Traumatic wound microbiome workshop.

On May 9-10, 2011, the Walter Reed Army Institute of Research, as the Army Center of Excellence for Infectious Disease, assembled over a dozen leaders in areas related to research into the communities of microorganisms which colonize and infect traumatic wounds. The objectives of the workshop were to obtain guidance for government researchers, to spur research community involvement in the field of traumatic wound research informed by a microbiome perspective, and to spark collaborative efforts serving the Wounded Warriors and similarly wounded civilians. During the discussions, it was made clear that the complexity of these infections will only be met by developing a new art of clinical practice that engages the numerous microbes and their ecology. It requires the support of dedicated laboratories and technologists who advance research methods such as community sequencing, as well as the kinds of data analysis expertise and facilities. These strategies already appear to be bearing fruit in the clinical management of chronic wounds. There are now funding announcements and programs supporting this area of research open to extramural collaborators.

Potential role of the EPEC translocated intimin receptor (Tir) in host apoptotic events.

Apoptosis, or programmed cell death, is a well-ordered process that allows damaged or diseased cells to be removed from an organism without severe inflammatory reactions. Multiple factors, including microbial infection, can induce programmed death and trigger reactions in both host and microbial cellular pathways. Whereas an ultimate outcome is host cell death, these apoptotic triggering mechanisms may also facilitate microbial spread and prolong infection. To gain a better understanding of the complex events of host cell response to microbial infection, we investigated the molecular role of the microorganism Enteropathogenic Escherichia coli (EPEC) in programmed cell death. We report that wild type strain of EPEC, E2348/69, induced apoptosis in cultured PtK2 and Caco-2 cells, and in contrast, infections by the intracellularly localized Listeria monocytogenes did not. Fractionation and concentration of EPEC-secreted proteins demonstrated that soluble protein factors expressed by the bacteria were capable of inducing the apoptotic events in the absence of organism attachment, suggesting adherence is not required to induce host cell death. Among the known EPEC proteins secreted via the Type III secretion (TTS) system, we identified the translocated intimin receptor (Tir) in the apoptosis-inducing protein sample. In addition, host cell ectopic expression of an EPEC GFP-Tir showed mitochondrial localization of the protein and produced apoptotic effects in transfected cells. Taken together, these results suggest a potential EPEC Tir-mediated role in the apoptotic signaling cascade of infected host cells.

Interaction of the enteropathogenic Escherichia coli protein, translocated intimin receptor (Tir), with focal adhesion proteins.

When enteropathogenic Escherichia coli (EPEC) attach and infect host cells, they induce a cytoskeletal rearrangement and the formation of cytoplasmic columns of actin filaments called pedestals. The attached EPEC and pedestals move over the surface of the host cell in an actin-dependent reaction [Sanger et al., 1996: Cell Motil Cytoskeleton 34:279-287]. The discovery that EPEC inserts the protein, translocated intimin receptor (Tir), into the membrane of host cells, where it binds the EPEC outer membrane protein, intimin [Kenny et al., 1997: Cell 91:511-520], suggests Tir serves two functions: tethering the bacteria to the host cell and providing a direct connection to the host's cytoskeleton. The sequence of Tir predicts a protein of 56.8 kD with three domains separated by two predicted trans-membrane spanning regions. A GST-fusion protein of the N-terminal 233 amino acids of Tir (Tir1) binds to alpha-actinin, talin, and vinculin from cell extracts. GST-Tir1 also coprecipitates purified forms of alpha-actinin, talin, and vinculin while GST alone does not bind these three focal adhesion proteins. Biotinylated probes of these three proteins also bound Tir1 cleaved from GST. Similar associations of alpha-actinin, talin, and vinculin were also detected with the C-terminus of Tir, i.e., Tir3, the last 217 amino acids. Antibody staining of EPEC-infected cultured cells reveals the presence of focal adhesion proteins beneath the attached bacteria. Our experiments support a model in which the cytoplasmic domains of Tir recruit a number of focal adhesion proteins that can bind actin filaments to form pedestals. Since pedestals also contain villin, tropomyosin and myosin II [Sanger et al., 1996: Cell Motil. Cytoskeleton 34:279-287], the pedestals appear to be a novel structure sharing properties of both focal adhesions and microvilli.

Characterization of a new class of DNA delivery complexes formed by the local anesthetic bupivacaine.

Bupivacaine, a local anesthetic and cationic amphiphile, forms stable liposomal-like structures upon direct mixing with plasmid DNA in aqueous solutions. These structures are on the order of 50-70 nm as determined by scanning electron microscopy, and are homogeneous populations as analyzed by density gradient centrifugation. The DNA within these structures is protected from nuclease degradation and UV-induced damage in vitro. Bupivacaine:DNA complexes have a negative zeta potential (surface charge), homogeneous nature, and an ability to rapidly assemble in aqueous solutions. Bupivacaine:DNA complexes, as well as similar complexes of DNA with other local anesthetics, have the potential to be a novel class of DNA delivery agents for gene therapy and DNA vaccines.