Rapid cytopathic effects of Clostridium perfringens beta-toxin on porcine endothelial cells

Clostridium perfringens type C isolates cause fatal, segmental necro-hemorrhagic enteritis in animals and humans. Typically, acute intestinal lesions result from extensive mucosal necrosis and hemorrhage in the proximal jejunum. These lesions are frequently accompanied by microvascular thrombosis in affected intestinal segments. In previous studies we demonstrated that there is endothelial localization of C. perfringens type C beta-toxin (CPB) in acute lesions of necrotizing enteritis. This led us to hypothesize that CPB contributes to vascular necrosis by directly damaging endothelial cells. By performing additional immunohistochemical studies using spontaneously diseased piglets, we confirmed that CPB binds to the endothelial lining of vessels showing early signs of thrombosis. To investigate whether CPB can disrupt the endothelium, we exposed primary porcine aortic endothelial cells to C. perfringens type C culture supernatants and recombinant CPB. Both treatments rapidly induced disruption of the actin cytoskeleton, cell border retraction, and cell shrinkage, leading to destruction of the endothelial monolayer in vitro. These effects were followed by cell death. Cytopathic and cytotoxic effects were inhibited by neutralization of CPB. Taken together, our results suggest that CPB-induced disruption of endothelial cells may contribute to the pathogenesis of C. perfringens type C enteritis.

Neonatal hemolytic uremic syndrome after mother-to-child transmission of a low-pathogenic stx2b harboring shiga toxin-producing Escherichia coli

This case describes evidence for a Shiga toxin-producing Escherichia coli (STEC) O146:H28 infection leading to hemolytic uremic syndrome in a neonate. STEC O146:H28 was linked hitherto with asymptomatic carriage in humans. Based on strain characteristics and genotyping data, the mother is a healthy carrier who transmitted the STEC during delivery. STEC strains belonging to the low-pathogenic STEC group must also be considered in the workup of neonatal hemolytic uremic syndrome.

Distribution of RTX toxin genes in strains of [Actinobacillus] rossii and [Pasteurella] mairii

Strains of [Actinobacillus] rossii, [Pasteurella] mairii and [Pasteurella] aerogenes can be isolated from abortion in swine. The RTX toxin Pax has previously been found only in those [P.] aerogenes strains isolated from abortion. Nothing is known about RTX toxins in field isolates of the other two species. To gain insight into the distribution of selected RTX toxin genes and their association with abortion, PCR screening for the pax, apxII and apxIII operons on 21 [A.] rossii and seven [P.] mairii isolates was done. Since species can be phenotypically misidentified, the study was backed up by a phylogenetic analysis of all strains based on 16S rRNA, rpoB and infB genes. The pax gene was detected in all [P.] mairii but not in [A.] rossii strains. No apx genes were found in [P.] mairii but different gene combinations for apx were detected in [A.] rossii strains. Most of these strains were positive for apxIII, either alone or in combination with apxII. Whereas pax was found to be associated to strains from abortion no such indication could be found with apx in [A.] rossii strains. Phylogenetically [A.] rossii strains formed a heterogeneous cluster separated from Actinobacillus sensu stricto. [P.] mairii strains clustered with [P.] aerogenes but forming a separate branch. The fact that [P.] aerogenes, [P.] mairii and [A.] rossii can phylogenetically clearly be identified and might contain distinct RTX toxin genes allows their proper diagnosis and will further help to investigate their role as pathogens.

Real-time multiplex PCR assays for reliable detection of Clostridium perfringens toxin genes in animal isolates

Typing of Clostridium perfringens strains by PCR-based determination of toxin genes proved to be a reliable method for diagnosis of enterotoxaemia in various animal species. We report the establishment and validation of three real-time fluorogenic (TaqMan) multiplex PCRs for the detection of C. perfringens alpha-, beta-, beta2-, epsilon-, entero- and iota-toxin genes. The composition of the PCRs was chosen with regard to robustness of the assays and in order to increase sensitivity compared to the conventional simplex PCRs. The combination of probe dyes selected for the real-time assays (FAM/TAMRA, Cy-5/BHQ-2 and VIC/TAMRA) as well as the designation of the chromosome-borne alpha-toxin as internal positive control allowed the creation of highly specific and sensitive, as well as time and cost effective PCRs. One hundred and three strains of C. perfringens isolated in Switzerland derived from clinical or suspected cases of enterotoxaemia in 10 different animal species were tested. The toxin genotypes were in agreement in both the conventional PCRs and the newly designed multiplex PCRs. Furthermore, the real-time PCR carried out as simplex allows to quantitate the copy numbers of plasmid-borne toxin genes in relation to the chromosomally located alpha-toxin gene.

CSTX-1, a toxin from the venom of the hunting spider Cupiennius salei, is a selective blocker of L-type calcium channels in mammalian neurons

The inhibitor cystine-knot motif identified in the structure of CSTX-1 from Cupiennius salei venom suggests that this toxin may act as a blocker of ion channels. Whole-cell patch-clamp experiments performed on cockroach neurons revealed that CSTX-1 produced a slow voltage-independent block of both mid/low- (M-LVA) and high-voltage-activated (HVA) insect Ca(v) channels. Since C. salei venom affects both insect as well as rodent species, we investigated whether Ca(v) channel currents of rat neurons are also inhibited by CSTX-1. CSTX-1 blocked rat neuronal L-type, but no other types of HVA Ca(v) channels, and failed to modulate LVA Ca(v) channel currents. Using neuroendocrine GH3 and GH4 cells, CSTX-1 produced a rapid voltage-independent block of L-type Ca(v) channel currents. The concentration-response curve was biphasic in GH4 neurons and the subnanomolar IC(50) values were at least 1000-fold lower than in GH3 cells. L-type Ca(v) channel currents of skeletal muscle myoballs and other voltage-gated ion currents of rat neurons, such as I(Na(v)) or I(K(v)) were not affected by CSTX-1. The high potency and selectivity of CSTX-1 for a subset of L-type channels in mammalian neurons may enable the toxin to be used as a molecular tool for the investigation of this family of Ca(v) channels.

Modulation of chemokine gene expression by Shiga-toxin producing Escherichia coli belonging to various origins and serotypes

Infection with Shiga-toxin producing Escherichia coli (STEC) may result in the development of the haemolytic-uremic syndrome (HUS), the main cause of acute renal failure in children. While O157:H7 STEC are associated with large outbreaks of HUS, it is difficult to predict whether a non-O157:H7 isolate can be pathogenic for humans. The mucosal innate immune response plays a central role in the pathogenesis of HUS; therefore, we compared the induction of IL-8 and CCL20 in human colon epithelial cells infected with strains belonging to different serotypes, isolated from cattle or from HUS patients. No correlation was observed between strain virulence and chemokine gene expression. Rather, the genetic background of the strains seems to determine the chemokine gene expression profile. Investigating the contribution of different bacterial factors in this process, we show that the type III secretion system of O157:H7 bacteria, but not the intimate adhesion, is required to stimulate the cells. In addition, H7, H10, and H21 flagellins are potent inducers of chemokine gene expression when synthesized in large amount.

Differential expression of stx2 variants in Shiga toxin-producing Escherichia coli belonging to seropathotypes A and C

Only a subset of Shiga toxin (Stx)-producing Escherichia coli (STEC) are human pathogens, but the characteristics that account for differences in pathogenicity are not well understood. In this study, we investigated the distribution of the stx variants coding for Stx2 and its variants in highly virulent STEC of seropathotype A and low-pathogenic STEC of seropathotype C. We analysed and compared transcription of the corresponding genes, production of Shiga toxins, and stx-phage release in basal as well as in induced conditions. We found that the stx(2) variant was mainly associated with strains of seropathotype A, whereas most of the strains of seropathotype C possessed the stx(2-vhb) variant, which was frequently associated with stx(2), stx(2-vha) or stx(2c). Levels of stx(2) and stx(2)-related mRNA were higher in strains belonging to seropathotype A and in those strains of seropathotype C that express the stx(2) variant than in the remaining strains of seropathotype C. The stx(2-vhb) genes were the least expressed, in basal as well as in induced conditions, and in many cases did not seem to be carried by an inducible prophage. A clear correlation was observed between stx mRNA levels and stx-phage DNA in the culture supernatants, suggesting that most stx(2)-related genes are expressed only when they are carried by a phage. In conclusion, some relationship between stx(2)-related gene expression in vitro and the seropathotype of the STEC strains was observed. A higher expression of the stx(2) gene and a higher release of its product, in basal as well as in induced conditions, was observed in pathogenic strains of seropathotype A. A subset of strains of seropathotype C shows the same characteristics and could be a high risk to human health.

Human microbiota-secreted factors inhibit shiga toxin synthesis by enterohemorrhagic Escherichia coli O157:H7

Escherichia coli O157:H7 is a food-borne pathogen causing hemorrhagic colitis and hemolytic-uremic syndrome, especially in children. The main virulence factor responsible for the more serious disease is the Shiga toxin 2 (Stx2), which is released in the gut after oral ingestion of the organism. Although it is accepted that the amount of Stx2 produced by E. coli O157:H7 in the gut is critical for the development of disease, the eukaryotic or prokaryotic gut factors that modulate Stx2 synthesis are largely unknown. In this study, we examined the influence of prokaryotic molecules released by a complex human microbiota on Stx2 synthesis by E. coli O157:H7. Stx2 synthesis was assessed after growth of E. coli O157:H7 in cecal contents of gnotobiotic rats colonized with human microbiota or in conditioned medium having supported the growth of complex human microbiota. Extracellular prokaryotic molecules produced by the commensal microbiota repress stx(2) mRNA expression and Stx2 production by inhibiting the spontaneous and induced lytic cycle mediated by RecA. These molecules, with a molecular mass of below 3 kDa, are produced in part by Bacteroides thetaiotaomicron, a predominant species of the normal human intestinal microbiota. The microbiota-induced stx(2) repression is independent of the known quorum-sensing pathways described in E. coli O157:H7 involving SdiA, QseA, QseC, or autoinducer 3. Our findings demonstrate for the first time the regulatory activity of a soluble factor produced by the complex human digestive microbiota on a bacterial virulence factor in a physiologically relevant context.

Long-term cellular and regional specificity of the photoreceptor toxin, iodoacetic acid (IAA), in the rabbit retina

This study investigated the anatomical consequences of a photoreceptor toxin, iodoacetic acid (IAA), in the rabbit retina. Retinae were examined 2 weeks, 1, 3, and 6 months after systemic IAA injection. The retinae were processed using standard histological methods to assess the gross morphology and topographical distribution of damage, and by immunohistochemistry to examine specific cell populations in the retina. Degeneration was restricted to the photoreceptors and was most common in the ventral retina and visual streak. In damaged regions, the outer nuclear layer was reduced in thickness or eliminated entirely, with a concomitant loss of immunoreactivity for rhodopsin. However, the magnitude of the effect varied between animals with the same IAA dose and survival time, suggesting individual differences in the bioavailability of the toxin. In all eyes, the inner retina remained intact, as judged by the thickness of the inner nuclear layer, and by the pattern of immunoreactivity for protein kinase C-alpha (rod bipolar cells) and calbindin D-28 (horizontal cells). Müller cell stalks became immunoreactive for glial fibrillary acidic protein (GFAP) even in IAA-treated retinae that had no signs of cell loss, indicating a response of the retina to the toxin. However, no marked hypertrophy or proliferation of Müller cells was observed with either GFAP or vimentin immunohistochemistry. Thus the selective, long lasting damage to the photoreceptors produced by this toxin did not lead to a reorganization of the surviving cells, at least with survival as long as 6 months, in contrast to the remodeling of the inner retina that is observed in inherited retinal degenerations such as retinitis pigmentosa and retinal injuries such as retinal detachment.

Clostridium perfringens beta-toxin binding to vascular endothelial cells in a human case of enteritis necroticans

Clostridium perfringens type C-induced enteritis necroticans is a rare but often fatal disease in humans. A consistent histopathological finding is an acute, deep necrosis of the small intestinal mucosa associated with acute vascular necrosis and massive haemorrhage in the lamina propria and submucosa. Retrospective immunohistochemical investigations of tissues from a diabetic adult who died of enteritis necroticans revealed endothelial localization of C. perfringens beta-toxin in small intestinal lesions. Our results indicate that vascular necrosis might be induced by a direct interaction between C. perfringens beta-toxin and endothelial cells and that targeted disruption of endothelial cells plays a role in the pathogenesis of enteritis necroticans.

Mesenteric lymphangitis and sepsis due to RTX toxin-producing Actinobacillus spp in 2 foals with hypothyroidism-dysmaturity syndrome

Actinobacillus suis-like organisms (ASLOs) have been isolated from the genital, respiratory, and digestive tracts of healthy adult horses, horses with respiratory disease, and septic foals. Two foals with congenital hypothyroidism-dysmaturity syndrome from separate farms developed ASLO infection. At necropsy, both had contracted carpal flexor tendons, thyroid hyperplasia, and thrombotic and necrotizing mesenteric lymphangitis and lymphadenitis; one foal also had mandibular prognathism. Numerous ASLOs were isolated from tissues from both foals, including intestine. Biochemical testing and mass spectrometric analysis of the two Actinobacillus isolates did not allow unequivocal identification. Comparative genetic analysis was done on these and similar isolates, including phylogeny based on 16S rRNA, rpoB and recN genes, as well as RTX (repeat in toxin) toxin typing of apxIA-apxIVA and aqxA genes. One isolate was identified as Actinobacillus suis sensu stricto, based on the presence of apxIA and apxIIA but not aqxA, whereas the other isolate had aqxA but neither apxIA nor apxIIA, consistent with A equuli ssp haemolyticus. Based on genotypic analysis of the isolates included for comparison, 3 of 3 equine ASLOs and 2 of 5 A equuli isolates were reclassified as A equuli subsp haemolyticus, emphasizing the importance of toxin genotyping in accurate classification of actinobacilli.

Characterization of the type I secretion system of the RTX toxin ApxII in "Actinobacillus porcitonsillarum"

Strains of Actinobacillus porcitonsillarum are regularly isolated from the tonsils of healthy pigs. A. porcitonsillarum is non pathogenic but phenotypically it strongly resembles the pathogenic species Actinobacillus pleuropneumoniae, thereby interfering with the diagnosis of the latter. A. porcitonsillarum is hemolytic but unlike A. pleuropneumoniae, it contains only apxII genes and not apxI or apxIII genes. In contrast to the truncated apxII operon of A. pleuropneumoniae, which lacks the type I secretion genes BD, characterization of the apxII operon in A. porcitonsillarum revealed that it contains an intact and complete apxII operon. This shows a typical RTX operon structure with the gene arrangement apxIICABD. The region upstream of the apxII operon is also different from that in A. pleuropneumoniae and contains an additional gene, aspC, encoding a putative aspartate aminotransferase. Trans-complementation experiments in Escherichia coli and A. pleuropneumoniae indicated that the entire apxII operon of A. porcitonsillarum is sufficient to express and secrete the ApxIIA toxin and that the ApxIIA toxin of A. pleuropneumoniae can be secreted by the type I secretion system encoded by apxIIBD. These findings suggest that the complete apxII operon found in A. porcitonsillarum might be an ancestor of the truncated homologue found in A. pleuropneumoniae. The genetic context of the apxII locus in A. porcitonsillarum and A. pleuropneumoniae suggests that in the latter, the contemporary truncated operon is the result of a recombination event within the species, rather than a horizontal transfer of an incomplete operon.

Antibodies to Aqx toxin of Actinobacillus equuli in horses and foals

Actinobacillus equuli is found in the normal oral flora of horses, but has been associated with several diseases, and particularly with the usually fatal septicaemia in neonatal foals which is thought to be associated with a failure of the passive transfer of immunoglobulins via the colostrum. The Aqx protein of A equuli, belonging to the RTX family of pore-forming toxins, is also cytotoxic to horse lymphocytes. The presence of antibodies to Aqx was investigated in sera from individual horses from different regions; the sera from adult horses and foals 24 hours after birth reacted with Aqx, and sera from foals sampled shortly after an intake of colostrum also reacted with Aqx, but sera from foals taken before an intake of colostrum did not react with Aqx.

Phylogenetic relationship of equine Actinobacillus species and distribution of RTX toxin genes among clusters

Equine Actinobacillus species were analysed phylogenetically by 16S rRNA gene (rrs) sequencing focusing on the species Actinobacillus equuli, which has recently been subdivided into the non-haemolytic A. equuli subsp. equuli and the haemolytic A. equuli subsp. haemolyticus. In parallel we determined the profile for RTX toxin genes of the sample of strains by PCR testing for the presence of the A. equuli haemolysin gene aqx, and the toxin genes apxI, apxII, apxIII and apxIV, which are known in porcine pathogens such as Actinobacillus pleuropneumoniae and Actinobacillus suis. The rrs-based phylogenetic analysis revealed two distinct subclusters containing both A. equuli subsp. equuli and A. equuli subsp. haemolyticus distributed through both subclusters with no correlation to taxonomic classification. Within one of the rrs-based subclusters containing the A. equuli subsp. equuli type strain, clustered as well the porcine Actinobacillus suis strains. This latter is known to be also phenotypically closely related to A. equuli. The toxin gene analysis revealed that all A. equuli subsp. haemolyticus strains from both rrs subclusters specifically contained the aqx gene while the A. suis strains harboured the genes apxI and apxII. The aqx gene was found to be specific for A. equuli subsp. haemolyticus, since A. equuli subsp. equuli contained no aqx nor any of the other RTX genes tested. The specificity of aqx for the haemolytic equine A. equuli and ApxI and ApxII for the porcine A. suis indicates a role of these RTX toxins in host species predilection of the two closely related species of bacterial pathogens and allows PCR based diagnostic differentiation of the two.