We present the genomic sequence of Legionella pneumophila, the bacterial agent of Legionnaires' disease, a potentially fatal pneumonia acquired from aerosolized contaminated fresh water. The genome includes a 45-kilobase pair element that can exist in chromosomal and episomal forms, selective expansions of important gene families, genes for unexpected metabolic pathways, and previously unknown candidate virulence determinants. We highlight the genes that may account for Legionella's ability to survive in protozoa, mammalian macrophages, and inhospitable environmental niches and that may define new therapeutic targets.

Intracellular pathogens exploit host cell functions to create a replication niche inside eukaryotic cells. The causative agent of Legionnaires' disease, the ␥-proteobacterium Legionella pneumophila, resides and replicates within a modified vacuole of protozoan and mammalian cells. L. pneumophila translocates effector proteins into host cells through the Icm-Dot complex, a specialized type IVB secretion system that is required for intracellular growth. To find out if some effector proteins may have been acquired through interdomain horizontal gene transfer (HGT), we performed a bioinformatic screen that searched for eukaryotic motifs in all open reading frames of the L. pneumophila Philadelphia-1 genome. We found 44 uncharacterized genes with many distinct eukaryotic motifs. Most of these genes contain G؉C biases compared to other L. pneumophila genes, supporting the theory that they were acquired through HGT. Furthermore, we found that several of them are expressed and up-regulated in stationary phase in an RpoS-dependent manner. In addition, at least seven of these gene products are translocated into host cells via the Icm-Dot complex, confirming their role in the intracellular environment. Reminiscent of the case with most Icm-Dot substrates, most of the strains containing mutations in these genes grew comparably to the parent strain intracellularly. Our findings suggest that in L. pneumophila, interdomain HGT may have been a major mechanism for the acquisition of determinants of infection.The ␥-proteobacterium Legionella pneumophila is an opportunistic human pathogen that multiplies within alveolar macrophages and causes the nosocomial and community-acquired pneumonia known as Legionnaires' disease (18,25,48). Human disease occurs when aerosolized L. pneumophila is inhaled from man-made or natural freshwater reservoirs harboring the bacteria. L. pneumophila poses a significant worldwide public health problem, particularly for individuals with compromised immune systems (19,38,40). Eradication of the pathogen from freshwater, industrial settings has proven difficult, since L. pneumophila thrives in environments that exclude antibacterial agents, such as biofilms and the intracellular compartments of protozoa (8,46,47).In order to create a replicative niche inside eukaryotic cells suitable for replication, L. pneumophila is believed to modulate host cell functions by the delivery of effector proteins through a type IVB secretion system known as the Icm-Dot complex (52, 59). Effector proteins presumably regulate several pathways in the host, including up-regulation of phagocytosis (23), delay in phagosome-lysosome fusion (24), recruitment of ARF1 to the phagosome (43), acquisition of endoplasmic reticulum-derived vesicles (29), and nonlytic egress from the host cell (11).Several laboratories found Icm-Dot substrates through genetic screens and bioinformatic approaches (3,7,11,13,36,43,44,57). Most of the known effector proteins are not individually required for intracellular multiplication, since knocking out th...

Legionella pneumophila is the causative agent of the severe and potentially fatal pneumonia Legionnaires' disease. L. pneumophila is able to replicate within macrophages and protozoa by establishing a replicative compartment in a process that requires the Icm/Dot type IVB secretion system. The signals and regulatory pathways required for Legionella infection and intracellular replication are poorly understood. Mutation of the rpoS gene, which encodes S , does not affect growth in rich medium but severely decreases L. pneumophila intracellular multiplication within protozoan hosts. To gain insight into the intracellular multiplication defect of an rpoS mutant, we examined its pattern of gene expression during exponential and postexponential growth. We found that S affects distinct groups of genes that contribute to Legionella intracellular multiplication. We demonstrate that rpoS mutants have a functional Icm/Dot system yet are defective for the expression of many genes encoding Icm/Dot-translocated substrates. We also show that S affects the transcription of the cpxR and pmrA genes, which encode two-component response regulators that directly affect the transcription of Icm/Dot substrates. Our characterization of the L. pneumophila small RNA csrB homologs, rsmY and rsmZ, introduces a link between S and the posttranscriptional regulator CsrA. We analyzed the network of S -controlled genes by mutational analysis of transcriptional regulators affected by S . One of these, encoding the L. pneumophila arginine repressor homolog gene, argR, is required for maximal intracellular growth in amoebae. These data show that S is a key regulator of multiple pathways required for L. pneumophila intracellular multiplication.Legionella pneumophila is a gram-negative opportunistic human pathogen that causes the severe and potentially fatal pneumonia Legionnaires' disease (30,47,67,83). L. pneumophila's ability to replicate within human alveolar macrophages is essential for its capacity to cause disease (44-46). Transmission of L. pneumophila to the human lung occurs as a result of the inhalation of aerosolized contaminated water droplets (74), often from exposure to showers or whirlpool baths (96). Legionella species are ubiquitous in most naturally occurring and man-made aquatic systems, where the organism replicates within a variety of unicellular protozoan hosts (28,38,96). It has been suggested that the interaction of Legionella species with environmental protozoa has selected for the bacterium's evolutionary adaptation to intracellular life in mammalian cells (99).Intracellular multiplication of L. pneumophila requires a series of ordered events that disrupt normal endocytic trafficking in both macrophage and protozoan host cells. These include preventing phagolysosome fusion and the acidification of the Legionella-containing vacuole (LCV), followed by the acquisition of membrane material derived from the Golgi and endoplasmic reticulum compartments of the host (71,85,93,95). These events are dependent upon the Icm/Dot type ...

Macrophages play important roles in recycling iron derived from the clearance of red blood cells (RBCs). They are also a critically important component of host defense, protecting against invading pathogens. However, the effects on macrophage biology of acutely ingesting large numbers of RBCs are not completely understood. To investigate this issue, we used a mouse model of RBC transfusion and clearance, which mimics the clinical setting. In this model, transfusions of refrigerator storage-damaged (ie, "old") RBCs led to increased erythrophagocytosis by splenic red pulp macrophages (RPMs). This robust erythrophagocytosis induced ferroptosis, an iron-dependent form of cell death, in RPMs. This was accompanied by increases in reactive oxygen species and lipid peroxidation in vivo, which were reduced by treatment in vitro with ferrostatin-1, a ferroptosis inhibitor. Old RBC transfusions also induced RPM-dependent chemokine expression by splenic Ly6C monocytes, which signaled Ly6C monocyte migration from bone marrow to spleen, where these cells subsequently differentiated into RPMs. The combination of cell division among remaining splenic RPMs, along with the influx of bone marrow-derived Ly6C monocytes, suggests that, following RPM depletion induced by robust erythrophagocytosis, there is a coordinated effort to restore homeostasis of the RPM population by local self-maintenance and contributions from circulating monocytes. In conclusion, these findings may be clinically relevant to pathological conditions that can arise as a result of increased erythrophagocytosis, such as transfusion-related immunomodulation and impaired host immunity.

The cytokinesis-block micronucleus (CBMN) assay has become a fully-validated and standardized method for radiation biodosimetry. The assay is typically performed using microscopy, which is labor intensive, time consuming and impractical after a large-scale radiological/nuclear event. Imaging flow cytometry (IFC), which combines the statistical power of traditional flow cytometry with the sensitivity and specificity of microscopy, has been recently used to perform the CBMN assay. Since this technology is capable of automated sample acquisition and multi-file analysis, we have integrated IFC into our Rapid Automated Biodosimetry Technology (RABiT-II). Assay development and optimization studies were designed to increase the yield of binucleated cells (BNCs), and improve data acquisition and analysis templates to increase the speed and accuracy of image analysis. Human peripheral blood samples were exposed ex vivo with up to 4 Gy of c rays at a dose rate of 0.73 Gy/min. After irradiation, samples were transferred to microtubes (total volume of 1 ml including blood and media) and organized into a standard 8 3 12 plate format. Sample processing methods were modified by increasing the blood-to-media ratio, adding hypotonic solution prior to cell fixation and optimizing nuclear DRAQ5 staining, leading to an increase of 81% in BNC yield. Modification of the imaging processing algorithms within IFC software also improved BNC and MN identification, and reduced the average time of image analysis by 78%. Finally, 50 ll of irradiated whole blood was cultured with 200 ll of media in 96-well plates. All sample processing steps were performed automatically using the RABiT-II cell::explorer robotic system adopting the optimized IFC-CBMN assay protocol. The results presented here detail a novel, high-throughput RABiT-IFC CBMN assay that possesses the potential to increase capacity for triage biodosimetry during a large-scale radiological/nuclear event.