Anna Brzostek

Mycobacterium tuberculosis is able to accumulate and utilize cholesterol.

It is expected that the obligatory human pathogen Mycobacterium tuberculosis must adapt metabolically to the various nutrients available during its cycle of infection, persistence, and reactivation. Cholesterol, which is an important part of the mammalian cytoplasmic membrane, is a potential energy source. Here, we show that M. tuberculosis grown in medium containing a carbon source other than cholesterol is able to accumulate cholesterol in the free-lipid zone of its cell wall. This cholesterol accumulation decreases the permeability of the cell wall for the primary antituberculosis drug, rifampin, and partially masks the mycobacterial surface antigens. Furthermore, M. tuberculosis was able to grow on mineral medium supplemented with cholesterol as the sole carbon source. Targeted disruption of the Rv3537 (kstD) gene inhibited growth due to inactivation of the cholesterol degradation pathway, as evidenced by accumulation of the intermediate, 9-hydroxy-4-androstene-3,17-dione. Our findings that M. tuberculosis is able to accumulate cholesterol in the presence of alternative nutrients and use it when cholesterol is the sole carbon source in vitro may facilitate future studies into the pathophysiology of this important deadly pathogen.

Mycobacterium tuberculosis ClpX Interacts with FtsZ and Interferes with FtsZ Assembly

FtsZ assembly at the midcell division site in the form of a Z-ring is crucial for initiation of the cell division process in eubacteria. It is largely unknown how this process is regulated in the human pathogen Mycobacterium tuberculosis. Here we show that the expression of clpX was upregulated upon macrophage infection and exposure to cephalexin antibiotic, the conditions where FtsZ-ring assembly is delayed. Independently, we show using pull-down, solid-phase binding, bacterial two-hybrid and mycobacterial protein fragment complementation assays, that M. tuberculosis FtsZ interacts with ClpX, the substrate recognition domain of the ClpXP protease. Incubation of FtsZ with ClpX increased the critical concentration of GTP-dependent polymerization of FtsZ. Immunoblotting revealed that the intracellular ratio of ClpX to FtsZ in wild type M. tuberculosis is approximately 1∶2. Overproduction of ClpX increased cell length and modulated the localization of FtsZ at midcell sites; however, intracellular FtsZ levels were unaffected. A ClpX-CFP fusion protein localized to the cell poles and midcell sites and colocalized with the FtsZ-YFP protein. ClpX also interacted with FtsZ mutant proteins defective for binding to and hydrolyzing GTP and possibly for interactions with other proteins. Taken together, our results suggest that M. tuberculosis ClpX interacts stoichiometrically with FtsZ protomers, independent of its nucleotide-bound state and negatively regulates FtsZ activities, hence cell division.

Toxoplasma gondii: The immunogenic and protective efficacy of recombinant ROP2 and ROP4 rhoptry proteins in murine experimental toxoplasmosis

Toxoplasmosis is a one of the most world-wide spread zoonosis representing a very serious clinical and veterinary problem. In the presented study, we evaluated the protective efficacy of a combined recombinant ROP2 and ROP4 subunit vaccine in a chronic Toxoplasma gondii infection in mice. The recombinant ROP2 (rROP2) and ROP4 (rROP4) proteins were cloned and expressed in Escherichia coli and then used for the immunization of C3H/HeJ mice. Both antigens generated a strong systemic mixed Th1/Th2 response polarized towards IgG1 antibody isotype. In contrast to rROP2 stimulating only the specific IL-2 release, rROP4 and crude toxoplasma lysate antigen (TLA) used as a source of native forms of the parasite proteins induced significant proliferation of splenocytes and specific production of IFN-gamma as well as IL-2, the Th1-type cytokines. Challenge of rROP2 and rROP4-vaccinated mice with cysts of low virulent T. gondii DX strain resulted in a partial protection effect with a significantly lower brain parasites load when compared with control animals. In the immunized group of mice the brain cysts number was reduced by nearly 46% as was determined in two independent experiments. These results suggest that, similar to ROP2, rhoptry protein ROP4 could be a very good candidate for future anti-T. gondii multicomponent vaccine based on the recombinant forms of different parasite proteins.

Methodological and Clinical Aspects of the Molecular Epidemiology of Mycobacterium tuberculosis and Other Mycobacteria

SUMMARY Molecular typing has revolutionized epidemiological studies of infectious diseases, including those of a mycobacterial etiology. With the advent of fingerprinting techniques, many traditional concepts regarding transmission, infectivity, or pathogenicity of mycobacterial bacilli have been revisited, and their conventional interpretations have been challenged. Since the mid-1990s, when the first typing methods were introduced, a plethora of other modalities have been proposed. So-called molecular epidemiology has become an essential subdiscipline of modern mycobacteriology. It serves as a resource for understanding the key issues in the epidemiology of tuberculosis and other mycobacterial diseases. Among these issues are disclosing sources of infection, quantifying recent transmission, identifying transmission links, discerning reinfection from relapse, tracking the geographic distribution and clonal expansion of specific strains, and exploring the genetic mechanisms underlying specific phenotypic traits, including virulence, organ tropism, transmissibility, or drug resistance. Since genotyping continues to unravel the biology of mycobacteria, it offers enormous promise in the fight against and prevention of the diseases caused by these pathogens. In this review, molecular typing methods for Mycobacterium tuberculosis and nontuberculous mycobacteria elaborated over the last 2 decades are summarized. The relevance of these methods to the epidemiological investigation, diagnosis, evolution, and control of mycobacterial diseases is discussed.

Evaluation of three recombinant multi-antigenic vaccines composed of surface and secretory antigens of Toxoplasma gondii in murine models of experimental toxoplasmosis.

The great clinical and economical impact of Toxoplasma gondii infections makes the development of an effective vaccine for controlling toxoplasmosis an extremely important aim. In the presented study, we evaluate the protective and immunogenic properties of three recombinant subunit vaccines composed of rROP2+rGRA4+rSAG1, rROP2+rROP4+rGRA4 and rROP2+rROP4+rSAG1 proteins of T. gondii in an experimental toxoplasmosis model in the C3H/HeJ and C57BL/6 mouse strains. All three recombinant vaccines induced partial protection as measured by the reduction of brain cyst burden following challenge with five tissue cysts of the low virulence DX T. gondii strain. The level of protection was dependent on the antigen composition of the vaccine and the genetic background of the laboratory animals. The strongest protection against chronic toxoplasmosis was induced in both C3H/HeJ and C57BL/6 mice by the mixture of rhoptry proteins rROP2 and rROP4 combined with tachyzoite major protein rSAG1. The average parasite burden in these groups of mice was reduced by 71% and 90%, respectively, compared to non-vaccinated mice. The observed protective effect was related to the vaccine-induced cellular and humoral immune responses, as measured by the antigen-induced release of the Th1 cytokines IFN-γ and IL-2, the antigen-stimulated proliferation of spleen cells of vaccinated animals in comparison to control animals and the development of systemic antigen-specific IgG1 and IgG2a (C3H/HeJ) or IgG2c (C57BL/6) antibodies. Our studies show that recombinant rROP2, rROP4, rGRA4 and rSAG1 antigens may be promising candidates for a subunit vaccine against toxoplasmosis. Additionally, we demonstrate that the ideal composition of vaccine antigens can be equally effective in mice with different genetic backgrounds and variable levels of innate resistance to toxoplasmosis, resulting in strong protection against T. gondii invasion.

Evaluation of NAD+-Dependent DNA Ligase of Mycobacteria as a Potential Target for Antibiotics

ABSTRACT Mycobacteria contain genes for several DNA ligases, including ligA, which encodes a NAD+-dependent enzyme that has been postulated to be a target for novel antibacterial compounds. Using a homologous recombination system, direct evidence is presented that wild-type ligA cannot be deleted from the chromosome of Mycobacterium smegmatis. Deletions of native ligA in M. smegmatis could be obtained only after the integration of an extra copy of M. smegmatis or Mycobacterium tuberculosis ligA into the attB site of the chromosome, with expression controlled by chemically inducible promoters. The four ATP-dependent DNA ligases encoded by the M. smegmatis chromosome were unable to replace the function of LigA. Interestingly, the LigA protein from M. smegmatis could be substituted with the NAD+-dependent DNA ligase of Escherichia coli or the ATP-dependent ligase of bacteriophage T4. The conditional mutant strains allowed the analysis of the effect of LigA depletion on the growth of M. smegmatis. The protein level of the conditional mutants was estimated by Western blot analysis using antibodies raised against LigA of M. tuberculosis. This revealed that a strong overproduction or depletion of LigA did not affect the growth or survival of mycobacteria under standard laboratory conditions. In conclusion, although NAD+-dependent DNA ligase is essential for mycobacterial viability, only low levels of protein are required for growth. These findings suggest that very efficient inhibition of enzyme activity would be required if NAD+-dependent DNA ligase is to be useful as an antibiotic target in mycobacteria. The strains developed here will provide useful tools for the evaluation of the efficacy of any appropriate compounds in mycobacteria.

Cholesterol oxidase is indispensable in the pathogenesis of Mycobacterium tuberculosis.

Despite considerable research effort, the molecular mechanisms of Mycobacterium tuberculosis (Mtb) virulence remain unclear. Cholesterol oxidase (ChoD), an extracellular enzyme capable of converting cholesterol to its 3-keto-4-ene derivative, cholestenone, has been proposed to play a role in the virulence of Mtb. Here, we verified the hypothesis that ChoD is capable of modifying the bactericidal and pro-inflammatory activity of human macrophages. We also sought to determine the contribution of complement receptor 3 (CR3)- and Toll-like receptor 2 (TLR2)-mediated signaling pathways in the development of macrophage responses to Mtb. We found that intracellular replication of an Mtb mutant lacking a functional choD gene (ΔchoD) was less efficient in macrophages than that of the wild-type strain. Blocking CR3 and TLR2 with monoclonal antibodies enhanced survival of ΔchoD inside macrophages. We also showed that, in contrast to wild-type Mtb, the ΔchoD strain induced nitric oxide production in macrophages, an action that depended on the TLR2, but not the CR3, signaling pathway. Both wild-type and mutant strains inhibited the production of reactive oxygen species (ROS), but the ΔchoD strain did so to a significantly lesser extent. Blocking TLR2-mediated signaling abolished the inhibitory effect of wild-type Mtb on ROS production by macrophages. Wild-type Mtb, but not the ΔchoD strain, decreased phorbol myristate acetate-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which are involved in both TLR2- and CR3-mediated signaling pathways. Our finding also revealed that the production of interleukin 10 by macrophages was significantly lower in ΔchoD-infected macrophages than in wild-type Mtb-infected macrophages. However, tumor necrosis factor-α production by macrophages was the same after infection with mutant or wild-type strains. In summary, we demonstrate here that ChoD is required for Mtb interference with the TLR2-mediated signaling pathway and subsequent intracellular growth and survival of the pathogen in human macrophages.

AccD6, a Key Carboxyltransferase Essential for Mycolic Acid Synthesis in Mycobacterium tuberculosis, Is Dispensable in a Nonpathogenic Strain

Acetyl coenzyme A carboxylase (ACC) is a key enzyme providing a substrate for mycolic acid biosynthesis. Although in vitro studies have demonstrated that the protein encoded by accD6 (Rv2247) may be a functional carboxyltransferase subunit of ACC in Mycobacterium tuberculosis, the in vivo function and regulation of accD6 in slow- and fast-growing mycobacteria remain elusive. Here, directed mutagenesis demonstrated that although accD6 is essential for M. tuberculosis, it can be deleted in Mycobacterium smegmatis without affecting its cell envelope integrity. Moreover, we showed that although it is part of the type II fatty acid synthase operon, the accD6 gene of M. tuberculosis, but not that of M. smegmatis, possesses its own additional promoter (P(acc)). The expression level of accD6(Mtb) placed only under the control of P(acc) is 10-fold lower than that in wild-type M. tuberculosis but is sufficient to sustain cell viability. Importantly, this limited expression level affects growth, mycolic acid content, and cell morphology. These results provide the first in vivo evidence for AccD6 as a key player in the mycolate biosynthesis of M. tuberculosis, implicating AccD6 as the essential ACC subunit in pathogenic mycobacteria and an excellent target for new antitubercular compounds. Our findings also highlight important differences in the mechanism of acetyl carboxylation between pathogenic and nonpathogenic mycobacterial species.

The role of 3-ketosteroid 1(2)-dehydrogenase in the pathogenicity of Mycobacterium tuberculosis

BackgroundA growing body of evidence suggests that Mycobacterium tuberculosis (Mtb) uses the host’s cholesterol as a source of carbon and energy during infection. Strains defective in cholesterol transport or degradation exhibit attenuated growth in activated macrophages and diminished infectivity in animal models. The aim of this study was to evaluate intracellular replication of a cholesterol degradation-deficient Mtb mutant in human macrophages (MØ) in vitro and assess the functional responses of Mtb mutant-infected MØ.ResultsA mutant Mtb H37Rv strain containing an inactivated kstD gene (∆kstD), which encodes 3-ketosteroid 1(2)-dehydrogenase (KstD), was previously prepared using the homologous recombination-based gene-replacement technique. A control strain carrying the kstD gene complemented with an intact kstD was also previously constructed. In this study, human resting MØ were obtained after overnight differentiation of the human monocyte-macrophage cell line THP-1. Resting MØ were further activated with interferon-γ (IFN-γ). The ability of the kstD-defective Mtb mutant strain to replicate intracellularly in human MØ was evaluated using a colony-forming assay. Nitric oxide (NO) and reactive oxygen species (ROS) production by MØ infected with wild-type or ∆kstD strains was detected using Griess reagent and chemiluminescence methods, respectively. The production of tumor necrosis factor-α and interleukin-10 by MØ after infection with wild-type or mutant Mtb was examined using enzyme-linked immunosorbent assays.We found that replication of mutant Mtb was attenuated in resting MØ compared to the wild-type or complemented strains. Moreover, the mutant was unable to inhibit the NO and ROS production induced through Toll-like receptor 2 (TLR2) signaling in infected resting MØ. In contrast, mutant and wild-type Mtb behaved similarly in MØ activated with IFN-γ before and during infection.ConclusionsThe Mtb mutant ∆kstD strain, which is unable to use cholesterol as a source of carbon and energy, has a limited ability to multiply in resting MØ following infection, reflecting a failure of the ∆kstD strain to inhibit the TLR2-dependent bactericidal activity of resting MØ.

A Novel Role of the PrpR as a Transcription Factor Involved in the Regulation of Methylcitrate Pathway in Mycobacterium tuberculosis

Mycobacterium tuberculosis, the pathogen that causes tuberculosis, presumably utilizes fatty acids as a major carbon source during infection within the host. Metabolism of even-chain-length fatty acids yields acetyl-CoA, whereas metabolism of odd-chain-length fatty acids additionally yields propionyl-CoA. Utilization of these compounds by tubercle bacilli requires functional glyoxylate and methylcitrate cycles, respectively. Enzymes involved in both pathways are essential for M. tuberculosis viability and persistence during growth on fatty acids. However, little is known about regulatory factors responsible for adjusting the expression of genes encoding these enzymes to particular growth conditions. Here, we characterized the novel role of PrpR as a transcription factor that is directly involved in regulating genes encoding the key enzymes of methylcitrate (methylcitrate dehydratase [PrpD] and methylcitrate synthase [PrpC]) and glyoxylate (isocitrate lyase [Icl1]) cycles. Using cell-free systems and intact cells, we demonstrated an interaction of PrpR protein with prpDC and icl1 promoter regions and identified a consensus sequence recognized by PrpR. Moreover, we showed that an M. tuberculosis prpR-deletion strain exhibits impaired growth in vitro on propionate as the sole carbon source. Real-time quantitative reverse transcription-polymerase chain reaction confirmed that PrpR acts as a transcriptional activator of prpDC and icl1 genes when propionate is the main carbon source. Similar results were also obtained for a non-pathogenic Mycobacterium smegmatis strain. Additionally, we found that ramB, a prpR paralog that controls the glyoxylate cycle, is negatively regulated by PrpR. Our data demonstrate that PrpR is essential for the utilization of odd-chain-length fatty acids by tubercle bacilli. Since PrpR also acts as a ramB repressor, our findings suggest that it plays a key role in regulating expression of enzymes involved in both glyoxylate and methylcitrate pathways.