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In reference to gene annotation, more than half of the tRNA species synthesized by Mycobacterium tuberculosis require the enzymatic addition of the cytosine-cytosine-adenine (CCA) tail, which is indispensable for amino acid charging and tRNA functionality. It makes the mycobacterial CCA-adding enzyme essential for survival of the bacterium and a potential target for novel pipelines in drug discovery avenues. Here, we described the rv3907c gene product, originally annotated as poly(A)polymerase ( rv3907c , PcnA) as a functional CCA-adding enzyme (CCA Mtb ) essential for viability of M. tuberculosis . The depletion of the enzyme affected tRNAs maturation, inhibited bacilli growth, and resulted in abundant accumulation of polyadenylated RNAs. We determined the enzymatic activities displayed by the mycobacterial CCA Mtb in vitro and studied the effects of inhibiting of its transcription in bacterial cells. We are the first to properly confirm the existence of RNA polyadenylation in mycobacteria, a previously controversial phenomenon, which we found promoted upon CCA-adding enzyme downexpression.

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.

invasion relies on the battery of specialized proteins, such as ROP8, which plays a key role in formation of parasitophorous vacuoles within host cells. The aim of the work was to evaluate diagnostic, immunoprophylactic and therapeutic potential of recombinant ROP8 (rROP8) protein as despite many years of research this parasitosis still is considered a significant medical and economic problem and the main areas requiring improvement are the diagnostics of the infection, vaccine development especially for humans and safe, specific chemotherapy. Recombinant ROP8 protein (rROP8) of was cloned, expressed in Rosetta(DE3)pLysS, and purified by nickel-affinity chromatography. Its diagnostic potential was evaluated using ELISA on sera from experimentally infected mice and human patients. The immunogenic properties of rROP8 were assessed through stimulation of human monocytes, mouse macrophages, and monocyte-derived dendritic cells, followed by cytokine profiling and co-culture with autologous CD4 lymphocytes. Finally, drug affinity responsive target stability (DARTS) assays and confocal microscopy were used to investigate the interaction of thiazolidin-4-one derivatives with rROP8 and their inhibitory effects on parasitophorous vacuole formation. The ROP8 proved to be promising as a potential diagnostic tool enabling the detection of specific IgG antibodies in mouse and human immune sera comparably to the native parasite antigen (TLA). The protein was also capable of activating human monocytes and mouse macrophages leading to the enhanced clearance of tachyzoites via phagocytosis. The protein was also able to activate human monocyte-derived dendritic cells as shown by the cytometric analysis of surface markers and multiplex detection of specific cytokines, also in co-cultures with homologous lymphocytes, leading to Th1/Th17 lymphocyte phenotype. Finally, the proteolytic degradation of rROP8 was significantly inhibited by the presence of all thiazolidin-4-one derivatives, which exhibited high activity against in previous studies.

IntroductionIn 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.MethodsWe 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.FindingsTranscriptomes 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-1β, 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, TGFβ, 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.ConclusionElevated 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.

Toxoplasmosis is caused by the opportunistic, cosmopolitan protozoan is one of the most common parasitoses in the world. This parasite can pose a threat to people with immunodeficiency but also to the fetus, since the invasion can lead to miscarriages. Moreover, this parasite can contribute to economic losses in livestock farming. These problems lead to the implementation of new, safe solutions for the development of effective toxoplasmosis immunoprophylaxis. In this work, newly produced recombinant trivalent chimeric proteins of , based on SAG1-SAG2 recombinant chimeric antigen that differ in one terminal antigenic component, were tested in terms of their ability to induce an effective post-vaccination response. Antigens were tested to assess their ability to elicit APC cells response and further mice of the C3H/HeOuJ strain were immunized using those antigens, to evaluate their immunogenicity and immunoprotective effect . Two weeks after the last dose mice were either sacrificed to assess selected parameters of the immune response or infected with DX strain to determine the degree of protection one month later. The results of serological tests revealed a high level of serum IgG antibodies specific for the native TLA antigens. TLA-stimulated splenocytes produced cytokines that are important in inhibiting protozoal invasion. Additionally, CD3 CD4 and CD3 CD8 T cell subpopulations of splenocytes were analysed by flow cytometry. One month after experimental infection mice were sacrificed, and their brains were isolated to count tissue cyst. Immunization of mice with recombinant trivalent chimeric proteins of resulted in reduction of tissue cyst burden rates reaching even 74%. The obtained results demonstrate strong immunogenicity of the studied proteins and will allow to select candidates for further research aimed at increasing the immunoprotective properties of experimental vaccines against toxoplasmosis based on chimeric antigens.

Fluoroquinolones (FQs) are pharmacological cornerstone agents in global tuberculosis (TB) management, critical for both accelerating drug-sensitive regimens and defining the resistance spectrum of multidrug-resistant TB. To evaluate the therapeutic potential of this class across the mycobacterial spectrum, we conducted a comprehensive phenotypic screening campaign utilizing OTAVA and ECBD pilot-libraries against reference strains of ( ) and ( ). This initial screen, followed by detailed bactericidal concentration (BC) and cytotoxicity assessments, identified and selected potent FQ derivatives for in-depth characterization. These FQ compounds exhibited exceptionally high potency against , with several demonstrating robust bactericidal activity at concentrations far below toxic levels to mammalian cells. This activity was retained in clinically relevant, challenging states, as the compounds effectively eliminated residing within human macrophages and persisted within protective biofilm structures. In stark contrast, displayed marked intrinsic resistance. Most FQ derivatives highly potent against required significantly higher concentrations for inhibition and bactericidal effect against . For instance, a leading clinical FQ showed substantially diminished activity against compared to . Analysis of acquired resistance mechanisms demonstrated that both and primarily rely on similar acquired point mutations in the Quinolone Resistance Determining Regions (QRDRs) of the and genes. However, molecular docking indicated a slight but significant reduction in theoretical binding affinity to the gyrase complex. We hypothesize that the profound differential in intrinsic susceptibility is at least partially driven by non-target factors. This work supports the continued critical role of FQs in therapy while highlighting the importance of structural modifications that could address hypothesized intrinsic resistance mechanisms in .

This study presents an evaluation of four tetravalent recombinant chimeric proteins containing fragments of the Toxoplasma gondii antigens, SAG2, GRA1, ROP1 and AMA1, as potential replacements of a the soluble, whole-cell tachyzoite lysate (TLA) used in serological assays. Recombinant chimeric proteins (SAG2-GRA1-ROP1-AMA1N, AMA1N-SAG2-GRA1-ROP1, AMA1C-SAG2-GRA1-ROP1, and AMA1-SAG2-GRA1-ROP1) obtained by genetic engineering were tested for their reactivity with specific IgM and IgG antibodies from sera of experimentally infected mice and humans with T. gondii infection using an enzyme-linked immunosorbent assay (ELISA). In total 192 serum samples from patients with acquired T. gondii infection and 137 sera from seronegative individuals were examined. The reactivity of chimeric antigens with antibodies generated during T. gondii invasion was measured and compared to the results obtained in assays based on whole-cell Toxoplasma antigen. Chimeric proteins proved effective in differentiation between T. gondii-infected and uninfected individuals (100% sensitivity and specificity in the IgG ELISAs) which shows their potential usefulness as a replacements for TLA in standardized commercial tests for the serodiagnosis of toxoplasmosis. In addition, the chimeric proteins were tested for use in avidity determination. Obtained results were comparable to those of the corresponding commercial assays, suggesting the utility of these proteins for avidity assessment. Furthermore, this study demonstrated that the AMA1-SAG2-GRA1-ROP1 chimeric protein has the potential to distinguish specific antibodies from serum samples of individuals with the early and chronic phase of T. gondii infection.

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.

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.

Two-component signal transduction systems enable mycobacterial cells to quickly adapt and adequately respond to adverse environmental conditions encountered at various stages of host infection. We attempted to determine the role of the Rv3143 “orphan” response regulator in the physiology of Mycobacterium tuberculosis and its orthologue Msmeg_2064 in Mycobacterium smegmatis . We identified the Rv3143 protein as an interaction partner for NuoD, a member of the type I NADH dehydrogenase complex involved in oxidative phosphorylation. The mutants Δ rv3143 and Δ msmeg_2064 were engineered in M. tuberculosis and M. smegmatis cells, respectively. The Δ msmeg_2064 strain exhibited a significant reduction in growth and viability in the presence of reactive nitrogen species. The Rv3143-deficient strain was sensitive to valinomycin, which is known to reduce the electrochemical potential of the cell and overexpressed genes required for nitrate respiration. An increased level of reduction of the 2,3,5-triphenyltetrazolium chloride (TTC) electron acceptor in Δ rv3143 and Δ msmeg_2064 cells was also evident. The silencing of ndh expression using CRISPRi/dCas9 affected cell survival under limited oxygen conditions. Oxygen consumption during entry to hypoxia was most severely affected in the double-mutant Δ msmeg_2064 ndh CRISPRi/dCas9 . We propose that the regulatory protein Rv3143 is a component of the Nuo complex and modulates its activity.