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1.
Emerg Infect Dis ; 23(3): 423-429, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28221129

RESUMO

Tuberculosis is a leading cause of illness and death in Congo. No data are available about the population structure and transmission dynamics of the Mycobacterium tuberculosis complex strains prevalent in this central Africa country. On the basis of single-nucleotide polymorphisms detected by whole-genome sequencing, we phylogenetically characterized 74 MTBC isolates from Brazzaville, the capital of Congo. The diversity of the study population was high; most strains belonged to the Euro-American lineage, which split into Latin American Mediterranean, Uganda I, Uganda II, Haarlem, X type, and a new dominant sublineage named Congo type (n = 26). Thirty strains were grouped in 5 clusters (each within 12 single-nucleotide polymorphisms), from which 23 belonged to the Congo type. High cluster rates and low genomic diversity indicate recent emergence and transmission of the Congo type, a new Euro-American sublineage of MTBC.


Assuntos
Mycobacterium tuberculosis/genética , Tuberculose/epidemiologia , Tuberculose/microbiologia , Adulto , Análise por Conglomerados , Congo/epidemiologia , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/classificação , Filogenia , Adulto Jovem
2.
Chembiochem ; 18(13): 1172-1176, 2017 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-28249101

RESUMO

Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (Tb), has a complex cell envelope which forms an efficient barrier to antibiotics, thus contributing to the challenges of anti-tuberculosis therapy. However, the unique Mtb cell wall can be considered an advantage and be utilized to selectively label Mtb bacteria. Here we introduce three azido pentoses as new compounds for metabolic labeling of Mtb: 3-azido arabinose (3AraAz), 3-azido ribose (3RiboAz), and 5-azido arabinofuranose (5AraAz). 5AraAz demonstrated the highest level of Mtb labeling and was efficiently incorporated into the Mtb cell wall. All three azido pentoses can be easily used to label a variety of Mtb clinical isolates without influencing Mtb-dependent phagosomal maturation arrest in infection studies with human macrophages. Thus, this metabolic labeling method offers the opportunity to attach desired molecules to the surface of Mtb bacteria in order to facilitate investigation of the varying virulence characteristics of different Mtb clinical isolates, which influence the outcome of a Tb infection.


Assuntos
Azidas/química , Parede Celular/química , Mycobacterium tuberculosis/química , Pentoses/química , Coloração e Rotulagem/métodos , Biomarcadores/metabolismo , Parede Celular/metabolismo , Citometria de Fluxo , Expressão Gênica , Humanos , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Mycobacterium tuberculosis/metabolismo , Fagocitose , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/imunologia
3.
J Immunol ; 189(7): 3575-84, 2012 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-22925928

RESUMO

Streptococcus pneumoniae (pneumococcus) is a pathogen that causes severe local and life-threatening invasive diseases, which are associated with high mortality rates. Pneumococci have evolved several strategies to evade the host immune system, including complement to disseminate and to survive in various host niches. Thus, pneumococci bind complement inhibitors such as C4b-binding protein (C4BP) and factor H via pneumococcal surface protein C, thereby inhibiting the classical and alternative complement pathways. In this study, we identified the pneumococcal glycolytic enzyme enolase, a nonclassical cell surface and plasminogen-binding protein, as an additional pneumococcal C4BP-binding protein. Furthermore, we demonstrated that human, but not mouse, C4BP bound pneumococci. Recombinant enolase bound in a dose-dependent manner C4BP purified from plasma, and the interaction was reduced by increasing ionic strength. Enolase recruited C4BP and plasminogen, but not factor H, from human serum. Moreover, C4BP and plasminogen bound to different domains of enolase as they did not compete for the interaction with enolase. In direct binding assays with recombinant C4BP mutants lacking individual domains, two binding sites for enolase were identified on the complement control protein (CCP) domain 1/CCP2 and CCP8 of the C4BP α-chains. C4BP bound to the enolase retained its cofactor activity as determined by C4b degradation. Furthermore, in the presence of exogenously added enolase, an increased C4BP binding to and subsequently decreased C3b deposition on pneumococci was observed. Taken together, pneumococci specifically interact with human C4BP via enolase, which represents an additional mechanism of human complement control by this versatile pathogen.


Assuntos
Proteína de Ligação ao Complemento C4b/metabolismo , Inativadores do Complemento/metabolismo , Evasão da Resposta Imune , Fosfopiruvato Hidratase/metabolismo , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/imunologia , Proteína de Ligação ao Complemento C4b/antagonistas & inibidores , Glicólise , Humanos , Plasminogênio/metabolismo , Ligação Proteica/imunologia , Streptococcus pneumoniae/metabolismo
4.
PLoS One ; 17(7): e0265554, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35839162

RESUMO

BACKGROUND: Pulmonary Tuberculosis (TB) is diagnosed through sputum samples. As sputum sampling is challenging in children and cachexic patients, the development of diagnostic tests using saliva appears promising but has been discouraged due to low bacterial load and poor sensitivity. Here, we present a novel and rapid method to enrich Mycobacterium tuberculosis (Mtb) from saliva, which may serve as a basis for a diagnostic saliva test. METHODS: Lipobiotin-functionalized magnetic beads (LMBs) were incubated with Mtb-spiked PBS and saliva from healthy donors as well as with saliva from TB patients. Flow cytometry was used to evaluate the capacity of the beads to bind Mtb, while real-time quantitative polymerase chain reaction (qPCR) was utilized to detect Mtb and determine the amount of mycobacterial DNA in different sample types. RESULTS: We found that LMBs bind Mtb efficiently when compared to non-functionalized beads. The development of an qPCR assay based on the use of LMBs (LMB assay) allowed us to enrich mycobacterial DNA in spiked sample types, including PBS and saliva from healthy donors (enrichment of up to ~8.7 fold). In Mtb-spiked saliva samples, we found that the LMB assay improved the detection rate of 102 bacteria in a volume of 5 ml from 0 out of 15 (0%) to 6 out of 15 (40%). Consistent with that, the LMB assay increased the rate of correctly identified saliva samples from TB patients in two independent cohorts. CONCLUSIONS: Implementation of the principle of the LMB-based assay may improve the sensitivity of existing diagnostic techniques, e.g. by functionalizing materials that facilitate Mtb sampling from the oral cavity.


Assuntos
Mycobacterium tuberculosis , Tuberculose dos Linfonodos , Tuberculose Pulmonar , Criança , Humanos , Fenômenos Magnéticos , Mycobacterium tuberculosis/genética , Saliva , Sensibilidade e Especificidade , Escarro/microbiologia , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/microbiologia
5.
ACS Infect Dis ; 8(7): 1303-1315, 2022 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-35763439

RESUMO

One-fourth of the global human population is estimated to be infected with strains of the Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB). Using lipidomic approaches, we show that tuberculostearic acid (TSA)-containing phosphatidylinositols (PIs) are molecular markers for infection with clinically relevant MTBC strains and signify bacterial burden. For the most abundant lipid marker, detection limits of ∼102 colony forming units (CFUs) and ∼103 CFUs for bacterial and cell culture systems were determined, respectively. We developed a targeted lipid assay, which can be performed within a day including sample preparation─roughly 30-fold faster than in conventional methods based on bacterial culture. This indirect and culture-free detection approach allowed us to determine pathogen loads in infected murine macrophages, human neutrophils, and murine lung tissue. These marker lipids inferred from mycobacterial PIs were found in higher levels in peripheral blood mononuclear cells of TB patients compared to healthy individuals. Moreover, in a small cohort of drug-susceptible TB patients, elevated levels of these molecular markers were detected at the start of therapy and declined upon successful anti-TB treatment. Thus, the concentration of TSA-containing PIs can be used as a correlate for the mycobacterial burden in experimental models and in vitro systems and may prospectively also provide a clinically relevant tool to monitor TB severity.


Assuntos
Mycobacterium tuberculosis , Tuberculose , Animais , Humanos , Leucócitos Mononucleares , Camundongos , Fosfatidilinositóis , Ácidos Esteáricos , Tuberculose/microbiologia
6.
Front Microbiol ; 13: 832054, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35350619

RESUMO

"Ancestral" Mycobacterium tuberculosis complex (MTBC) strains of Lineage 1 (L1, East African Indian) are a prominent tuberculosis (TB) cause in countries around the Indian Ocean. However, the pathobiology of L1 strains is insufficiently characterized. Here, we used whole genome sequencing (WGS) of 312 L1 strains from 43 countries to perform a characterization of the global L1 population structure and correlate this to the analysis of the synthesis of phenolic glycolipids (PGL) - known MTBC polyketide-derived virulence factors. Our results reveal the presence of eight major L1 sub-lineages, whose members have specific mutation signatures in PGL biosynthesis genes, e.g., pks15/1 or glycosyltransferases Rv2962c and/or Rv2958c. Sub-lineage specific PGL production was studied by NMR-based lipid profiling and strains with a completely abolished phenolphthiocerol dimycoserosate biosynthesis showed in average a more prominent growth in human macrophages. In conclusion, our results show a diverse population structure of L1 strains that is associated with the presence of specific PGL types. This includes the occurrence of mycoside B in one sub-lineage, representing the first description of a PGL in an M. tuberculosis lineage other than L2. Such differences may be important for the evolution of L1 strains, e.g., allowing adaption to different human populations.

7.
Int J Med Microbiol ; 298(7-8): 645-55, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18585090

RESUMO

In Mycobacterium marinum, the homologue of Rv1500 of M. tuberculosis encodes a glycosyltransferase. Initially, it was suggested that this gene is involved in the synthesis of phosphatidylinositol mannosides (PIMs), generating Ac(2)PIM(7) from Ac(2)PIM(5). Phosphatidylinositol mannoside and its related compounds lipomannan (LM) and lipoarabinomannan (LAM) have been shown to modulate the host response to an infection with M. tuberculosis. Here, we generated a deletion mutant of Rv1500 in M. tuberculosis H37Rv, and analyzed the mutant using a biochemical approach as well as in vitro and in vivo infection models. Inactivation of Rv1500 did not lead to an altered expression pattern of PIMs in M. tuberculosis H37Rv. We found phosphatidylinositol (PI), PIM(2), AcPIM(2), Ac(2)PIM(2), and AcPIM(6) in both strains, but were unable to detect Ac(2)PIM(7) or Ac(2)PIM(5) either in the wild type or the mutant strain. Uptake and growth of H37Rv and Rv1500 mutant strains in murine bone marrow-derived macrophages was identical, and TNFalpha and IL-12p40 production in mouse macrophages and dendritic cells was induced to similar levels following infection with either strain. Aerosol challenge of mice showed that wild type and Rv1500 mutant strains had identical growth rates in infected organs over time. We verified mRNA expression of Rv1500 in H37Rv and conclude that Rv1500 must serve a redundant role in viability and virulence of M. tuberculosis.


Assuntos
Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/patogenicidade , Animais , Células Dendríticas/imunologia , Feminino , Deleção de Genes , Subunidade p40 da Interleucina-12/biossíntese , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Fosfatidilinositóis/análise , Fator de Necrose Tumoral alfa/biossíntese , Virulência
8.
PLoS One ; 7(4): e34852, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22514678

RESUMO

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.


Assuntos
Proteína de Ligação ao Complemento C4b/metabolismo , Fator H do Complemento/metabolismo , Fator I do Complemento/metabolismo , Prevotella intermedia/metabolismo , Humanos , Ligação Proteica
9.
Microbiology (Reading) ; 155(Pt 4): 1332-1339, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19332834

RESUMO

Mycobacterium tuberculosis can utilize various nutrients including nitrate as a source of nitrogen. Assimilation of nitrate requires the reduction of nitrate via nitrite to ammonium, which is then incorporated into metabolic pathways. This study was undertaken to define the molecular mechanism of nitrate assimilation in M. tuberculosis. Homologues to a narGHJI-encoded nitrate reductase and a nirBD-encoded nitrite reductase have been found on the chromosome of M. tuberculosis. Previous studies have implied a role for NarGHJI in nitrate respiration rather than nitrate assimilation. Here, we show that a narG mutant of M. tuberculosis failed to grow on nitrate. A nirB mutant of M. tuberculosis failed to grow on both nitrate and nitrite. Mutant strains of Mycobacterium smegmatis mc(2)155 that are unable to grow on nitrate were isolated. The mutants were rescued by screening a cosmid library from M. tuberculosis, and a gene with homology to the response regulator gene glnR of Streptomyces coelicolor was identified. A DeltaglnR mutant of M. tuberculosis was generated, which also failed to grow on nitrate, but regained its ability to utilize nitrate when nirBD was expressed from a plasmid, suggesting a role of GlnR in regulating nirBD expression. A specific binding site for GlnR within the nirB promoter was identified and confirmed by electrophoretic mobility shift assay using purified recombinant GlnR. Semiquantitative reverse transcription PCR, as well as microarray analysis, demonstrated upregulation of nirBD expression in response to GlnR under nitrogen-limiting conditions. In summary, we conclude that NarGHJI and NirBD of M. tuberculosis mediate the assimilatory reduction of nitrate and nitrite, respectively, and that GlnR acts as a transcriptional activator of nirBD.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/crescimento & desenvolvimento , Nitrato Redutase/metabolismo , Nitratos/metabolismo , Nitrito Redutases/metabolismo , Proteínas de Bactérias/genética , Meios de Cultura , Humanos , Dados de Sequência Molecular , Mutação , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Nitrato Redutase/genética , Nitrito Redutases/genética , Nitritos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA
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