 as well as a single
member of EBL have been shown to be key mediators of virulence enabling the
parasite to invade a wider range of host erythrocytes. One member of Py235,
PY01365 is most abundantly transcribed in parasite
populations and the protein specifically binds to erythrocytes and is recognized
by the protective monoclonal antibody 25.77, suggesting a key role of this
particular member in virulence. Recent studies have indicated that overall
levels of Py235 expression are essential for parasite virulence. Here we show
that disruption of PY01365 in the virulent YM line directly
impacts parasite virulence. Furthermore the disruption of
PY01365 leads to a reduction in the number of schizonts
that express members of Py235 that react specifically with the mcAb 25.77.
Erythrocyte binding assays show reduced binding of Py235 to red blood cells in
the PY01365 knockout parasite as compared to YM. While our
results identify PY01365 as a mediator of parasite virulence,
they also confirm that other members of Py235 are able to substitute for
Brucella neotomae is not known to be associated with clinical disease in any host species. Previous research suggested that B. neotomae might not express detectable levels of Cu/Zn superoxide dismutase (SOD), a periplasmic enzyme known to be involved in protecting Brucella from oxidative bactericidal effects of host phagocytes. This study was undertaken to investigate the genetic basis for the disparity in SOD expression in B. neotomae. Our Western blot and SOD enzyme assay analyses indicated that B. neotomae does express SOD, but at a substantially reduced level. Nucleotide sequence analysis of region upstream to the sodC gene identified a single-nucleotide insertion in the potential promoter region. The same single-nucleotide insertion was also detected in the sodC promoter of B. suis strain Thomsen, belonging to biovar 2 in which SOD expression was undetectable previously. Examination of the sodC promoter activities using translational fusion constructs with E. coli β-galactosidase demonstrated that the B. neotomae and B. suis biovar 2 promoters were very weak in driving gene expression. Site-directed mutation studies indicated that the insertion of A in the B. neotomae sodC promoter reduced the promoter activity. Increasing the level of SOD expression in B. neotomae through complementation with B. abortus sodC gene did not alter the bacterial survival in J774A.1 macrophage-like cells and in tissues of BALB/c and C57BL/6 mice. These results for the first time demonstrate the occurrence of a single-nucleotide polymorphism affecting promoter function and gene expression in Brucella.
Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics.
The objective of this work was to develop an alternative to conventional phage lysis tests – a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages ϕA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. ϕA1122-specific qPCR enabled the detection of an initial bacterial concentration of 103 CFU/ml (equivalent to as few as one Y. pestis cell per 1-µl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, ϕA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR.
Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria.
Recombinant proteins and in particular single domains or peptides are often poorly immunogenic unless conjugated to a carrier protein. Virus-like-particles are a very efficient means to confer high immunogenicity to antigens. We report here the development of virus-like-particles (VLPs) derived from the RNA bacteriophage AP205 for epitope-based vaccines.
Peptides of angiotensin II, S.typhi outer membrane protein (D2), CXCR4 receptor, HIV1 Nef, gonadotropin releasing hormone (GnRH), Influenza A M2-protein were fused to either N- or C-terminus of AP205 coat protein. The A205-peptide fusions assembled into VLPs, and peptides displayed on the VLP were highly immunogenic in mice. GnRH fused to the C-terminus of AP205 induced a strong antibody response that inhibited GnRH function in vivo. Exposure of the M2-protein peptide at the N-terminus of AP205 resulted in a strong M2-specific antibody response upon immunization, protecting 100% of mice from a lethal influenza infection.
AP205 VLPs are therefore a very efficient and new vaccine system, suitable for complex and long epitopes, of up to at least 55 amino acid residues in length. AP205 VLPs confer a high immunogenicity to displayed epitopes, as shown by inhibition of endogenous GnRH and protective immunity against influenza infection.
In a mouse model of respiratory tract infection by Bordetella pertussis, bacteria multiply in the airways over the first week and are then cleared over the next 3–4 weeks by the host immune response. Pertussis toxin (PT), a virulence factor secreted exclusively by B. pertussis, promotes bacterial growth in the airways by suppression and modulation of host immune responses. By comparison of wild type and PT-deficient strains, we examined the role of PT in modulating airway cytokine and chemokine responses affecting neutrophil recruitment during B. pertussis infection in mice. We found that, despite early inhibition of neutrophil recruitment by PT, high numbers of neutrophils were recruited to the airways by 4 days post-infection with the wild type strain, but not with the PT-deficient strain, and that this correlated with upregulation of neutrophil-attracting chemokine gene expression. In addition, there was similar upregulation of genes expressing the cytokines IL-17A (IL-17), TNF-α and IFN-γ, indicating a mixed Th1/Th17 response. Expression of IL-6, a cytokine involved in Th17 induction, was upregulated earlier than the IL-17 response. We showed that PT, rather than bacterial numbers, was important for induction of these responses. Flow cytometric analysis revealed that the IL-17-producing cells were macrophages and neutrophils as well as T cells, and were present predominantly in the airways rather than the lung tissue. Antibody neutralization of IL-17 significantly reduced chemokine gene expression and neutrophil recruitment to the airways, but only modestly increased peak bacterial loads. These data indicate that PT stimulates inflammatory responses by induction of Th1- and Th17-associated cytokines, including IL-17, during B. pertussis infection in mice, but a role for IL-17 in protection against the infection remains to be established.
Peptide libraries or antigenic determinants can be displayed on the surface of bacteria through insertion in a suitable outer membrane scaffold protein. Here, we inserted the well-known antibody epitopes 3xFLAG and 2xmyc in exterior loops of the transmembrane (TM) domain of OmpA. Although these highly charged epitopes were successfully displayed on the cell surface, their levels were 10-fold reduced due to degradation. We verified that the degradation was not caused by the absence of the C-terminal domain of OmpA. In contrast, a peptide that was only moderately charged (SA-1) appeared to be stably incorporated in the outer membrane at normal protein levels. Together, these results suggest that the display efficiency is sensitive to the charge of the inserted epitopes. In addition, the high-level expression of OmpA variants with surface-displayed epitopes adversely affected growth in a strain dependent, transient manner. In a MC4100 derived strain growth was affected, whereas in MC1061 derived strains growth was unaffected. Finally, results obtained using a gel-shift assay to monitor β-barrel folding in vivo show that the insertion of small epitopes can change the heat modifiability of the OmpA TM domain from ‘aberrant’ to normal, and predict that some β-barrels will not display any significant heat-modifiability at all.
High-throughput genomics and the emerging field of synthetic biology demand ever more convenient, economical, and efficient technologies to assemble and clone genes, gene libraries and synthetic pathways. Here, we describe the development of a novel and extremely simple cloning method, circular polymerase extension cloning (CPEC). This method uses a single polymerase to assemble and clone multiple inserts with any vector in a one-step reaction in vitro. No restriction digestion, ligation, or single-stranded homologous recombination is required. In this study, we elucidate the CPEC reaction mechanism and demonstrate its usage in demanding synthetic biology applications such as one-step assembly and cloning of complex combinatorial libraries and multi-component pathways.
The macrophage is the natural niche of Mycobacterium tuberculosis infection. In order to combat oxidative and nitrosative stresses and persist in macrophages successfully, M. tuberculosis is endowed with a very efficient antioxidant complex. Amongst these antioxidant enzymes, TpX is the only one in M. tuberculosis with sequence homology to thiol peroxidase. Previous reports have demonstrated that the M. tuberculosis TpX protein functions as a peroxidase in vitro. It is the dominant antioxidant which protects M. tuberculosis against oxidative and nitrosative stresses. The level of the protein increases in oxidative stress. To determine the roles of tpx gene in M. tuberculosis survival and virulence in vivo, we constructed an M. tuberculosis strain lacking the gene. The characteristics of the mutant were examined in an in vitro stationary phase model, in response to stresses; in murine bone marrow derived macrophages and in an acute and an immune resistant model of murine tuberculosis. The tpx mutant became sensitive to H2O2 and NO compared to the wild type strain. Enzymatic analysis using bacterial extracts from the WT and the tpx mutant demonstrated that the mutant contains reduced peroxidase activity. As a result of this, the mutant failed to grow and survive in macrophages. The growth deficiency in macrophages became more pronounced after interferon-γ activation. In contrast, its growth was significantly restored in the macrophages of inducible nitric oxide synthase (iNOS or NOS2) knockout mice. Moreover, the tpx mutant was impaired in its ability to initiate an acute infection and to maintain a persistent infection. Its virulence was attenuated. Our results demonstrated that tpx is required for M. tuberculosis to deal with oxidative and nitrosative stresses, to survive in macrophages and to establish acute and persistent infections in animal tuberculosis models.
LipL32 is the major leptospiral outer membrane lipoprotein expressed during infection and is the immunodominant antigen recognized during the humoral immune response to leptospirosis in humans. In this study, we investigated novel aspects of LipL32. In order to define the immunodominant domains(s) of the molecule, subfragments corresponding to the N-terminal, intermediate, and C-terminal portions of the LipL32 gene were cloned and the proteins were expressed and purified by metal affinity chromatography. Our immunoblot results indicate that the C-terminal and intermediate domains of LipL32 are recognized by sera of patients with laboratory-confirmed leptospirosis. An immunoglobulin M response was detected exclusively against the LipL32 C-terminal fragment in both the acute and convalescent phases of illness. We also evaluated the capacity of LipL32 to interact with extracellular matrix (ECM) components. Dose-dependent, specific binding of LipL32 to collagen type IV and plasma fibronectin was observed, and the binding capacity could be attributed to the C-terminal portion of this molecule. Both heparin and gelatin could inhibit LipL32 binding to fibronectin in a concentration-dependent manner, indicating that the 30-kDa heparin-binding and 45-kDa gelatin-binding domains of fibronectin are involved in this interaction. Taken together, our results provide evidence that the LipL32 C terminus is recognized early in the course of infection and is the domain responsible for mediating interaction with ECM proteins.
Heat shock proteins (Hsp) are families of highly conserved molecules and immunodominant antigens in some infections and in autoimmune diseases. Some reports suggest that different regions of the Hsp60 molecule induce distinct immune responses. However, there are no reports comparing physiological T-cell reactivity to Hsp60 in mice. In this study, we have analyzed T-cell proliferation and cytokine production induced by Hsp60, under physiological conditions, in three mouse strains bearing distinct major histocompatibility complex (MHC) backgrounds. Proliferative response predominantly was found in C57BL/6 mice, mostly induced by N-terminal and intermediate Hsp60 peptides (P < 0.0001). Interferon-γ (IFNγ) production was broadly induced by different regions of Hsp60 in all three mouse strains, although response was focused in different peptide groups in each strain. We did not observe an exclusive Th1 or Th2 cytokine profile induced by any particular region of Hsp60. However, we identified a strain hierarchy in IL-10 production induced by Hsp60 peptides from different regions, mostly detected in C3H/HePas, and in BALB/c, but not in C57BL/6 mice. In contrast, IL-4 production only was induced by the intermediate and C-terminal region peptides in both C3H/HePas and BALB/c mice. Our data give original information on physiological cellular reactivity to Hsp60. We also have identified peptides with the capacity to induce the production of anti-inflammatory cytokines, bringing perspectives for their use in immunotherapy of chronic inflammatory diseases and allograft rejection.
Infections with human papillomavirus type 16 (HPV-16) are closely associated with the development of human cervical carcinoma, which is one of the most common causes of cancer death in women worldwide. At present, the most promising vaccine against HPV-16 infection is based on the L1 major capsid protein, which self-assembles in virus-like particles (VLPs). In this work, we used a lactose-inducible system based on the Lactobacillus casei lactose operon promoter (plac) for expression of the HPV-16 L1 protein in L. casei. Expression was confirmed by Western blotting, and an electron microscopy analysis of L. casei expressing L1 showed that the protein was able to self-assemble into VLPs intracellularly. The presence of conformational epitopes on the L. casei-produced VLPs was confirmed by immunofluorescence using the anti-HPV-16 VLP conformational antibody H16.V5. Moreover, sera from mice that were subcutaneously immunized with L. casei expressing L1 reacted with Spodoptera frugiperda-produced HPV-16 L1 VLPs, as determined by an enzyme-linked immunosorbent assay. The production of L1 VLPs by Lactobacillus opens the possibility for development of new live mucosal prophylactic vaccines.
Intranasal challenge of C57BL/6 mice with Streptococcus pneumoniae serotypes 6B, 14, and 23F produced colonization of the middle ear and NP. Intranasal vaccination with ethanol-killed nonencapsulated cells with adjuvant protected both sites. Of four nontoxic adjuvants tested, the cholera toxin B subunit was most effective and least nonspecifically protective.
The main keratinase (kerA) gene from the Bacillus licheniformis S90 was optimized by two codon optimization strategies and expressed in Pichia pastoris in order to improve the enzyme production compared to the preparations with the native kerA gene. The results showed that the corresponding mutations (synonymous codons) according to the codon bias in Pichia pastoris were successfully introduced into keratinase gene. The highest keratinase activity produced by P. pastoris pPICZαA-kerAwt, pPICZαA-kerAopti1 and pPICZαA-kerAopti2 was 195 U/ml, 324 U/ml and 293 U/ml respectively. In addition, there was no significant difference in biomass concentration, target gene copy numbers and relative mRNA expression levels of every positive strain. The molecular weight of keratinase secreted by recombinant P. pastori was approx. 39 kDa. It was optimally active at pH 7.5 and 50°C. The recombinant keratinase could efficiently degrade both α-keratin (keratin azure) and β-keratin (chicken feather meal). These properties make the P. pastoris pPICZαA-kerAopti1 a suitable candidate for industrial production of keratinases.
Recombinant virus-like particles (VLP) antigenically similar to rabbit hemorrhagic disease virus (RHDV) were recently expressed at high levels inside Pichia pastoris cells. Based on the potential of RHDV VLP as platform for diverse vaccination purposes we undertook the design, development and scale-up of a production process. Conformational and stability issues were addressed to improve process control and optimization. Analyses on the structure, morphology and antigenicity of these multimers were carried out at different pH values during cell disruption and purification by size-exclusion chromatography. Process steps and environmental stresses in which aggregation or conformational instability can be detected were included. These analyses revealed higher stability and recoveries of properly assembled high-purity capsids at acidic and neutral pH in phosphate buffer. The use of stabilizers during long-term storage in solution showed that sucrose, sorbitol, trehalose and glycerol acted as useful aggregation-reducing agents. The VLP emulsified in an oil-based adjuvant were subjected to accelerated thermal stress treatments. None to slight variations were detected in the stability of formulations and in the structure of recovered capsids. A comprehensive analysis on scale-up strategies was accomplished and a nine steps large-scale production process was established. VLP produced after chromatographic separation protected rabbits against a lethal challenge. The minimum protective dose was identified. Stabilized particles were ultimately assayed as carriers of a foreign viral epitope from another pathogen affecting a larger animal species. For that purpose, a linear protective B-cell epitope from Classical Swine Fever Virus (CSFV) E2 envelope protein was chemically coupled to RHDV VLP. Conjugates were able to present the E2 peptide fragment for immune recognition and significantly enhanced the peptide-specific antibody response in vaccinated pigs. Overall these results allowed establishing improved conditions regarding conformational stability and recovery of these multimers for their production at large-scale and potential use on different animal species or humans.
Batrachochytrium dendrobatidis (Bd) is a fungus that causes the disease chytridiomycosis and is associated with widespread amphibian declines. Populations vary in their susceptibility to Bd infections, and the virulence of the infecting lineage can also vary. Both of these factors may manifest as a differential physiological stress response. In addition, variation in disease susceptibility across amphibian populations may be influenced by immunosuppression caused by chronic stress imposed by environmental factors. Here, we use a non-invasive water-borne hormone technique to assess stress levels (corticosterone) of free-living tadpole populations that are infected by Bd. We found that corticosterone release rates were higher in infected populations of two species of tadpoles (Alytes obstetricans and A. muletensis) than in an uninfected population for both species. The relationship between corticosterone and the intensity of infection differed between species, with only the infected A. obstetricans population showing a significant positive correlation. The higher corticosterone release rates found in A. obstetricans may be an outcome of infection by a highly virulent lineage of Bd (BdGPL), whereas A. muletensis is infected with a less virulent lineage (BdCAPE). These results suggest that different lineages of Bd impose different levels of stress on the infected animals, and that this may influence survival. The next step is to determine whether higher corticosterone levels make individuals more susceptible to Bd or if Bd infections drive the higher corticosterone levels.
L-amino acid oxidase (LAAO) is attracting increasing attention due to its important functions. Diverse detection methods with their own properties have been developed for characterization of LAAO. In the present study, a simple, rapid, sensitive, cost-effective and reproducible method for quantitative in-gel determination of LAAO activity based on the visualization of Prussian blue-forming reaction is described. Coupled with SDS-PAGE, this Prussian blue agar assay can be directly used to determine the numbers and approximate molecular weights of LAAO in one step, allowing straightforward application for purification and sequence identification of LAAO from diverse samples.
Bacteriophage lytic enzymes have recently attracted considerable interest as novel antimicrobials against Gram-positive bacteria. In this work, antimicrobial activity in milk of HydH5 [a virion-associated peptidoglycan hydrolase (VAPGH) encoded by the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88], and three different fusion proteins created between HydH5 and lysostaphin has been assessed. The lytic activity of the five proteins (HydH5, HydH5Lyso, HydH5SH3b, CHAPSH3b and lysostaphin) was confirmed using commercial whole extended shelf-life milk (ESL) in challenge assays with 104 CFU/mL of the strain S. aureus Sa9. HydH5, HydH5Lyso and HydH5SH3b (3.5 µM) kept the staphylococcal viable counts below the control cultures for 6 h at 37°C. The effect is apparent just 15 minutes after the addition of the lytic enzyme. Of note, lysostaphin and CHAPSH3b showed the highest staphylolytic protection as they were able to eradicate the initial staphylococcal challenge immediately or 15 min after addition, respectively, at lower concentration (1 µM) at 37°C. CHAPSH3b showed the same antistaphyloccal effect at room temperature (1.65 µM). No re-growth was observed for the remainder of the experiment (up to 6 h). CHAPSH3b activity (1.65 µM) was also assayed in raw (whole and skim) and pasteurized (whole and skim) milk. Pasteurization of milk clearly enhanced CHAPSH3b staphylolytic activity in both whole and skim milk at both temperatures. This effect was most dramatic at room temperature as this protein was able to reduce S. aureus viable counts to undetectable levels immediately after addition with no re-growth detected for the duration of the experiment (360 min). Furthermore, CHAPSH3b protein is known to be heat tolerant and retained some lytic activity after pasteurization treatment and after storage at 4°C for 3 days. These results might facilitate the use of the peptidoglycan hydrolase HydH5 and its derivative fusions, particularly CHAPSH3b, as biocontrol agents for controlling undesirable bacteria in dairy products.