In vitro evidence for a bacterial pathogenesis of equine laminitis

Utilizing an in vitro laminitis explant model, we have investigated how bacterial broth cultures and purified bacterial proteases activate matrix metalloproteinases (MMPs) and alter structural integrity of cultured equine lamellar hoof explants. Four Gram-positive Streptococcus spp. and three Gram-negative bacteria all induced a dose-dependent activation of MMP-2 and MMP-9 and caused lamellar explants to separate. MMP activation was deemed to have occurred if a specific MMP inhibitor, batimastat, blocked MMP activity and prevented lamellar separation. Thermolysin and streptococcal pyrogenic exotoxin B (SpeB) both separated explants dose-dependently but only thermolysin was inhibitable by batimastat or induced MMP activation equivalent to that seen with bacterial broths. Additionally, thermolysin and broth MMP activation appeared to be cell dependent as MMP activation did not occur in isolation. These results suggest the rapid increase in streptococcal species in the caecum and colon observed in parallel with carbohydrate induced equine laminitis may directly cause laminitis via production of exotoxin(s) capable of activating resident MMPs within the lamellar structure. Once activated, these MMPs can degrade key components of the basement membrane (BM) hemidesmosome complex, ultimately separating the BM from the epidermal basal cells resulting in the characteristic laminitis histopathology of hoof lamellae. While many different causative agents have been evaluated in the past, the results of this study provide a unifying aetiological mechanism for the development of carbohydrate induced equine laminitis. (C) 2001 Elsevier Science B.V. All rights reserved.

An extensive repertoire of type III secretion effectors in Escherichia coli O157 and the role of lambdoid phages in their dissemination

Several pathogenic strains of Escherichia coli exploit type III secretion to inject effector proteins into human cells, which then subvert eukaryotic cell biology to the bacterium's advantage. We have exploited bioinformatics and experimental approaches to establish that the effector repertoire in the Sakai strain of enterohemorrhagic E. coli (EHEC) O157:H7 is much larger than previously thought. Homology searches led to the identification of > 60 putative effector genes. Thirteen of these were judged to be likely pseudogenes, whereas 49 were judged to be potentially functional. In total, 39 proteins were confirmed experimentally as effectors: 31 through proteomics and 28 through translocation assays. At the protein level, the EHEC effector sequences fall into > 20 families. The largest family, the NleG family, contains 14 members in the Sakai strain alone. EHEC also harbors functional homologs of effectors from plant pathogens (HopPtoH, HopW, AvrA) and from Shigella (OspD, OspE, OspG), and two additional members of the Map/IpgB family. Genes encoding proven or predicted effectors occur in > 20 exchangeable effector loci scattered throughout the chromosome. Crucially, the majority of functional effector genes are encoded by nine exchangeable effector loci that lie within lambdoid prophages. Thus, type III secretion in E. coli is linked to a vast phage metagenome, acting as a crucible for the evolution of pathogenicity.

Alkyl halides, super hydrogen production and the pathogenesis of pneumatosis cystoides coli

Background and aims-The colons of patients with pneumatosis cystoides coli produce excessive H-2. Exposure to alkyl halides could explain this. Six consecutive patients who had pneumatosis cystoides coli while taking chloral hydrate (1-5+ g/day) are reported. Patients 2 and 3 were investigated after they had ceased chloral hydrate treatment. One produced methane, the other did not. (Pneumatosis cystoides coli patients are non-methanogenic according to the literature.) Both had overnight fasting breath H-2 of less than 10 ppm. A literature review disclosed just one patient who was using chloral at the time of diagnosed pneumatosis cystoides coli, but an epidemic of the disease in workers exposed to trichloroethylene. Methods-(i) In vitro experiments with human faeces: chloral or closely related alkyl halides were added to anaerobic faecal cultures derived from four methane-producing and three non-methanogenic human subjects. H-2 and CH4 gases were measured. (ii) In vivo animal experiment: chloral hydrate was added to drinking water of four Wistar rats, and faecal HI compared with control rats. Results-Alkyl halides increased H-2 up to 900 times in methanogenic and 10 times in non-methanogenic faecal cultures. The K-i of chloral was 0.2 mM. Methanogenesis was inhibited in concert with the increase in net H-2. In the rat experiment, chloral hydrate increased H-2 10 times, but did not cause pneumatosis. Conclusions-Chloral and trichloroethylene are alkyl halides chemically similar to chloroform, a potent inhibitor of H-2 consumption by methanogens and acetogens. These bacteria are the most important H-2-consuming species in the colon. It is postulated that exposure to these alkyl halides increases net H-2 production, which sets the scene for counterperfusion supersaturation and the formation of gas cysts. In recent times, very low prescribing rates for chloral have caused primary pneumatosis cystoides to become extremely rare. As with primary pneumatosis, secondary pneumatosis cystoides, which occurs if there is small bowel bacterial overgrowth distal to a proximally located gut obstruction, is predicted by counterperfusion supersaturation. Inherent unsaturation due to metabolism of O-2 is a safety factor, which could explain why gas bubbles do not form more often in tissue with high H-2 tension.

Role of capsule in the pathogenesis of Fowl Cholera caused by Pasteurella multocida Serogroup A

We have constructed a defined acapsular mutant inPasteurella multocida X-73 (serogroup A:1) by disrupting the hexA gene through the insertion of a tetracycline resistance cassette. The genotype of thehexA::tet(M) strain was confirmed by PCR and Southern hybridization, and the acapsular phenotype of this strain was confirmed by electron microscopy. ThehexA::tet(M) strain was attenuated in both mice and chickens. Complementation of the mutant with an intact hexAB fragment restored lethality in mice but not in chickens. In contrast to the results described previously for P. multocida serogroup B (J. D. Boyce and B. Adler, Infect. Immun. 68:3463–3468, 2000), thehexA::tet(M) strain was sensitive to the bactericidal action of chicken serum, whereas the wild-type and complemented strains were both resistant. Following inoculation into chicken muscle, the bacterial count of thehexA::tet(M) strain decreased significantly, while the wild-type and complemented strains both grew rapidly over 4 h. The capsule is thus an essential virulence determinant in the pathogenesis of fowl cholera.

Wolbachia pipientis: intracellular infection and pathogenesis in Drosophila

Wolbachia pipientis is a vertically transmitted, obligate intracellular symbiont of arthropods. The bacterium is best known for its ability to manipulate host reproductive biology where it can induce cytoplasmic incompatibility, parthenogenesis, feminization and male-killing. In addition to the various reproductive phenotypes it generates through interaction with host reproductive tissue it is also known to infect somatic tissues. However, relatively little is known about the consequences of infection of these tissues with the exception that in some hosts Wolbachia acts as a classical mutualist and in others a pathogen, dramatically shortening adult insect lifespan. Manipulation experiments have demonstrated that the severity of Wolbachia-induced effects on the host is determined by a combination of host genotype, Wolbachia strain, host tissue localization, and interaction with the environment. The recent completion of the whole genome sequence of Wolbachia pipientis wMel strain indicates that it is likely to use a type IV secretion system to establish and maintain infection in its host. Moreover, an unusual abundance of genes encoding proteins with eukaryotic-like ankyrin repeat domains suggest a function in the various described phenotypic effects in hosts.

Wolbachia genomes and the many faces of symbiosis

How could scientists working on the pathogenesis of filarial diseases, speciation and parthenogenesis in insects, sex-ratio deviations in crustaceans, pest control, and the evolution of bacterial genomes be united? How could a common research project attract the interest of these scientists? How could parasitology be made even more multi-disciplinary? Two workshops organized by New England Biolabs Inc. (Beverly, MA, USA) provide a simple answer to these questions: studying the genomes of Wolbachia endosymbionts.

Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis

The maternally inherited intracellular symbiont Wolbachia pipientis is well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility, parthenogenesis, and the feminization of genetic males in different hosts. The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been determined. In this paper, we report on (i) the purification of one of the most abundantly expressed Wolbachia proteins from infected Drosophila eggs and (ii) the subsequent cloning and characterization of the gene (wsp) that encodes it. The functionality of the wsp promoter region was also successfully tested in Escherichia coli. Comparison of sequences of this gene from different strains of Wolbachia revealed a high level of variability. This sequence variation correlated with the ability of certain Wolbachia strains to induce or rescue the cytoplasmic incompatibility phenotype in infected insects. As such, this gene will be a very useful tool for Wolbachia strain typing and phylogenetic analysis, as well as understanding the molecular basis of the interaction of Wolbachia with its host.

Wolbachia pipientis: bacterial density and unidirectional cytoplasmic incompatibility between infected populations of Aedes albopictus

Unidirectional cytoplasmic incompatibility is seen when certain Wolbachia-infected insect populations are crossed. Two hypotheses might explain this phenomenon: superinfections with mutually incompatible strains of Wolbachia producing incompatibility when crossed to individuals infected with only a single bacterial strain or, alternatively, a bacterial dosage model, with differences in Wolbachia densities responsible for the incompatibility. A quantitative PCR assay was set up as a general method to compare Wolbachia densities between populations. Using this assay in unidirectionally incompatible stocks of the mosquito Aedes albopictus, we have determined that densities are significantly higher in Houston than in the Mauritius and Koh Samui stocks. This is consistent with a dosage model for the observed crossing patterns, but does not rule out the possibility that superinfection is the primary cause of the incompatibility.

16S rRNA phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects

Bacterial endosymbionts of insects have long been implicated in the phenomenon of cytoplasmic incompatibility, in which certain crosses between symbiont-infected individuals lead to embryonic death or sex ratio distortion. The taxonomic position of these bacteria has, however, not been known with any certainty. Similarly, the relatedness of the bacteria infecting various insect hosts has been unclear. The inability to grow these bacteria on defined cell-free medium has been the major factor underlying these uncertainties. We circumvented this problem by selective PCR amplification and subsequent sequencing of the symbiont 16S rRNA genes directly from infected insect tissue. Maximum parsimony analysis of these sequences indicates that the symbionts belong in the α-subdivision of the Proteobacteria, where they are most closely related to the Rickettsia and their relatives. They are all closely related to each other and are assigned to the type species Wolbachia pipientis. Lack of congruence between the phylogeny of the symbionts and their insect hosts suggests that horizontal transfer of symbionts between insect species may occur. Comparison of the sequences for W. pipientis and for Wolbachia persica, an endosymbiont of ticks, shows that the genus Wolbachia is polyphyletic. A PCR assay based on 16S primers was designed for the detection of W. pipientis in insect tissue, and initial screening of insects indicates that cytoplasmic incompatibility may be a more general phenomenon in insects than is currently recognized.

Bacterial expression of two human aryl sulfotransferases

The effect of replacing a single codon in the N-terminal of human aryl sulfotransferase (HAST) 1 and 3 with one that is more commonly found in E. coli genes was assessed. The pKK233-2 E. coli expression vector was employed and the polymerase chain reaction (PCR) was used to introduce the 5' nucleotide substitution, at the same time maintaining the fidelity of the amino acid sequence. The data indicates that this change had a minimal effect on protein production, subcellular localization or, in the case of HAST3, catalytic activity. In general, the pKK233-2 E. coli vector has been less than optimal for expressing human sulfotransferase cDNAs. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

Insect peptides with improved protease-resistance protect mice against bacterial infection

At a time of the emergence of drug-resistant bacterial strains, the development of antimicrobial compounds with novel mechanisms of action is of considerable interest. Perhaps the most promising among these is a family of antibacterial peptides originally isolated from insects. These were shown to act in a stereospecific manner on an as-yet unidentified target bacterial protein. One of these peptides, drosocin, is inactive in vivo due to the rapid decomposition in mammalian sera. However, another family member, pyrrhocoricin, is significantly more stable, has increased in vitro efficacy against Gram-negative bacterial strains, and if administered alone, as we show here, is devoid of in vitro or in vivo toxicity. At low doses, pyrrhocoricin protected mice against Escherichia call infection, but at a higher dose augmented the infection of compromised animals. Analogs of pyrrhocoricin were, therefore, synthesized to further improve protease resistance and reduce toxicity. A linear derivative containing unnatural amino acids at both termini showed high potency and lack of toxicity in vivo and an expanded cyclic analog displayed broad activity spectrum in vitro. The bioactive conformation of native pyrrhocoricin was determined by nuclear magnetic resonance spectroscopy, and similar to drosocin, reverse turns were identified as pharmacologically important elements at the termini, bridged by an extended peptide domain. Knowledge of the primary and secondary structural requirements for in vivo activity of these peptides allows the design of novel antibacterial drug leads.