Crude Extracellular Products Research Articles

Toxicity of Crude Extracellular Products of Aeromonas hydrophila on Rohu, Labeo rohita (Ham.)

In the present study the haemolytic and proteolytic activity of extracellular products (ECP) secreted from Aeromonas hydrophila (CAHH14 strain) were studied with respect to temperature and different time of incubation as well as its lethal toxicity on rohu, Labeo rohita. The strain was isolated from Catla catla (showing abdominal dropsy symptom) collected from the pond of Central Institute of Freshwater Aquaculture (CIFA), Bhubaneswar, India and was characterized on the basis of biochemical tests. The highest production of haemolysin was achieved when the bacteria was grown at 35°C for 30h. The proteolytic activity was found to be highest when the bacterium was grown at 30°C for 36h. The haemolytic and proteolytic toxin produced by Aeromonas hydrophila was found to be lethal to rohu (LD(50) 1.7×10(4)cfu/ml). The lethality of ECP was decreased by heating and completely inactivated by boiling at 100°C for 10min. This indicates that protease activity and haemolytic activity of A. hydrophila ECP was temperature dependant.

A highly virulent pathogen, Aeromonas hydrophila, from the freshwater crayfish Pacifastacus leniusculus

Aeromonas spp. are characteristic bacteria of freshwaters and many of them can be components of the bacterial flora of aquatic animals and may become pathogens on animals including humans. In this study Aeromonas hydrophila was isolated from the freshwater crayfish, Pacifastacus leniusculus, and was found to be a highly pathogenic bacterium among many isolated bacteria. Mortality reached 100% within 6 h when 200 μl of 1.24 × 10 7 CFU/ml was applied by injection. Histopathological studies of moribund crayfish showed that extensive necrotic nuclei and clump-infiltrated hemocytes were found in observed tissues including gill, heart, hepatopancreas and the circulatory system. To verify how crayfish are susceptible to this bacterium, crude extracellular products (ECPs) obtained from culture supernatant of A. hydrophila was studied either in vivo or in vitro. ECPs (200 μl) were able to kill crayfish by injection. In an in vitro study, ECPs induced cytotoxicity of hemocytes as well as hematopoietic cells in a dose- and time-dependent manner after 30 min post inoculation. Two genes coding for endotoxins were also found in this isolate of A. hydrophila. This indicates that the bacterial endotoxins are the causative agents of crayfish mortality. Moreover, the effect of temperature on the infectivity of A. hydrophila to crayfish was also studied. At 4 °C, all crayfish survived, whereas at 20 °C the animals died rapidly after bacterial challenge. At this low temperature A. hydrophila did not replicate or replicated at a very low degree and hence crayfish could probably mount effective cellular reactions towards A. hydrophila.

Effects of extracellular products of Vibrio alginolyticus on penaeid prawn plasma components.

The effects of both crude extracellular products (ECP) and a partially purified protease of Vibrio alginolyticus on the plasma components of kuruma prawn (Penaeus japonicus) and tiger prawn (P. monodon) were studied using crossed immunoelectrophoresis (CIE). A component of the plasma, tentatively identified as coagulogen, apparently disappeared after incubation with the ECP, while the amount of a component tentatively identified as haemocyanin decreased. The coagulogen and an unknown component (component 1) in the penaeid plasma showed an increased migration rate after incubation with a partially purified 33 kDa protease of the bacterium. In contrast, incubation with protease had no detectable effect on the amount of haemocyanin. These events may significantly contribute to the pathogenicity of Vibrio alginolyticus in penaeids.

Alkaline serine protease is an exotoxin of Vibrio alginolyticus in kuruma prawn, Penaeus japonicus.

An extracellular lethal toxin produced by Vibrio alginolyticus strain Swy originally isolated from diseased kuruma prawn(Penaeus japonicus) was partially purified by Fast Protein Liquid Chromatography with hydrophobic interaction (Phenyl Sepharose Hig hPerformance) chromatography and gel filtration columns. The toxin is an alkaline serine protease, inhibited by phenyl-methylsulfonyl fluoride (PMSF),and showed maximal activity at pH 10, having a molecular weight of about 33kDa estimated by SDS-PAGE and gel filtration chromatography. In addition, the toxin was also completely inhibited by FeCl2 but partially inhibited by CaCl2, CuCl2, CoCl2,MnCl2, and ZnCl2, and not inhibited by ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(beta-amino-ethyl ether)N,N,N',N'-tetraacetic acid (EGTA), iodoacetamide, pepstatin A, sodium dodecylsulfate (SDS), and N-tosyl-l-phenyl-alanine chloromethyl ketone (TPCK). Both the crude extracellular products (ECP) and the partially purified toxin are lethal for kuruma prawn at LD50 values of 0.30 and 0.27 microg protein/g body weight, respectively. The addition of PMSF completely inhibited the lethal toxicity of both the ECP and the partially purified toxin, indicating that this serine protease is a lethal factor produced by the bacterium. The 33-kDa protease is, therefore, suggested to be a new toxic protease produced by V. alginolyticus strain Swy.

The trapping of intravenously injected extracellular products from Aeromonas salmonicidain head kidney and spleen of vaccinated and nonvaccinated Atlantic salmon, Salmo salarL.

Trapping of intravenously injected crude extracellular products from Aeromonas salmonicidawas studied in Atlantic salmon, Salmo salarL. Frozen tissue from head kidney and spleen was collected 1 h and 8 h after the injection and localisation of A. salmonicidaantigens, complement factor 3 and immunoglobulin was investigated in vaccinated and nonvaccinated fish using a double immunofluorescence staining technique. In the head kidney of vaccinated fish, A. salmonicidaantigens was colocalised with complement factor 3 and immunoglobulin in sinusoidal cells. A weaker reactivity to A. salmonicidaantigens was detected in colocalisation with complement factor 3 alone in nonvaccinated fish. In the spleen, ellipsoidal sheaths showed reactivity to A. salmonicidaantigens in fish from both groups. In addition, the ellipsoids that trapped A. salmonicidaantigens were stained for complement factor 3 and immunoglobulin in the vaccinated fish but stained for complement factor 3 alone in nonvaccinated fish. Computer-assisted morphometric analysis was further used to provide semi-quantitative estimates of A. salmonicidaantigens and complement factor 3 in the head kidney. The estimated stained areas were significantly larger for the two antigens in vaccinated fish compared with nonvaccinated fish.

Staphylococcus hyicus-skin reactions in piglets caused by crude extracellular products and by partially purified exfoliative toxin

Staphylococcus hyicus may cause a spontaneous generalized exudative epidermitis in piglets. The progression and regression of macroscopical and histopathological lesions in piglet skin after subcutaneous injection of sterile concentrated culture supernatant (CCS) from one virulent and one avirulent strain of S. hyicus was studied every 24 h until 144 h post-injection. CCS from the virulent strain caused local alterations of the epidermis comparable to those of spontaneous exudative epidermitis: exfoliation, crust formation, exocytosis, formation of vesicles, and pustules and acanthosis. CCS from the avirulent strain only caused a transient erythema of the skin and no histopathological alterations of the epidermis. Additionally, proteins in CCS from the virulent strain were fractionated by column chromatography. Skin reactions similar to those caused by CCS from the virulent strain were induced by one fraction of proteins that contained eight protein bands in SDS-PAGE analysis. Two of these protein bands, with molecular weights of approximately 27 kDa and 30 kDa, were unique to the virulent strain compared to the avirulent strain. The results of this study indicate that one of these two proteins or both is a heat-labile exfoliative toxin, and that the toxin is a significant factor in the pathogenesis of exudative epidermitis in piglets.

Effects of the acetylcholinesterase toxin of Aeromonas hydrophila on the central nervous system of fish

The purified acetylcholinesterase (AcChE) toxin, crude extracellular products (ECP) or viable virulent Aeromonas hydrophila were injected intraperitoneally into rainbow trout in different sublethal and lethal doses. When fish showed signs of morbidity, brain tissue was excised and assayed for acetylcholinesterase activity. In all cases there was a large increase in AcChE activity (about 40-fold for purified AcChE-toxin). This was shown to be due to an accumulation of the fish's own AcChE and not the bacterial toxin. Nevertheless, the latter was detected in brain homogenates from fish in all treatment groups using a rabbit antiserum to the purified toxin to probe Western blots of brain homogenates, demonstrating that the toxin does gain access to brain tissue and is produced during in vivo infection. The results strongly suggest that this toxin plays a central role in the pathogenesis of A. hydrophila infection.

An extracellular acetylcholinesterase produced by Aeromonas hydrophila is a major lethal toxin for fish

A hitherto unrecognised lethal toxin from the extracellular products (ECP) of Aeromonas hydrophila is described. The pure toxin was 300 times more toxic than the crude ECP and is the most toxic substance so far described from this bacterium, with a minimum lethal dose of 0.05 μg g −1 fish. The toxin had high acetylcholinesterase activity and occurred in native ECP as a monomeric 15.5 kDa polypeptide. The purified toxin had five isoelectric focusing forms ranging from pl 4.45 to 4.70. The ECP of each of six strains of A. hydrophila isolated from fish possessed acetylcholinesterase activity suggesting that the toxin is common in this species. The toxin was not a cytolysin and produced no gross pathology in injected fish. Its enzymic nature, low lethal dose, lack of tissue pathology and its apparent narcotic effect suggest that this toxin may act upon the central nervous system of the fish.

Do extracellular products of Aeromonas salmonicida induce thrombosis by entering the fish coagulation system at factor X?

Abstract. Thrombosis of minute vessels is a common feature of acute furunculosis in Atlantic salmon. Crude extracellular products (F.CP) of Aeromans, salmonicida injected into the dorsal aorta of cannulated fish elicited an activation of the coagulations systems of the fish When screened with a number of chromogenic substrates, the ECP protease revealed a factor‐X like activity. Thus, it may he hypothesized that the ECP induces thrombosis by entering the fish coagulation systems at factor X. and that thrombus formation is a way of creating a site of infection.

Glycerophospholipid:cholesterol acyltransferase complexed with lipopolysaccharide (LPS) is a major lethal exotoxin and cytolysin of Aeromonas salmonicida: LPS stabilizes and enhances toxicity of the enzyme

An extracellular lethal toxin produced by Aeromonas salmonicida was purified by fast-protein liquid ion-exchange chromatography. The toxin is composed of glycerophospholipid:cholesterol acyltransferase (GCAT) (molecular mass, 25 kilodaltons) aggregated with lipopolysaccharide (LPS), the GCAT/LPS complex having a molecular mass of about 2,000 kilodaltons, estimated by gel filtration chromatography. The toxin is lethal for Atlantic salmon (Salmo salar L.) at a concentration of 0.045 micrograms of protein per g of body weight. The toxin is a hemolysin (T-lysin, active on fish erythrocytes), leukocytolysin, and cytotoxin. Antiserum to the purified toxin neutralized the lethal toxicity of the crude extracellular toxins, indicating this toxin to be the major lethal factor produced by A. salmonicida. In the crude extracellular products, small amounts of free GCAT were also present. This has been purified, and its activities and properties have been compared with those of the GCAT/LPS complex. The presence of LPS did not influence the GCAT activity of the enzyme with egg yolk or phosphatidylcholine (lecithin) as a substrate, but the specific hemolytic activity and lethal toxicity was about eightfold higher in the complexed form. Furthermore, the free GCAT was more susceptible to proteolytic and heat inactivation than was the GCAT/LPS complex. Recombination of LPS (phenol extracted from extracellular products of A. salmonicida) with free GCAT enhanced the hemolytic activity, lethal toxicity, and heat stability of the latter but did not influence its lecithinase activity. In native polyacrylamide gel electrophoresis, the GCAT/LPS complex and the recombined GCAT-LPS both showed a high-molecular-mass band which did not enter the gel, while the free GCAT produced a single band with low molecular mass. In isoelectric focusing gels, the GCAT/LPS and recombined GCAT-LPS produced a nonfocusing smear with pIs from pI 5.0 to 5.8, while the free GCAT produced a single band with pI 4.3. These data show that free GCAT can combine with LPS to produce a high-molecular-mass complex with enhanced toxicity and heat stability compared with those of free GCAT, similar to the preexisting GCAT/LPS complex, and indicate that the LPS moiety of the toxin plays an active role in toxicity.

The Humoral Immune Response of Rainbow Trout ( Salmo gairdneri) Immunised by Various Regimes and Preparations of Aeromonas salmonicida Antigens

Antibody production in rainbow trout to extracellular antigens was investigated. The following antigen preparations and immunisation regimes were used: native extracellular products (ECP) in Freund's complete adjuvant (FCA), intraperitoneally (i.p.) with and without booster; formalinized ECP in FCA, i.p., with and without booster; washed, formalinized A. salmonicida cells in FCA, i.p., with booster; native ECP in saline, i.m., four weekly injections at two different doses, 45 μg and 6 μg each injection (after the protocol of Shieh, 1985) and native ECP neutralised with normal rainbow trout serum, i.p., single injection (after the protocol of Sakai, 1985). Using crossed immunoelectrophoresis all antisera contained precipitating antibodies to three to five ECP components except that from fish immunised i.m. with 6 μg protein where antibodies were undetectable. In no case were specific antibodies to ECP protease or haemolysin detected. In a rabbit immunised with formalinized ECP in FCA under a similar regime to the rainbow trout, antibodies to at least 15 ECP components, including protease and haemolysin, were detected. The assumption of a specific immune response to the protease, at least in respect of antibody production, in recent reports of protection afforded by vaccines composed of either crude ECP or partially purified protease (Shieh, 1985) or partially purified protease inactivated by normal serum (Sakai, 1985) is not supported by the present findings.

Characteristics and Distribution of Extracellular Proteases from Aeromonas hydrophila

SUMMARY: The major extracellular proteases of Aeromonas hydrophila NRC 505 and ATCC 7966 are a thermostable metallo-protease (TSMP) and a thermolabile serine-protease (TLSP), respectively. The purified enzymes differed in sensitivity to heating at 56 °C for 30 min, in response to the inhibitors EDTA and phenylmethylsulphonyl fluoride (PMSF), and were antigenically distinct. When crude extracellular products (ECP) of 47 strains of A. hydrophila were screened, 27 strains produced both TSMP and TLSP. TSMP was the only extracellular protease produced by 19 strains, whereas only A. hydrophila ATCC 7966 produced TLSP as its sole protease. This distribution of protease enzymes among strains explains conflicting reports from studies in which single strains of A. hydrophila were examined. The TLSP of A. hydrophila was similar to the protease of A. salmonicida. Either or both TSMP and TLSP were produced by some strains of A. sobria and A. caviae.

Semi-purification of an immunosuppressor substance secreted by Streptococcus mutans that plays a role in the protection of the bacteria in the host.

Crude extracellular products of Streptococcus mutans (CEP-Sm) suppress the proliferative response to phytohaemagglutinin of human peripheral blood mononuclear cells and the primary immune response of C57BL/6 mice to sheep erythrocytes. This immunosuppressive effect favours the survival of the microorganism, and the bacteria lose the ability to secrete immunosuppressor substance if previously subcultured several times. Cells incubated with CEP-Sm and subsequently washed recover the ability to proliferate. Traces of CEP-Sm or to short a time of contact between CEP-Sm and the target immune system induced higher proliferative ratios or higher in vivo immune responses than controls, respectively. The proliferative values of cultures supplemented with CEP-Sm were parallel to the control values up to a certain time, after which they dropped abruptly. This drop is followed by a proliferation, and the higher the amount of CEP-Sm added to the cultures, the shorter the time until the proliferation. CEP-Sm was fractionated by means of ion exchange chromatography followed by double preparative isoelectrofocusing, ending in a subfraction of isoelectric point between 3.9 and 4.2, containing a heat-unstable material stainable by Coomassie blue but unstainable by periodic acid Schiff or methylene blue, and having a maximum optical density of 280 nm.

Characterization of extracellular metallo- and serine-proteases of Aeromonas hydrophila strain B51.

The extracellular proteases of Aeromonas hydrophila B51 were stable on heating (56 degrees C) and on storage at 4 degrees C or -20 degrees C. Inhibitor studies showed that 72% of the total activity was inhibited by EDTA (a metalloprotease inhibitor) and 26% was inhibited by phenylmethanesulphonyl fluoride (a serine protease inhibitor). Analytical isoelectric focussing revealed the presence of 33 proteins in the crude extracellular products. Using a casein overlay technique three separate zones of proteolytic activity were detected: a zone with pI 6.5-6.8, formed of two closely focussed bands (possibly isomers of the same protease) and completely inhibited by EDTA; a single band with pI 7.0, which was inhibited by EDTA; and a diffuse zone with pI 8.3-8.5, which was only partially inhibited by EDTA. It is concluded that the serine protease activity focussed in this latter zone. These results indicate the presence of at least four, and possibly five proteases. Our results differ substantially from those reported by other workers using different isolates and it is suggested that significant differences in the character of extracellular products and extracellular proteases exist between different isolates of A. hydrophila.

Effects of short-term cold storage on recovery of proteases from extracellular products of Aeromonas hydrophila.

Three protease-containing fractions were recovered by gel filtration from concentrated crude extracellular products produced by Aeromonas hydrophila grown in a defined medium. The recovery of a heat-stable protease was differentially prevented when the crude preparation was stored for 48 h at -20 degrees C but was unaffected by storage of the crude preparation at either 4 or -70 degrees C. Once fractionated, the heat-stable protease appeared to be unaffected by subsequent storage at 4, -20, or -70 degrees C.

Fractionation and characterization of the immunosuppressive substance in crude extracellular products released by Streptococcus intermedius.

The noncytotoxic immunosuppressive substance detected in crude extracellular products of Streptococcus intermedius (CEP-SI) was fractionated by two steps of preparative isoelectric focusing in sucrose gradients using ampholytes of pH range from 3.5 to 6 and 4 to 5, respectively. The in vitro and in vivo suppressor effects of the most highly purified fraction of CEP-Si, designated fraction 3' (F3'EP-Si), corresponded well with those of the original CEP-Si. F3'EP-Si was sensitive to the effects of alpha, gamma, and delta chymotrypsin, trypsin, and heating. It contained approximately 1% of the total amount of protein found in the original CEP-Si, corresponding to a single band on analytical isoelectric focusing, stainable by Coomassie Blue and of isoelectric point of 4.25. The absorption spectrum of F3'EP-Si had a maximum at 260 nm but its biological activity was resistant to deoxyribonuclease and ribonuclease A and it did not contain material stainable by methylene blue. It was also resistant to neuraminidase and did not contain material stainable by periodic acid schiff. We conclude that the substance responsible for the suppressor activity of CEP-Si is a protein of molecular weight approximately 90,000, which adheres to Sephadex of cellulose acetate and forms complexes with other, nonactive constituents of CEP-Si.

Evidence for the synthesis and release of strongly immunosuppressive, noncytotoxic substances by Streptococcus intermedius.

Products secreted by Streptococcus intermedius were studied for their effects on the immune response. Three different preparations of crude extracellular products from S. intermedius (CEP-Si) were found to have powerful suppressor activity in vitro as shown by inhibition of human lymphocyte proliferation (uptake of [3H]thymidine) and protein synthesis in response to a wide variety of stimulants, including mitogens and antigens, and suppression of plaque formation by human cells in response to sheep erythrocytes. CEP-Si was noncytotoxic, because cells incubated with high concentrations of CEP-Si and subsequently washed were viable and recovered their ability to respond to mitogens, and because leukocyte migration was not inhibited by CEP-Si, nor was the release of leukocyte migration inhibitory factor from sensitized lymphocytes. The possibility of antigen or mitogen competition was excluded. The effects of CEP-Si in vitro were time dependent and did not require the presence of monocytes. Cells pretreated with CEP-Si and then washed suppressed plaque formation by fresh autologous cells in highly stimulated cultures. CEP-Si injected into C57BL/6 mice also strongly suppressed their immune response to sheep erythrocytes, and the in vivo suppression was correlated with the effects of CEP-Si in vitro.

Antigens of group A streptococci involved in passive hemagglutination reactions.

Antibodies to streptococcal extracellular products were detected in rabbit sera by passive hemagglutination tests as early as 1 week after intravenous injection of live group A streptococci (strain C203S). Using these antibodies in immunoadsorbent columns, we prepared an antigenic fraction from crude concentrates of streptococcal extracellular products. The specific activity of this fraction in sensitizing glutaraldehyde-treated erythrocytes for passive hemagglutination tests and in absorbing passive hemagglutination antibodies from immune sera was increased by 80-fold and at least 24-fold, respectively, as compared with crude extracellular products. The fraction has been found to contain at least three serologically active antigens, which are of considerable interest because: (i) they gave rise to early and consistent immune responses both in humans and in experimental animals; (ii) they appear so far to be produced only by beta-hemolytic streptococci; and (iii) antibodies to these antigens are present in high titers in patients with acute rheumatic fever. Our data suggest that passive hemagglutination antigens may be distinct from those extracellular enzymes and hemolysins ordinarily employed in streptococcal serological studies.