Arcanolysin is a cholesterol-dependent cytolysin of the human pathogen Arcanobacterium haemolyticum.

BACKGROUND: Arcanobacterium haemolyticum is an emerging human pathogen that causes pharyngitis, wound infections, and a variety of occasional invasive diseases. Since its initial discovery in 1946, this Gram positive organism has been known to have hemolytic activity, yet no hemolysin has been previously reported. A. haemolyticum also displays variable hemolytic activity on laboratory blood agar that is dependent upon which species the blood is derived. RESULTS: Here we describe a cholesterol-dependent cytolysin (CDC) secreted by A. haemolyticum, designated arcanolysin (aln), which is present in all strains (n = 52) tested by DNA dot hybridization. Among the known CDCs, ALN is most closely related to pyolysin (PLO) from Trueperella (formerly Arcanobacterium) pyogenes. The aln probe, however, did not hybridize to DNA from T. pyogenes. The aln open reading frame has a lower mol %G+C (46.7%) than the rest of the A. haemolyticum genome (53.1%) and is flanked by two tRNA genes, consistent with probable acquisition by horizontal transfer. The ALN protein (~ 64 kDa) contains a predicted signal sequence, a putative PEST sequence, and a variant undecapeptide within domain 4, which is typically important for function of the toxins. The gene encoding ALN was cloned and expressed in Escherichia coli as a functional recombinant toxin. Recombinant ALN had hemolytic activity on erythrocytes and cytolytic activity on cultured cells from human, rabbit, pig and horse origins but was poorly active on ovine, bovine, murine, and canine cells. ALN was less sensitive to inhibition by free cholesterol than perfringolysin O, consistent with the presence of the variant undecapeptide. CONCLUSIONS: ALN is a newly identified CDC with hemolytic activity and unique properties in the CDC family and may be a virulence determinant for A. haemolyticum.

Cholesterol microcrystals and cochleate cylinders: attachment of pyolysin oligomers and domain 4.

Using an established organic solvent injection procedure for the preparation of aqueous cholesterol microcrystal suspensions, it has now been shown that a new, hollow, cylindrical, tightly-coiled, multi-bilayer form of cholesterol can be generated, termed the cochleate cylinder. Cholesterol cochleate cylinders are formed in larger numbers at intermediate temperatures (40-75°C) but are not formed at 100°C. The structure of the cholesterol microcrystals and cochleate cylinders is shown in negatively stained electron micrographs. Oligomerization and attachment of pyolysin to cholesterol microcrystals and cochleate cylinders is shown, as is the attachment of the pyolysin "cholesterol-binding" domain 4 (D4) fragment. The bound D4 domain forms a linear array on the two planar surfaces and edges of the cholesterol microcrystals and a quasi helical array on the surface of the cochleate cylinders. Little evidence has been obtained to support the possibility that interaction or hetero-oligomerization can occur between intact pyolysin and the pyolysin D4 fragment on the surface of cholesterol microcrystals. Using immobilized cholesterol crystals attached to a carbon support film, single-sided linear labelling of the cholesterol surface with pyolysin D4 has been achieved, which correlates well with the images from the microcrystal suspensions and our earlier data using non-cytolytic streptolysin O mutants.

Phospholipase D promotes Arcanobacterium haemolyticum adhesion via lipid raft remodeling and host cell death following bacterial invasion.

BACKGROUND: Arcanobacterium haemolyticum is an emerging bacterial pathogen, causing pharyngitis and more invasive infections. This organism expresses an unusual phospholipase D (PLD), which we propose promotes bacterial pathogenesis through its action on host cell membranes. The pld gene is found on a genomic region of reduced %G + C, suggesting recent horizontal acquisition. RESULTS: Recombinant PLD rearranged HeLa cell lipid rafts in a dose-dependent manner and this was inhibited by cholesterol sequestration. PLD also promoted host cell adhesion, as a pld mutant had a 60.3% reduction in its ability to adhere to HeLa cells as compared to the wild type. Conversely, the pld mutant appeared to invade HeLa cells approximately two-fold more efficiently as the wild type. This finding was attributable to a significant loss of host cell viability following secretion of PLD from intracellular bacteria. As determined by viability assay, only 15.6% and 82.3% of HeLa cells remained viable following invasion by the wild type or pld mutant, respectively, as compared to untreated HeLa cells. Transmission electron microscopy of HeLa cells inoculated with A. haemolyticum strains revealed that the pld mutant was contained within intracellular vacuoles, as compared to the wild type, which escaped the vacuole. Wild type-infected HeLa cells also displayed the hallmarks of necrosis. Similarly inoculated HeLa cells displayed no signs of apoptosis, as measured by induction of caspase 3/7, 8 or 9 activities. CONCLUSIONS: These data indicate that PLD enhances bacterial adhesion and promotes host cell necrosis following invasion, and therefore, may be important in the disease pathogenesis of A. haemolyticum infections.

Transcriptional regulation of pyolysin production in the animal pathogen, Arcanobacterium pyogenes.

Arcanobacterium pyogenes is an opportunistic pathogen of a number of important livestock species, and usually infects from an endogenous, commensal source. Thus, as with other normal flora opportunistic pathogens, the regulation of A. pyogenes virulence factors is likely important during both commensal and pathogenic interactions with the host. The aim of this study was to investigate the regulation of a key A. pyogenes virulence factor, the cholesterol dependent cytolysin, pyolysin (PLO), under in vitro conditions, as a first step to understanding its regulation during the disease process. Analysis of PLO production in broth culture indicated that expression of PLO was induced during early stationary phase, and that this correlated with an increase in plo-specific mRNA. Analysis of a plo-cat transcriptional fusion indicated that transcription of plo was also induced during early stationary phase. Primer extension analysis and 5' RACE suggested that two putative promoter sequences, P1 and P2 were active. Analysis of site-directed mutants of these promoters in the plo-cat fusion indicated that P2 was the major stationary phase promoter. Deletions of the plo promoter region from the plo-cat fusion implicated three direct repeat (DR) sequences as important for plo transcription. Mutagenesis of both DR1 and DR2 resulted in reduction in plo transcription, while the presence of only DR3 in deletions of the plo promoter region repressed transcription from P2. Gel shift experiments indicated that a soluble factor from A. pyogenes binds to the plo promoter region and that the DRs may act as binding sites for a transcriptional regulator.

Lethal effects of Clostridium perfringens epsilon toxin are potentiated by alpha and perfringolysin-O toxins in a mouse model.

Epsilon toxin (ETX) is the most important virulence factor of Clostridium perfringens type D. Two other important toxins, alpha toxin (CPA) and perfringolysin-O (PFO), are encoded and potentially produced by most C. perfringens type D isolates. The biological effects of these toxins are dissimilar although they are all lethal. Since the possible interaction of these toxins during infection is unknown, the effects of CPA and PFO on the lethal activity of ETX were studied in a mouse model. Mice were injected intravenously or intragastrically with CPA or PFO with or without ETX. Sublethal doses of CPA or PFO did not affect the lethality of ETX when either was injected together with the latter intravenously. However, sublethal or lethal doses of CPA or PFO resulted in reduction of the survival time of mice injected simultaneously with ETX when compared with the intravenous effect of ETX injected alone. When PFO was inoculated intragastrically with ETX, a reduction of the survival time was observed. CPA did not alter the survival time when inoculated intragastrically with ETX. The results of the present study suggest that both CPA and PFO have the potential to enhance the ETX lethal effects during enterotoxemia in natural hosts such as sheep and goats.

The genome of epsilon15, a serotype-converting, Group E1 Salmonella enterica-specific bacteriophage.

The genome sequence of the Salmonella enterica serovar Anatum-specific, serotype-converting bacteriophage epsilon15 has been completed. The nonredundant genome contains 39,671 bp and 51 putative genes. It most closely resembles the genome of phiV10, an Escherichia coli O157:H7-specific temperate phage, with which it shares 36 related genes. More distant relatives include the Burkholderia cepacia-specific phage, BcepC6B (8 similar genes), the Bordetella bronchiseptica-specific phage, BPP-1 (8 similar genes) and the Photobacterium profundum prophage, P Pphipr1 (6 similar genes). epsilon15 gene identifications based on homologies with known gene families include the terminase small and large subunits, integrase, endolysin, two holins, two DNA methylase enzymes (one adenine-specific and one cytosine-specific) and a RecT-like enzyme. Genes identified experimentally include those coding for the serotype conversion proteins, the tail fiber, the major capsid protein and the major repressor. epsilon15's attP site and the Salmonella attB site with which it interacts during lysogenization have also been determined.

Characterization of Campylobacter jejuni biofilms under defined growth conditions.

Campylobacter jejuni is a major cause of human diarrheal disease in many industrialized countries and is a source of public health and economic burden. C. jejuni, present as normal flora in the intestinal tract of commercial broiler chickens and other livestock, is probably the main source of human infections. The presence of C. jejuni in biofilms found in animal production watering systems may play a role in the colonization of these animals. We have determined that C. jejuni can form biofilms on a variety of abiotic surfaces commonly used in watering systems, such as acrylonitrile butadiene styrene and polyvinyl chloride plastics. Furthermore, C. jejuni biofilm formation was inhibited by growth in nutrient-rich media or high osmolarity, and thermophilic and microaerophilic conditions enhanced biofilm formation. Thus, nutritional and environmental conditions affect the formation of C. jejuni biofilms. Both flagella and quorum sensing appear to be required for maximal biofilm formation, as C. jejuni flaAB and luxS mutants were significantly reduced in their ability to form biofilms compared to the wild-type strain.

Multiple genetic elements carry the tetracycline resistance gene tet(W) in the animal pathogen Arcanobacterium pyogenes.

The tet(W) gene is associated with tetracycline resistance in a wide range of bacterial species, including obligately anaerobic rumen bacteria and isolates from the human gut and oral mucosa. However, little is known about how this gene is disseminated and the types of genetic elements it is carried on. We examined tetracycline-resistant isolates of the animal commensal and opportunistic pathogen Arcanobacterium pyogenes, all of which carried tet(W), and identified three genetic elements designated ATE-1, ATE-2, and ATE-3. These elements were found in 25%, 35%, and 60% of tetracycline-resistant isolates, respectively, with some strains carrying both ATE-2 and ATE-3. ATE-1 shows characteristics of a mobilizable transposon, and the tet(W) genes from strains carrying this element can be transferred at low frequencies between A. pyogenes strains. ATE-2 has characteristics of a simple transposon, carrying only the resistance gene and a transposase, while in ATE-3, the tet(W) gene is associated with a streptomycin resistance gene that is 100% identical at the DNA level with the aadE gene from the Campylobacter jejuni plasmid pCG8245. Both ATE-2 and ATE-3 show evidence of being carried on larger genetic elements, but conjugation to other strains was not observed under the conditions tested. ATE-1 was preferentially associated with A. pyogenes strains of bovine origin, while ATE-2 and ATE-3 elements were primarily found in porcine isolates, suggesting that these elements may circulate in different environments. In addition, four alleles of the tet(W) gene, primarily associated with different elements, were detected among A. pyogenes isolates.

Skewed genomic variability in strains of the toxigenic bacterial pathogen, Clostridium perfringens.

Clostridium perfringens is a Gram-positive, anaerobic spore-forming bacterium commonly found in soil, sediments, and the human gastrointestinal tract. C. perfringens is responsible for a wide spectrum of disease, including food poisoning, gas gangrene (clostridial myonecrosis), enteritis necroticans, and non-foodborne gastrointestinal infections. The complete genome sequences of Clostridium perfringens strain ATCC 13124, a gas gangrene isolate and the species type strain, and the enterotoxin-producing food poisoning strain SM101, were determined and compared with the published C. perfringens strain 13 genome. Comparison of the three genomes revealed considerable genomic diversity with >300 unique "genomic islands" identified, with the majority of these islands unusually clustered on one replichore. PCR-based analysis indicated that the large genomic islands are widely variable across a large collection of C. perfringens strains. These islands encode genes that correlate to differences in virulence and phenotypic characteristics of these strains. Significant differences between the strains include numerous novel mobile elements and genes encoding metabolic capabilities, strain-specific extracellular polysaccharide capsule, sporulation factors, toxins, and other secreted enzymes, providing substantial insight into this medically important bacterial pathogen.

Association of genes encoding beta2 toxin and a collagen binding protein in Clostridium perfringens isolates of porcine origin.

Clostridium perfringens is a cause of economically significant enteritis in livestock. Beta2 toxin, encoded by one of two cpb2 alleles, is implicated as a virulence factor in this disease. Previous studies determined that the consensus cpb2 allele is preferentially associated with C. perfringens isolated from pigs. In C. perfringens strain 13, the consensus cpb2 allele is found on the plasmid pCP13, which also carries cna, encoding a putative collagen binding protein, CpCna. This protein was shown to be a bona fide collagen adhesin, as recombinant, HIS-tagged CpCna bound collagen type I as determined by Far Western blotting. Genomic DNA from C. perfringens isolated from a variety of host species were subjected to PCR to determine the prevalence of cna in these strains and correlate its carriage with the presence and type of cpb2 allele. The cna gene was found in 55.8% of isolates from all host species (n=208) and 68.1% of porcine isolates (n=119). In cpb2+ isolates, cna was present in 69.9% of isolates from all hosts (n=153), but was found in 98.7% of porcine isolates (n=75). Furthermore in porcine isolates, the consensus cpb2 allele and cna were absolutely correlated with the presence of pcp12, a pCP13-encoded gene, and pcp12 was never found in any isolate that lacks either cpb2 allele. The finding that CpCna binds collagen and that the cna gene is associated with the consensus cpb2 allele implicates CpCna as a potential virulence factor in porcine enteritis caused by C. perfringens.

Arcanobacterium pyogenes: molecular pathogenesis of an animal opportunist.

Arcanobacterium pyogenes is a commensal and an opportunistic pathogen of economically important livestock, causing diseases as diverse as mastitis, liver abscessation and pneumonia. This organism possesses a number of virulence factors that contribute to its pathogenic potential. A. pyogenes expresses a cholesterol-dependent cytolysin, pyolysin, which is a haemolysin and is cytolytic for immune cells, including macrophages. Expression of pyolysin is required for virulence and this molecule is the most promising vaccine candidate identified to date. A. pyogenes also possesses a number of adherence mechanisms, including two neuraminidases, the action of which are required for full adhesion to epithelial cells, and several extracellular matrix-binding proteins, including a collagen-binding protein, which may be required for adhesion to collagen-rich tissue. A. pyogenes also expresses fimbriae, which are similar to the type 2 fimbriae of Actinomyces naeslundii, and forms biofilms. However, the role of these factors in the pathogenesis of A. pyogenes infections remains to be elucidated. A. pyogenes also invades and survives within epithelial cells and can survive within J774A.1 macrophages for up to 72 h, suggesting an important role for A. pyogenes interaction with host cells during pathogenesis. The two component regulatory system, PloSR, up-regulates pyolysin expression and biofilm formation but down-regulates expression of proteases, suggesting that it may act as a global regulator of A. pyogenes virulence. A. pyogenes is a versatile pathogen, with an arsenal of virulence determinants. However, most aspects of the pathogenesis of infection caused by this important opportunistic pathogen remain poorly characterized.

Genotypes and antibiotic resistance of salmonella newport isolates from U.S. market oysters.

The consumption of raw oysters contributes to food-borne diseases such as salmonellosis. Prevalence studies in our laboratory have shown that Salmonella spp. were present in 7.4% of U.S. market oysters, with the majority (78/101) of isolates being of the Salmonella enterica Newport serovar. E-tests were performed to assess the susceptibility of the S. Newport oyster isolates to antibiotics used for treatment of Salmonella infections. The oyster isolates were susceptible to Gentamicin, Trimethoprim/Sulphamethoxazole, and Ciprofloxacin, while for the most part they were resistant to Ampicillin and Tetracycline. Consistent with the uniform antibiotic susceptibility profile of these isolates, only three pulsed-field gel electrophoresis (PFGE) profiles were identified among the isolates. Of these three profiles, one was present in 63 of the 78 isolates, indicating not only the presence of S. Newport in oysters, but the predominance of a single PFGE type.

Prevalence of Salmonella spp. in oysters in the United States.

Food-borne diseases such as salmonellosis can be attributed, in part, to the consumption of raw oysters. To determine the prevalence of Salmonella spp. in oysters, oysters harvested from 36 U.S. bays (12 each from the West, East, and Gulf coasts in the summer of 2002, and 12 bays, four per coast, in the winter of 2002-2003) were tested. Salmonella was isolated from oysters from each coast of the United States, and 7.4% of all oysters tested contained Salmonella. Isolation tended to be bay specific, with some bays having a high prevalence of Salmonella, while other bays had none. Differences in the percentage of oysters from which Salmonella was isolated were observed between the summer and winter months, with winter numbers much lower probably due to a variety of weather-related events. The vast majority (78/101) of Salmonella isolates from oysters were Salmonella enterica serovar Newport, a major human pathogen, confirming the human health hazard of raw oyster consumption. Contrary to previous findings, no relationship was found between the isolation of fecal coliforms and Salmonella from oysters, indicating a necessity for specific monitoring for Salmonella and other pathogens rather than the current reliance on fecal coliform testing.

Atypical cpb2 genes, encoding beta2-toxin in Clostridium perfringens isolates of nonporcine origin.

Beta2-toxin, encoded by cpb2, is implicated in the pathogenesis of Clostridium perfringens enteritis. However, cpb2 genes from nonporcine C. perfringens isolates were not always expressed, at least in vitro. Nucleotide sequencing identified atypical cpb2 genes with 70.2 to 70.7% DNA identity to previously identified (consensus) cpb2. Atypical beta2-toxin displayed 62.3% identity and 80.4% similarity to consensus beta2-toxin. No porcine type C isolates (n = 16) and only 3.3% of porcine type A isolates (n = 60) carried atypical cpb2 genes. However, 88.5% of nonporcine isolates carried atypical cpb2 (n = 78), but beta2-toxin was not expressed. Almost half of the nonporcine consensus cpb2 genes (44.4%) carried a frameshift mutation (n = 9), resulting in an absence of beta2-toxin expression. These findings strengthen the role of beta2-toxin in the pathogenesis of enteritis in neonatal pigs. However, the identification of apparently nonexpressed, atypical cpb2 genes raises the question of whether this protein plays the same role in enteritis in other animal species.

Ribosomal mutations in Arcanobacterium pyogenes confer a unique spectrum of macrolide resistance.

Four macrolide-resistant Arcanobacterium pyogenes isolates contained A2058T, A2058G, or C2611G (Escherichia coli numbering) mutations in their 23S rRNA genes. While these mutations conferred resistance to erythromycin, oleandomycin, and spiramycin, they did not confer resistance to tylosin.

A second tylosin resistance determinant, Erm B, in Arcanobacterium pyogenes.

Arcanobacterium pyogenes, a common inhabitant of the mucosal surfaces of livestock, is also a pathogen associated with a variety of infections. In livestock, A. pyogenes is exposed to antimicrobial agents used for prophylaxis and therapy, notably tylosin, a macrolide used extensively for the prevention of liver abscessation in feedlot cattle in the United States. Many, but not all, tylosin-resistant A. pyogenes isolates carry erm(X), suggesting the presence of other determinants of tylosin resistance. Oligonucleotide primers designed for conserved regions of erm(B), erm(C), and erm(T) were used to amplify a 404-bp fragment from a tylosin-resistant A. pyogenes isolate, OX-7. DNA sequencing revealed that the PCR product was 100% identical to erm(B) genes, and the erm(B) gene region was cloned in Escherichia coli. The A. pyogenes Erm B determinant had the most DNA identity with an Erm B determinant carried by the Clostridium perfringens plasmid pIP402. However, the A. pyogenes determinant lacked direct repeat DR1 and contained a deletion in DR2. Flanking the A. pyogenes erm(B) gene were partial and entire genes similar to those found on the Enterococcus faecalis multiresistance plasmid pRE25. This novel architecture suggests that the erm(B) element may have arisen by recombination of two distinct genetic elements. Ten of 32 tylosin-resistant isolates carried erm(B), as determined by DNA hybridization, and all 10 isolates carried a similar element. Insertion of the element was site specific, as PCR and Southern blotting analysis revealed that the erm(B) element was inserted into orfY, a gene of unknown function. However, in three strains, this insertion resulted in a partial duplication of orfY.

Spider and bacterial sphingomyelinases D target cellular lysophosphatidic acid receptors by hydrolyzing lysophosphatidylcholine.

Bites by Loxosceles spiders can produce severe clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, hemolysis, and persistent inflammation. The causative factor is a sphingomyelinase D (SMaseD) that cleaves sphingomyelin into choline and ceramide 1-phosphate. A similar enzyme, showing comparable bioactivity, is secreted by certain pathogenic corynebacteria and acts as a potent virulence factor. However, the molecular basis for SMaseD toxicity is not well understood, which hampers effective therapy. Here we show that the spider and bacterial SMases D hydrolyze albumin-bound lysophosphatidylcholine (LPC), but not sphingosylphosphorylcholine, with K(m) values ( approximately 20-40 microm) well below the normal LPC levels in blood. Thus, toxic SMases D have intrinsic lysophospholipase D activity toward LPC. LPC hydrolysis yields the lipid mediator lysophosphatidic acid (LPA), a known inducer of platelet aggregation, endothelial hyperpermeability, and pro-inflammatory responses. Introduction of LPA(1) receptor cDNA into LPA receptor-negative cells renders non-susceptible cells susceptible to SmaseD, but only in LPC-containing media. Degradation of circulating LPC to LPA with consequent activation of LPA receptors may have a previously unappreciated role in the pathophysiology of secreted SMases D.

Potential virulence and antimicrobial susceptibility of Campylobacter jejuni isolates from food and companion animals.

Infection in humans with Campylobacter jejuni is commonly associated with exposure to food animal fecal material. In this study, we report on the recovery, potential for virulence and antimicrobial resistance levels of C. jejuni isolated from food and companion animals. Three hundred and seventy-eight fecal samples from food and companion animals and surface swabs from beef carcasses were tested for the presence of C. jejuni. C. jejuni was isolated from 13.8% (11/80) of dogs, 5% (1/20) of goats, 28.3% (17/60) of dairy cattle, 0% (0/65) of range cattle, 73.5% (36/49) of feedlot cattle, and 94.7% (18/19) of beef carcasses. Beef cattle from a single Arizona herd showed a considerable increase in fecal shedding of C. jejuni from pasture to feedlot and over time on the feedlot. Forty-two isolates were tested for susceptibility to four antimicrobial agents, each representing a class of antimicrobial drug approved for use in both humans and animals. None of the isolates were found to be resistant to erythromycin or gentamicin, whereas 2.4% of isolates were resistant to ciprofloxacin and 28.6% of isolates were resistant to tetracycline. The presence of virulence traits among the 42 isolates was assessed using in vitro macrophage survival and epithelial cell adherence and invasion assays. Of the isolates examined, 17 were able to survive within macrophages through 72 h at viable counts of >/=10(3)/well and 12 were capable of invading epithelial cells at viable counts of >/=10(3)/well. Data from these studies suggests that many of the isolates recovered from the non-poultry animal sources have the capacity to cause disease if transmitted to humans.

Valvular endocarditis associated with Helcococcus ovis infection in a bovine.

A 12-month-old Angus bull calf with a history of fever and lethargy of several weeks' duration was necropsied. Macroscopic findings included general dehydration, congestion, and edema within the craniodorsal lobes of the lung, multifocal ecchymotic hemorrhages on the dorsal epaxial and gluteal muscles, bloody ingesta within the gastrointestinal tract, and a 4- x 4- x 5-cm irregular plaque located on the right atrioventricular heart valve. Microscopically, there were focally extensive pulmonary alveolar infiltrates of histiocytes and neutrophils, large numbers of necrotic hypereosinophilic hepatocytes located within the centrilobular and midzonal regions of the liver, and, within the plaque from the right atrioventricular valve, a large mass formed by abundant laminated fibrin that contained numerous small multifocal aggregates of gram-positive cocci. This report describes the first apparent isolation of Helcococcus ovis from cattle.