Swapna Samanta

Borrelia burgdorferi Gene Expression In Vivo and Spirochete Pathogenicity

ABSTRACT Borrelia burgdorferi spirochetes that do not cause arthritis or carditis were developed and used to investigate Lyme disease pathogenesis. A clonal isolate of B. burgdorferiN40 (cN40), which induces disease in C3H/HeN (C3H) mice, was repeatedly passaged in vitro to generate nonpathogenic spirochetes. The passage 75 isolate (N40-75) was infectious for C3H mice but did not cause arthritis or carditis, and spirochetes were at low levels or absent in the joints or hearts, respectively. N40-75 could, however, cause disease in severe combined immunodeficient (SCID) mice, suggesting that the response in immunocompetent mice prevented effective spirochete dissemination and the subsequent development of arthritis and carditis. Administration of immune sera at 4 days after spirochete challenge aborted N40-75, but not cN40, infection in SCID mice. A B. burgdorferi genomic expression library was differentially probed with sera from cN40- and N40-75-infected mice, to identify genes that may not be effectively expressed by N40-75 in vivo. N40-75 was defective in the up-regulation of several genes that are preferentially expressed during mammalian infection, including dbpAB,bba64, and genes that map to the cp32 family of plasmids. These data suggest that adaptation and gene expression may be required for B. burgdorferi to effectively colonize the host, evade humoral responses, and cause disease.

Borrelia burgdorferi-Infected, Interleukin-6-Deficient Mice Have Decreased Th2 Responses and Increased Lyme Arthritis

Recently, interleukin (IL)-6 was shown to be one of the earliest factors that trigger the differentiation of naive T cells into effector Th2 cells in vitro. Lyme arthritis was studied in IL-6-deficient mice, since joint inflammation is influenced by the T helper cell response against Borrelia burgdorferi. Arthritis incidence increased in B. burgdorferi-infected IL-6-deficient mice compared with that in controls. Furthermore, splenocytes of B. burgdorferi-infected IL-6-deficient mice produced significantly less IL-4 in response to Borrelia antigens than did C57BL/6 (B6) mice, and B. burgdorferi-specific IgG2b levels were significantly reduced in IL-6-deficient mice at 60 days of infection. These results extend previous in vitro observations by demonstrating an in vivo role for IL-6 in the differentiation of CD4 T cells toward a Th2 phenotype and further show that CD4 T cell responses influence murine Lyme arthritis.

Borrelia burgdorferi basic membrane proteins A and B participate in the genesis of Lyme arthritis

Lyme arthritis results from colonization of joints by Borrelia burgdorferi and the ensuing host response. Using gene array–based differential analysis of B. burgdorferi gene expression and quantitative reverse trancription-polymerase chain reaction, we identified two paralogous spirochete genes, bmpA and bmpB, that are preferentially up-regulated in mouse joints compared with other organs. Transfer of affinity-purified antibodies against either BmpA or BmpB into B. burgdorferi–infected mice selectively reduced spirochete numbers and inflammation in the joints. B. burgdorferi lacking bmpA/B were therefore generated to further explore the role of these proteins in the pathogenesis of Lyme disease. B. burgdorferi lacking bmpA/B were infectious in mice, but unable to persist in the joints, and they failed to induce severe arthritis. Complementation of the mutant spirochetes with a wild-type copy of the bmpA and bmpB genes partially restored the original phenotype. These data delineate a role for differentially produced B. burgdorferi antigens in spirochete colonization of mouse joints, and suggest new strategies for the treatment of Lyme arthritis.

Borrelia burgdorferi-Induced Inflammation Facilitates Spirochete Adaptation and Variable Major Protein-Like Sequence Locus Recombination

Spirochete adaptation in vivo is associated with preferential Borrelia burgdorferi gene expression. In this paper, we show that the administration of B. burgdorferi-immune sera to IFN-γR-deficient mice that have been infected with B. burgdorferi N40 for 4 days causes spirochete clearance. In contrast, immune sera-mediated clearance of B. burgdorferi N40 is not apparent in immunocompetent mice, suggesting a role for IFN-γ-mediated responses in B. burgdorferi N40 host adaptation. B. burgdorferi-immune sera also induces clearance of B. burgdorferi N40 that have been passaged in vitro 75 times (B. burgdorferi N40-75), a derivative of B. burgdorferi N40 that does not rapidly adapt in vivo in immunocompetent mice. B. burgdorferi N40-75 produce lower levels of IFN-γ and IL-12 in mice than does B. burgdorferi N40, and the administration of these cytokines to B. burgdorferi N40-75-infected mice results in an increased spirochetal burden, further indicating that IFN-γ-mediated events promote B. burgdorferi survival. Differential immunoscreening and RT-PCR demonstrate that IFN-γ-mediated signals facilitate spirochete recombination at the variable major protein like sequence locus, a site for early antigenic variation in vivo, and that recombination rates by B. burgdorferi N40 are lower in IFN-γR-deficient mice than in control animals. These results suggest that the murine immune response can promote the in vivo adaptation of B. burgdorferi.

Anaplasma phagocytophilum modulates gp91phox gene expression through altered interferon regulatory factor 1 and PU.1 levels and binding of CCAAT displacement protein.

ABSTRACT Infection of neutrophil precursors with Anaplasma phagocytophilum, the causative agent of human granulocytic ehrlichiosis, results in downregulation of the gp91phox gene, a key component of NADPH oxidase. We now show that repression of gp91phox gene transcription is associated with reduced expression of interferon regulatory factor 1 (IRF-1) and PU.1 in nuclear extracts of A. phagocytophilum-infected cells. Loss of PU.1 and IRF-1 correlated with increased binding of the repressor, CCAAT displacement protein (CDP), to the promoter of the gp91phox gene. Reduced protein expression of IRF-1 was observed with or without gamma interferon (IFN-γ) stimulation, and the defect in IFN-γ signaling was associated with diminished binding of phosphorylated Stat1 to the Stat1 binding element of the IRF-1 promoter. The diminished levels of activator proteins and enhanced binding of CDP account for the transcriptional inhibition of the gp91phox gene during A. phagocytophilum infection, providing evidence of the first molecular mechanism that a pathogen uses to alter the regulation of genes that contribute to an effective respiratory burst.

Plasmodium falciparum phosphoethanolamine methyltransferase is essential for malaria transmission

Significance Malaria, caused by intraerythrocytic protozoan parasites of the genus Plasmodium, is by far the deadliest and most prevalent parasitic disease. Most fatalities are attributable to infection by Plasmodium falciparum. The transmission of P. falciparum into Anopheles mosquitoes is absolutely dependent on the ability of the parasite to differentiate into mature gametocytes. Here we show that P. falciparum requires the plant-like phosphatidylcholine synthesis machinery, which is fueled by host serine, and its key enzyme, phosphoethanolamine N-methyltransferase (PfPMT), for gametocyte development, maturation, and transmission. We identified several compounds that inhibit PfPMT activity and gametocyte development. These compounds also inhibited parasite asexual replication. These new chemical entities provide scaffolds for future development of dual-function antimalarials that can block both infection and transmission. Efficient transmission of Plasmodium species between humans and Anopheles mosquitoes is a major contributor to the global burden of malaria. Gametocytogenesis, the process by which parasites switch from asexual replication within human erythrocytes to produce male and female gametocytes, is a critical step in malaria transmission and Plasmodium genetic diversity. Nothing is known about the pathways that regulate gametocytogenesis and only few of the current drugs that inhibit asexual replication are also capable of inhibiting gametocyte development and blocking malaria transmission. Here we provide genetic and pharmacological evidence indicating that the pathway for synthesis of phosphatidylcholine in Plasmodium falciparum membranes from host serine is essential for parasite gametocytogenesis and malaria transmission. Parasites lacking the phosphoethanolamine N-methyltransferase enzyme, which catalyzes the limiting step in this pathway, are severely altered in gametocyte development, are incapable of producing mature-stage gametocytes, and are not transmitted to mosquitoes. Chemical screening identified 11 inhibitors of phosphoethanolamine N-methyltransferase that block parasite intraerythrocytic asexual replication and gametocyte differentiation in the low micromolar range. Kinetic studies in vitro as well as functional complementation assays and lipid metabolic analyses in vivo on the most promising inhibitor NSC-158011 further demonstrated the specificity of inhibition. These studies set the stage for further optimization of NSC-158011 for development of a class of dual activity antimalarials to block both intraerythrocytic asexual replication and gametocytogenesis.

Selective Anti-Inflammatory Action of Interleukin-11 in Murine Lyme Disease: Arthritis Decreases while Carditis Persists

The role of interleukin (IL)-11, a cytokine with potent anti-inflammatory properties, in murine Lyme disease was investigated. Borrelia burgdorferi-infected mice treated with IL-11 developed less arthritis than did control animals. In contrast, IL-11 blocking antibodies increased Lyme arthritis. Murine Lyme carditis was not affected by either IL-11 or IL-11 antibodies. Administration of IL-11 was associated with increased production of mRNA for IL-12 and inducible nitric oxide synthase but not interferon-gamma or IL-4 in B. burgdorferi-infected mice, suggesting a predominant effect of IL-11 on the innate immune response. These data show that IL-11 selectively reduced joint but not cardiac inflammation caused by B. burgdorferi in mice.

Dissociation of Infectivity and Pathogenicity in Borrelia burgdorferi

ABSTRACT Clonal Borrelia burgdorferi N40 (cN40) passaged 75 times in vitro (N40-75) infects mice but does not cause disease. N40-75 passaged 45 times further in vitro (N40-120) was no longer infectious and lacked genes encoded on linear plasmids 38 and 28-1, among other differences. These data suggest that B. burgdorferi cN40, N40-75, and N40-120 have distinct phenotypes that can be used to dissect the genetic elements responsible for pathogenicity and infectivity.

Anaplasma phagocytophilum Increases Cathepsin L Activity, Thereby Globally Influencing Neutrophil Function

ABSTRACT Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, is an unusual obligate intracellular pathogen that persists in neutrophils. A. phagocytophilum increases the binding of a repressor, CCAAT displacement protein (CDP), to the gp91phox promoter, thereby diminishing the host oxidative burst. We now show that A. phagocytophilum infection also enhances the binding of CDP to the promoters of human neutrophil peptide 1 and C/EBPε—molecules important for neutrophil defense and maturation—suggesting that this is a general strategy used by this pathogen to alter polymorphonuclear leukocyte function. To explore the mechanism by which A. phagocytophilum increases CDP activity, we assessed the effects of this microbe on cathepsin L, a protease that cleaves CDP into a form with increased DNA binding ability. A. phagocytophilum infection resulted in elevated cathepsin L activity and the proteolysis of CDP. Blocking the action of cathepsin L with a chemical inhibitor or small interfering RNA targeting of this molecule caused a marked reduction in the degree of A. phagocytophilum infection. These data demonstrate that increasing cathepsin L activity is a strategy used by A. phagocytophilum to alter CDP activity and thereby globally influence neutrophil function. As therapeutic options for A. phagocytophilum and related organisms are limited, these results also identify a cellular pathway that may be targeted for the treatment of A. phagocytophilum infection.

The ModERN Resource: Genome-Wide Binding Profiles for Hundreds of Drosophila and Caenorhabditis elegans Transcription Factors

The model organism Encylopedia of Regulatory Elements (modERN) project was designed to generate genome-wide binding profiles for the majority of transcription... To develop a catalog of regulatory sites in two major model organisms, Drosophila melanogaster and Caenorhabditis elegans, the modERN (model organism Encyclopedia of Regulatory Networks) consortium has systematically assayed the binding sites of transcription factors (TFs). Combined with data produced by our predecessor, modENCODE (Model Organism ENCyclopedia Of DNA Elements), we now have data for 262 TFs identifying 1.23 M sites in the fly genome and 217 TFs identifying 0.67 M sites in the worm genome. Because sites from different TFs are often overlapping and tightly clustered, they fall into 91,011 and 59,150 regions in the fly and worm, respectively, and these binding sites span as little as 8.7 and 5.8 Mb in the two organisms. Clusters with large numbers of sites (so-called high occupancy target, or HOT regions) predominantly associate with broadly expressed genes, whereas clusters containing sites from just a few factors are associated with genes expressed in tissue-specific patterns. All of the strains expressing GFP-tagged TFs are available at the stock centers, and the chromatin immunoprecipitation sequencing data are available through the ENCODE Data Coordinating Center and also through a simple interface (http://epic.gs.washington.edu/modERN/) that facilitates rapid accessibility of processed data sets. These data will facilitate a vast number of scientific inquiries into the function of individual TFs in key developmental, metabolic, and defense and homeostatic regulatory pathways, as well as provide a broader perspective on how individual TFs work together in local networks and globally across the life spans of these two key model organisms.