Richard P. Darveau

A novel Escherichia coli lipid A mutant that produces an antiinflammatory lipopolysaccharide.

A unique screen was used to identify mutations in Escherichia coli lipid A biosynthesis that result in a decreased ability to stimulate E-selectin expression by human endothelial cells. A mutation was identified in the msbB gene of E. coli that resulted in lipopolysaccharide (LPS) that lacks the myristoyl fatty acid moiety of the lipid A. Unlike all previously reported lipid A mutants, the msbB mutant was not conditionally lethal for growth. Viable cells or purified LPS from an msbB mutant had a 1000-10,000-fold reduction in the ability to stimulate E-selectin production by human endothelial cells and TNF alpha production by adherent monocytes. The cloned msbB gene was able to functionally complement the msbB mutant, restoring both the LPS to its native composition and the ability of the strain to stimulate immune cells. Nonmyristoylated LPS acted as an antagonist for E-selectin expression when mixed with LPS obtained from the parental strain. These studies demonstrate a significant role for the myristate component of LPS in immune cell activation and antagonism. In addition, the msbB mutant allowed us to directly examine the crucial role that the lipid A structure plays when viable bacteria are presented to host defense cells.

Beta-lactam antibiotics potentiate magainin 2 antimicrobial activity in vitro and in vivo.

The ability of magainin 2 to augment antibiotic therapy was examined. Susceptibility to magainin 2 was determined on Escherichia coli incubated in the presence and absence of sublethal concentrations of antibiotics both in vitro and in vivo. Experiments in buffer and normal human serum revealed that E. coli exposed to sublethal amounts of cefepime, a beta-lactam antibiotic, was significantly more susceptible to the antimicrobial activity of magainin 2. Bacteria incubated with subinhibitory concentrations of other beta-lactam type antibiotics, but not amikacin (an aminoglycoside) or ciprofloxacin (a quinolone), were also more susceptible to magainin 2 in normal human serum. Bacteria were less susceptible to magainin 2 when they were examined in heat-inactivated serum. Complement was shown to be required for magainin 2 activity in serum by using C8-deficient sera. The combination of magainin 2 and cefepime was shown to be more antimicrobial in normal human serum for a variety of bacterial strains. Magainin 2 was completely inactive as a therapeutic agent when it was administered alone (2 mg per mouse) but significantly increased the survival of mice when it was administered with a low level of cefepime.

Peptides related to the carboxyl terminus of human platelet factor IV with antibacterial activity.

A peptide (C13) corresponding to the last 13 amino acids of the carboxyl terminus of human platelet factor IV was found to be antibacterial. Amino acid substitutions predicted to disrupt either the amphipathic or alpha-helical nature of C13 rendered the peptide inactive. Antibacterial activity was demonstrated in normal human serum on bacteria which had been previously exposed to low levels of cefepime, a beta-lactam antibiotic. Peptide analogues were examined for more potent antibacterial activity in an antibacterial assay that employed normal human serum and low levels of cefepime. A peptide analogue (C18G) with 80-fold more antibacterial activity than C13 was identified. Studies in C8-deficient sera confirmed an essential role of human serum complement for optimal antibacterial activity. Additional studies showed low levels of cefepime, although not essential, enhanced the antibacterial activity of C18G. Animal protection experiments demonstrated that either peptide C18G or an analogue with all D amino acids (C18X) significantly increased the survival of neutropenic mice when coadministered with a low level of cefepime. This work has resulted in the identification of a new group of antibacterial peptides.

Identification of serum components that inhibit the tumoricidal activity of amphiphilic alpha helical peptides

Antimicrobial peptides that can form amphiphilic alpha helices were tested for their ability to lyse various human tumor cell lines in vitro. These peptides include C18G, whose sequence is a derivative of the carboxyl terminus of human platelet factor IV, and 399, an idealized amphiphilic alpha helix. Both peptides exhibited potent antitumor activity against all cell lines tested, unlike magainin 2, a naturally occurring antimicrobial peptide of similar structure, which was relatively inactive under the same conditions. Also, the lytic activity of C18G is specific for tumor cells versus human red blood cells. The effects of serum can be important when evaluating the potency of lytic peptides, since other tumoricidal peptides have been shown to be completely inactivated by low serum levels. Experiments with C18G and 399 revealed that their activity was indeed reduced in the presence of human serum, but that significant lytic activity remained even at relatively high serum concentrations. Various serum components were tested for their inhibitory activity. Whereas albumin and high-density lipoprotein had only slight inhibitory properties, low-density lipoprotein was found to be a potent inhibitor of peptide-mediated cell lysis. The peptide 399, which is more sensitive to serum inhibition than C18G, also binds more extensively to all serum components tested.