wIRA: hyperthermia as a treatment option for intracellular bacteria, with special focus on Chlamydiae and Mycobacteria

The emergence of antibiotic-resistant bacteria in the last century is alarming and calls for alternative, nonchemical treatment strategies. Thermal medicine uses heat for the treatment of infectious diseases but its use in facultative and obligate intracellular bacteria remains poorly studied. In this review, we summarize previous research on reducing the infectious burden of; Mycobacterium ulcerans; and; Chlamydia trachomatis; by using water-filtered infrared A-radiation (wIRA), a special form of heat radiation with high tissue penetration and low thermal load on the skin surface.; Mycobacterium ulcerans; is a thermosensitive bacterium causing chronic necrotizing skin disease. Therefore, previous data on wIRA-induced improvement of wound healing and reduction of wound infections is summarized first. Then, pathogenesis and treatment of infections with; M. ulcerans; causing Buruli ulcer and of those with; C. trachomatis; infecting the ocular conjunctiva and resulting in blinding trachoma are discussed. Both bacteria cause neglected tropical diseases and have similar geographical distributions. Results of previous; in vitro; and; in vivo; studies using wIRA on; M. ulcerans; and; C. trachomatis; infections are presented. Finally, technical aspects of using wIRA in patients are critically reviewed and open questions driving future research are highlighted. In conclusion, wIRA is a promising tool for reducing infectious burden due to intracellular bacteria such as; M. ulcerans; and; C. trachomatis;

Susceptibility to Mycobacterium ulcerans Disease (Buruli ulcer) Is Associated with IFNG and iNOS Gene Polymorphisms

Buruli ulcer (BU) is a chronic necrotizing disease of the skin and subcutaneous fat tissue. The causative agent, Mycobacterium ulcerans, produces mycolactone, a macrolide toxin, which causes apoptosis of mammalian cells. Only a small proportion of individuals exposed to M. ulcerans develop clinical disease, as surrounding macrophages may control the infection by bacterial killing at an early stage, while mycolactone concentration is still low. Otherwise, bacterial multiplication leads to in higher concentrations of mycolactone, with formation of necrotizing lesions that are no more accessible to immune cells. By typing a cohort of 96 Ghanaian BU patients and 384 endemic controls without BU, we show an association between BU and single nucleotide polymorphisms (SNPs) in iNOS (rs9282799) and IFNG (rs2069705). Both polymorphisms influence promoter activity in vitro. A previously reported SNP in SLC11A1 (NRAMP, rs17235409) tended to be associated with BU. Altogether, these data reflect the importance of IFNG signaling in early defense against M. ulcerans infection

Antibody-Mediated Neutralization of the Exotoxin Mycolactone, the Main Virulence Factor Produced by Mycobacterium ulcerans

Mycolactone, the macrolide exotoxin produced by Mycobacterium ulcerans, causes extensive tissue destruction by inducing apoptosis of host cells. In this study, we aimed at the production of antibodies that could neutralize the cytotoxic activities of mycolactone.; Using the B cell hybridoma technology, we generated a series of monoclonal antibodies with specificity for mycolactone from spleen cells of mice immunized with the protein conjugate of a truncated synthetic mycolactone derivative. L929 fibroblasts were used as a model system to investigate whether these antibodies can inhibit the biological effects of mycolactone. By measuring the metabolic activity of the fibroblasts, we found that anti-mycolactone mAbs can completely neutralize the cytotoxic activity of mycolactone.; The toxin neutralizing capacity of anti-mycolactone mAbs supports the concept of evaluating the macrolide toxin as vaccine target

Pan-Bcl-2 inhibitor Obatoclax is a potent late stage autophagy inhibitor in colorectal cancer cells independent of canonical autophagy signaling

Background: Colorectal cancer is the third most common malignancy in humans and novel therapeutic approaches are urgently needed. Autophagy is an evolutionarily highly conserved cellular process by which cells collect unnecessary organelles or misfolded proteins and subsequently degrade them in vesicular structures in order to refuel cells with energy. Dysregulation of the complex autophagy signaling network has been shown to contribute to the onset and progression of cancer in various models. The Bcl-2 family of proteins comprises central regulators of apoptosis signaling and has been linked to processes involved in autophagy. The antiapoptotic members of the Bcl-2 family of proteins have been identified as promising anticancer drug targets and small molecules inhibiting those proteins are in clinical trials. Methods: Flow cytometry and colorimetric assays were used to assess cell growth and cell death. Long term 3D cell culture was used to assess autophagy in a tissue mimicking environment in vitro. RNA interference was applied to modulate autophagy signaling. Immunoblotting and q-RT PCR were used to investigate autophagy signaling. Immunohistochemistry and fluorescence microscopy were used to detect autophagosome formation and autophagy flux. Results: This study demonstrates that autophagy inhibition by obatoclax induces cell death in colorectal cancer (CRC) cells in an autophagy prone environment. Here, we demonstrate that pan-Bcl-2 inhibition by obatoclax causes a striking, late stage inhibition of autophagy in CRC cells. In contrast, ABT-737, a Mcl-1 sparing Bcl-2 inhibitor, failed to interfere with autophagy signaling. Accumulation of p62 as well as Light Chain 3 (LC3) was observed in cells treated with obatoclax. Autophagy inhibition caused by obatoclax is further augmented in stressful conditions such as starvation. Furthermore, our data demonstrate that inhibition of autophagy caused by obatoclax is independent of the essential pro-autophagy proteins Beclin-1, Atg7 and Atg12. Conclusions: The objective of this study was to dissect the contribution of Bcl-2 proteins to autophagy in CRC cells and to explore the potential of Bcl-2 inhibitors for autophagy modulation. Collectively, our data argue for a Beclin-1 independent autophagy inhibition by obatoclax. Based on this study, we recommend the concept of autophagy inhibition as therapeutic strategy for CRC

Targeting the Mycobacterium ulcerans cytochrome bc1:aa3 for the treatment of Buruli ulcer

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a neglected tropical skin disease that is most commonly found in children from West and Central Africa. Despite the severity of the infection, therapeutic options are limited to antibiotics with severe side effects. Here, we show that M. ulcerans is susceptible to the anti-tubercular drug Q203 and related compounds targeting the respiratory cytochrome bc; 1; :aa; 3; . While the cytochrome bc; 1; :aa; 3; is the primary terminal oxidase in Mycobacterium tuberculosis, the presence of an alternate bd-type terminal oxidase limits the bactericidal and sterilizing potency of Q203 against this bacterium. M. ulcerans strains found in Buruli ulcer patients from Africa and Australia lost all alternate terminal electron acceptors and rely exclusively on the cytochrome bc; 1; :aa; 3; to respire. As a result, Q203 is bactericidal at low dose against M. ulcerans replicating in vitro and in mice, making the drug a promising candidate for Buruli ulcer treatment

Geometagenomics illuminates the impact of agriculture on the distribution and prevalence of plant viruses at the ecosystem scale

Disease emergence events regularly result from human activities such as agriculture, which frequently brings large populations of genetically uniform hosts into contact with potential pathogens. Although viruses cause nearly 50% of emerging plant diseases, there is little systematic information about virus distribution across agro-ecological interfaces and large gaps in understanding of virus diversity in nature. Here we applied a novel landscape-scale geometagenomics approach to examine relationships between agricultural land use and distributions of plantassociated viruses in two Mediterranean-climate biodiversity hotspots (Western Cape region of South Africa and Rhône river delta region of France). In total, we analysed 1725 geo-referenced plant samples collected over two years from 4.5 × 4.5 km2 grids spanning farmlands and adjacent uncultivated vegetation. We found substantial virus prevalence (25.8–35.7%) in all ecosystems, but prevalence and identified family-level virus diversity were greatest in cultivated areas, with some virus families displaying strong agricultural associations. Our survey revealed 94 previously unknown virus species, primarily from uncultivated plants. This is the first effort to systematically evaluate plant-associated viromes across broad agro-ecological interfaces. Our findings indicate that agriculture substantially influences plant virus distributions and highlight the extent of current ignorance about the diversity and roles of viruses in nature

Excision of HIV-1 Proviral DNA by Recombinant Cell Permeable Tre-Recombinase

Over the previous years, comprehensive studies on antiretroviral drugs resulted in the successful introduction of highly active antiretroviral therapy (HAART) into clinical practice for treatment of HIV/AIDS. However, there is still need for new therapeutic approaches, since HAART cannot eradicate HIV-1 from the infected organism and, unfortunately, can be associated with long-term toxicity and the development of drug resistance. In contrast, novel gene therapy strategies may have the potential to reverse the infection by eradicating HIV-1. For example, expression of long terminal repeat (LTR)-specific recombinase (Tre-recombinase) has been shown to result in chromosomal excision of proviral DNA and, in consequence, in the eradication of HIV-1 from infected cell cultures. However, the delivery of Tre-recombinase currently depends on the genetic manipulation of target cells, a process that is complicating such therapeutic approaches and, thus, might be undesirable in a clinical setting. In this report we demonstrate that E.coli expressed Tre-recombinases, tagged either with the protein transduction domain (PTD) from the HIV-1 Tat trans-activator or the translocation motif (TLM) of the Hepatitis B virus PreS2 protein, were able to translocate efficiently into cells and showed significant recombination activity on HIV-1 LTR sequences. Tre activity was observed using episomal and stable integrated reporter constructs in transfected HeLa cells. Furthermore, the TLM-tagged enzyme was able to excise the full-length proviral DNA from chromosomal integration sites of HIV-1-infected HeLa and CEM-SS cells. The presented data confirm Tre-recombinase activity on integrated HIV-1 and provide the basis for the non-genetic transient application of engineered recombinases, which may be a valuable component of future HIV eradication strategies

Studies on the structure and function of protein kinase G, a virulence factor of "Mycobacterium tuberculosis"

The genome of M. tuberculosis comprises eleven serine/threonine protein kinases which carry out various functions, e.g. in cell division, metabolism and pathogenicity. Nine of these eleven kinases are membrane-bound and have a C-terminal extracellular domain and an intracellular Hanks-type kinase domain located at the N-terminus. Protein kinase G (PknG) differs from these kinases, because it is predicted to be a cytosolic protein since it lacks a transmembrane domain. The kinase domain is preceded by a long N-terminal stretch containing two CXXC motifs. Moreover, PknG possesses a tetratricopeptide repeat within its C-terminal domain. PknG, expressed by pathogenic mycobacteria upon infection, has been shown to be secreted into the macrophage cytosol where it modulates and prevents phagosome-lysosome fusion, finally resulting in survival of the bacilli. A potent inhibitor, termed AX20017, was identified which specifically inhibited the catalytic activity of PknG in vitro. Infection of J774 macrophages with mycobacteria treated with AX20017 led to an increase rate of lysosomol delivery events. The same effect was observed when macrophages were infected with mycobacteria expressing a kinase-dead-version of PknG which strongly supports the idea that PknG activity is required for mycobacterial survival. A broad-ranged kinase assay screen including 25 kinases representatively chosen from the six major kinase families showed that the inhibitor was highly selective by inhibiting only PknG with high efficiency. Normal cellular processes within host cells were not affected. To elucidate the structural basis of inhibition of PknG and to learn more about the function of the kinase, the structure of PknG in complex with its inhibitor was solved. Since PknG full length was shown to be unstable, limited analysis was performed and a truncated version of PknG missing 8 kDa of the N-terminus was constructed. A purification protocol was established that allowed the purification of pure and stable protein. Crystallization assays were performed to screen for optimal conditions. The structure of dimeric PknG in complex with its specific inhibitor AX20017 was solved at a resolution of 2.4 Å using SIRAS. Three domains of PknG were defined: a) The N-terminal region encompassing the two CXXC motifs, which were shown to complex iron, thus forming a rubredoxin domain b) The kinase domain displaying a closed configuration, with the inhibitor occupying the nucleotide-binding pocket c) and the C-terminal domain with the tetratricopeptide repeat mediating dimerization of the two PknG molecules. The structure explained the high specificity of inhibition by AX20017. In total, 15 polar and non-polar interactions between the inhibitor and the kinase- or N-terminal domain were determined. Sequence alignments with all 518 human kinases derived from six major kinase families revealed that 6 of these interactions were not detected in any other kinase underlining the high specificity of inhibition. By expressing proteins with mutated inhibitor binding sites, these sites of interaction were further confirmed. Moreover, the molecular model of the kinase allowed insights into the regulation of PknG. Site-directed mutagenesis on the four cysteines forming the rubredoxin motif completely abolished PknG activity. This, in combination with the observed interactions between the rubredoxin domain and the lobes of the kinase domain, suggested that PknG activity might be regulated by the N-terminal globular domain. PknG undergoes autophosphorylation, similar to most mycobacterial kinases studied thus far. However, in-depth analysis of PknG autophosphorylation revealed significant differences. Whereas most mycobacterial kinases display a conserved autophosphorylation pattern on the activation loop, phosphorylated residues in PknG were exclusively identified at the Nterminus. For PknG, classified as the unique mycobacterial non-RD kinase due to a missing arginine residue in the catalytic loop, absence of autophosphorylation in the activation loop was suggested. By performing kinase assays using constructs lacking the potential Nterminal phosphorylation sites, this assumption was confirmed. Moreover, by analyzing these mutants on an endogenous substrate, it was demonstrated that autophosphorylation is not a prerequisite for activating the catalytic activity of PknG. Infection experiments with mycobacteria expressing mutated unphosphorylated PknG protein indicated that autophosphorylation is crucial for the role of PknG in preventing lysosomal delivery. Preliminary results of survival assays showed that mycobacteria which express PknG devoid of autophosphorylation sites were rapidly transferred to lysosomes and degraded. To further understand the activity of PknG within host macrophages, PknG was localized in eukaryotic cells. Upon transfection, PknG was detected in the nucleus of HeLa, Mel JuSo and HEK cells. Transfection assays with different truncated PknG constructs point to the Cterminal TPR containing domain of PknG for being responsible for nuclear translocation. In addition, Ag84, a downstream target of PknA, was studied. To address its function, Ag84 was overproduced in mycobacteria. Overexpression resulted in drastic changes in cell morphology, particularly in cell size and shape. Localization studies revealed that wildtype Ag84 localized equally to the cell poles, whereas overexpressed Ag84 was asymmetrically distributed at the poles leading to unbalanced cell wall extension. Furthermore, overexpressed Ag84 was found to affect cell division by preventing septum formation adjacent to the cell pole with higher Ag84 concentrations. Biochemical analysis revealed that Ag84 is a cytosolic protein. As an alternative explanation for the polar localization, oligomerization of Ag84 was analyzed. Chemical crosslinking and size-exclusion chromatography analysis showed that Ag84 is able to form oligomers of >6 molecules which might act as a complex scaffold fitting to the curvature of the cell poles. This complex structure then might allow recruiting other proteins involved in processes such as cell wall synthesis. Ag84 is the first effector protein, for which a role in mycobacterial cell shape control has been described. In addition, Ag84 was found to be involved in septum formation and therefore has a similar role as B. subtilis DivIVA, which in Bacillus however interacts with the MinCD system that is absent in mycobacteria. To conclude, the results presented in this thesis contribute to a better understanding of mycobacterial virulence. The knowledge of the structure of PknG might be particular useful with regard to the design of more potent drugs and the biochemistry data obtained for PknG allowed important insights into function and regulation of this eukaryotic-like serine/threonine protein kinase. Taken together, the results add more information to the complex network of mycobacteria-host interactions

Selamectin is the avermectin with the best potential for Buruli ulcer treatment

A comprehensive analysis was done to evaluate the potential use of anti-parasitic macrocyclic lactones (including avermectins and milbemycins) for Buruli ulcer (BU) therapy. A panel containing nearly all macrocyclic lactones used in human or in veterinary medicine was analyzed for activity in vitro against clinical isolates of Mycobacterium ulcerans. Milbemycin oxime and selamectin were the most active drugs against M. ulcerans with MIC values from 2 to 8 μg/mL and 2 to 4 μg/mL, respectively. In contrast, ivermectin and moxidectin, which are both in clinical use, showed no significant activity (MIC< 32 μg/mL). Time-kill kinetic assays showed bactericidal activity of selamectin and in vitro pharmacodynamic studies demonstrated exposure-dependent activity. These data together with analyses of published pharmacokinetic information strongly suggest that selamectin is the most promising macrocyclic lactone for BU treatment

Subversion of host immune responses by Mycobacterium tuberculosis

Tuberculosis, one of the world's oldest diseases has nowadays reached a pandemic prevalence. Despite its long history and intense research, efficient drugs against its causative agent, M. tuberculosis, are still limited. One reason for the pathogen's success lies within its capability to evade host immune defense mechanisms and to create a niche within host cells enabling the bacterium to persist for long periods. M. tuberculosis has evolved a diversified set of strategies to manipulate the immune response of the host. In this communication, we discuss some of the strategies employed by M. tuberculosis in order to survive within the hostile environment of the macrophage. A detailed analysis of the molecular basis of host-pathogen interactions will unravel novel mechanisms and might contribute to finding novel approaches to treat and combat tuberculosis