Resistance of pathogens to antimicrobial therapeutics has become a widespread problem. Resistance can emerge naturally, but it can also be engineered intentionally, which is an important consideration in designing therapeutics for bioterrorism agents. Blocking host receptors used by pathogens represents a powerful strategy to overcome this problem, because extensive alterations to the pathogen may be required to enable it to switch to a new receptor that can still support pathogenesis. Here, we demonstrate a facile method for producing potent receptor-directed antitoxins. We used phage display to identify a peptide that binds both anthraxtoxin receptors and attached this peptide to a synthetic scaffold. Polyvalency increased the potency of these peptides by >50,000-fold in vitro and enabled the neutralization of anthrax toxin in vivo. This work demonstrates a receptor-directed anthrax-toxin inhibitor and represents a promising strategy to combat a variety of viral and bacterial diseases.antimicrobial resistance ͉ phage display ͉ therapeutics P athogens can develop resistance to drugs directed against microbial targets by modifying the drug, by lowering the concentration of drug that reaches the target, or by mutating the target (1, 2). There is also an increasing concern that therapeutics developed for bioterrorism agents may be rendered ineffective if the microbial target is altered intentionally. This problem could be overcome, however, by designing inhibitors that block host proteins used by the pathogen or its toxins to cause disease.Microbial pathogens and their products interact with host structures to facilitate colonization or to promote cellular uptake. Many of these interactions are polyvalent, meaning that they involve the simultaneous binding of multiple ligands on one entity to multiple receptors on another (3). The design of synthetic polyvalent (4-8) or oligovalent (9, 10) molecules also represents a promising approach to enhance the potency of inhibitors of microbial pathogens and toxins. Current examples of this approach have involved the design of molecules that bind directly to the pathogen or toxin. Inhibitors that bind host proteins would represent an effective way to attenuate virulence that may be less susceptible to resistance mechanisms, and the use of polyvalency could provide a significant enhancement in the potency of these inhibitors.ANTXR1 and ANTXR2 are host receptors that bind and internalize anthrax toxin (11,12). These proteins are likely important for anthrax pathogenesis because the toxin impairs the immune response and is responsible for the major symptoms and death associated with anthrax. Thus, blocking these receptors could represent a promising approach to anthrax therapy.ANTXR1 and ANTXR2 are widely expressed type I membrane proteins that bind components of the extracellular matrix (13). They both contain an extracellular I domain, which binds the protective antigen (PA) component of anthrax toxin. The two proteins are 40% identical overall and share 60% identity within ...

Hsp10 (10-kDa heat shock protein, also known as chaperonin 10 or Cpn10) is a co-chaperone for Hsp60 in the protein folding process. This protein has also been shown to be identical to the early pregnancy factor, which is an immunosuppressive growth factor found in maternal serum. In this study we have used immunogold electron microscopy to study the subcellular localization of Hsp10 in rat tissues sections embedded in LR Gold resin employing polyclonal antibodies raised against different regions of human Hsp10. In all rat tissues examined including liver, heart, pancreas, kidney, anterior pituitary, salivary gland, thyroid, and adrenal gland, antibodies to Hsp10 showed strong labeling of mitochondria. However, in a number of tissues, in addition to the mitochondrial labeling, strong and highly specific labeling with the Hsp10 antibodies was also observed in several extramitochondrial compartments. These sites included zymogen granules in pancreatic acinar cells, growth hormone granules in anterior pituitary, and secretory granules in PP pancreatic islet cells. Additionally, the mature red blood cells which lack mitochondria, also showed strong reactivity with the Hsp10 antibodies. The observed labeling with the Hsp10 antibodies, both within mitochondria as well as in other compartments/cells, was abolished upon omission of the primary antibodies or upon preadsorption of the primary antibodies with the purified recombinant human Hsp10. These results provide evidence that similar to a number of other recently described mitochondrial proteins (viz., Hsp60, tumor necrosis factor receptor-associated protein-1, P32 (gC1q-R) protein, and cytochrome c), Hsp10 is also found at a variety of specific extramitochondrial sites in normal rat tissue. These results raise important questions as to how these mitochondrial proteins are translocated to other compartments and their possible function(s) at these sites. The presence of these proteins at extramitochondrial sites in normal tissues has important implications concerning the role of mitochondria in apoptosis and genetic diseases.

We have identified an optimized peptide inhibitor that can be used to develop potent anthrax toxin therapeutics. Anthrax toxin, an essential virulence factor of Bacillus anthracis, elicits many of the symptoms associated with the disease, and is responsible for death. The toxin is composed of a cell-binding component, protective antigen, and two enzymatic components, edema factor and lethal factor. The three proteins are secreted individually by the bacterium and then assemble into functional complexes on the surface of mammalian cells. These complexes are endocytosed, and the enzymatic components are translocated into the cytosol, where they exert their activities. We screened a phage display library for peptides that can bind the heptameric cell-binding subunit of anthrax toxin, and identified a novel peptide that can block toxin assembly. We made a series of mutant peptides and attached these peptides to polymer backbones to assess their inhibitory activities in vitro. This series of truncated peptide mutants was used to identify a minimal peptide sequence, TYWWLD, that can be used to develop potent polyvalent inhibitors of anthrax toxin.

Cytochrome c oxidase (COX) complex is an integral part of the electron transport chain. Three subunits of this complex (COX I, COX II and COX III) are encoded by mitochondrial (mit-) DNA. High-resolution immunogold electron microscopy has been used to study the subcellular localization of COX I and COX II in rat tissue sections, embedded in LR Gold resin, using monoclonal antibodies for these proteins. Immunofluorescence labeling of BS-C-1 monkey kidney cells with these antibodies showed characteristic mitochondrial labeling. In immunogold labeling studies, the COX I and COX II antibodies showed strong and specific mitochondrial labeling in the liver, kidney, heart and pancreas. However, in rat pancreatic acinar tissue, in addition to mitochondrial labeling, strong and specific labeling was also observed in the zymogen granules (ZGs). In the anterior pituitary, strong labeling with these antibodies was seen in the growth hormone secretory granules. In contrast to these compartments, the COX I or COX II antibodies showed only minimal labeling (five- to tenfold lower) of the cytoplasm, endoplasmic reticulum and the nucleus. Strong labeling with the COX I or COX II antibodies was also observed in highly purified ZGs from bovine pancreas. The observed labeling, in all cases, was completely abolished upon omission of the primary antibodies. These results provide evidence that, similar to a number of other recently studied mit-proteins, COX I and COX II are also present outside the mitochondria. The presence of mit-DNA encoded COX I and COX II in extramitochondrial compartments, provides strong evidence that proteins can exit, or are exported, from the mitochondria. Although the mechanisms responsible for protein exit/export remain to be elucidated, these results raise fundamental questions concerning the roles of mitochondria and mitochondrial proteins in diverse cellular processes in different compartments.

Despite the extensive structural characterization of glycosphingolipids (GSLs), their functions in cell physiology and pathobiology remain elusive. This is largely owing to the fact that they are difficult to handle, being insoluble in aqueous media, and that no one gene alone determines their synthesis. The heterogeneity of the lipid moiety provides a further confounding factor. GSLs are central components within lipid rafts, which are major foci for transmembrane signaling and interactions between eukaryotic cells and microbial pathogens. GSL receptor function often requires the lipid moiety, and lipid-free sugar analogs are ineffective inhibitors. In order to overcome some of these problems, we have synthesized adamantyl GSL analogs which, in part, mimic GSL membrane receptor function in solution. These compounds are made by replacing the endogenous fatty acid with an adamantan frame. This rigid hydrophobic structure surprisingly increases the water solubility of the conjugate and retains receptor function. These GSL mimics provide probes to study GSL receptor function within cells. They compete with native GSLs for ligand binding and are taken up by cells to potentially alter GSL-mediated interaction. We are focused on two derivatives, adamantyl globotriaosyl ceramide and adamantyl sulfogalactosyl ceramide, and have used these analogs to probe GSL function in microbial pathology and hsp70 function. This chapter describes the syntheses and uses of these mimics.

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