• Lentivirus-induced dendritic cells for immunization against high-risk WT1(+) acute myeloid leukemia.

      Sundarasetty, Bala Sai; Singh, Vijay Kumar; Salguero, Gustavo; Geffers, Robert; Rickmann, Mareike; Macke, Laura; Borchers, Sylvia; Figueiredo, Constanca; Schambach, Axel; Gullberg, Urban; et al. (2013-02)
      Wilms' tumor 1 antigen (WT1) is overexpressed in acute myeloid leukemia (AML), a high-risk neoplasm warranting development of novel immunotherapeutic approaches. Unfortunately, clinical immunotherapeutic use of WT1 peptides against AML has been inconclusive. With the rationale of stimulating multiantigenic responses against WT1, we genetically programmed long-lasting dendritic cells capable of producing and processing endogenous WT1 epitopes. A tricistronic lentiviral vector co-expressing a truncated form of WT1 (lacking the DNA-binding domain), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-4 (IL-4) was used to transduce human monocytes ex vivo. Overnight transduction induced self-differentiation of monocytes into immunophenotypically stable "SmartDC/tWT1" (GM-CSF(+), IL-4(+), tWT1(+), IL-6(+), IL-8(+), TNF-α(+), MCP-1(+), HLA-DR(+), CD86(+), CCR2(+), CCR5(+)) that were viable for 3 weeks in vitro. SmartDC/tWT1 were produced with peripheral blood mononuclear cells (PBMC) obtained from an FLT3-ITD(+) AML patient and surplus material from a donor lymphocyte infusion (DLI) and used to expand CD8(+) T cells in vitro. Expanded cytotoxic T lymphocytes (CTLs) showed antigen-specific reactivity against WT1 and against WT1(+) leukemia cells. SmartDC/tWT1 injected s.c. into Nod.Rag1(-/-).IL2rγc(-/-) mice were viable in vivo for more than three weeks. Migration of human T cells (huCTLs) to the immunization site was demonstrated following adoptive transfer of huCTLs into mice immunized with SmartDC/tWT1. Furthermore, SmartDC/tWT1 immunization plus adoptive transfer of T cells reactive against WT1 into mice resulted in growth arrest of a WT1(+) tumor. Gene array analyses of SmartDC/tWT1 demonstrated upregulation of several genes related to innate immunity. Thus, SmartDC/tWT1 can be produced in a single day of ex vivo gene transfer, are highly viable in vivo, and have great potential for use as immunotherapy against malignant transformation overexpressing WT1.
    • LINT, a novel dL(3)mbt-containing complex, represses malignant brain tumour signature genes.

      Meier, Karin; Mathieu, Eve-Lyne; Finkernagel, Florian; Reuter, L Maximilian; Scharfe, Maren; Doehlemann, Gunther; Jarek, Michael; Brehm, Alexander; Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Marburg, Germany. (2012-05)
      Mutations in the l(3)mbt tumour suppressor result in overproliferation of Drosophila larval brains. Recently, the derepression of different gene classes in l(3)mbt mutants was shown to be causal for transformation. However, the molecular mechanisms of dL(3)mbt-mediated gene repression are not understood. Here, we identify LINT, the major dL(3)mbt complex of Drosophila. LINT has three core subunits-dL(3)mbt, dCoREST, and dLint-1-and is expressed in cell lines, embryos, and larval brain. Using genome-wide ChIP-Seq analysis, we show that dLint-1 binds close to the TSS of tumour-relevant target genes. Depletion of the LINT core subunits results in derepression of these genes. By contrast, histone deacetylase, histone methylase, and histone demethylase activities are not required to maintain repression. Our results support a direct role of LINT in the repression of brain tumour-relevant target genes by restricting promoter access.
    • Liposome-encapsulated antigens induce a protective CTL response against Listeria monocytogenes independent of CD4+ T cell help.

      Grenningloh, R; Darj, A; Bauer, H; zur Lage, S; Chakraborty, T; Jacobs, T; Weiss, S; Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany. (2008-06)
      Protection against intracellular pathogens is usually mediated by cytotoxic T lymphocytes (CTL). Induction of a protective CTL response for vaccination purposes has proven difficult because of the limited access of protein antigens or attenuated pathogens to the MHC class I presentation pathway. We show here that pH-sensitive PE/CHEMS liposomes can be used as a vehicle to efficiently deliver intact proteins for presentation by MHC class I. Mice immunized with listerial proteins encapsulated in such liposomes launched a strong CTL response and were protected against a subsequent challenge with L. monocytogenes. Remarkably, the CTL response was induced independently of detectable CD4(+) T cell help.
    • Listeria monocytogenes desensitizes immune cells to subsequent Ca2+ signaling via listeriolysin O-induced depletion of intracellular Ca2+ stores.

      Gekara, Nelson O; Groebe, Lothar; Viegas, Nuno; Weiss, Siegfried; Department of Molecular Immunology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany. nelson.gekara@helmholtz-hzi.de (2008-02)
      Listeriolysin O (LLO), the pore-forming toxin of Listeria monocytogenes, is a prototype of the cholesterol-dependent cytolysins (CDCs) secreted by several pathogenic and nonpathogenic gram-positive bacteria. In addition to mediating the escape of the bacterium into the cytosol, this toxin is generally believed to be a central player in host-pathogen interactions during L. monocytogenes infection. LLO triggers the influx of Ca(2+) into host cells as well as the release of Ca(2+) from intracellular stores. Thus, many of the cellular responses induced by LLO are related to calcium signaling. Interestingly, in this study, we report that prolonged exposure to LLO desensitizes cells to Ca(2+) mobilization upon subsequent stimulations with LLO. Cells preexposed to LLO-positive L. monocytogenes but not to the LLO-deficient Deltahly mutant were found to be highly refractory to Ca(2+) induction in response to receptor-mediated stimulation. Such cells also exhibited diminished Ca(2+) signals in response to stimulation with LLO and thapsigargin. The presented results suggest that this phenomenon is due to the depletion of intracellular Ca(2+) stores. The ability of LLO to desensitize immune cells provides a significant hint about the possible role played by CDCs in the evasion of the immune system by bacterial pathogens.
    • Listeria monocytogenes induces T cell receptor unresponsiveness through pore-forming toxin listeriolysin O.

      Gekara, Nelson O; Zietara, Natalia; Geffers, Robert; Weiss, Siegfried; Molecular Immunology, Helmholtz Center for Infection Research, Braunschweig, Germany. nelson.gekara@mims.umu.se (2010-12-01)
      The success of many pathogens relies on their ability to circumvent the innate and adaptive immune defenses. How bacterial pathogens subvert adaptive immune defenses is not clear. Cholesterol-dependent cytolysins (CDCs) represent an expansive family of homologous pore-forming toxins that are produced by more than 20 gram-positive bacterial species. Listeriolysin O (LLO), a prototype CDC, is the main virulence factor of Listeria monocytogenes.
    • Local application of bacteria improves safety of Salmonella-mediated tumor therapy and retains advantages of systemic infection.

      Kocijancic, Dino; Felgner, Sebastian; Schauer, Tim; Frahm, Michael; Heise, Ulrike; Zimmermann, Kurt; Erhardt, Marc; Weiss, Siegfried; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-06-07)
      Cancer is a devastating disease and a large socio-economic burden. Novel therapeutic solutions are on the rise, although a cure remains elusive. Application of microorganisms represents an ancient therapeutic strategy, lately revoked and refined via simultaneous attenuation and amelioration of pathogenic properties. Salmonella Typhimurium has prevailed in preclinical development. Yet, using virulent strains for systemic treatment might cause severe side effects. In the present study, we highlight a modified strain based on Salmonella Typhimurium UK-1 expressing hexa-acylated Lipid A. We corroborate improved anti-tumor properties of this strain and investigate to which extent an intra-tumoral (i.t.) route of infection could help improve safety and retain advantages of systemic intravenous (i.v.) application. Our results show that i.t. infection exhibits therapeutic efficacy against CT26 and F1.A11 tumors similar to a systemic route of inoculation. Moreover, i.t. application allows extensive dose titration without compromising tumor colonization. Adverse colonization of healthy organs was generally reduced via i.t. infection and accompanied by less body weight loss of the murine host. Despite local application, adjuvanticity remained, and a CT26-specific CD8+ T cell response was effectively stimulated. Most interestingly, also secondary tumors could be targeted with this strategy, thereby extending the unique tumor targeting ability of Salmonella. The i.t. route of inoculation may reap the benefits of systemic infection and aid in safety assurance while directing potency of an oncolytic vector to where it is most needed, namely the primary tumor.
    • Mast cells initiate early anti-Listeria host defences.

      Gekara, Nelson O; Weiss, Siegfried; Helmholtz Centre for Infection Research, Department of Molecular Immunology, Inhoffenstrasse 7, 38124 Braunschweig, Germany. Nelson.Gekara@helmholtz-hzi.de (2008-01)
      The Gram-positive bacterium Listeria monocytogenes (L. m.) is the aetiological agent of listeriosis. The early phase listeriosis is characterized by strong innate host responses that play a major role in bacterial clearance. This is emphasized by the fact that mice deficient in T and B cells have a remarkable ability to control infection. Mast cells, among the principal effectors of innate immunity, have largely been studied in the context of hyper-reactive conditions such as allergy and autoimmune diseases. In the present study, we evaluated the significance of mast cells during the early phase of listeriosis. Compared with controls, mice depleted of mast cells showed hundred-fold higher bacterial burden in spleen and liver and were significantly impaired in neutrophil mobilization. Although L. m. interacts with and triggers mast cell degranulation, bacteria were hardly found within such cells. Mainly neutrophils and macrophages phagozytosed L. m. Thus, mast cells control infection not via direct bacterial uptake, but by initiating neutrophils influx to the site of infection. We show that this is initiated by pre-synthesized TNF-alpha, rapidly secreted by mast cell upon activation by L. m. We also show that upon recruitment, neutrophils also become activated and additionally secrete TNF-alpha thus amplifying the anti-L. m. inflammatory response.
    • MatrixCatch--a novel tool for the recognition of composite regulatory elements in promoters.

      Deyneko, Igor V; Kel, Alexander E; Kel-Margoulis, Olga V; Deineko, Elena V; Wingender, Edgar; Weiss, Siegfried; Department of Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany. Igor.Deyneko@helmholtz-hzi.de (2013)
      Accurate recognition of regulatory elements in promoters is an essential prerequisite for understanding the mechanisms of gene regulation at the level of transcription. Composite regulatory elements represent a particular type of such transcriptional regulatory elements consisting of pairs of individual DNA motifs. In contrast to the present approach, most available recognition techniques are based purely on statistical evaluation of the occurrence of single motifs. Such methods are limited in application, since the accuracy of recognition is greatly dependent on the size and quality of the sequence dataset. Methods that exploit available knowledge and have broad applicability are evidently needed.
    • Metatranscriptome Analysis of the Vaginal Microbiota Reveals Potential Mechanisms for Protection against Metronidazole in Bacterial Vaginosis.

      Deng, Zhi-Luo; Gottschick, Cornelia; Bhuju, Sabin; Masur, Clarissa; Abels, Christoph; Wagner-Döbler, Irene; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-06-27)
      Bacterial vaginosis (BV) is a prevalent multifactorial disease of women in their reproductive years characterized by a shift from the
    • Methylome analysis and integrative profiling of human HCCs identify novel protumorigenic factors.

      Neumann, Olaf; Kesselmeier, Miriam; Geffers, Robert; Pellegrino, Rossella; Radlwimmer, Bernhard; Hoffmann, Katrin; Ehemann, Volker; Schemmer, Peter; Schirmacher, Peter; Lorenzo Bermejo, Justo; et al. (2012-11)
      To identify new tumor-suppressor gene candidates relevant for human hepatocarcinogenesis, we performed genome-wide methylation profiling and vertical integration with array-based comparative genomic hybridization (aCGH), as well as expression data from a cohort of well-characterized human hepatocellular carcinomas (HCCs). Bisulfite-converted DNAs from 63 HCCs and 10 healthy control livers were analyzed for the methylation status of more than 14,000 genes. After defining the differentially methylated genes in HCCs, we integrated their DNA copy-number alterations as determined by aCGH data and correlated them with gene expression to identify genes potentially silenced by promoter hypermethylation. Aberrant methylation of candidates was further confirmed by pyrosequencing, and methylation dependency of silencing was determined by 5-aza-2'-deoxycytidine (5-aza-dC) treatment. Methylation profiling revealed 2,226 CpG sites that showed methylation differences between healthy control livers and HCCs. Of these, 537 CpG sites were hypermethylated in the tumor DNA, whereas 1,689 sites showed promoter hypomethylation. The hypermethylated set was enriched for genes known to be inactivated by the polycomb repressive complex 2, whereas the group of hypomethylated genes was enriched for imprinted genes. We identified three genes matching all of our selection criteria for a tumor-suppressor gene (period homolog 3 [PER3], insulin-like growth-factor-binding protein, acid labile subunit [IGFALS], and protein Z). PER3 was down-regulated in human HCCs, compared to peritumorous and healthy liver tissues. 5-aza-dC treatment restored PER3 expression in HCC cell lines, indicating that promoter hypermethylation was indeed responsible for gene silencing. Additionally, functional analysis supported a tumor-suppressive function for PER3 and IGFALS in vitro. CONCLUSION: The present study illustrates that vertical integration of methylation data with high-resolution genomic and transcriptomic data facilitates the identification of new tumor-suppressor gene candidates in human HCC.
    • More than just a metabolic regulator--elucidation and validation of new targets of PdhR in Escherichia coli.

      Göhler, Anna-Katharina; Kökpinar, Öznur; Schmidt-Heck, Wolfgang; Geffers, Robert; Guthke, Reinhard; Rinas, Ursula; Schuster, Stefan; Jahreis, Knut; Kaleta, Christoph; Department of Genetics, University of Osnabrück, Osnabrück, Germany. (2011)
      The pyruvate dehydrogenase regulator protein (PdhR) of Escherichia coli acts as a transcriptional regulator in a pyruvate dependent manner to control central metabolic fluxes. However, the complete PdhR regulon has not yet been uncovered. To achieve an extended understanding of its gene regulatory network, we combined large-scale network inference and experimental verification of results obtained by a systems biology approach.
    • Mouse SAMHD1 Has Antiretroviral Activity and Suppresses a Spontaneous Cell-Intrinsic Antiviral Response.

      Behrendt, Rayk; Schumann, Tina; Gerbaulet, Alexander; Nguyen, Laura A; Schubert, Nadja; Alexopoulou, Dimitra; Berka, Ursula; Lienenklaus, Stefan; Peschke, Katrin; Gibbert, Kathrin; et al. (2013-08-29)
      Aicardi-Goutières syndrome (AGS), a hereditary autoimmune disease, clinically and biochemically overlaps with systemic lupus erythematosus (SLE) and, like SLE, is characterized by spontaneous type I interferon (IFN) production. The finding that defects of intracellular nucleases cause AGS led to the concept that intracellular accumulation of nucleic acids triggers inappropriate production of type I IFN and autoimmunity. AGS can also be caused by defects of SAMHD1, a 3' exonuclease and deoxynucleotide (dNTP) triphosphohydrolase. Human SAMHD1 is an HIV-1 restriction factor that hydrolyzes dNTPs and decreases their concentration below the levels required for retroviral reverse transcription. We show in gene-targeted mice that also mouse SAMHD1 reduces cellular dNTP concentrations and restricts retroviral replication in lymphocytes, macrophages, and dendritic cells. Importantly, the absence of SAMHD1 triggered IFN-β-dependent transcriptional upregulation of type I IFN-inducible genes in various cell types indicative of spontaneous IFN production. SAMHD1-deficient mice may be instrumental for elucidating the mechanisms that trigger pathogenic type I IFN responses in AGS and SLE.
    • Multiplex profiling of inflammation-related bioactive lipid mediators in Toxocara canis- and Toxocara cati-induced neurotoxocarosis.

      Waindok, Patrick; Janecek-Erfurth, Elisabeth; Lindenwald, Dimitri; Wilk, Esther; Schughart, Klaus; Geffers, Robert; Balas, Laurence; Durand, Thierry; Rund, Katharina Maria; Schebb, Nils Helge; et al. (PLOS, 2019-09-01)
      BACKGROUND: Somatic migration of Toxocara canis- and T. cati-larvae in humans may cause neurotoxocarosis (NT) when larvae accumulate and persist in the central nervous system (CNS). Host- or parasite-induced immunoregulatory processes contribute to the pathogenesis; however, detailed data on involvement of bioactive lipid mediators, e.g. oxylipins or eico-/docosanoids, which are involved in the complex molecular signalling network during infection and inflammation, are lacking. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate if T. canis- and T. cati-induced NT affects the homeostasis of oxylipins during the course of infection, a comprehensive lipidomic profiling in brains (cerebra and cerebella) of experimentally infected C57BL/6J mice was conducted at six different time points post infection (pi) by liquid-chromatography coupled to electrospray tandem mass spectrometry (LC-ESI-MS/MS). Only minor changes were detected regarding pro-inflammatory prostaglandins (cyclooxygenase pathway). In contrast, a significant increase of metabolites resulting from lipoxygenase pathways was observed for both infection groups and brain regions, implicating a predominantly anti-inflammatory driven immune response. This observation was supported by a significantly increased 13-hydroxyoctadecadienoic acid (HODE)/9-HODE ratio during the subacute phase of infection, indicating an anti-inflammatory response to neuroinfection. Except for the specialised pro-resolving mediator (SPM) neuroprotectin D1 (NPD1), which was detected in mice infected with both pathogens during the subacute phase of infection, no other SPMs were detected. CONCLUSIONS/SIGNIFICANCE: The obtained results demonstrate the influence of Toxocara spp. on oxylipins as part of the immune response of the paratenic hosts. Furthermore, this study shows differences in the alteration of the oxylipin composition between T. canis- and T. cati-brain infection. Results contribute to a further understanding of the largely unknown pathogenesis and mechanisms of host-parasite interactions during NT.
    • Murine solid tumours as a novel model to study bacterial biofilm formation in vivo.

      Pawar, V; Crull, K; Komor, U; Kasnitz, N; Frahm, M; Kocijancic, D; Westphal, K; Leschner, S; Wolf, K; Loessner, H; et al. (2014-08)
      Bacteria of many species are able to invade and colonize solid tumours in mice. We have focused on Salmonella enterica serovar Typhimurium. Detailed analysis revealed that such tumour-invading Salmonella form biofilms, thus providing a versatile in vivo test system for studying bacterial phenotypes and host-pathogen interactions. It appears that biofilm formation by S. typhimurium is induced as a defence against the immune system of the host, and in particular against neutrophils. Further, we extended our work to the clinically more relevant biofilm infection by Pseudomonas aeruginosa. The induction of P. aeruginosa biofilms in neoplastic tissue appears to be elicited as a reaction against the immune system. Reconstitution experiments reveal that T cells are responsible for biofilm induction. Isogenic mutants that are no longer able to form biofilms can be used for comparison studies to determine antimicrobial resistance, especially therapeutic efficacy against P. aeruginosa located in biofilms.
    • Murine toll-like receptor 2 activation induces type I interferon responses from endolysosomal compartments.

      Dietrich, Nicole; Lienenklaus, Stefan; Weiss, Siegfried; Gekara, Nelson O; Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany. (2010)
      BACKGROUND: Toll-like receptors (TLRs) are among the first-line sentinels for immune detection and responsiveness to pathogens. The TLR2 subfamily of TLRs (TLR1, TLR2, TLR6) form heterodimers with each other and are thus able to recognize a broad range of components from several microbes such as yeast, Gram-positive bacteria and protozoa. Until now, TLR2 activation by bacterial ligands has long been associated with pro-inflammatory cytokines but not type I interferon responses. METHODOLOGY/PRINCIPAL FINDINGS: Using a variety of transgenic mice, here we provide in vivo and in vitro data showing that TLR2 activation does in fact induce interferon-beta and that this occurs via MyD88-IRF1 and -IRF7 pathways. Interestingly, by microscopy we demonstrate that although a cell surface receptor, TLR2 dependent induction of type I interferons occurs in endolysosomal compartments where it is translocated to upon ligand engagement. Furthermore, we could show that blocking receptor internalization or endolysosomal acidification inhibits the ability of TLR2 to trigger the induction type I interferon but not pro-inflammatory responses. CONCLUSION/SIGNIFICANCE: The results indicate that TLR2 activation induces pro-inflammatory and type I interferon responses from distinct subcellular sites: the plasma membrane and endolysosomal compartments respectively. Apart from identifying and characterizing a novel pathway for induction of type I interferons, the present study offers new insights into how TLR signaling discriminates and regulates the nature of responses to be elicited against extracellular and endocytosed microbes. These findings may also have clinical implication. Excessive production of pro-inflammatory cytokines and type I IFNs following activation of TLRs is a central pathologic event in several hyper-inflammatory conditions. The discovery that the induction of pro-inflammatory and type I IFN responses can be uncoupled through pharmacological manipulation of endolysosomal acidification suggests new avenues for potential therapeutic intervention against inflammations and sepsis.
    • The mycobacterial thioredoxin peroxidase can act as a one-cysteine peroxiredoxin.

      Trujillo, Madia; Mauri, PierLuigi; Benazzi, Louise; Comini, Marcelo; De Palma, Antonella; Flohé, Leopold; Radi, Rafael; Stehr, Matthias; Singh, Mahavir; Ursini, Fulvio; et al. (2006-07-21)
      Thioredoxin peroxidase (TPx) has been reported to dominate the defense against H(2)O(2), other hydroperoxides, and peroxynitrite at the expense of thioredoxin (Trx) B and C in Mycobacterium tuberculosis (Mt). By homology, the enzyme has been classified as an atypical 2-C-peroxiredoxin (Prx), with Cys(60) as the "peroxidatic" cysteine (C(P)) forming a complex catalytic center with Cys(93) as the "resolving" cysteine (C(R)). Site-directed mutagenesis confirms Cys(60) to be C(P) and Cys(80) to be catalytically irrelevant. Replacing Cys(93) with serine leads to fast inactivation as seen by conventional activity determination, which is associated with oxidation of Cys(60) to a sulfinic acid derivative. However, in comparative stopped-flow analysis, WT-MtTPx and MtTPx C93S reduce peroxynitrite and react with TrxB and -C similarly fast. Reduction of pre-oxidized WT-MtTPx and MtTPx C93S by MtTrxB is demonstrated by monitoring the redox-dependent tryptophan fluorescence of MtTrxB. Furthermore, MtTPx C93S remains stable for 10 min at a morpholinosydnonimine hydrochloride-generated low flux of peroxynitrite and excess MtTrxB in a dihydrorhodamine oxidation model. Liquid chromatography-tandem mass spectrometry analysis revealed disulfide bridges between Cys(60) and Cys(93) and between Cys(60) and Cys(80) in oxidized WT-MtTPx. Reaction of pre-oxidized WT-MtTPx and MtTPx C93S with MtTrxB C34S or MtTrxC C40S yielded dead-end intermediates in which the Trx mutants are preferentially linked via disulfide bonds to Cys(60) and never to Cys(93) of the TPx. It is concluded that neither Cys(80) nor Cys(93) is required for the catalytic cycle of the peroxidase. Instead, MtTPx can react as a 1-C-Prx with Cys(60) being the site of attack for both the oxidizing and the reducing substrate. The role of Cys(93) is likely to conserve the oxidation equivalents of the sulfenic acid state of C(P) as a disulfide bond to prevent overoxidation of Cys(60) under a restricted supply of reducing substrate.
    • The Mycobacterium avium ssp. paratuberculosis specific mptD gene is required for maintenance of the metabolic homeostasis necessary for full virulence in mouse infections.

      Meißner, Thorsten; Eckelt, Elke; Basler, Tina; Meens, Jochen; Heinzmann, Julia; Suwandi, Abdulhadi; Oelemann, Walter M R; Trenkamp, Sandra; Holst, Otto; Weiss, Siegfried; et al. (2014)
      Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease, a chronic granulomatous enteritis in ruminants. Furthermore, infections of humans with MAP have been reported and a possible association with Crohn's disease and diabetes type I is currently discussed. MAP owns large sequence polymorphisms (LSPs) that were exclusively found in this mycobacteria species. The relevance of these LSPs in the pathobiology of MAP is still unclear. The mptD gene (MAP3733c) of MAP belongs to a small group of functionally uncharacterized genes, which are not present in any other sequenced mycobacteria species. mptD is part of a predicted operon (mptABCDEF), encoding a putative ATP binding cassette-transporter, located on the MAP-specific LSP14. In the present study, we generated an mptD knockout strain (MAPΔmptD) by specialized transduction. In order to investigate the potential role of mptD in the host, we performed infection experiments with macrophages. By this, we observed a significantly reduced cell number of MAPΔmptD early after infection, indicating that the mutant was hampered with respect to adaptation to the early macrophage environment. This important role of mptD was supported in mouse infection experiments where MAPΔmptD was significantly attenuated after peritoneal challenge. Metabolic profiling was performed to determine the cause for the reduced virulence and identified profound metabolic disorders especially in the lipid metabolism of MAPΔmptD. Overall our data revealed the mptD gene to be an important factor for the metabolic adaptation of MAP required for persistence in the host.
    • Mycobacterium tuberculosis Is a Natural Ornithine Aminotransferase (rocD) Mutant and Depends on Rv2323c for Growth on Arginine.

      Hampel, Annegret; Huber, Claudia; Geffers, Robert; Spona-Friedl, Marina; Eisenreich, Wolfgang; Bange, Franz-Christoph; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2015)
      Mycobacterium tuberculosis (Mtb) possesses a genetic repertoire for metabolic pathways, which are specific and fit to its intracellular life style. Under in vitro conditions, Mtb is known to use arginine as a nitrogen source, but the metabolic pathways for arginine utilization have not been identified. Here we show that, in the presence of arginine, Mtb upregulates a gene cluster which includes an ornithine aminotransferase (rocD) and Rv2323c, a gene of unknown function. Isotopologue analysis by using 13C- or 15N-arginine revealed that in Mtb arginine is not only used as nitrogen source but also as carbon source for the formation of amino acids, in particular of proline. Surprisingly, rocD, which is widespread in other bacteria and is part of the classical arginase pathway turned out to be naturally deleted in Mtb, but not in non-tuberculous mycobacteria. Mtb lacking Rv2323c showed a growth defect on arginine, did not produce proline from arginine, and incorporated less nitrogen derived from arginine in its core nitrogen metabolism. We conclude that the highly induced pathway for arginine utilization in Mtb differs from that of other bacteria including non-tuberculous mycobacteria, probably reflecting a specific metabolic feature of intracellular Mtb.
    • Mycobacterium tuberculosis isolates from Rio de Janeiro reveal unusually low correlation between pyrazinamide resistance and mutations in the pncA gene.

      Bhuju, Sabin; Fonseca, Leila de Souza; Marsico, Anna Grazia; de Oliveira Vieira, Gisele Betzler; Sobral, Luciana Fonseca; Stehr, Matthias; Singh, Mahavir; Saad, Maria Helena Féres; Department of Genome Analytics, Helmholtz Centre for Infection Research, Braunschweig, Germany. (2013-06-14)
      It has been widely accepted, that pyrazinamide (PZA) resistance in Mycobacterium tuberculosis is correlated with mutations in the pncA gene. But since years researchers have been puzzled by the fact that up to 30% of PZA resistant strains do not show any correlation between PZA resistance and mutations in the pncA gene, and thus may vary with geographic area. The objective of the study was to investigate the correlation between PZA susceptibility and mutations in pncA gene in M. tuberculosis isolates from individuals living in a highly endemic area. Therefore we analyzed drug resistant and multidrug resistant (MDR) isolates from patients in Rio de Janeiro, Brazil. From a total of 97 clinical isolates of M. tuberculosis 35 were identified as PZA resistant, 24/35 strains did not show PZase activity and 15/24 (62.5%) strains possess mutation in the pncA gene. This is a low correlation between PZA resistance and PZase activity (68.6%) and even lower correlation between PZA resistance and the presence of mutation in pncA gene (45.7%). Most of the mutations found were conserved near the active site or metal binding site of PZase. The 146A>C mutation was found both in PZA resistant and susceptible isolates, suggesting that this mutation may not fully associated with PZA resistance. Of the mutations found, three have not been previously described. The insertions 192-193 TCCTCGTC and 388-389 AGGTCGATG, although found before, here was found to be a short tandem repeat and in one strain, insertion of the IS6110 was observed 55nt upstream of the gene. All PZA resistant isolates had no mutation in the gene coding ribosomal protein S1 (rpsA), which has recently been proposed as alternate target for pyrazinoic acid (POA). The results show a low association of PZA resistance and pncA gene mutations in a selected patient group from an highly endemic area. Our findings point out that the phenotypic susceptibility testing remains important for the detection of PZA-resistant M. tuberculosis.
    • The mycolyltransferase 85A, a putative drug target of Mycobacterium tuberculosis: development of a novel assay and quantification of glycolipid-status of the mycobacterial cell wall.

      Elamin, Ayssar A; Stehr, Matthias; Oehlmann, Wulf; Singh, Mahavir; Department of Genome Analysis, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany. (2009-12)
      The enzymes of the antigen 85 complex (Ag85A, B, and C) possess mycolyltransferase activity and catalyze the synthesis of the most abundant glycolipid of the mycobacterial cell wall, the cord factor. The cord factor (trehalose 6,6'-dimycolate, TDM) is essential for the integrity of the mycobacterial cell wall and pathogenesis of the bacillus. Thus, TDM biosynthesis is regarded as a potential drug target for control of Mycobacterium tuberculosis infections. Trehalose 6,6'-dimycolate (TDM) is synthesized from two molecules of trehalose-6'-monomycolate (TMM) by antigen 85A. We report here a novel enzyme assay using the natural substrate TMM. The novel colorimetric assay is based on the quantification of glucose from the degradation of trehalose, which is the product from catalytic activity of antigen 85A. Using the new assay, K(m) and K(cat) were determined with values of 129.6+/-8.1 microM and 65.4+/-4.1 min(-1), respectively. This novel assay is also suitable for robust high-throughput screening (HTS) for compound library screening against mycolyltransferase (antigen 85A). The assay is significantly faster and more convenient to use than all assays currently in use. The assay has a very low coefficient of variance (0.04) in 96-well plates and shows a Z' factor of 0.67-0.73, indicating the robustness of the assay. In addition, this new assay is highly suitable for the quantification of total TMM of the mycobacterial cell envelope.