Legionella Pneumophila Transcriptome during Intracellular Multiplication in Human Macrophages

Abstract

Legionella pneumophila is the causative agent of Legionnaires' disease, an acute pulmonary infection. L. pneumophila is able to infect and multiply in both phagocytic protozoa, such as Acanthamoeba castellanii, and mammalian professional phagocytes. The best-known L. pneumophila virulence determinant is the Icm/Dot type IVB secretion system, which is used to translocate more than 150 effector proteins into host cells. While the transcriptional response of Legionella to the intracellular environment of A. castellanii has been investigated, much less is known about the Legionella transcriptional response inside human macrophages. In this study, the transcriptome of L. pneumophila was monitored during exponential and post-exponential phase in rich AYE broth as well as during infection of human cultured macrophages. This was accomplished with microarrays and an RNA amplification procedure called selective capture of transcribed sequences to detect small amounts of mRNA from low numbers of intracellular bacteria. Among the genes induced intracellularly are those involved in amino acid biosynthetic pathways leading to l-arginine, l-histidine, and l-proline as well as many transport systems involved in amino acid and iron uptake. Genes involved in catabolism of glycerol are also induced during intracellular growth, suggesting that glycerol could be used as a carbon source. The genes encoding the Icm/Dot system are not differentially expressed inside cells compared to control bacteria grown in rich broth, but the genes encoding several translocated effectors are strongly induced. Moreover, we used the transcriptome data to predict previously unrecognized Icm/Dot effector genes based on their expression pattern and confirmed translocation for three candidates. This study provides a comprehensive view of how L. pneumophila responds to the human macrophage intracellular environment.

Keywords: Icm/Dot effectors; SCOTS; THP-1; iron; microarray.

Figure 1

Kinetics of THP-1 macrophage infection by Legionella pneumophila. (A) Ten million human cultured THP-1 macrophages were infected with L. pneumophila at a MOI of 1. At each time point, cells were washed three times and a fraction was resuspended in distilled water to release intracellular bacteria. Shown is the number of colony forming units (CFU) present inside the totality of cells. (B) CFU was normalized against the number of CFU at T0 to show change in intracellular multiplication.

Figure 2

Effects of SCOTS on the cDNA population. (A) Southern blot of L. pneumophila gDNA digested with HincII and hybridized with labeled cDNA from the T0 time point obtained before (lane 1) and after the first (lane 2), the second (lane 3), and the third (lane 4) round of SCOTS. Lane 5 was hybridized with labeled rDNA. (B) qPCR was used to validate the expression profiles obtained by microarray for eight genes in all the conditions. (C) Comparison of microarray data obtained when using the SCOTS amplification method and when using a standard microarray protocol. Shown is the normalized signal intensity obtained from E phase growth in rich AYE broth. See text for details.

Figure 3

Analysis of the normalized signal intensities for each condition reveals similarity between in vivo conditions. (A) Hierarchical clustering of the normalized signal intensity of each replicate for each condition. The red line marks a cluster of genes highly expressed in all conditions tested. (B) The Bioconductor package was used to generate paired correlation matrix showing the degree of similarity between conditions. The correlation value is displayed in the lower right part of each graph. The x and y axis represent the median of the log2 transform of the normalized signal intensity. (C) Normalized signal intensities for genes encoding proteins of the Icm/Dot Type IVB secretion system. Putative location of each gene products is shown on the left side of the annotation: OM, outer membrane; P, periplasm; IM, inner membrane; C, cytoplasm. (D) Normalized signal intensities for genes encoding known and putative regulators.

Figure 4

Genes differentially expressed compared to the E phase control. (A) Hierarchical clustering of the relative expression of L. pneumophila genes during intracellular multiplication in macrophages and during PE phase in rich AYE broth compared to exponential growth in broth. Only genes with significant change in expression (−2 > log2 > 2, P < 0.001) in at least one condition are shown. (B) The number of genes positively or negatively affected during intracellular growth compared to E phase is displayed in Venn diagrams. (C) Genes with significant changes in expression were grouped according to their published COG class. The percentage of genes that change positively (red line) or negatively (green line) during intracellular growth, compared to the E phase control, is shown for selected COG classes. Refer to Figure 5 for the results of other COG classes.

Figure 5

Fraction of genes in orthologous groups deferentially expressed during infection. Number of genes significantly induced (red bar) or repressed (green bar) intracellularly at T0 (A), T6 (B), and T18 (C) compared to the E phase control.

Figure 6

Some metabolic pathways are induced during intracellular growth. The synthesis pathways were drawn according to the Biocyc database (http://biocyc.org). A series of four circles next to each gene display its relative expression value compared to the E phase control. NA, There is no known or predicted gene mediating this reaction in L. pneumophila; *glycolysis genes are not differentially expressed.

Figure 7

Relative expression of genes involved in various functions. Heat map of the expression ratio to E phase of (A) genes involved in iron acquisition, (B) known virulence factors other than the Icm/Dot system, (C) genes encoding regulators, and (D) genes encoding proteins involved in defense mechanisms against oxidative stress and antimicrobial peptides.

Figure 8

Icm/Dot effectors are differentially expressed inside human cells. (A) Hierarchical clustering of the normalized signal intensity of the genes encoding Icm/Dot effectors for each replicate and each condition. (B) Hierarchical clustering of the expression ratio of the genes encoding Icm/Dot effectors compared to the E phase control. Only genes with significant change in expression (−2 > log2 > 2, P < 0.001) in at least one condition are shown.

Figure 9

Identification of new Icm/Dot effectors. (A) Translocation of the TEM-effector fusions leads to the cleavage of CCF4/AM. Translocation was determined for each TEM-effector fusion by measuring the ratio of cleaved (460 nm) to uncleaved (530 nm) CCF4/AM in wild type KS79 or KS79 dotA (Type IV secretion deficient). FabI serves as negative control; RalF and LepA are known Legionella effector and serves as positive control. The number of biological replicates analyzed is shown. (B) Immunoblot on whole cell lysate using an anti-TEM rabbit polyclonal antibody showing expression of the TEM-effector fusions. Moleular weight of fusion proteins: TEM-FabI: 59.3 kDa, TEM-RalF: 73 kDa, TEM-LepA: 161.9 kDa, TEM-lpg1959: 106 kDa, TEM-lpg1961: 88 kDa, TEM-lpg2827: 66.7 kDa, TEM-lpg2828: 78.5 kDa.

Figure 10

Identification of genes differentially expressed compared to the T0 control. (A) Hierarchical clustering of the transcriptome of L. pneumophila during intracellular multiplication in THP-1 macrophages compared to intracellular growth at T0. PE phase compared to E phase is shown as a reference. Only genes with significant change in expression (−2 > log2 > 2, P < 0.001) in at least one condition are shown. (B) The number of genes positively or negatively affected during intracellular growth compared to T0 is displayed in Venn diagrams. (C) Heat map of the Icm/Dot effectors differentially expressed compared to T0. PE phase compared to E phase is shown as a reference.