SummaryThe facultative intracellular bacterial pathogen Listeria monocytogenes is capable of replicating within a broad range of host cell types and host species. We report here the establishment of the fruit fly Drosophila melanogaster as a new model host for the exploration of L. monocytogenes pathogenesis and host response to infection. Listeria monocytogenes was capable of establishing lethal infections in adult fruit flies and larvae with extensive bacterial replication occurring before host death. Bacteria were found in the cytosol of insect phagocytic cells, and were capable of directing host cell actin polymerization. Bacterial gene products necessary for intracellular replication and cell-to-cell spread within mammalian cells were similarly found to be required within insect cells, and although previous work has suggested that L. monocytogenes virulence gene expression requires temperatures above 30 ∞ ∞ ∞ ∞ C, bacteria within insect cells were found to express virulence determinants at 25 ∞ ∞ ∞ ∞ C. Mutant strains of Drosophila that were compromised for innate immune responses demonstrated increased susceptibility to L. monocytogenes infection. These data indicate L. monocytogenes infection of fruit flies shares numerous features of mammalian infection, and thus that Drosophila has the potential to serve as a genetically tractable host system that will facilitate the analysis of host cellular responses to L. monocytogenes infection.

Despite the wealth of knowledge regarding the mechanisms of action and the mechanisms of resistance to azole antifungals, very little is known about how the azoles are imported into pathogenic fungal cells. Here the in-vitro accumulation and import of Fluconazole (FLC) was examined in the pathogenic fungus, Candida albicans. In energized cells, FLC accumulation correlates inversely with expression of ATP-dependent efflux pumps. In de-energized cells, all strains accumulate FLC, suggesting that FLC import is not ATP-dependent. The kinetics of import in de-energized cells displays saturation kinetics with a Km of 0.64 uM and Vmax of 0.0056 pmol/min/108 cells, demonstrating that FLC import proceeds via facilitated diffusion through a transporter rather than passive diffusion. Other azoles inhibit FLC import on a mole/mole basis, suggesting that all azoles utilize the same facilitated diffusion mechanism. An analysis of related compounds indicates that competition for azole import depends on an aromatic ring and an imidazole or triazole ring together in one molecule. Import of FLC by facilitated diffusion is observed in other fungi, including Cryptococcus neoformans, Saccharomyces cerevisiae, and Candida krusei, indicating that the mechanism of transport is conserved among fungal species. FLC import was shown to vary among Candida albicans resistant clinical isolates, suggesting that altered facilitated diffusion may be a previously uncharacterized mechanism of resistance to azole drugs.

LpxC [UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase] is a metalloamidase that catalyzes the first) identified a series of synthetic LpxC-inhibitory molecules that were bactericidal for Escherichia coli. These molecules did not inhibit the growth of Pseudomonas aeruginosa and were therefore not developed further as antibacterial drugs. The inactivity of the LpxC inhibitors for P. aeruginosa raised the possibility that LpxC activity might not be essential for all gram-negative bacteria. By placing the lpxC gene of P. aeruginosa under tight control of an arabinose-inducible promoter, we demonstrated the essentiality of LpxC activity for P. aeruginosa. It was found that compound L-161,240, the most potent inhibitor from the previous study, was active against a P. aeruginosa construct in which the endogenous lpxC gene was inactivated and in which LpxC activity was supplied by the lpxC gene from E. coli. Conversely, an E. coli construct in which growth was dependent on the P. aeruginosa lpxC gene was resistant to the compound. The differential activities of L-161,240 against the two bacterial species are thus the result primarily of greater potency toward the E. coli enzyme rather than of differences in the intrinsic resistance of the bacteria toward antibacterial compounds due to permeability or efflux. These data validate P. aeruginosa LpxC as a target for novel antibiotic drugs and should help direct the design of inhibitors against clinically important gram-negative bacteria.Lipopolysaccharide has a critical function in gram-negative bacterial membrane integrity and resistance to host defenses, and therefore, the conserved lipopolysaccharide biosynthetic enzymes are attractive targets for novel antibacterial drugs. A drug targeting enzymes of this biosynthetic pathway would need to be active against Pseudomonas aeruginosa and other nonfermenting gram-negative bacterial species, as well as against Escherichia coli and other enteric bacteria, to be clinically useful. The P. aeruginosa outer membrane is less permeable to small molecules than that of E. coli, and P. aeruginosa has several multidrug efflux pumps. As a result of both of these factors, P. aeruginosa is less susceptible than E. coli to many antibiotics (24). Several laboratories have focused on the metalloenzyme LpxC [UDP-(3-O-acyl)-N-acetylglucosamine deacetylase], since it catalyzes the first committed step in lipid A synthesis (Fig. 1) and has been demonstrated to be essential for the growth of E. coli (3,12,38). P. aeruginosa LpxC is similar in sequence (Fig. 2) and catalyzes the same activity (11). While the essentiality of LpxC activity for P. aeruginosa has not been formally proven, the lpxC gene was not inactivated in a saturating transposon mutagenesis study (15). These data suggest that it might be possible to discover LpxC inhibitors active against both E. coli and P. aeruginosa. However, none of the early LpxC inhibitors, some of which showed antibacterial activity against E. coli and certain other organisms, were able to inhibit growth of...