Jun Fujii

Rapid Apoptosis Induced by Shiga Toxin in HeLa Cells

ABSTRACT Apoptosis was induced rapidly in HeLa cells after exposure to bacterial Shiga toxin (Stx1 and Stx2; 10 ng/ml). Approximately 60% of HeLa cells became apoptotic within 4 h as detected by DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and electron microscopy. Stx1-induced apoptosis required enzymatic activity of the Stx1A subunit, and apoptosis was not induced by the Stx2B subunit alone or by the anti-globotriaosylceramide antibody. This activity was also inhibited by brefeldin A, indicating the need for toxin processing through the Golgi apparatus. The intracellular pathway leading to apoptosis was further defined. Exposure of HeLa cells to Stx1 activated caspases 3, 6, 8, and 9, as measured both by an enzymatic assay with synthetic substrates and by detection of proteolytically activated forms of these caspases by Western immunoblotting. Preincubation of HeLa cells with substrate inhibitors of caspases 3, 6, and 8 protected the cells against Stx1-dependent apoptosis. These results led to a more detailed examination of the mitochondrial pathway of apoptosis. Apoptosis induced by Stx1 was accompanied by damage to mitochondrial membranes, measured as a reduced mitochondrial membrane potential, and increased release of cytochrome c from mitochondria at 3 to 4 h. Bid, an endogenous protein known to permeabilize mitochondrial membranes, was activated in a Stx1-dependent manner. Caspase-8 is known to activate Bid, and a specific inhibitor of caspase-8 prevented the mitochondrial damage. Although these data suggested that caspase-8-mediated cleavage of Bid with release of cytochrome c from mitochondria and activation of caspase-9 were responsible for the apoptosis, preincubation of HeLa cells with a specific inhibitor of caspase-9 did not protect against apoptosis. These results were explained by the discovery of a simultaneous Stx1-dependent increase in endogenous XIAP, a direct inhibitor of caspase-9. We conclude that the primary pathway of Stx1-induced apoptosis and DNA fragmentation in HeLa cells is unique and includes caspases 8, 6, and 3 but is independent of events in the mitochondrial pathway.

Autophagy induced by 2-deoxy-D-glucose suppresses intracellular multiplication of Legionella pneumophila in A/J mouse macrophages.

Legionella pneumophila Philadelphia-1 (Lp-1) can grow intracellularly in A/J mouse peritoneal macrophages (A/J Mφ). We previously reported that 2-deoxy-D-glucose (2dG), when added in macrophage culture media, inhibited the intracellular multiplication of Lp-1 in A/J Mφ. We found that 1mM of 2dG caused LC3-II-conversion that reflects an induction of autophagy and that 1 and 10mM of 2dG induced apoptosis associated with caspase-4 activation. We therefore investigated whether 2dG-induced autophagy or apoptosis suppresses the replication of Lp-1 in 2dG-treated A/J Mφ. When autophagy-related 5 (Atg5) was knocked down by RNA interference, the Atg5-siRNA-transfected cells revealed an enhanced replication of Lp-1 in A/J Mφ compared with the nontargetting siRNA-transfected cells. However, caspase-4 inhibitor did not affect the 2dG-induced inhibition of intracellular multiplication of Lp-1 in A/J Mφ. These findings suggested that autophagy, not apoptosis, suppressed the intracellular growth of Lp-1 in A/J Mφ when 1 or 10 mM of 2dG were added to the culture media.

Toxicity of Shiga Toxin 1 in the Central Nervous System of Rabbits

ABSTRACT The action of Shiga toxin (Stx) on the central nervous system was examined in rabbits. Intravenous Stx1 was 44 times more lethal than Stx2 and acted more rapidly than Stx2. However, Stx1 accumulated more slowly in the cerebrospinal fluid than did Stx2. Magnetic resonance imaging demonstrated a predominance of Stx1-dependent lesions in the spinal cord. Pretreatment of the animals with anti-Stx1 antiserum intravenously completely protected against both development of brain lesions and mortality.

Protection by a Recombinant Mycobacterium bovis Bacillus Calmette-Guérin Vaccine Expressing Shiga Toxin 2 B Subunit against Shiga Toxin-Producing Escherichia coli in Mice

ABSTRACT We have developed a novel vaccine against Shiga toxin (Stx)-producing Escherichia coli (STEC) infection using a recombinant Mycobacterium bovis BCG (rBCG) system. Two intraperitoneal vaccinations with rBCG expressing the Stx2 B subunit (Stx2B) resulted in an increase of protective serum IgG and mucosal IgA responses to Stx2B in BALB/c mice. When orally challenged with 103 CFU of STEC strain B2F1 (O91: H21), the immunized mice survived statistically significantly longer than the nonvaccinated mice. We suggest that intraperitoneal immunization with rBCG expressing Stx2B would be a potential vaccine strategy for STEC.

Effects of Shiga Toxin 2 on Lethality, Fetuses, Delivery, and Puerperal Behavior in Pregnant Mice

ABSTRACT Shiga toxin 2 (Stx2) is produced by enterohemorrhagicEscherichia coli (EHEC) and is known as the major virulence factor of EHEC. The aim of this study was to evaluate the effects of Stx2 on (i) maternal lethality, (ii) fetuses, (iii) delivery period, and (iv) maternal behavior after delivery. Timed pregnant ICR mice were injected intravenously with Stx2 on day 5 of pregnancy (early stage) or on day 15 (late stage). In early-stage experiments, the number of normal fetuses of mice injected with Stx2 was significantly lower than that of control mice. In late-stage experiments, mothers injected with Stx2 delivered normal numbers of neonates, but could not take care of them. The lethal doses of Stx2 were not different for pregnant and nonpregnant female mice at either stage. We conclude that Stx2 is toxic to the fetus in early pregnancy and affects maternal puerperal behavior in late pregnancy.

Brainstem mechanisms of autonomic dysfunction in encephalopathy‐associated Shiga toxin 2 intoxication

Acute encephalopathy is the major determinant of death in an early stage of Shiga toxin (Stx)‐producing Escherichia coli infection. Rapid progress toward refractory hypotension and dysfunction of breathing implies autonomic center dysfunction of patients. To clarify whether autonomic dysfunction becomes an ultimate cause of death in Shiga toxemia, we injected purified Stx2 (20 μg/kg) intravenously into rabbits, and monitored changes in cardiovascular and respiratory function together with renal sympathetic nerve activity (RSNA) in the conscious state. After an approximately 24‐hour silent (lag) period, all rabbits given Stx2 developed hemorrhagic diarrhea (25.7 ± 1.1 hours) and limb paralysis (31.2 ± 1.3 hours). This limb paralysis was observed initially in the hind legs, and then it gradually extended to the forelegs. After 23.2 ± 2.3 hours, RSNA increased gradually, and arterial blood pressure was maintained within normal limits together with an increase in the maximum gain of baroreflex response. Severe hypotension developed within 34.8 ± 2.2 hours, without any increase in heart rate; RSNA significantly increased by 39.5 ± 0.9 hours. In the final stage, RSNA decreased concurrently with decreases in arterial blood pressure, heart rate, and baroreflex response, suggesting dysfunction of the baroreflex control system. Thereafter, all rabbits died within 47.8 ± 1.2 hours after the intravenous Stx2 injection. Magnetic resonance imagings of the central nervous system (T2‐weighted images) showed high‐intensity areas in the dorsal two‐thirds of the cervical spinal cord and brainstem 48 hours after Stx2 administration. These results show that the cause of death is circulatory failure caused by impairment of the cardiovascular center in the medulla. We believe that this animal model helps to clarify the mechanism of rapid progress to death of patients with Shiga toxin–producing E coli infection. Ann Neurol 1999;45:716–723

Successful steroid pulse therapy for brain lesion caused by Shiga toxin 2 in rabbits

Betamethasone sodium phosphate (BSP) is usually used as a steroid therapy for human brain edema. High doses of BSP (36mg/kg) twice a day for two days statistically reduced the mortality rate and improved the survival period of Stx2 (1.4mug/kg; 1.6LD(50))-toxemic rabbits. We made evaluations on three kinds of magnetic resonance images (MRI) including T1-weighted, T2-weighted, and enhanced MRI using gadopentetate dimeglumine (Gd-DTPA) to detect brain lesion induced by an intravenous injection of Stx2 in rabbits. Enhanced T1-weighted MRI was the most sensitive tool to find leakage of Gd-DTPA suggesting impairment of the blood brain barrier caused by Stx2. Enhanced MRI revealed that BSP treatment inhibited the leakage of Gd-DTPA, as directly evidenced by the protective effect of BSP against brain edema induced by intravenous injection of Stx2. Interleukin 1beta was not induced after Stx2 treatment in brain primary mixed cell culture.

Investigation of Encephalopathy Caused by Shiga Toxin 2c-Producing Escherichia coli Infection in Mice

A large outbreak of Shiga toxin (Stx)-producing enteroaggregative Escherichia coli (EAEC) O104:H4 occurred in northern Germany. From this outbreak, at least 900 patients developed hemolytic uremic syndrome (HUS), resulting in more than 50 deaths. Thirty percent of the HUS patients showed encephalopathy. We previously established a mouse model with encephalopathy associated with blood brain barrier (BBB) damage after oral infection with the Shiga toxin (Stx) 2c-producing Escherichia coli O157: H- strain E32511 (E32511). In this model, we detected high expression of the Stx receptor synthase enzyme, glycosphingolipid globotriaosylceramide (Gb3) synthase, in endothelial cells (ECs) and neurons in the reticular formation of the medulla oblongata by in situ hybridization. Caspase-3 was activated in neurons in the reticular formation of the medulla oblongata and the anterior horn of the spinal cord. Astrocytes (ASTs) were activated in the medulla oblongata and spinal cord, and a decrease in aquaporin 4 around the ECs suggested that BBB integrity was compromised directly by Stx2c or through the activation of ASTs. We also report the effectiveness of azithromycin (AZM) in our model. Moreover, AZM strongly inhibited the release of Stx2c from E32511 in vitro.

LEGIONELLA INFECTION AND CONTROL IN OCCUPATIONAL AND ENVIRONMENTAL HEALTH

This review describes the microbiological characteristics of Legionella species, their habits in the environment, source and route of infection, pathogenesis, symptoms and treatment of legionellosis, and disease outbreaks worldwide. We also describe prevention measures to deal with this organism in the work environment. Particularly in Japan, the present measures and countermeasures against legionellosis are inadequate when compared with those Europe and the United States because occupational and environmental medicine in Japan has not been structured from a microbiological viewpoint. As a result, workplace inspections have not covered cooling towers. We surveyed the cooling towers provided in work environments and in hospitals in Kitakyushu City for contamination by Legionella species. The surveillance definitely revealed that we are surrounded by cooling towers contaminated with Legionella. We conclude that in Japan, occupational and environmental physicians should routinely monitor the water in cooling towers.

Adipose tissue is the first colonization site of Leptospira interrogans in subcutaneously infected hamsters

Leptospirosis is one of the most widespread zoonoses in the world, and its most severe form in humans, “Weil’s disease,” may lead to jaundice, hemorrhage, renal failure, pulmonary hemorrhage syndrome, and sometimes,fatal multiple organ failure. Although the mechanisms underlying jaundice in leptospirosis have been gradually unraveled, the pathophysiology and distribution of leptospires during the early stage of infection are not well understood. Therefore, we investigated the hamster leptospirosis model, which is the accepted animal model of human Weil’s disease, by using an in vivo imaging system to observe the whole bodies of animals infected with Leptospira interrogans and to identify the colonization and growth sites of the leptospires during the early phase of infection. Hamsters, infected subcutaneously with 104 bioluminescent leptospires, were analyzed by in vivo imaging, organ culture, and microscopy. The results showed that the luminescence from the leptospires spread through each hamster’s body sequentially. The luminescence was first detected at the injection site only, and finally spread to the central abdomen, in the liver area. Additionally, the luminescence observed in the adipose tissue was the earliest detectable compared with the other organs, indicating that the leptospires colonized the adipose tissue at the early stage of leptospirosis. Adipose tissue cultures of the leptospires became positive earlier than the blood cultures. Microscopic analysis revealed that the leptospires colonized the inner walls of the blood vessels in the adipose tissue. In conclusion, this is the first study to report that adipose tissue is an important colonization site for leptospires, as demonstrated by microscopy and culture analyses of adipose tissue in the hamster model of Weil’s disease.