Nakaba Sugimoto

Clostridium perfringens Enterotoxin Utilizes Two Structurally Related Membrane Proteins as Functional Receptors in Vivo

Human and mouse cDNAs showing homology to theClostridium perfringens enterotoxin (CPE) receptor gene (CPE-R) from Vero cells (DDBJ/EMBL/GenBankTMaccession no. D88492) (Katahira, J., Inoue, N., Horiguchi, Y., Matsuda, M., and Sugimoto, N. (1997) J. Cell Biol. 136, 1239–1247) were cloned. They were classified into two groups, the Vero cell CPE receptor homologues and rat androgen withdrawal apoptosis protein (RVP1; accession no. M74067) homologues, based on the similarities of primary amino acid sequences. L929 cells that were originally insensitive to CPE became sensitive to CPE on their transfection with cDNAs encoding either the CPE receptor or RVP1 homologues, indicating that these gene products are not only structurally similar but also functionally active as receptors for CPE. By binding assay, the human RVP1 homologue showed differences in affinity and capacity of binding from those of the human CPE receptor. Northern blot analysis showed that mouse homologues of the CPE receptor and RVP1 are expressed abundantly in mouse small intestine. The expression ofCPE-R mRNA in the small intestine was restricted to cryptic enterocytes, indicating that the CPE receptor is expressed in intestinal epithelial cells. These results are consistent with reports that CPE binds to the small intestinal cells via two different kinds of receptors. High levels of expression of CPE-R and/orRVP1 mRNA were also detected in other organs, including the lungs, liver, and kidneys, but only low levels were expressed in heart and skeletal muscles. These results indicate that CPE uses structurally related cellular proteins as functional receptors in vivo and that organs that have not so far been recognized as CPE-sensitive have the potential to be targets of CPE.

ppGpp with DksA controls gene expression in the locus of enterocyte effacement (LEE) pathogenicity island of enterohaemorrhagic Escherichia coli through activation of two virulence regulatory genes.

For a new pathogen to emerge, it must acquire both virulence genes and a system for responding to changes in environmental conditions. Starvation of nutrients or growth arrest induces the stringent response in Escherichia coli, via increased ppGpp. We found the adherence capacity of enterohaemorrhagic E. coli (EHEC) and gene expression in the locus of enterocyte effacement (LEE) were enhanced by a downshift in nutrients or by entry into the stationary growth phase, both of which increase the ppGpp concentration. The activation was dependent on relA and spoT, which encode enzymes for the synthesis and degradation of ppGpp, and on dksA, which encodes an RNA polymerase accessory protein required for the stringent response. Upon induction of RelA expression, LEE gene transcription was activated within 20 min, even without starvation. The expression of two LEE transcriptional regulators, Ler and Pch, was activated by ppGpp and essential for the enhancement of LEE gene expression. In addition, the ler and pch promoters were directly activated by ppGpp in an in vitro transcription system. These findings suggest that the regulation of virulence genes in EHEC is integrated with E. colis stringent response system, through the regulation of virulence regulatory genes.

NleC, a type III secretion protease, compromises NF-κB activation by targeting p65/RelA.

The NF-κB signaling pathway is central to the innate and adaptive immune responses. Upon their detection of pathogen-associated molecular patterns, Toll-like receptors on the cell surface initiate signal transduction and activate the NF-κB pathway, leading to the production of a wide array of inflammatory cytokines, in attempt to eradicate the invaders. As a countermeasure, pathogens have evolved ways to subvert and manipulate this system to their advantage. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are closely related bacteria responsible for major food-borne diseases worldwide. Via a needle-like protein complex called the type three secretion system (T3SS), these pathogens deliver virulence factors directly to host cells and modify cellular functions, including by suppressing the inflammatory response. Using gain- and loss-of-function screenings, we identified two bacterial effectors, NleC and NleE, that down-regulate the NF-κB signal upon being injected into a host cell via the T3SS. A recent report showed that NleE inhibits NF-κB activation, although an NleE-deficient pathogen was still immune-suppressive, indicating that other anti-inflammatory effectors are involved. In agreement, our present results showed that NleC was also required to inhibit inflammation. We found that NleC is a zinc protease that disrupts NF-κB activation by the direct cleavage of NF-κBs p65 subunit in the cytoplasm, thereby decreasing the available p65 and reducing the total nuclear entry of active p65. More importantly, we showed that a mutant EPEC/EHEC lacking both NleC and NleE (ΔnleC ΔnleE) caused greater inflammatory response than bacteria carrying ΔnleC or ΔnleE alone. This effect was similar to that of a T3SS-defective mutant. In conclusion, we found that NleC is an anti-inflammatory bacterial zinc protease, and that the cooperative function of NleE and NleC disrupts the NF-κB pathway and accounts for most of the immune suppression caused by EHEC/EPEC.

Requirement of N-glycan on GPI-anchored proteins for efficient binding of aerolysin but not Clostridium septicum α-toxin

Aerolysin of the Gram‐negative bacterium Aeromonas hydrophila consists of small (SL) and large (LL) lobes. The α‐toxin of Gram‐positive Clostridium septicum has a single lobe homologous to LL. These toxins bind to glycosylphosphatidylinositol (GPI)‐anchored proteins and generate pores in the cells plasma membrane. We isolated CHO cells resistant to aerolysin, with the aim of obtaining GPI biosynthesis mutants. One mutant unexpectedly expressed GPI‐anchored proteins, but nevertheless bound aerolysin poorly and was 10‐fold less sensitive than wild‐type cells. A cDNA of N‐acetylglucosamine transferase I (GnTI) restored the binding of aerolysin to this mutant. Therefore, N‐glycan is involved in the binding. Removal of mannoses by α‐mannosidase II was important for the binding of aerolysin. In contrast, the α‐toxin killed GnTI‐deficient and wild‐type CHO cells equally, indicating that its binding to GPI‐anchored proteins is independent of N‐glycan. Because SL bound to wild‐type but not to GnTI‐deficient cells, and because a hybrid toxin consisting of SL and the α‐toxin killed wild‐type cells 10‐fold more efficiently than GnTI‐ deficient cells, SL with its binding site for N‐glycan contributes to the high binding affinity of aerolysin.

Regulation of virulence by butyrate sensing in enterohaemorrhagic Escherichia coli.

Enterohaemorrhagic Escherichia coli (EHEC) colonizes and proliferates at the mucosal surface, inducing severe diarrhoea. Short-chain fatty acids (SCFAs) are abundant in the intestine owing to the metabolic activity of microflora, and are important for colonic health. We found that, although a high concentration of SCFAs inhibited the growth of EHEC, at low concentrations, the SCFAs markedly enhanced the expression of the virulence genes required for cell adherence and the induction of attaching and effacing (A/E) lesions. Of the SCFAs tested, butyrate markedly enhanced the expression of these virulence-associated genes, even at the low concentration of 1.25 mM, but acetate and propionate showed only a small effect at concentrations higher than 40 mM. Butyrate enhanced the promoter activity of the LEE1 operon, which encodes a global regulator of the LEE genes, Ler. This enhancement was dependent on a regulator, PchA. Butyrate sensing was completely abrogated by the deletion of lrp, the gene for the leucine-responsive regulatory protein, Lrp. Expression of a constitutively active mutant of Lrp enhanced the expression of the LEE genes in the absence of butyrate, and a response-defective Lrp derivative reduced the response to butyrate. Thus, upon entering the distal ileum, EHEC may respond to the higher butyrate level via Lrp by increasing its virulence expression, leading to efficient colonization of the target niche.

Dual regulatory pathways integrating the RcsC–RcsD–RcsB signalling system control enterohaemorrhagic Escherichia coli pathogenicity

Bacterial pathogenesis is strictly regulated in response to changes in environmental conditions. A His–Asp phosphorelay system consisting of a sensor kinase and response regulator is used by Gram‐positive and Gram‐negative bacteria to control gene expression in response to environmental stimuli. We screened His–Asp phosphorelay systems for their effect on virulence expression in enterohaemorrhagic Escherichia coli (EHEC), and found rcsD or rcsB overexpression enhanced locus for enterocyte effacement (LEE) gene transcription and adherence to Caco‐2 cells through transcriptional activation of the ler regulatory gene. An EHEC‐specific regulator GrvA, encoded by ECs1274, was required for ler transcription activation by RcsB. Furthermore, GrvA activated ler transcription in E. coli K12. Stimulation of the RcsDCB regulatory system by RcsF overexpression slightly increased EspB expression in the wild type but not the ECs1274 mutant. However, EspB expression in an rcsB deletion mutant increased compared with wild type, suggesting that RcsB negatively regulates LEE gene expression and that active RcsB protein is present under normal growth conditions. Deletion of pchA, which encodes a positive regulator for ler, abolished the effect of the rcsB deletion, suggesting that pchA mediated the negative RcsB effect. pchA transcript levels decreased when RcsB expression increased. Thus, LEE gene transcription may be regulated by RcsB through two oppositely regulated O157‐specific regulators, PchA and GrvA.

Activation of Motility by Sensing Short-Chain Fatty Acids via Two Steps in a Flagellar Gene Regulatory Cascade in Enterohemorrhagic Escherichia coli

ABSTRACT The regulated expression of virulence genes is critical for successful infection by an intestinal pathogen. Bacteria rely on sensing environmental signals to find preferable niches and reach the infectious state. Orally ingested enterohemorrhagic Escherichia coli (EHEC) travels through the gastrointestinal tract and encounters a variety of environmental factors, some of which act as triggering signals for the induction of virulence genes. Butyrate, one of the main short-chain fatty acids (SCFAs), is such a signal, enhancing the expression of genes for intimate attachment and type III secretion. We further explored the role of SCFAs and found a positive effect of SCFAs on flagellar expression. Although EHEC did not produce flagella when grown in Dulbeccos modified Eagles medium (DMEM), a tissue culture medium that enhances virulence gene expression, the addition of SCFAs to the medium induced the production of flagella, and the EHEC bacteria became motile. Among SCFAs, butyrate simultaneously activates both virulence and flagellar genes. Flagella did not affect initial adherence, and they were not expressed in adherent bacteria during microcolony formation. SCFAs activated flagellar genes via two regulatory steps. Butyrate activated the flhDC regulatory genes through leucine-responsive regulatory protein (Lrp), which is also a regulator of virulence genes. However, butyrate, acetate, and propionate also activated downstream genes independently of flhDC activation. Consequently, when encountering increased concentrations of SCFAs, which are abundant in acetate, in the intestine, EHEC first activates flagellar production and motility, followed by genes involved in adherence and type III secretion, which leads to efficient adherence in a preferable niche.

Primary action of Clostridium perfringens type a enterotoxin on hela and vero cells in the absence of extracellular calcium: rapid and characteristic changes in membrane permeability

Clostridium perfringens type A enterotoxin bound rapidly to HeLa and Vero cells in the absence of extracellular Ca2+ at 37 degrees C. The bound toxin rapidly (within 2 min) caused influx of Na+ and efflux of K+ and Mg2+. Changes in membrane permeability occurred in the absence or presence of extracellular Ca2+ and to the similar extents at 37 degrees C and 4 degrees C, in contrast to the subsequent bleb and balloon formation, which required both extracellular Ca2+ and incubation at 37 degrees C. Substances with molecular weights of over ca. 200 protected the cells from the morphological alterations induced by the toxin, whereas substances with molecular weights of less than ca. 200 did not. The mechanism of the primary action of the enterotoxin is discussed.

Comparison of effects of botulinum toxin subtype A1 and A2 using twitch tension assay and rat grip strength test

Botulinum toxin type A is used as a therapeutic agent for some spastic neurological disorders. Type A organisms have been classified into four subtypes (A1 to A4) based on the amino acid sequence variability of the produced neurotoxin. At present, commercially available preparations of the toxin belong to subtype A1. To date, no study has compared the characteristics of the biological activity of toxins from different subtypes. We compared the efficacy of A1 toxin (LL toxin or neurotoxin: NTX) with that of A2 toxin (NTX) employing the twitch tension assay using the mouse phrenic nerve hemidiaphragm and grip strength test in rats. The inhibitory effects on neuromuscular transmission of A2NTX at pH 7.4 and pH 6.8 were 1.95 and 3.73 times more potent than those of A1LL, respectively. The 50% effective doses for the administered limb, the dose which caused a 50% reduction in grip strength, i.e. ED(50), of A1LL, A1NTX, and A2NTX were calculated as 0.087, 0.060, and 0.040 U/head, respectively. These doses for the contralateral limb, i.e. TD(50), of A1LL, A1NTX, and A2NTX were calculated as 6.35, 7.54, and 15.62 U/head, respectively. In addition, the time required for A2NTX-injected rats to recover the grip strength of the contralateral limb was 17 days, while that for rats injected with A1LL was 35 days. The results indicated that A2NTX is a more potent neuromuscular blocker than A1 toxins, and suggested that A2NTX will provide a preferentical therapeutic agent for neurological disorders.

Acute botulinum-like intoxication by tetanus neurotoxin in mice

Abstract Intravenous injection of purified tetanus toxin(1000-0.06 μg) killed mice within minutes(20–450 min), causing flaccid paralysis indistinguishable from that in botulinum intoxication: a linear relation was found between the log of the toxin dose and that of death time(survival time). The dose and route dependences of the manifestations of the spastic paralysis typical of classical tetanus and of the acute botulinum-like flaccid paralysis were studied in relation to the death time. Treatment of the toxin with trypsin or gangliosides did not affect its acute botulinum-like toxicity. Theophylline delayed the time of acute death due to the botulinum-like intoxication in mice caused by tetanus toxin and provided some protection.