Inhibition of LpxC Protects Mice from Resistant Acinetobacter baumannii by Modulating Inflammation and Enhancing Phagocytosis

Abstract

New treatments are needed for extensively drug-resistant (XDR) Gram-negative bacilli (GNB), such as Acinetobacter baumannii. Toll-like receptor 4 (TLR4) was previously reported to enhance bacterial clearance of GNB, including A. baumannii. However, here we have shown that 100% of wild-type mice versus 0% of TLR4-deficient mice died of septic shock due to A. baumannii infection, despite having similar tissue bacterial burdens. The strain lipopolysaccharide (LPS) content and TLR4 activation by extracted LPS did not correlate with in vivo virulence, nor did colistin resistance due to LPS phosphoethanolamine modification. However, more-virulent strains shed more LPS during growth than less-virulent strains, resulting in enhanced TLR4 activation. Due to the role of LPS in A. baumannii virulence, an LpxC inhibitor (which affects lipid A biosynthesis) antibiotic was tested. The LpxC inhibitor did not inhibit growth of the bacterium (MIC > 512 µg/ml) but suppressed A. baumannii LPS-mediated activation of TLR4. Treatment of infected mice with the LpxC inhibitor enhanced clearance of the bacteria by enhancing opsonophagocytic killing, reduced serum LPS concentrations and inflammation, and completely protected the mice from lethal infection. These results identify a previously unappreciated potential for the new class of LpxC inhibitor antibiotics to treat XDR A. baumannii infections. Furthermore, they have far-reaching implications for pathogenesis and treatment of infections caused by GNB and for the discovery of novel antibiotics not detected by standard in vitro screens.

Severe Acinetobacter baumannii Sepsis Is Associated with Elevation of Pentraxin 3

“…Studies have attributed septic shock associated with A. baumannii infections to bacterial LPS and outer membrane shedding in secreted outer membrane vesicles by the bacterium (14,44,45). However, data presented here suggest that this may not be the case, but rather, elevated PTX3 production induced by select A. baumannii strains appears to correspond with increased susceptibility of mice to infection.…”

“…A similar association between elevated PTX3 production and disease severity has been observed following both Gram-positive and Gram-negative sepsis in human patients (29)(30)(31)(32)(33)35); however, at this time we cannot discern if the effects observed here are due in part to other entities, e.g., inflammatory molecules. Interestingly, although studies examining A. baumannii bloodstream infections involving hypercoagulability and DIC have attributed these symptoms to bacterial LPS and outer membrane shedding (14,44,45), data presented here indicate that disease severity was dependent on interaction of live bacteria with the immune system, as evidenced by the complete lack of mortality in mice challenged with UV-killed or HK bacteria administered at high inocula (Fig. 2).…”

“…Traditionally, bacterial sepsis models used for study of A. baumannii utilize porcine mucin to enhance virulence of the bacterium through inhibition of phagocytosis (13,43,44). Consequently, LD 50 values as low as 10 3 to 10 4 CFU have been reported, with time to death (TTD) near 1 week postchallenge (13,14). However, given that published data (18,24,26,27) indicate that PTX3 opsonizes the bacteria to promote bacterial phagocytosis, contributing to clearance, we chose not to use porcine mucin in the bacterial preparation for our sepsis model.…”

“…Consequently, mortality rates range from 30 to 75% depending on the route of infection (8). Although specific virulence factors (lipopolysaccharide [LPS] and membrane glycosylation) and a robust cellular innate immune response (neutrophil infiltration) contribute to disease severity and clearance during A. baumannii sepsis, respectively, very little is known about differences in virulence or mortality between strains and the overall protective host immune response necessary for protection against A. baumannii infection (9)(10)(11)(12)(13)(14)(15).…”

Effects of silver nanoparticles in combination with antibiotics on the resistant bacteria <em>Acinetobacter baumannii</em>

“…Lin et al found that an LpxC inhibitor blocked the ability of bacteria to activate the sepsis cascade, enhanced opsonophagocytic killing of A. baumannii, and protected mice from lethal infection. 38 Moreover, the potential contributions of PMB and AgNPs both involve Lipid A of LPS, a convergence which supports their synergistic effects.…”