Extensive recombination events and horizontal gene transfer shaped the Legionella pneumophila genomes

<p>Abstract</p> <p>Background</p> <p><it>Legionella pneumophila </it>is an intracellular pathogen of environmental protozoa. When humans inhale contaminated aerosols this bacterium may cause a severe pneumonia called Legionnaires' disease. Despite the abundance of dozens of <it>Legionella </it>species in aquatic reservoirs, the vast majority of human disease is caused by a single serogroup (Sg) of a single species, namely <it>L. pneumophila </it>Sg1. To get further insights into genome dynamics and evolution of Sg1 strains, we sequenced strains Lorraine and HL 0604 1035 (Sg1) and compared them to the available sequences of Sg1 strains Paris, Lens, Corby and Philadelphia, resulting in a comprehensive multigenome analysis.</p> <p>Results</p> <p>We show that <it>L. pneumophila </it>Sg1 has a highly conserved and syntenic core genome that comprises the many eukaryotic like proteins and a conserved repertoire of over 200 Dot/Icm type IV secreted substrates. However, recombination events and horizontal gene transfer are frequent. In particular the analyses of the distribution of nucleotide polymorphisms suggests that large chromosomal fragments of over 200 kbs are exchanged between <it>L. pneumophila </it>strains and contribute to the genome dynamics in the natural population. The many secretion systems present might be implicated in exchange of these fragments by conjugal transfer. Plasmids also play a role in genome diversification and are exchanged among strains and circulate between different <it>Legionella </it>species.</p> <p>Conclusion</p> <p>Horizontal gene transfer among bacteria and from eukaryotes to <it>L. pneumophila </it>as well as recombination between strains allows different clones to evolve into predominant disease clones and others to replace them subsequently within relatively short periods of time.</p

Molecular Mimicry by an F-Box Effector of Legionella pneumophila Hijacks a Conserved Polyubiquitination Machinery within Macrophages and Protozoa

The ability of Legionella pneumophila to proliferate within various protozoa in the aquatic environment and in macrophages indicates a remarkable evolution and microbial exploitation of evolutionarily conserved eukaryotic processes. Ankyrin B (AnkB) of L. pneumophila is a non-canonical F-box-containing protein, and is the only known Dot/Icm-translocated effector of L. pneumophila essential for intra-vacuolar proliferation within both macrophages and protozoan hosts. We show that the F-box domain of AnkB and the 9L10P conserved residues are essential for intracellular bacterial proliferation and for rapid acquisition of polyubiquitinated proteins by the Legionella-containing vacuole (LCV) within macrophages, Dictyostelium discoideum, and Acanthamoeba. Interestingly, translocation of AnkB and recruitment of polyubiquitinated proteins in macrophages and Acanthamoeba is rapidly triggered by extracellular bacteria within 5 min of bacterial attachment. Ectopically expressed AnkB within mammalian cells is localized to the periphery of the cell where it co-localizes with host SKP1 and recruits polyubiquitinated proteins, which results in restoration of intracellular growth to the ankB mutant similar to the parental strain. While an ectopically expressed AnkB-9L10P/AA variant is localized to the cell periphery, it does not recruit polyubiquitinated proteins and fails to trans-rescue the ankB mutant intracellular growth defect. Direct in vivo interaction of AnkB but not the AnkB-9L10P/AA variant with the host SKP1 is demonstrated. Importantly, RNAi-mediated silencing of expression of SKP1 renders the cells non-permissive for intracellular proliferation of L. pneumophila. The role of AnkB in exploitation of the polyubiquitination machinery is essential for intrapulmonary bacterial proliferation in the mouse model of Legionnaires' disease. Therefore, AnkB exhibits a novel molecular and functional mimicry of eukaryotic F-box proteins that exploits conserved polyubiquitination machinery for intracellular proliferation within evolutionarily distant hosts

Uncoupling protein-2 mRNA expression in mice subjected to intermittent hypoxia

Objetivo: Investigar o efeito da hipóxia intermitente com um modelo de apneia obstrutiva do sono (AOS) sobre a expressão de uncoupling protein-2 (UCP2), assim como sobre perfis glicêmicos e lipídicos, em camundongos C57BL. Métodos: Camundongos C57BL machos foram expostos a hipóxia intermitente ou hipóxia simulada (grupo controle) 8 h/dia durante 35 dias. A condição de hipóxia intermitente envolveu a exposição dos camundongos a uma atmosfera de 92% de N e 8% de CO2 por 30 s, com redução progressiva de fração de O2 inspirado até 8 ± 1%, seguida por exposição a ar ambiente por 30 s e repetições do ciclo (480 ciclos no período experimental de 8 h). Os pâncreas foram dissecados para isolar as ilhotas. Foi realizada PCR em tempo real utilizando o método TaqMan. Resultados: A expressão do mRNA da UCP2 nas ilhotas pancreáticas foi 20% maior no grupo controle que no grupo hipóxia (p = 0,11). A insulina sérica de jejum foi maior no grupo hipóxia do que no grupo controle (p = 0,01). O modelo de avaliação da homeostase de resistência à insulina indicou que, em comparação com os camundongos controle, aqueles expostos à hipóxia intermitente apresentaram 15% menor resistência à insulina (p = 0,09) e 21% maior função das células beta (p = 0,01). A coloração das ilhotas pancreáticas por imuno-histoquímica não mostrou diferenças significativas entre os grupos em termos da área ou da intensidade das células alfa e beta, marcadas por insulina e glucagon. Conclusões: Segundo nosso conhecimento, esta é a primeira descrição do efeito da hipóxia intermitente sobre a expressão da UCP2. Nossos achados sugerem que UCP2 regula a produção de insulina na AOS. Futuras investigações sobre o papel da UCP2 no controle glicêmico em pacientes com AOS são justificadas.Objective: To investigate the effect of intermittent hypoxia—a model of obstructive sleep apnea (OSA)—on pancreatic expression of uncoupling protein-2 (UCP2), as well as on glycemic and lipid profiles, in C57BL mice. Methods: For 8 h/day over a 35-day period, male C57BL mice were exposed to intermittent hypoxia (hypoxia group) or to a sham procedure (normoxia group). The intermittent hypoxia condition involved exposing mice to an atmosphere of 92% N and 8% CO2 for 30 s, progressively reducing the fraction of inspired oxygen to 8 ± 1%, after which they were exposed to room air for 30 s and the cycle was repeated (480 cycles over the 8-h experimental period). Pancreases were dissected to isolate the islets. Real-time PCR was performed with TaqMan assays. Results: Expression of UCP2 mRNA in pancreatic islets was 20% higher in the normoxia group than in the hypoxia group (p = 0.11). Fasting serum insulin was higher in the hypoxia group than in the normoxia group (p = 0.01). The homeostasis model assessment of insulin resistance indicated that, in comparison with the control mice, the mice exposed to intermittent hypoxia showed 15% lower insulin resistance (p = 0.09) and 21% higher pancreatic β-cell function (p = 0.01). Immunohistochemical staining of the islets showed no significant differences between the two groups in terms of the area or intensity of α- and β-cell staining for insulin and glucagon. Conclusions: To our knowledge, this is the first report of the effect of intermittent hypoxia on UCP2 expression. Our findings suggest that UCP2 regulates insulin production in OSA. Further study of the role that UCP2 plays in the glycemic control of OSA patients is warranted

The Coxiella burnetii Dot/Icm System Delivers a Unique Repertoire of Type IV Effectors into Host Cells and Is Required for Intracellular Replication

Coxiella burnetii, the causative agent of human Q fever, is an intracellular pathogen that replicates in an acidified vacuole derived from the host lysosomal network. This pathogen encodes a Dot/Icm type IV secretion system that delivers bacterial proteins called effectors to the host cytosol. To identify new effector proteins, the functionally analogous Legionella pneumophila Dot/Icm system was used in a genetic screen to identify fragments of C. burnetii genomic DNA that when fused to an adenylate cyclase reporter were capable of directing Dot/Icm-dependent translocation of the fusion protein into mammalian host cells. This screen identified Dot/Icm effectors that were proteins unique to C. burnetii, having no overall sequence homology with L. pneumophila Dot/Icm effectors. A comparison of C. burnetii genome sequences from different isolates revealed diversity in the size and distribution of the genes encoding many of these effectors. Studies examining the localization and function of effectors in eukaryotic cells provided evidence that several of these proteins have an affinity for specific host organelles and can disrupt cellular functions. The identification of a transposon insertion mutation that disrupts the dot/icm locus was used to validate that this apparatus was essential for translocation of effectors. Importantly, this C. burnetii Dot/Icm-deficient mutant was found to be defective for intracellular replication. Thus, these data indicate that C. burnetii encodes a unique subset of bacterial effector proteins translocated into host cells by the Dot/Icm apparatus, and that the cumulative activities exerted by these effectors enables C. burnetii to successfully establish a niche inside mammalian cells that supports intracellular replication

Type IV Secretion-Dependent Activation of Host MAP Kinases Induces an Increased Proinflammatory Cytokine Response to Legionella pneumophila

The immune system must discriminate between pathogenic and nonpathogenic microbes in order to initiate an appropriate response. Toll-like receptors (TLRs) detect microbial components common to both pathogenic and nonpathogenic bacteria, whereas Nod-like receptors (NLRs) sense microbial components introduced into the host cytosol by the specialized secretion systems or pore-forming toxins of bacterial pathogens. The host signaling pathways that respond to bacterial secretion systems remain poorly understood. Infection with the pathogen Legionella pneumophila, which utilizes a type IV secretion system (T4SS), induced an increased proinflammatory cytokine response compared to avirulent bacteria in which the T4SS was inactivated. This enhanced response involved NF-κB activation by TLR signaling as well as Nod1 and Nod2 detection of type IV secretion. Furthermore, a TLR- and RIP2-independent pathway leading to p38 and SAPK/JNK MAPK activation was found to play an equally important role in the host response to virulent L. pneumophila. Activation of this MAPK pathway was T4SS-dependent and coordinated with TLR signaling to mount a robust proinflammatory cytokine response to virulent L. pneumophila. These findings define a previously uncharacterized host response to bacterial type IV secretion that activates MAPK signaling and demonstrate that coincident detection of multiple bacterial components enables immune discrimination between virulent and avirulent bacteria

Prolonged Survival of Allografts Induced by Mycobacterial Hsp70 Is Dependent on CD4+CD25+ Regulatory T Cells

Background: Heat shock proteins (Hsps) are stress induced proteins with immunomodulatory properties. The Hsp70 of Mycobacterium tuberculosis (TBHsp70) has been shown to have an anti-inflammatory role on rodent autoimmune arthritis models, and the protective effects were demonstrated to be dependent on interleukin-10 (IL-10). We have previously observed that TBHsp70 inhibited maturation of dendritic cells (DCs) and induced IL-10 production by these cells, as well as in synovial fluid cells. Methodology/Principal Findings: We investigated if TBHsp70 could inhibit allograft rejection in two murine allograft systems, a transplanted allogeneic melanoma and a regular skin allograft. In both systems, treatment with TBHsp70 significantly inhibited rejection of the graft, and correlated with regulatory T cells (Tregs) recruitment. This effect was not tumor mediated because injection of TBHsp70 in tumor-free mice induced an increase of Tregs in the draining lymph nodes as well as inhibition of proliferation of lymph node T cells and an increase in IL-10 production. Finally, TBHsp70 inhibited skin allograft acute rejection, and depletion of Tregs using a monoclonal antibody completely abolished this effect. Conclusions/Significance: We present the first evidence for an immunosuppressive role for this protein in a graft rejection system, using an innovative approach - immersion of the graft tissue in TBHsp70 solution instead of protein injection. Also, this is the first study that demonstrates dependence on Treg cells for the immunosuppressive role of TBHsp70. This finding is relevant for the elucidation of the immunomodulatory mechanism of TBHsp70. We propose that this protein can be used not only for chronic inflammatory diseases, but is also useful for organ transplantation management.Pontificia Universidade Catolica do Rio Grande do Sul (PUCRS)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Financiadora de Estudos e Projetos (FINEP

Comprehensive Identification of Protein Substrates of the Dot/Icm Type IV Transporter of Legionella pneumophila

A large number of proteins transferred by the Legionella pneumophila Dot/Icm system have been identified by various strategies. With no exceptions, these strategies are based on one or more characteristics associated with the tested proteins. Given the high level of diversity exhibited by the identified proteins, it is possible that some substrates have been missed in these screenings. In this study, we took a systematic method to survey the L. pneumophila genome by testing hypothetical orfs larger than 300 base pairs for Dot/Icm-dependent translocation. 798 of the 832 analyzed orfs were successfully fused to the carboxyl end of β-lactamase. The transfer of the fusions into mammalian cells was determined using the β-lactamase reporter substrate CCF4-AM. These efforts led to the identification of 164 proteins positive in translocation. Among these, 70 proteins are novel substrates of the Dot/Icm system. These results brought the total number of experimentally confirmed Dot/Icm substrates to 275. Sequence analysis of the C-termini of these identified proteins revealed that Lpg2844, which contains few features known to be important for Dot/Icm-dependent protein transfer can be translocated at a high efficiency. Thus, our efforts have identified a large number of novel substrates of the Dot/Icm system and have revealed the diverse features recognizable by this protein transporter

CD34+/M-cadherin+ Bone Marrow Progenitor Cells Promote Arteriogenesis in Ischemic Hindlimbs of ApoE−/− Mice

BACKGROUND: Cell-based therapy shows promise in treating peripheral arterial disease (PAD); however, the optimal cell type and long-term efficacy are unknown. In this study, we identified a novel subpopulation of adult progenitor cells positive for CD34 and M-cadherin (CD34⁺/M-cad⁺ BMCs) in mouse and human bone marrow. We also examined the long-lasting therapeutic efficacy of mouse CD34⁺/M-cad⁺ BMCs in restoring blood flow and promoting vascularization in an atherosclerotic mouse model of PAD. METHODS AND FINDINGS: Colony-forming cell assays and flow cytometry analysis showed that CD34⁺/M-cad⁺ BMCs have hematopoietic progenitor properties. When delivered intra-arterially into the ischemic hindlimbs of ApoE⁻/⁻ mice, CD34⁺/M-cad⁺ BMCs alleviated ischemia and significantly improved blood flow compared with CD34⁺/M-cad⁻ BMCs, CD34⁻/M-cad⁺ BMCs, or unselected BMCs. Significantly more arterioles were seen in CD34⁺/M-cad⁺ cell-treated limbs than in any other treatment group 60 days after cell therapy. Furthermore, histologic assessment and morphometric analyses of hindlimbs treated with GFP⁺ CD34⁺/M-cad⁺ cells showed that injected cells incorporated into solid tissue structures at 21 days. Confocal microscopic examination of GFP⁺ CD34⁺/M-cad⁺ cell-treated ischemic legs followed by immunostaining indicated the vascular differentiation of CD34⁺/M-cad⁺ progenitor cells. A cytokine antibody array revealed that CD34⁺/M-cad⁺ cell-conditioned medium contained higher levels of cytokines in a unique pattern, including bFGF, CRG-2, EGF, Flt-3 ligand, IGF-1, SDF-1, and VEGFR-3, than did CD34⁺/M-cad⁻ cell-conditioned medium. The proangiogenic cytokines secreted by CD34⁺/M-cad⁺ cells induced oxygen- and nutrient-depleted endothelial cell sprouting significantly better than CD34⁺/M-cad⁻ cells during hypoxia. CONCLUSION: CD34⁺/M-cad⁺ BMCs represent a new progenitor cell type that effectively alleviates hindlimb ischemia in ApoE⁻/⁻ mice by consistently improving blood flow and promoting arteriogenesis. Additionally, CD34⁺/M-cad⁺ BMCs contribute to microvascular remodeling by differentiating into vascular cells and releasing proangiogenic cytokines and growth factors

Chemical Genetics Reveals Bacterial and Host Cell Functions Critical for Type IV Effector Translocation by Legionella pneumophila

Delivery of effector proteins is a process widely used by bacterial pathogens to subvert host cell functions and cause disease. Effector delivery is achieved by elaborate injection devices and can often be triggered by environmental stimuli. However, effector export by the L. pneumophila Icm/Dot Type IVB secretion system cannot be detected until the bacterium encounters a target host cell. We used chemical genetics, a perturbation strategy that utilizes small molecule inhibitors, to determine the mechanisms critical for L. pneumophila Icm/Dot activity. From a collection of more than 2,500 annotated molecules we identified specific inhibitors of effector translocation. We found that L. pneumophila effector translocation in macrophages requires host cell factors known to be involved in phagocytosis such as phosphoinositide 3-kinases, actin and tubulin. Moreover, we found that L. pneumophila phagocytosis and effector translocation also specifically require the receptor protein tyrosine phosphate phosphatases CD45 and CD148. We further show that phagocytosis is required to trigger effector delivery unless intimate contact between the bacteria and the host is artificially generated. In addition, real-time analysis of effector translocation suggests that effector export is rate-limited by phagocytosis. We propose a model in which L. pneumophila utilizes phagocytosis to initiate an intimate contact event required for the translocation of pre-synthesized effector molecules. We discuss the need for host cell participation in the initial step of the infection and its implications in the L. pneumophila lifestyle. Chemical genetic screening provides a novel approach to probe the host cell functions and factors involved in host–pathogen interactions