Citation Details

Journal Article

Anti-Bacterial Agents; Bioware; Blotting, Southern; Chromatography, Thin Layer; Computational Biology; Cytochromes c; Genetic Complementation Test; Humans; Microscopy, Electron, Scanning; Microscopy, Immunoelectron; Mutagenesis; Peptides; Phospholipids; Polymerase Chain Reaction; Staphylococcus aureus; Xen36

Antimicrobial peptides (AMPs) form an important part of the innate host defense. In contrast to most AMPs, human dermcidin has an anionic net charge. To investigate whether bacteria have developed specific mechanisms of resistance to dermcidin, we screened for mutants of the leading human pathogen, Staphylococcus aureus, with altered resistance to dermcidin. To that end, we constructed a plasmid for use in mariner-based transposon mutagenesis and developed a high-throughput cell viability screening method based on luminescence. In a large screen, we did not find mutants with strongly increased susceptibility to dermcidin, indicating that S. aureus has no specific mechanism of resistance to this AMP. Furthermore, we detected a mutation in a gene of unknown function that resulted in significantly increased resistance to dermcidin. The mutant strain had an altered membrane phospholipid pattern and showed decreased binding of dermcidin to the bacterial surface, indicating that dermcidin interacts with membrane phospholipids. The mode of this interaction was direct, as shown by assays of dermcidin binding to phospholipid preparations, and specific, as the resistance to other AMPs was not affected. Our findings indicate that dermcidin has an exceptional value for the human innate host defense and lend support to the idea that it evolved to evade bacterial resistance mechanisms targeted at the cationic character of most AMPs. Moreover, they suggest that the antimicrobial activity of dermcidin is dependent on the interaction with the bacterial membrane and might thus assist with the determination of the yet unknown mode of action of this important human AMP.

Journal Article

Animals; Bioware; Chronic Disease; Disease Models, Animal; Immunity, Innate; Killer Cells, Natural; Macrophages; Mice; Mice, Inbred C3H; Mice, Inbred Strains; Monocytes; Neoplasms; Neovascularization, Pathologic; Osteomyelitis; Osteosarcoma; Staphylococcal Infections; Xen36

Clinical studies over the past several years have reported that metastasis-free survival times in humans and dogs with osteosarcoma are significantly increased in patients that develop chronic bacterial osteomyelitis at their surgical site. However, the immunological mechanism by which osteomyelitis may suppress tumor growth has not been investigated. Therefore, we used a mouse model of osteomyelitis to assess the effects of bone infection on innate immunity and tumor growth. A chronic Staphylococcal osteomyelitis model was established in C3H mice and the effects of infection on tumor growth of syngeneic DLM8 osteosarcoma were assessed. The effects of infection on tumor angiogenesis and innate immunity, including NK cell and monocyte responses, were assessed. We found that osteomyelitis significantly inhibited the growth of tumors in mice, and that the effect was independent of the infecting bacterial type, tumor type, or mouse strain. Depletion of NK cells or monocytes reversed the antitumor activity elicited by infection. Moreover, infected mice had a significant increase in circulating monocytes and numbers of tumor associated macrophages. Infection suppressed tumor angiogenesis but did not affect the numbers of circulating endothelial cells. Therefore, we concluded that chronic localized bacterial infection could elicit significant systemic antitumor activity dependent on NK cells and macrophages.

Journal Article

Acetates; Alginates; Animals; Anti-Infective Agents; Bandages; Bioware; Chitosan; Glucuronic Acid; Hexuronic Acids; Male; Mice; Mice, Inbred BALB C; Occlusive Dressings; Proteus mirabilis; Pseudomonas aeruginosa; Silver Sulfadiazine; Staphylococcus aureus; Wound Healing; Wound Infection; Xen8.1, Xen5, Xen44

HemCon bandage is an engineered chitosan acetate preparation used as a hemostatic control dressing, and its chemical structure suggests that it should also be antimicrobial. We tested its ability to rapidly kill bacteria in vitro and in mouse models of infected wounds. We used the Gram-negative species Pseudomonas aeruginosa and Proteus mirabilis and the Gram-positive Staphylococcus aureus that had all been stably transduced with the entire bacterial lux operon to allow in vivo bioluminescence imaging. An excisional wound in Balb/c mice was inoculated with 50-250 million cells followed after 30 min by application of HemCon bandage, alginate sponge bandage, silver sulfadiazine cream or no treatment. HemCon was more adhesive to the wound and conformed well to the injury compared to alginate. Animal survival was followed over 15 days with observations of bioluminescence emission and animal activity daily. Chitosan acetate treated mice infected with P. aeruginosa and P. mirabilis all survived while those receiving no treatment, alginate and silver sulfadiazine demonstrated 25-100% mortality. Chitosan acetate was much more effective than other treatments in rapidly reducing bioluminescence in the wound consistent with its rapid bactericidal activity in vitro as well as its light-scattering properties. S. aureus formed only non-lethal localized infections after temporary immunosuppression of the mice but HemCon was again more effective in reducing bioluminescence. The data suggest that chitosan acetate rapidly kills bacteria in the wound before systemic invasion can take place, and is superior to alginate bandage and silver sulfadiazine that may both encourage bacterial growth in the short term.

Journal Article

Aging; Animals; Antigens, CD36; Cell Line; Dimerization; Ethylnitrosourea; Gene Deletion; Glycerides; Homozygote; Humans; Immunologic Deficiency Syndromes; Lipopeptides; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutagenesis; Mutation; Oligopeptides; Peptidoglycan; Phenotype; Receptors, Cell Surface; Signal Transduction; Staphylococcal Infections; Staphylococcus aureus; Toll-Like Receptor 2; Toll-Like Receptors; Tumor Necrosis Factor-alpha; Zymosan

Toll-like receptor 2 (TLR2) is required for the recognition of numerous molecular components of bacteria, fungi and protozoa. The breadth of the ligand repertoire seems unusual, even if one considers that TLR2 may form heteromers with TLRs 1 and 6 (ref. 12), and it is likely that additional proteins serve as adapters for TLR2 activation. Here we show that an N-ethyl-N-nitrosourea-induced nonsense mutation of Cd36 (oblivious) causes a recessive immunodeficiency phenotype in which macrophages are insensitive to the R-enantiomer of MALP-2 (a diacylated bacterial lipopeptide) and to lipoteichoic acid. Homozygous mice are hypersusceptible to Staphylococcus aureus infection. Cd36(obl) macrophages readily detect S-MALP-2, PAM(2)CSK(4), PAM(3)CSK(4) and zymosan, revealing that some--but not all--TLR2 ligands are dependent on CD36. Already known as a receptor for endogenous molecules, CD36 is also a selective and nonredundant sensor of microbial diacylglycerides that signal via the TLR2/6 heterodimer.

Journal Article

Abscess; Acetamides; Animals; Anti-Bacterial Agents; Bioware; Catheterization; Colony Count, Microbial; Dose-Response Relationship, Drug; Female; Foreign Bodies; Luminescent Measurements; Mice; Mice, Inbred BALB C; Muscle, Skeletal; Oxazolidinones; Staphylococcal Infections; Staphylococcus aureus; Thigh; Time Factors; Wound Infection; Xen8.1

Staphylococcal infections associated with catheter and prosthetic implants are difficult to eradicate and often lead to chronic infections. Development of novel antibacterial therapies requires simple, reliable, and relevant models for infection. Using bioluminescent Staphylococcus aureus, we have adapted the existing foreign-body and deep-wound mouse models of staphylococcal infection to allow real-time monitoring of the bacterial colonization of catheters or tissues. This approach also enables kinetic measurements of bacterial growth and clearance in each infected animal. Persistence of infection was observed throughout the course of the study until termination of the experiment at day 16 in a deep-wound model and day 21 in the foreign-body model, providing sufficient time to test the effects of antibacterial compounds. The usefulness of both animal models was assessed by using linezolid as a test compound and comparing bioluminescent measurements to bacterial counts. In the foreign-body model, a three-dose antibiotic regimen (2, 5, and 24 h after infection) resulted in a decrease in both luminescence and bacterial counts recovered from the implant compared to those of the mock-treated infected mice. In addition, linezolid treatment prevented the formation of subcutaneous abscesses, although it did not completely resolve the infection. In the thigh model, the same treatment regimen resulted in complete resolution of the luminescent signal, which correlated with clearance of the bacteria from the thighs.