forward-genetics-screens-using-macrophages-to-identify-toxoplasma

Forward Genetics Screens Using Macrophages to Identify <em>Toxoplasma gondii </em>Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

Toxoplasma gondii is an obligate intracellular protozoa parasite that causes the disease toxoplasmosis. It resides within host cells in a parasitophorous vacuole distinct from the host cell endocytic system. T. gondii was used as a model to investigate how obligate intracellular parasites alter their gene expression in response to the host immune response during infection compared to growth in host cells in vitro. While bacterial pathogens clearly alter gene expression to adapt to the host environment during infection, the degree to which the external environment affects gene expression by obligate intracellular pathogens sequestered within host cells is less clear. The global transcriptome of T. gondii was analyzed in vivo in the presence and absence of the IFN-γ-dependent host innate immune response. The parasites' in vivo transcriptome was also compared to its transcriptome in vitro in fibroblast cells. Our results indicate that the parasite transcriptome is significantly altered during in vivo infection in the presence, but not absence, of IFN-γ-dependent immunity compared with fibroblasts infected in vitro. Many of the parasite genes increased in vivo appear to be common to an early general stress response by the parasite; surprisingly putative oocyst stage specific genes were also disproportionately increased during infection.

Identification of Toxoplasma gondii genes responsive to the host immune response during in vivo infection.

A global sample of group A streptococci (GAS) revealed > or =80 separate acquisitions of tetracycline resistance. Of 244 clones, 38 and 25% displayed resistance to tetracycline and erythromycin, respectively; a relatively high proportion (15%) were resistant to both classes of drugs. tet(M) displayed a highly significant association with erm(B).

Tetracycline resistance in group a streptococci: emergence on a global scale and influence on multiple-drug resistance.

Toxoplasma gondii mutants identified as defective in the establishment of chronic infection were screened to isolate those specifically impaired in their ability to replicate within activated macrophages. One of the identified mutants contains an insertion in the hypothetical gene TGME49_111670. Genetic complementation restores the ability of the mutant to replicate in immune cells and produce cysts in the brains of mice. While the mutant is more sensitive to nitric oxide than is its parental strain, it is not defective in its ability to suppress nitric oxide. The disrupted protein has no significant homology to proteins with known functions, but is predicted to have one transmembrane domain. Immunofluorescence shows the protein on the parasite surface, even in activated macrophages, colocalizing with a tachyzoite surface antigen, SAG1, and oriented with its C-terminal end external. Western analysis reveals that the protein is downregulated in bradyzoites. Despite the tachyzoite specificity of this protein, mice infected with the mutant succumb to acute infection similarly to those infected with the parent strain. Serum samples from mice with chronic T. gondii infection react to a polypeptide from TGME49_11670, indicating that the protein is seen by the immune system during infection. This study is the first to characterize a T. gondii surface protein that contains a transmembrane domain and show that the protein contributes to parasite replication in activated immune cells and the establishment of chronic infection.

A transmembrane domain-containing surface protein from Toxoplasma gondii augments replication in activated immune cells and establishment of a chronic infection.

Toxoplasma Gondii Mutanten als defekt an der Gr&uuml;ndung einer chronischen Infektion identifiziert wurden gezeigt, um die speziell beeintr&auml;chtigt in ihrer F&auml;higkeit, in aktivierten Makrophagen replizieren zu isolieren. Eines der identifizierten Mutanten enth&auml;lt eine Einf&uuml;gung im hypothetischen gen TGME49_111670. Genetische Erg&auml;nzung stellt die F&auml;higkeit von den Mutanten in Immunzellen replizieren und Zysten in den Gehirnen der M&auml;use zu produzieren. W&auml;hrend der Mutant empfindlicher auf Stickstoffmonoxid ist als seine elterliche Belastung ist, ist es nicht Defekt in seiner F&auml;higkeit, Stickstoffmonoxid zu unterdr&uuml;cken. Das gest&ouml;rte Protein hat keine deutliche Homologie zu Proteinen mit bekannten Funktionen, sondern wird voraussichtlich eine transmembrane-Dom&auml;ne verwenden. Immunofluoreszenz zeigt das Protein auf der Oberfl&auml;che des Parasiten, auch in aktivierten Makrophagen, mit Tachyzoite Oberfl&auml;che Antigen, SAG1, colocalizing und mit seinem C-terminalen Ende externe orientiert. Westliche Analyse zeigt, dass das Protein Downregulated in Bradyzoites ist. Trotz der Tachyzoite Besonderheit dieses Proteins erliegen M&auml;use mit den Mutanten infizierte akute Infektion &auml;hnlich der Stammkultur-infizierten. Serumproben von M&auml;usen mit chronischen T. Gondii Infektion reagieren auf ein Polypeptid aus TGME49_11670, die darauf hinweist, dass das Protein w&auml;hrend der Infektion vom Immunsystem gesehen wird. Diese Studie ist die erste charakterisieren ein T. Gondii-Oberfl&auml;chen-Protein, das eine transmembrane-Dom&auml;ne enth&auml;lt und zeigen, dass das Protein zur Parasiten-Replikation in aktivierten Immunzellen und die Einrichtung einer chronischen Infektion beitr&auml;gt.

Ein Dom&auml;ne-haltige Oberfl&auml;chen Transmembraneprotein von Toxoplasma Gondii erweitert die Replikation im aktivierten Immunzellen und Aufbau einer chronischen Infektion.

Apicomplexa are primarily obligate intracellular protozoa that have evolved complex developmental stages important for pathogenesis and transmission. Toxoplasma gondii, responsible for the disease toxoplasmosis, has the broadest host range of the Apicomplexa as it infects virtually any warm-blooded vertebrate host. Key to T. gondii's pathogenesis is its ability to differentiate from a rapidly replicating tachyzoite stage during acute infection to a relatively non-immunogenic, dormant bradyzoite stage contained in tissue cysts. These bradyzoite cysts can reconvert back to tachyzoites years later causing serious pathology and death if a person becomes immune-compromised. Like the sexual stage sporozoites, bradyzoites are also orally infectious and a major contributor to transmission. Because of the critical role of stage conversion to pathogenesis and transmission, a major research focus is aimed at identifying molecular mediators and pathways that regulate differentiation. Tachyzoite to bradyzoite development can occur spontaneously in vitro and be induced in response to exogenous stress including but not limited to host immunity. The purpose of this review is to explore the potential contributors to stage differentiation in infection and how a determination is made by the parasite to differentiate from tachyzoites to bradyzoites.

Toxoplasma gondii: determinants of tachyzoite to bradyzoite conversion.

SLT-VEGF is a recombinant cytotoxin comprised of Shiga-like toxin (SLT) subunit A fused to human vascular endothelial growth factor (VEGF). It is highly cytotoxic to tumor endothelial cells overexpressing VEGF receptor-2 (VEGFR-2/KDR/Flk1) and inhibits the growth of primary tumors in subcutaneous models of breast and prostate cancer and inhibits metastatic dissemination in orthotopic models of pancreatic cancer. We examined the efficacy of SLT-VEGF in limiting tumor growth and metastasis in an orthotopic melanoma model, using NCR athymic nude mice inoculated with highly metastatic Line IV Cl 1 cultured human melanoma cells. Twice weekly injections of SLT-VEGF were started when tumors became palpable at one week after intradermal injection of 1 × 10(6) cells/mouse. Despite selective depletion of VEGFR-2 overexpressing endothelial cells from the tumor vasculature, SLT-VEGF treatment did not affect tumor growth. However, after primary tumors were removed, continued SLT-VEGF treatment led to fewer tumor recurrences (p = 0.007), reduced the incidence of lung metastasis (p = 0.038), and improved survival (p = 0.002). These results suggest that SLT-VEGF is effective at the very early stages of tumor development, when selective killing of VEGFR-2 overexpressing endothelial cells can still prevent further progression. We hypothesize that SLT-VEGF could be a promising adjuvant therapy to inhibit or prevent outgrowth of metastatic foci after excision of aggressive primary melanoma lesions.

SLT-VEGF reduces lung metastases, decreases tumor recurrence, and improves survival in an orthotopic melanoma model.

Toxoplasma gondii modifies its host cell to suppress its ability to become activated in response to IFN-γ and TNF-α and to develop intracellular antimicrobial effectors, including NO. Mechanisms used by T. gondii to modulate activation of its infected host cell likely underlie its ability to hijack monocytes and dendritic cells during infection to disseminate to the brain and CNS where it converts to bradyzoites contained in tissue cysts to establish persistent infection. To identify T. gondii genes important for resistance to the effects of host cell activation, we developed an in vitro murine macrophage infection and activation model to identify parasite insertional mutants that have a fitness defect in infected macrophages following activation but normal invasion and replication in naive macrophages. We identified 14 independent T. gondii insertional mutants out of >8000 screened that share a defect in their ability to survive macrophage activation due to macrophage production of reactive nitrogen intermediates (RNIs). These mutants have been designated counter-immune mutants. We successfully used one of these mutants to identify a T. gondii cytoplasmic and conoid-associated protein important for parasite resistance to macrophage RNIs. Deletion of the entire gene or just the region encoding the protein in wild-type parasites recapitulated the RNI-resistance defect in the counter-immune mutant, confirming the role of the protein in resistance to macrophage RNIs.

Discovery of a novel Toxoplasma gondii conoid-associated protein important for parasite resistance to reactive nitrogen intermediates.

BackgroundHuman Papilloma Virus (HPV) infection is the major cause of cervical cancer worldwide. With limited data available on HPV prevalence in the Arab countries, this study aimed to identify the prevalence and genotypic distribution of HPV in the State of Qatar.Methods3008 cervical samples, exclusively of women with Arabic origin residing in Qatar were collected from the Women¿s Hospital and Primary Health Care Corporation in Doha, State of Qatar. HPV DNA detection was done using GP5+/6+ primers based real time-polymerase chain reaction (RT-PCR) assay followed by the usage of HPV type specific primers based RT- PCR reactions and Sanger sequencing for genotype identification.ResultsSimilar prevalence rates of HPV infection was identified in both Qatari and non-Qatari women at 6.2% and 5.9% respectively. HPV prevalence rate of 5.8% and 18.4% was identified in women with normal cytology and in women with abnormal cytology respectively. HPV 81, 11 and 16, in decreasing order were the most commonly identified genotypes. HPV 81 was the most frequent low-risk genotype among women with both normal (74.0%) and abnormal (33.3%) cytology. HPV 16 (4.6%) was identified as the predominant high-risk HPV genotype among women with normal cytology and HPV 16, HPV 18, and HPV 56 (22.2% each) were the most common identified high-risk genotypes in women with abnormal cytology.ConclusionsThe overall HPV prevalence in Arab women in Qatar was identified as 6.1% with an increased HPV prevalence seen in women with abnormal cytology results and no significant trends seen with age. In contrast to Western countries, we report a varied genotypic profile of HPV with a high prevalence of low-risk HPV genotype 81 among the Arab women residing in Qatar.

Article	Year
Identification of Toxoplasma gondii genes responsive to the host immune response during in vivo infection. PloS one\| PubMed ID: 23071600	2012
Tetracycline resistance in group a streptococci: emergence on a global scale and influence on multiple-drug resistance. Antimicrobial agents and chemotherapy\| PubMed ID: 17307980	2007
A transmembrane domain-containing surface protein from Toxoplasma gondii augments replication in activated immune cells and establishment of a chronic infection. Infection and immunity\| PubMed ID: 19581395	2009
Toxoplasma gondii: determinants of tachyzoite to bradyzoite conversion. Parasitology research\| PubMed ID: 20514494	2010
SLT-VEGF reduces lung metastases, decreases tumor recurrence, and improves survival in an orthotopic melanoma model. Toxins\| PubMed ID: 22069683	2010
Discovery of a novel Toxoplasma gondii conoid-associated protein important for parasite resistance to reactive nitrogen intermediates. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 22387554	2012
Molecular epidemiology and genotype distribution of Human Papillomavirus (HPV) among Arab women in the state of Qatar. Journal of translational medicine\| PubMed ID: 25424736	2014

Sini Skariah

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