Priority Research Area Infections

Microbial Interface Biology

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Projects

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))

 

 

 

 

 

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