Levels of Alpha-Toxin Correlate with Distinct Phenotypic Response Profiles of Blood Mononuclear Cells and with agr Background of Community-Associated Staphylococcus aureus Isolates

Epidemiological studies of Staphylococcus aureus have shown a relation between certain clones and the presence of specific virulence genes, but how this translates into virulence-associated functional responses is not fully elucidated. Here we addressed this issue by analyses of community-acquired S. aureus strains characterized with respect to antibiotic resistance, ST types, agr types, and virulence gene profiles. Supernatants containing exotoxins were prepared from overnight bacterial cultures, and tested in proliferation assays using human peripheral blood mononuclear cells (PBMC). The strains displayed stable phenotypic response profiles, defined by either a proliferative or cytotoxic response. Although, virtually all strains elicited superantigen-mediated proliferative responses, the strains with a cytotoxic profile induced proliferation only in cultures with the most diluted supernatants. This indicated that the superantigen-response was masked by a cytotoxic effect which was also confirmed by flow cytometry analysis. The cytotoxic supernatants contained significantly higher levels of alpha-toxin than did the proliferative supernatants. Addition of alpha-toxin to supernatants characterized as proliferative switched the response into cytotoxic profiles. In contrast, no effect of Panton Valentine Leukocidin, delta-toxin or phenol soluble modulin alpha-3 was noted in the proliferative assay. Furthermore, a significant association between agr type and phenotypic profile was found, where agrII and agrIII strains had predominantly a proliferative profile whereas agrI and IV strains had a predominantly cytotoxic profile. The differential response profiles associated with specific S. aureus strains with varying toxin production could possibly have an impact on disease manifestations, and as such may reflect specific pathotypes.

Modelling staphylococcal pneumonia in a human 3D lung tissue model system delineates toxin-mediated pathology

Staphylococcus aureus necrotizing pneumonia is recognized as a toxin-mediated disease, yet the tissue-destructive events remain elusive, partly as a result of lack of mechanistic studies in human lung tissue. In this study, a three-dimensional (3D) tissue model composed of human lung epithelial cells and fibroblasts was used to delineate the role of specific staphylococcal exotoxins in tissue pathology associated with severe pneumonia. To this end, the models were exposed to the mixture of exotoxins produced by S. aureus strains isolated from patients with varying severity of lung infection, namely necrotizing pneumonia or lung empyema, or to purified toxins. The necrotizing pneumonia strains secreted high levels of alpha-toxin and Panton-Valentine leukocidin (PVL), and triggered high cytotoxicity, inflammation, necrosis and loss of E-cadherin from the lung epithelium. In contrast, the lung empyema strain produced moderate levels of PVL, but negligible amounts of alpha-toxin, and triggered limited tissue damage. alpha-toxin had a direct damaging effect on the epithelium, as verified using toxin-deficient mutants and pure alpha-toxin. Moreover, PVL contributed to pathology through the lysis of neutrophils. A combination of alpha-toxin and PVL resulted in the most severe epithelial injury. In addition, toxin-induced release of pro-inflammatory mediators from lung tissue models resulted in enhanced neutrophil migration. Using a collection of 31 strains from patients with staphylococcal pneumonia revealed that strains producing high levels of alpha-toxin and PVL were cytotoxic and associated with fatal outcome. Also, the strains that produced the highest toxin levels induced significantly greater epithelial disruption. Of importance, toxin-mediated lung epithelium destruction could be inhibited by polyspecific intravenous immunoglobulin containing antibodies against alpha-toxin and PVL. This study introduces a novel model system for study of staphylococcal pneumonia in a human setting. The results reveal that the combination and levels of alpha-toxin and PVL correlate with tissue pathology and clinical outcome associated with pneumonia.

Comparative study on the toxin production and profiles Raphidophytes at different salinities

PLEASE ALSO CHECK THE FULL TEXT ABSTRACT. Toxin production and toxin profiles of four Raphidophytes grown under different salinities were compared to investigate the influence of salinity on cellular content of neurotoxin. In Chatonella andqua CaTx-1, CaTx-11, and CaTx-111 peaked at 25 pplt with yields of 0.99, 0.42, and 2.90 pg/ceU, but the highest yields (2.35 pg/cell) of CaTx-IV was attained at 30 ppt. On the other hand, Chatonella marina yielded higher proportions of CmTx-1 (0.55 pg/ceH) and CmTx-111 (2.50 pg/cell) at 25 ppt. However, CmTx-IV was present in its highest amount (1.65 pg/cell) at 30 ppt, as seen in C anriqua. A smaH amount of CmTx-11 was also detected at 20-35 ppt. The toxin compositions indicate that H. akashiwo is more sensitive to higher salinities than the other three raphidophytes. Substantial compositional change was observed in case of H. akashiwo. HaTx-11 (corresponding to PbTx-9) was detected only as a trace at 20 and 25 ppt. Toxin HaTx-IV (corresponding to oxidized PbTx-2) was most dominant and peaked at 20 ppt with a yield of 0.3 pg/cell. Considerable amounts of HaTx-1 and III (corresponding to PbTx-2 and 3) were also detected. At higher salinities of above 25 ppt HaTx-11 was not detected. F. japonica gave highest yields of FjTx-11 (PbTx-2) and FjTx-IV (Oxidized PbTx-2) at 20 ppt with yields of 0.95, 1.54 pg/cell while the production of toxic profiles FjTx-1 (PbTx- 1) and FjTx-111 (PbTx-3) peaked at 25 ppt with yields of 0.99, 2.54 pg/ceU. A sharp decrease in all toxins profiles (CaTx, CmTx, HaTX and FjTx) was found at salinities of above 30 ppt.

Toxicity test: Sponge toxin on tilapia fry

The research was conducted to determine the toxicity of extracts from five Philippine species of marine sponges on tilapia Oreochromis niloticus fry. It was found out that the most potent was the methanol extract of Dysidea herbacea, it kills with the least toxin and at the shortest time.

Effects of cyanobacterial toxin microcystin-LR on the transcription levels of immune-related genes in grass carp Ctenopharyngodon idella

Recent studies in mammals have revealed that the cyanobacterial toxin MC-LR suppresses immune functions. Nevertheless, immunotoxic effects of microcystins have been little studied in fish. In this paper, we present the profiles of the immune modulation of MC-LR in grass carp, and quantitative real-time PCR methodology was developed for the measurement of relative transcription changes of six immune-related genes in the spleen and head kidney of the grass carp Ctenopharyngodon idella, which were intraperitoneally injected with 50 mu g MC-LR center dot kg(-1) body weight in a three-week period. This study was focused exclusively on gene transcription level changes at different time points after MC-LR exposure, so, only one dose was given. The investigated genes were interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), type I interferon (Type I IFN), peptidoglycan recognition protein-L (PGRP-L), immunoglobulin M (IgM) and major histocompatibility complex class I (MHC-I) genes. The results demonstrated that the transcription levels of the TNF-alpha, type I IFN, and PGRP-L genes in the spleen and head kidney were significantly low at all time points, and those of IL-1 beta were significantly low in the head kidney at different time points. In addition, IgM and MHC-I transcription levels were only significantly low in the spleen and head kidney at 21 d postinjection. The changes in the transcription levels of immune-related genes induced by MC-LR confirmed its effect on inhibiting immune function at the transcription level.

Improvement in growth and toxin production of Alexandrium tamarense by two-step culture method

The growth and toxin content of the dinoflagellate Alexandrium tamarense ATHK was markedly affected by culture methods. In early growth phase at lower cell density static or mild agitation methods were beneficial to growth, but continuous agitation or aeration, to some extent, had an adverse effect on cell growth. Static culture in 2 L Erlenmeyer flasks had the highest growth rate (0.38 d(-1)) but smaller cell size compared with other culture conditions. Cells grown under aerated conditions possessed low nitrogen and phosphorus cell yields, namely high N and P cell-quota. At day 18, cells grown in continuous agitated and 1 h aerated culture entered the late stationary phase and their cellular toxin contents were higher (0.67 and 0.54 pg cell(-1)) compared with cells grown by other culture methods (0.27-0.49 pg cell(-1)). The highest cell density and cellular toxin content were 17190 cells mL(-1) and 1.26 pg cell(-1) respectively in an airlift photobioreactor with two-step culture. The results indicate that A. tamarense could be grown successfully in airlift photobioreactor by a two-step culture method, which involved cultivating the cells statically for 4 days and then aerating the medium. This provides an efficient way to enhance cell and toxin yield of A. tamarense.

Analysis of paralytic shellfish toxins in Aphanizomenon DC-1 from Lake Dianchi, China

Lake Dianchi is in Yunnan Province in southwestern China. In recent years, significant cyanobacterial blooms have occurred in this lake nearly every year because of eutrophication. Monitoring data for the past 5 years acquired by our research group showed that phytoplankton composition alternated between species of Microcystis sp. during warm seasons and those of Aphanizomenon sp. during cool seasons. In March 2003, when phytoplankton composition was highly dominated by Aphanizomenon sp., samples were taken from the lake for toxin detection and immediate strain isolation. A mouse bioassay with extracts from the lyophilized field material showed obvious intoxication from paralytic shellfish poisons (PSPs), and all mice died within 30 min. Further analysis of both field and isolated algal strain Aphanizomenon DC-1 by the postcolumn HPLC-FLD method confirmed its PSP-producing ability The analogues found in the extracts from the field material were neoSTX, dcSTX, and dcGTX3, with contents of 2.279, 1.135, and 0.547 ng/mg DW, respectively. Under laboratory culture condition, toxin content in the Aphanizomenon strain DC-1 varied greatly during different growth phases, with two peaks: in the early-exponential and late-stationary growth phases. When the culture grew at a relatively high rate during the mid- to late-exponential growth phase, toxin content declined gradually. Moreover, the types of toxin in the DC-1 strain varied greatly during a single culture cycle. The HPLC results showed that dcSTX was the only toxin isomer detected throughout the culture period, and its level remained stable. On the other hand, dcGTX2 and GTX4 were the major toxins during the early-exponential and stationary phases, respectively. This article presents the first data on the identification and detection of paralytic shellfish toxins from cyanobacteria in Lake Dianchi. As far as we know, this is also the first report of this type of toxin in inland water bodies in China. Our study indicates the threat associated with PSP toxins in Lake Dianchi and suggests that necessary measures and programs for control are urgently needed to prevent the spread of toxic cyanobacterial blooms. (c) 2006 Wiley Periodicals, Inc.

In vivo studies on toxin accumulation in liver and ultrastructural changes of hepatocytes of the phytoplanktivorous bighead carp i.p.-injected with extracted microcystins

Phytoplanktivorous bighead carp were injected i.p. with extracted microcystins (mainly MC-RR and -LR) at two doses, 200 and 500 MC-LReq. mu g kg(-1) bw, and the changes in extractable MCs in liver and in the ultrastructure of hepatocytes were studied at 1, 3, 12, 24 and 48 h after injection. Quantitative and qualitative determinations of MCs in the liver were conducted by HPLC and LC-MS, respectively. MC concentration in the liver reached the maxima at 12 It (2.89 mu g MCs g(-1) dry weight at the lower dose) or at 3 h (5.43 mu g MCs g(-1) dry weight at the higher dose) post-injection, followed by sharp declines afterwards, whereas the ultrastructural changes of hepatocytes in both dose groups suggest progressive increases in severity toward the directions of apoptosis and necrosis from I to 24 h, respectively. There were two new findings in fish: widening of intercellular spaces was among the early ultrastructural changes induced by MCs and ultrastructural recovery of hepatocytes was evident at 48 h post-injection in both dose groups. Both the present and previous studies suggest that with in vivo or in vitro exposure to microcystins, hepatocyte damage in fish tends to proceed toward the direction of apoptosis at lower MC concentrations but toward the direction of necrosis at high MC concentrations. The temporal dynamics of MCs in the liver suggest that bighead carp may have a mechanism to degrade or bind MC-LR actively after it enters the blood system. (c) 2005 Elsevier Ltd. All rights reserved.

Effects of nitrogen forms on the production of cyanobacterial toxin microcystin-LR by an isolated Microcystis aeruginosa

A cyanobacterial strain, which produced high content of microcystin-LR (MC-LR) but no rnicrocystin-RR (MC-RR), was isolated from the hypertrophic Dianchi Lake in China and identified as Microcystis aeruginosa DC-1. Effects of nitrogen containing chemicals and trace elements on the growth and the production of MC-LR by this strain were Studied. In the presence of bicine, compared with urea and ammonium, nitrate greatly promoted the growth and the production of MC-LR. However, leucine and arginine, which were the constitutional components in the molecular structure of MC-LR or RR, inhibited the production of MC-LR. Iron and silicon up to 10mg/L had little effects on the growth of M. aeruginosa DC-1, but the production of MC-LR was apparently enhanced. Under all conditions studied here, only MC-LR but no RR was detected within the cells of M. aeruginosa DC-1. Thus, chemical forms of nitrogen, rather than the usually concerned the total nitrogen, Lind trace elements played important roles in the production of MC toxins during cyanobacterial blooms.

杀伤血管内皮生长因子受体1阳性细胞的靶向毒素

白喉毒素(diphtheria toxin DT) 是棒状白喉杆菌被茁噬菌体感染后分泌的一种外毒素. 它可以阻断真核细胞的蛋白质合成,杀死细胞. 血管内皮生长因子(VEGF) 的R82A,K84A,H86A突变体可以和肿瘤血管上高表达的VEGF受体1 (VEGFR-1) 特异性结合. 首先从白喉杆菌中提取基因组DNA,扩增出白喉毒素C区、T区基因. 并运用点突变技术,制成VEGF的R82A,K84A,H86A突变体. 利用这个可以和肿瘤血管上特异性受体相结合的VEGF的突变体,代替白喉毒素上的受体结合区,制成了针对VEGFR-1的靶向融合毒素——DT391-mVEGF. 以去除了受体结合区的DT391为阴性对照,细胞实验表明,融合毒素对VEGFR-1阳性的肿瘤细胞有特异性杀伤作用.

Microbial modulation in the biomass and toxin production of a red-tide causing alga

The effect of S-10, a strain of marine bacteria isolated from sediment in the Western Xiamen Sea, on the growth and paralytic shellfish poison (PSP) production in the alga Alexandrium tamarense (A. tamarense) was studied under controlled experimental conditions. The results of these experiments have shown that the growth of A. tamarense is obviously inhibited by S-10 at high concentrations, however no evident effect on its growth was observed at low concentrations. Its PSP production was also inhibited by S 10 at different concentrations, especially at low concentrations. The toxicity of this strain of A. tamarense is about (0.9512.14) x 10(-6) MU/cell, a peak toxicity value of 12.14 x 10(-6) MU/cell appeared on the 14th day, after which levels decreased gradually. The alga grew well in conditions of pH 6-8 and salinities of 20-34 parts per thousand. The toxicity of the alga varied markedly at different pH and salinity levels. Toxicity decreased as pH increased, while it increased with salinity and reached a peak value at a salinity of 30 parts per thousand, after which it declined gradually. S-10 at a concentration of 1.02 x 10(9) cells/ml inhibited growth and the PSP production of A. tamarense at different pH and salinity levels. S-10 had the strongest inhibitory function on the growth of A. tamarense under conditions of pH 7 and a salinity of 34 parts per thousand. The best inhibitory effect on PSP production by A. tamarense was at pH 7, this inhibitory effect on PSP production did not relate to salinity. Interactions between marine bacteria and A. tamarense were also investigated using the flow cytometer technique (FCM) as well as direct microscope counting. S-10 was identitied as being a member of the genus Bacillus, the difference in 16S rDNA between S-10 and Bacillus halmapalus was only 2%. The mechanism involved in the inhibition of growth and PSP production of A. tamarense by this strain of marine bacteria, and the prospect of using it and other marine bacteria in the biocontrol of red-tides was discussed. (c) 2005 Elsevier Ltd. All rights reserved.

A recent shellfish toxin investigation in China

A shellfish toxin investigation along the Chinese coast has recently been conducted using both HPLC and mouse assay methods. The results showed that DSP was widely distributed in different shellfish species in China. 26 out of 89 samples had DTX1 (dinophysistoxin-1) or OA (okadaic acid) but the DSP content in most shellfish samples did not reach the regulatory limit for human consumption adopted in many countries (20 mu g/100 g soft tissue). PSP was also found in 5 out of 96 samples along the coast. One sample, Chlamys nobilis from Hong Kong contained high levels of PSP (320 mu g STX equivalent/100 g soft tissue), compared to the regulatory limit (80 mu g STX equivalent/100 g soft tissue). After the recent outbreak of red tide in Hong Kong waters, three further shellfish samples were collected within 40 days to investigate the impacts of this event, It was shown that high levels of PSP continued to exist in Hong Kong waters. This report provides the first report of DSP and PSP distribution along the Chinese coast. (C) 1999 Elsevier Science Ltd, All rights reserved.

Toxin-screening and identification of bacteria isolated from highly toxic marine gastropod Nassarius semiplicatus

Bacteria isolated from a highly toxic sample of gastropod Nassarius semiplicatus in Lianyungang, Jiangsu Province in July 2007, were studied to probe into the relationship between bacteria and toxicity of nassariid gastropod. The toxicity of the gastropod sample was 2 x 10(2) mouse unit (MU) Per gram Of tissue (wet weight). High concentration of tetrodotoxin (TTX) and its analogues (TTXs) were found in the digestive gland and muscle of the gastropod, using high performance liquid chromatography coupled with mass chromatography (LC-MS). Bacterial strains isolated from the digestive gland were cultured and screened for TTX with a competitive ELISA method. Tetrodotoxin was detected in a proportion of bacterial strains, but the toxin content was low. Partial 16S ribosomal DNA (rDNA) of the TTX-producing strains was then sequenced and compared with those published in the GenBank to tentatively identify the toxic strains. It was found that most of the toxic strains were closely affiliated with genus Vibrio, and the others were related to genus Shewanella, Marinomonas, Tenacibaculum and Aeromonas. These findings suggest that tetrodotoxin-producing bacteria might play an important role in tetrodotoxin accumulation/production in N. semiplicatus. (C) 2008 Elsevier Ltd. All rights reserved.

Overexpression of the cardiac beta(2)-adrenergic receptor and expression of a beta-adrenergic receptor kinase-1 (betaARK1) inhibitor both increase myocardial contractility but have differential effects on susceptibility to ischemic injury.

Cardiac beta(2)-adrenergic receptor (beta(2)AR) overexpression is a potential contractile therapy for heart failure. Cardiac contractility was elevated in mice overexpressing beta(2)ARs (TG4s) with no adverse effects under normal conditions. To assess the consequences of beta(2)AR overexpression during ischemia, perfused hearts from TG4 and wild-type mice were subjected to 20-minute ischemia and 40-minute reperfusion. During ischemia, ATP and pH fell lower in TG4 hearts than wild type. Ischemic injury was greater in TG4 hearts, as indicated by lower postischemic recoveries of contractile function, ATP, and phosphocreatine. Because beta(2)ARs, unlike beta(1)ARs, couple to G(i) as well as G(s), we pretreated mice with the G(i) inhibitor pertussis toxin (PTX). PTX treatment increased basal contractility in TG4 hearts and abolished the contractile resistance to isoproterenol. During ischemia, ATP fell lower in TG4+PTX than in TG4 hearts. Recoveries of contractile function and ATP were lower in TG4+PTX than in TG4 hearts. We also studied mice that overexpressed either betaARK1 (TGbetaARK1) or a betaARK1 inhibitor (TGbetaARKct). Recoveries of function, ATP, and phosphocreatine were higher in TGbetaARK1 hearts than in wild-type hearts. Despite basal contractility being elevated in TGbetaARKct hearts to the same level as that of TG4s, ischemic injury was not increased. In summary, beta(2)AR overexpression increased ischemic injury, whereas betaARK1 overexpression was protective. Ischemic injury in the beta(2)AR overexpressors was exacerbated by PTX treatment, implying that it was G(s) not G(i) activity that enhanced injury. Unlike beta(2)AR overexpression, basal contractility was increased by betaARK1 inhibitor expression without increasing ischemic injury, thus implicating a safer potential therapy for heart failure.