Plesiomonas shigelloides exports a lethal cytotoxic-enterotoxin (LCE) by membrane vesicles

ABSTRACT Plesiomonas shigelloides isolated from water in Brazil was previously described as a hemorrhagic heat-labile cytotoxic-enterotoxin producer. We purified this toxin from culture supernatants using ion metallic affinity chromatography (IMAC) followed by molecular exclusion chromatography. The pure toxin presented molecular mass of 50 kDa and isoelectric point (pI) around 6.9 by 2D electrophoresis. When injected intravenously, the purified cytotoxic-enterotoxin induced also severe spasms followed by sudden death of mice. Hence, we entitled it as lethal cytotoxic-enterotoxin (LCE). The presence of membrane vesicles (MVs) on cell surfaces of P. shigelloides was observed by scan electron microscopy (SEM). From these MVs the LCE toxin was extracted and confirmed by biological and serological assays. These data suggest that P. shigelloides also exports this cytotoxic-enterotoxin by membrane vesicles, a different mechanism of delivering extra cellular virulence factors, so far not described in this bacterium.

Perfil de producción de citoquinas en linfocitos T de sangre periférica de pacientes con poliposis nasal y asma bronquial en respuesta a la estimulación con enterotoxina estafilocócica B / Cytokine production profile in peripheral blood T lymphocytes from patients with nasal poliposis and bronchial asthma in response to staphylococcal enterotoxin B

Introducción: La poliposis nasal (PN) se presenta frecuentemente asociada a asma bronquial (AB). La enterotoxina estafilocócica B (SEB) jugaría un papel en su patogenia. No se ha estudiado si el perfil de citoquinas inducido por SEB en linfocitos T (LT) de pacientes con PNyAB difiere del de controles sanos. Objetivo: Comparar el perfil de citoquinas de LT de sangre periférica de pacientes con PN-AByde controles, estimulados con SEB o concanavalina A (ConA). Material y método: Células mononucleares de sangre periférica de 9 pacientes con PN-AB y de 6 controles se estimularon con SEB o ConA. El porcentaje LT CD4+ productores de interferón (IFN)-y, interleuquina (IL) IL-4, IL-5, IL-17 e IL-21 se determinó mediante citometrfa de flujo. Resultados: El grupo PN-AB presentó un menor porcentaje de LT productores de IL-5 que los controles al estimularse con SEB y con ConA. No hubo diferencia en las otras citoquinas estudiadas. Discusión: Nuestros resultados en sangre periférica difieren de lo descrito en tejido de pólipos nasales. Conclusión: Se sugiere que la respuesta inflamatoria de la PN se originaría localmente ya que los LT de sangre de pacientes con PN-AB no muestran una polarización hacia perfiles proinflamatorios con los estímulos utilizados.

Introduction: Nasal poliposis (NP) is frequently associated with bronchial asthma (BA) and its pathogenesis is still unknown. Staphylococcal enterotoxin B (SEB) has been implicated in the development of NP, however if the SEB-induced cytoklne profile of peripheral blood T lymphocytes (TL) of PN-BA patients differs from that of normal controls has not been studied. Aim: To compare the cytoklne profile of CD4+ TL from NP-BA and controls stimulated with SEB or concanavalin A (ConA). Material and method: Peripheral blood mononuclear cells from 9 NP-BA patients and from 6 controls were stimulated with SEB or ConA. The percentage of interferon (IFN)-y, interleukin {II) 11-4,11-5,11-17, and 11-21 producing TL was analyzed by flow cytometry Results: The percentage of SEB and ConA stimulated CD4+ IL-5-producing TLs was lower in the NP-BA group compared to the control group. There were no differences in the other cytokine-producing populations. Discussion: Unlike what is described in nasal polyp tissue, our findings show a diminished production of IL-5 by peripheral TL from the NP-AB group. Conclusion: A local sinonasal origin of the chronic inflammation is suggested since peripheral blood TL of NP-BA patients do not show a pro-inflammatory polarization with the tested stimuli.

Impact of zinc deficiency on vibrio cholerae enterotoxin-stimulated water and electrolyte transport in animal model.

The effect of zinc deficiency on the function of the intestine to absorb water and electrolytes was studied in animal models, stimulated by Vibrio cholerae enterotoxin. Sprague-Dawley rats, used in the study, were divided into four groups: Zinc-deficient, ad libitum zinc-fed control, zinc weight-matched control, and zinc-deficient acutely-repleted. 14C-labelled polyethylene glycol solution was used for measuring the absorption capacity of the small intestine. Significantly lower absorption of water and sodium per cm of the intestine was observed in the zinc-deficient animals compared to the ad libitum zinc-fed control animals (p < 0.01). An improved absorption capacity was equally observed in the zinc-deficient acutely-repleted animals and ad libitum zinc-fed control group. The zinc-deficient animals showed four times greater cholera toxin-induced net secretions of water and sodium compared to the ad libitum zinc-fed group (p < 0.01), while a 40% reduction was observed in the zinc-deficient acutely-repleted group. The results suggest that zinc deficiency is associated with reduced absorption of water and electrolytes and increased secretion of the same stimulated by cholera toxin.

Unique features of bovine lymphocytes exposed to a staphylococcal enterotoxin

We previously demonstrated that stimulation of bovine peripheral blood mononuclear cells (PBMCs) with staphylococcal enterotoxin C (SEC), led to an inversion of the CD4(+):CD8(+) T cell ratio and generation of an atypical CD8(+) T cell subpopulation expressing CD26. In the present study, we examined T cell apoptosis and proliferation profiles of PBMC subpopulations in cultures stimulated with SEC. Unlike when stimulated with concanavalin A, nucleic acid synthesis in bovine PBMC cultures stimulated with SEC was low during the first four days but increased greatly on day 5. In contrast, nucleic acid synthesis in human PBMC cultures stimulated with SEC increased continuously. To investigate the mechanism of delayed bovine T cell proliferation, various cell phenotypes were monitored. The inversion of the bovine CD4(+):CD8(+) T cell ratio in PBMC cultures stimulated by SEC was associated with higher proliferation and lower apoptosis of CD8(+) T cells compared to CD4(+) T cells. The mRNA levels for interleukin (IL)-4 and IL-13 were sustained over 4 days but IL-12 mRNA levels dropped to background on day 2. These data suggest that SEC induces a prolonged Th-2- biased microenvironment, and together with the inversion of the bovine CD4(+):CD8(+) T cell ratios in bovine PBMC cultures with SEC, may in part explain the inability of the mammary immune system to establish an effective response to Staphylococcus aureus infections.

Water permeability properties of the human small intestine in vitro: effects of Escherichia coli heat-stable enterotoxin

The net absorptive water flux (Jw), the transepithelial potential difference (PD) and the short-circuit current (Isc) were simultaneously measured in the human small intestine in vitro with the following results: 1) An absorptive Jw was observed when the jejunum or the ileum were mounted between two identical standard solutions in the presence of an hydrostatic pressure gradient (delta P) of 13 cm of water (mucosal side positive). 2) The absorptive Jw was a linear function of the applied delta P or the imposed osmotic transepithelial gradient (deltaOsm) in both intestinal segments. The hydrostatic (Phydr) and osmotic (Posm) permeabilities to water for jejunum and ileum were: 0.349 ñ 0.049 cm/s vs. 0.156 ñ 0.022 cm/s and 0.0012 ñ 0.0001 cm/s vs. 0.0019 ñ O.0003, respectively. 3) A fraction of this absorptive Jw was independent of the presence of any hydrostatic, osmotic or chemical gradient and represented the transport associated to movement of water (Jwt). 4) PD and Isc values were similar in the jejunum and in the ileum but the transepithelial resistance (Rt) was significantly greater in ileum than in jejunum. 5) 2 mug/ml of E. coli heat-stable enterotoxin (STa) caused a significant inhibition of the absorptive Jw without modification of Phydr, Posm or Isc. 6) After STa treatment, the absorptive Jwt reverted to a secretory one in the jejunum. In the ileum, STa action caused a 48 per cent decrease in the absorptive Jwt values.

Vascular and glomerular effects of Clostridium difficile toxin A peptide on the isolated rat kidney

Toxin A peptide from Clostridium difficile caused damage and secretion in the intestinal mucosa. These effects are mediated in part by pro-inflammatory substances. In order to evaluate and compare the biologic effect of toxin A on renal vascular, glomerular and tubular functions, we studied this toxin in isolated rat kidneys. Isolated kidneys from adult male Wistar rats (260-320 g) were perfused with Krebs-Henseleit solution containing 60 mg/ml dialyzed bovine serum albumin. We studied the effect of toxin A peptide (3.2 x 10(-6) M, injected into perfusate) on glomerular filtration rate (GFR), urinary flow rate (UF) and total sodium reabsorption (TNa+, per cent ). All experiments were preceded by a 30-min basal period, and in another group of kidneys the time course of the variables was followed without toxin infusion for unpaired control. Toxin A (TxA) reduced the perfusion pressure (PP), from PPcontrol/30min = 124.89 +/- 1.91 to PPTxA/120min = 88.13 +/- 5.1 mmHg (N = 6, P < 0.01) with a maximal effect at 120 min after toxin infusion. TxA also caused a significant decrease in GFR with maximal effect at 90 min after toxin infusion (GFRcontrol/30min = 0.53 +/- 0.05 to GFRTxA/90min = 0.30 + 0.05 ml min-1g-1; N = 6, P < 0.01). TxA did not alter renal tubular sodium transport when compared with a control without toxin infusion. In addition, toxin-treated kidneys caused a time-dependent increase in urinary flow from UFcontrol/30min = 0.16 +/- 0.08 to UFTxA/120min = 0.35 +/- 0.1 ml min-1g-1 (N = 6, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Vibrio cholerae enterotoxin peptide on glomerular filtration rate and renal proximal tubular sodium transport

Cholera toxin peptide stimulates adenylyl cyclase activity in several tissues and causes severe intestinal water and electrolyte secretion. To evaluate the regulatory function of sodium transport in renal tubules, we studied the effect of cholera toxin peptide on rat kidneys. Isolated kidneys from adult male hooded rats weighting 240-335 g were perfused with Krebs-Henseleit solution containing 60 mg/ml dialyzed bovine serum albumin (BSA). The effects of Vibrio cholerae peptide (CT; molecular weight, approximately 82,000 Dalton) on glomerular filtration rate (GFR), proximal sodium reabsorption ( per cent pTNa+) and urinary flow rate (UF) were studied. All experiments were preceded by a 30-min control period and in another group of kidneys the time course of the variables was followed without toxin infusion, for a paired control. Control kidneys perfused with Krebs-Henseleit solution plus 60 mg/ml BSA presented stable GFR (paired internal control GFR30 min = 0.596 +/- 0.248 ml g-1 min-1 vs GFR120 min = 0.694 +/- 0.362, N = 32; P > 0.05) and per cent pTNa+ ( per cent pTNa+ 30 min = 75 +/- 8.3 vs 84 +/- 1.6 for the internal control, N = 32; P > 0.05). CT caused a dose (0.03, 0.75 and 1.0 microgram/ml)-dependent decrease in GFR starting at 30 min and with a maximal peak of effect at 90 min after toxin infusion (GFRCT = 0.130 +/- 0.086 ml g-1 min-1, N = 12, vs paired internal control GFRControl/30 min = 0.660 +/- 0.132, N = 12; P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of Bacillus cereus enteropathy.

Three strains of Bacillus cereus isolated from sausages (Salami and Trekker, RANBAC, Ranchi) produced enterotoxin which caused vascular permeability in skin and haemorrhage in the ligated ileal loops of rabbits. Histopathological studies revealed haemorrhage and congestion in submucosa, mononuclear cell infiltration in lamina propria and submucosa and villous atrophy. Histochemical studies ruled out the effect on mitochondrial enzymes of intestinal epithelial cells. Purified enterotoxin given intradermally to rabbits caused severe necrotic reaction at the site of injection and death within 4 hr. Histopathological changes observed in liver included congestion of portal veins and sinusoids, vacuolar degeneration of hepatocytes, and hyperplasia of bile ducts. These suggested that B. cereus enterotoxin affected the capillaries of blood vessels locally and also systemically resulting into release of proteinaceous exudates and red blood cells.

Evaluation of Biken test for detection of Salmonella enterotoxin.

Biken test by using antiserum to purified Salmonella enterotoxin (SE) was standardized and carried out to screen salmonellae for their enterotoxigenicity. As many as 101 strains of Salmonella belonging to 15 different serotypes isolated from foods of animal origin were subjected to Biken test. Of these, 68 (67.32%) were found seropositive. The test correlated with the rabbit ligated ileal loop (RLIL) test completely for the detection of enterotoxin producing salmonellae with 24 test strains. However, 5 of the 13 strains which were negative in the RLIL test, yielded positive results with the Biken test.