Derivatives of the Antimicrobial Peptide BP100 for Expression in Plant Systems

Production of antimicrobial peptides in plants constitutes an approach for obtaining them in high amounts. However, their heterologous expression in a practical and efficient manner demands some structural requirements such as a minimum size, the incorporation of retention signals to assure their accumulation in specific tissues, and the presence of protease cleavage amino acids and of target sequences to facilitate peptide detection. Since any sequence modification may influence the biological activity, peptides that will be obtained from the expression must be screened prior to the synthesis of the genes for plant transformation. We report herein a strategy for the modification of the antimicrobial undecapeptide BP100 that allowed the identification of analogues that can be expressed in plants and exhibit optimum biological properties. We prepared 40 analogues obtained by incorporating repeated units of the antimicrobial undecapeptide, fragments of natural peptides, one or two AGPA hinges, a Gly or Ser residue at the N-terminus, and a KDEL fragment and/or the epitope tag54 at the C-terminus. Their antimicrobial, hemolytic and phytotoxic activities, and protease susceptibility were evaluated. Best sequences contained a magainin fragment linked to the antimicrobial undecapeptide through an AGPA hinge. Moreover, since the presence of a KDEL unit or of tag54 did not influence significantly the biological activity, these moieties can be introduced when designing compounds to be retained in the endoplasmic reticulum and detected using a complementary epitope. These findings may contribute to the design of peptides to be expressed in plants

Comparative effects of macro-sized aluminum oxide and aluminum oxide nanoparticles on erythrocyte hemolysis: influence of cell source, temperature and size

Al2O3 is the most abundantly produced nanomaterial and has been used in diverse fields, including the medical, military and industrial sectors. As there are concerns about the health effects of nanoparticles, it is important to understand how they interact with cells, and specifically with red blood cells. The hemolysis induced by three commercial nano-sized aluminum oxide particles (nanopowder 13 nm, nanopowder <50 nm and nanowire 2-6 nm × 200-400 nm) was compared to aluminum oxide and has been studied on erythrocytes from humans, rats and rabbits, in order to elucidate the mechanism of action and the influence of size and shape on hemolytic behavior. The concentrations inducing 50% hemolysis (HC50) were calculated for each compound studied. The most hemolytic aluminum oxide particles were of nanopowder 13, followed by nanowire and nanopowder 50. The addition of albumin to PBS induced a protective effect on hemolysis in all the nano-forms of Al2O3, but not on Al2O3. The drop in HC50 correlated to a decrease in nanomaterial size, which was induced by a reduction of aggregation Aluminum oxide nanoparticles are less hemolytic than other oxide nanoparticles, and behave differently depending on the size and shape of the nanoparticles. The hemolytic behavior of aluminum oxide nanoparticles differs from that of aluminum oxide.

Virulence factors and antimicrobial resistance of Staphylococcus aureus isolated from bovine mastitis in Rio de Janeiro

The study was conducted to characterize pheno-genotypically the virulence factors and resistance pattern of Staphylococcus aureus isolates from milk samples of cows with subclinical mastitis. All hemolytic isolates presented beta-hemolysin, and 38% of the non-hemolytic isolates were able to express hemolysins in the presence of a beta-hemolytic strain. The amplification of the coa-gene displayed four different size polymorphisms with about 400 bp, 600 bp, 700 bp and 900 bp. The spaA gene that encodes the IgG-binding region of protein A revealed sizes of 700 bp and 900 bp. The amplification of region X from spaA yielded a single amplicon for each isolate with the prevalent amplicon size being of 180 bp. Amplification of sae gene yielded an amplicon size of 920 bp in 71% of the isolates. Antibiotic resistance pattern revealed that 42% S. aureus were susceptible to all antimicrobials tested. Seven different antibiotic patterns were observed. Our results indicated that 47% and 25% of S. aureus strains exhibited resistance to penicillin and oxacillin respectively. All oxacillin-resistant isolates were mecA-positive.

Matched case-control study evaluating the frequency of the main agents associated with neonatal diarrhea in piglets

A case-control study was carried out in litters of 1 to 7-day-old piglets to identify the main infectious agents involved with neonatal diarrhea in pigs. Fecal samples (n=276) from piglets were collected on pig farms in the State of Rio Grande do Sul, Brazil, from May to September 2007. Litters with diarrhea were considered cases (n=129) and normal litters (n=147) controls. The samples were examined by latex agglutination test, PAGE, conventional isolating techniques, ELISA, PCR, and microscopic methods in order to detect rotavirus, bacterial pathogens (Escherichia coli, Clostridium perfringens type A and C, and Clostridium difficile), and parasites (Coccidian and Cryptosporidium spp.). Outbreaks of diarrhea were not observed during sampling. At least one agent was detected in fecal samples on 25 out of 28 farms (89.3%) and in 16 farms (57.1%) more than one agent was found. The main agents diagnosed were Coccidia (42.86%) and rotavirus (39.29%). The main agents identified in litters with diarrhea were Clostridium difficile (10.6%), Clostridium perfringens type A (8.8%) and rotavirus (7.5%); in control litters, Clostridium difficile (16.6%) and Coccidian (8.5%). Beta hemolytic Escherichia coli and Clostridium perfringens type C were not detected. When compared with controls, no agent was significantly associated with diarrhea in case litters. These findings stress the need for caution in the interpretation of laboratorial diagnosis of mild diarrhea in neonatal pigs, as the sole detection of an agent does not necessarily indicate that it is the cause of the problem.

Enterohemorrhagic Escherichia coli O157: H7 from healthy dairy cattle in Mid-West Brazil: occurrence and molecular characterization

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 represents the major Shiga toxin-producing E. coli (STEC) strain related to large outbreaks and severe diseases such as hemorrhagic colitis (HC) and the potentially lethal hemolytic uremic syndrome (HUS). The aim of this study was to report the occurrence and molecular characterization of O157:H7 isolates obtained by rectal swab from 52 healthy dairy cattle belonging to 21 farms in Mid-West of Brazil. Detection of 16SrRNA, stx1, stx2, rfbO157, fliCh7, eae, ehxA, saa, cnf1, chuA, yjaA and TSPE4.C2 genes was performed by PCR. The isolates were further characterized by serotyping. Two hundred and sixty E. coli isolates were obtained, of which 126 were characterized as STEC. Two isolates from the same cow were identified as serotype O157:H7. Both isolates presented the stx2, eae, ehxA, saa and cnf1 virulence factor genes and the chuA gene in the phylogenetic classification (virulent group D), suggesting that they were clones. The prevalence of O157:H7 was found to be 1.92% (1/52 animals), demonstrating that healthy dairy cattle from farms in the Mid-West of Brazil are an important reservoir for highly pathogenic E. coli O157:H7.

Is there correlation between the turbulent eddies size and mechanical hemolysis ?

Hemolytic profile of an artificial device chronically implanted in the cardiovascular system may represent the difference between the success and failure in its long-term performance. Last decades have witnessed efforts on the development of methods capable of predicting red blood cell damage in artificial organs. However, all of them have had limited success to predict hemolysis. The primary cause of this problem is that such models do not take into consideration structures of turbulent flow. The present paper demonstrates that microscopic measurable occurrences of the turbulent flow may be linked to red blood cell trauma. This study suggests that if the smallest turbulent eddies dimension is under 10 m m hemolysis is not dependent on the exposure time and the red blood cells damage depends only on the dissipation of the turbulent energy in the erythrocyte membrane. The analysis reported here opens the possibility of mapping the flow field in artificial assist devices based on the smallest eddy length scales. This is a promising new trend and should be considered in the designing requirements of the next generations of artificial organs.

Single-step purification of crotapotin and crotactine from Crotalus durissus terrificus venom using preparative isoelectric focusing

We describe the isolation of crotoxin, a presynaptic B-neurotoxin, as well as its subunits B (crotactine) and A (crotapotin) from lyophilized Crotalus durissus terrificus venom by a single-step preparative isoelectric focusing procedure. From 98 mg of dried venom protein 20.1 mg of crotactine and 13.1 mg of crotapotin were recovered in the first step of focalization and 4.2 mg in a second run. These values correspond to 35.7% of the total venom protein applied. Crotactine separated in the 9.3-7.0 pH range (tubes 1-6) and crotapotin in the 1.8-2.8 pH range (tubes 15-19) and both were homogeneous by SDS-PAGE and N-terminal amino acid analysis. Crotactine, a 12-kDa protein, presented hemolytic and phospholipase A2 activity. Thus, using isoelectric focusing we simultaneously purified both toxins in high yields. This method can be used as an alternative for the purification and characterization of proteins from other snake venoms under conditions in which biological activity is retained

The calcium-dependent protease of Loxosceles gaucho venom acts preferentially upon red cell band 3 transmembrane protein

Eighty micrograms red blood cell (RBC) ghosts from patients who had previously exhibited the cutaneous form of loxoscelism (presenting localized dermonecrosis) and the viscerocutaneous form of loxoscelism (presenting dermonecrosis, hemoglobinuria, hematuria, and jaundice) and from controls were incubated with 2.5 µg crude Loxosceles gaucho venom in 5 mM phosphate buffer, pH 7.4, at 37ºC. Among all membrane proteins, quantitative proteolysis of the important integral transmembrane protein 3 increased with venom dose and with incubation time from 30 to 120 min, as demonstrated by gel densitometry. Similar quantitative data were obtained for RBC ghosts from patients and from control subjects, a fact that argues against the possibility of genetic factors favoring the hemolytic viscerocutaneous form. These data suggest that the clinical forms may be different types of the same disease, with the viscerocutaneous form being the result of large amounts of intravascularly injected venom and the superficial form being the result of in situ venom action. Since protein 3 is a housekeeping integral membrane protein, whose genetic deficiency leads to hemolytic anemia, it is reasonable to relate it to the hemolysis which occurs in the viscerocutaneous form of loxoscelism. The venom protease responsible for the process was not inhibited after 120-min incubation by 0.2 mM paramethylsulfonyl fluoride or by 0.2 mM N-ethylmaleimide but was inhibited by 25 mM ethylenediaminetetraacetic acid (a calcium-chelating agent) in 5 mM phosphate buffer at pH 7.4, which suggests that the enzyme is a calcium-dependent metalloprotease.

Biological activity of Serratia marcescens cytotoxin

Serratia marcescens cytotoxin was purified to homogeneity by ion-exchange chromatography on a DEAE Sepharose Fast Flow column, followed by gel filtration chromatography on a Sephadex G100 column. The molecular mass of the cytotoxin was estimated to be about 50 kDa. Some biological properties of the cytotoxin were analyzed and compared with well-characterized toxins, such as VT1, VT2 and CNF from Escherichia coli and hemolysin produced by S. marcescens. The sensitivity of the cell lines CHO, HeLa, HEp-2, Vero, BHK-21, MA 104 and J774 to the cytotoxin was determined by the cell viability assay using neutral red. CHO and HEp-2 were highly sensitive, with massive cellular death after 1 h of treatment, followed by BHK-21, HeLa, Vero and J774 cells, while MA 104 was insensitive to the toxin. Cytotoxin induced morphological changes such as cell rounding with cytoplasmic retraction and nuclear compactation which were evident 15 min after the addition of cytotoxin. The cytotoxic assays show that 15 min of treatment with the cytotoxin induced irreversible intoxication of the cells, determined by loss of cell viability. Concentrations of 2 CD50 (0.56 µg/ml) of purified cytotoxin did not present any hemolytic activity, showing that the cytotoxin is distinct from S. marcescens hemolysin. Antisera prepared against S. marcescens cytotoxin did not neutralize the cytotoxic activity of VT1, VT2 or CNF toxin, indicating that these toxins do not share antigenic determinants with cytotoxin. Moreover, we did not detect gene sequences for any of these toxins in S. marcescens by PCR assay. These results suggest that S. marcescens cytotoxin is not related to any of these toxins from E. coli.

An experimental model of hemolysis-induced acute pancreatitis

The literature indicates that acute pancreatitis is a complication of massive hemolysis with a prevalence of about 20%. We describe an experimental model of hemolysis-induced acute pancreatitis. Hemolytic anemia was induced in rats by a single ip injection of 60 mg/kg of 20 mg/ml acetylphenylhydrazine (APH) in 20% (v/v) ethanol on the first experimental day (day 0). One hundred and fifty Wistar albino rats weighing 180-200 g were divided into three groups of 50 animals each: groups 1, 2 and 3 were injected ip with APH, 20% ethanol, and physiological saline, respectively. Ten rats from each group were sacrificed on study days 1, 2, 3, 4 and 5. Serum amylase, lipase levels and pancreatic tissue tumor necrosis factor-alpha (TNF-alpha) and platelet-activating factor (PAF) contents were determined and a histological examination of the pancreas was performed. No hemolysis or pancreatitis was observed in any of the rats in groups 2 and 3. In group 1, massive hemolysis was observed in 35 (70%) of 50 rats, moderate hemolysis in seven (14%), and no hemolysis in eight (16%). Thirty-three of 35 (94.2%) rats with massive hemolysis had hyperamylasemia, and 29 of these rats (82.8%) had histologically proven pancreatitis. The most severe pancreatitis occurred on day 3, as demonstrated by histology. Tissue TNF-alpha and PAF levels were statistically higher in group 1 than in groups 2 and 3. Acute massive hemolysis induced acute pancreatitis, as indicated by histology, in almost 80% of cases. Hemolysis may induce acute pancreatitis by triggering the release of proinflammatory and immunoregulatory cytokines.

Changes in red blood cell osmotic fragility induced by total plasma and plasma fractions obtained from rats bearing progressive and regressive variants of the Walker 256 tumor

Two variants (A and B) of the widely employed Walker 256 rat tumor cells are known. When inoculated sc, the A variant produces solid, invasive, highly metastasizing tumors that cause severe systemic effects and death. We have obtained a regressive variant (AR) whose sc growth is slower, resulting in 70-80% regression followed by development of immunity against A and AR variants. Simultaneously with the beginning of tumor regression, a temporary anemia developed (~8 days duration), accompanied by marked splenomegaly (~300%) and changes in red blood cell osmotic fragility, with mean corpuscular fragility increasing from 4.1 to 6.5 g/l NaCl. The possibility was raised that plasma factors associated with the immune response induced these changes. In the present study, we identify and compare the osmotic fragility increasing activity of plasma fractions obtained from A and AR tumor bearers at different stages of tumor development. The results showed that by day 4 compounds precipitating in 60% (NH4)2SO4 and able to increase red blood cell osmotic fragility appeared in the plasma of A and AR tumor bearers. Later, these compounds disappeared from the plasma of A tumor bearers but slightly increased in the plasma of AR tumor bearers. Furthermore, by day 10, compounds precipitating between 60 and 80% (NH4)2SO4 and with similar effects appeared only in plasma of AR tumor bearers. The salt solubility, production kinetics and hemolytic activity of these compounds resemble those of the immunoglobulins. This, together with their preferential increase in rats bearing the AR variant, suggest their association with an immune response against this tumor.

The role of complement in the early phase of Leishmania (Leishmania) amazonensis infection in BALB/c mice

Complement-depleted and -non-depleted BALB/c mice were inoculated with Leishmania (Leishmania) amazonensis promastigotes into the hind footpad to study the role of the complement system in cutaneous leishmaniasis. Total serum complement activity was measured by hemolytic assay and C3 fragment deposit at the inoculation site was determined by direct immunofluorescence in the early period of infection, i.e., at 3, 24, 48 h and 7 days post-infection. The inflammatory reaction and the parasite burden were evaluated in the skin lesion at 7 and 30 days post-infection. Total serum complement activity decreased in the early phase of infection, from 3 to 24 h, in non-depleted mice compared to non-infected and non-depleted mice. C3 fragment deposit at the site of parasite inoculation was present throughout the period of infection in non-depleted mice. In contrast, no C3 fragment deposit was observed at the inoculation site in complement-depleted mice. Complement-depleted mice showed a significant decrease in the inflammatory response and a significant increase in the number of parasites (70.0 ± 5.3 vs 5.3 ± 1.5) at 7 days of infection (P < 0.05). A higher number of parasites were also present at 30 days of infection at the inoculation site of complement-depleted mice (78.5 ± 24.9 vs 6.3 ± 5.7). These experiments indicate that complement has an important role at the beginning of experimental cutaneous leishmaniasis caused by L. (L.) amazonensis by controlling the number of parasites in the lesion.

The Shiga toxin 2 B subunit inhibits net fluid absorption in human colon and elicits fluid accumulation in rat colon loops

Shiga toxin (Stx)-producing Escherichia coli (STEC) colonizes the large intestine causing a spectrum of disorders, including watery diarrhea, bloody diarrhea (hemorrhagic colitis), and hemolytic-uremic syndrome. It is estimated that hemolytic-uremic syndrome is the most common cause of acute renal failure in infants in Argentina. Stx is a multimeric toxin composed of one A subunit and five B subunits. In this study we demonstrate that the Stx2 B subunit inhibits the water absorption (Jw) across the human and rat colonic mucosa without altering the electrical parameters measured as transepithelial potential difference and short circuit current. The time-course Jw inhibition by 400 ng/ml purified Stx2 B subunit was similar to that obtained using 12 ng/ml Stx2 holotoxin suggesting that both, A and B subunits of Stx2 contributed to inhibit the Jw. Moreover, non-hemorrhagic fluid accumulation was observed in rat colon loops after 16 h of treatment with 3 and 30 ng/ml Stx2 B subunit. These changes indicate that Stx2 B subunit induces fluid accumulation independently of A subunit activity by altering the usual balance of intestinal absorption and secretion toward net secretion. In conclusion, our results suggest that the Stx2 B subunit, which is non-toxic for Vero cells, may contribute to the watery diarrhea observed in STEC infection. Further studies will be necessary to determine whether the toxicity of Stx2 B subunit may have pathogenic consequences when it is used as a component in an acellular STEC vaccine or as a vector in cancer vaccines.

A role for histone-like protein H1 (H-NS) in the regulation of hemolysin expression by Serratia marcescens

The histone-like protein H1 (H-NS) is an abundant structural component of the bacterial nucleoid and influences many cellular processes including recombination, transcription and transposition. Mutations in the hns gene encoding H-NS are highly pleiotropic, affecting the expression of many unrelated genes. We have studied the role of H-NS on the regulation of hemolysin gene expression in Serratia marcescens. The Escherichia coli hns mutant carrying S. marcescens hemolysin genes on a plasmid constructed by ligation of the 3.2-kb HindIII-SacI fragment of pR02 into pBluescriptIIKS, showed a high level of expression of this hemolytic factor. To determine the osmoregulation of wild-type and hns defective mutants the cells were grown to mid-logarithmic phase in LB medium with 0.06 or 0.3 M NaCl containing ampicillin and kanamycin, whereas to analyze the effect of pH on hemolysin expression, the cells were grown to late-logarithmic phase in LB medium buffered with 0.1 M Tris-HCl, pH 4.5 to 8.0. To assay growth phase-related hemolysin production, bacterial cells were grown in LB medium supplemented with ampicillin and kanamycin. The expression of S. marcescens hemolysin genes in wild-type E. coli and in an hns-defective derivative at different pH and during different growth phases indicated that, in the absence of H-NS, the expression of hemolysin did not vary with pH changes or growth phases. Furthermore, the data suggest that H-NS may play an important role in the regulation of hemolysin expression in S. marcescens and its effect may be due to changes in DNA topology influencing transcription and thus the amount of hemolysin expression. Implications for the mechanism by which H-NS influences gene expression are discussed.

Analysis of five streptokinase formulations using the euglobulin lysis test and the plasminogen activation assay

Streptokinase, a 47-kDa protein isolated and secreted by most group A, C and G ß-hemolytic streptococci, interacts with and activates human protein plasminogen to form an active complex capable of converting other plasminogen molecules to plasmin. Our objective was to compare five streptokinase formulations commercially available in Brazil in terms of their activity in the in vitro tests of euglobulin clot formation and of the hydrolysis of the plasmin-specific substrate S-2251™. Euglobulin lysis time was determined using a 96-well microtiter plate. Initially, human thrombin (10 IU/ml) and streptokinase were placed in individual wells, clot formation was initiated by the addition of plasma euglobulin, and turbidity was measured at 340 nm every 30 s. In the second assay, plasminogen activation was measured using the plasmin-specific substrate S-2251™. Streptase™ was used as the reference formulation because it presented the strongest fibrinolytic activity in the euglobulin lysis test. The Unitinase™ and Solustrep™ formulations were the weakest, showing about 50% activity compared to the reference formulation. All streptokinases tested activated plasminogen but significant differences were observed. In terms of total S-2251™ activity per vial, Streptase™ (75.7 ± 5.0 units) and Streptonase™ (94.7 ± 4.6 units) had the highest activity, while Unitinase™ (31.0 ± 2.4 units) and Strek™ (32.9 ± 3.3 units) had the weakest activity. Solustrep™ (53.3 ± 2.7 units) presented intermediate activity. The variations among the different formulations for both euglobulin lysis test and chromogenic substrate hydrolysis correlated with the SDS-PAGE densitometric results for the amount of 47-kDa protein. These data show that the commercially available clinical streptokinase formulations vary significantly in their in vitro activity. Whether these differences have clinical implications needs to be investigated.