Legionella dumoffii DjlA, a member of the DnaJ family, is required for intracellular growth

Abstract

Legionella dumoffii is one of the common causes of Legionnaires' disease and is capable of replicating in macrophages. To understand the mechanism of survival within macrophages, transposon mutagenesis was employed to isolate the genes necessary for intracellular growth. We identified four defective mutants after screening 790 transposon insertion mutants. Two transposon insertions were in genes homologous to icmB or dotC, within dot/icm loci, required for intracellular multiplication of L. pneumophila. The third was in a gene whose product is homologous to the 17-kDa antigen forming part of the VirB/VirD4 type IV secretion system of Bartonella henselae. The fourth was in the djlA (for "dnaj-like A") gene. DjlA is a member of the DnaJ/Hsp40 family. Transcomplementation of the djlA mutant restored the parental phenotype in J774 macrophages, A549 human alveolar epithelial cells, and the amoeba Acanthamoeba culbertsoni. Using confocal laser-scanning microscopy and transmission electron microscopy, we revealed that in contrast to the wild-type strain, L. dumoffii djlA mutant-containing phagosomes were unable to inhibit phagosome-lysosome fusion. Transmission electron microscopy also showed that in contrast to the virulent parental strain, the djlA mutant was not able to recruit host cell rough endoplasmic reticulum. Furthermore, the stationary-phase L. dumoffii djlA mutants were more susceptible to H2O2, high osmolarity, high temperature, and low pH than was their parental strain. These results indicate that DjlA is required for intracellular growth and organelle trafficking, as well as bacterial resistance to environmental stress. This is the first report demonstrating that a single DjlA-deficient mutant exhibits a distinct phenotype.

FIG. 1.

Intracellular growth of L. dumoffii strains within J774 mouse macrophages (A) and A549 human epithelial cells (B). The formation of colonies (CFU per milliliter) was determined at the times indicated, in triplicate, for at least two independent experiments. Error bars indicate the standard deviations determined from samples taken from one experiment. Symbols: ○, L. dumoffii wild-type strain; ▪, HOLD254; □, HOLD491; •, HMLD4001; ▵, HMLD4002.

FIG. 2.

Complementation of intracellular growth defects of djlA mutant HOLD254 in J774 macrophages (A), in A549 epithelial cells (B), and in A. culbertsoni (C). Growth was measured over 72 h (A and B) or 48 h (C). The data points and error bars represent the mean CFU/well for triplicate samples from a typical experiment (performed at least twice) and their standard deviations. Symbols: ○, L. dumoffii wild-type strain; ▪, HOLD254; ▵, HOLD254-1 (djlA/pHRO18); □, HOLD254-2 (djlA/pHRO25).

FIG. 3.

Chromosomal arrangement of the region surrounding the djlA gene and sequence alignment of DjlA proteins. (A) At the top is a plasmid used for complementation studies (pHRO18) and an 8-kb region of the L. dumoffii cosmid clone including the djlA gene, along with the location of relevant restriction enzyme sites. The thick line represents the DNA region that we sequenced. Below these diagrams, the distance between the djlA gene and neighboring genes and the orientation and size of the transcribed genes are delineated by the arrows below the 4.2-kb sequenced region. Another plasmid used for complementation studies (pHRO25) is also shown. The site of the Tn903dIIlacZ insertion (Tn) is indicated by the inverted arrowhead. The full names of the gene mapped are as follows: waaA, Kdo transferase gene; djlA, dnaJ-like A gene; plaA, lysophospholipase A gene. Orf1 is a putative open reading frame which showed no homology to known genes. (B) Sequence similarity of the predicted Dj1A protein of L. dumoffii (L.d, top line), L. pneumophila (L.p, middle line) and E. coli (E.c, bottom line). Amino acid residues conserved in the three sequences, appear in bold type. Gaps marked by dashes are introduced to reveal the maximal similarity among the sequences. The C-terminal J-domain and the N-terminal TMD are shown schematically above the sequences.

FIG. 4.

Colocalization of the intracellular growth mutant with late endosomal/lysosomal marker LAMP-1 or LAMP-2 in J774 mouse macrophage cells by confocal laser-scanning microscopy. J774 macrophages were incubated with the L. dumoffii mutant or wild-type strain for 4 h. (A) Late endosomes and lysosomes stained with rat monoclonal antibody 1D4B, specific for LAMP-1, and Cy3-labeled anti-rat secondary antibody (red) are shown on the left. Bacteria stained with rabbit polyclonal antibody specific for L. dumoffii Tex-KL and Alexa488-labeled anti-rabbit secondary antibody (green) are shown in the middle. Merged images showing LAMP-1-positive bacteria (yellow) and LAMP-1-negative bacteria (green) are shown on the right. (B) Data were collected from about 100 intracellular bacteria in total. The percentage that is LAMP-1 or LAMP-2 positive was calculated by dividing the number of colocalizing intracellular bacteria by the total number of intracellular bacteria scored. The average and standard deviation described here were calculated from three coverslips per strain in two independent experiments.

FIG. 5.

Distribution of a lysosomal marker, BSA-gold, in phagosomes containing the wild-type strain or the djlA mutant strain. To label the lysosomal compartment, J774 cells were incubated with 15-nm BSA-gold overnight, washed, and then chased for 3 h. Cells were then infected with wild-type strain (A) or djlA mutant strain (B). At 4 h postinfection, the cells were fixed and processed for electron microscopy. Arrows in panel A indicate phagosomes containing no detectable gold; arrowheads in panel B indicate phagolysosomes containing BSA-gold; asterisks indicate, lysosomes containing BSA-gold. Bar, 0.5 μm.

FIG. 6.

Transmission electron micrographs of J774 mouse macrophages infected by the wild-type L. dumoffii (A) and the djlA mutant HOLD254 (B) at 8 h after infection. (A) Wild-type L. dumoffii-containing phagosomes were surrounded by RER (arrows). (B) HOLD254-containing phagosomes appeared to harbor much debris resulting from fusing lysosomes. Bar, 1.0 μm.

FIG. 7.

Sensitivities of in vitro-grown stationary-phase wild-type L. dumoffii (open bars), the djlA mutant strain (hatched bars), and the djlA complemented strain (solid bars) to oxidative stress, osmotic stress, acid stress, and heat shock (10 mM hydrogen peroxide for 30 min, 5 M sodium chloride for 30 min, pH 3 for 5 min, and 48°C for 60 min, respectively). Stationary-phase cultures were exposed to each stress as described in Materials and Methods. The percentage of viable bacteria was calculated by dividing the CFU obtained from plating the bacteria onto BCYE agar plates following exposure to the indicated stress by the CFU of the bacteria obtained from plating the bacteria onto BCYE agar plates prior to exposure to the stress and multiplying by 100. Experiments were performed at least three times, and the results represent the mean and standard deviation. Results were analyzed for significance by analysis of variance and by a two-tailed, unpaired t test. Asterisks indicate significant differences between the djlA mutant and two other strains. (*, P < 0.01; **, P < 0.001).