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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

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

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 approach is to interfere with the formation of lipid-laden "foamy" macrophages in the host, as they provide a nutrient-rich host cell environment for Mycobacterium tuberculosis (Mtb). Here, we provide evidence that Wnt family member 6 (WNT6), a ligand of the evolutionarily conserved Wingless/Integrase 1 (WNT) signaling pathway, 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 Mtb survival in macrophages. Moreover, treatment of Mtb-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 Mtb 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.

Anti-Infective and Anti-Inflammatory Mode of Action of Peptide 19-2.5.

The polypeptide Pep19-2.5 (Aspidasept®) has been described to act efficiently against infection-inducing bacteria by binding and neutralizing their most potent toxins, i.e., lipopolysaccharides (LPS) and lipoproteins/peptides (LP), independent of the resistance status of the bacteria. The mode of action was described to consist of a primary Coulomb/polar interaction of the N-terminal region of Pep19-2.5 with the polar region of the toxins followed by a hydrophobic interaction of the C-terminal region of the peptide with the apolar moiety of the toxins. However, clinical development of Aspidasept as an anti-sepsis drug requires an in-depth characterization of the interaction of the peptide with the constituents of the human immune system and with other therapeutically relevant compounds such as antibiotics and non-steroidal anti-inflammatory drugs (NSAIDs). In this contribution, relevant details of primary and secondary pharmacodynamics, off-site targets, and immunogenicity are presented, proving that Pep19-2.5 may be readily applied therapeutically against the deleterious effects of a severe bacterial infection.

Inactivation of Bacteria by γ-Irradiation to Investigate the Interaction with Antimicrobial Peptides.

The activity of antimicrobial peptides (AMPs) has been investigated extensively using model membranes composed of phospholipids or lipopolysaccharides in aqueous environments. However, from a biophysical perspective, there is a large scientific interest regarding the direct interaction of membrane-active peptides with whole bacteria. Working with living bacteria limits the usability of experimental setups and the interpretation of the resulting data because of safety risks and the overlap of active and passive effects induced by AMPs. We killed or inactivated metabolic-active bacteria using γ-irradiation or sodium azide, respectively. Microscopy, flow cytometry, and SYTOX green assays showed that the cell envelope remained intact to a high degree at the minimal bactericidal dose. Furthermore, the tumor-necrosis-factor-α-inducing activity of the lipopolysaccharides and the chemical lipid composition was unchanged. Determining the binding capacity of AMPs to the bacterial cell envelope by calorimetry is difficult because of an overlapping of the binding heat and metabolic activities of the bacteria-induced by the AMPs. The inactivation of all active processes helps to decipher the complex thermodynamic information. From the isothermal titration calorimetry (ITC) results, we propose that the bacterial membrane potential (Δψ) is possibly an underestimated modulator of the AMP activity. The negative surface charge of the outer leaflet of the outer membrane of Gram-negative bacteria is already neutralized by peptide concentrations below the minimal inhibitory concentration. This proves that peptide aggregation on the bacterial membrane surface plays a decisive role in the degree of antimicrobial activity. This will not only enable many biophysical approaches for the investigation between bacteria and membrane-active peptides in the future but will also make it possible to compare biophysical parameters of active and inactive bacteria. This opens up new possibilities to better understand the active and passive interaction processes between AMPs and bacteria.

The Multi-Modal Effect of the Anti-fibrotic Drug Pirfenidone on NSCLC.

Although immune checkpoint and targeted therapies offer remarkable benefits for lung cancer treatment, some patients do not qualify for these regimens or do not exhibit consistent benefit. Provided that lung cancer appears to be driven by transforming growth factor beta signaling, we investigated the single drug potency of Pirfenidone, an approved drug for the treatment of lung fibrosis. Five human lung cancer cell lines and one murine line were investigated for transforming growth factor beta inhibition via Pirfenidone by using flow cytometry, In-Cell western analysis, proliferation assays as well as comprehensive analyses of the transcriptome with subsequent bioinformatics analysis. Overall, Pirfenidone induced cell cycle arrest, down-regulated SMAD expression and reduced proliferation in lung cancer. Furthermore, cell stress pathways and pro-apoptotic signaling may be mediated by reduced expression of Survivin. A murine subcutaneous model was used to assess the in vivo drug efficacy of Pirfenidone and showed reduced tumor growth and increased infiltration of T cells and NK cells. This data warrant further clinical evaluation of Pirfenidone with advanced non-small cell lung cancer. The observed in vitro and in vivo effects point to a substantial benefit for using Pirfenidone to reactivate the local immune response and possible application in conjunction with current immunotherapies.

Shaping the niche in macrophages: Genetic diversity of the M. tuberculosis complex and its consequences for the infected host.

Pathogenic mycobacteria of the Mycobacterium tuberculosis complex (MTBC) have co-evolved with their individual hosts and are able to transform the hostile environment of the macrophage into a permissive cellular habitat. The impact of MTBC genetic variability has long been considered largely unimportant in TB pathogenesis. Members of the MTBC can now be distinguished into three major phylogenetic groups consisting of 7 phylogenetic lineages and more than 30 so called sub-lineages/subgroups. MTBC genetic diversity indeed influences the transmissibility and virulence of clinical MTBC isolates as well as the immune response and the clinical outcome. Here we review the genetic diversity and epidemiology of MTBC strains and describe the current knowledge about the host immune response to infection with MTBC clinical isolates using human and murine experimental model systems in vivo and in vitro. We discuss the role of innate cytokines in detail and portray two in our group recently developed approaches to characterize the intracellular niches of MTBC strains. Characterizing the niches and deciphering the strategies of MTBC strains to transform an antibacterial effector cell into a permissive cellular habitat offers the opportunity to identify strain- and lineage-specific key factors which may represent targets for novel antimicrobial or host directed therapies for tuberculosis.

Azido Pentoses: A New Tool To Efficiently Label Mycobacterium tuberculosis Clinical Isolates.

Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (Tb), has a complex cell envelope which forms an efficient barrier to antibiotics, thus contributing to the challenges of anti-tuberculosis therapy. However, the unique Mtb cell wall can be considered an advantage and be utilized to selectively label Mtb bacteria. Here we introduce three azido pentoses as new compounds for metabolic labeling of Mtb: 3-azido arabinose (3AraAz), 3-azido ribose (3RiboAz), and 5-azido arabinofuranose (5AraAz). 5AraAz demonstrated the highest level of Mtb labeling and was efficiently incorporated into the Mtb cell wall. All three azido pentoses can be easily used to label a variety of Mtb clinical isolates without influencing Mtb-dependent phagosomal maturation arrest in infection studies with human macrophages. Thus, this metabolic labeling method offers the opportunity to attach desired molecules to the surface of Mtb bacteria in order to facilitate investigation of the varying virulence characteristics of different Mtb clinical isolates, which influence the outcome of a Tb infection.

The bacillary and macrophage response to hypoxia in tuberculosis and the consequences for T cell antigen recognition.

Mycobacterium tuberculosis is a facultative anaerobe and its characteristic pathological hallmark, the granuloma, exhibits hypoxia in humans and in most experimental models. Thus the host and bacillary adaptation to hypoxia is of central importance in understanding pathogenesis and thereby to derive new drug treatments and vaccines.

The Wnt Blows: On the Functional Role of Wnt Signaling in Mycobacterium tuberculosis Infection and Beyond.

In recent years, it has become apparent that the Wnt signaling pathway, known for its essential functions in embryonic development and tissue homeostasis, exerts immunomodulatory functions during inflammation and infection. Most functional studies indicate that Wnt5a exerts pro-inflammatory functions on its cellular targets, which include various types of immune and non-immune cells. Wnt5a expression has also been linked to the pathogenesis of chronic inflammatory diseases. Activation of beta-catenin-dependent Wnt signaling, e.g., by Wnt3a, has however been shown to limit inflammation by interfering with the nuclear factor kappa-light chain-enhancer of activated B-cells (NF-kappaB) pathway. This review focuses on the regulation of Wnt5a, Wnt3a, and the recently identified Wnt6 and their functional role in bacterial infections with a primary focus on pulmonary tuberculosis, a leading infectious cause of morbidity and mortality worldwide.

Wnt6 is expressed in granulomatous lesions of Mycobacterium tuberculosis-infected mice and is involved in macrophage differentiation and proliferation.

The Wnt signaling network, an ancient signaling system governing ontogeny and homeostatic processes, has recently been identified to exert immunoregulatory functions in a variety of inflammatory and infectious disease settings including tuberculosis. In this study, we show that Wnt6 is expressed in granulomatous lesions in the lung of Mycobacterium tuberculosis-infected mice. We identified foamy macrophage-like cells as the primary source of Wnt6 in the infected lung and uncovered a TLR-MyD88-NF-κB-dependent mode of induction in bone marrow-derived macrophages. Analysis of Wnt6-induced signal transduction revealed a pertussis toxin-sensitive, ERK-mediated, but ß-catenin-independent induction of c-Myc, a master regulator of cell proliferation. Increased Ki-67 mRNA expression levels and enhanced thymidine incorporation in Wnt6-treated macrophage cultures demonstrate a proliferation-promoting effect on murine macrophages. Further functional studies in M. tuberculosis-infected macrophages using Wnt6 conditioned medium and Wnt6-deficient macrophages uncovered a Wnt6-dependent induction of macrophage Arginase-1 and downregulation of TNF-α. This identifies Wnt6 as a novel factor driving macrophage polarization toward an M2-like phenotype. Taken together, these findings point to an unexpected role for Wnt6 in macrophage differentiation in the M. tuberculosis-infected lung.

Clade-specific virulence patterns of Mycobacterium tuberculosis complex strains in human primary macrophages and aerogenically infected mice.

UNLABELLED: In infection experiments with genetically distinct Mycobacterium tuberculosis complex (MTBC) strains, we identified clade-specific virulence patterns in human primary macrophages and in mice infected by the aerosol route, both reflecting relevant model systems. Exclusively human-adapted M. tuberculosis lineages, also termed clade I, comprising "modern" lineages, such as Beijing and Euro-American Haarlem strains, showed a significantly enhanced capability to grow compared to that of clade II strains, which include "ancient" lineages, such as, e.g., East African Indian or M. africanum strains. However, a simple correlation of inflammatory response profiles with strain virulence was not apparent. Overall, our data reveal three different pathogenic profiles: (i) strains of the Beijing lineage are characterized by low uptake, low cytokine induction, and a high replicative potential, (ii) strains of the Haarlem lineage by high uptake, high cytokine induction, and high growth rates, and (iii) EAI strains by low uptake, low cytokine induction, and a low replicative potential. Our findings have significant implications for our understanding of host-pathogen interaction and factors that modulate the outcomes of infections. Future studies addressing the underlying mechanisms and clinical implications need to take into account the diversity of both the pathogen and the host. IMPORTANCE: Clinical strains of the Mycobacterium tuberculosis complex (MTBC) are genetically more diverse than previously anticipated. Our analysis of mycobacterial growth characteristics in primary human macrophages and aerogenically infected mice shows that the MTBC genetic differences translate into pathogenic differences in the interaction with the host. Our study reveals for the first time that "TB is not TB," if put in plain terms. We are convinced that it is very unlikely that a single molecular mechanism may explain the observed effects. Our study refutes the hypothesis that there is a simple correlation between cytokine induction as a single functional parameter of host interaction and mycobacterial virulence. Instead, careful consideration of strain- and lineage-specific characteristics must guide our attempts to decipher what determines the pathological potential and thus the outcomes of infection with MTBC, one of the most important human pathogens.

Preclinical investigations reveal the broad-spectrum neutralizing activity of peptide Pep19-2.5 on bacterial pathogenicity factors.

Bacterial infections are known to cause severe health-threatening conditions, including sepsis. All attempts to get this disease under control failed in the past, and especially in times of increasing antibiotic resistance, this leads to one of the most urgent medical challenges of our times. We designed a peptide to bind with high affinity to endotoxins, one of the most potent pathogenicity factors involved in triggering sepsis. The peptide Pep19-2.5 reveals high endotoxin neutralization efficiency in vitro, and here, we demonstrate its antiseptic/anti-inflammatory effects in vivo in the mouse models of endotoxemia, bacteremia, and cecal ligation and puncture, as well as in an ex vivo model of human tissue. Furthermore, we show that Pep19-2.5 can bind and neutralize not only endotoxins but also other bacterial pathogenicity factors, such as those from the Gram-positive bacterium Staphylococcus aureus. This broad neutralization efficiency and the additive action of the peptide with common antibiotics makes it an exceptionally appropriate drug candidate against bacterial sepsis and also offers multiple other medication opportunities.

Lipid-labeling facilitates a novel magnetic isolation procedure to characterize pathogen-containing phagosomes.

Here we describe a novel approach for the isolation and biochemical characterization of pathogen-containing compartments from primary cells: We developed a lipid-based procedure to magnetically label the surface of bacteria and visualized the label by scanning and transmission electron microscopy (SEM, TEM). We performed infection experiments with magnetically labeled Mycobacterium avium, M. tuberculosis and Listeria monocytogenes and isolated magnetic bacteria-containing phagosomes using a strong magnetic field in a novel free-flow system. Magnetic labeling of M. tuberculosis did not affect the virulence characteristics of the bacteria during infection experiments addressing host cell activation, phagosome maturation delay and replication in macrophages in vitro. Biochemical analyses of the magnetic phagosome-containing fractions provided evidence of an enhanced presence of bacterial antigens and a differential distribution of proteins involved in the endocytic pathway over time as well as cytokine-dependent changes in the phagosomal protein composition. The newly developed method represents a useful approach to characterize and compare pathogen-containing compartments, in order to identify microbial and host cell targets for novel anti-infective strategies.