Search

In the present study we investigated the influence of pentoxifylline (POF) on bacillus CalmetteGuérin (BCG)- and phytohaemagglutinin (PHA)-induced DNA synthesis and cytokine release, and BCG-induced cytotoxicity of human peripheral blood mononuclear cells (PBMC). DNA synthesis of PBMC stimulated with either BCG or PHA was inhibited by POF. We also demonstrated that the addition of POF led to a POF dose-dependent decrease of the release of the cytokines interleukin (IL)-2, interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α). The release of IL-6 remained unaffected. With respect to the inhibition of BCG-induced IL-2 and IFN-γ release POF is active at the transcriptional (mRNA) level, as found by polymerase chain reaction (PCR). However, PHA induced mRNA expression of these lymphokines is not affected by POF. Thus, the existence of a post-transcriptional regulation of PHA-induced cytokine release by POF can be assumed. The observed inhibition of cytokine release is correlated with a potent inhibitory effect of POF on BCG-induced cytotoxicity against bladder tumour cell lines. This effect is reversible. [ABSTRACT FROM AUTHOR]

In the search for new antitubercular compounds, we leveraged target-directed dynamic combinatorial chemistry (tdDCC) as an efficient hit-identification method. In tdDCC, the target selects its own binders from a dynamic library generated in situ , reducing the number of compounds that require synthesis and evaluation. We combined a total of 12 hydrazides and six aldehydes to generate 72 structurally diverse N -acylhydrazones. To amplify the best binders, we employed anti-infective target 4-diphosphocytidyl-2 C -methyl-d-erythritol kinase (IspE) from Mycobacterium tuberculosis ( Mtb ). We successfully validated the use of tdDCC as hit-identification method for IspE and optimized the analysis of tdDCC hit determination. From the 72 possible N -acylhydrazones, we synthesized 12 of them, revealing several new starting points for the development of IspE inhibitors as antibacterial agents., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Background: It was previously shown that GlnA3 sc enabled Streptomyces coelicolor to survive in excess polyamines. However, subsequent studies revealed that Rv1878, the corresponding Mycobacterium tuberculosis (M.tb) ortholog, was not essential for the detoxification of spermine (Spm), in M.tb. On the other hand, the multi-drug efflux pump Rv1877 was previously shown to enable export of a wide range of compounds, while Rv0191 was shown to be more specific to chloramphenicol., Rationale: Therefore, we first wanted to determine if detoxification of Spm by efflux can be achieved by any efflux pump, or if that was dependent upon the function of the pump. Next, since Rv1878 was found not to be essential for the detoxification of Spm, we sought to follow-up on the investigation of the physiological role of Rv1878 along with Rv1877 and Rv0191., Approach: To evaluate the specificity of efflux pumps in the mycobacterial tolerance to Spm, we generated unmarked ∆ rv1877 and ∆ rv0191 M.tb mutants and evaluated their susceptibility to Spm. To follow up on the investigation of any other physiological roles they may have, we characterized them along with the ∆ rv1878 M.tb mutant., Results: The ∆ rv1877 mutant was sensitive to Spm stress, while the ∆ rv0191 mutant was not. On the other hand, the ∆ rv1878 mutant grew better than the wild-type during iron starvation yet was sensitive to cell wall stress. The proteins Rv1877 and Rv1878 seemed to play physiological roles during hypoxia and acidic stress. Lastly, the ∆ rv0191 mutant was the only mutant that was sensitive to oxidative stress., Conclusion: The multidrug MFS-type efflux pump Rv1877 is required for Spm detoxification, as opposed to Rv0191 which seems to play a more specific role. Moreover, Rv1878 seems to play a role in the regulation of iron homeostasis and the reconstitution of the cell wall of M.tb. On the other hand, the sensitivity of the ∆ rv0191 mutant to oxidative stress, suggests that Rv0191 may be responsible for the transport of low molecular weight thiols., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Sao Emani and Reiling.)

Importance: This is the first study that attempted to demonstrate the mechanisms of reactive oxygen species (ROS) generation by spermine (Spm) in Mycobacterium tuberculosis (M.tb). Furthermore, this is the first study to demonstrate that it is able to enhance the activity of currently available and World Health Organization (WHO)-approved tuberculosis (TB) drugs. Spermine can easily be obtained since it is already found in our diet. Moreover, as opposed to conventional antibiotics, it is less toxic to humans since it is found in millimolar concentrations in the body. Finally, with the difficulty of curing TB with conventional antibiotics, this study suggests that less toxic molecules, such as Spm, could in a long-term perspective be incorporated in a TB regimen to boost the treatment., Competing Interests: The authors declare no conflict of interest.

Mycobacterium avium is one of the prominent disease-causing bacteria in humans. It causes lymphadenitis, chronic and extrapulmonary, and disseminated infections in adults, children, and immunocompromised patients. M. avium has ∼4500 predicted protein-coding regions on average, which can help discover several variants at the proteome level. Many of them are potentially associated with virulence; thus, identifying such proteins can be a helpful feature in developing panel-based theranostics. In line with such a long-term goal, we carried out an in-depth proteomic analysis of M. avium with both data-dependent and data-independent acquisition methods. Further, a set of proteogenomic investigations were carried out using (i) a protein database for Mycobacterium tuberculosis, (ii) an M. avium genome six-frame-translated database, and (iii) a variant protein database of M. avium. A search of mass spectrometry data against M. avium protein database resulted in identifying 2954 proteins. Further, proteogenomic analyses aided in identifying 1301 novel peptide sequences and correcting translation start sites for 15 proteins. Ultimately, we created a spectral library of M. avium proteins, including novel genome search-specific peptides and variant peptides detected in this study. We validated the spectral library by a data-independent acquisition of the M. avium proteome. Thus, we present an M. avium spectral library of 29,033 peptide precursors supported by 0.4 million fragment ions for further use by the biomedical community., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

In the present manuscript, we describe how we successfully used ligand-based virtual screening (LBVS) to identify two small-molecule, drug-like hit classes with excellent ADMET profiles against the difficult to address microbial enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS). In the fight against antimicrobial resistance (AMR), it has become increasingly important to address novel targets such as DXPS, the first enzyme of the 2- C -methyl-d-erythritol-4-phosphate (MEP) pathway, which affords the universal isoprenoid precursors. This pathway is absent in humans but essential for pathogens such as Mycobacterium tuberculosis , making it a rich source of drug targets for the development of novel anti-infectives. Standard computer-aided drug-design tools, frequently applied in other areas of drug development, often fail for targets with large, hydrophilic binding sites such as DXPS. Therefore, we introduce the concept of pseudo-inhibitors, combining the benefits of pseudo-ligands (defining a pharmacophore) and pseudo-receptors (defining anchor points in the binding site), for providing the basis to perform a LBVS against M. tuberculosis DXPS. Starting from a diverse set of reference ligands showing weak inhibition of the orthologue from Deinococcus radiodurans DXPS, we identified three structurally unrelated classes with promising in vitro (against M. tuberculosis DXPS) and whole-cell activity including extensively drug-resistant strains of M. tuberculosis . The hits were validated to be specific inhibitors of DXPS and to have a unique mechanism of inhibition. Furthermore, two of the hits have a balanced profile in terms of metabolic and plasma stability and display a low frequency of resistance development, making them ideal starting points for hit-to-lead optimization of antibiotics with an unprecedented mode of action., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

8-Nitro-1,3-benzothiazin-4-ones (BTZs) are known as potent antitubercular agents. BTZ043 as one of the most advanced compounds has reached clinical trials. The putative oxidation products of BTZ043, namely, the corresponding BTZ sulfoxide and sulfone, were reported in this journal (Tiwari et al. ACS Med. Chem Lett. 2015 , 6 , 128-133). The molecular structures were later revised to the constitutionally isomeric benzisothiazolone and its 1-oxide, respectively. Here, we report two BTZ043-derived benzisothiazolinones (BITs) with in vitro activity against mycobacteria. The constitutionally isomeric O -acyl benzisothiazol-3-ols, in contrast, show little or no antimycobacterial activity in vitro. The structures of the four compounds were investigated by X-ray crystallography and NMR spectroscopy. Molecular covalent docking of the new compounds to Mycobacerium tuberculosis decaprenylphosphoryl-β-d-ribose 2'-epimerase (DprE1) suggests that the active BITs exert antimycobacterial activity through inhibition of DprE1 like BTZs., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

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., Competing Interests: CH reports that he obtains personal fees from Janssen outside the submitted work. The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

"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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gisch, Utpatel, Gronbach, Kohl, Schombel, Malm, Dobos, Hesser, Diel, Götsch, Gerdes, Shuaib, Ntinginya, Khosa, Viegas, Kerubo, Ali, Al-Hajoj, Ndung’u, Rachow, Hoelscher, Maurer, Schwudke, Niemann, Reiling and Homolka.)

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.

Target-directed dynamic combinatorial chemistry (tdDCC) enables identification, as well as optimization of ligands for un(der)explored targets such as the anti-infective target 1-deoxy-d-xylulose-5-phosphate synthase (DXPS). We report the use of tdDCC to first identify and subsequently optimize binders/inhibitors of the anti-infective target DXPS. The initial hits were also optimized for their antibacterial activity against E. coli and M. tuberculosis during subsequent tdDCC runs. Using tdDCC, we were able to generate acylhydrazone-based inhibitors of DXPS. The tailored tdDCC runs also provided insights into the structure-activity relationship of this novel class of DXPS inhibitors. The competition tdDCC runs provided important information about the mode of inhibition of acylhydrazone-based inhibitors. This approach holds the potential to expedite the drug-discovery process and should be applicable to a range of biological targets., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

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.

Shigella flexneri invades host cells by entering within a bacteria-containing vacuole (BCV). In order to establish its niche in the host cytosol, the bacterium ruptures its BCV. Contacts between S. flexneri BCV and infection-associated macropinosomes (IAMs) formed in situ have been reported to enhance BCV disintegration. The mechanism underlying S. flexneri vacuolar escape remains however obscure. To decipher the molecular mechanism priming the communication between the IAMs and S. flexneri BCV, we performed mass spectrometry-based analysis of the magnetically purified IAMs from S. flexneri-infected cells. While proteins involved in host recycling and exocytic pathways were significantly enriched at the IAMs, we demonstrate more precisely that the S. flexneri type III effector protein IpgD mediates the recruitment of the exocyst to the IAMs through the Rab8/Rab11 pathway. This recruitment results in IAM clustering around S. flexneri BCV. More importantly, we reveal that IAM clustering subsequently facilitates an IAM-mediated unwrapping of the ruptured vacuole membranes from S. flexneri, enabling the naked bacterium to be ready for intercellular spread via actin-based motility. Taken together, our work untangles the molecular cascade of S. flexneri-driven host trafficking subversion at IAMs to develop its cytosolic lifestyle, a crucial step en route for infection progression at cellular and tissue level., Competing Interests: The authors have declared that no competing interests exist.

Mycobacterium tuberculosis subverts host immunity to proliferate within host tissues. Non-selective transient receptor potential (TRP) ion channels are involved in host responses and altered upon bacterial infections. Altered expression and localization of TRPV4 in macrophages upon virulent M. tuberculosis infection together with differential distribution of TRPV4 in human tuberculosis (TB) granulomas indicate a role of TRPV4 in TB. Compared with wild-type mice, Trpv4-deficient littermates showed transiently higher mycobacterial burden and reduced proinflammatory responses. In the absence of TRPV4, activation failed to render macrophages capable of controlling mycobacteria. Surprisingly, Trpv4-deficient mice were superior to wild-type ones in controlling M. tuberculosis infection in the chronic phase. Thus, Trpv4 is important in host responses to mycobacteria, although with opposite functions early versus late in infection. Ameliorated chronic infection in the absence of Trpv4 and its expression in human TB lesions indicate TRPV4 as putative target for host-directed therapy., Competing Interests: Declaration of Interests The authors have no conflict of interest., (Copyright © 2020. Published by Elsevier Inc.)

Translation of consecutive proline motifs causes ribosome stalling and requires rescue via the action of a specific translation elongation factor, EF-P in bacteria and archaeal/eukaryotic a/eIF5A. In Eukarya, Archaea, and all bacteria investigated so far, the functionality of this translation elongation factor depends on specific and rather unusual post-translational modifications. The phylum Actinobacteria, which includes the genera Corynebacterium, Mycobacterium, and Streptomyces, is of both medical and economic significance. Here, we report that EF-P is required in these bacteria in particular for the translation of proteins involved in amino acid and secondary metabolite production. Notably, EF-P of Actinobacteria species does not need any post-translational modification for activation. While the function and overall 3D structure of this EF-P type is conserved, the loop containing the conserved lysine is flanked by two essential prolines that rigidify it. Actinobacteria's EF-P represents a unique subfamily that works without any modification., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2020 Marwitz, Turkowski, Nitschkowski, Weigert, Brandenburg, Reiling, Thomas, Reck, Drömann, Seeger, Rabe, Savai and Goldmann.)

Salmonella is a human and animal pathogen that causes gastro-enteric diseases. The key to Salmonella infection is its entry into intestinal epithelial cells, where the bacterium resides within a Salmonella-containing vacuole (SCV). Salmonella entry also induces the formation of empty macropinosomes, distinct from the SCV, in the vicinity of the entering bacteria. A few minutes after its formation, the SCV increases in size through fusions with the surrounding macropinosomes. Salmonella also induces membrane tubules that emanate from the SCV and lead to SCV shrinkage. Here, we show that these antipodal events are utilized by Salmonella to either establish a vacuolar niche or to be released into the cytosol by SCV rupture. We identify the molecular machinery underlying dynamic SCV growth and shrinkage. In particular, the SNARE proteins SNAP25 and STX4 participate in SCV inflation by fusion with macropinosomes. Thus, host compartment size control emerges as a pathogen strategy for intracellular niche regulation., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

In cutaneous Leishmaniasis the parasitic control in human host macrophages is still poorly understood. We found an increased expression of the human cathelicidin CAMP in skin lesions of Ethiopian patients with cutaneous leishmaniasis. Vitamin D driven, Cathelicidin-type antimicrobial peptides (CAMP) play an important role in the elimination of invading microorganisms. Recombinant cathelicidin was able to induce cell-death characteristics in Leishmania in a dose dependent manner. Using human primary macrophages, we demonstrated pro-inflammatory macrophages (hMDM1) to express a higher level of human cathelicidin, both on gene and protein level, compared to anti-inflammatory macrophages (hMDM2). Activating the CAMP pathway using Vitamin D in hMDM1 resulted in a cathelicidin-mediated- Leishmania restriction. Finally, a reduction of cathelicidin in hMDM1, using a RNA interference (RNAi) approach, increased Leishmania parasite survival. In all, these data show the human cathelicidin to contribute to the innate immune response against Leishmaniasis in a human primary cell model., (Copyright © 2019 Crauwels, Bank, Walber, Wenzel, Agerberth, Chanyalew, Abebe, König, Ritter, Reiling and van Zandbergen.)

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., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The number of effective antituberculotic drugs is strongly limited to four first-line drugs in standard therapy. In case of resistances second-line antibiotics are used with a poor efficacy and tolerability. Therefore, novel antituberculotic drugs are urgently needed. We synthesized novel nonclassical 1,4-dihydropyridines and evaluated their antituberculotic properties depending on substituent effects. Preferred substituents could be identified. As related classical 1,4-dihydropyridines are known as inhibitors of the transmembrane efflux pump ABCB1 in cancer cells, we wondered whether a use of our compounds may be of favour to enhance the antituberculotic drug efficacy of the second-line antituberculotic drug clofazimine, which is a known substrate of ABCB1 by a suggested inhibition of a corresponding efflux pump in Mycobacterium tuberculosis (Mtb). For this, we determined the ABCB1 inhibiting properties of our compounds in a mouse T-lymphoma cell line model and then evaluated the drug-enhancing properties of selected compounds in a co-application with clofazimine in our Mtb strain. We identified novel enhancers of clofazimine toxicity which could prevent clofazimine resistance development mediated by an efflux pump activity.

Wollamides are cyclic hexapeptides, recently isolated from an Australian soil Streptomyces isolate, that exhibit promising in vitro antimycobacterial activity against Mycobacterium bovis Bacille Calmette Guérin without displaying cytotoxicity against a panel of mammalian cells. Here, we report the synthesis and antimycobacterial activity of 36 new synthetic wollamides, collated with all known synthetic and natural wollamides, to reveal structure characteristics responsible for in vitro growth-inhibitory activity against Mycobacterium tuberculosis (H37Rv, H37Ra, CDC1551, HN878, and HN353). The most potent antimycobacterial wollamides were those where residue VI d-Orn (wollamide B) was replaced by d-Arg (wollamide B1) or d-Lys (wollamide B2), with all activity being lost when residue VI was replaced by Gly, l-Arg, or l-Lys (wollamide B3). Substitution of other amino acid residues mainly reduced or ablated antimycobacterial activity. Significantly, whereas wollamide B2 was the most potent in restricting M. tuberculosis in vitro , wollamide B1 restricted M. tuberculosis intracellular burden in infected macrophages. Wollamide B1 synergized with pretomanid (PA-824) in inhibiting M. tuberculosis in vitro growth but did not antagonize prominent first- and second-line tuberculosis antibiotics. Furthermore, wollamide B1 exerted bactericidal activity against nonreplicating M. tuberculosis and impaired growth of multidrug- and extensively drug-resistant clinical isolates. In vivo pharmacokinetic profiles for wollamide B1 in rats and mice encourage further optimization of the wollamide pharmacophore for in vivo bioavailability. Collectively, these observations highlight the potential of the wollamide antimycobacterial pharmacophore., (Copyright © 2019 American Society for Microbiology.)

The importance of bacterial lectins for adhesion, pathogenicity, and biofilm formation is well established for many Gram-positive and Gram-negative bacteria. However, there is very little information available about lectins of the tuberculosis-causing bacterium, Mycobacterium tuberculosis ( Mtb ). In this paper we review previous studies on the carbohydrate-binding characteristics of mycobacteria and related Mtb proteins, discussing their potential relevance to Mtb infection and pathogenesis.

Introduction: With regard to the anti-mycobacterial activity of 2-pyrazinoic acid esters (POEs), recent studies have shown that both pyrazine core and alkyl part of POE interact with the fatty acid synthase type (I) (FAS (I)) precluding a complex formation between NADPH and FAS (I). Methods: Considering this interaction at the reductase site of FAS (I) responsible for reduction of β-ketoacyl-CoA to β-hydroxyacyl-CoA, we hypothesized that POE containing a bioreducible center in its alkyl part might show an increased anti-tubercular activity due to the involvement of FAS (I) in extra bio-reduction reaction. Thus, we synthesized novel POEs, confirmed their structures by spectral data, and subsequently evaluated their anti-mycobacterial activity against Mycobacterium tuberculosis (Mtb) (H37Rv) strain at 10 μg/mL concentration. Results: Compounds 3c, 3j , and 3m showed higher activity with regard to the inhibition of Mtb growth by 45.4, 45.7, and 51.2% respectively. Unexpectedly, the maltol derived POE 3l having the lowest log p value among the POEs indicated the highest anti-mycobacterial growth activity with 56% prevention. Compounds 3c and 3l showed no remarkable cytotoxicity on human macrophages at 10 μg/mL concentration as analyzed by xCELLigence real-time cell analysis. In further experiments, some of the tested POEs, unlike pyrazinamide (PZA), exhibited significant antibacterial and also anti-fungal activities. POEs showed an enhanced bactericidal activity on gram-positive bacteria as shown for Staphylococcus aureus , e.g. compound 3b with a MIC value of 125 μg/mL but not E. coli as a gram-negative bacteria, except for maltol derived POE ( 3l ) that showed an inverse activity in the susceptibility test. In the anticancer activity test against the human leukemia K562 cell lines using MTT assay, compounds 3e and 3j showed the highest cytotoxic effect with IC50 values of 25±8.0 μΜ and 25±5.0 μΜ, respectively. Conclusion: It was found that the majority of POEs containing a bioreducible center showed higher inhibitory activities on Mtb growth when compared to the similar compounds without a bio-reducible functional group., (© 2019 The Author(s).)

Background: In order to eliminate tuberculosis (TB), an effective vaccine is urgently needed to prevent infection with Mycobacterium tuberculosis . A key obstacle for the development of novel TB vaccines is the lack of surrogate markers for immune protection against M. tuberculosis ., Methods: We investigated growth rates of M. tuberculosis in the mycobacterial growth inhibition assay (MGIA) as a marker for mycobacterial growth control of human bronchoalveolar lavage (BALC) and peripheral blood mononuclear cells (PBMC) before and after vaccination with Mycobacterium bovis Bacille Calmette-Guérin (BCG) of healthy adult volunteers., Results: Vaccination induced a positive response ( p  < 0.001) to purified protein derivate (PPD) in 58.8% of the individuals in an interferon-γ release assay-ELISpot. Intraindividual evaluation of the MGIA growth rates before and after M. bovis BCG-vaccination revealed no significant difference in time to culture positivity before and after vaccination in BALC ( p  = 0.604) and PBMC ( p  = 0.199). The magnitude of the PPD-response induced by M. bovis BCG-vaccination did not correlate with growth control in BALC and PBMC (correlation = 0.468, 95% CI: -0.016 to 0.775)., Conclusion: In conclusion, M. bovis BCG-vaccination-induced mycobacterial-specific cytokine immune response does not result in functional immune control against M. tuberculosis in the MGIA.

The number of effective first-line antibiotics for the treatment of Mycobacterium tuberculosis infection is strongly limited to a few drugs. Due to emerging resistance against those drugs, second- and third-line antibiotics have been established in therapy with certain problems and also increasing mycobacterial resistance. An alternative to such novel drugs or combined therapeutic regimes which may reduce resistance development is finding enhancers of mycobacterial drug effectiveness, especially enhancers that counteract causative resistance mechanisms. Such enhancers may reduce the extracellular drug efflux mediated by bacterial efflux pumps and thus enhance the intracellular drug toxicity. We developed novel 1,4-dihydropyridines (DHPs) as potential efflux pump inhibitors with some determined P-gp affinities. The influence on the antituberculotic drug toxicity has been investigated for three prominent antituberculotic drugs. Exclusive and selective toxicity enhancing effects have been detected for isoniazid (INH) which could be related to certain substituent effects of the 1,4-DHPs. So, structure-dependent activities have been found. Thus, promising enhancers could be identified and a suggested efflux pump inhibition is discussed.

Background: Blood based Interferon-(IFN)-γ release assays (IGRAs) have a poor predictive value for the development of tuberculosis. This study aimed to investigate the correlation between IGRAs and pulmonary immune responses in tuberculosis contacts in Germany., Methods: IGRAs were performed on bronchoalveolar lavage (BAL) cells and peripheral blood from close healthy contacts of patients with culturally confirmed tuberculosis. Cellular BAL composition was determined by flow cytometry. BAL cells were co-cultured with three strains of Mycobacterium tuberculosis (Mtb) and Mtb derived antigens including Purified Protein Derivative (PPD), 6 kD Early Secretory Antigenic Target (ESAT-6) and 10 kD Culture Filtrate Protein (CFP-10). Levels of 29 cytokines and chemokines were analyzed in the supernatants by multiplex assay. Associations and effects were examined using linear mixed-effects models., Results: There were wide variations of inter-individual cytokine levels in BAL cell culture supernatants. Mycobacterial infection and stimulation with PPD showed a clear induction of several macrophage and lymphocyte associated cytokines, reflecting activation of these cell types. No robust correlation between cytokine patterns and blood IGRA status of the donor was observed, except for slightly higher Interleukin-2 (IL-2) responses in BAL cells from IGRA-positive donors upon mycobacterial infection compared to cells from IGRA-negative donors. Stronger correlations were observed when cytokine patterns were stratified according to BAL IGRA status. BAL cells from donors with BAL IGRA-positive responses produced significantly more IFN-γ and IL-2 upon PPD stimulation and mycobacterial infection than cells from BAL IGRA-negative individuals. Correlations between BAL composition and basal cytokine release from unstimulated cells were suggestive of pre-activated lymphocytes but impaired macrophage activity in BAL IGRA-positive donors, in contrast to BAL IGRA-negative donors., Conclusions: In vitro BAL cell cytokine responses to M. tuberculosis antigens or infection do not reflect blood IGRA status but do correlate with stronger cellular responses in BAL IGRA-positive donors. The cytokine patterns observed suggest a pre-activated state of lymphocytes and suppressed macrophage responsiveness in BAL cells from BAL IGRA-positive individuals.

The signaling pathways that sustain the disease process of chronic rhinosinusitis with nasal polyps (CRSwNP) remain poorly understood. We sought to determine the expression levels of Wnt signaling genes in CRSwNP and to study the role of the Wnt pathway in inflammation and epithelial remodeling in the nasal mucosa. Microarrays and real time-quantitative polymerase chain reaction comparing gene expression in matched NPs and inferior turbinates revealed that WNT2B, WNT3A, WNT4, WNT7A, WNT7B, and FZD2 were up-regulated and that FZD1, LRP5, LRP6, and WIF1 were down-regulated in NPs. Immunolabeling showed robust expression of Wnt ligands, nuclear β-catenin, and Axin-2 in NP tissue, suggesting that Wnt/β-catenin signaling is activated in NPs. We used primary human nasal epithelial cell (HNEpC) cultures to test the functional consequences of Wnt pathway activation. Monolayer HNEpCs treated with recombinant human WNT (rhWNT) 3A, but not with rhWNT4, had altered epithelial morphology and decreased adhesion, without loss of viability. We found that neither rhWNT3A nor rhWNT4 treatment induced proliferation. The expression and release of inflammatory cytokines IL-6 and granulocyte-macrophage colony-stimulating factor were increased after rhWNT3A exposure of HNEpCs. When differentiated at an air-liquid interface, rhWNT3A- and WNT agonist-, but not rhWNT4-treated HNEpCs, had abnormal epithelial architecture, failed to undergo motile ciliogenesis, and had defective noncanonical Wnt (planar cell polarity) signaling. On the basis of these results, we propose a model in which Wnt/β-catenin signaling sustains mucosal inflammation and leads to a spectrum of changes consistent with those seen during epithelial remodeling in NPs.

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., (Copyright © 2016 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

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.

NF-κB transcription factors are key regulators of pulmonary inflammatory disorders and repair. Constitutive lung cell type- and microenvironment-specific NF-κB/inhibitor κBα (IκB-α) regulation, however, is poorly understood. Surfactant protein (SP)-A provides both a critical homeostatic and lung defense control, in part by immune instruction of alveolar macrophages (AMs) via clathrin-mediated endocytosis. The central endocytic proteins, clathrin heavy chain (CHC) and the clathrin adaptor protein (AP) complex AP2, have pivotal alternative roles in cellular homeostasis that are endocytosis independent. Here, we dissect endocytic from alternative functions of CHC, the α-subunit of AP2, and dynamin in basal and SP-A-modified LPS signaling of macrophages. As revealed by pharmacological inhibition and RNA interference in primary AMs and RAW264.7 macrophages, respectively, CHC and α-adaptin, but not dynamin, prevent IκB-α degradation and TNF-α release, independent of their canonical role in membrane trafficking. Kinetics studies employing confocal microscopy, Western analysis, and immunomagnetic sorting revealed that SP-A transiently enhances the basal protein expression of CHC and α-adaptin, depending on early activation of protein kinase CK2 (former casein kinase II) and Akt1 in primary AMs from rats, SP-A(+/+), and SP-A(-/-) mice, as well as in vivo when intratracheally administered to SP-A(+/+) mice. Constitutive immunomodulation by SP-A, but not SP-A-mediated inhibition of LPS-induced NF-κB activity and TNF-α release, requires CHC, α-adaptin, and dynamin. Our data demonstrate that endocytic proteins constitutively restrict NF-κB activity in macrophages and provide evidence that SP-A enhances the immune regulatory capacity of these proteins, revealing a previously unknown pathway of microenvironment-specific NF-κB regulation in the lung.

Toll like receptors (TLR) are key elements of the innate immune response and involved in the recognition of pathogens. To test common and rare TLR variants involved in susceptibility or resistance to infection with Mycobacterium tuberculosis we screened the exons of the genes encoding TLR 1, 2, 4, and the adaptor molecule TIRAP in more than 4500 tuberculosis (TB) cases and controls from Ghana. The analysis yielded 109 variants with possible functional impact, including 101 non-synonymous variants, three stop-variants, and five indels. Association analyses yielded a significant result for the TLR1 variant rs3923647, conferring strong protection against TB (Odds ratio [OR] 0.21, CI confidence interval [CI] 0.05-0.6, Pnominal 1 x 10-3) when applying a recessive model of inheritance. Replication analyses with an additional 3370 Ghanaian cases and control samples, and with data from a recent TB study of 533 African-Americans confirmed the protective effect and resulted in a combined OR of 0.19, with a nominal P value of 2.2 x 10-5, and a corrected P value of 4.1 x 10-4. The SNP is located near the binding pocket of TLR1 and causes an amino acid exchange from histidine to leucine at position 305. The observed effect may, therefore, be attributable to structural changes in the recognition site of the TLR1 molecule, allowing to bind those mycobacterial ligands which preferentially may induce a protective immune response. This is supported by the analysis of BCG-stimulated peripheral blood mononuclear cells, showing increased induction of the proinflammatory cytokine IFN-γ in carriers of the mutant TLR1 rs3923647 TT genotype, compared to the IFN-γ levels of individuals with the AT and AA genotypes.

Background: Mycobacterial infections remain a significant cause of morbidity and mortality worldwide. Due to limitations of the currently available model systems, there are still comparably large gaps in the knowledge about the pathogenesis of these chronic inflammatory diseases in particular with regard to the human host. Therefore, we aimed to characterize the initial phase of mycobacterial infections utilizing a human ex vivo lung tissue culture model designated STST (Short-Term Stimulation of Tissues)., Methods: Human lung tissues from 65 donors with a size of 0.5-1 cm(3) were infected each with two strains of three different mycobacterial species (M. tuberculosis, M. avium, and M. abscessus), respectively. In order to preserve both morphology and nucleic acids, the HOPE® fixation technique was used. The infected tissues were analyzed using histo- and molecular-pathological methods. Immunohistochemistry was applied to identify the infected cell types., Results: Morphologic comparisons between ex vivo incubated and non-incubated lung specimens revealed no noticeable differences. Viability of ex vivo stimulated tissues demonstrated by TUNEL-assay was acceptable. Serial sections verified sufficient diffusion of the infectious agents deep into the tissues. Infection was confirmed by Ziel Neelsen-staining and PCR to detect mycobacterial DNA. We observed the infection of different cell types, including macrophages, neutrophils, monocytes, and pneumocytes-II, which were critically dependent on the mycobacterial species used. Furthermore, different forms of nuclear alterations (karyopyknosis, karyorrhexis, karyolysis) resulting in cell death were detected in the infected cells, again with characteristic species-dependent differences., Conclusion: We show the application of a human ex vivo tissue culture model for mycobacterial infections. The immediate primary infection of a set of different cell types and the characteristic morphologic changes observed in these infected human tissues significantly adds to the current understanding of the initial phase of human pulmonary tuberculosis. Further studies are ongoing to elucidate the molecular mechanisms involved in the early onset of mycobacterial infections in the human lung.

Sepsis is still a major cause of death and many efforts have been made to improve the physical condition of sepsis patients and to reduce the high mortality rate associated with this disease. While achievements were implemented in the intensive care treatment, all attempts within the field of novel therapeutics have failed. As a consequence new medications and improved patient stratification as well as a thoughtful management of the support therapies are urgently needed. In this study, we investigated the simultaneous administration of ibuprofen as a commonly used nonsteroidal anti-inflammatory drug (NSAID) and Pep19-2.5 (Aspidasept), a newly developed antimicrobial peptide. Here, we show a synergistic therapeutic effect of combined Pep19-2.5-ibuprofen treatment in an endotoxemia mouse model of sepsis. In vivo protection correlates with a reduction in plasma levels of both tumor necrosis factor α and prostaglandin E, as a likely consequence of Pep19-2.5 and ibuprofen-dependent blockade of TLR4 and COX pro-inflammatory cascades, respectively. This finding is further characterised and confirmed in a transcriptome analysis of LPS-stimulated human monocytes. The transcriptome analyses showed that Pep19-2.5 and ibuprofen exerted a synergistic global effect both on the number of regulated genes as well as on associated gene ontology and pathway expression. Overall, ibuprofen potentiated the anti-inflammatory activity of Pep19-2.5 both in vivo and in vitro, suggesting that NSAIDs could be useful to supplement future anti-sepsis therapies.

Direct interaction of Chlamydiae with the endoplasmic reticulum (ER) is essential in intracellular productive infection. However, little is known about the interplay between Chlamydiae and the ER under cellular stress conditions that are observed in interferon gamma (IFN-γ) induced chlamydial persistent infection. ER stress responses are centrally regulated by the unfolded protein response (UPR) under the control of the ER chaperone BiP/GRP78 to maintain cellular homeostasis. In this study, we could show that the ER directly contacted with productive and IFN-γ-induced persistent inclusions of Chlamydia pneumoniae (Cpn). BiP/GRP78 induction was observed in the early phase but not in the late phase of IFN-γ-induced persistent infection. Enhanced BiP/GRP78 expression in the early phase of IFN-γ-induced persistent Cpn infection was accompanied by phosphorylation of the eukaryotic initiation factor-2α (eIF2α) and down-regulation of the vesicle-associated membrane protein-associated protein B. Loss of BiP/GRP78 function resulted in enhanced phosphorylation of eIF2α and increased host cell apoptosis. In contrast, enhanced BiP/GRP78 expression in IFN-γ-induced persistent Cpn infection attenuated phosphorylation of eIF2α upon an exogenous ER stress inducer. In conclusion, ER-related BiP/GRP78 plays a key role to restore cells from stress conditions that are observed in the early phase of IFN-γ-induced persistent infection., (© 2015 John Wiley & Sons Ltd.)

Candida glabrata currently ranks as the second most frequent cause of invasive candidiasis. Our previous work has shown that C. glabrata is adapted to intracellular survival in macrophages and replicates within non-acidified late endosomal-stage phagosomes. In contrast, heat killed yeasts are found in acidified matured phagosomes. In the present study, we aimed at elucidating the processes leading to inhibition of phagosome acidification and maturation. We show that phagosomes containing viable C. glabrata cells do not fuse with pre-labeled lysosomes and possess low phagosomal hydrolase activity. Inhibition of acidification occurs independent of macrophage type (human/murine), differentiation (M1-/M2-type) or activation status (vitamin D3 stimulation). We observed no differential activation of macrophage MAPK or NFκB signaling cascades downstream of pattern recognition receptors after internalization of viable compared to heat killed yeasts, but Syk activation decayed faster in macrophages containing viable yeasts. Thus, delivery of viable yeasts to non-matured phagosomes is likely not triggered by initial recognition events via MAPK or NFκB signaling, but Syk activation may be involved. Although V-ATPase is abundant in C. glabrata phagosomes, the influence of this proton pump on intracellular survival is low since blocking V-ATPase activity with bafilomycin A1 has no influence on fungal viability. Active pH modulation is one possible fungal strategy to change phagosome pH. In fact, C. glabrata is able to alkalinize its extracellular environment, when growing on amino acids as the sole carbon source in vitro. By screening a C. glabrata mutant library we identified genes important for environmental alkalinization that were further tested for their impact on phagosome pH. We found that the lack of fungal mannosyltransferases resulted in severely reduced alkalinization in vitro and in the delivery of C. glabrata to acidified phagosomes. Therefore, protein mannosylation may play a key role in alterations of phagosomal properties caused by C. glabrata.

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.

Immunoresponsive gene 1 (Irg1) is highly expressed in mammalian macrophages during inflammation, but its biological function has not yet been elucidated. Here, we identify Irg1 as the gene coding for an enzyme producing itaconic acid (also known as methylenesuccinic acid) through the decarboxylation of cis-aconitate, a tricarboxylic acid cycle intermediate. Using a gain-and-loss-of-function approach in both mouse and human immune cells, we found Irg1 expression levels correlating with the amounts of itaconic acid, a metabolite previously proposed to have an antimicrobial effect. We purified IRG1 protein and identified its cis-aconitate decarboxylating activity in an enzymatic assay. Itaconic acid is an organic compound that inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt, a pathway essential for bacterial growth under specific conditions. Here we show that itaconic acid inhibits the growth of bacteria expressing isocitrate lyase, such as Salmonella enterica and Mycobacterium tuberculosis. Furthermore, Irg1 gene silencing in macrophages resulted in significantly decreased intracellular itaconic acid levels as well as significantly reduced antimicrobial activity during bacterial infections. Taken together, our results demonstrate that IRG1 links cellular metabolism with immune defense by catalyzing itaconic acid production.

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., (© 2012 John Wiley & Sons A/S.)

Human TLR1 plays an important role in host defense against Mycobacterium tuberculosis. Our aim was to analyze the association of the loss of TLR1 surface expression and TLR1 SNPs with susceptibility to TB. TLR1neg and TLR1pos cells from healthy individuals were identified by flow cytometry and compared by sequencing. TLR1 expression was measured using quantitative real-time PCR and immunoblotting. TLR1 SNP analyses of healthy individuals and TB patients from EU-C and Ghana were performed, and association of the TLR1 genotypes with increased risk of developing TB was statistically evaluated. Lack of TLR1 surface expression accompanied by impaired function was strongly associated with TLR1 SNP G743A. Genotyping of EU-C controls and TB patients revealed an association of TLR1 743A/1805G alleles [OR 2.37 (95% CI 1.13, 4.93), P=0.0219; OR 2.74 (95% CI 1.26, 6.05), P=0.0059] as well as TLR1neg 743AA/1805GG versus TLR1pos genotypes 743AG/1805TG [OR 4.98 (95% CI 1.64, 15.15), P=0.0034; OR 5.70 (95% CI 1.69, 20.35), P=0.0015] and 743AG + GG/1805TG + TT [OR 3.54 (95% CI 1.29, 9.90), P=0.0086; OR 4.17 (95% CI 1.52, 11.67), P=0.0025] with increased susceptibility to TB. No association of G743A with TB was found in Ghana as a result of a low frequency of genotype 743AA. Our data gain new insights in the role of TLR1 in M. tuberculosis defense and provide the first evidence that TLR1 variants are associated with susceptibility to TB in a low-incidence country.

Interferon (IFN)-gamma release assays (IGRA) have improved tuberculosis contact tracing, but discrimination of recent from remote Mycobacterium tuberculosis contacts is not possible by IGRA alone. We present results of a tuberculosis contact investigation with a new early-secretory-antigenic-target (ESAT)-6 and culture-filtrate-protein (CFP)-10 specific interleukin (IL)-2 ELISpot in addition to ESAT-6 and CFP-10 specific IFN-gamma ELISpot and tuberculin skin testing (TST). Results of the TST, IFN-gamma ELISpot and IL-2 ELISpot were positive in 6/172 (3.4%), 7/167 (4.2%) and 6/196 (3.1%) of contacts, respectively. Close contact (> or =100 hours) to the index case increased the risk of positive results in the IFN-gamma ELISpot, TST, and IL-2 ELISpot by 40.8, 19.3, and 2.5 times, respectively. Individuals with a positive IFN-gamma ELISpot/negative IL-2 ELISpot result had a median (IQR) duration of index case exposure of 568 hours (133&#95;1000) compared to individuals with a positive IFN-gamma ELISpot/positive IL-2 ELISpot result (median = 24 hours; 20&#95;130; p-value = 0.047). Combination of a M. tuberculosis specific IFN-gamma ELISpot with a M. tuberculosis specific IL-2 ELISpot significantly improved the identification of individuals with the highest risk of recent M. tuberculosis infection and is a promising method that should be explored to target tuberculosis preventive chemotherapy.

In a mouse model of mycobacteria-induced immunopathology, wild-type C57BL/6 (WT), IL-18-knockout (KO) and IFN-alphabeta receptor-KO mice developed circumscript, centrally necrotizing granulomatous lesions in response to aerosol infection with M. avium, whereas mice deficient in the IFN-gamma receptor, STAT-1 or IRF-1 did not exhibit granuloma necrosis. Comparative, microarray-based gene expression analysis in the lungs of infected WT and IRF-1-KO mice identified a set of genes whose differential regulation was closely associated with granuloma necrosis, among them cathepsin K, cystatin F and matrix metalloprotease 10. Further microarray-based comparison of gene expression in the lungs of infected WT, IFN-gamma-KO and IRF-1-KO mice revealed four distinct clusters of genes with variable dependence on the presence of IFN-gamma, IRF-1 or both. In particular, IRF-1 appeared to be directly involved in the differentiation of a type I immune response to mycobacterial infection. In summary, IRF-1, rather than being a mere transcription factor downstream of IFN-gamma, may be a master regulator of mycobacteria-induced immunopathology.

The capsules of two colony morphotypes of Mycobacterium avium strain 2151 were investigated, i.e. the virulent smooth-transparent (SmT1) and the nonvirulent smooth-opaque (SmO) types. From both morphotypes we separated a nonacylated arabinomannan (AM) from an acylated polysaccharide fraction by affinity chromatography, of which the AMs were structurally characterized. The AMs from the virulent morphotype, in contrast to that from the nonvirulent form, possessed a larger mannan chain and a shorter arabinan chain. Incubation of murine bone marrow-derived macrophages and human dendritic cells showed that the acylated polysaccharide fractions were potent inducers of tumor necrosis factor-alpha, interleukin-12, and interleukin-10 compared with nonacylated AMs, which led to only a marginal cytokine release. Further in vitro experiments showed that both the acylated polysaccharide fractions and the nonacylated AMs were able to induce in vitro anti-tumor cytotoxicity of human peripheral blood mononuclear cells. Thus, morphotype-specific structural differences in the capsular AMs of M. avium do not correlate with biological activity; however, their acylation is a prerequisite for effective stimulation of murine macrophages and human dendritic cells.

Interleukin 15 (IL-15) is a pleiotropic cytokine that is hardly detectable in biological fluids. Here, we show that IL-15 forms functional heterocomplexes with soluble high affinity IL-15 receptor alpha (IL-15Ralpha) chain in mouse serum and cell-conditioned medium, which prevents IL-15 detection by ELISA. We also demonstrate that two soluble IL-15Ralpha (sIL-15Ralpha) sushi domain isoforms are generated through a novel alternative splicing mechanism within the IL-15Ralpha gene. These isoforms potentiate IL-15 action by promoting the IL-15-mediated proliferation of the CTLL cell line and interferon gamma production by murine NK cells, which suggests a role in IL-15 transpresentation. Conversely, a full-length sIL-15Ralpha ectodomain released by tumor necrosis factor-alpha-converting enzyme (TACE)-dependent proteolysis inhibits IL-15 activity. Thus, a dual mechanism of sIL-15Ralpha generation exists in mice, giving rise to polypeptides with distinct properties, which regulate IL-15 function.

Differential induction of cell death in mycobacteria-infected monocytes and macrophages has been invoked as one possible mechanism by which some tumor necrosis factor (TNF)-targeted biologicals reactivate tuberculosis more frequently than others. We infected primary human monocytes and monocyte-derived macrophages with the virulent Mycobacterium tuberculosis strain H37Rv and followed the rate of cell death in the absence or presence of a wide concentration range of four different TNF-targeted biologicals: infliximab and adalimumab (both monoclonal antibodies to human TNF) and etanercept and polyethylene-glycols TNFR1 (fusion constructs of human TNFR2 and TNFR1, respectively). None of the TNF-targeted biologicals used modulated the death rate of monocytes/macrophages induced by infection with M. tuberculosis alone. Our data support the view that mycobacteria-induced cell death is largely independent of TNF and that the primary target for differential modulation by TNF-targeted biologicals during tuberculosis is not a recently recruited monocyte or freshly differentiated macrophage.

Microarray--assisted gene--expression screens of human macrophages revealed WNT5A, a homolog of Wingless, a key regulator of Drosophila melanogaster embryonic segmentation and patterning, to be consistently up-regulated following stimulation with different mycobacterial species and conserved bacterial structures. The expression of WNT5A required Toll-like receptor signaling and NF-kappaB activation, which identifies a novel induction pathway for a Wingless homolog. We show that human peripheral-blood mononuclear cells express the WNT5A receptor Frizzled-5 (FZD5). Both WNT5A and FZD5 also were detected in granulomatous lesions in the lungs of Mycobacterium tuberculosis-infected patients. Functional studies showed that WNT5A and FZD5 regulate the microbially induced interleukin-12 response of antigen-presenting cells and interferon-gamma production by mycobacterial antigen-stimulated T cells. Our findings implicate the evolutionarily conserved WNT/Frizzled signaling system in bridging innate and adaptive immunity to infections.

Classification of pathogenic species according to the distinct host transcriptional responses that they elicit may become a relevant tool for microarray-based diagnosis of infection. Individual strains of Mycobacterium avium, an opportunistic pathogen in humans, have previously been shown to differ in terms of growth and persistence. In order to cover a wide spectrum of virulence, we selected four M. avium isolates (2151SmO, 2151SmT, SE01, TMC724) that have distinct intramacrophage replication characteristics and cause differential activation in human macrophages. Following infection with each of these strains, the expression of 12,558 genes in human macrophages was systematically analyzed by microarray technology. Fifty genes (including genes encoding proinflammatory cytokines, chemokines, signaling, and adhesion molecules) were differentially expressed more than twofold in response to all of the M. avium isolates investigated and therefore constitute a common macrophage signature in response to M. avium. The magnitude of regulation of most of these genes was directly correlated with the host cell-activating capacity of the particular M. avium strain. The regulation of a number of genes not previously associated with mycobacterial infections was apparent; these genes included genes encoding lymphocyte antigen 64 and myosin X. In addition, individual response patterns typical for some M. avium isolates could be defined by the pronounced upregulation of interleukin-12p40 (IL-12p40) (in the case of 2151SmO) or the specific upregulation of SOCS-1 and IL-10 (in the case of SE01) in macrophages. TMC724, a strain of avian origin, could not be classified by any one of these schemes, possibly indicating the limits of pathogen categorization solely by immune response signatures.