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Mannheimia haemolytica leukotoxin binds to lipid rafts in bovine lymphoblastoid cells and is internalized in a dynamin-2- and clathrin-dependent manner.

Department of Pathobiological Sciences, University of Wisconsin, 2015, Linden Drive, West, Madison, WI 53706, USA.
Infection and Immunity (Impact Factor: 4.16). 11/2007; 75(10):4719-27. DOI: 10.1128/IAI.00534-07
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ABSTRACT Mannheimia haemolytica is the principal bacterial pathogen of the bovine respiratory disease complex. Its most important virulence factor is a leukotoxin (LKT), which is a member of the RTX family of exotoxins produced by many gram-negative bacteria. Previous studies demonstrated that LKT binds to the beta(2)-integrin LFA-1 (CD11a/CD18) on bovine leukocytes, resulting in cell death. In this study, we demonstrated that depletion of lipid rafts significantly decreases LKT-induced bovine lymphoblastoid cell (BL-3) death. After binding to BL-3 cells, some of the LKT relocated to lipid rafts in an LFA-1-independent manner. We hypothesized that after binding to LFA-1 on BL-3 cells, LKT moves to lipid rafts and clathrin-coated pits via a dynamic process that results in LKT internalization and cytotoxicity. Knocking down dynamin-2 by small interfering RNA reduced both LKT internalization and cytotoxicity. Similarly, expression of dominant negative Eps15 protein expression, which is required for clathrin coat formation, reduced LKT internalization and LKT-mediated cytotoxicity to BL-3 cells. Finally, we demonstrated that inhibiting actin polymerization reduced both LKT internalization and LKT-mediated cytotoxicity. These results suggest that both lipid rafts and clathrin-mediated mechanisms are important for LKT internalization and cytotoxicity in BL-3 cells and illustrate the complex nature of LKT internalization by the cytoskeletal network.

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    • "Additionally, LPS binds to and stabilizes Lkt, preventing its degradation and augmenting its activity (Jeyaseelan et al., 2002). Lkt, a calcium-dependent cytotoxin, is a member of the RTX family of toxins produced by many Gram negative bacteria (Atapattu & Czuprynski, 2007; Baluyut et al., 1981; Clinkenbeard et al., 1989; Dassanayake et al., 2007a; Dassanayake et al., 2008; Deshpande et al., 2002; Zecchinon et al., 2004; Zecchinon et al., 2005). Lkt activity is responsible for much of the gross and histopathologic tissue damage seen with M. haemolytica and B. trehalosi infections (Clinkenbeard et al., 1989; Dassanayake et al., 2007b; Jeyaseelan et al., 2002; Leite et al., 2005; Zecchinon et al., 2005). "
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    ABSTRACT: Bacterial pneumonia is a common and often life-threatening respiratory problem in both meat and dairy goats. Options for approved antibiotic therapy in goats to combat these bacterial infections are severely limited and frequently drugs must be used in an extra-label manner. Tulathromycin, a triamilide macrolide antimicrobial drug shown to be effective against swine and cattle respiratory bacterial agents, has been identified as a potentially useful drug in caprines. The present study was conducted to determine the susceptibility of recognized bacterial respiratory pathogens to commonly prescribed antimicrobials, with a particular emphasis on the efficacy of tulathromycin against these agents. Minimum inhibitory concentration (MIC) testing using microbroth dilution was performed on a collection of 45 Mannheimia haemolytica, 11 Pasteurella multocida, and 11 Bibersteinia trehalosi isolates from the lungs of goats with clinical pneumonia. To further characterize efficacy of tulathromycin against these pathogens, minimum bactericidal concentration (MBC) testing and kinetic killing assays were conducted. Most isolates were susceptible to the antimicrobials tested; however, increased resistance as demonstrated by higher MIC values was seen in all species to penicillin, in P. multocida to sulfadimethoxine, and in B. trehalosi to the tetracyclines. All isolates were susceptible to tulathromycin, which demonstrated a high killing efficiency in both bactericidal assays. Results of this study indicate that most goat pneumonic bacterial pathogens remain susceptible to commonly prescribed antibiotics, although some evidence of resistance was seen to certain drugs; and that tulathromycin is highly effective against goat respiratory pathogens which could make it a valuable medication in this species.
    Veterinary Microbiology 10/2011; 156(1-2):178-82. DOI:10.1016/j.vetmic.2011.10.025 · 2.73 Impact Factor
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    • "Acylation, especially covalent linking of saturated fatty acids, represents a targeting signal for many proteins that interact with membrane microdomains [17]. The requirement of lipid microdomains for the cytotoxity induced by various RTX toxins, particularly leukotoxins from Mannheimia haemolytica and Actinobacillus actinomycetemcomitans, has been pointed out in the last few years [18], [19]. Binding of proteins to lipid rafts may result in internalisation of such proteins into cells. "
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    ABSTRACT: Bordetella pertussis, the bacterium that causes whooping cough, secretes an adenylate cyclase toxin (ACT) that must be post-translationally palmitoylated in the bacterium cytosol to be active. The toxin targets phagocytes expressing the CD11b/CD18 integrin receptor. It delivers a catalytic adenylate cyclase domain into the target cell cytosol producing a rapid increase of intracellular cAMP concentration that suppresses bactericidal functions of the phagocyte. ACT also induces calcium fluxes into target cells. Biochemical, biophysical and cell biology approaches have been applied here to show evidence that ACT and integrin molecules, along with other raft components, are rapidly internalized by the macrophages in a toxin-induced calcium rise-dependent process. The toxin-triggered internalisation events occur through two different routes of entry, chlorpromazine-sensitive receptor-mediated endocytosis and clathrin-independent internalisation, maybe acting in parallel. ACT locates into raft-like domains, and is internalised, also in cells devoid of receptor. Altogether our results suggest that adenylate cyclase toxin, and maybe other homologous pathogenic toxins from the RTX (Repeats in Toxin) family to which ACT belongs, may be endowed with an intrinsic capacity to, directly and efficiently, insert into raft-like domains, promoting there its multiple activities. One direct consequence of the integrin removal from the cell surface of the macrophages is the hampering of their adhesion ability, a fundamental property in the immune response of the leukocytes that could be instrumental in the pathogenesis of Bordetella pertussis.
    PLoS ONE 02/2011; 6(2):e17383. DOI:10.1371/journal.pone.0017383 · 3.23 Impact Factor
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    • "Additionally, LPS binds to and stabilizes Lkt, preventing its degradation and augmenting its activity (Jeyaseelan et al., 2002). Lkt, a calcium-dependent cytotoxin, is a member of the RTX family of toxins produced by many Gram negative bacteria (Atapattu & Czuprynski, 2007; Baluyut et al., 1981; Clinkenbeard et al., 1989; Dassanayake et al., 2007a; Dassanayake et al., 2008; Deshpande et al., 2002; Zecchinon et al., 2004; Zecchinon et al., 2005). Lkt activity is responsible for much of the gross and histopathologic tissue damage seen with M. haemolytica and B. trehalosi infections (Clinkenbeard et al., 1989; Dassanayake et al., 2007b; Jeyaseelan et al., 2002; Leite et al., 2005; Zecchinon et al., 2005). "
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    ABSTRACT: The number of antimicrobials available to treat bacterial respiratory disease in goats is extremely limited. Currently, only ceftiofur has been approved for use in this species. Concerns over the development of antimicrobial resistance in bacteria that infect humans have led to proposed restrictions on the use of this drug in food-producing animals. Tulathromycin, a triamilide macrolide antibiotic found to be safe and effective against respiratory bacterial pathogens in cattle and swine, was formulated to enhance persistence in lung tissue. The Minor Use Animal Drug Program (National Research Support Project-7) identifies and supports research directed at meeting the Food and Drug Administration (FDA) requirements for drug approval in minor species or for minor uses in major species. The current work was undertaken to support FDA approval of tulathromycin in the caprine species by providing an assessment of target animal drug safety, drug tissue elimination, and drug efficacy. Tulathromycin had no detrimental clinical effects, even in animals treated with 5X the proposed label dose for three times the proposed label duration. Tissue elimination and pharmacokinetic behavior paralleled that found in both cattle and swine. Efficacy analysis was conducted on the clinical effect and pharmacokinetic behavior of tulathromycin in goats as well as in vitro antimicrobial effects on bacterial isolates recovered from goats. Bacteria isolated from goats with clinical cases of pneumonia were highly susceptible to this drug in several in vitro assays. Overall, this research demonstrated that tulathromycin would be a safe, effective, and valuable medication in the treatment of bacterial pneumonia in goats.
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