Identification of the aspartate-beta-semialdehyde dehydrogenase gene of Legionella pneumophila and characterization of a null mutant

Abstract

The ability of Legionella pneumophila to cause Legionnaires' disease is dependent on its capacity to survive in the intracellular environment of its host cells. Furthermore, outbreaks of this disease have been associated with contaminated water sources where L. pneumophila survives as a parasite of protozoa. In this study, we determined the effect of nutritional auxotrophy on the ability of L. pneumophila to survive in the intracellular environment of its host cells. We generated a diaminopimelic acid (DAP) auxotroph (AA400) of L. pneumophila by disruption of the aspartate-beta-semialdehyde (asd) gene. The ability of AA400 to survive within macrophages and protozoa was found to be defective. This defect was due solely to the asd disruption since complementation of the mutant with the wild-type asd gene restored its capacity for intracellular survival. Furthermore, the defect was not completely complemented by DAP supplementation to the culture media. Thus, our results suggest that disruption of the asd gene may prove to be useful in the design of attenuated vaccines against Legionnaires' disease.

FIG. 1

(A) Restriction map of the L. pneumophila 4.3-kb fragment containing the asd gene. The relative location of a mini-Tn10::kan insertion in the asd gene is indicated. (B) Southern hybridization of DNA from wild-type L. pneumophila (AA100), Δasd L. pneumophila (AA400), and plasmid pOHBOC1K harboring the 4.3-kb L. pneumophila fragment with the mini-Tn10::kan insertion. All DNA samples were digested with PstI and probed with pOH1 harboring the 4.3-kb L. pneumophila fragment. The 1.8-kb increase in the size of the band in pOHBOC1K and AA400 corresponds to the size of the kan cassette insertion.

FIG. 2

DNA sequence of the L. pneumophila asd gene. The start codon is indicated in bold.

FIG. 3

(A) In vitro growth kinetics of AA400 in BYE medium. Cell density was determined at an optical density of 550 nm (OD 550). (B) AA400 grown in BYE medium in the absence of DAP. Error bars cannot be seen due to their small values.

FIG. 4

Cytopathogenicity of L. pneumophila AA400 (Δasd), AA400^c, and AA100 (wild type) to U937 macrophage-like cells. Error bars cannot be seen due to their small values.

FIG. 5

(A and B) Intracellular growth kinetics of L. pneumophila AA400 (Δasd), AA400^c, and AA100 (wild type). ○, AA400 plus 40 μg of DAP per ml; □, AA100; ◊, AA400^c; ▵, AA400, no DAP. (C and D) Comparison of intracellular survival of AA400 complemented with the PCR-generated asd (AA400^cc) to that of AA100 and AA400^c. CFU levels were determined by plating dilutions from each time point on BCYE agar. Some error bars cannot be seen due to their small values. □, AA100; ◊, AA400^cc; ○, AA400^c.