Figure 5 - uploaded by Janet E Hill
Content may be subject to copyright.
Mucohaemorrhagic diarrhea associated with ''Brachyspira hampsonii'' strain 30446. Fecal consistency following inoculation with pure broth ''Brachyspira hampsonii'' strain 30446 culture ranged from that similar to wet cement (A, right side), clots of blood (A, left side) or mucus (B), or severe watery mucohaemorrhagic diarrhea (C). Images A, B, C were taken on days 5, 6 and 8 PI respectively. doi:10.1371/journal.pone.0057146.g005
Source publication
Mucohaemorrhagic diarrhea caused by swine dysentery, is a severe production limiting disease of swine. Recently, pigs in western Canada with clinical signs indistinguishable from swine dysentery were observed. Despite the presence of spirochetes on fecal smears, recognized spp. including could not be identified. A phylogenetically distinct , called...
Context in source publication
Context 1
... examination in feces of CTRL pigs, no sample was positive by qPCR or culture during following inoculation. Of the 12 INOC pigs, eight developed mucohaemorrhagic diarrhea between 5 and 9 days PI (Figures 4, 5, 6). Of the remaining four INOC pigs (#683, #684, #688 and #695) soft feces were variably observed between days 1 and 14. ...
Similar publications
Background
The anaerobic spirochetes Brachyspira hyodysenteriae and Brachyspira pilosicoli cause diarrheal diseases in pigs. Their fastidious nature has hampered standardization of methods for antimicrobial susceptibility testing. For monitoring of antimicrobial susceptibility wild type cutoff values are needed to define where the wild type distrib...
Brachyspira (B.) hyodysenteriae is widespread globally, and can cause mucohaemorrhagic colitis (swine dysentery, SD) with severe economic impact in infected herds. Typical strains of B. hyodysenteriae are strongly haemolytic on blood agar, and the haemolytic activity is believed to contribute to virulence in vivo. However, recently there have been...
Diet has been implicated as a major factor impacting clinical disease expression of swine dysentery and Brachyspira hyodysenteriae colonization. However, the impact of diet on novel pathogenic strongly beta-hemolytic Brachyspira spp. including "B. hampsonii" has yet to be investigated. In recent years, distillers dried grains with solubles (DDGS),...
Avian intestinal spirochaetosis (AIS) is a condition arising from colonization of the caeca and colon of birds with anaerobic spirochaetes of the genus Brchyspira. The purpose of the present study was to evaluate PCR amplification from formalin-fixed, paraffin-embedded tissue method for detecting infected laying hens with Brachyspira pilosicoli wit...
Brachyspira is associated with diarrhea and colitis in pigs, and control of these pathogens is complicated by their complex ecology. Identification of wildlife reservoirs of Brachyspira requires the discrimination of colonized animals and those simply contaminated through environmental exposure. Lesser snow geese (Chen caerulescens caerulescens) we...
Citations
... SD is characterized by mucohaemorrhagic diarrhea and colitis. It is caused by Brachyspira hyodysenteriae (Harris et al. 1972), B. suanatina (Råsbäck et al. 2007) or B. hampsonii (Rubin et al. 2013). Brachyspira pilosicoli is the causative agent of SC, clinically described as mucoid, watery diarrhea linked to mild colitis when compared to SD (Taylor et al. 1980). ...
Swine dysentery, spirochetal colitis, and salmonellosis are production-limiting enteric diseases of global importance to the swine industry. Despite decades of efforts, mitigation of these diseases still relies on antibiotic therapy. A common knowledge gap among the 3 agents is the early B-cell response to infection in pigs. Thus, this study aimed to characterize the porcine B-cell response to Brachyspira hyodysenteriae, Brachyspira hampsonii (virulent and avirulent strains), Brachyspira pilosicoli, and Salmonella Typhimurium, the agents of the syndromes mentioned above. Immortalized porcine B-cell line derived from a crossbred pig with lymphoma were co-incubated for 8 h with each pathogen, as well as E. coli lipopolysaccharide (LPS) and a sham-inoculum (n = 3/treatment). B-cell viability following treatments was evaluated using trypan blue, and the expression levels of B-cell activation-related genes was profiled using reverse transcription quantitative PCR. Only S. Typhimurium and LPS led to increased B-cell mortality. B. pilosicoli downregulated B-lymphocyte antigen (CD19), spleen associated tyrosine Kinase (syk), tyrosine-protein kinase (lyn), and Tumour Necrosis Factor alpha (TNF-α), and elicited no change in immunoglobulin-associated beta (CD79b) and swine leukocyte antigen class II (SLA-DRA) expression levels, when compared to the sham-inoculated group. In contrast, all other treatments significantly upregulated CD79b and stimulated responses in other B-cell downstream genes. These findings suggest that B. pilosicoli does not elicit an immediate T-independent B-cell response, nor does it trigger antigen-presenting mechanisms. All other agents activated at least one trigger within the T-independent pathways, as well as peptide antigen presenting mechanisms. Future research is warranted to verify these findings in vivo.
... After growth on solid medium, the agar plates were washed with sterile PBS and incubated in trypticase soy growth broth (TSB), enriched with 0.5% glucose, 0.2% NaHCO 3 , 0.05% L-cysteine-HCl, 1.0% yeast extract, 10% FBS and 5% porcine fecal extract [31] in a proportion of 1:100 mL (wash: broth) for 21 h at 37 • C in a shaker incubator, followed by inoculation of the animals. Pigs from the BRA and CO groups were intragastrically inoculated for three consecutive days at 7, 8, and 9 days post infection (dpi) with 50 mL of inoculum containing 5.31 × 10 6 bacteria/mL, as described by Jacobson et al. [14] and Rubin et al. [32]. The NEG and LAW groups received 50 mL of sterile TBS via the same route on the same day. ...
... Feces DNA was extracted using a commercial QIAamp DNA Stool Mini Kit (Qiagen Inc., Toronto, ON, Canada) according to the manufacturer's instructions. For B. hyodysenteriae, the primers JH0073 (5 -AGT GAA ATA GTT GCT CAT ATC AAA-3 ) and the JH0074 (5 -GCA TCA CTG ATT AAA GAA CCA ATT-3 )-targeting Nox gene were used to perform qPCR according to Rubin et al. [32]. ...
... V2002), 25 mg/L of colistin (Colistin, Sigma-Aldrich, cat no. C1511) [32], and incubated for at least three days at 42 • C in jars with an anaerobic atmosphere. Anaerobic conditions were generated using a vacuum pump filled with a mixture of N 2 (80%), CO 2 (10%), and H 2 (10%) gases. ...
Brachyspira hyodysenteriae and Lawsonia intracellularis coinfection has been observed in the diagnostic routine; however, no studies have evaluated their interaction. This study aimed to characterize lesions and possible synergisms in experimentally infected pigs. Four groups of piglets, coinfection (CO), B. hyodysenteriae (BRA), L. intracellularis (LAW), and negative control (NEG), were used. Clinical signals were evaluated, and fecal samples were collected for qPCR. At 21 days post infection (dpi), all animals were euthanized. Gross lesions, bacterial isolation, histopathology, immunohistochemistry, and fecal microbiome analyses were performed. Diarrhea started at 12 dpi, affecting 11/12 pigs in the CO group and 5/11 pigs in the BRA group. Histopathological lesions were significantly more severe in the CO than the other groups. B. hyodysenteriae was isolated from 11/12 pigs in CO and 5/11 BRA groups. Pigs started shedding L. intracellularis at 3 dpi, and all inoculated pigs tested positive on day 21. A total of 10/12 CO and 7/11 BRA animals tested positive for B. hyodysenteriae by qPCR. A relatively low abundance of microbiota was observed in the CO group. Clinical signs and macroscopic and microscopic lesions were significantly more severe in the CO group compared to the other groups. The presence of L. intracellularis in the CO group increased the severity of swine dysentery.
... In experimental settings, multiple strategies have been used to induce SD in naïve pigs including direct contact with infected pigs (seeder pig model) [11], intragastric inoculation with homogenates of large intestine and its contents from affected pigs [12], or inoculation with bacterial cultures grown on solid or liquid media [13][14][15]. However, success of these experimental models in our lab and others has been variable in terms of inducing SD, which could be related to the additive effect of multiple animal factors including but not limited to pig age, weight, housing, diet [6,7], genetics [16], events such as environmental and social stresses, dietary changes, or prior exposure to pathogenic Brachyspira spp. ...
... After the oral feeding tube is passed through into the stomach, the inoculum is administered, followed by a sterile volume of PBS to empty the inoculant remnant from the gastric tube. For B. hampsonii (genomovar I or II) incidence rates have ranged from 20 to 72%, whereas incubation period and duration of clinical signs ranged from 5 to 13 days, and 3 to 12 days, respectively [12,13,22]. It is clear from these studies that the results of experimental inoculation are highly variable, which is probably a reflection of the variety conditions used across the experiments. ...
... The inocula were prepared as described previously [11]. Briefly, the frozen purified isolates of B. hampsonii, B. hyodysenteriae G44 or D19 were cultured in JBS [12] or BHIS (trial A only) broth, anaerobically incubated, and scaled up every 24-h using a 1:9 dilution of broth culture (v/v) in JBS or BHIS until the required volume for inoculation was achieved. The purity of each inoculum aliquot was assessed by PCR amplification and sequencing of the nox gene [28]. ...
Swine dysentery (SD) caused by pathogenic Brachyspira spp. is an economic challenge for the swine industry. In research settings, experimental reproduction of swine dysentery typically relies on intragastric inoculation which has shown variable success. This project aimed to improve the consistency of the experimental inoculation protocol used for swine dysentery in our laboratory. Over six experiments, we evaluated the influence of group housing in inoculated pigs using a frozen-thawed broth culture of strongly hemolytic B. hyodysenteriae strain D19 (Trial A), compared the relative virulence of B. hyodysenteriae strains D19 and G44 (Trial B), compared inoculum volumes (50 mL vs 100 mL) for G44 and B. hampsonii 30446 (Trial C), and performed three independent trials evaluating intragastric inoculation using different oral inoculation methods: oral feed balls (Trial D), and oral syringe bolus of 100 mL (Trial E) or 300 mL (Trial F). Intragastric inoculation with a fresh broth culture of B. hyodysenteriae strain G44 resulted in a shorter incubation period and a higher proportionate duration of mucohemorrhagic diarrhea (MMHD) compared to D19. Intragastric inoculation with either 50 or 100 mL of B. hampsonii 30446 or B. hyodysenteriae (G44) were statistically equivalent. Oral inoculation with 100 mL or 300 mL also yielded similar results to intragastric inoculation but was more expensive due to the additional work and supplies associated with syringe training. Our future research will use intragastric inoculation with 100 mL of a fresh broth culture containing B. hyodysenteriae strain G44 as it yields a high incidence of mucohaemorrhagic diarrhea with a reasonable cost.
Supplementary Information
The online version contains supplementary material available at 10.1186/s13567-023-01180-y.
... The collected samples were stored at − 20°C until further analysis. Fecal consistency score was determined weekly with 0 = formed, normal; 1 = soft, wet cement consistency; 2 = runny or watery; 3 = mucoid diarrhea; 4 = bloody diarrhea, as previously described [33]. As we described previously [14] at the end of the experiment, blood samples were taken from the anterior jugular vein of the pigs into lithium heparin and serum tubes when pigs were at the ad lib fed state (BD Vacutainer®, Franklin Lakes, NJ, USA). ...
Background
Very low-protein (VLP) diets negatively impact calcium (Ca) metabolism and absorption. The objective of this study was to investigate the effect of supplemental branched-chain amino acids (BCAA) and limiting amino acids (LAA) on Ca digestibility, absorption and reabsorption in pigs fed with VLP diets. Forty-eight piglets were assigned to six treatments: positive control (PC), negative control (NC), and NC containing LAA 25%, LAA 50%, LAA + BCAA 25% (LB25) and LAA + BCAA 50% (LB50) more than recommendations.
Results
Relative to PC or NC, LB25 and LB50 had higher digestibility of Ca and plasma Ca and phosphorus (P), but lower plasma vitamin D 3 . LB50 tended to increase vitamin D receptor transcript and protein in the gut, but decreased mRNA or protein abundance of parathyroid hormone 1 receptor (PTH1R), calbindin 1 (CALB1), cytochrome P450 family 27 subfamily B member 1 and occludin in small intestine. LB50 increased the transcript of cytochrome P450 family 24 subfamily A member 1 and PTH1R but decreased the transcript of transient receptor potential cation channel subfamily V member 5, CALB1 and solute carrier family 17 member 4 in kidney.
Conclusion
Overall, BCAA increased Ca digestibility through regulating the transcellular and paracellular Ca absorption in the gut and reabsorption in kidney during protein restriction.
... Pigs were challenged with B. hyodysenteriae field strain D16-37945, which was originally isolated from pigs with clinical SD (gift from Dr. Janet Hill, Univ. of Saskatchewan). Cultures were incubated at 39 • C on a magnetic stir plate under anaerobic conditions (Thermo Fisher Scientific, #OXAN0025A) in brain heart infusion containing 5% v/v fetal bovine serum, 5% v/v sheep's blood, and 1% w/v glucose [23]. Motility was checked by dark field microscopy before pig challenge. ...
... Sigma-Aldrich #S9132-1 g, R3501-1 g, V2002-250 mg, S9007-5 g, C1511-10MU, respectively). After 4 days at 42 • C in anaerobic conditions (Thermo Fisher Scientific, #OXAN0025A), positive cultures were identified by colony-free zones of hemolysis with motile spirochetes visible by dark field microscopy [3,23,24]. ...
... Pigs were checked twice daily to assess lethargy, solitary behaviour, sunken eyes (dehydration), and thinness/hollow flanks. Fecal consistency was scored once daily as 0 = normal, 1 = wet cement, 2 = watery/runny, 3 = mucoid diarrhea, 4 = hemorrhagic diarrhea, with half-points assigned if between two scores [23]. Ten pigs (10 of 12 from B. hyodysenteriae group) developed bloody diarrhea (fecal score of 4) and were terminated within 12 h of onset (8-15 dpc) by sodium pentobarbital (humane endpoint) alongside 3 sham pigs terminated at 13 dpc as controls (N = 3 of 5 Sham group). ...
Brachyspira hyodysenteriae, an etiologic agent of swine dysentery (SD), is known for causing colitis. Although some aspects of colonic defenses during infection have been described previously, a more comprehensive picture of the host and microbiota interaction in clinically affected animals is required. This study aimed to characterize multiple aspects of colonic innate defenses and microbiome factors in B. hyodysenteriae-infected pigs that accompany clinical presentation of hemorrhagic diarrhea. We examined colonic mucus barrier modifications, leukocyte infiltration, cathelicidin expression, as well as microbiome composition. We showed that B. hyodysenteriae infection caused microscopic hemorrhagic colitis with abundant neutrophil infiltration in the colonic lamina propria and lumen, with minor macrophage infiltration. Mucus hypersecretion with abundant sialylated mucus in the colon, as well as mucosal colonization by [Acetivibrio] ethanolgignens, Lachnospiraceae, and Campylobacter were pathognomonic of B. hyodysenteriae infection. These findings demonstrate that B. hyodysenteriae produces clinical disease through multiple effects on host defenses, involving alterations of mucosal innate immunity and microbiota. Given that B. hyodysenteriae is increasingly resistant to antimicrobials, this understanding of SD pathogenesis may lead to future development of non-antibiotic and anti-inflammatory alternative therapeutics.
... Source of B. hampsonii 30446 and B. hyodysenteriae G44. The source of B. hampsonii strain 30446 was described by Rubin [11]. Briefly, B. hampsonii strain 30446 originated from a frozen stock (-80˚C) of colonic and cecal mucosa samples of clinically affected 13-week-old pigs from a porcine reproductive and respiratory syndrome virus (PRRSV) negative farm. ...
... An aliquot of each inocula was assessed for purity using nox PCR and sequencing. The concentrations of B. hampsonii strain 30446 and B. hyodysenteriae G44 in the inoculum were determined by quantitative PCR [11,12]. ...
... Preparation of inocula. Frozen, purified isolates of either B. hampsonii strain 30446 or B. hyodysenteriae strain G44 were cultured in JBS broth [11] and anaerobically incubated in sterile jars using gas-packs (Anaerogen TM 2.5 L, Thermo Scientific1 Oxoide Sachet) at 37˚C for 24 hours on a magnetic stirrer. After each 24-hours of incubation, a 1:9 broth culture dilution (v/v) in JBS broth was prepared scaling up from 10 mL to 500 mL with a concentration target of 1× 10 8 to 10 9 GE/mL. ...
Swine dysentery is causally associated with Brachyspira hampsonii and B . hyodysenteriae infection. Given the importance of transmission models in understanding re-emergent diseases and developing control strategies such as vaccines, the objective of this experiment was to evaluate two experimental natural transmission (seeder pig) models in grower pigs, each with 24 animals. Seeder pigs were intragastrically inoculated using broth cultures of either B . hampsonii strain 30446 (genomovar II) or B . hyodysenteriae strain G44. In trial 1, three seeder pigs were placed into two pens containing nine susceptible contact pigs creating a 1:3 seeder:contact ratio. This was sufficient to achieve natural B . hampsonii infection of 13/18 (72%) contact pigs, however, the incidence of mucoid or mucohemorrhagic diarrhea (MMHD) in contact pigs differed significantly between pens (4/9 versus 9/9; P = 0.03). In trial 2, eight seeder pigs inoculated intragastrically with B . hampsonii did not develop MMHD but when re-inoculated with B . hyodysenteriae 14 days later, all developed mucohemorrhagic diarrhea within 13 days of re-inoculation. Two seeder pigs were placed into each of 4 contact pens each containing 4 pigs. This 1:2 seeder:contact ratio resulted in natural infection of 14/16 (87%) contact pigs with incubation period ranging from 9–15 days. There were no significant differences among pens in incubation period, duration, clinical period or severity of diarrhea. These trials demonstrated that a 1:2 seeder:contact ratio with groups of six grower pigs per pen sustained natural transmission of B . hyodysenteriae G44 with greater consistency in the incidence of MMHD among pens compared to a B . hampsonii 30446 transmission model using 1:3 seeder:contact ratio in pens of 12. Understanding why B . hampsonii intragastric inoculation failed in one experiment warrants additional research.
... Inocula averaged 3.2 × 10 8 CFU/mL for S. Typhimurium, 7.9 × 10 7 genome copies/mL for B. hyodysenteriae, and 1 × 10 4 cells/mL for L. intracellularis. B, hyodysenteriae and S. Typhimurium inocula were quantified using previously described methods (4,76). L. intracellularis dose was provided by the vendor. ...
Swine dysentery, ileitis, and porcine salmonellosis are production-limiting diseases of global importance for swine production. They are caused by infection with Brachyspira hyodysenteriae, Lawsonia intracellularis, and Salmonella enterica serovar Typhimurium, respectively. Currently, the prevention, treatment, and control of these diseases still relies on antimicrobials. The goal of this study was to evaluate the effectiveness of four commercially available non-antimicrobial compounds in preventing lesions caused by the bacteria cited above using an in vitro intestinal culture model. A total of five pigs per pathogen were used and multiple compounds were evaluated. For compound F (a fungal fermented rye), S (a blend of short and medium chain fatty acids), and P (a synergistic blend of short and medium chain fatty acids, including coated butyrates), a total of four explants/pig for each treatment were used, while for compound D (an extract of carob and thyme) only 12 explants/pig for each treatment were used. Explants were exposed to a combination of pathogen only (n = 4/compound/pig), compound only (n = 4/compound/pig), or pathogen and compound (n = 4/compound/pig) and sampled at two time-points. Histopathology and gene expression levels were evaluated to investigate the treatment effect on explants. Short and medium-chain fatty acids, and an extract of carob and thyme, was found to mitigate lesions due to B. hyodysenteriae exposure. A fungal fermented prebiotic increased healthy epithelial coverage when explants were exposed to L. intracellularis or S. Typhimurium. These findings represent a step towards finding alternatives to antimicrobials usage and control of swine dysentery, ileitis, and salmonellosis in pork production.
... Piglet diarrhea is one of the most common microbial diseases causing high mortality and serious economic losses (1). Piglet diarrhea is usually caused by bacterial pathogens, such as Clostridia (2), Escherichia coli (3), Salmonella choleraesuis (4), and hyodysenteriae (5). Clostridium perfringens (C. ...
Clostridium perfringens beta2 (CPB2) toxin, one of the virulence factors of Clostridium perfringens (C. perfringens), can cause necrotizing enterocolitis in piglets. Accumulating pieces of evidence indicate that microRNAs (miRNAs) refer to the regulation of inflammatory processes. Previously, we have discovered that miR-30d was differentially expressed between the ileum of normal piglets and C. perfringens type C-infected diarrheal piglets. Here, we found that miR-30d expression was lowered in CPB2 toxin-treated intestinal porcine epithelial cells (IPEC-J2) at different time points. Subsequently, we determined that miR-30d inhibitor attenuated CPB2 toxin revulsive inflammatory damage in IPEC-J2 cells and promoted cell proliferation and cell cycle progression, whereas miR-30d mimic had opposite results. In addition, we confirmed that Proteasome activator subunit 3 (PSME3) was a downstream target gene of miR-30d via a dual luciferase reporter assay, qPCR, and western blot. We also found that overexpression of PSME3 suppressed CPB2 toxin-induced inflammatory damage and promoted cell proliferation and cycle progression. Our results demonstrate that miR-30d aggravates CPB2 toxin revulsive IPEC-J2 cells inflammatory injury via targeting PSME3, thereby providing a novel perspective for the prevention and treatment of piglet diarrhea at the molecular level.
... Lactobacillus rhamnosus CRL1505 also augments the Th1 response triggered by TLR2 signaling and increases the expression of MHC class II molecules as well as IL-1β, IL-6, IL-10, and IFN-γ in APCs for modulating intestinal immunity and thereby improving immune-health status [40]. In contrast, the phylum Spirochaetes, which is responsible for mucohaemorrhagic colitis in swine [41], was abundant in unsuccessful donors and in unsuccessful recipients throughout the experiment even after FMT. Further studies are required to explore the role of Spirochaetes in the occurrence or progression of CD; nevertheless, these results suggest that this phylum may negatively affect the efficacy of FMT. ...
Background
Establishing fecal microbiota transplantation (FMT) to prevent multifactorial diarrhea in calves is challenging because of the differences in farm management practices, the lack of optimal donors, and recipient selection. In this study, the underlying factors of successful and unsuccessful FMT treatment cases are elucidated, and the potential markers for predicting successful FMT are identified using fecal metagenomics via 16S rRNA gene sequencing, fecal metabolomics via capillary electrophoresis time-of-flight mass spectrometry, and machine learning approaches.
Results
Specifically, 20 FMT treatment cases, in which feces from healthy donors were intrarectally transferred into recipient diarrheal calves, were conducted with a success rate of 70%. Selenomonas was identified as a microorganism genus that showed significant donor–recipient compatibility in successful FMT treatments. A strong positive correlation between the microbiome and metabolome data, which is a prerequisite factor for FMT success, was confirmed by Procrustes analysis in successful FMT (r = 0.7439, P = 0.0001). Additionally, weighted gene correlation network analysis confirmed the positively or negatively correlated pairs of bacterial taxa (family Veillonellaceae) and metabolomic features (i.e., amino acids and short-chain fatty acids) responsible for FMT success. Further analysis aimed at establishing criteria for donor selection identified the genus Sporobacter as a potential biomarker in successful donor selection. Low levels of metabolites, such as glycerol 3-phosphate, dihydroxyacetone phosphate, and isoamylamine, in the donor or recipients prior to FMT, are predicted to facilitate FMT.
Conclusions
Overall, we provide the first substantial evidence of the factors related to FMT success or failure; these findings could improve the design of future microbial therapeutics for treating diarrhea in calves.
1SZe_1Amjvg5rTHsSJMvRbVideo abstract
... Swine dysentery is an infectious disease of pigs characterized by severe mucohaemmorhagic diarrhea, resulting in massive production losses due to decreased weight gain and increased mortality in infected animals [16,17]. Initially attributed to infection with the spirochete B. hyodysenteriae [16][17][18][19], recent outbreaks of swine dysentery in North America have been attributed to the recently identified species Brachyspira hampsonii [20,21] while sporadic outbreaks in Northern Germany and Scandinavia have been associated with Brachyspira suanatina [22][23][24]. While these species are not known to infect humans, the weakly β-hemolytic species Brachyspira pilosicoli and Brachyspira aalborgi can cause mild diarrhea and colitis in immunocompromised patients [25][26][27]. ...
... While these species are not known to infect humans, the weakly β-hemolytic species Brachyspira pilosicoli and Brachyspira aalborgi can cause mild diarrhea and colitis in immunocompromised patients [25][26][27]. Regardless of the specific species, a common observation with strongly pathogenic Brachyspira isolates is a strong β-hemolytic phenotype when these bacteria are cultured on blood agar in contrast to the weak β-hemolytic phenotype exhibited by less virulent Brachyspira species [3,20,21,23,[28][29][30]. To date four putative hemolysin genes have been identified in Brachyspira hampsonii and B. hyodysenteriae, termed tlyA/B/C and hlyA [3,[30][31][32][33]. ...
... Site directed mutagenesis was used to mutate serine 9 to lysine (S9K TlyA-His) and histidine 40 to glutamine (H40Q TlyA-His). In both cases the mutations from B. innocens/murdochii/aalborgi were chosen due to the fact that these bacteria are weakly hemolytic and are only capable of causing mild disease [41,42] in comparison to the strongly β-hemolytic, highly virulent Brachyspira hampsonii/hyodysenteriae [16,[18][19][20][21]43]. The S9K and H40Q TlyA mutants were expressed and purified with a C-terminal His-tag as described previously, and the hemolytic activity of the mutants against pig erythrocytes was quantified (Fig. 6B). ...
Background
TlyA proteins are expressed in a variety of pathogenic bacteria and possess dual hemolytic and ribosomal RNA methyltransferase functions. While the mechanism of TlyA mediated rRNA methylation is well understood, relatively little is known about the mechanism of TlyA induced hemolysis.
Methods
TlyA protein from the pig pathogen Brachyspira hampsonii was heterologously expressed and purified from an E. coli host. Hemolytic activity and rRNA methylation were assessed in vitro. Site-directed mutagenesis was used to mutate amino acids believed to be involved in TlyA mediated hemolysis.
Results
Purified TlyA-His protein exhibited both hemolytic and rRNA methyltransferase activities in vitro, with partial inhibition of hemolysis observed under reducing conditions. Mutation of cysteine 80 to alanine impaired hemolytic activity. A C27A/C93A mutant was capable of dimerizing under non-reducing conditions, indicating that a C80-C80 disulfide bond is involved in TlyA oligomerization. A mutation conserved in several avirulent Brachyspira species (S9K) completely abolished hemolytic activity of TlyA. This loss of activity was attributed to impaired oligomerization in the S9K mutant, as assessed by ITC and size-exclusion chromatography experiments.
Conclusions
Oligomeric assembly and hemolytic activity of TlyA from Brachyspira hampsonii is dependent on the formation of an intermolecular C80-C80 disulfide bond and noncovalent interactions involving serine 9. The conservation of these amino acids in TlyA proteins from pathogenic bacteria suggests a correlation between tlyA gene mutations and bacterial virulence.
General significance
Our results further elucidate the mechanisms underlying TlyA mediated hemolysis and provide evidence of a conserved mechanism of oligomerization for TlyA family proteins.
