Essential Role for the Legionella pneumophila Rep Helicase Homologue in Intracellular Infection of Mammalian Cells

Abstract

We have previously isolated 32 mutants of Legionella pneumophila that are defective in the infection of mammalian cells but not protozoa. The mutated loci have been designated macrophage-specific infectivity (mil) loci. In this study we characterized the mil mutant GK11. This mutant was incapable of growth within U937 macrophage-like cells and WI-26 alveolar epithelial cells. This defect in intracellular replication correlated with a defect in cytopathogenicity to these cells. Sequence analysis of the GK11 locus revealed it to be highly similar to rep helicase genes of other bacteria. Since helicase mutants of Escherichia coli are hypersensitive to thymine starvation, we examined the sensitivity of GK11 to thymineless death (TLD). In the absence of thymine and thymidine, mutant GK11 did not undergo TLD but was defective for in vitro growth, and the defect was partially restored when these compounds were added to the growth medium. In addition, supplementation with thymidine or thymine partially restored the ability of GK11 to grow within and kill U937 macrophage-like cells. The data suggested that the low levels of thymine or thymidine in the L. pneumophila phagosome contributed to the defect of GK11 within macrophages. Using confocal laser scanning microscopy, we determined the effect of the mutation in the Rep helicase homologue on the intracellular trafficking of GK11 within macrophages. In contrast to the wild-type strain, phagosomes harboring GK11 colocalized with several late endosomal/lysosomal markers, including LAMP-1, LAMP-2, and cathepsin D. In addition, only 50% of the GK11 phagosomes colocalized with the endoplasmic reticulum marker BiP 4 h postinfection. Colocalization of BiP with GK11 phagosomes was absent 6 h postinfection, while 90% of the wild-type phagosomes colocalized with this marker at both time points. We propose that the low level of thymine within the L. pneumophila phagosome in combination with simultaneous exposure to multiple stress stimuli results in deleterious mutations that cannot be repaired in the rep helicase homologue mutant, rendering it defective in intracellular replication.

HtrA Homologue of Legionella pneumophila : an Indispensable Element for Intracellular Infection of Mammalian but Not Protozoan Cells

“…Our data favor the prediction that it is more likely that L. pneumophila is exposed to a significantly lesser degree of stress stimuli within protozoa than within macrophages. We have recently shown that the Rep helicase of L. pneumophila, which is required for DNA repair following damage by stress stimuli, is also indispensable with mammalian cells but not within protozoa (30). These observations provide compelling evidence that although the intracellular infection is similar at the molecular and ultrastructural levels (3, 13, 26, 41), significant differences exist between the phagosomal microenvironments within protozoan and mammalian host cells.…”

Identification of Legionella pneumophila rcp , a pagP -Like Gene That Confers Resistance to Cationic Antimicrobial Peptides and Promotes Intracellular Infection

“…This suggests adaptation of the rcp mutants to their environment, possibly involving the induction of other CAMP resistance mechanisms. Interestingly, a typical cytopathic effect was not elicited by the mutant; i.e., the extent of cytopathicity did not closely correlate with numbers of recovered CFU (3,32,42,50). Recently, mutants have been characterized which are defective for cytopathicity but have no intracellular growth defect and thus remain trapped inside the macrophage (1).…”

Biofilm-derived Legionella pneumophila evades the innate immune response in macrophages

“…These communities have been identified as a causative source of infection in susceptible hosts who inhale aerosols of contaminated water containing L. pneumophila . In the human lung environment, L. pneumophila replicates exponentially within alveolar macrophages prior to lysing the host cell and invading other macrophages causing a type of walking pneumonia called Legionnaire's disease or Legionellosis (Horwitz and Silverstein, 1980; Harb and Abu Kwaik, 2000). Legionellosis has two clinically distinct forms: Legionnaires' disease, a severe type of infection, which includes pneumonia and Pontiac fever, a milder self-limiting illness (Lau and Ashbolt, 2009).…”