Isolation, purification and characterization of GPI-anchored membrane proteins from Trypanosoma rangeli and Trypanosoma cruzi

GPI-anchored proteins from plasma membrane of Trypanosoma rangeli and Trypanosoma cruzi epimastigotes were isolated and characterized using the partition Triton X-114 method. The detection by Western blot of specific proteins of 90, 85 and 56 kDa molecular mass in T. rangeli compared to those of 30, 70 and 100 kDa detected in T. cruzi demonstrates specific discrimination between these two species of Trypanosoma. The potential diagnostic value of the here reported proteins to differentiate mixed infections by T. cruzi and T. rangeli is evaluated and its potential for epidemiological studies of Chagas disease in endemic areas is also discussed.     

Inhibition of adherence of Mycobacterium avium complex and Mycobacterium tuberculosis to fibronectin on the respiratory mucosa

Mycobacterium species adhere to the respiratory mucosa via mucus and fibronectin of extracellular matrix exposed by damaged epithelium. We have investigated whether inhibiting adherence to fibronectin influences subsequent infection of human respiratory tissue by Mycobacterium avium complex and Mycobacterium tuberculosis. Human respiratory tissue was pretreated with mycobacterial fibronectin attachment proteins prior to infection with M. avium complex and M. tuberculosis and the number of recoverable bacteria over time was compared to untreated controls. Inhibition significantly reduced recovery of M. avium complex at 15min (P= 0.02), 7days (P = 0.04), and 14 days (P= 0.03); whereas recovery of M. tuberculosis was only reduced at 15 min (P = 0.01) and not at later timepoints. We conclude that M. avium complex and M. tuberculosis infection of the mucosa proceeds by different mechanisms, since M. tuberculosis infection is independent of fibronectin adherence.     

Low oral BCG doses fail to protect cattle against an experimental challenge with Mycobacterium bovis

Studies were undertaken to determine whether a dose of oral Mycobacterium bovis bacillus Calmette–Guérin (BCG) which did not induce skin test reactivity could protect cattle against bovine tuberculosis (TB). Groups of calves (n = 9) were vaccinated by administering 10⁸, 10⁷ or 10⁶ colony forming units (CFU) of BCG orally or 10⁶ CFU subcutaneous (s.c.) BCG. A control group (n = 10) was not vaccinated. All animals were challenged with M. bovis 18 weeks after vaccination and euthanized and necropsied at 16 weeks following challenge. Positive responses in the single cervical tuberculin skin test (severe interpretation) at 15 weeks post-vaccination were only observed in the s.c. BCG and 10⁸ CFU oral BCG groups (four of nine animals/group). Following experimental challenge with M. bovis, both these BCG-vaccinated groups had significant reductions in lesion scores and bacterial counts whereas there was no protection in calves vaccinated with oral doses of 10⁶ or 10⁷ CFU of BCG. In conclusion, low oral doses of BCG did not induce skin test responses, IFN-γ responses or protection against TB, however, in the BCG vaccine groups where protection was observed, there was no correlation between protection and skin test responses or IFN-γ responses.     

Human Mycobacterium bovis infection in ten Latin American countries

The aim of this work was to obtain the best possible estimate of the relevance of bovine tuberculosis (BTB) in humans in Argentina, Brazil, Chile, Colombia, Costa Rica, Dominican Republic, Ecuador, Peru, Uruguay and Venezuela. Sources of information were a questionnaire filled by the participant laboratories, and a search of published literature (1970-2007). Only four of these countries reported bacteriologically confirmed cases of BTB in humans. Most of these were diagnosed in Argentina, where the mean percentage of Mycobacterium bovis cases in relation to those due to Mycobacterium tuberculosis (2000-2006) ranged from 0.34% to 1.0%, according to the region. A slowly decreasing trend was observed in non HIV as well as in HIV/AIDS patients in Buenos Aires. In most of these countries, the low coverage of culture methods, especially of those including pyruvate-containing media, appropriate to isolate M. bovis, contributes to an underestimate of the problem. It was confirmed that BTB in humans exists, even though its relevance seems to be low. Milk pasteurization, sanitary controls to dairy products, and meat inspection at slaughterhouses contribute to the protection of human health. However, occupational aerogenous exposure to TB cattle and their carcasses remains a source of infection in the region.     

Expression of foreign genes in Mycobacterium bovis BCG strains using different promoters reveals instability of the hsp60 promoter for expression of foreign genes in Mycobacterium bovis BCG strains

SETTING: Optimization of BCG as a vehicle for live recombinant vaccines requires improved strategies for stable antigen expression. OBJECTIVES: To investigate the effects of various combinations of post-translational signals and promoters on expression and stability in different BCG strains. DESIGN: Plasmids were constructed using mycobacterial promoters (hsp60, 19-kDa antigen, 85A antigen--from the Mycobacterium tuberculosis complex--and the 18-kDa antigen from Mycobacterium leprae) and post-translation signals (85A antigen secretion and 19-kDa antigen acylation signals), coupled with reporter genes. RESULTS: The 19-kDa acylation signal had little effect on expression, while the 85A secretion signal enhanced markedly the levels of cell-associated product. Inclusion of the hsp60 promoter caused plasmid instability; various deletions affecting the promoter region occurred during or soon after transformation, but not during subsequent growth of the transformants, nor with other promoters. BCG Moreau appeared to be more susceptible to deletions than other BCG strains. CONCLUSIONS: The 85A signal may prove useful in optimizing gene expression in BCG, irrespective of secretion of the product. Deletions associated with the hsp60 promoter may be due to a transient lethal induction of the hsp60 promoter associated with electroporation. With intact plasmid there was no marked difference in expression between BCG strains.     

Modeling early bactericidal activity in murine tuberculosis provides insights into the activity of isoniazid and pyrazinamide

Standard tuberculosis (TB) treatment includes an initial regimen containing drugs that are both rapidly bactericidal (isoniazid) and sterilizing (rifampin and pyrazinamide), and ethambutol to help prevent the emergence of drug resistance. Antagonism between isoniazid and pyrazinamide has been demonstrated in a TB treatment mouse model. Because isoniazid's bactericidal activity is greatest during the initial two treatment days, we hypothesized that removing isoniazid after the second day would increase the effectiveness of the standard regimen. To test this hypothesis, we developed a mouse model to measure the early bactericidal activity (EBA) of drug regimens designed to analyze the essentiality of both isoniazid and pyrazinamide during the first 14 d of therapy. Our results clearly indicate that discontinuation of isoniazid after the second day of treatment increases the EBA of standard therapy in the mouse model, whereas omitting pyrazinamide during the first 14 d was detrimental. Substitution of moxifloxacin for isoniazid on day 3 did not increase the EBA compared with only removing isoniazid after day 2. Our data show that a mouse model can be used to analyze the EBA of TB drugs, and our findings support pursuing clinical trials to evaluate the possible benefit of removing isoniazid after the first 2 treatment days.     

Mycobacterium chelonae tenosynovitis of the hand

OBJECTIVE: Tenosynovitis of the hand due to atypical mycobacteria is an uncommon condition. We present a case of tenosynovitis of the hand due to Mycobacterium chelonae in a patient without a recognized penetrating injury, who was treated successfully with clarithromycin and antituberculous medications and without debridement. We reviewed the available literature to summarize the experience with this infectious entity. METHODS: Case report and review of the literature (MEDLINE 1976-2003). Only cases that were sufficiently detailed were included. RESULTS: Twelve cases of upper extremity infection due to M. chelonae have been reported: hand tenosynovitis in most and arthritis in a few. These infections resulted from percutaneous inoculation or hematogenous seeding. The clinical course was indolent initially but insidiously destructive. Previously, treatment always included surgical excision of the infected tissues and antibiotic therapy. This is the first case of M. chelonae musculoskeletal infection that resolved with only antimicrobial therapy. CONCLUSIONS: Musculoskeletal infections by nontuberculous mycobacteria are clinically indistinguishable from those of tuberculosis and diagnosis is usually delayed. Prompt diagnosis of atypical mycobacteria with appropriate antimicrobial treatment may avoid the need for surgical debridement. Relevance We recommend a trial of antibiotics for M. chelonae before surgical debridement.     

Vaccination of European badgers (Meles meles) with BCG by the subcutaneous and mucosal routes induces protective immunity against endobronchial challenge with Mycobacterium bovis

Mycobacterium bovis is endemic in badger (Meles meles) populations of Ireland and the United Kingdom and infected badgers are a potential source of infection for cattle. In domestic livestock tuberculosis causes economic losses from lost production and the costs associated with eradication programmes, and in addition there is a risk of zoonotic infection. Whereas culling is currently used to control tuberculous badger populations in Ireland, vaccination, if it were available, would be preferred. A study was undertaken to examine the protective responses of badgers vaccinated either by the subcutaneous or mucosal (intranasal and conjunctival) routes with bacille Calmette-Guérin (BCG), when challenged with M. bovis by the endobronchial route. Three groups of badgers were used. The first group (n = 4) was vaccinated with 5 × 10⁵ colony forming units (cfu) of BCG by subcutaneous injection. In the second group (n = 5) badgers were vaccinated via the mucosal route by instilling 1.0 × 10⁵ cfu into each conjunctival sac and spraying 1.0 × 10⁵ cfu into each nostril (final vaccine dose of 4 × 10⁵ cfu). The control (n = 5) badgers served as a non-vaccinated group. Twelve weeks post-vaccination all badgers in the three groups were challenged with 10⁴ cfu of M. bovis by endobronchial inoculation. At 12 weeks post-infection all badgers were examined post-mortem to assess the pathological and bacteriological responses to challenge. Gross and histological lesions of tuberculosis were seen in all challenged badgers and M. bovis was recovered from all challenged badgers. However, across six of the eight parameters used to measure disease severity, the infection in the vaccinated badgers was significantly less severe than in the control group. The BCG vaccine induced a significant protective effect in the badgers and the protective immunity was generated by subcutaneous and mucosal vaccination.     

Vaccine and skin testing properties of two avirulent Mycobacterium bovis mutants with and without an additional esat-6 mutation

SETTING: Molecular techniques are now available to develop new live tuberculosis vaccines by producing avirulent strains of the Mycobacterium tuberculosis complex with known genes deleted. OBJECTIVES: Determine if removal of esat-6 from new live tuberculosis vaccines with known attenuating mutations affects their vaccine efficacy and if it could enable the development of discriminating diagnostic tests. DESIGN: Remove the esat-6 gene by allelic exchange from two illegitimate mutants of Mycobacterium bovis that had previously been shown to have similar vaccine efficacy to BCG in a guinea pig vaccination model. Determine the effect this removal has on virulence, vaccine efficacy and skin test reactivity in guinea pigs. RESULTS: Two double knockout strains of M. bovis were produced and their virulence and vaccine efficacy were compared to their parent strains. Removal of the esat-6 gene had no significant effect on vaccine efficacy. In skin tests, animals inoculated with the double knockout strains reacted to PPD but not ESAT-6, whereas those inoculated with the parent strains had similar skin test reactivity to both PPD and esat-6. CONCLUSION: Removal of esat-6 from new live tuberculosis vaccine candidates has no significant effect on vaccine properties but does enable the use of skin tests to distinguish between vaccination and infection.     

The scent of Mycobacterium tuberculosis

Worldwide, tuberculosis (TB) kills nearly 2 million people annually, yet rapid diagnosis still relies on a 100-year-old method of sputum staining for acid-fast bacilli. The advent of solid phase micro-extraction and gas chromatography/mass spectrometry makes it possible to systematically investigate whether volatile metabolites from organisms belonging to the genus Mycobacterium can be used as a rapid and highly selective alternative to the traditional diagnostic methods. We have identified four specific compounds (methyl phenylacetate, methyl p-anisate, methyl nicotinate and o-phenylanisole) from Mycobacterium tuberculosis and Mycobacterium bovis cultures grown in vitro that are distinctive volatile markers. These compounds are detectable before the visual appearance of colonies, potentially useful as the basis of a non-invasive diagnostic test for TB and have characteristic odors.     

Tuberculosis in ruminants: characteristics of intra-tonsilar Mycobacterium bovis infection models in cattle and deer

Mycobacterium bovis infection produces tubercular lymphadenitis in the head lymphatics of cattle and deer, in addition to pulmonary disease. A low-dose intra-tonsilar infection model that establishes tuberculosis (Tb) lymphadenitis in cattle and deer is characterised in this study. Intra-tonsilar infection of red deer (500 cfus of M. bovis) was monitored longitudinally at 6-week intervals over a period of 23 weeks. Lesion characteristics, bacteriological and immunological parameters were assessed, and compared against those observed in cattle at 20 weeks post-infection, where the latter were infected with 500 or 5000 cfus of M. bovis. Intra-tonsilar inoculation of M. bovis established infection in >90% of deer and cattle, with lesion frequencies at the draining sentinel lymphatic site (left medial retropharyngeal node) of 68-86% and tissue bacterial burdens >3.5 logs/g of tissue, the tonsil being a major site of M. bovis persistence in deer only. Mineralisation occurred at lesion sites in both species in the later stages (18-23 weeks) of infection, with extensive coarse mineralisation observed mainly in cattle. The severity of infection or disease in cattle that received the higher or lower dose of M. bovis did not differ markedly. Pathogen-induced cellular immune response (lymphocyte transformation) and humoral responses (IgG and IgG(1) anti-mycobacterial antibodies) were recorded in both species, and the magnitude of these was noticeably amplified by skin tuberculin testing. IgG(1) antibodies were detectable within 6 weeks post-inoculation in deer and could be associated with early detection of lymphadenitis. Deer and cattle show similar levels of susceptibility to M. bovis infection.     

Torins are potent antimalarials that block replenishment of Plasmodium liver stage parasitophorous vacuole membrane proteins

Residence within a customized vacuole is a highly successful strategy used by diverse intracellular microorganisms. The parasitophorous vacuole membrane (PVM) is the critical interface between Plasmodium parasites and their possibly hostile, yet ultimately sustaining, host cell environment. We show that torins, developed as ATP-competitive mammalian target of rapamycin (mTOR) kinase inhibitors, are fast-acting antiplasmodial compounds that unexpectedly target the parasite directly, blocking the dynamic trafficking of the Plasmodium proteins exported protein 1 (EXP1) and upregulated in sporozoites 4 (UIS4) to the liver stage PVM and leading to efficient parasite elimination by the hepatocyte. Torin2 has single-digit, or lower, nanomolar potency in both liver and blood stages of infection in vitro and is likewise effective against both stages in vivo, with a single oral dose sufficient to clear liver stage infection. Parasite elimination and perturbed trafficking of liver stage PVM-resident proteins are both specific aspects of torin-mediated Plasmodium liver stage inhibition, indicating that torins have a distinct mode of action compared with currently used antimalarials.The population at risk for developing malaria is vast, comprising some 3.3 billion people particularly in sub-Saharan Africa and Southeast Asia, with mortality estimates ranging from 655,000 to 1,200,000 (1). Widespread resistance has limited the therapeutic utility of most existing antimalarial drugs, and artemisinin, the highly efficacious cornerstone of artemisinin combination therapies, appears to be at risk for the same fate (2). The need for new antimalarial chemotherapeutic strategies is thus acute.Plasmodium spp., the causative agents of malaria, have a complex life cycle with alternating motile-nonreplicative and sessile-replicative forms in both mammal and mosquito. In the mammalian host, Plasmodium invades and replicates inside two very distinct cell types: hepatocytes and red blood cells (RBCs). In mammals, the Plasmodium life cycle is initiated by a motile sporozoite that invades a hepatocyte, where it resides for 2–14 d, multiplying into >10,000 merozoites in a single cycle (3). Once released into the bloodstream, each of these motile merozoites will infect an RBC and, within 1–3 d, generate 10–30 new merozoites, which will contribute to the continuous cycle of blood stage infection that causes the symptoms, morbidity, and mortality of malaria.These two stages of mammalian infection, despite taking place in distinct cell types and having an orders-of-magnitude difference in parasite replication, do share common features. In both, the motile “zoite” invades the host cell through formation of a parasitophorous vacuole (PV). Both stages grow and replicate exclusively within the confines of the PV, and the parasitophorous vacuole membrane (PVM), which is populated with parasite proteins, constitutes the physical host–parasite interface throughout development. Unlike the vacuoles of many intracellular pathogens including Leishmania, Chlamydia, Mycobacteria, and Legionella (4, 5), the Plasmodium vacuole, like that of Toxoplasma gondii, does not fuse with host lysosomes and is not acidified (6). This is not unsurprising in the context of Plasmodium development in an RBC, which lacks endomembrane system trafficking and, indeed, lysosomes. The highly polarized hepatocyte, however, has extensive vesicular transport networks (7) and can target intracellular pathogens residing in a vacuole (8), suggesting that the exoerythrocytic form (EEF) may need to resist host cell attack.Although the PVM is thought to be critical for Plasmodium growth in both the hepatocyte and the RBC contexts, its cellular roles remain elusive. The importance of several Plasmodium PVM-resident proteins, however, has been conclusively demonstrated in both blood and liver stages. Attempts to generate exported (exp)1 and Plasmodium translocon of exported protein (ptex)150 knockout parasites in Plasmodium falciparum failed (9, 10), revealing that these are both essential proteins for the blood stage, whereas Plasmodium berghei and Plasmodium yoelii mutants lacking up-regulated in sporozoites (uis)3 or uis4 fail to complete liver stage development (11, 12). These PVM-resident proteins, and thus the PVM itself, are performing functions that are crucial for Plasmodium growth, but delineating the functions of individual PVM-resident proteins has proven as difficult as identifying the cellular processes mediated by the PVM.The one process in which both the centrality of the PVM is known and evidence for the participation of specific PVM proteins exists is the export of parasite proteins to the RBC. A cohort of parasite proteins that are involved in extensive physiological and structural modifications of the infected RBC (iRBC) is exported into the iRBC cytoplasm and beyond (13). Five proteins have been identified as components of PTEX, the proposed export machinery at the iRBC PVM (9). Although liver stage protein export has been shown for the Circumsporozite (CS) protein (14) and PTEX components are expressed in P. falciparum EEFs (15), a role for parasite protein export into the hepatocyte remains speculative; the host hepatocyte may not require the extensive structural remodeling that the iRBC does.Conversely, however, the hepatocyte, with its extensive vesicular transport network, intuitively constitutes a more hostile host environment than the RBC, and there is evidence that the liver stage PVM may play a crucial role in preventing host cell-mediated parasite killing, as it does in Toxoplasma gondii (16). Support for a protective role for the liver stage PVM comes from knockout parasites that fail in the earliest steps of PVM formation and remodeling. Sporozoites lacking the p52/p36 gene pair invade hepatocytes successfully, but fail in PVM formation (17, 18) and are severely reduced in abundance midway through liver stage development. Parasites lacking slarp/sap (19, 20), a regulator of early liver stage development, fail to express UIS4 and exported protein 1 (EXP-1), along with other parasite proteins, and are also eliminated at the beginning of infection.Acquisition of resources from the host-cell environment, an unambiguous requirement for an obligate intracellular parasite like Plasmodium, is a function ascribed to the PVM in both mammalian stages. The PVM allows the free passage of molecules (21, 22), presumably through proteinaceous pores, which may contribute to acquisition of host nutrients and disposal of parasite waste products. Members of the early transcribed membrane protein (ETRAMP) family, single-pass transmembrane proteins conserved among Plasmodium spp., which are highly expressed and developmentally regulated in both blood and liver stage parasites (23, 24), could be candidates for mediating uptake of host resources. Such a role in lipid uptake has indeed been proposed for the P. berghei ETRAMP UIS3 on the basis of its interaction with host-cell L-FABP (liver fatty acid binding protein) (25).Although Plasmodium parasites must use host resources to support their own growth in both mammalian stages, the single cycle replicative output of the liver stage parasite is vastly greater than that of the blood stage, which may reflect a similarly increased need for host resources. In this respect, the hepatocyte constitutes far superior “raw material” compared with the RBC; hepatocytes are not only metabolically active, but also highly versatile cells, which are capable of altering uptake, storage, production, and degradation of a wide array of macromolecules in response to cellular and organismal requirements. The presence of a growing Plasmodium parasite is sensed by the host hepatocyte, which responds with activation of cellular stress responses and altered metabolism (26, 27). The mammalian target of rapamycin (mTOR) kinase integrates signals from amino acids, stress, oxygen, energy, and growth factors and responds by altering cellular protein and lipid synthesis, as well as autophagy (28). As such, we sought to determine how inhibition of host mTOR signaling would affect Plasmodium liver stage development. Here we show that torins, a single structural class of mTOR inhibitors, are highly potent antiplasmodial compounds targeting both mammalian stages in vitro and in vivo. Independent of host-cell mTOR, torins impair trafficking of Plasmodium liver stage PVM-resident proteins, revealing the fast turnover of these proteins at the liver stage PVM, and provoke elimination of liver stage parasites.     

Early, nonciliary role for microtubule proteins in left-right patterning is conserved across kingdoms

Many types of embryos' bodyplans exhibit consistently oriented laterality of the heart, viscera, and brain. Errors of left-right patterning present an important class of human birth defects, and considerable controversy exists about the nature and evolutionary conservation of the molecular mechanisms that allow embryos to reliably orient the left-right axis. Here we show that the same mutations in the cytoskeletal protein tubulin that alter asymmetry in plants also affect very early steps of left-right patterning in nematode and frog embryos, as well as chirality of human cells in culture. In the frog embryo, tubulin α and tubulin γ-associated proteins are required for the differential distribution of maternal proteins to the left or right blastomere at the first cell division. Our data reveal a remarkable molecular conservation of mechanisms initiating left-right asymmetry. The origin of laterality is cytoplasmic, ancient, and highly conserved across kingdoms, a fundamental feature of the cytoskeleton that underlies chirality in cells and multicellular organisms.     

寄生虫分泌/膜蛋白的生物信息学特征及研究策略

分泌/膜蛋白足寄生虫与宿主相互作用的主要分子,由于其直接暴露于宿主免疫系统及体内药物,因此是寄生虫诊断试剂、疫苗及药物作用靶标主要的候选分子,对其系统深入的研究有助于阐明寄生虫与宿主相瓦作用的分子机制.该文概述了寄生虫分泌/膜蛋白的牛物信息学特征及研究策略,并用生物信息学方法对部分寄生虫一些重婴的诊断及疫苗候选分子进行了分泌/膜篮白特征的分析.     

Quality control of overexpressed membrane proteins

Overexpression of membrane proteins in Escherichia coli frequently leads to the formation of aggregates or inclusion bodies, which is undesirable for most studies. Ideally, one would like to optimize the expression conditions by monitoring simultaneously and rapidly both the amounts of properly folded and aggregated membrane protein, a requirement not met by any of the currently available methods. Here, we describe a simple gel-based approach with green fluorescent protein as folding indicator to detect well folded and aggregated proteins simultaneously. The method allows for rapid screening and, importantly, pinpointing the most likely bottlenecks in protein production.     

Subtractive screening with the Mycobacterium tuberculosis surface protein phage display library

Surface proteins consist of secreted and membrane proteins and play a central role in the interaction of the pathogen with its environment, especially in the pathogenicity of Mycobacterium tuberculosis (MTB). Research on surface proteins in MTB has focused on 2D electrophoresis of culture filtrate proteins (CFP), extraction of transmembrane proteins with detergent and predicting their properties with a range of available algorithms. However, functional analysis of these secretomes is possible only if many proteins are expressed and purified individually, which limits a large number of studies to the function of the proteome. Here, we utilized a phage display system to construct a whole genomic surface protein phage display library of MTB, which can complete direct selection, identification, expression, purification and functional research of surface proteins of MTB. With this system we made a new serological approach involving iterative subtraction screening. Cross-reactivity of antibodies was reduced by preadsorption of the surface protein phage display library with the sera of healthy BCG-vaccinated individuals prior to studying their reactivity against the sera of tuberculosis (TB) patients. As a result six antigens were identified, three of which have not previously been reported as diagnosis antigens. The surface protein phage display library shows great promise in the study of MTB.