c Acinetobacter baumannii, a Gram-negative multidrug-resistant (MDR) bacterium, is now recognized as one of the more common nosocomial pathogens. Because most clinical isolates are found to be multidrug resistant, alternative therapies need to be developed to control this pathogen. We constructed a bacteriophage genomic library based on prophages induced from 13 A. baumannii strains and screened it for genes encoding bacteriolytic activity. Using this approach, we identified 21 distinct lysins with different activities and sequence diversity that were capable of killing A. baumannii. The lysin (PlyF307) displaying the greatest activity was further characterized and was shown to efficiently kill (>5-log-unit decrease) all tested A. baumannii clinical isolates. Treatment with PlyF307 was able to significantly reduce planktonic and biofilm A. baumannii both in vitro and in vivo. Finally, PlyF307 rescued mice from lethal A. baumannii bacteremia and as such represents the first highly active therapeutic lysin specific for Gram-negative organisms in an array of native lysins found in Acinetobacter phage. Members of Acinetobacter are soil bacteria that frequently colonize the human skin without harm (1). However, in environments in which individuals are immunocompromised or suffer from a variety of wounds (e.g., in hospital settings or on battlefields), Acinetobacter baumannii can cause severe life-threatening infections (2-4). Symptoms of A. baumannii infections range from mild skin wounds and urinary tract infections to more severe conditions, including pneumonia, meningitis, and sepsis (5). A. baumannii is now one of the most common causes of hospital-acquired pneumonia (2) and sepsis; while not common (only 1.3% of all sepsis cases), it is associated with mortality rates of up to 58% (6).One of the main threats from A. baumannii is the high rate of resistance to antibiotics commonly used to treat Gram-negative infections. More than 80% of Acinetobacter species are considered to be multidrug resistant (MDR) (i.e., resistant to at least three classes of antibiotics), resulting in infections with poor clinical outcomes, including high rates of morbidity and death, prolonged hospital stays, and substantial health care expenses (3, 7). In addition, several strains of pan-drug-resistant A. baumannii have been isolated, showing resistance to a wide variety of clinically used antibiotics (8). A. baumannii is also capable of surviving treatments with detergents and disinfectants, dehydration, and UV radiation and thus is difficult to eradicate from surfaces in hospital environments (9, 10). The organism not only is intrinsically resistant to many antibiotics (owing to ␤-lactamases, weak membrane permeability, and efficient efflux systems) but also can readily acquire foreign plasmids and is considered to have a high degree of genetic plasticity (11). Outbreaks caused by MDR Acinetobacter have been reported from hospitals worldwide; more recently, they have become a serious problem in military medical facilities (4). One of ...

Chemokines, like stromal cell-derived factor-1 (SDF1/CXCL12), are small secreted proteins that signal cells to migrate. Because SDF1 and its receptor CXCR4 play important roles in embryonic development, cancer metastasis, and HIV/AIDS, this chemokine signaling system is the subject of intense study. However, it is not known whether the monomeric or dimeric structure of SDF1 is responsible for signaling in vivo. Previous structural studies portrayed the SDF1 structure as either strictly monomeric in solution or dimeric when crystallized. Here, we report two-dimensional NMR, pulsed-field gradient diffusion and fluorescence polarization measurements at various SDF1 concentrations, solution conditions, and pH. These results demonstrate that SDF1 can form a dimeric structure in solution, but only at nonacidic pH when stabilizing counterions are present. Thus, while the previous NMR structural studies were performed under acidic conditions that strongly promote the monomeric state, crystallographic studies used nonacidic buffer conditions that included divalent anions shown here to promote dimerization. This pH-sensitive aggregation behavior is explained by a dense cluster of positively charged residues at the SDF1 dimer interface that includes a histidine side chain at its center. A heparin disaccharide shifts the SDF1 monomer-dimer equilibrium in the same manner as other stabilizing anions, suggesting that glycosaminoglycan binding may be coupled to SDF1 dimerization in vivo.Keywords: chemokines; heparin; NMR; fluorescence polarization; monomer-dimer equilibrium Chemokines are small, secreted proteins that induce cell migration through activation of G protein-coupled receptors (GPCR), and also bind extracellular matrix glycosaminoglycans (GAG) in order to direct chemotaxis along a gradient of increasing chemokine concentration. Chemokines are categorized into four families, C, CC, CXC, and CX 3 C, based on the arrangement of conserved cysteine residues near the amino terminus. The well-characterized chemokine tertiary fold consists of a flexible amino terminus, followed by a three-stranded anti-parallel ␤-sheet and a carboxyl terminal ␣-helix. Chemokine quaternary structure is more varied; some chemokines are constitutively monomeric, whereas others self-associate to form homodimers mediated by residues of the amino terminus (CC chemokines) or the first ␤-strand (CXC chemokines). Dimerization was initially thought to be an artifact of the high concentrations necessary for structural studies, since chemokines are fully functional in chemotaxis and calcium flux assays at low nanomolar concentrations where the monomeric species should predominate (Rajarathnam et al. 1994(Rajarathnam et al. , 1995Paavola et al. 1998). Moreover, disruption of the IL-8 dimer through mutagenesis does not alter its properties as a receptor agonist in vitro (Rajarathnam et al. 1994(Rajarathnam et al. , 1995. In contrast, a recent study showed that dimerization is critical for the in vivo leukocyte recruitment activity of three dimeri...

Chemokines adopt a conserved tertiary structure stabilized by two disulfide bridges and direct the migration of leukocytes. Lymphotactin (Ltn) is a unique chemokine in that it contains only one disulfide and exhibits large-scale structural heterogeneity. Under physiological solution conditions (37°C, 150 mM NaCl) Ltn is in equilibrium between the canonical chemokine fold (Ltn10) and a distinct 4-stranded β-sheet (Ltn40). Consequently, it has not been possible to address the biological significance of each structural species independently. To stabilize the Ltn10 structure independent of specific solution conditions, Ltn variants containing a second disulfide bridge were designed. Placement of the new cysteines was based on a sequence alignment of Ltn with either the first (Ltn-CC1) or third disulfide (Ltn-CC3) in the CC chemokine, HCC-2. NMR data demonstrate that both CC1 and CC3 retain the Ltn10 chemokine structure and no longer exhibit structural rearrangement. The ability of each mutant to activate the Ltn receptor, XCR1, has been tested using an intracellular Ca 2+ flux assay. These data support the conclusion that the chemokine fold of Ltn10 is responsible for receptor activation. We also examined the role of amino-and carboxyl-terminal residues in Ltnmediated receptor activation. In contrast to previous reports, we find that the 25 residues comprising the novel C-terminal extension do not participate in receptor activation, while the native N-terminus is absolutely required for Ltn function.Chemokines are small, secreted proteins that signal leukocytes to migrate during an immune response. Binding of chemokines to G-protein coupled receptors (GPCRs) 1 stimulates cell chemotaxis, while diffusion and glycosaminoglycan (GAG) binding are believed to establish the chemokine gradient that migrating cells follow. Approximately 50 chemokines have been identified and are classified into four subfamilies (CXC, CC, CX 3 C, and C) based on the spacing of two cysteine residues near the N-terminus, each of which contributes to a pair of conserved disulfide bonds (1).Lymphotactin (Ltn), the only example of a C-type chemokine, lacks the first and third cysteine residues found in all other chemokines and contains a unique extended C-terminal sequence that is conserved across species (2). We have previously shown that under physiological conditions (37 °C, 150 mM NaCl), Ltn exhibits reversible conformational heterogeneity, converting between two distinct structural species. The relative population of the two conformations is highly dependent on the temperature and ionic strength of the solution (3).*Address correspondence to: bvolkman@mcw.edu, . ‖ Present Address: The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399.1 The abbreviations used are: GCPR, G-coupled protein receptor; GAG, glycosaminoglycan; Ltn, lymphotactin; MALDI-MS, matrixassisted laser desorption ionization mass spectrometry; CNBr, cyanogen bromide; RANTES, regulated on activation normal T cell expressed and secreted; HCC-2, human che...

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