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.

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.

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.

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-ROP1_L (SGR), SAG1_L-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.

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—AMA1^domainI-SAG2-GRA1-ROP1_L (A^NSGR), AMA1^{domainsII,III}-SAG2-GRA1-ROP1_L (A^CSGR) and AMA1^fullprotein-SAG2-GRA1-ROP1_L (A^FSGR)—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.