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Microbial Interface Biology

PD Dr. Norbert Reiling
PD Dr. Norbert Reiling
+49 4537 / 188-4860
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Fig0 triangle“All you have to do is breathe.” -  Infection with the causative agent of tuberculosis (TB), Mycobacterium tuberculosis, usually occurs through the air by inhalation of pathogen-containing aerosols from an infected person who coughs, sneezes, or talks. After inhalation, the pathogenic bacteria come into contact with alveolar macrophages in the lungs. Whether these cells are able to kill the bacteria depends primarily on processes that occur at the interface between M. tuberculosis and the macrophage. This points directly to the research focus of the Microbial Interface Biology Research Group (RG): the detailed characterization of the interaction of primary macrophages with pathogenic strains of the Mycobacterium tuberculosis complex (MTBC). Several new methods developed in the RG are used to isolate and characterize intracellular compartments of infected macrophages (phagosomes, macropinosomes, and lipid bodies). In addition, the RG has developed several assay systems for the rapid identification of new anti-mycobacterial agents. The discovery of truly new chemical entities with activity against MTBC strains is urgently needed because the treatment of TB requires a combination of antibiotics over several months, a strategy that is becoming increasingly complicated in times of increasing numbers of multidrug-resistant M. tuberculosis isolates. In addition, it is our goal to identify, decipher and modulate the signaling pathways induced by M. tuberculosis in its main host cell to limit the growth of the bacteria in the cells. The latter is a first and important step in the development of adjunct host-directed therapies for the treatment of pulmonary TB. This approach is of advantage since it does not bear the risk of resistance development of the bacteria. In this context, the analysis and modulation of lipid metabolism in the interaction between host cell and pathogen is an important research focus of the group. In recent years, the RG has been instrumental in several patent applications involving novel antifungal or anti-inflammatory strategies. Thus, the RG's basic science-oriented work has a clear translational perspective.

 
I. WNT signaling in tuberculosis infection - From novel mediators to host directed therapy

Building on previous work on the functional significance of pattern recognition receptors in M. tuberculosis infection, we were the first to demonstrate a regulatory function for components of the evolutionarily highly conserved Wingless/Integrase 1 (WNT) signaling pathway in pulmonary tuberculosis by systematic analyses of mycobacteria-infected macrophages. Several factors such as WNT5a, WNT3a and, as recently shown, WNT6 are important for the interaction of the innate immune system with the adaptive immune system in the pathogenesis of tuberculosis, but also in other inflammatory and infectious diseases. In this context, WNT proteins have both pro- and anti-inflammatory effects on macrophages and other cells of the immune system as well as on bacterial number development during infection.

WNT6/ACC2-induced storage of triacylglycerols in macrophages is exploited by Mycobacterium tuberculosis

We previously demonstrated a strong expression of WNT6 in granulomatous lesions in the lung of M. tuberculosis-infected mice. Detailed analysis revealed an unexpected novel role for WNT6 in macrophage function, as WNT6 impacts differentiation and proliferation of macrophages. Our observation that the majority of WNT6-expressing macrophages contain lipid vesicles led us to suggest that M. tuberculosis may induce WNT6 to promote the formation of foamy macrophages. These foam cells, which are full of lipid bodies, represent an important habitat for M. tuberculosis during tuberculosis infection. In view of emerging drug-resistant tuberculosis (TB), host-directed adjunct therapies are urgently needed to improve treatment outcomes with currently available anti-TB therapies. One option is to interfere with the above mentioned formation of lipid-laden “foamy” macrophages in the host, as they provide a nutrient-rich host cell environment for Mycobacterium tuberculosis. We now provided evidence that WNT6 promotes foam cell formation by regulating key lipid metabolic genes including acetyl-CoA carboxylase 2 (ACC2) during pulmonary TB. Using genetic and pharmacological approaches, we demonstrated that lack of functional WNT6 or ACC2 significantly reduced intracellular triacylglycerol (TAG) levels and M. tuberculosis survival in macrophages. Moreover, treatment of M. tuberculosis-infected mice with a combination of a pharmacological ACC2 inhibitor and the anti-TB drug isoniazid (INH) reduced lung TAG and cytokine levels, as well as lung weights, compared with treatment with INH alone. This combination also reduced Mtb bacterial numbers and the size of mononuclear cell infiltrates in livers of infected mice. In summary, our findings demonstrate that M. tuberculosis exploits WNT6/ACC2-induced storage of TAGs in macrophages to facilitate its intracellular survival, a finding that opens new perspectives for host-directed adjunctive treatment of pulmonary TB.

Figure 1: (A) CFU analysis of Mtb-infected (MOI 0.5:1) wild-type (WT), ACC1-KO, and ACC2-KO human macrophage–like cells (BLaER1 macrophages) at days 0 (4 h) and 3 p.i. (d 3); n = 3. (B–D) CFU analysis of Mtb-infected (MOI 1:1) hMDMs day 7 p.i. after incubation in the absence (solvent) and presence of ACC2 inhibitors; n = 3. (From Brandenburg J, et al., J Clin Invest (2022))

 

 

 

II. „TB is not TB“ – The impact of pathogen variability

Clinical isolates of the Mycobacterium tuberculosis complex (MTBC) have been shown to differ genetically significantly more than previously anticipated. In an earlier study performing infection experiments with human primary macrophages and aerosol-infected mice, we identified clade-specific virulence patterns of clinical isolates of MTBC. Exclusively human-adapted M. tuberculosis lines, also referred to as clade I or "modern" lines, such as Beijing and Haarlem isolates, show significantly increased ability to grow in human macrophages compared with “ancestral” clade II strains, which include East African Indian (EAI) and M. africanum isolates. However, a simple correlation between the virulence of the MTBC strain used and the inflammatory potential of such an isolate was not observed. Our data reveal different pathogenicity profiles that will be investigated in detail. Our work also demonstrates the need to consider pathogen-specific characteristics in addition to host-specific factors in further studies to understand host-pathogen interactions in tuberculosis.

Sub-Lineage Specific Phenolic Glycolipid Patterns in the Mycobacterium tuberculosis Complex Lineage 1

"Ancestral" Mycobacterium tuberculosis complex (MTBC) strains of Lineage 1 (L1, East African Indian) are a prominent tuberculosis (TB) cause in countries around the Indian Ocean. However, the pathobiology of L1 strains is insufficiently characterized. Here, we used whole genome sequencing (WGS) of 312 L1 strains from 43 countries to perform a characterization of the global L1 population structure and correlate this to the analysis of the synthesis of phenolic glycolipids (PGL) - known MTBC polyketide-derived virulence factors. Our results reveal the presence of eight major L1 sub-lineages, whose members have specific mutation signatures in PGL biosynthesis genes, e.g., pks15/1 or glycosyltransferases Rv2962c and/or Rv2958c. Sub-lineage specific PGL production was studied by NMR-based lipid profiling and strains with a completely abolished phenolphthiocerol dimycoserosate biosynthesis showed in average a more prominent growth in human macrophages. In conclusion, our results show a diverse population structure of L1 strains that is associated with the presence of specific PGL types. This includes the occurrence of mycoside B in one sub-lineage, representing the first description of a PGL in an M. tuberculosis lineage other than L2. Such differences may be important for the evolution of L1 strains, e.g., allowing adaption to different human populations.

Figure 2: L1 MTBC strains with a completely abolished phenolphthiocerol dimycoserosate biosynthesis due to a 1 bp insertion in Rv2946c (yes: PGL group 2; no: all other PGL groups) shown an enhanced growth in human macrophages. Shown are the means ± SEM of strains analyzed (**P = 0.0083, Mann-Whitney U test). (From Gisch N, et al., Front Microbiol. (2022))

Tuberculostearic Acid-Containing Phosphatidylinositols as Markers of Bacterial Burden in Tuberculosis

One-fourth of the global human population is estimated to be infected with strains of the Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB). Using lipidomic approaches, we show that tuberculostearic acid (TSA)-containing phosphatidylinositols (PIs) are molecular markers for infection with clinically relevant MTBC strains and signify bacterial burden. For the most abundant lipid marker, detection limits of ∼102 colony forming units (CFUs) and ∼103 CFUs for bacterial and cell culture systems were determined, respectively. We developed a targeted lipid assay, which can be performed within a day including sample preparation-roughly 30-fold faster than in conventional methods based on bacterial culture. This indirect and culture-free detection approach allowed us to determine pathogen loads in infected murine macrophages, human neutrophils, and murine lung tissue. These marker lipids inferred from mycobacterial PIs were found in higher levels in peripheral blood mononuclear cells of TB patients compared to healthy individuals. Moreover, in a small cohort of drug-susceptible TB patients, elevated levels of these molecular markers were detected at the start of therapy and declined upon successful anti-TB treatment. Thus, the concentration of TSA-containing PIs can be used as a correlate for the mycobacterial burden in experimental models and in vitro systems and may prospectively also provide a clinically relevant tool to monitor TB severity.

Figure 3: (Above) Structure of the sn-1 isomer of PI 16:0_19:0 (TSA) used as a marker for mycobacterial loads and CFUs. (left) Detection of Mtb in culture using PI 16:0_19:0 and metabolic labeling of TSA. Correlation between the amount of PI 16:0_19:0 (TSA) and CFUs (n = 3) of Mtb H37Rv (mCherry). (From Brandenburg J, et al., ACS Infect Dis. (2022))ACS Infect Dis. (2022))

 

III. “A magnet helps“ – On the use of an unusual lipopeptide and magnetic beads in tuberculosis infection biology

Pathogenic mycobacteria, after uptake by macrophages, are able to delay and block the normal maturation of the phagosome containing them. Fusion with lysosomal compartments does not occur and the pathogen survives in the cell.  To now be able to analyze structural features of phagosomes containing pathogenic mycobacteria, we have developed an immunomagnetic method - employing a biotinylated lipopeptide termed Lipobiotin – to isolate and functionally characterize these phagosomes from primary cell. The short time requirement and versatility of the method developed in the RG allows comparative biochemical and mass spectrometric analysis of mycobacteria-containing phagosomes. Our goal is to identify essential factors and mechanisms that are important for the survival of pathogenic mycobacteria and the successful killing of the pathogen by the host cell, respectively. Based on this approach we then wondered whether the same lipid used in the phagosome isolation procedure, could also be of help to enrich mycobacteria from buffers and more complex fluids.

Lipobiotin-capture magnetic bead assay for isolation, enrichment and detection of Mycobacterium tuberculosis from saliva

Pulmonary Tuberculosis (TB) is diagnosed through sputum samples. As sputum sampling is challenging in children and cachexic patients, the development of diagnostic tests using saliva appears promising but has been discouraged due to low bacterial load and poor sensitivity. Here, we present a novel and rapid method to enrich Mycobacterium tuberculosis (Mtb) from saliva, which may serve as a basis for a diagnostic saliva test. Lipobiotin-functionalized magnetic beads (LMBs) were incubated with Mtb-spiked PBS and saliva from healthy donors as well as with saliva from TB patients. Flow cytometry was used to evaluate the capacity of the beads to bind Mtb, while real-time quantitative polymerase chain reaction (qPCR) was utilized to detect Mtb and determine the amount of mycobacterial DNA in different sample types. We found that LMBs bind Mtb efficiently when compared to non-functionalized beads. The development of an qPCR assay based on the use of LMBs (LMB assay) allowed us to enrich mycobacterial DNA in spiked sample types, including PBS and saliva from healthy donors (enrichment of up to ~8.7 fold). In Mtb-spiked saliva samples, we found that the LMB assay improved the detection rate of 102 bacteria in a volume of 5 ml from 0 out of 15 (0%) to 6 out of 15 (40%). Consistent with that, the LMB assay increased the rate of correctly identified saliva samples from TB patients in two independent cohorts. Implementation of the principle of the LMB-based assay may improve the sensitivity of existing diagnostic techniques, e.g. by functionalizing materials that facilitate Mtb sampling from the oral cavity.

Figure 4. The effect of LMB assay on qPCR-based detection of Mtb in saliva. Saliva samples from healthy subjects were spiked with Mtb H37Rv and either left untreated or underwent pre-treatment with LMBs (LMB assay). The same volume of untreated or LMB pre-treated sample was subjected to heat treatment, DNA purification and qPCR analysis. Line at each condition indicates the median of data from five independent experiments each consisting of three technical replicates. Above each condition, the percentage (%) of samples in which no signal could be detected (not detectable, n.d.) is given. Ctrl, control, no bacteria added. A non-parametric analysis of variance for repeated measurements was used as a statistical test, which included data from all doses of bacteria (untreated vs LMB assay; p = 6.038205 x 10−35). (From Hansen J et al., PLOS ONE (2022))

 

 
IV. „New drugs urgently needed“ – Rapid and relevant test systems to identify novel compounds against M. tuberculosis

Due to the fact that TB is still the leading cause from a single bacterial agent worldwide, there is an urgent need for novel antibiotics to improve treatment of TB patients, in particular of those infected with drug-resistant  and multi drug resistant strains. Our expertise in dealing with primary macrophages has led us to also use our in vitro infection models with M. tuberculosis to identify and characterize the efficacy of new anti-TB lead structures. Compounds of interest are first tested for their activity against mCherry10-expressing M. tuberculosis bacteria in a 96 well-based medium throughput system. Only a one-digit mg amount of a compound is needed for the initial tests. This system, established in 2013 and continuously improved allows us to analyze small and medium sized component libraries for putative novel antiTb compounds. Following studies on the cytotoxic effect of the compounds on host cells (primary macrophages), the effect of the new compounds on intracellular bacteria is addressed using M. tuberculosis-infected human macrophages in particular will be analyzed in order to identify promising anti-TB lead structures. We are involved in targeted screens as well as phenotypic screens with different cooperation partners. We are proud to be part of the Thematic Translational Transfer Unit Tuberculosis (TTU-TB) of the German Center for Infection Research (DZIF) dealing with “New drugs and regimen” and have become partners in EU-funded Marie Skłodowska-Curie Action “MepAnti”, (representing a training network for the development of novel anti-infective drugs) as well as several BMBF funded research consortia. Recently we have set up a high-content imaging system, which will also allow us identify compounds do not target the bacteria but the host in order to identify compounds to be used in future host-directed therapy approaches.

Figure 5: Rapid and relevant test systems to identify new compounds against M. tuberculosis

 

 


 
  • “New drugs & regimens: drug screening and improving the predictive value of preclinical models” – TTU TB, Deutsches Zentrum für Infektionsforschung (DZIF), BMBF, 2021-2025
  • “Validation of the confirmed γ-glutamylspermine synthetase GlnA3 as a target for development of novel anti-tubercular drugs” - Sub project: "Relevance of the γ-glutamylspermine synthetase GlnA3 for successful growth inhibition of M. tuberculosis" (GSS-TUBTAR) – BMBF, Programm: Targetvalidierung für die pharmazeutische Wirkstoffentwicklung II, 2021-2023
  • “Isolation and Characterization of M. tuberculosis-induced lipid droplets from human primary macrophages“; Leibniz Center Infection (LCI), 2018-2021
  • “Discovering new therapeutic targets and drugs to combat AMR tuberculosis: proteomics characterization and drug screening of mycobacterium-infected macrophages" –  Olav Thon Foundation, Norwegen (in Kooperation with T. Flo (N),  M. Lerm (SE) und K. Prasad (IND), 2018-2022
  • „Exploiting the methylerythritol phosphate pathway as a source of drug targets for novel anti-infectives“ - MepAnti - Marie Skodowska-Curie Innovative Training Networks, 2020-2024
  • „NaPAnti: Development of natural product-based antibacterials targeting the sliding clamp DnaN“ - Bundesministerium für Bildung und Forschung, 2019-2022
  • „InhibitMycoRex: Optimization of mycobacterial thioredoxin reductase inhibitors, novel lead compounds against M. tuberculosis“ – Deutsches Zentrum für Infektionsforschung, 2019-2021
  • „ETBRA: European tuberculosis regimen accelerator“ – EU / Horizon 2020 (IMI), 2020-

 
  • Isolation and cultivation of primary immune cells  
    • murine bone marrow derived macrophages
    • human monocytes
    • human monocyte-derived macrophages
    • humane alveolar macrophages
  • Infection of primary cells with M. tuberculosis (Mtb) under BSL3 conditions  in vitro
  • Magnetic isolation and characterization
    • Mtb-containing intracellular  compartments
    • macropinosomes
  • Isolation und functional characterisation of lipid droplets 
  • CRISPR/CAS9 in human immune cells
  • Transfection of human macrophages
  • Quantitative RT-PCR (Light Cycler)
  • ELISA
  • Histology and immunohistochemistry
  • Flow cytometry of mycobacteria and Mtb infected immune cells (BSL3)
  • Aerosol infection of immunocompetent and gene deficient mice with Mtb
  • Step wise identification of novel compounds with antimycobacterial activity
    • 96-well format based anti-Tb activity tests using GFP- und mCherry-expr. Mtb (H37Rv)
    •  Macrophage cytotoxicity tests using real time impedance measurements (xCelligence)
    • Activity tests using M. tuberculosis-infected human primary macrophages
    • Activity tests against drug susceptible, MDR and XDR-Mtb clinical isolates (in coop. with Dr. Doris Hillemann, National Reference Center) (MGIT format)

2024

Braun-Cornejo, M, Ornago, C, Sonawane, V, Haupenthal, J, Kany, AM, Diamanti, E, Jézéquel, G, Reiling, N, Blankenfeldt, W & Maas, P et al. 2024, 'Target-Directed Dynamic Combinatorial Chemistry Affords Binders of Mycobacterium tuberculosis IspE', ACS omega, Jg. 9, Nr. 36, S. 38160-38168. https://doi.org/10.1021/acsomega.4c05537

Kotimoole, CN, Ramya, VK, Kaur, P, Reiling, N, Shandil, RK, Narayanan, S, Flo, TH & Prasad, TSK 2024, 'Discovery of Species-Specific Proteotypic Peptides To Establish a Spectral Library Platform for Identification of Nontuberculosis Mycobacteria from Mass Spectrometry-Based Proteomics', JOURNAL OF PROTEOME RESEARCH , Jg. 23, Nr. 3, S. 1102-1117. https://doi.org/10.1021/acs.jproteome.3c00850

Sao Emani, C & Reiling, N 2024, 'The efflux pumps Rv1877 and Rv0191 play differential roles in the protection of Mycobacterium tuberculosis against chemical stress', Frontiers in Microbiology, Jg. 15, S. 1359188. https://doi.org/10.3389/fmicb.2024.1359188

 

2023

Du, X, Sonawane, V, Zhang, B, Wang, C, de Ruijter, B, Dömling, ASS, Reiling, N & Groves, MR 2023, 'Inhibitors of Aspartate Transcarbamoylase inhibit Mycobacterium tuberculosis growth', ChemMedChem, S. e202300279. https://doi.org/10.1002/cmdc.202300279

Johannsen, S, Gierse, RM, Olshanova, A, Smerznak, E, Laggner, C, Eschweiler, L, Adeli, Z, Hamid, R, Alhayek, A, Reiling, N, Haupenthal, J & Hirsch, AKH 2023, 'Not Every Hit-Identification Technique Works on 1-Deoxy-d-Xylulose 5-Phosphate Synthase (DXPS): Making the Most of a Virtual Screening Campaign', ChemMedChem, Jg. 18, Nr. 11, S. e202200590. https://doi.org/10.1002/cmdc.202200590

Kotimoole, C, Antil, N, Kasaragod, S, Behera, S, Arvind, A, Reiling, N, Flo, T & Prasad, T 2023, 'Development of a spectral library for the discovery of altered genomic events in Mycobacterium avium associated with virulence using mass spectrometry-based proteogenomic analysis', MOLECULAR & CELLULAR PROTEOMICS, Jg. 22, Nr. 5, S. 100533. https://doi.org/10.1016/j.mcpro.2023.100533

Seitz, L, Reiling, N & Hilgeroth, A 2023, 'Synthesis and Evaluation of Novel Substituted N-Aryl 1,4-Dihydropyridines as An-tituberculostatic Agents', Medicinal Chemistry. https://doi.org/10.2174/1573406419666230622121512

 

2022

Aryal N, Chen J, Bhattarai K, Hennrich O, Handayani I, Kramer M, Straetener J, Wommer T, Berscheid A, Peter S, Reiling N, Brötz-Oesterhelt H, Geibel C, Lämmerhofer M, Mast Y, Gross H. High Plasticity of the Amicetin Biosynthetic Pathway in Streptomyces sp. SHP 22-7 Led to the Discovery of Streptcytosine P and Cytosaminomycins F and G and Facilitated the Production of 12F-Plicacetin. J NAT PROD. 2022 Mar 25;85(3):530-539. doi: 10.1021/acs.jnatprod.1c01051. Epub 2022 Mar 9.

Brandenburg J, Heyckendorf J, Marwitz F, Zehethofer N, Linnemann L, Gisch N, Karaköse H, Reimann M, Kranzer K, Kalsdorf B, Sanchez-Carballo P, Weinkauf M, Scholz V, Malm S, Homolka S, Gaede KI, Herzmann C, Schaible UE, Hölscher C, Reiling N, Schwudke D. Tuberculostearic Acid-Containing Phosphatidylinositols as Markers of Bacterial Burden in Tuberculosis. ACS INFECT DIS. 2022 Jul 8;8(7):1303-1315. doi: 10.1021/acsinfecdis.2c00075.

Gisch N, Utpatel C, Gronbach LM, Kohl TA, Schombel U, Malm S, Dobos KM, Hesser DC, Diel R, Götsch U, Gerdes S, Shuaib YA, Ntinginya NE, Khosa C, Viegas S, Kerubo G, Ali S, Al-Hajoj SA, Ndung'u PW, Rachow A, Hoelscher M, Maurer FP, Schwudke D, Niemann S, Reiling N, Homolka S. Sub-Lineage Specific Phenolic Glycolipid Patterns in the Mycobacterium tuberculosis Complex Lineage 1. FRONT MICROBIOL. 2022 Mar 8;13:832054. doi: 10.3389/fmicb.2022.832054.

Hansen J, Kolbe K, König IR, Scherließ R, Hellfritzsch M, Malm S, Müller-Loennies S, Zallet J, Hillemann D, Wiesmüller KH, Herzmann C, Brandenburg J, Reiling N. Lipobiotin-capture magnetic bead assay for isolation, enrichment and detection of Mycobacterium tuberculosis from saliva. PLOS ONE. 2022 Jul 15;17(7):e0265554. doi: 10.1371/journal.pone.0265554.

Richter A, Seidel RW, Goddard R, Eckhardt T, Lehmann C, Dörner J, Siersleben F, Sondermann T, Mann L, Patzer M, Jäger C, Reiling N, Imming P. BTZ-Derived Benzisothiazolinones with In Vitro Activity against Mycobacterium tuberculosis. ACS MED. CHEM. LETT. 2022, July 25, 2022 epub ahead of print. https://doi.org/10.1021/acsmedchemlett.2c00215

Zhu D, Johannsen S, Masini T, Simonin C, Haupenthal J, Illarionov B, Andreas A, Awale M, Gierse RM, van der Laan T, van der Vlag R, Nasti R, Poizat M, Buhler E, Reiling N, Müller R, Fischer M, Reymond JL & Hirsch A. Discovery of novel drug-like antitubercular hits targeting the MEP pathway enzyme DXPS by strategic application of ligand-based virtual screening. CHEM SCI. 2022, doi: 10.1039/D2SC02371G Epub 2022 Aug 8.

 

Head

PD Dr. Norbert Reiling
PD Dr. Norbert Reiling
+49 4537 / 188-4860
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Scientific staff

Dr. Gareth Prosser
Dr. Gareth Prosser
+49 4537 / 188-5855
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Vidhisha Sonawane
Vidhisha Sonawane
+49 4537 / 188-5855
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Linda Zemke
Linda Zemke
+49 4537 / 188-5855
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Technical staff

 
Merve Breiholz
+49 4537 / 188-5940
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Svenja Goldenbaum
Svenja Goldenbaum
+49 4537 / 188-5940
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Carolin Möller (geb. Golin)
Carolin Möller (geb. Golin)
+49 4537 / 188-6870
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Lisa Niwinski
Lisa Niwinski
+49 4537 / 188-6870
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Last Update: 29.11.2023