Cell cycle regulation and novel structural features of thymidine kinase,an essential enzyme in Trypanosoma brucei

Thymidine kinase (TK) is a key enzyme in the pyrimidine salvage pathway which catalyzes the transfer of the γ‐phosphate of ATP to 2′‐deoxythymidine (dThd) forming thymidine monophosphate (dTMP). Unlike other type II TKs, the Trypanosoma brucei enzyme (TbTK) is a tandem protein with two TK homolog domains of which only the C‐terminal one is active. In this study, we establish that TbTK is essential for parasite viability and cell cycle progression, independently of extracellular pyrimidine concentrations. We show that expression of TbTK is cell cycle regulated and that depletion of TbTK leads to strongly diminished dTTP pools and DNA damage indicating intracellular dThd to be an essential intermediate metabolite for the synthesis of thymine‐derived nucleotides. In addition, we report the X‐ray structure of the catalytically active domain of TbTK in complex with dThd and dTMP at resolutions up to 2.2 Å. In spite of the high conservation of the active site residues, the structures reveal a widened active site cavity near the nucleobase moiety compared to the human enzyme. Our findings strongly support TbTK as a crucial enzyme in dTTP homeostasis and identify structural differences within the active site that could be exploited in the process of rational drug design.     

Double drugging of prolyl-tRNA synthetase provides a new paradigm for anti-infective drug development

Toxoplasmosis is caused by Toxoplasma gondii and in immunocompromised patients it may lead to seizures, encephalitis or death. The conserved enzyme prolyl-tRNA synthetase (PRS) is a validated druggable target in Toxoplasma gondii but the traditional ‘single target–single drug’ approach has its caveats. Here, we describe two potent inhibitors namely halofuginone (HFG) and a novel ATP mimetic (L95) that bind to Toxoplasma gondii PRS simultaneously at different neighbouring sites to cover all three of the enzyme substrate subsites. HFG and L95 act as one triple-site inhibitor in tandem and form an unusual ternary complex wherein HFG occupies the 3’-end of tRNA and the L-proline (L-pro) binding sites while L95 occupies the ATP pocket. These inhibitors exhibit nanomolar IC₅₀ and EC₅₀ values independently, and when given together reveal an additive mode of action in parasite inhibition assays. This work validates a novel approach and lays a structural framework for further drug development based on simultaneous targeting of multiple pockets to inhibit druggable proteins.     

Diverging substrate specificity of pure human thymidine kinases 1 and 2 against antiviral dideoxynucleosides

The two thymidine (dThd) kinases in human cells, the cytosolic, S-phase-specific TK1 and the mitochondrial, constitutively expressed TK2 were purified to homogeneity as judged from sodium dodecyl sulfate-gel electrophoresis. The substrate specificity of TK1 and TK2 toward natural substrates and important nucleoside analogues was compared. With TK1, the Km values for 5-fluorodeoxyuridine (FdUrd), 3'-azido-2',3'-dideoxythymidine (AZT), and 3'-fluoro-2',3'-dideoxythymidine (FLT) were 2.2, 0.6, and 2.1 microM as compared to 0.5 microM for dThd and 9 microM for deoxyuridine (dUrd). With TK2, dUrd, deoxycytidine (dCyd), and 5-fluorodeoxyuridine (FdUrd) were efficiently phosphorylated, but with distinctly different kinetics: Michaelis-Menten kinetics with dCyd, dUrd, and FdUrd; negative cooperativity with dThd. Negative cooperativity was also observed with AZT, although this drug was a very poor substrate for TK2 with a Vmax of 5-6% of that with dThd. FLT, 2',3'-dideoxycytidine (ddCyd), and arabinofuranosylcytosine (araC) were not substrates for TK2, and 2',3'-didehydrodideoxy-thymidine (D4T) was not a substrate for TK1 or TK2. On the other hand, AZT, FLT, and D4T were competitive inhibitors with Ki values of 0.6, 6, and 2073 microM for TK1, and 2, 10, and 78 microM for TK2, respectively. The much lower tolerance for modifications of the deoxyribose moiety of TK2 as compared to TK1 is important for the design of new antiviral nucleoside analogues intended for use in cells with different expression of TK1 and TK2.     

3′-(1,2,3-Triazol-1-yl)-3′-deoxythymidine analogs as substrates for human and Ureaplasma parvum thymidine kinase for structure–activity investigations

The pathogenic mycoplasma Ureaplasma parvum (Up) causes opportunistic infections and relies on salvage of nucleosides for DNA synthesis and Up thymidine kinase (UpTK) provides the necessary thymidine nucleotides. The anti-HIV compound 3?-azido-3′-deoxythymidine (AZT) is a good substrate for TK. Methods for a rapid and efficient synthesis of new 3′-α-[1,2,3]triazol-3′-deoxythymidine analogs from AZT under Huisgen conditions are described. Thirteen 3′-analogues were tested with human cytosolic thymidine kinase (hTK1) and UpTK. The new analogs showed higher efficiencies (K_m/V_max values) in all cases with UpTK than with hTK1. Still, hTK1 was preferentially inhibited by 9 out of 10 tested analogs. Structural models of UpTK and hTK1 were constructed and used to explain the kinetic results. Two different binding modes of the nucleosides within the active sites of both enzymes were suggested with one predominating in the bacterial enzyme and the other in hTK1. These results will aid future development of anti-mycoplasma nucleosides.     

A Glucose Transporter Can Mediate Ribose Uptake: DEFINITION OF RESIDUES THAT CONFER SUBSTRATE SPECIFICITY IN A SUGAR TRANSPORTER*

Sugars, the major energy source for many organisms, must be transported across biological membranes. Glucose is the most abundant sugar in human plasma and in many other biological systems and has been the primary focus of sugar transporter studies in eukaryotes. We have previously cloned and characterized a family of glucose transporter genes from the protozoan parasite Leishmania. These transporters, called LmGT1, LmGT2, and LmGT3, are homologous to the well characterized glucose transporter (GLUT) family of mammalian glucose transporters. We have demonstrated that LmGT proteins are important for parasite viability. Here we show that one of these transporters, LmGT2, is a more effective carrier of the pentose sugar d-ribose than LmGT3, which has a 6-fold lower relative specificity (V_max/K_m) for ribose. A pair of threonine residues, located in the putative extracellular loops joining transmembrane helices 3 to 4 and 7 to 8, define a filter that limits ribose approaching the exofacial substrate binding pocket in LmGT3. When these threonines are substituted by alanine residues, as found in LmGT2, the LmGT3 permease acquires ribose permease activity that is similar to that of LmGT2. The location of these residues in hydrophilic loops supports recent suggestions that substrate recognition is separated from substrate binding and translocation in this important group of transporters.     

A Target-Based High Throughput Screen Yields Trypanosoma brucei Hexokinase Small Molecule Inhibitors with Antiparasitic Activity

Background

The parasitic protozoan Trypanosoma brucei utilizes glycolysis exclusively for ATP production during infection of the mammalian host. The first step in this metabolic pathway is mediated by hexokinase (TbHK), an enzyme essential to the parasite that transfers the γ-phospho of ATP to a hexose. Here we describe the identification and confirmation of novel small molecule inhibitors of bacterially expressed TbHK1, one of two TbHKs expressed by T. brucei, using a high throughput screening assay.

Methodology/Principal Findings

Exploiting optimized high throughput screening assay procedures, we interrogated 220,233 unique compounds and identified 239 active compounds from which ten small molecules were further characterized. Computation chemical cluster analyses indicated that six compounds were structurally related while the remaining four compounds were classified as unrelated or singletons. All ten compounds were ∼20-17,000-fold more potent than lonidamine, a previously identified TbHK1 inhibitor. Seven compounds inhibited T. brucei blood stage form parasite growth (0.03≤EC₅₀<3 µM) with parasite specificity of the compounds being demonstrated using insect stage T. brucei parasites, Leishmania promastigotes, and mammalian cell lines. Analysis of two structurally related compounds, ebselen and SID 17387000, revealed that both were mixed inhibitors of TbHK1 with respect to ATP. Additionally, both compounds inhibited parasite lysate-derived HK activity. None of the compounds displayed structural similarity to known hexokinase inhibitors or human African trypanosomiasis therapeutics.

Conclusions/Significance

The novel chemotypes identified here could represent leads for future therapeutic development against the African trypanosome.     

Characterization of a highly diverged mitochondrial ATP synthase Fo subunit in Trypanosoma brucei

The mitochondrial F₁F_o ATP synthase of the parasite Trypanosoma brucei has been previously studied in detail. This unusual enzyme switches direction in functionality during the life cycle of the parasite, acting as an ATP synthase in the insect stages, and as an ATPase to generate mitochondrial membrane potential in the mammalian bloodstream stages. Whereas the trypanosome F₁ moiety is relatively highly conserved in structure and composition, the F_o subcomplex and the peripheral stalk have been shown to be more variable. Interestingly, a core subunit of the latter, the normally conserved subunit b, has been resistant to identification by sequence alignment or biochemical methods. Here, we identified a 17 kDa mitochondrial protein of the inner membrane, Tb927.8.3070, that is essential for normal growth, efficient oxidative phosphorylation, and membrane potential maintenance. Pull-down experiments and native PAGE analysis indicated that the protein is both associated with the F₁F_o ATP synthase and integral to its assembly. In addition, its knockdown reduced the levels of F_o subunits, but not those of F₁, and disturbed the cell cycle. Finally, analysis of structural homology using the HHpred algorithm showed that this protein has structural similarities to F_o subunit b of other species, indicating that this subunit may be a highly diverged form of the elusive subunit b.     

Crystal structure of Trypanosoma cruzi heme peroxidase and characterization of its substrate specificity and compound I intermediate

The protozoan parasite Trypanosoma cruzi is the causative agent of American trypanosomiasis, otherwise known as Chagas disease. To survive in the host, the T. cruzi parasite needs antioxidant defense systems. One of these is a hybrid heme peroxidase, the T. cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP). TcAPx-CcP has high sequence identity to members of the class I peroxidase family, notably ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). The aim of this work was to solve the structure and examine the reactivity of the TcAPx-CcP enzyme. Low temperature electron paramagnetic resonance spectra support the formation of an exchange-coupled [Fe(IV)=O Trp₂₃₃^•+] compound I radical species, analogous to that used in CcP and LmP. We demonstrate that TcAPx-CcP is similar in overall structure to APX and CcP, but there are differences in the substrate-binding regions. Furthermore, the electron transfer pathway from cytochrome c to the heme in CcP and LmP is preserved in the TcAPx-CcP structure. Integration of steady state kinetic experiments, molecular dynamic simulations, and bioinformatic analyses indicates that TcAPx-CcP preferentially oxidizes cytochrome c but is still competent for oxidization of ascorbate. The results reveal that TcAPx-CcP is a credible cytochrome c peroxidase, which can also bind and use ascorbate in host cells, where concentrations are in the millimolar range. Thus, kinetically and functionally TcAPx-CcP can be considered a hybrid peroxidase.     

A Homolog of CcpA Mediates Catabolite Control in Listeria monocytogenes but Not Carbon Source Regulation of Virulence Genes

Readily utilizable sugars down-regulate virulence gene expression in Listeria monocytogenes, which has led to the proposal that this regulation may be an aspect of global catabolite regulation (CR). We recently demonstrated that the metabolic enzyme α-glucosidase is under CR in L. monocytogenes. Here, we report the cloning and characterization from L. monocytogenes of an apparent ortholog of ccpA, which encodes an important mediator of CR in several low-G+C-content gram-positive bacteria. L. monocytogenes ccpA (ccpA_Lm) is predicted to encode a 335-amino-acid protein with nearly 65% identity to the gene product of Bacillus subtilis ccpA (ccpA_Bs). Southern blot analysis with a probe derived from ccpA_Lm revealed a single strongly hybridizing band and also a second band of much lower intensity, suggesting that there may be other closely related sequences in the L. monocytogenes chromosome, as is the case in B. subtilis. Disruption of ccpA_Lm resulted in the inability of the mutant to grow on glucose-containing minimal medium or increase its growth rate in the presence of preferred sugars, and it completely eliminated CR of α-glucosidase activity in liquid medium. However, α-glucosidase activity was only partially relieved from CR on solid medium. These results suggest that ccpA is an important element of carbon source regulation in L. monocytogenes. Nevertheless, utilizable sugars still down-regulate the expression of hly, which encodes the virulence factor hemolysin, in a ccpA_Lm mutant, indicating that CcpA is not involved in carbon source regulation of virulence genes.     

Structural features of Cryptococcus neoformans bifunctional GAR/AIR synthetase may present novel antifungal drug targets

Cryptococcus neoformans is a fungus that causes life-threatening systemic mycoses. During infection of the human host, this pathogen experiences a major change in the availability of purines; the fungus can scavenge the abundant purines in its environmental niche of pigeon excrement, but must employ de novo biosynthesis in the purine-poor human CNS. Eleven sequential enzymatic steps are required to form the first purine base, IMP, an intermediate in the formation of ATP and GTP. Over the course of evolution, several gene fusion events led to the formation of multifunctional purine biosynthetic enzymes in most organisms, particularly the higher eukaryotes. In C. neoformans, phosphoribosyl-glycinamide synthetase (GARs) and phosphoribosyl-aminoimidazole synthetase (AIRs) are fused into a bifunctional enzyme, while the human ortholog is a trifunctional enzyme that also includes GAR transformylase. Here we functionally, biochemically, and structurally characterized C. neoformans GARs and AIRs to identify drug targetable features. GARs/AIRs are essential for de novo purine production and virulence in a murine inhalation infection model. Characterization of GARs enzymatic functional parameters showed that C. neoformans GARs/AIRs have lower affinity for substrates glycine and PRA compared with the trifunctional metazoan enzyme. The crystal structure of C. neoformans GARs revealed differences in the glycine- and ATP-binding sites compared with the Homo sapiens enzyme, while the crystal structure of AIRs shows high structural similarity compared with its H. sapiens ortholog as a monomer but differences as a dimer. The alterations in functional and structural characteristics between fungal and human enzymes could potentially be exploited for antifungal development.     

The Tyrosine Kinase Btk Regulates the Macrophage Response to Listeria monocytogenes Infection

In this study we investigated the role of Bruton''s tyrosine kinase (Btk) in the immune response to the Gram-positive intracellular bacterium Listeria monocytogenes (Lm). In response to Lm infection, Btk was activated in bone marrow-derived macrophages (BMMs) and Btk ^−/− BMMs showed enhanced TNF-α, IL-6 and IL-12p40 secretion, while type I interferons were produced at levels similar to wild-type (wt) BMMs. Although Btk-deficient BMMs displayed reduced phagocytosis of E. coli fragments, there was no difference between wt and Btk ^−/− BMMs in the uptake of Lm upon infection. Moreover, there was no difference in the response to heat-killed Lm between wt and Btk ^−/− BMMs, suggesting a role for Btk in signaling pathways that are induced by intracellular Lm. Finally, Btk ^−/− mice displayed enhanced resistance and an increased mean survival time upon Lm infection in comparison to wt mice. This correlated with elevated IFN-γ and IL-12p70 serum levels in Btk ^−/− mice at day 1 after infection. Taken together, our data suggest an important regulatory role for Btk in macrophages during Lm infection.     

Molecular characterization of thymidine kinase from Ureaplasma urealyticum: nucleoside analogues as potent inhibitors of mycoplasma growth

Ureaplasma urealyticum (U. urealyticum), belonging to the class Mollicutes, is a human pathogen colonizing the urogenital tract and causes among other things respiratory diseases in premature infants. We have studied the salvage of pyrimidine deoxynucleosides in U. urealyticum and cloned a key salvage enzyme, thymidine kinase (TK) from U. urealyticum. Recombinant Uu-TK was expressed in E. coli, purified and characterized with regards to substrate specificity and feedback inhibition. Uu-TK efficiently phosphorylated thymidine (dThd) and deoxyuridine (dUrd) as well as a number of pyrimidine nucleoside analogues. All natural ribonucleoside/deoxyribonucleoside triphosphates, except dTTP, served as phosphate donors, while dTTP was a feedback inhibitor. The level of Uu-TK activity in U. urealyticum extracts increased upon addition of dUrd to the growth medium. Fluoropyrimidine nucleosides inhibited U. urealyticum and M. pneumoniae growth and this inhibitory effect could be reversed by addition of dThd, dUrd or deoxytetrahydrouridine to the growth medium. Thus, the mechanism of inhibition was most likely the depletion of dTTP, either via a blocked thymidine kinase reaction and/or thymidylate synthesis step and these metabolic reactions should be suitable targets for antimycoplasma chemotherapy.     

The kinetic properties of a human PPIP5K reveal that its kinase activities are protected against the consequences of a deteriorating cellular bioenergetic environment

We obtained detailed kinetic characteristics–stoichiometry, reaction rates, substrate affinities and equilibrium conditions–of human PPIP5K2 (diphosphoinositol pentakisphosphate kinase 2). This enzyme synthesizes ‘high-energy’ PP-InsPs (diphosphoinositol polyphosphates) by metabolizing InsP₆ (inositol hexakisphosphate) and 5-InsP₇ (5-diphosphoinositol 1,2,3,4,6-pentakisphosphate) to 1-InsP₇ (1-diphosphoinositol 2,3,4,5,6-pentakisphosphate) and InsP₈ (1,5-bis-diphosphoinositol 2,3,4,6-tetrakisphosphate), respectively. These data increase our insight into the PPIP5K2 reaction mechanism and clarify the interface between PPIP5K catalytic activities and cellular bioenergetic status. For example, stochiometric analysis uncovered non-productive, substrate-stimulated ATPase activity (thus, approximately 2 and 1.2 ATP molecules are utilized to synthesize each molecule of 1-InsP₇ and InsP₈, respectively). Impaired ATPase activity of a PPIP5K2-K248A mutant increased atomic-level insight into the enzyme''s reaction mechanism. We found PPIP5K2 to be fully reversible as an ATP-synthase in vitro, but our new data contradict previous perceptions that significant ‘reversibility’ occurs in vivo. PPIP5K2 was insensitive to physiological changes in either [AMP] or [ATP]/[ADP] ratios. Those data, together with adenine nucleotide kinetics (ATP K_m=20–40 μM), reveal how insulated PPIP5K2 is from cellular bioenergetic challenges. Finally, the specificity constants for PPIP5K2 revise upwards by one-to-two orders of magnitude the inherent catalytic activities of this enzyme, and we show its equilibrium point favours 80–90% depletion of InsP₆/5-InsP₇.     

Trypanosoma brucei thymidine kinase is tandem protein consisting of two homologous parts, which together enable efficient substrate binding

Trypanosoma brucei causes African sleeping sickness, a disease for which existing chemotherapies are limited by their toxicity or lack of efficacy. We have found that four parasites, including T. brucei, contain genes where two or four thymidine kinase (TK) sequences are fused into a single open reading frame. The T. brucei full-length enzyme as well as its two constituent parts, domain 1 and domain 2, were separately expressed and characterized. Of potential interest for nucleoside analog development, T. brucei TK was less discriminative against purines than human TK1 with the following order of catalytic efficiencies: thymidine > deoxyuridine ≫ deoxyinosine > deoxyguanosine. Proteins from the TK1 family are generally dimers or tetramers, and the quaternary structure is linked to substrate affinity. T. brucei TK was primarily monomeric but can be considered a two-domain pseudodimer. Independent kinetic analysis of the two domains showed that only domain 2 was active. It had a similar turnover number (k_cat) as the full-length enzyme but could not self-dimerize efficiently and had a 5-fold reduced thymidine/deoxyuridine affinity. Domain 1, which lacks three conserved active site residues, can therefore be considered a covalently attached structural partner that enhances substrate binding to domain 2. A consequence of the non-catalytic role of domain 1 is that its active site residues are released from evolutionary pressure, which can be advantageous for developing new catalytic functions. In addition, nearly identical 89-bp sequences present in both domains suggest that the exchange of genetic material between them can further promote evolution.     

Cryptosporidium Lactate Dehydrogenase Is Associated with the Parasitophorous Vacuole Membrane and Is a Potential Target for Developing Therapeutics

The apicomplexan, Cryptosporidium parvum, possesses a bacterial-type lactate dehydrogenase (CpLDH). This is considered to be an essential enzyme, as this parasite lacks the Krebs cycle and cytochrome-based respiration, and mainly–if not solely, relies on glycolysis to produce ATP. Here, we provide evidence that in extracellular parasites (e.g., sporozoites and merozoites), CpLDH is localized in the cytosol. However, it becomes associated with the parasitophorous vacuole membrane (PVM) during the intracellular developmental stages, suggesting involvement of the PVM in parasite energy metabolism. We characterized the biochemical features of CpLDH and observed that, at lower micromolar levels, the LDH inhibitors gossypol and FX11 could inhibit both CpLDH activity (K _i = 14.8 μM and 55.6 μM, respectively), as well as parasite growth in vitro (IC₅₀ = 11.8 μM and 39.5 μM, respectively). These observations not only reveal a new function for the poorly understood PVM structure in hosting the intracellular development of C. parvum, but also suggest LDH as a potential target for developing therapeutics against this opportunistic pathogen, for which fully effective treatments are not yet available.     

Biochemical and Kinetic Characterization of the Recombinant ADP-Forming Acetyl Coenzyme A Synthetase from the Amitochondriate Protozoan Entamoeba histolytica

Entamoeba histolytica, an amitochondriate protozoan parasite that relies on glycolysis as a key pathway for ATP generation, has developed a unique extended PP_i-dependent glycolytic pathway in which ADP-forming acetyl-coenzyme A (CoA) synthetase (ACD; acetate:CoA ligase [ADP-forming]; EC 6.2.1.13) converts acetyl-CoA to acetate to produce additional ATP and recycle CoA. We characterized the recombinant E. histolytica ACD and found that the enzyme is bidirectional, allowing it to potentially play a role in ATP production or in utilization of acetate. In the acetate-forming direction, acetyl-CoA was the preferred substrate and propionyl-CoA was used with lower efficiency. In the acetyl-CoA-forming direction, acetate was the preferred substrate, with a lower efficiency observed with propionate. The enzyme can utilize both ADP/ATP and GDP/GTP in the respective directions of the reaction. ATP and PP_i were found to inhibit the acetate-forming direction of the reaction, with 50% inhibitory concentrations of 0.81 ± 0.17 mM (mean ± standard deviation) and 0.75 ± 0.20 mM, respectively, which are both in the range of their physiological concentrations. ATP and PP_i displayed mixed inhibition versus each of the three substrates, acetyl-CoA, ADP, and phosphate. This is the first example of regulation of ACD enzymatic activity, and possible roles for this regulation are discussed.     

Uridine phosphorylase from Schistosoma mansoni

Uridine phosphorylase is the only pyrimidine nucleoside cleaving activity that can be detected in extracts of Schistosoma mansoni. The enzyme is distinct from the two purine nucleoside phosphorylases contained in this parasite. Although Urd is the preferred substrate, uridine phosphorylase can also catalyze the reversible phosphorolysis of dUrd and dThd, but not Cyd, dCyd, or orotidine. The enzyme was purified 170-fold to a specific activity of 2.76 nmol/min/mg of protein with a 16% yield. It has a Mr of 56,000 as determined by molecular sieving on Sephadex G-100. The mechanism of uridine phosphorylase is sequential. When Urd was the substrate, the KUrd = 13 microM and the KPi = 533 +/- 78 microM. When dThd was used as a substrate, the KdThd = 54 microM and the KPi = 762 +/- 297 microM. The Vmax with dThd was 53 +/- 9.8% that of Urd. dThd was a competitive inhibitor when Urd was used as a substrate. The enzyme showed substrate inhibition by Urd, dThd (greater than 0.125 mM) and phosphate (greater than 10 mM). 5-(Benzyloxybenzyloxybenzyl)acyclouridine was identified as a potent and specific inhibitor of parasite (Ki = 0.98 microM) but not host uridine phosphorylase. Structure-activity relationship studies suggest that uridine phosphorylase from S. mansoni has a hydrophobic pocket adjacent to the 5-position of the pyrimidine ring and indicate differences between the binding sites of the mammalian and parasite enzymes. These differences may be useful in designing specific inhibitors for schistosomal uridine phosphorylase which will interfere selectively with nucleic acids synthesis in this parasite.     

A Leishmania major protein with extensive homology to silent information regulator 2 of Saccharomyces cerevisiae

We have isolated a cDNA from the protozoan parasite Leishmania major (Lm) that encodes a protein homologous to the Saccharomyces cerevisiae and Kluyveromyces marxianus silent information regulator 2 (SIR2) proteins. The deduced Lm SIR2-related protein (termed LmSIR2rp) consists of 381 amino acids that share 40.5% identity with yeast SIR2, increasing to 60% when substitutions are included. Moreover, the LmSIR2rp aa sequence contains a single potential zinc-binding domain with a CysXaa₂CysXaa₂₀CysXaa₂Cys motif, and its C-terminal part is rich in Ser (16 Ser residues over 40 aa) which constitute potential sites for phosphorylation. The characterization of a novel Lm gene product which shows considerable similarity to a yeast mating-type regulatory protein provides a new tool to investigate the parasite differentiation control mechanisms and gene expression regulation     

CD14-Dependent Monocyte Isolation Enhances Phagocytosis of Listeria monocytogenes by Proinflammatory,GM-CSF-Derived Macrophages

Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages.