NF-κB signaling pathway mechanism in cow intertoe skin inflammation caused by Fusobacterium necrophorum.

Background: Fusobacterium necrophorum is the main pathogen inducing bovine foot rot. The infected site is often accompanied by a strong inflammatory response, but the specific inflammatory regulatory mechanism remains unclear. Aim: A cow skin explants model was established to elucidate the mechanism of F. necrophorum bacillus causing foot rot in cows, and to provide reference for future clinical practice. Methods: Cow intertoe skin explants were cultured in vitro, and F. necrophorum bacteria solution and nuclear factor-κB (NF-κB) inhibitor BAY 1-7082 were added to establish an in vitro infection model. Hematoxylin and eosin staining, terminal - deoxynucleotidyl transferase mediated nick end labeling, and immunohistochemistry were used to detect the pathological changes of the skin explants infected with F. necrophorum, the degree of tissue cell apoptosis, and the expression of the apoptosis-related protein Caspase-3, respectively. RT-qPCR, Western blot, and ELISA were used to detect the activation of the NF-κB pathway and inflammatory cytokines by F. necrophorum. Results: The intertoe skin structure of cows infected with F. necrophorum changed with different degrees of inflammation, and the degree of tissue cell apoptosis was significantly increased (P < 0.01). In addition, infection with F. necrophorum significantly increased the phosphorylation level of IκBα protein and up-regulated the expression level of NF-κB p65. The high expression and transcriptional activity of NF-κB p65 significantly increased the expression and concentration of the inflammatory cytokines TNF-α, IL-1ß, and IL-8, thus inducing the occurrence of an inflammatory response. However, inhibition of NF-κB p65 activity significantly decreased the expression of inflammatory factors in the intertoe skin of cows infected with F. necrophorum. Conclusion: F. necrophorum activates NF-κB signaling pathway by increasing the expression of TNF-α, IL-1ß, IL-8 and other inflammatory factors, leading to foot rot in dairy cows.

Leukotoxin production by Fusobacterium necrophorum strains in relation to severity of liver abscesses in cattle.

Fusobacterium necrophorum, a Gram-negative anaerobe, is the primary etiologic agent of liver abscesses of beef cattle. The bacterium, a member of the microbial community of the rumen, travels to the liver via portal circulation to cause abscesses. The severity of liver abscesses vary from mild with one or two small abscesses to severe with medium to large multiple abscesses. Leukotoxin, a secreted protein, is the critical virulence factor involved in the infection. Our objective was to compare leukotoxin production between strains of F. necrophorum isolated from mild and severe liver abscesses collected from slaughtered cattle. The quantification of leukotoxin was based on assays to measure cytotoxicity and protein antigen concentration. One-hundred strains, 50 from mild and 50 from severe abscesses, were utilized in the study. Cell-free supernatants were prepared from cultures grown in anaerobic broth at 9 and 24 h incubations. The leukotoxic activity was quantified by measuring cytotoxicity based on the release of lactic dehydrogenase from bovine lymphocyte cells, BL3, treated with the culture supernatant. Leukotoxin protein concentration was quantified by a sandwich ELISA assay with a leukotoxin-specific monoclonal antibody as the capture antibody. The leukotoxin activity and concentration were highly variable among the strains within each severity of liver abscesses. Although the leukotoxic activity was unaffected by incubation time, leukotoxin protein concentration was consistently higher at 24 h compared to 9 h incubation. Strains from severe liver abscesses had significantly higher leukotoxic activity and higher protein concentration compared to strains from mild liver abscesses (P < 0.0001) at both 9 and 24 h culture supernatants. Across all strains, the correlation coefficients between leukotoxic activity and leukotoxin concentration at 9 and 24 h were 0.14 (P = 0.17) and 0.47 (P < 0.0001), respectively. In conclusion, strains isolated from severe liver abscesses had significantly higher leukotoxic activities and leukotoxin protein concentrations compared to strains isolated from mild liver abscesses.

Screening of BHK-21 cellular proteins that interact with outer membrane protein 43K OMP of Fusobacterium necrophorum.

Fusobacterium necrophorum is a Gram negative, spore-free, anaerobic bacterium that can cause pyogenic and necrotic infections in animals and humans. It is a major bovine pathogen and causes hepatic abscesses, foot rot, and necrotic laryngitis. The 43K OMP of F. necrophorum is an outer membrane protein with molecular weight of 43 kDa, exhibiting similarity to pore-forming proteins of other Fusobacterium species that plays an important role in bacterial infections. However, the role of 43K OMP in F. necrophorum adhesion remains unknown. In this study, we evaluated whether the 43K OMP of F. necrophorum mediates adhesion to BHK-21 cells and performed a preliminary screen of the proteins that interact with 43K OMP of F. necrophorum by immunoprecipitation-mass spectrometry. The results showed that the natural 43K OMP and recombinant 43K OMP could bind to BHK-21 cells, and preincubation of F. necrophorum with an antibody against the recombinant 43K OMP of F. necrophorum decreased binding to BHK-21 cells. Seventy differential interacting proteins were successfully screened by immunoprecipitation-mass spectrometry. Among these seventy differential interacting proteins, seven cell membrane proteins and four extracellular matrix proteins shown to be relevant to bacteria adhesion through subcellular localization and single-molecule function analysis. These data increase our understanding of the pathogenesis of F. necrophorum and provide a new theoretical basis for the design of antimicrobial drugs against F. necrophorum.

[Design of artificial foetor flatus based on bacterial volatile compounds].

INTRODUCTION: Excessive flatulence can be a huge social problem. The purpose of this study was to design artificial flatus from bacterial volatile compounds to stimulate research into neutralizing measures. MATERIAL AND METHODS: Anaerobic bacteria, representing a broad spectrum, from a recognized international culture collection were included. The strains were incubated in an anaerobic jar. After 24 hours the lid was removed, and the odour was evaluated by a specialist in clinical microbiology. RESULTS: Four different anaerobic strains were chosen for further studies based on their individual odours. In total, seven different combinations of two or three strains were tested. The combination of Bacteroides fragilis ATCC 25285, Clostridium difficile ATCC 700057 and Fusobacterium necrophorum ATCC 25286 was chosen as it had a suitably foul odour. CONCLUSION: It is possible to design artificial flatus from bacterial volatile compounds. The method is easy and inexpensive and can stimulate further research into neutralizing measures. TRIAL REGISTRATION: none. FUNDING: none.

The effects of iron limitation and cell density on prokaryotic metabolism and gene expression: Excerpts from Fusobacterium necrophorum strain 774 (sheep isolate).

Fusobacterium necrophorum is a Gram-negative obligate anaerobe associated with several diseases in humans and animals. Despite its increasing clinical significance, there is little or no data on the relationship between its metabolism and virulence. Previous studies have shown that bacteria grown under iron-limitation express immunogenic antigens similar to those generated in vivo. Thus, this paper describes the relationship between F. necrophorum subsp. necrophorum (Fnn) metabolism and the expression of the encoded putative virulence factors under iron-restricted conditions. At the midlog phase, iron limitation reduced Fnn growth but the cell density was dependent on the size of the inoculum. Preferential utilization of glucose-1-phosphate, d-mannitol and l-phenylalanine; production of 2-hydroxycaproic acid and termination of dimethyl sulphide production were major Fnn response-factors to iron limitation. Ultimately, iron restriction resulted in an increased ability of Fnn to metabolize diverse carbon sources and in the expression of stress-specific virulence factors. Iron starvation in low Fnn cell density was associated with the up-regulation of haemagglutinin (HA) and leukotoxin (lktA) genes (2.49 and 3.72 fold change respectively). However, Fnn encoded Haemolysin (Hly), yebN homologue (febN) and tonB homologue, were down-regulated (0.15, 0.79 and 0.33, fold changes respectively). Interestingly, cell density appeared to play a regulatory role in the final bacteria cell biomass, induction of a metabolic gene expression and the expression pattern virulence factors in Fnn suggesting the role of a cell density-associated regulatory factor. This report suggest that future studies on differential expression of bacterial genes under altered environmental condition(s) should consider testing the effect of cell concentrations as this is often neglected in such studies. In conclusion, iron restriction induces preferential utilization of carbon sources and altered metabolism in Fnn with associated changes in the expression pattern of the virulence factors.

Identification of an outer membrane protein of Fusobacterium necrophorum subsp. necrophorum that binds with high affinity to bovine endothelial cells.

Fusobacterium necrophorum, a Gram-negative anaerobe, is the primary etiologic agent of liver abscesses in cattle. There are two subspecies; subsp. necrophorum and subsp. funduliforme, which differ in morphological, biochemical, molecular characteristics, and virulence. The subsp. necrophorum, which is more virulent, occurs more frequently in liver abscesses than the subsp. funduliforme. Bacterial adhesion to the host cell surface is critical to the pathogenesis of several bacterial infections, and in F. necrophorum, outer membrane proteins (OMP) have been shown to mediate adhesion to bovine endothelial cells. The objective of this study was to identify potential adhesins that are involved in adhesion of F. necrophorum subsp. necrophorum to the host cells. An OMP of 42.4 kDa, which binds with high affinity to the bovine endothelial cells and is recognized by the sera from cattle with liver abscesses, was identified. N-terminal sequencing of the protein showed 96% homology to the FomA protein of F. nucleatum. The PCR analysis showed that this fomA gene was present in several strains of subsp. necrophorum, subsp. funduliforme of bovine and subsp. funduliforme of human origin. The purified native and recombinantly expressed protein when preincubated with the endothelial cells, prevented the attachment of subsp. necrophorum significantly. In addition, the polyclonal antibody produced against the protein prevented the binding of subsp. necrophorum to bovine endothelial cells.

Outer membrane proteins of Fusobacterium necrophorum subsp. necrophorum and subsp. funduliforme.

Fusobacterium necrophorum, classified into subsp. necrophorum (Fnn) and subsp. funduliforme (Fnf), is frequently associated with necrotic infections of animals and humans. The outer membrane proteins (OMP) of many Gram negative bacteria play an important role in bacterial adhesion and establishment of infection. The OMP profile of F. necrophorum has not been well characterized. We analyzed OMP of bovine strains of Fnn and Fnf and human strains of F. necrophorum. Electrophoretic separations of extracted OMP of Fnn and Fnf strains of cattle showed a total of 19 and 20 protein bands, respectively. The most prominent protein band was 40 kDa in Fnn and 37.5 kDa in Fnf. The four human clinical strains examined had more heterogeneous banding patterns and had different profiles than those of bovine Fnf strains. A total of 11 protein bands in Fnn and 13 protein bands in Fnf were recognized by sera from cattle with liver abscesses. The intensities of many of the bands in Fnn were higher than that of Fnf. We conclude that the two subspecies of F. necrophorum differ in their OMP profiles and the difference may account for differences in their virulence and involvement in the pathogenesis of necrotic infections.

Binding and activation of plasminogen at the surface of Fusobacterium necrophorum.

Fusobacterium necrophorum is a gram-negative, anaerobic bacterium, which has been suggested to be a normal inhabitant of the oral flora. In rare cases, it can invade the tonsils and deeper tissues, causing the serious condition Lemièrre's syndrome. Recruitment of host plasminogen is a well-known bacterial virulence mechanism, and plasmin activity at the bacterial surface is thought to be important for bacterial invasion. Herein we show that plasminogen can be recruited to the surface of F. necrophorum, that surface-bound plasminogen is more easily converted to active plasmin than plasminogen in buffer, and that bound plasminogen is protected against inactivation by α2-antiplasmin. These findings add to the understanding of the pathogenesis of Lemièrre's syndrome.

Adhesion of Fusobacterium necrophorum to bovine endothelial cells is mediated by outer membrane proteins.

Fusobacterium necrophorum, a Gram-negative anaerobe, is frequently associated with suppurative and necrotic infections of animals and humans. The organism is a major bovine pathogen, and in cattle, the common fusobacterial infections are hepatic abscesses, foot rot, and necrotic laryngitis. The species comprises two subspecies: F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme. Bacterial adhesion to the host cell surface is a critical initial step in the pathogenesis, and outer membrane proteins (OMP) play an important role in adhesion and establishment of certain Gram-negative bacterial infections. The means by which F. necrophorum attaches to epithelial or endothelial cells has not been determined. We evaluated whether OMP of F. necrophorum, isolated from a liver abscess, mediated adhesion to bovine endothelial cells (adrenal gland capillary endothelial cell line). The extent of binding of subsp. necrophorum to the endothelial cells was higher than that of F. necrophorum subsp. funduliforme. Trypsin treatment of bacterial cells decreased their binding to endothelial cells indicating the protein nature of adhesins. Preincubation of endothelial cells with OMP extracted from F. necrophorum decreased the binding of bacterial cells. In addition, binding of each subspecies to endothelial cells was inhibited by polyclonal antibodies raised against respective OMP and the antibody-mediated inhibition was subspecies specific. The western blot analysis of OMP bound to endothelial cells with anti-OMP antibodies showed four OMP of 17, 24, 40 and 74 kDa. We conclude that OMP of F. necrophorum play a role in adhesion of bacterial cells to the endothelial cells.

In vitro degradation of lysine by ruminal fluid-based fermentations and by Fusobacterium necrophorum.

The objective of these studies was to characterize some factors affecting lysine degradation by mixed ruminal bacteria and by ruminal Fusobacterium necrophorum. Mixed ruminal bacteria degraded lysine, and addition of pure cultures of F. necrophorum did not increase lysine degradation. Addition of acetic or propionic acid strikingly reduced NH(3) production from lysine by mixed ruminal bacteria at pH 6, but not at pH 7. Although typical ruminal environments with acidic pH and normal concentrations of volatile fatty acids might inhibit lysine degradation by F. necrophorum, ruminal fluid contained enough bacteria with a lysine-degrading capacity to ferment 50 mM lysine in vitro. Of 7 strains of ruminal F. necrophorum tested, all grew on both lactate and lysine as the primary energy source. Both subspecies of ruminal F. necrophorum (necrophorum and funduliforme) used lysine as a primary C and energy source. Lysine and glutamic acid were effectively fermented by F. necrophorum, but alanine and tryptophan were not, and histidine and methionine were fermented only to a minor extent. The end products of lactate fermentation by F. necrophorum were propionate and acetate, and those of lysine degradation were butyrate and acetate. Fermentation of glutamic acid by F. necrophorum yielded acetate and butyrate in a ratio near to 2:1. The minimum inhibitory concentration of tylosin for F. necrophorum was not dependent on whether bacteria were grown with lactate or lysine, but F. necrophorum was more susceptible to monensin when grown on lysine than on lactate. Although F. necrophorum is generally resistant to monensin, the ionophore may reduce lysine degradation by F. necrophorum in the rumen. The essential oil components limonene, at 20 or 100 µg/mL, and thymol, at 100 µg/mL, inhibited F. necrophorum growth, whereas eugenol, guaiacol, and vanillin had no effect. Our findings may lead to ways to minimize ruminal lysine degradation and thus increase its availability to the animal.

Effects of the collagenolytic cell wall component of Fusobacterium necrophorum subsp. necrophorum on bovine hepatocytes.

The effects of the collagenolytic cell wall component (CCWC) of Fusobacterium necrophorum subsp. necrophorum on bovine hepatic cell and cytoskeletons were investigated. Scanning electron microscopy (SEM) demonstrated that CCWC damaged the cell surfaces, forming tiny holes on the cell membranes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) profiles revealed that CCWC degraded bovine cytokeratin and vimentin and by indirect fluorescent antibody (IFA) method, it was shown that CCWC caused the deformation of hepatocellular vimentin. This suggested that CCWC contributes to bovine hepatic injury and it may be as important pathogenic factor in the development of bovine hepatic abscesses.

Factors affecting lysine degradation by ruminal fusobacteria.

Fusobacterium necrophorum can readily be enriched from the rumen with lysine, and its deamination rate is very rapid. The addition of F. necrophorum JB2 to mixed ruminal bacteria significantly increased lysine degradation, but only if the ratio of ruminal fluid to basal medium was less than 25%. If more ruminal fluid (pH 6.1) was added, ammonia production decreased by as much as 80%. Clarified, autoclaved ruminal fluid was also inhibitory. When F. necrophorum JB2 was grown in a lysine-limited continuous culture (0.1 h(-1) dilution rate) and pH was decreased using HCl, optical density decreased linearly, and the culture washed out at pH 5.6. Batch cultures of F. necrophorum JB2 deaminated as much lysine at pH 6.1 as at pH 6.6, but only if fermentation acids were not present. Sodium acetate (100 mM) had little effect at pH 6.6, but the same concentration inhibited ammonia production by 80% at pH 6.1. The idea that fermentation acids could prevent the enrichment of fusobacteria in vivo was supported by the observation that dietary lysine supplementation did not enhance the lysine deamination rate of the mixed ruminal bacteria.

Leukotoxins of gram-negative bacteria.

Leukotoxins are a group of exotoxins that produce their primary toxic effects against leukocytes, especially polymorphonuclear cells (PMNs). Leukotoxins include a variety of chemicals ranging from 9,10-epoxy 12-octadecenoate, a fatty acid derivative secreted by leukocytes themselves, to proteins such as RTX (repeats in toxin). This review focuses on leukotoxins of three species of gram-negative bacteria, Mannheimia (Pasteurella) haemolytica, Actinobacillus actinomycetemcomitans, and Fusobacterium necrophorum.

Cloning, sequencing, and expression of the leukotoxin gene from Fusobacterium necrophorum.

Fusobacterium necrophorum is a gram-negative, rod-shaped, anaerobic bacterium that is a primary or secondary etiological agent in a variety of necrotic purulent infections in animals and humans. Included are diseases of cattle such as liver abscesses and foot rot, which have economically important consequences for the cattle industry. The major virulence factor of this bacterium is leukotoxin, a secreted protein of high molecular weight active against leukocytes from ruminants. The screening of a genomic DNA library with polyclonal antisera raised against native affinity-purified leukotoxin and further extension of the sequence using inverse PCR led to the cloning of the entire leukotoxin gene. The leukotoxin gene open reading frame (ORF; lktA) consists of 9,726 bp and encodes a protein of 3,241 amino acids with an overall molecular weight of 335,956. The leukotoxin does not have sequence similarity with any other bacterial leukotoxin. Five truncated overlapping polypeptides covering the whole lktA ORF were used to immunize rabbits. In Western blot assays, polyclonal antisera raised against all five truncated polypeptides recognized affinity-purified leukotoxin from F. necrophorum culture supernatant in a Western blot assay. Antisera directed against two of the five polypeptides had neutralizing activity against the toxin. The entire leukotoxin ORF was expressed in Escherichia coli. Flow-cytometric analysis showed that the recombinant leukotoxin was active against bovine polymorphonuclear leukocytes and was inhibited with antiserum raised against the F. necrophorum leukotoxin. Southern blot hybridization analysis revealed different patterns of lktA hybridizing bands between isolates of the two subspecies of F. necrophorum.

Effects of Fusobacterium necrophorum subspecies necrophorum on extracellular matrix of tissue-cultured bovine kidney cells.

The effects of Fusobacterium necrophorum subsp. necrophorum on the extracellular matrix were investigated. The toxic preparation from the culture induced reduction in the number of tissue-cultured bovine kidney cells. The exposed cells often manifested partial loss of cytoplasm and were morphologically irregular. Scanning electron microscopy demonstrated partial loss of the microvilli on the exposed cells and roughness of the cell surfaces. Finally, sodium dodecyl sulphate polyacrylamide gel electrophoresis profiles revealed complete degradation of bovine collagen type 1 after treatment with the toxic preparation. This degradation was inhibited by the addition of homologous antiserum. These findings indicate that the degradation may contribute to the establishment of the infection caused by F.n. subsp. necrophorum.

Adherence of Fusobacterium necrophorum subspecies necrophorum to different animal cells.

The adherence of Fusobacterium necrophorum subsp. necrophorum to the surfaces of animal cells was studied in order to elucidate the differences between the bacterial appearance in clinical specimens from various animals. The bacterial cells had a strong affinity for murine and rabbit cheek cell surfaces. The bacterium showed a moderate affinity for goat cells, whereas it adhered not so well to canine, feline, human or porcine cells. Treatment of the bacterial cells with haemagglutinin antiserum prior to the binding assay reduced the degree of attachment to murine and rabbit cells. Scanning electron microscopy revealed that the adherent fusobacteria often penetrated into murine and rabbit cell membranes. These observations indicate that the bacterial attachment contributes to the establishment of the infection in mice and rabbits. It is suggested that the weak binding ability resulted in a low incidence of the bacterium in canine, feline and porcine lesions.

The erythrocyte receptor for Fusobacterium necrophorum hemolysin: phosphatidylcholine as a possible candidate.

An attempt was made to determine the receptor for the hemolysin of Fusobacterium necrophorum using horse erythrocyte or its membranes as target. The spectrum of erythrocyte sensitivity has indicated that horse, dog and mouse erythrocytes are highly sensitive whereas cattle, sheep, goat and chicken red blood cells are insensitive to this hemolysin. A high correlation between sensitivity and phosphatidylcholine content of the erythrocyte membranes was noted. Binding of hemolysin to horse erythrocyte membranes was reduced significantly by prior treatment of membranes with phospholipase A2 but not with phospholipase C. Pretreatment of erythrocyte membranes with pronase, proteinase K, trypsin or neuraminidase did not alter binding of hemolysin to the membranes, suggesting that protein or sialyl residues are not involved as receptors. Gas liquid chromatography analysis showed that the fatty acid profile from hydrolysis of bovine liver phosphatidylcholine by hemolysin and phospholipase A2 were similar. In conclusion, this report presents evidence that phosphatidylcholine may be acting as a possible receptor for the hemolysin of F. necrophorum.

Endopeptidase activities of selected Porphyromonas spp., Prevotella spp. and Fusobacterium spp. of oral and non-oral origin.

The ability of three Porphyromonas spp., seven Prevotella spp., seven Fusobacterium spp. and two related Bacteroides spp. (B. levii and B. macacae) to degrade an extensive range of synthetic endo-, amino- and diamino peptidase substrates linked to the fluorescent leaving group 7-amido-4-methylcoumarin (NHMec) was investigated. Many more species than was previously recognized exhibited peptidase activities, albeit at lower levels than those already described for Porphyromonas gingivalis. Detection of chymotrypsin-like activity was dependent on which of three NHMec-linked substrates was used, but all species exhibited degradative activity with at least one of these substrates. Elastase-like activity was detected in all species though not all species reacted with each of the elastase substrates. Glycylprolyl peptidase activity was detected in all of the species tested with the exception of F. mortiferum, F. gonidiaformans, F. naviforme and F. necrophorum. While the detection of peptidase activities does not appear to be useful for the differentiation of species within the genera Bacteroides and Prevotella, its ability to differentiate species of the genus Porphyromonas or Fusobacterium warrants further investigation.

The effects of physical and chemical agents on the secretion and stability of a Fusobacterium necrophorum hemolysin.

The effect of various agents as enhancers or inhibitors of hemolysin secretion by Fusobacterium necrophorum was investigated. The hemolysin secreted in phosphate buffered saline (PBS) alone was inactivated shortly after secretion. Tween-80 or albumin preserved the hemolytic activity in PBS in which cultures of F. necrophorum had been suspended. Hemoglobin was found to enhance hemolysin secretion. However, higher concentrations diminished secretion. Chloramphenicol, an inhibitor of protein synthesis, exhibited no effect on the hemolytic activity. However, the addition of sodium azide, an energy metabolic inhibitor, significantly reduced the hemolytic activity. Lower temperatures and pH above 9 and below 6 all yielded a low hemolytic activity. Cells suspended in Tween-80 prior to sonication yielded a substantial amount of extracellular hemolytic activity with low intracellular activity detected. However, cells suspended in PBS alone yielded a low extracellular activity but rather a high intracellular activity. The same spectrum of red blood cells of different species were found to be sensitive to both the extracellular and intracellular hemolysins.