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mBio ; 10(6)2019 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-31848273


New approaches are needed to control leprosy, but understanding of the biology of the causative agent Mycobacterium leprae remains rudimentary, principally because the pathogen cannot be grown in axenic culture. Here, we applied 13C isotopomer analysis to measure carbon metabolism of M. leprae in its primary host cell, the Schwann cell. We compared the results of this analysis with those of a related pathogen, Mycobacterium tuberculosis, growing in its primary host cell, the macrophage. Using 13C isotopomer analysis with glucose as the tracer, we show that whereas M. tuberculosis imports most of its amino acids directly from the host macrophage, M. leprae utilizes host glucose pools as the carbon source to biosynthesize the majority of its amino acids. Our analysis highlights the anaplerotic enzyme phosphoenolpyruvate carboxylase required for this intracellular diet of M. leprae, identifying this enzyme as a potential antileprosy drug target.IMPORTANCE Leprosy remains a major problem in the world today, particularly affecting the poorest and most disadvantaged sections of society in the least developed countries of the world. The long-term aim of research is to develop new treatments and vaccines, and these aims are currently hampered by our inability to grow the pathogen in axenic culture. In this study, we probed the metabolism of M. leprae while it is surviving and replicating inside its primary host cell, the Schwann cell, and compared it to a related pathogen, M. tuberculosis, replicating in macrophages. Our analysis revealed that unlike M. tuberculosis, M. leprae utilized host glucose as a carbon source and that it biosynthesized its own amino acids, rather than importing them from its host cell. We demonstrated that the enzyme phosphoenolpyruvate carboxylase plays a crucial role in glucose catabolism in M. leprae Our findings provide the first metabolic signature of M. leprae in the host Schwann cell and identify novel avenues for the development of antileprosy drugs.

Carbono/metabolismo , Glucosa/metabolismo , Mycobacterium leprae/fisiología , Células de Schwann/metabolismo , Células de Schwann/microbiología , Metabolismo de los Hidratos de Carbono , Línea Celular , Interacciones Huésped-Patógeno , Humanos , Lepra/metabolismo , Lepra/microbiología , Macrófagos/metabolismo , Macrófagos/microbiología , Redes y Vías Metabólicas
BMC Genomics ; 20(1): 506, 2019 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-31215404


BACKGROUND: Klebsiella pneumoniae (KP) is an opportunistic pathogen that mainly causes respiratory and urinary tract infections. The frequent occurrence of simultaneously virulent and multiple drug-resistant isolates led WHO to include this species in the list of top priorities for research and development of therapeutic alternatives. The comprehensive knowledge of the molecular mechanisms underlying KP virulence may lead to the proposal of more efficient and specific drugs. One of its virulence factors is the Type VI Secretion System (T6SS), which contributes to bacterial competition, cell invasion and in vivo colonisation. Despite the few studies showing the involvement of T6SS in KP pathogenesis, little is known concerning the regulation of its expression. The understanding of regulatory mechanisms may give more clues about the function of the system and the possibilities of future interference in this process. This work aimed to standardise the annotation of T6SS genes in KP strains and identify mechanisms of their transcriptional regulation through computational predictions. RESULTS: We analyzed the genomes of Kp52.145, HS11286 and NTUH-K2044 strains to perform a broad prediction and re-annotation of T6SS genes through similarity searches, comparative and linear discriminant analysis. 38 genes were found in Kp52.145, while 29 in HS11286 and 30 in NTUH-K2044. Genes coding for iron uptake systems are encoded in adjacencies of T6SS, suggesting that KP T6SS might also play a role in ion import. Some of the T6SS genes are comprised in syntenic regions. 17 sigma 70-dependent promoter regions were identified in Kp52.145, 12 in HS11286 and 12 in NTUH-K2044. Using VirtualFootprint algorithm, binding sites for 13 transcriptional regulators were found in Kp52.145 and 9 in HS11286 and 17 in NTUH-K2044. Six of them are common to the 3 strains: OxyR, H-NS, RcsAB, GcvA, Fis, and OmpR. CONCLUSIONS: The data presented herein are derived from computational analysis. Although future experimental studies are required to confirm those predictions, they suggest that KP T6SS might be regulated in response to environmental signals that are indeed sensed by the bacteria inside the human host: temperature (H-NS), nutrition-limitation (GcvA and Fis), oxidative stress (OxyR) and osmolarity (RscAB and OmpR).

Regulación Bacteriana de la Expresión Génica , Klebsiella pneumoniae/genética , Transcripción Genética/genética , Sistemas de Secreción Tipo VI/genética , Secuencia de Aminoácidos , Sitios de Unión , Genoma Bacteriano/genética , Anotación de Secuencia Molecular , Sintenía , Factores de Transcripción/metabolismo , Sistemas de Secreción Tipo VI/química , Sistemas de Secreción Tipo VI/metabolismo
J Proteomics ; 80: 34-42, 2013 Mar 27.
Artículo en Inglés | MEDLINE | ID: mdl-23159400


Trypanosoma cruzi proteins with molecular weight between 30 and 34 kDa have shown high reactivity in western blot assays with serum samples from chagasic individuals. However, in-depth analysis of the constituents of these protein fractions has not been performed. This is the first report of an immunoaffinity proteomic approach to identify the immunodominant 30-34 kDa proteins of T. cruzi that could eventually be used for the diagnosis of Chagas disease. We used two different sample preparation protocols for protein digestion coupled to mass spectrometry to identify proteins in the protein fraction. The immunodominant proteins and their respective epitopes were then identified by co-immunoprecipitation and excision-epitope mapping/mass spectrometry, using human sera followed by the prediction and three-dimensional structural modeling of reactive epitopes. The use of different sample preparation methods allowed the identification of a relatively high number of proteins, some of which were only identified after one or multiple sample preparation and digestion protocols. Seven immunodominant proteins were identified by co-immunoprecipitation with purified IgGs from chagasic serum samples. Moreover, six reactive peptide epitopes were detected in four of these proteins by excision-epitope mapping/mass spectrometry. Three-dimensional structural models were obtained for the immunoreactive peptides, which correlated well with the linear B-cell epitope prediction tools.

Anticuerpos/química , Enfermedad de Chagas/sangre , Enfermedad de Chagas/parasitología , Epítopos/química , Trypanosoma cruzi/metabolismo , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Electroforesis en Gel de Poliacrilamida , Mapeo Epitopo , Epítopos de Linfocito B/química , Humanos , Inmunoglobulina G/química , Inmunoprecipitación , Espectrometría de Masas , Peso Molecular , Péptidos/química , Conformación Proteica , Proteoma , Proteómica/métodos
J Bacteriol ; 193(24): 6929-38, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21984792


The control of Vibrio cholerae phoBR expression by PhoB involves its binding to Pho boxes at -35 (box 1), -60 (box 2), and -80 (box 3) from the putative phoB translation start site. These loci were located in the sense (box 1) and antisense (boxes 2 and 3) strands of the phoBR regulatory region, and PhoB binds to these individual boxes with distinct affinities. Fusions of sequences containing different combinations of these boxes upstream of the lacZ reporter in a plasmid demonstrated that only those carrying boxes 1, 2, and 3, or 1 alone, activated transcription under inorganic phosphate (P(i)) limitation. When a fragment, including only boxes 1 and 2, was fused to lacZ, expression was no longer induced by low P(i), suggesting a repressive role for PhoB~box2 (PhoB bound to box 2) over the transcriptional activity induced by PhoB~box1. The similarity between lacZ expression levels from promoter fragments containing the three boxes or box 1 alone showed that PhoB~box3 eliminated the repressive effect imposed by PhoB~box2 on phoBR transcription. Complementation assays with a phoBR-containing plasmid demonstrated that the 234-bp promoter fragment carrying the three boxes is absolutely required for operon expression in Vibrio cholerae ΔphoBR cells. This was observed under P(i) abundance, when phoBR was expressed at a basal level and, also in low P(i) conditions, when Pho regulon genes were fully expressed. Thus, under P(i) limitation, PhoB exerts dual regulatory functions by binding sequentially distinct Pho boxes to enable the fine-tuning and precise control of phoBR expression in V. cholerae cells.

Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Operón , Vibrio cholerae/metabolismo , Proteínas Bacterianas/química , Secuencia de Bases , Sitios de Unión , Datos de Secuencia Molecular , Fosfatos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Vibrio cholerae/química , Vibrio cholerae/genética
Microbiology ; 156(Pt 10): 3011-20, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20688821


The putative phosphoporin encoded by vca1008 of Vibrio cholerae O1 is expressed in vivo during infection and is essential for the intestinal colonization of infant mice. In vitro, its expression is induced under inorganic phosphate (P(i)) limitation in a PhoB/R-dependent manner. In this work we demonstrated that VCA1008 has a strain-specific role in the physiology and pathogenicity of V. cholerae O1. Disruption of vca1008 led to a growth defect, an inability to colonize and a high susceptibility to sodium deoxycholate (DOC; the major bile compound) in the El Tor biotype strain N16961, but did not affect the classical strain O395 in the same way. Furthermore, vca1008 promoter activity was higher in N16961 cells grown under a low P(i) supply in the presence of DOC than in the absence of the detergent. In the P(i)-limited cells, vca1008 was positively regulated by PhoB, but when DOC was added to the medium, it negatively affected the PhoB-mediated activation of the gene, and enhanced vca1008 expression in a ToxR-dependent manner. These findings reveal for the first time a complex strain-specific interplay between ToxR and PhoB/R systems to control porin genes, as well as the influence of DOC on the expression of PhoB- and ToxR-regulated genes and pathogenesis in pandemic strains of V. cholerae.

Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Ácido Desoxicólico/farmacología , Porinas/metabolismo , Factores de Transcripción/metabolismo , Vibrio cholerae/patogenicidad , Animales , Proteínas Bacterianas/genética , Clonación Molecular , Proteínas de Unión al ADN/genética , Prueba de Complementación Genética , Ratones , Mutación , Fosfatos/metabolismo , Porinas/genética , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Vibrio cholerae/genética , Vibrio cholerae/crecimiento & desarrollo , Vibrio cholerae/metabolismo , Virulencia
Proteomics ; 6(5): 1495-511, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16447160


A proteomic analysis of a wild-type and of a phoB mutant showed that Vibrio cholerae expresses genes of two major regulons in response to phosphate starvation. The Pho regulon, expressed by the wild-type, allowed the cells to adapt to the new environment. Induction of the general stress regulon was mainly observed in the phoB mutant as a strategy to resist stress and survive. Some functions of the adaptative and survival responses play roles in the pathogenicity of the bacteria. Among the members of the Pho regulon, we found a porin described as an important factor for the intestinal colonisation. Other functions not obviously related to phosphate metabolism, expressed preferentially by the wild-type cells, have also been implicated in virulence. These findings might explain the lack of virulence of the phoB mutant. The Pho regulon picture of V. cholerae, however, will not be complete until minor members and membrane proteins are identified. Among the phosphate-starvation induced genes we have found 13 hypothetical ones and for some of them functions have been assigned. The majority of the genes identified here have not been described before, thus they could be used to expand the proteomic reference map of V. cholerae El Tor.

Adaptación Fisiológica , Proteínas Bacterianas/metabolismo , Fosfatos/metabolismo , Proteoma/análisis , Regulón , Vibrio cholerae O1/fisiología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Electroforesis en Gel Bidimensional , Datos de Secuencia Molecular , Operón , Vibrio cholerae O1/genética , Vibrio cholerae O1/patogenicidad