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. 2010 Nov;192(22):6001-16.
doi: 10.1128/JB.00778-10. Epub 2010 Sep 10.

Legionella Pneumophila Strain 130b Possesses a Unique Combination of Type IV Secretion Systems and Novel Dot/Icm Secretion System Effector Proteins

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Legionella Pneumophila Strain 130b Possesses a Unique Combination of Type IV Secretion Systems and Novel Dot/Icm Secretion System Effector Proteins

Gunnar N Schroeder et al. J Bacteriol. .
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Abstract

Legionella pneumophila is a ubiquitous inhabitant of environmental water reservoirs. The bacteria infect a wide variety of protozoa and, after accidental inhalation, human alveolar macrophages, which can lead to severe pneumonia. The capability to thrive in phagocytic hosts is dependent on the Dot/Icm type IV secretion system (T4SS), which translocates multiple effector proteins into the host cell. In this study, we determined the draft genome sequence of L. pneumophila strain 130b (Wadsworth). We found that the 130b genome encodes a unique set of T4SSs, namely, the Dot/Icm T4SS, a Trb-1-like T4SS, and two Lvh T4SS gene clusters. Sequence analysis substantiated that a core set of 107 Dot/Icm T4SS effectors was conserved among the sequenced L. pneumophila strains Philadelphia-1, Lens, Paris, Corby, Alcoy, and 130b. We also identified new effector candidates and validated the translocation of 10 novel Dot/Icm T4SS effectors that are not present in L. pneumophila strain Philadelphia-1. We examined the prevalence of the new effector genes among 87 environmental and clinical L. pneumophila isolates. Five of the new effectors were identified in 34 to 62% of the isolates, while less than 15% of the strains tested positive for the other five genes. Collectively, our data show that the core set of conserved Dot/Icm T4SS effector proteins is supplemented by a variable repertoire of accessory effectors that may partly account for differences in the virulences and prevalences of particular L. pneumophila strains.

Figures

FIG. 1.
FIG. 1.
Circular map of the L. pneumophila 130b draft genome. From the outside in, the green regions in the first circle show the positions of the T4SSs (detailed in Table 3) and the hypervariable region (shown in detail in Fig. 5). The second circle shows the scale in Mbp. The third and fourth circles show the predicted CDSs transcribed clockwise and counterclockwise, respectively. The fifth circle shows known Legionella T4SS effector genes (described in Table 4) and their paralogues (colored red) and new putative effectors (dark blue) (Table 5). The sixth circle shows the 159 contigs of the draft genome, colored brown and orange alternately, and circle 7 shows the 4 scaffolds that link 145 of these contigs (scaffold 1, light green; scaffold 2, light blue; scaffold 3, light gray; scaffold 4, light pink). Circle 8 shows a plot of the GC content (percent), and the innermost circle is a plot of GC deviation (G − C/G + C).
FIG. 2.
FIG. 2.
Phylogeny of Legionella showing the phylogenetic relationship of L. pneumophila 130b to the other sequenced L. pneumophila strains, Corby, Paris, Lens, and Philadelphia-1, with L. longbeachae NSW150 as an outgroup. The tree was built using 26 housekeeping genes (frr, infC, nusA, pgk, rplA, rplB, rplC, rplD, rplE, rplK, rplL, rplM, rplN, rplP, rplS, rplT, rpmA, rpoB, rpsB, rpsC, rpsE, rpsI, rpsK, rpsM, smpB, and tsf) that were found in single copies in all six genomes. The numbers next to the branches show percentages of support from a bootstrap analysis of 500 replicates. The tree shows a highly supported (99.8%) grouping of Lens and 130b. The scale bar represents the number of substitutions per site.
FIG. 3.
FIG. 3.
Organization of the Dot/Icm T4SSB (A), Lvh1 T4SSA (B), and Trb1-like T4SSA (C) loci on the L. pneumophila 130b chromosome. The color coding represents shared gene names.
FIG. 4.
FIG. 4.
Organization of the putative genomic-island-associated T4SS gene clusters LGI-1 and LGI-2 on the L. pneumophila 130b chromosome. The color coding shows the sequence and predicted structural homology of the encoded proteins to T4SS-associated proteins, conjugative-transfer/integrative-element-associated proteins, and the Legionella Lvr proteins or the presence of transmembrane domains and/or signal peptides that are conserved between the two clusters (blue). The scheme illustrates the high degree of organizational conservation of the two clusters. Plus and minus indicate that the clusters are on opposite strands of the chromosome.
FIG. 5.
FIG. 5.
A region of high genome variability in L. pneumophila encodes several new and known Dot/Icm T4SS effectors. Shown is a genome comparison of the five sequenced L. pneumophila genomes (130b, Lens, Philadelphia-1, Corby, and Paris). The CDSs for each genome are shown (130b lpw_19681 to lpw_20471, Lens lpl1893 to lpl1966, Philadelphia-1 lpg1930 to lpg1990, Corby lpc1384 to lpc1473, and Paris lpp1904 to lpp1971). For 130b, CDSs encoding homologues of known effectors are colored red (from left to right, lem15, lem16, lem17, ralF, legC4, lirAB, pieCDEFG, and setA), new effectors are dark blue (ltpG and ltpH), and transposases are black. tRNA-Phe is represented by an orange bar. Regions with significant nucleotide similarity between the genomes are linked by shading (the percent identity [BLASTn] is indicated on the right). The scale bar indicates genome length. The genome comparison shows that the previously defined hypervariable effector region (98) (brown CDSs) and plasticity island of effectors (72) (pink CDSs) partially overlap (gray CDSs) and can be significantly extended.
FIG. 6.
FIG. 6.
L. pneumophila 130b translocates 10 novel Dot/Icm T4SS effector proteins into Raw264.7 macrophages. L. pneumophila 130b wild type (black bars) or the T4S-deficient L. pneumophila 130b ΔDotA mutant (white bars) harboring pXDC61 TEM1-effector gene constructs were grown for 21 h in the presence of 0.5 mM IPTG to induce expression of the TEM1-effector fusion proteins and used to infect macrophages (MOI, 40) (A) or grown for 16 h, and expression of TEM1-effector fusions was induced 5 h prior to and parallel to the infection (1 mM IPTG; MOI, 40) (B). The translocation of the TEM1 fusions of the proven Dot/Icm T4SS effector LegC2 (positive control), the new putative effectors, and the negative control Fab1 was measured using a Fluostar Optima plate reader (410-nm [10-nm band-pass] excitation and 450-nm and 520-nm emission filters). The translocation rate is expressed for each sample in relation to the emission ratio of uninfected cells. The fusion proteins of LegC2 and the Legionella translocated proteins LtpA to LtpJ, but not the negative control (Fab1) or the effector candidates Lpw_21901 and Lpw_28221, were translocated into host cells. The error bars represent standard deviations (SD). Similar results were obtained in three independent experiments.
FIG. 7.
FIG. 7.
Distribution of the 10 novel Dot/Icm T4SS effectors in 87 L. pneumophila isolates. Genomic DNA from 87 clinical and environmental L. pneumophila isolates was analyzed for the presence of homologues of the novel L. pneumophila 130b effectors ltpA to ltpJ by PCR. The dotA gene was included in the screen as a positive control for the presence of the Dot/Icm T4SSB. The new effectors fall in two classes. Class 1 is formed by the rare effectors ltpB, ltpC, ltpE, ltpF, and ltpJ, which were detected in less than 15% of the analyzed strains. Class 2 contains the more prevalent effectors ltpA, ltpD, ltpG, ltpH, and ltpI, which were found in 34% to 62% of the isolates.

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