The dps gene of symbiotic "Candidatus Legionella jeonii" in Amoeba proteus responds to hydrogen peroxide and phagocytosis

Abstract

To survive in host cells, intracellular pathogens or symbiotic bacteria require protective mechanisms to overcome the oxidative stress generated by phagocytic activities of the host. By genomic library tagging, we cloned a dps (stands for DNA-binding protein from starved cells) gene of the symbiotic "Candidatus Legionella jeonii" organism (called the X bacterium) (dps(X)) that grows in Amoeba proteus. The gene encodes a 17-kDa protein (pI 5.19) with 91% homology to Dps and DNA-binding ferritin-like proteins of other organisms. The cloned gene complemented the dps mutant of Escherichia coli and conferred resistance to hydrogen peroxide. Dps(X) proteins purified from E. coli transformed with the dps(X) gene were in oligomeric form, formed a complex with pBlueskript SKII DNA, and protected the DNA from DNase I digestion and H(2)O(2)-mediated damage. The expression of the dps(X) gene in "Candidatus Legionella jeonii" was enhanced when the host amoeba was treated with 2 mM H(2)O(2) and by phagocytic activities of the host cell. These results suggested that the Dps protein has a function protective of the bacterial DNA and that its gene expression responds to oxidative stress generated by phagocytic activities of the host cell. With regard to the fact that invasion of Legionella sp. into respiratory phagocytic cells causes pneumonia in mammals, further characterization of dps(X) expression in the Legionella sp. that multiplies in a protozoan host in the natural environment may provide valuable information toward understanding the protective mechanisms of intracellular pathogens.

FIG. 1.

Alignment of the deduced amino acid sequence of the Dps_X protein with those of other organisms. Asterisks indicate identical residues in all compared Dps proteins, periods show conserved residues, and dashes show gaps inserted for an optimal alignment of amino acids. The amino acids for the DNA-binding signature are in bold (7). Positively charged amino acids at the N or C terminus reported to be involved in DNA binding are underlined (7). Amino acids of Dps_X that have been mutated are shown in bold and underlined. Dps proteins are from Bacillus anthracis (Dlp1 and Dlp2), Listeria innocua (FLis), Streptococcus mutans (SmDps), Helicobacter pylori (NAP), Agrobacterium tumefaciens (AtDps), Escherichia coli (EcDps), Mycobacterium smegmatis (MsDps), Legionella pneumophila (LpDps), and “Ca. Legionella jeonii” (Dps_X).

FIG. 2.

Phylogenetic relationships between Dps_X (AAT09106) of “Ca. Legionella jeonii” and its homologues of other bacterial species. Tree relationships were achieved using the neighbor-joining method, and the scale bar denotes 10 substitutions per 100 amino acids. The resultant phylogenetic tree was generated using the Treeview program, version 1.61. Ferritin (Ftn), bacterioferritin (Bft), and neutrophil-activating protein (NAP) are Dps homologues. The database accession numbers are shown with species names.

FIG. 3.

The dps_X gene complements the dps::kan mutant of E. coli in the assay of numbers of CFU (A) and colony patches (B). The presence of the dps_X gene increases the survival of the dps::kan mutant strain, but the pBluescript SKII vector alone has no effect on survival. The wild-type strain was ZK126 (E. coli K-12); the dps::kan mutant was ZK1058 (E. coli K-12). Error bars represent the standard deviations based on three experiments.

FIG. 4.

Purification of His-tagged Dps_X and E. coli Dps proteins. An SDS-polyacrylamide gel (10%) was stained with BCB, and Western blotting (WB) with antiserum against His-tagged Dps_X protein was performed. Lanes: M, molecular mass markers (masses in kilodaltons appear at the left); 1, crude extract for Dps_X; 2, purified Dps_X; 3, crude extract for E. coli Dps; 4, purified E. coli Dps. The arrow and the arrowhead indicate the positions of His-tagged E. coli Dps (23 kDa) and Dps_X (21 kDa), respectively.

FIG. 5.

Comparisons of oligomeric properties of His-tagged Dps_X and E. coli Dps in vitro. Purified His-tagged Dps_X and E. coli Dps proteins were analyzed by nondenaturing gel (6%) electrophoresis and stained with BCB and polyclonal antiserum against Dps_X protein (Western blotting [WB]). Lanes: 1, BSA (66 kDa, pI 4.8); 2, horse spleen ferritin (450 kDa, pI 4.5); 3, His-tagged Dps_X; 4, His-tagged E. coli Dps. Masses in kilodaltons appear at the left. Bands I, II, and III represent two putative lower-number oligomers (I and II) and one higher-number oligomer (III) of Dps_X. The arrow and arrowhead indicate the putative low- and high-number oligomers of E. coli Dps, respectively.

FIG. 6.

Comparisons of His-tagged Dps_X and E. coli Dps in DNA binding and protection of DNA. (A) Formation of protein-DNA complexes. The pBluescript SKII DNA incubated with proteins at 30°C for 30 min was analyzed for gel retardation in 1% agarose gel and stained with ethidium bromide. Lanes: 1, pBluescript SKII DNA alone; 2 to 6, pBluescript SKII DNA incubated with BSA, Dps_X, and E. coli Dps, respectively. (B) Protection of DNA from DNase I digestion. The pBluescript SKII DNA (1 μg) was incubated with proteins at 30°C for 30 min and then treated with 1 U DNase I for 5 min. (C) Protection of DNA from H₂O₂-mediated damage. The pBluescript SKII DNA (1 μg) was incubated with proteins at 30°C for 30 min and then processed for the Fenton reaction. The DNA-to-protein molar ratios were 1:1,000 for additions marked “a” and 1:200 for additions marked “b.”

FIG. 7.

Effects of 2 mM H₂O₂ on the expression of the dps, groEL, and dnaK genes from “Ca. Legionella jeonii” of xD amoebae. Total RNA was extracted from xD amoebae treated with 2 mM H₂O₂ and analyzed by RT-PCR using primers specific to the dps_X, groEL_X, dnaK_X, and 16S rRNA_X genes of the symbiotic “Ca. Legionella jeonii” organism. The PCR products were analyzed by electrophoresis on a 1% agarose gel, and the band density was read by a Kodak model 440CF image station (Kodak Digital Science, NY) for comparisons of the levels of gene expression to that of 16S rRNA. The bars represent the means ± standard deviations from three experiments.

FIG. 8.

Effects of the phagocytosis of the host on the expression of the dps, groEL, and dnaK genes from “Ca. Legionella jeonii” of xD amoebae. Total RNA was extracted from xD amoebae at time intervals after feeding with Tetrahymena organisms, and gene expression was analyzed by RT-PCR using specific primers for the dps_X, groEL_X, dnaK_X, and 16S rRNA_X genes. The PCR products were analyzed by electrophoresis in a 1% agarose gel, and the relative levels of gene expression were the same as those shown in Fig. 7.