Sensitization of Gram-Negative Bacilli to Host Antibacterial Proteins

Abstract

To address the need for novel alternatives to antibiotics, we attempted to sensitize gram-negative bacilli to innate antibacterial protagonists. We report a lipopeptide-like sequence (C10OOc12O) that inflicted outer membrane damage at a low micromolar range, whereas measurable bacterial growth inhibition in broth medium required >10-fold higher concentrations. In serum, however, C10OOc12O induced antibacterial activity in a manner suppressible by anticomplement antibodies or heat treatment and acted synergistically with exogenous lysozyme in broth and serum media. Upon subcutaneous administration, C10OOc12O exhibited high circulating levels that correlated with significant therapeutic efficacies, using either the mouse peritonitis-sepsis model or the thigh infection model. These findings are consistent with the view that, by damaging the outer membrane, C10OOc12O was able to enhance gram-negative bacilli susceptibility to antibacterial components of the immune humoral arm. Such lipopeptides may therefore be useful in fighting gram-negative bacilli threats through sensitization to endogenous and/or exogenous antibacterial proteins such as lysozyme and complements.

Eliciting improved antibacterial efficacy of host proteins in the presence of antibiotics

“…MICs were determined using the microdilution assay, as described in Jammal et al (29). Briefly, bacteria were grown overnight and were adjusted to 10 6 colony-forming units (CFU)/ml.…”

“…The lipopeptide's postulated aptitude to alter the OM structure at subinhibitory concentrations (29) was revisited here by using various complementing methodologies. First we attempted to visualize the alleged membrane damage resulting from E. coli exposure to C 10 OOc 12 O in PBS at concentrations likely to be achieved in mouse blood at nontoxic doses [i.e., at least 10 mM (29)] by using electron microscopy to compare the contour of untreated and treated bacteria.…”

“…In contrast, two distinct biochemical assays support the notion that C 10 OOc 12 O significantly altered the E. coli outermost permeability barrier. As previously reported (29), we have used the E. coli mutant strain ML-35p (37) to monitor the selective permeation of GNB outer and/or cytoplasmic membrane (CM). That study provided initial evidence for the lipopeptide's ability to damage both membranes, although asymmetrically (i.e., some CM permeabilization occurred only at .10 mM) (29).…”

“…As previously reported (29), we have used the E. coli mutant strain ML-35p (37) to monitor the selective permeation of GNB outer and/or cytoplasmic membrane (CM). That study provided initial evidence for the lipopeptide's ability to damage both membranes, although asymmetrically (i.e., some CM permeabilization occurred only at .10 mM) (29). To confirm the occurrence of such damage specifically in the ATCC strain 25922 used in animal studies, we now used the OM-impermeable hydrophobic fluorescent dye NPN that, upon OM disruption, becomes able to bind the CM, thereby enhancing the fluorescence emission intensity.…”

“…Using a synthetic library of cationic MACs (26,27), we previously generated a lipopeptide-like short sequence (decanoyl-dilysyl-aminododecanoyl-lysylamide, C 10 KKc 12 K) (28) that sensitized gram-negative bacilli (GNB) to various antibiotics in correlation with mild membrane damage. More recently we proposed that an ornithyl-based analog (C 10 OOc 12 O) with enhanced bioavailability can sensitize GNB to host plasma immune factors (29). In the present work, we follow on these studies by further challenging the lipopeptide approach, asking whether the newly gained bioavailability of C 10 OOc 12 O can further improve the therapeutic outcome in combination with ineffective antibiotics.…”