Revealing the Mechanism of NLRP3 Inflammatory Pathway Activation through K+ Efflux Induced by PLO via Signal Point Mutations.

Trueperella pyogenes is an important opportunistic pathogenic bacterium widely distributed in the environment. Pyolysin (PLO) is a primary virulence factor of T. pyogenes and capable of lysing many different cells. PLO is a member of the cholesterol-dependent cytolysin (CDC) family of which the primary structure only presents a low level of homology with other members from 31% to 45%. By deeply studying PLO, we can understand the overall pathogenic mechanism of CDC family proteins. This study established a mouse muscle tissue model infected with recombinant PLO (rPLO) and its single-point mutations, rPLO N139K and rPLO F240A, and explored its mechanism of causing inflammatory damage. The inflammatory injury abilities of rPLO N139K and rPLO F240A are significantly reduced compared to rPLO. This study elaborated on the inflammatory mechanism of PLO by examining its unit point mutations in detail. Our data also provide a theoretical basis and practical significance for future research on toxins and bacteria.

Bilateral cavernous sinus and ophthalmic vein thrombosis secondary to Trueperella pyogenes rhinosinusitis.

Ophthalmic vein thrombosis is a severe clinical entity with proptosis, eyelid swelling, orbital pain and reduction of visual acuity; its incidence is rare with 3-4 cases /million /year. Clinical manifestations result from venous congestion caused by septic (orbital cellulitis) or aseptic aetiologies (coagulopathies, trauma) and in some cases it could be associated with cavernous sinus thrombosis. In this paper, we describe a case report unique in the literature, of bilateral cavernous sinus and ophthalmic veins thrombosis due to both septic and aseptic causes characterized by unilateral sphenoid sinusitis sustained by Trueperella pyogenes infection. Trueperella pyogenes is an opportunistic animal pathogen, and its infections occur in both domestic and wild animals worldwide but are rare in humans; this is the first instance of human infection in the head and neck with an unknown hypercoagulable state.

Case report: Omphalitis caused by Trueperella pyogenes infection in a Korean indigenous calf.

Omphalitis, commonly caused by opportunistic bacteria has been significantly associated with morbidity and mortality in neonatal calves. Trueperella pyogenes is a commensal and opportunistic pathogen that can cause suppurative infection in farm animals. Our case involved a 10-day-old female Korean indigenous calf that presented with umbilical enlargement accompanied by a greenish-yellow purulent discharge and right forelimb lameness. The calf was diagnosed with failure of passive transfer at 24 h of age. Physical examination found hypothermia (38.1°C), tachycardia (110 beats/min), tachypnea (47 cycles/min), and open mouth breathing. Ultrasonography revealed hyperechoic pus in the 9th and 10th right intercostals, for which a liver abscess due to omphalophlebitis was suspected. After 3 days, the calf died. T. pyogenes was detected in the umbilical cord, lung, liver, kidney, intestine, mesenteric lymph node, urinary bladder, and bladder ligament. All genes related to the virulent factors (i.e., plo, cbpA, fimA, fimC, fimG, nanH, and nanP) were also identified, with plo and fimA being associated with pathogenicity. A final diagnosis of omphalitis was established based on the identification of virulent T. pyogenes and umbilical cord dilatation on ultrasonography. Antimicrobial susceptibility tests showed that the isolated T. pyogenes was susceptible to amoxicillin, ceftiofur, florfenicol, enrofloxacin, ofloxacin, and ciprofloxacin, suggesting the suitability of these antibiotics for treating T. pyogenes-induced omphalitis. Hence, accurate and rapid diagnosis of the involved bacteria and antimicrobial susceptibility patterns can help guide therapeutic decisions. Our case provides useful information that could aid large animal clinicians in the diagnosis and treatment of T. pyogenes-induced omphalitis.

Whole genome-based antimicrobial resistance, virulence, and phylogenetic characteristics of Trueperella pyogenes clinical isolates from humans and animals.

Trueperella pyogenes is an opportunistic zoonotic bacterial pathogen, whose antimicrobial resistance, virulence, and genetic relatedness between strains from animals and humans are barely studied. These characteristics were therefore analyzed for clinical T. pyogenes strains from 31 animals of 11 different species and 8 humans determining their complete circular genome sequence and antimicrobial susceptibility. The MICs of 19 antimicrobials including 3 antiseptics correlated to the resistance genes identified in silico within the genomes revealing a predominance of resistance to streptomycin (aadA9), sulfamethoxazole (sul1), and tetracycline (tet(33), tet(W/N/W)) among strains from humans and cattle. Additional resistance genes (erm(X), erm(56), cmx, drfA1, aadA1, aph(3'')-Ib (strA), aph(6)-Id (strB), aac(3)-IVa, aph(4)-Ia) were found only sporadically. The resistance genes were localized on genetic elements integrated into the chromosome. A cgMLST-based phylogenetic analysis revealed two major clusters each containing genetically diverse strains. The human strains showed the closest relatedness to strains from cattle. Virulence genes coding for fimbriae (fimA, fimC), neuroamidase (nanP, nanH), pyolysin (plo), and collagen binding protein (cbpA) were identified in strains from different hosts, but no correlation was observed between virulence factors and strain origin. The existence of resistance genes typically found in Gram-negative bacteria within the Gram-positive T. pyogenes indicates a wider capacity to adapt to antimicrobial selective pressure. Moreover, the presence of similar antimicrobial resistance profiles found in cattle and human strains as well as their closest relatedness suggests common zoonotic features and cattle as the potential source for human infections.

Molecular Characterization of Resistance and Virulence Factors of Trueperella pyogenes Isolated from Clinical Bovine Mastitis Cases in China.

Purpose: The present study was designed to investigate the resistance determinants and virulence factors of 45 Trueperella pyogenes isolates from clinical bovine mastitis in Hexi Corridor of Gansu, China. Methods: Minimum inhibitory concentrations (MICs) was tested by E-test method. Gene of antimicrobial resistance, virulence integrase and integron gene cassettes were determined by PCR and DNA sequencing. Results: The T. pyogenes isolates exhibited high resistance to streptomycin (88.9%) and tetracycline (64.4%), followed by erythromycin (15.6%) and gentamicin (13.3%). Resistance to streptomycin was most commonly encoded by aadA9 (88.9%); and to tetracycline, by tetW (64.4%). Importantly, all streptomycin-resistant isolates carried aadA9 alone or in combination with aadA1, aadA11 and strA-strB. Similarly, all tetracycline-resistant isolates harbored tetW alone or in combination with tetA33. Meanwhile, ermX was detected in 13.3% isolates, only one erythromycin-resistant isolate was not identified for this gene. Moreover, all T. pyogenes isolates carried class 1 integrons, and 17.8% of them contained gene cassettes, including arrays aadA1-aadB (4.4%), aad A24-dfrA1-ORF1 (2.2%) and aadA1 (2.2%). Furthermore, all tested isolates harbored virulent genes plo and fimA, followed by fimC (88.9%), fimE (86.6%) nanP (75.6%), nanH (40.0%), cbpA (35.6%) and fimG (6.7%). Conclusion: To our knowledge, this is the first report of integron gene cassettes of T. pyogenes isolates from bovine mastitis cases in China. These findings are useful for developing the prevention and the virulence factors of T. pyogenes could be promising candidates for vaccine antigens for bovine mastitis caused by T. pyogenes in China.

Phenotypic Characteristics, Antimicrobial Susceptibility and Virulence Genotype Features of Trueperella pyogenes Associated with Endometritis of Dairy Cows.

Trueperella pyogenes can cause various infections in the organs and tissues of different livestock (including pigs, cows, goats, and sheep), including mastitis, endometritis, pneumonia, or abscesses. Moreover, diseases induced by T. pyogenes cause significant economic losses in animal husbandry. In recent large-scale investigations, T. pyogenes has been identified as one of the main pathogens causing endometritis in lactating cows. However, the main treatment for the above-mentioned diseases is still currently antibiotic therapy. Understanding the impact of endometritis associated with T. pyogenes on the fertility of cows can help optimize antibiotic treatment for uterine diseases, thereby strategically concentrating the use of antimicrobials on the most severe cases. Therefore, it is particularly important to continuously monitor the prevalence of T. pyogenes and test its drug resistance. This study compared the uterine microbiota of healthy cows and endometritis cows in different cattle farms, investigated the prevalence of T. pyogenes, evaluated the genetic characteristics and population structure of isolated strains, and determined the virulence genes and drug resistance characteristics of T. pyogenes. An amount of 186 dairy cows were involved in this study and 23 T. pyogenes strains were isolated and identified from the uterine lavage fluid of dairy cows with or without endometritis.

Association of clinical respiratory disease signs and lower respiratory tract bacterial pathogens with systemic inflammatory response in pre-weaned dairy calves.

This study investigated the potential associations between 3 acute phase proteins (APPs) haptoglobin, serum amyloid A, and fibrinogen, clinical signs of respiratory disease, and the presence of bacterial pathogens in the lower respiratory tract (LRT) of pre-weaned dairy calves. This cross-sectional study included 150 pre-weaned calves (2-86 d old) from 15 large dairy herds in Estonia. Tracheobronchial lavage, blood, and fecal samples were collected from 5 calves showing clinical signs indicative of LRT disease, and samples from 5 calves without clinical signs of LRT disease per herd. All samples collected from these calves were analyzed for concentrations of systemic APPs, LRT bacteria, and intestinal pathogens. Heifer blood and bulk tank milk samples were collected for the detection of disease-specific antibodies against bovine herpesvirus 1, bovine viral diarrhea virus, bovine respiratory syncytial virus, and Mycoplasma bovis. Mixed-effects linear regression models were used to analyze the associations of clinical respiratory disease signs and LRT bacteria with APPs. Increased plasma fibrinogen concentrations in calves were associated with higher rectal temperature (>39.5°C), increased respiratory rate (>50 breaths/min), and coughing. Increased serum amyloid A concentrations were associated with higher rectal temperature (>39.5°C) and respiratory rate between 40 and 50 breaths/min. Calves with the presence of fecal Cryptosporidium spp. and rectal temperature of 39°C and above had increased serum haptoglobin concentrations. Increased fibrinogen concentrations were associated with the presence of Pasteurella multocida in the calf LRT, whereas increased concentrations of fibrinogen and serum amyloid A were associated with the presence of Trueperella pyogenes. In conclusion, APPs showed variable associations with clinical signs of respiratory disease and LRT bacteria. Plasma fibrinogen concentration could be used as a complementary calf-side test to assess systemic inflammation caused by LRT bacteria such as P. multocida and T. pyogenes in pre-weaned dairy calves.

Immunoinformatics design of novel multi-epitope vaccine against Trueperella Pyogenes using collagen adhesion protein, fimbriae, and pyolysin.

Trueperella pyogenes (T. pyogenes) is an opportunistic pathogen that causes infertility, mastitis, and metritis in animals. T. pyogenes is also a zoonotic disease and is considered an economic loss agent in the livestock industry. Therefore, vaccine development is necessary. Using an immunoinformatics approach, this study aimed to construct a multi-epitope vaccine against T. pyogenes. The collagen adhesion protein, fimbriae, and pyolysin (PLO) sequences were initially retrieved. The HTL, CTL, and B cell epitopes were predicted. The vaccine was designed by binding these epitopes with linkers. To increase vaccine immunogenicity, profilin was added to the N-terminal of the vaccine construct. The antigenic features and safety of the vaccine model were investigated. Docking, molecular dynamics simulation of the vaccine with immune receptors, and immunological simulation were used to evaluate the vaccine's efficacy. The vaccine's sequence was then optimized for cloning. The vaccine construct was designed based on 18 epitopes of T. pyogenes. The computational tools validated the vaccine as non-allergenic, non-toxic, hydrophilic, and stable at different temperatures with acceptable antigenic features. The vaccine model had good affinity and stability to bovine TLR2, 4, and 5 as well as stimulation of IgM, IgG, IL-2, IFN-γ, and Th1 responses. This vaccine also increased long-lived memory cells, dendritic cells, and macrophage population. In addition, codon optimization was done and cloned in the E. coli K12 expression vector (pET-28a). For the first time, this study introduced a novel multi-epitope vaccine candidate based on collagen adhesion protein, fimbriae, and PLO of T. pyogenes. It is expected this vaccine stimulates an effective immune response to prevent T. pyogenes infection.

Inflammation plays a critical role in damage to the bronchiolar epithelium induced by Trueperella pyogenes in vitro and in vivo.

Trueperella pyogenes can cause severe pulmonary disease in swine, but the mechanism of pathogenesis is not well defined. T. pyogenes-induced damage to porcine bronchial epithelial cells (PBECs), porcine precision-cut lung slices (PCLS), and respiratory epithelium of mice remains unknown. In this study, we used T. pyogenes 20121 to infect PBECs in air-liquid interface conditions and porcine PCLS. T. pyogenes could adhere to, colonize, and induce cytotoxic effect on PBECs and the luminal surface of bronchi in PCLS, which damaged the bronchiolar epithelium. Moreover, bronchiolar epithelial cells showed extensive degeneration in the lungs of infected mice. Furthermore, western blot showed that the NOD-like receptor (NLR)/C-terminal caspase recruitment domain (ASC)/caspase-1 axis and nuclear factor-kappa B pathway were involved in inflammation in PCLS and lungs of mice, which also confirms that porcine PCLS provide a platform to analyze the pulmonary immune response. Meanwhile, the levels of p-c-Jun N-terminal kinase, p-extracellular signal-regulated kinase, and p-protein kinase B (AKT) were increased significantly, which indicated the mitogen-activated protein kinase and Akt pathways were also involved in inflammation in T. pyogenes-infected mice. In addition, we used T. pyogenes 20121 to infect tumor necrosis factor-alpha (tnf-α-/-) mice, and the results indicated that apoptosis and injury in respiratory epithelium of infected tnf-α-/- mice were alleviated. Thus, the pro-inflammatory cytokine TNF-α played a role in apoptosis and the respiratory epithelium injury in mouse lungs. Collectively, our study provides insight into the inflammatory injury induced by T. pyogenes and suggests that blocking NLR may be a potential therapeutic strategy against T. pyogenes infection.

Preparation and evaluation of recombinant pyolysin, fimbriae E and HtaA based protein vaccines against Trueperella pyogenes.

Trueperella pyogenes (T. pyogenes) could cause zoonotic disease in various mammals, resulting in significant economic losses. Due to the lack of effective vaccine and the emergence of bacterial resistance, there is a big need for new and improved vaccines. In this study, the non-hemolytic pyolysin mutant (PLOW497F), fimbriae E (FimE) and a truncated cell wall protein (HtaA-2) were selected to generate single or multivalent protein vaccines and their efficacies against lethal T. pyogenes challenge were evaluated in a mouse model. The results showed that the levels of specific antibody were significantly higher than the PBS control group after the booster vaccination. Compared to PBS treated mice, vaccinated mice had upregulated expressions of the inflammatory cytokine genes after the first vaccination. There was a downward trend thereafter, but return to the similar or even higher levels after challenge. Furthermore, co-immunization with rFimE or rHtaA-2 could significantly enhance the anti-hemolysis antibodies induced by rPLOW497F. The supplement of rHtaA-2 induced higher agglutinating antibodies compared with single administration with rPLOW497F or rFimE. Apart from these, the pathological lesions of lung were alleviated in rHtaA-2, rPLOW497F or their combinations immunized mice. Notably, immunization with rPLOW497F, rHtaA-2, combinations of rPLOW497F and rHtaA-2 or rHtaA-2 and rFimE completely protected mice from challenge, whereas the PBS immunized mice could not survive past 1 day post challenge. Thus, PLOW497F and HtaA-2 might be useful in developing efficient vaccines to prevent T. pyogenes infection.

Construction of live-attenuated Trueperella pyogenes by antibiotic treatment and sequential passage: methods for vaccine development.

Trueperella pyogenes (T. pyogenes) is a zoonotic pathogen that is cause a variety of pyogenic diseases in animals. The complex pathogenicity and various virulence factors are important challenges to produce an effective vaccine. According to previous trials, inactivated whole-cell bacteria or recombinant vaccines were unsuccessful in preventing disease. Thus, this study aims to introduce a new vaccine candidate based on a live-attenuated platform. For this purpose, first T. pyogenes was subjected to sequential passage (SP) and antibiotic treatment (AT) to lose their pathogenicity. Second, Plo and fimA expressions as virulence genes were evaluated by qPCR and then mice were challenged with bacteria from SP and AT culture by intraperitoneal route. Compared to the control group (T. pyogenes-wild type), plo and fimA gene expressions were downregulated and vaccinated mice have a normal spleen appearance in contrast to the control group. In addition, there was no significant difference between bacterial count from spleen, liver, heart and peritoneal fluid in vaccinated mice and the control group. In conclusion, this study introduces a new T. pyogenes vaccine candidate based on a live-attenuated strategy that mimics natural infection without pathogenicity for further investigation on vaccines against T. pyogenes infections.

Antimicrobial susceptibility of Trueperella pyogenes isolated from cattle and pigs with septicemia in southern Kyushu, Japan.

We investigated the antimicrobial susceptibility of Trueperella pyogenes isolates from cattle and pigs diagnosed with septicemia at slaughter. The strains investigated in this study were found to be susceptible to ampicillin, cefazolin, gentamicin, and levofloxacin. However, 53.3% of the strains were resistant to tetracycline, 49.7% to streptomycin, 13.8% to erythromycin, 13.2% to clindamycin, and 1.2% to oxacillin and kanamycin. We also found that the resistance of pig-derived strains of T. pyogenes to tetracycline, streptomycin, erythromycin, and clindamycin was higher than that of cattle-derived strains, and these strains were resistant to up to four antimicrobial agents used simultaneously. The present results provide new data on the range of the minimum inhibitory concentration (MIC) values of various antimicrobial agents for T. pyogenes isolated from livestock with septicemia. The breakpoint values used in this study are generally considered applicable.

Involvement of the E3 ubiquitin ligase Cblb in host defense and evaluation of transcriptome during Trueperella pyogenes infection.

Trueperella pyogenes (T. pyogenes) is a versatile and ingenious bacterium that causes severe suppurative injuries in lots of economically important ruminants. The underlying pathogenesis of T. pyogenes infection remains poorly understood. In the current study, we performed transcriptome sequencing of mouse blood tissue infected with T. pyogenes. A total of 36.73 G clean data were collected, and 136 differentially expressed genes were obtained in the infection group compared to the control group. In addition, we found that the E3 ubiquitin ligase Cblb exhibited significant upregulation in the infection groups compared to the control group. Mechanistically, T. pyogenes infection markedly enhanced the expression of Cblb and regulated the host defense response. Inhibiting Cblb expression with Cblb siRNA impaired the inflammatory response and reduced the effect of phagocytosis in RAW264.7 murine macrophages. Intriguingly, overexpression of Cblb induced a strong inflammatory response and enhanced phagocytosis against T. pyogenes infection in macrophages. More importantly, the overexpression of Cblb significantly reduced the bacterial load and protected mice from the T. pyogenes infections. Therefore, our findings reveal that Cblb is a novel and potential regulator in response to T. pyogenes infection and shed new light on the development of promising treatments against T. pyogenes-related diseases.

Comparative phylogenomics of Trueperella pyogenes reveals host-based distinction of strains.

Trueperella pyogenes, an opportunistic pathogen causes various ailments in different animals. Different strains from different animals have distinct characters phenotypically and genotypically. Hence understanding the strains in a particular geographical location helps in framing the preventive measures. Comparative genomics of all the available T. pyogenes genome in the NCBI was conducted to understand the relatedness among strains. Whole genome phylogeny showed host associated clustering of strains recovered from swine lungs. Core genome phylogeny also showed host associated clustering mimicking whole genome phylogeny results. MLST analysis showed that there was higher diversity among cattle strains. Multidimensional scaling revealed five swine clusters, two cattle and buffalo clusters. Pangenome analysis also showed that T. pyogenes had an open genome with 57.09% accessory genome. Host specific genes were identified by pangenome analysis, and (R)-citramalate synthase was specific for swine strains of Asian origin. Host specifc genes identified by pangenome analysis can be exploited for developing a molecular assay to specifically identify the strains. The study shows that MLST having higher discriminatory power can be used as an epidemiological tool for strain discrimination of T. pyogenes.

Antimicrobial susceptibility, virulence genes and genomic characterization of Trueperella pyogenes isolated from abscesses in dairy cattle.

Trueperella pyogenes is an opportunistic animal pathogen mainly associated with various suppurative infections in wild and domestic animals. Limited studies have investigated the pathogenesis of diseases caused by this pathogen. The main objective of the current study was to investigate the prevalence, phenotypic properties, virulence genotypes, antimicrobial susceptibility and genetic characterization of T. pyogenes isolated from abscess lesions in different tissues of on-farm dairy cattle. The study was performed on 150 postpartum cattle with clinical abscess symptoms on 22 farms around Tehran, Iran. Classical and disk diffusion methods are used for phenotypic characterization and antibiotic susceptibility. Detection of virulence factor encoding genes and genomic characterization of the isolates also are carried out by conventional PCR and BOX-PCR assays, respectively. Sixty-eight T. pyogenes strains (45.3%) were isolated, 12 were identified as pure cultures and the other 56 strains were isolated from mixed cultures. Seven distinct biotypes were identified among the T. pyogenes isolates. The isolates were mostly resistance to trimethoprim-sulfamethoxazole (70.6%), erythromycin (36.7%), tetracycline (26.5%) and tylosin (23.5%) antibiotics. Also, the genes plo, nanH, nanP and fimA were detected in all isolates. Forty-two isolates (61.7%) carried all virulence factor genes detected in this study. Three isolates only carried plo, nanH, nanP and fimA genes were identified as the least frequent genotype. All sixty-eight isolates and the reference strain were categorized into seven main clusters (A-G). A strong association was observed between virulence factor encoding genes, pathogenicity and biochemical biotypes in some specific clonal relationships.

Effects of Luteolin on Biofilm of Trueperella pyogenes and Its Therapeutic Effect on Rat Endometritis.

Trueperella pyogenes is an opportunistic pathogen that causes suppurative infections in animals. The development of new anti-biofilm drugs will improve the current treatment status for controlling T. pyogenes infections in the animal husbandry industry. Luteolin is a naturally derived flavonoid compound with antibacterial properties. In this study, the effects and the mechanism of luteolin on T. pyogenes biofilm were analyzed and explored. The MBIC and MBEC of luteolin on T. pyogenes were 156 µg/mL and 312 µg/mL, respectively. The anti-biofilm effects of luteolin were also observed by a confocal laser microscope and scanning electron microscope. The results indicated that 312 µg/mL of luteolin could disperse large pieces of biofilm into small clusters after 8 h of treatment. According to the real-time quantitative PCR detection results, luteolin could significantly inhibit the relative expression of the biofilm-associated genes luxS, plo, rbsB and lsrB. In addition, the in vivo anti-biofilm activity of luteolin against T. pyogenes was studied using a rat endometritis model established by glacial acetic acid stimulation and T. pyogenes intrauterine infusion. Our study showed that luteolin could significantly reduce the symptoms of rat endometritis. These data may provide new opinions on the clinical treatment of luteolin and other flavonoid compounds on T. pyogenes biofilm-associated infections.

Comparative genomics of Trueperella pyogenes available in the genome database reveals multidrug resistance genomic islands.

OBJECTIVE: An opportunistic pathogen, Trueperella pyogenes can infect cattle, buffalo, pig, goat, cat, dog, forest musk deer, etc., affecting various organs. The aim of this study was to identify the multidrug resistance genomic islands of T. pyogenes genomes available in NCBI database and also in the recently isolated strain TN_CUL_2020. METHODS: The strain TN_CUL_2020 isolated from swine lung abscess was sequenced by Illumina platform, and all the available T. pyogenes genome in NCBI database was retrieved for the comparative analysis. The ABRicate searches was used to identify antimicrobial resistance genes, and genomic islands (GIs) were predicted using IslandViewer 4. RESULTS: The strains SH01, SH02, and TP1 were predicted with maximum number of drug resistance genes. Genomic islands identified had multidrug resistance genes along with the class I integron and/or IS6100 elements in SH01, SH02, TP1. Composite transposons of IS6100 were noted in T2849, T4479, and TP3 intercalating tet(33) resistance genes. Several strains were predicted with phage elements, type IV secretion system, the toxin-antitoxin system in the GIs. CONCLUSION: Swine strains SH01, SH02 were predicted with multidrug resistance genes along with class I integrons. The presence of class I integrons, insertional elements, type IV secretion system, toxin-antitoxin system, and phage elements may aid in the horizontal transfer of antimicrobial resistance genes.

Single Point Mutation and Its Role in Specific Pathogenicity to Reveal the Mechanism of Related Protein Families.

Pyolysin (PLO) is secreted by Trueperella pyogenes as a water-soluble monomer after forming transmembrane ß-barrel channels in the cell membrane by binding cholesterol. Two significantly conserved residues at domain 1 of PLO are mutated, which provides novel evidence of a relationship between conformational change and interaction with the cell membrane and uncovers the pore formation mechanism of the cholesterol-dependent cytolysin (CDC) family. Moreover, PLO is a special member of the CDCs, which the percentage of sequence identities between PLO and other CDC members is from 31% to 45%, while others are usually from 40% to 70%. It is important to understand that at very low sequence identities, models can be different in the pathogenic mechanisms of these CDC members, which are dedicated to a large number of Gram-positive bacterial pathogens. Our studies, for the first time, located and mutated two different highly conserved structural sites in the primary structure critical for PLO structure and function that proved the importance of these sites. Together, novel and repeatable observations into the pore formation mechanism of CDCs are provided by our findings. IMPORTANCE Postpartum disease of dairy cows caused by persistent bacterial infection is a global disease, which has a serious impact on the development of the dairy industry and brings huge economic losses. As one of the most relevant pathogenic bacteria for postpartum diseases in dairy cows, Trueperella pyogenes can secrete pyolysin (PLO), a member of the cholesterol-dependent cytolysin (CDC) family and recognized as the most important toxin of T. pyogenes. However, the current research work on PLO is still insufficient. The pathogenic mechanism of this toxin can be fully explored by changing the local structure and overall function of the toxin by a previously unidentified single point mutation. These studies lay the groundwork for future studies that will explore the contribution of this large family of CDC proteins to microbial survival and human disease.

Molecular Basis for Luteolin as a Natural TatD DNase Inhibitor in Trueperella pyogenes.

TatD960 and TatD825 are DNases that contribute to biofilm formation and virulence in Trueperella pyogenes (T. pyogenes). Luteolin is a natural flavonoid commonly found in plants that exhibits antimicrobial capacity. Our study aims to investigate the effects of luteolin on TatD DNases as a natural inhibitor. In this research, the expression of tatD genes and TatD proteins in T. pyogenes treated with luteolin was detected, and then the effect of luteolin on the hydrolysis of DNA by TatD DNases was analyzed using agarose gel electrophoresis. Moreover, the interactions between luteolin and TatD DNases were tested using surface plasmon resonance (SPR) assays and molecular docking analysis. After 1/2 MIC luteolin treatment, the transcription of tatD genes and expression of TatD proteins appeared to be reduced in 80-90% of T. pyogenes (n = 20). The gel assay revealed that luteolin can inhibit the activity of TatD DNases. The SPR assay showed that the KD values of luteolin to TatD960 and TatD825 were 6.268 × 10-6 M and 5.654 × 10-6 M, respectively. We found through molecular docking that hydrogen bonding is predominant in the interaction of luteolin and TatD DNases. Our data indicate that luteolin inhibited the ability of TatD DNases by decreasing their binding to DNA. The current study provides an insight into the development of luteolin as a DNase inhibitor in preventing biofilm formation and virulence in T. pyogenes.

Prevalence and Genetic Diversity of Trueperella pyogenes Isolated from Infections in European Bison (Bison bonasus).

Trueperella pyogenes is a Gram-positive bacterium causing purulent infections in many animal species, including the European bison. However, the data about the virulence and genetic relationships of T. pyogenes strains isolated from these wild ruminants are strongly limited. The aim of this study was to investigate the prevalence of T. pyogenes infections in the European bison, and to evaluate the genetic diversity of isolates from these animals. In the time span of 10 years, 328 European bison from 16 different locations were examined. The standard bacteriological methods were used for T. pyogenes isolation and identification from clinical specimens obtained from urogenital tract infections and abscesses of different locations. The presence of genes encoding known virulence factors was investigated by PCR, and the genetic diversity of T. pyogenes strains was examined with the RAPD-PCR method. The prevalence of T. pyogenes infections was 14.6%, and the pathogen was isolated from both female (47.9% of isolates) and male (52.1% of isolates) European bison. It should be highlighted that a considerable number of strains were isolated from the prepuce and penis infections. Therefore, the role of T. pyogenes in the pathogenesis of balanoposthitis should be seriously perceived. A total of 39 T. pyogenes strains were subjected to genetic characterization. All studied strains carried the plo gene, while the nanH (25.6%), nanP (23.1%), cbpA (7.7%), fimA (97.4%), fimC (69.2%), fimE (92.3%) and fimG (15.4%) genes were present with a variable frequency among the tested strains. The virulence genotype plo/fimA/fimC/fimE was dominant. RAPD-PCR typing showed a high level of genetic diversity among European bison T. pyogenes strains, and a total of 31 different RAPD profiles were distinguished. In a few cases, the same RAPD profile was found in strains obtained from animals living in the same area. This study provided the first data about the prevalence and genetic relationships of T. pyogenes in the Polish population of European bison. However, further epidemiological investigations are needed to understand the routes of transmission and dissemination of the pathogen in these wild animals.