New coumarin linked thiazole derivatives as antimycobacterial agents: Design, synthesis, enoyl acyl carrier protein reductase (InhA) inhibition and molecular modeling.

Tuberculosis is a global serious problem that imposes major health, economic and social challenges worldwide. The search for new antitubercular drugs is extremely important which could be achieved via inhibition of different druggable targets. Mycobacterium tuberculosis enoyl acyl carrier protein reductase (InhA) enzyme is essential for the survival of M. tuberculosis. In this investigation, a series of coumarin based thiazole derivatives was synthesized relying on a molecular hybridization approach and was assessed against thewild typeMtb H37Rv and its mutant strain (ΔkatG) via inhibiting InhA enzyme. Among the synthesized derivatives, compounds 2b, 3i and 3j were the most potent against wild type M. tuberculosis with MIC values ranging from 6 to 8 µg/ mL and displayed low cytotoxicity towards mouse fibroblasts at concentrations 8-13 times higher than the MIC values. The three hybrids could also inhibit the growth of ΔkatGmutant strain which is resistant to isoniazid (INH). Compounds 2b and 3j were able to inhibit the growth of mycobacteria inside human macrophages, indicating their ability to penetrate human professional phagocytes. The two derivatives significantly suppress mycobacterial biofilm formation by 10-15 %. The promising target compounds were also assessed for their inhibitory effect against InhA and showed potent effectiveness with IC50 values of 0.737 and 1.494 µM, respectively. Molecular docking studies revealed that the tested compounds occupied the active site of InhA in contact with the NAD+ molecule. The 4-phenylcoumarin aromatic system showed binding interactions within the hydrophobic pocket of the active site. Furthermore, H-bond formation and π -π stacking interactions were also recorded for the promising derivatives.

1H-Benzo[d]Imidazole Derivatives Affect MmpL3 in Mycobacterium tuberculosis.

1H-benzo[d]imidazole derivatives exhibit antitubercular activity in vitro at a nanomolar range of concentrations and are not toxic to human cells, but their mode of action remains unknown. Here, we showed that these compounds are active against intracellular Mycobacterium tuberculosis To identify their target, we selected drug-resistant M. tuberculosis mutants and then used whole-genome sequencing to unravel mutations in the essential mmpL3 gene, which encodes the integral membrane protein that catalyzes the export of trehalose monomycolate, a precursor of the mycobacterial outer membrane component trehalose dimycolate (TDM), as well as mycolic acids bound to arabinogalactan. The drug-resistant phenotype was also observed in the parental strain overexpressing the mmpL3 alleles carrying the mutations identified in the resistors. However, no cross-resistance was observed between 1H-benzo[d]imidazole derivatives and SQ109, another MmpL3 inhibitor, or other first-line antitubercular drugs. Metabolic labeling and quantitative thin-layer chromatography (TLC) analysis of radiolabeled lipids from M. tuberculosis cultures treated with the benzoimidazoles indicated an inhibition of trehalose dimycolate (TDM) synthesis, as well as reduced levels of mycolylated arabinogalactan, in agreement with the inhibition of MmpL3 activity. Overall, this study emphasizes the pronounced activity of 1H-benzo[d]imidazole derivatives in interfering with mycolic acid metabolism and their potential for therapeutic application in the fight against tuberculosis.

Evaluation of the Mycobactericidal Effect of Thio-functionalized Carbohydrate Derivatives.

Sugars with heteroatoms other than oxygen have attained considerable importance in glycobiology and in drug design since they are often more stable in blood plasma due to their resistance to enzymes, such as glycosidases, phosphorylases and glycosyltransferases. The replacement of oxygen atoms in sugars with sulfur forms thio-sugars, which are potentially useful for the treatment of diabetes and some bacterial and viral infections. Here, we evaluated the antibacterial activity of thio-functionalized carbohydrate derivatives. A set of 21 compounds was screened against acid-fast Mycobacterium tuberculosis (Mtb), gram-negative Escherichia coli and gram-positive Staphylococcus aureus. The tested carbohydrate derivatives were most effective against tubercle bacilli, with as many as five compounds (thioglycoside 6, thiosemicarbazone 16A, thiosemicarbazone 20, aminothiadiazole 23, and thiazoline 26) inhibiting its growth with MIC50 ≤ 50 µM/CFU. Only two compounds (aminothiadiazole 23 and thiazoline 26) were able to inhibit the growth of E. coli at concentrations below 1 mM, and one of them, aminothiadiazole 23, inhibited the growth of S. aureus at a concentration ≤1 mM. The five compounds affecting the growth of mycobacteria were either thiodisaccharides (6, 16A, and 20) or thioglycosides (23 and 26). All of these compounds (6, 16A, 20, 23, and 26) were able to inhibit the growth of Mtb deposited within human macrophages. However, three of the five selected compounds (6, 23, and 26) exhibited relatively high cytotoxicity in mouse fibroblasts at micromolar concentrations. The selected thio-sugars are very promising compounds, thus making them candidates for further modifications that would decrease their cytotoxicity against eukaryotic cells without affecting their antimycobacterial potential.

Two-Year Follow-up Study of Mycobacterium tuberculosis Antigen-Driven IFN-γ Responses and Macrophage sCD14 Levels After Tuberculosis Contact.

Clinical data regarding the prediction of active tuberculosis (TB) development in close TB contacts are scarce. To address this problem, we performed a 2-year follow-up study of Mycobacterium tuberculosis (M.tb) antigen-driven IFN-gamma responses and serum levels of soluble macrophage CD14 receptor in individuals with recent or prolonged M.tb exposure. Between June 2011 and June 2013, we studied 60 healthy Polish adults with recent household or long-term work TB contact and individuals without known M.tb exposure. All of them underwent baseline and repeated testing with IGRA (IFN-gamma release assay) and serum sCD14 ELISA quantification. Frequencies of IGRA results differed at the baseline and follow-up testing. IGRA reversions were noticed in almost one-third of Work TB Contacts and no participants from the Household TB Contact group. IGRA conversions were found in 40 % of Household TB Contacts. No correlation between the IGRA result and the sCD14 level was observed. IFN-γ variability has important implications for clinical practice and requires caution in interpreting the results to distinguish new infections from nonspecific inter-individual variations in cytokine responses. The impairment of IFN-γ response in some individuals with prolonged M.tb exposure representing a resistant immune status does not allow considering IGRA results as reliable and credible. Monitoring the serum sCD14 level can reduce the likelihood of a false prediction of active TB development in close TB contacts showing an M.tb-specific increase in the IFN-gamma production in repeated IGRA testing.

Evaluation of long-term immunity and protection against T. gondii after immunization with multivalent recombinant chimeric T. gondii proteins.

Toxoplasmosis caused by the opportunistic, cosmopolitan protozoan Toxoplasma gondii is one of the most common parasitoses in the world. Although it may prove dangerous or even fatal for immunocompromised individuals, immunoprophylaxis for humans is still nonexistent. Thus, the aim of the current work was to assess the ability of two immunogenic recombinant chimeric T. gondii proteins, SAG2-GRA1-ROP1 (SGR) and SAG1-MIC1-MAG1-GRA2 (SMMG), selected in previous experiments to induce long-lasting immunity when administered with a safe adjuvant. Thus, the determination of immunological parameters and parasite challenge were performed both two weeks after the last boost injection and 6 months postvaccination. Both experimental vaccines triggered specific humoral and cellular responses in immunized C3H/HeOuJ male mice, characterized by the production of specific IgG (IgG1/IgG2a) antibodies in vivo and the synthesis of key Th1/Th2 cytokines by Toxoplasma lysate antigen-stimulated splenocytes in vitro. Although the levels of specific antibodies and cytokine release were in most cases lower six months postimmunization, the protection rates conferred by the vaccination were comparable regardless of the time after the administration of the last vaccine dose. The results indicate that both preparations induce long-lasting immunity, which makes them attractive candidates for further research aimed at boosting their immunogenicity and immunoprotective capacity.

The functional response of human monocyte-derived macrophages to serum amyloid A and Mycobacterium tuberculosis infection.

Introduction: In the course of tuberculosis (TB), the level of major acute phase protein, namely serum amyloid A (hSAA-1), increases up to a hundredfold in the pleural fluids of infected individuals. Tubercle bacilli infecting the human host can be opsonized by hSAA-1, which affects bacterial entry into human macrophages and their intracellular multiplication. Methods: We applied global RNA sequencing to evaluate the functional response of human monocyte-derived macrophages (MDMs), isolated from healthy blood donors, under elevated hSAA-1 conditions and during infection with nonopsonized and hSAA-1-opsonized Mycobacterium tuberculosis (Mtb). In the same infection model, we also examined the functional response of mycobacteria to the intracellular environment of macrophages in the presence and absence of hSAA-1. The RNASeq analysis was validated using qPCR. The functional response of MDMs to hSAA-1 and/or tubercle bacilli was also evaluated for selected cytokines at the protein level by applying the Milliplex system. Findings: Transcriptomes of MDMs cultured in the presence of hSAA-1 or infected with Mtb showed a high degree of similarity for both upregulated and downregulated genes involved mainly in processes related to cell division and immune response, respectively. Among the most induced genes, across both hSAA-1 and Mtb infection conditions, CXCL8, CCL15, CCL5, IL-1ß, and receptors for IL-7 and IL-2 were identified. We also observed the same pattern of upregulated pro-inflammatory cytokines (TNFα, IL-6, IL-12, IL-18, IL-23, and IL-1) and downregulated anti-inflammatory cytokines (IL-10, TGFß, and antimicrobial peptide cathelicidin) in the hSAA-1 treated-MDMs or the phagocytes infected with tubercle bacilli. At this early stage of infection, Mtb genes affected by the inside microenvironment of MDMs are strictly involved in iron scavenging, adaptation to hypoxia, low pH, and increasing levels of CO2. The genes for the synthesis and transport of virulence lipids, but not cholesterol/fatty acid degradation, were also upregulated. Conclusion: Elevated serum hSAA-1 levels in tuberculosis enhance the response of host phagocytes to infection, including macrophages that have not yet been in contact with mycobacteria. SAA induces antigen processing and presentation processes by professional phagocytes reversing the inhibition caused by Mtb infection.

Mycobacterium tuberculosis Binds Human Serum Amyloid A, and the Interaction Modulates the Colonization of Human Macrophages and the Transcriptional Response of the Pathogen.

As a very successful pathogen with outstanding adaptive properties, Mycobacterium tuberculosis (Mtb) has developed a plethora of sophisticated mechanisms to subvert host defenses and effectively enter and replicate in the harmful environment inside professional phagocytes, namely, macrophages. Here, we demonstrated the binding interaction of Mtb with a major human acute phase protein, namely, serum amyloid A (SAA1), and identified AtpA (Rv1308), ABC (Rv2477c), EspB (Rv3881c), TB 18.6 (Rv2140c), and ThiC (Rv0423c) membrane proteins as mycobacterial effectors responsible for the pathogen-host protein interplay. SAA1-opsonization of Mtb prior to the infection of human macrophages favored bacterial entry into target phagocytes accompanied by a substantial increase in the load of intracellularly multiplying and surviving bacteria. Furthermore, binding of human SAA1 by Mtb resulted in the up- or downregulation of the transcriptional response of tubercle bacilli. The most substantial changes were related to the increased expression level of the genes of two operons encoding mycobacterial transporter systems, namely, mmpL5/mmpS5 (rv0676c), and rv1217c, rv1218c. Therefore, we postulate that during infection, Mtb-SAA1 binding promotes the infection of host macrophages by tubercle bacilli and modulates the functional response of the pathogen.

Imidazole-Thiosemicarbazide Derivatives as Potent Anti-Mycobacterium tuberculosis Compounds with Antibiofilm Activity.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogenic bacterium and the causative agent of tuberculosis. This disease is one of the most ancient and deadliest bacterial infections, as it poses major health, social and economic challenges at a global level, primarily in low- and middle-income countries. The lack of an effective vaccine, the long and expensive drug therapy, and the rapid spread of drug-resistant strains of Mtb have led to the re-emergence of tuberculosis as a global pandemic. Here, we assessed the in vitro activity of new imidazole-thiosemicarbazide derivatives (ITDs) against Mtb infection and their effects on mycobacterial biofilm formation. Cytotoxicity studies of the new compounds in cell lines and human monocyte-derived macrophages (MDMs) were performed. The anti-Mtb activity of ITDs was evaluated by determining minimal inhibitory concentrations of resazurin, time-kill curves, bacterial intracellular growth and the effect on biofilm formation. Mutation frequency and whole-genome sequencing of mutants that were resistant to ITDs were performed. The antimycobacterial potential of ITDs with the ability to penetrate Mtb-infected human macrophages and significantly inhibit the intracellular growth of tubercle bacilli and suppress Mtb biofilm formation was observed.

The Immunogenic and Immunoprotective Activities of Recombinant Chimeric T. gondii Proteins Containing AMA1 Antigen Fragments.

Toxoplasmosis, one of the most common parasitoses worldwide, is potentially dangerous for individuals with a weakened immune system, but specific immunoprophylaxis intended for humans is still lacking. Thus, efforts have been made to create an efficient universal vaccine for both animals and humans to overcome the shortcomings of currently used treatment methods and protect all hosts against toxoplasmosis. The current work represents a relatively new approach to vaccine development based on recombinant chimeric Toxoplasma gondii antigens. In the present research, three tetravalent chimeric proteins containing different portions of the parasite's AMA1 antigen-AMA1domainI-SAG2-GRA1-ROP1L (ANSGR), AMA1domainsII,III-SAG2-GRA1-ROP1L (ACSGR) and AMA1fullprotein-SAG2-GRA1-ROP1L (AFSGR)-were tested for their immunogenic and immunoprotective capacities. All tested proteins were immunogenic, as evidenced by the triggering of specific humoral and cellular immune responses in vaccinated C3H/HeOuJ mice, defined by the production of specific IgG (IgG1/IgG2a) antibodies in vivo and synthesis of key Th1/Th2 cytokines by Toxoplasma lysate antigen-stimulated splenocytes in vitro. Although all tested preparations provided partial protection against chronic toxoplasmosis in immunized and T. gondii-challenged mice, the intensity of the generated immunoprotection depended on the fragment of the AMA1 antigen incorporated into the chimeric antigen's structure.

PPE51 Is Involved in the Uptake of Disaccharides by Mycobacterium tuberculosis.

We have recently found that selected thio-disaccharides possess bactericidal effects against Mycobacterium tuberculosis but not against Escherichia coli or Staphylococcus aureus. Here, we selected spontaneous mutants displaying resistance against the investigated thio-glycoside. According to next-generation sequencing, four of six analyzed mutants which were resistant to high concentrations of the tested chemical carried nonsynonymous mutations in the gene encoding the PPE51 protein. The complementation of these mutants with an intact ppe51 gene returned their sensitivity to the wild-type level. The uptake of tritiated thio-glycoside was significantly more abundant in wild-type Mycobacterium tuberculosis compared to the strain carrying the mutated ppe51 gene. The ppe51 mutations or CRISPR-Cas9-mediated downregulation of PPE51 expression affected the growth of mutant strains on minimal media supplemented with disaccharides (maltose or lactose) but not with glycerol or glucose as the sole carbon and energy source. Taking the above into account, we postulate that PPE51 participates in the uptake of disaccharides by tubercle bacilli.

The Impact of the Antigenic Composition of Chimeric Proteins on Their Immunoprotective Activity against Chronic Toxoplasmosis in Mice.

Toxoplasmosis may pose a serious threat for individuals with weakened or undeveloped immune systems. However, to date, there is no specific immunoprophylaxis for humans. Thus, the aim of this study was to evaluate the immunogenicity of three trivalent-SAG2-GRA1-ROP1L (SGR), SAG1L-MIC1-MAG1 (SMM), and GRA1-GRA2-GRA6 (GGG)-and two tetravalent-SAG2-GRA1-ROP1-GRA2 (SGRG) and SAG1-MIC1-MAG1-GRA2 (SMMG)-chimeric T. gondii proteins, as well as their protective potential against chronic toxoplasmosis in laboratory mice. All three trivalent recombinant proteins possessed immunogenic properties, as defined by specific humoral and cellular responses in vaccinated mice characterized by the synthesis of specific IgG (IgG1/IgG2a) antibodies in vivo and the release of Th1/Th2 cytokines by stimulated splenocytes in vitro. Immunization with all three recombinant proteins provided partial protection against toxoplasmosis, although the protective capacity strongly depended on the individual antigenic composition of each preparation. The antigens providing the highest (86%) and lowest (45%) protection, SGR and SMM, respectively, were supplemented with GRA2 antigen fragment, to form the tetravalent chimeric proteins SGRG and SMMG. Further study revealed that the tetravalent preparations exhibited high immunogenic potential; however, the addition of another antigen to the recombinant protein structure had distinct effects on the protection generated, compared to that of the trivalent counterparts, depending on the antigen tested.