Bovine Lung Research Articles

Bovine Lung Peptides after Trypsinolysis Reveal Anti-Exudative Activity

Abstract Enzymatic hydrolysates of proteins isolated from cow lungs are used as anti-inflammatory agents for the growth and regeneration of new tissues. Hydrolysates were observed to inhibit the production of free radicals in cells and have a high anti-exudative activity even in low doses. In this study, we established the anti-exudative activity of peptides of protein hydrolysates isolated from bovine lungs. The anti-exudative activity of the peptides sum was compared with acetylsalicylic acid (ASA) and prednisolone, and the dynamics of inflammation were studied by tumour formation using carrageenan and formalin. Between 1 h and 24 h after administration of the drug, the effect of the peptides sum showed a significantly better indicator than the rest of the means. In general, the anti-exudative activity of the peptides sum was 2–3 times higher than the rest of the preparations. Biochemical indicators of prednisolone and peptides sum in two doses of 10−6 μg/kg and 10−4 μg/kg were studied. As a result of the study, it was found that the peptides sum at 10−6 μg/kg dose enhanced alanine aminotransferase (ALT) activity by 17.1% compared to those resulting from prednisolone treatment. In C-reactive protein (CRP) and antistreptolysin O (ASO) indicators, 10−4 μg/kg dose showed high activity. The sum of isolated peptides reduced ALT and ASO levels. This study provides additional support for preparing anti-inflammatory means from the peptides sum isolated from bovine lungs.

Ceapin-A7 suppresses the protective effects of Octreotide in human and bovine lung endothelial cells.

Endothelial injury can be the cause and consequence of severe inflammation and injury. Synthetic somatostatin analogs - which suppress Growth Hormone - are FDA-approved drugs associated with anti-inflammatory activities. In the present study we suggest that the protective activities of Octreotide in human and bovine endothelial cells are mitigated by Ceapin-A7, which is an activating transcription factor 6 suppressor. To study endothelial function, we assessed protein expression levels of key cytoskeletal proteins, as well as paracellular permeability. To evaluate inflammation, we measured factors which promote vascular leak, as well as reactive oxygen species generation. Collectively, our study supports the involvement of activating transcription factor 6 in the protective effects of Octreotide in endothelial barrier function.

Mycoplasma bovis Invades Non-Phagocytic Cells by Clathrin-Dependent Endocytic Pathways and Escapes from Phagocytic Vesicles.

Mycoplasma bovis (M. bovis) is capable of causing pneumonia, arthritis, mastitis, and various other ailments in cattle of all age groups, posing a significant threat to the healthy progression of the worldwide cattle industry. The invasion of non-phagocytic host cells serves as a pivotal mechanism enabling M. bovis to evade the immune system and penetrate mucosal barriers, thereby promoting its spread. To investigate the differences in M. bovis invasion into four types of non-phagocytic cells (Madin-Darby bovine kidney (MDBK) cells, embryonic bovine lung (EBL) cells, bovine embryo tracheal (EBTr) cells and bovine turbinate (BT) cells) and further elucidate its invasion mechanism, this study first optimized the experimental methods for M. bovis invasion into cells. Utilizing laser scanning confocal microscopy, transmission electron microscopy, and high-content live-cell imaging systems, the invasion process of M. bovis into four types of non-phagocytic cells was observed. The invasion rates of three different strains of M. bovis (PG45, 07801, 08M) were quantified through the plate counting method. In order to clarify the specific pathway of M. bovis invasion into cells, chlorpromazine (CPZ), amiloride (AMI), and methyl-β-cyclodextrin (M-β-CD) were used to inhibit CLR-mediated clathrin-dependent endocytosis (CDE) pathway, macropinocytosis, and lipid raft pathway, respectively. Subsequently, the invasion rates of PG45 into these four types of cells were measured. Using siRNA technology, the expression of clathrin (CLR) in EBL cells was knocked down to further verify the role of CLR in the invasion process of M. bovis. The results showed that the optimal conditions for M. bovis to invade non-phagocytic cells were a multiplicity of infection (MOI) of 1000 and an optimal invasion time of 4 h. All three strains of M. bovis have the ability to invade the four types of non-phagocytic cells, yet their invasion abilities vary significantly. Observations from transmission electron microscopy further confirmed that at 120 min post-infection, PG45 had successfully invaded EBL cells and was present within endocytic vesicles. It is noteworthy that almost all PG45 successfully escaped from the endocytic vesicles after 240 min of infection had passed. Through chemical inhibition experiments and CLR protein knockdown experiments, it was found that when the CDE and lipid raft pathways were blocked or CLR protein expression was reduced, the invasion rates of PG45, 07801, and 08M in MDBK, EBL, EBTr, and BT cells were significantly decreased (p < 0.05). The above results indicate that M. bovis can invade all types of non-phagocytic cells through endocytic pathways involving CDE (clathrin-dependent endocytosis) or lipid raft-mediated endocytosis, and possesses the ability to escape from phagosomes.

Octreotide protects against LPS-induced endothelial cell and lung injury

Growth hormone (GH) is a crucial regulator of growth, cell reproduction, and regeneration; and it is controlled by growth hormone-releasing hormone (GHRH) and somatostatin. Lipopolysaccharides (LPS) can compromise endothelial function, leading to increased inflammation and vascular leak. Octreotide (OCT) is an FDA-approved synthetic somatostatin analog (SSA) used to treat acromegaly and neuroendocrine tumors. The present study investigates the effects of OCT on LPS-induced injury in bovine and human lung endothelial cells, as well as in mouse lungs. Our in vitro observations suggest that OCT effectively counteracts LPS-induced endothelial leak, inflammation, and reactive oxygen species (ROS) generation. Furthermore, OCT reduces bronchoalveolar lavage fluid (BALF) protein concentration in an experimental model of Acute Lung Injury (ALI). Our study suggests that OCT mitigates LPS-induced endothelial cell and lung injury, suggesting that it may represent an exciting therapeutic possibility in diseases related to barrier dysfunction.

Evaluation of Bacterial Pneumonia and Pathological Changes in Lungs of Cattle Slaughtered in Maiduguri Central Abattoir, Borno State Nigeria

The study was designed to determine level of lungs inflammation and associated bacterial flora that influenced gross and histopathological lesions related to bovine pneumonic lungs amongst cattle slaughter in Maiduguri central abattoir. The study was conducted in Maiduguri, North-Eastern Nigeria. A total of 100 pneumonic lungs sampled from cattle slaughtered at Maiduguri central abattoir, 63 (63 %) were positive isolate and 37 were negative isolate (37 %). The results indicate isolates recovered were gram-positive and gram-negative bacteria with five bacterial genera isolated consist of 3 Gram-positive and 2 Gram-negative bacteria. The isolates were subjected to sensitivity test of various antibiotics like Ciprofloxacin, Pefloxacin, and Oflaxacinm and they were sensitive, while Chloramphenicol, Ciprofloxacin, Gentamicin, Streptomycin and Oflaxacin were Intermediate and Amoxicillin, Erythromycin, Cotrimoxazole, Gentamycin, and Ceftriaxone were resistance to the antimicrobial sensitivity tests. Pneumonic bacteria isolates based on sex and age of the cattle slaughtered at Maiduguri abattoir were recorded. Bull has the highest 58 (92.0%) compare to the cow 5 (8.0%). While age-wise, Isolates were recorded with highest 97.0 % among adult cattle when compare to the young cattle (3.0%). Three types of pneumonia were identified based on the Pathological examinations of 25 pneumonic lungs randomly selected from the 100 lungs samples which were recorded.

Rapid detection of causative bacteria including multiple infections of bovine respiratory disease using 16S rRNA amplicon-based nanopore sequencing.

Bovine respiratory disease (BRD) is a multifaceted condition that poses a primary challenge in calf rearing. Viruses and bacteria are etiological agents of BRD. Viral BRD is typically managed symptomatically, whereas bacterial BRD is predominantly managed through the empirical administration of antimicrobials. However, this empirical administration has raised concerns regarding the emergence of antimicrobial-resistant bacteria. Thus, rapid identification of pathogenic bacteria and judicious selection of antimicrobials are required. This study evaluated the usefulness of 16S rRNA analysis through nanopore sequencing for the rapid identification of BRD-causing bacteria. A comparative evaluation of nanopore sequencing and traditional culture method was performed on 100 calf samples detected with BRD. Nanopore sequencing facilitated the identification of bacteria at the species level in bovine nasal swabs, ear swabs, and lung tissue samples within approximately 6h. Of the 92 samples in which BRD-causing bacteria were identified via nanopore sequencing, 82 (89%) were concordant with the results of culture isolation. In addition, the occurrence of multiple infections exceeded that of singular infections. These results suggest that 16S rRNA sequencing via nanopore technology is effective in reducing analysis time and accurately identifying BRD-causing bacteria. This method is particularly advantageous for the initial detectable screening of BRD.

On the issue of prevention and eradication of minor viral bovine diseases in Ukraine

The study aimed to evaluate the epizootic status of livestock in Ukraine concerning the prevalence of bovine immunodeficiency virus and bovine foamy virus infections. A literature review was conducted to analyze the epizootic status of livestock farming in various countries regarding bovine immunodeficiency and spumavirus infections. To investigate this issue in Ukrainian livestock, blood samples were collected from 10–15 cows with further DNA extraction and studies via PCR, according to the developers’ recommendations. The biological characteristics of bovine foamy virus and bovine immunodeficiency virus were studied by infecting bovine fetal lung (LEK) and calf coronary vessels (KST) cell cultures, with each passage being visually monitored and examined through light microscopy. PCR was performed on the third and fifth passages to detect the genetic material. The genetic material of bovine leukemia virus, bovine immunodeficiency virus, and bovine foamy virus was confirmed in 12 farms across 8 regions of Ukraine. It was demonstrated that bovine immunodeficiency virus and bovine foamy virus can integrate into homologous cell cultures derived from cattle. The immunosuppressive effects of bovine foamy virus and its capability to inhibit components of the non-specific immune system were established on laboratory animal models. Emphasis is placed on the necessity to develop domestic tools for the retrospective diagnosis of bovine immunodeficiency and spumavirus infections and to implement a national anti-epizootic program

Microwave dielectric properties of normal, fibroelastotic, and malignant human lung tissue

Technological development of microwave treatment and detection techniques for lung cancer requires accurate and comprehensive knowledge of the microwave dielectric properties of human lung tissue. We characterize the dielectric properties of room temperature human lung tissue from 0.5 to 10 GHz for three lung tissue groups: normal, fibroelastotic, and malignant. We fit a two-pole Debye model to the measured frequency-dependent complex permittivity and calculate the median Debye parameters for the three groups. We find that malignant lung tissue is approximately 10% higher in relative permittivity and conductivity compared to normal lung tissue; this trend matches previously reported normal versus malignant data for other biological tissues. There is little contrast between benign lung tissue with fibroelastosis and malignant lung tissue. We extrapolate our data from room temperature to 37 °C using a temperature-dependence model for animal lung tissue and use the Maxwell-Garnett dielectric mixing model to predict the dielectric properties of inflation-dynamic human lung tissue; both approximations correspond with previously reported dielectric data of bovine and porcine lung tissue.

β-Adrenoceptor Agonists Attenuate Thrombin-Induced Impairment of Human Lung Endothelial Cell Barrier Function and Protect the Lung Vascular Barrier during Resuscitation from Hemorrhagic Shock.

β-adrenoceptor (β-AR) agonists are known to antagonize thrombin-induced impairment (TII) of bovine and ovine lung endothelial barrier function. The effects of adrenoceptor agonists and other vasoactive agents on human lung microvascular endothelial cell (HULEC-5a) barrier function upon thrombin exposure have not been studied. Furthermore, it is unknown whether the in vitro effects of adrenoceptor agonists translate to lung protective effects in vivo. We observed that epinephrine, norepinephrine, and phenylephrine enhanced normal and prevented TII of HULEC-5a barrier function. Arginine vasopressin and angiotensin II were ineffective. α1B-, α2A/B-, and β1/2-ARs were detectable in HULEC-5a by RT-PCR. Propranolol but not doxazosin blocked the effects of all adrenoceptor agonists. Phenylephrine stimulated β2-AR-mediated Gαs activation with 13-fold lower potency than epinephrine. The EC50 to inhibit TII of HULEC-5a barrier function was 1.8 ± 1.9 nM for epinephrine and >100 nM for phenylephrine. After hemorrhagic shock and fluid resuscitation in rats, Evans blue extravasation into the lung increased threefold (p < 0.01 vs. sham). Single low-dose (1.8 μg/kg) epinephrine administration at the beginning of resuscitation had no effects on blood pressure and reduced Evans blue extravasation by 60% (p < 0.05 vs. vehicle). Our findings confirm the effects of β-adrenoceptor agonists in HULEC-5a and suggest that low-dose β-adrenoceptor agonist treatment protects lung vascular barrier function after traumatic hemorrhagic shock.

Strain-dependent variations in replication of European clade 2.3.4.4b influenza A(H5N1) viruses in bovine cells and thermal inactivation in semi-skimmed or whole milk.

We investigated the thermostability of four European avian influenza A(H5N1) viruses in whole and semi-skimmed milk and their replication in bovine kidney and lung cells amid the current influenza A(H5N1) dairy cattle outbreak in the United States. Results showed strain-dependent differences in thermal inactivation, particularly in whole milk, and variable replication efficacy in lung cells. These findings support assessing the inactivation of European H5N1 viruses in milk and their replication in bovine cells, aiding biosafety protocols and public health measures.

Growth and organotypic branching of lung-specific microvascular cells on 2D and in 3D lung-derived matrices

Tissue-specific endothelial cells have vital roles in maintenance and functioning of native tissues with constant reciprocal crosstalk with resident cells. Three-dimensional (3D) physio-mimetic in vitro models which incorporate lung-specific microvasculature are needed to model lung-related diseases which involve modulation of endothelial cell behavior like cancer. In this study, we investigated the growth kinetics, morphological changes and responses to biological cues of lung microvasculature on two-dimensional (2D) and in lung matrix-derived 3D hydrogels. HUVEC and HULEC-5a cells were cultured on 2D and compared for their growth, morphologies, and responses to varying growth medium formulations. Brightfield and immunofluorescence imaging was performed to assess differences in morphology. For 3D cultures, native bovine lungs were decellularized, lyophilized, solubilized, and reconstituted into hydrogel form in which endothelial cells were embedded. Cell growth and organotypic branching was monitored in 3D hydrogels in the presence of varying biological cues including lung cancer cell secretome. HUVEC and HULEC-5a cells demonstrated comparable growth and morphology on 2D. However, in 3D lung-derived ECM hydrogels, tissue-specific HULEC-5a cells exhibited much better adaptation to their microenvironment, characterized by enhanced organotypic branching and longer branches. HULEC-5a growth was responsive to lung cancer cell-conditioned medium in both 2D and 3D conditions. In 3D, the concentration of ECM ligand significantly affected cell growth in long-term culture where molecular crowding had an inhibitory role. Our data reveals that HULEC-5a cells offer a reliable alternative to frequently pursued HUVECs with comparable growth and morphology. Due to their intrinsic program for cellular crosstalk with resident cells, the use of tissue-specific endothelium constitutes a vital aspect for modeling physiological and pathological processes. Furthermore, our study is the first demonstration of the synergy between lung-specific microvasculature with lung-specific ECM within a 3D in vitro model.

Enzymatic processing of animal by-products: production of antioxidant hydrolysates with Bacillus sp. CL18 crude protease.

Protein hydrolysates might display diverse bioactivities with potential relevance to human and animal health and food technology. Enzymatic hydrolysis of agro-industrial by-products is increasingly focused. In this study, a crude protease from Bacillus sp. CL18 was applied to obtain antioxidant protein hydrolysates from porcine, bovine, poultry, and fish by-products. The crude enzyme hydrolyzed all the twelve investigated by-products, as detected by increased soluble protein contents after 4h of proteolysis. Hydrolysates exhibited higher radical-scavenging, Fe2+-chelating and reducing power capacities than non-hydrolyzed by-products. Hydrolysis times (0-8h) and enzyme-to-substrate (E/S) ratios (384, 860, and 1,400 U/g) were assessed to produce antioxidant bovine lung hydrolysates. The highest E/S ratio accelerated both hydrolysis and increases in antioxidant activities; however, it did not result in bioactivities higher than hydrolysates obtained with the intermediate E/S ratio. Optimal antioxidant activities could be reached after 6h of hydrolysis using 860 U/g. Animal by-products are interesting sources of bioactive protein hydrolysates, which could be produced with a non-commercial bacterial protease. This might represent a promising strategy for the valorization of animal by-products generated in large amounts by the agri-food sector.

Study of the biological properties of new chemical compounds of the pyrimidine series

In order to study the effect of a number of 6-substituted uracil derivatives on bovine embryo lung cells, experiments were conducted. According to the study, the effect of the studied substances on the cultural and morphological properties of cell was determined by taking into account their viability and proliferation index. In %, it was calculated that viability was determined by the ratio of living cells to their total number. The proliferation index was determined as the ratio of the number of proliferating cells to the number of seeded cells. At 0.1 mM concentration of the compounds studied, a decrease in LEK cell viability was recorded compared to the control group, but this was only the case for compound No. 2. When other compounds were used at doses of 0.1 mM, LEK cell viability increased by 3.0-7.0% compared to controls. Increasing the dose of the compounds studied to 1.0 mM results in a 10.0% decrease in survival in the presence of compounds No. 3 and 6, 16.0% in the presence of compounds No. 2 and 7, 19.0% in the presence of compound No. 4 and 25.0% in the presence of compounds No. 8 and 9.

Host factor RBMX2 promotes epithelial cell apoptosis by downregulating APAF-1's Retention Intron after Mycobacterium bovis infection.

The Mycobacterium tuberculosis variant bovis (M. bovis) is a highly pathogenic environmental microorganism that causes bovine tuberculosis (bTB), a significant zoonotic disease. Currently, "test and culling" is the primary measure for controlling bTB, but it has been proven to be inadequate in animals due to their high susceptibility to the pathogen. Selective breeding for increased host resistance to bTB to reduce its prevalence is feasible. In this study, we found a vital host-dependent factor, RBMX2, that can potentially promote M. bovis infection. By knocking RBMX2 out, we investigated its function during M. bovis infection. Through transcriptome sequencing and alternative splicing transcriptome sequencing, we concluded that after M. bovis infection, embryo bovine lung (EBL) cells were significantly enriched in RNA splicing associated with apoptosis compared with wild-type EBL cells. Through protein/molecular docking, molecular dynamics simulations, and real-time quantitative PCR, we demonstrated that RBMX2 promotes the apoptosis of epithelial cells by upregulating and binding to apoptotic peptidase activating factor 1 (APAF-1), resulting in the alternative splicing of APAF-1 as a retention intron. To our knowledge, this is the first report of M. bovis affecting host epithelial cell apoptosis by hijacking RBMX2 to promote the intron splicing of downstream APAF-1. These findings may represent a significant contribution to the development of novel TB prevention and control strategies.

Associations of serostatus upon arrival with clinical respiratory disease, lung consolidation, and growth in veal calves

Respiratory tract infections remain a major problem during calf rearing, especially among milk (formula)-fed veal. Preconditioning of calves through appropriate colostrum management and vaccination could be helpful to address this issue. The objective of this study was to investigate whether the presence of serum antibodies against major respiratory tract pathogens (bovine respiratory syncytial virus, parainfluenza 3 virus, bovine coronavirus, Mycoplasmopsis bovis, Histophilus somni, Pasteurella multocida, and Mannheimia haemolytica) and total serum IgG concentration in calves upon arrival at the veal facility were associated with the occurrence of clinical bovine respiratory disease (BRD) or lung consolidation in the first 3 wk, as assessed by both the Wisconsin BRD scorecard (based on 5 clinical signs: cough, rectal temperature, ear position, and nasal and ocular discharge) and by quick thoracic ultrasound scanning. Additionally, the association between calves' serostatus production parameters were explored. A prospective cohort study was conducted among 442 male dairy calves on a large veal calf facility in Belgium. Both clinical scoring and quick thoracic ultrasound scanning were performed on all calves at 4 key moments in the production cycle: arrival at the facility, initiation of first metaphylactic antimicrobial treatment at peak incidence of BRD (wk 1), end of the first metaphylactic treatment (short-term evaluation) and at wk 10 (long-term evaluation). Mixed effects logit regression models were fitted to quantify relationships. The outcomes of interest were clinical respiratory disease (Wisconsin BRD scorecard positive), lung consolidation (≥1 cm or ≥ 3 cm), average daily weight gain, and cold carcass weight. In the first week of production, incidence of lung consolidation (≥1 cm) quickly increased from 14.9% upon arrival to 43.0% at the peak of the BRD incidence, while clinical BRD increased from 3.6% to 16.1%. The main finding of this study was that calves who were seropositive for bovine respiratory syncytial virus and bovine coronavirus at arrival had reduced odds of developing lung consolidation at the peak of the outbreak, 0.58 odds ratio (95% CI: 0.38-0.89) and 0.37 odds ratio (95% CI: 0.20-0.69), respectively. No relationships between serum IgG concentration at arrival and the development of lung consolidations or clinical respiratory disease were found. Nevertheless, on average, throughout the first 10 wk of the fattening cycle, calves with failed transfer of passive immunity (serum IgG < 7.5 g/L) gained 40 g/d (95% CI: 10-70 g/d) less weight (average daily gain). Hence, ensuring that calves have a positive serostatus for these respiratory tract pathogens before entering the facility may help lower the incidence of lung consolidations, subsequently reducing treatment incidence and the adverse effects on primary economic outcomes.

Validation of a multiplex-tandem RT-PCR for the detection of bovine respiratory disease complex using Scottish bovine lung samples

The welfare and economic impact of bovine respiratory disease complex (BRDC), and its associated antibiotic usage, are major challenges to cattle rearing and beef cattle finishing industries. Accurate pathogen diagnosis is important to undertake appropriate treatment and long-term management strategies, such as vaccine selection. Conventional diagnostic approaches have several limitations including high costs, long turnaround times and difficulty in test interpretation, which could delay treatment decisions and lead to unnecessary animal losses. We describe the validation of a multiplex-tandem (MT) reverse transcription-polymerase chain reaction (RT-PCR) for the detection of seven common pathogens associated with BRDC. This test has the potential to advance pathogen identification and to overcome many of the limitations of current testing methods. It requires a single sample and results are obtained quickly and not influenced by prior antimicrobial therapy or overgrowth of contaminating organisms. We demonstrated a test specificity of 100% and sensitivity ranging from 93.5% to 100% for these seven common pathogens. This test will be a useful addition to advance BRDC investigation and diagnosis.

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels.

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells' natural environment in vitro. However, maintaining the native biochemical content of the ECM, achieving mechanical stability, and comprehending the impact of the decellularization process on the mechanical properties of the ECM hydrogels are challenging. Here, a pipeline for decellularization of bovine lung tissue using two different protocols, downstream characterization of the effectiveness of decellularization, fabrication of reconstituted decellularized lung ECM hydrogels and assessment of their mechanical and cytocompatibility properties were described. Decellularization of the bovine lung was pursued using a physical (freeze-thaw cycles) or chemical (detergent-based) method. Hematoxylin and Eosin staining was performed to validate the decellularization and retention of major ECM components. For the evaluation of residual collagen and sulfated glycosaminoglycan (sGAG) content within the decellularized samples, Sirius red and Alcian blue staining techniques were employed, respectively. Mechanical properties of the decellularized lung ECM hydrogels were characterized by oscillatory rheology. The results suggest that decellularized bovine lung hydrogels can provide a reliable organotypicalternative to commercial ECM products by retaining most native ECM components. Furthermore, these findings reveal that the decellularization method of choice significantly affects gelation kinetics as well as the stiffness and viscoelastic properties of resulting hydrogels.

LppA is a novel plasminogen receptor of Mycoplasma bovis that contributes to adhesion by binding the host extracellular matrix and Annexin A2

Mycoplasma bovis is responsible for various inflammatory diseases in cattle. The prevention and control of M. bovis are complicated by the absence of effective vaccines and the emergence of multidrug-resistant strains, resulting in substantial economic losses worldwide in the cattle industry. Lipoproteins, vital components of the Mycoplasmas cell membrane, are deemed potent antigens for eliciting immune responses in the host upon infection. However, the functions of lipoproteins in M. bovis remain underexplored due to their low sequence similarity with those of other bacteria and the scarcity of genetic manipulation tools for M. bovis. In this study, the lipoprotein LppA was identified in all examined M. bovis strains. Utilizing immunoelectron microscopy and Western blotting, it was observed that LppA localizes to the surface membrane. Recombinant LppA demonstrated dose-dependent adherence to the membrane of embryonic bovine lung (EBL) cells, and this adhesion was inhibited by anti-LppA serum. In vitro binding assays confirmed LppA’s ability to associate with fibronectin, collagen IV, laminin, vitronectin, plasminogen, and tPA, thereby facilitating the conversion of plasminogen to plasmin. Moreover, LppA was found to bind and enhance the accumulation of Annexin A2 (ANXA2) on the cell membrane. Disrupting LppA in M. bovis significantly diminished the bacterium’s capacity to adhere to EBL cells, underscoring LppA’s function as a bacterial adhesin. In conclusion, LppA emerges as a novel adhesion protein that interacts with multiple host extracellular matrix proteins and ANXA2, playing a crucial role in M. bovis’s adherence to host cells and dissemination. These insights substantially deepen our comprehension of the molecular pathogenesis of M. bovis.

Terpene-Functionalized Fluoroquinolones as Potential Antimicrobials: Synthesis and Properties.

This study was the first to synthesize terpene-containing conjugates of fluoroquinolones, ciprofloxacin and norfloxacin, and to evaluate their antibacterial activity against gram-positive methicillin sensitive (MSSA) and methicillin resistant (MRSA) S. aureus, gram-negative P. aeruginosa as well as antifungal activity against C. albicans. The ability of obtained fluoroquinolones to inhibit S. aureus growth was found to depend upon the presence of a linker separating the bulky terpene and fluoroquinolone fragments, and this activity diminished with increasing its length. The highest activity against MSSA was demonstrated by ciprofloxacin derivatives with campholenic (MIC 1 μg/mL) and 2-(isobornan-2-yl-sulfanyl)acetyl (MIC 0.5 μg/mL) substituents. The compound with the last fragment showed high activity against MRSA (MIC 8 μg/mL). The terpene-functionalized norfloxacin derivatives generally proved to be less active than those containing ciprofloxacin fragment. Camphor-10-sulfonylamide derivative with the ciprofloxacin fragment was the only one of all compounds that showed high antifungal activity against C. albicans (8 μg/mL). The study presents data on docking fluoroquinolones to S. aureus DNA gyrase to explain the reasons for manifestation or disappearance of antibacterial activity. The cytotoxicity of fluoroquinolones that showed any antimicrobial activity was investigated against bovine primary lung cells, and they were found to be not toxic in most cases.

A genome-wide transposon mutagenesis screening identifies LppB as a key factor associated with Mycoplasma bovis colonization and invasion into host cells.

Mycoplasma spp., the smallest self-replicating and genome-reduced organisms, have raised a great concern in both the medical and veterinary fields due to their pathogenicity. The molecular determinants of these wall-less bacterium efficiently use their limited genes to ensure successful infection of the host remain unclear. In the present study, we used the ruminant pathogen Mycoplasma bovis as a model to identify the key factors for colonization and invasion into host cells. We constructed a nonredundant fluorescent transposon mutant library of M. bovis using a modified transposon plasmid, and identified 34 novel adhesion-related genes based on a high-throughput screening approach. Among them, the ΔLppB mutant exhibited the most apparent decrease in adhesion to embryonic bovine lung (EBL) cells. The surface-localized lipoprotein LppB, which is highly conserved in Mycoplasma species, was then confirmed as a key factor for M. bovis adhesion with great immunogenicity. LppB interacted with various components (fibronectin, vitronectin, collagen IV, and laminin) of host extracellular matrix (ECM) and promoted plasminogen activation through tPA to degrade ECM. The 439-502 amino acid region of LppB is a critical domain, and F465 and Y493 are important residues for the plasminogen activation activity. We further revealed LppB as a key factor facilitating internalization through clathrin- and lipid raft-mediated endocytosis, which helps the Mycoplasma invade the host cells. Our study indicates that LppB plays a key role in Mycoplasma infection and is a potential new therapeutic and vaccine target for Mycoplasma species.