RESUMEN
A major virulence trait of Mycobacterium tuberculosis (M. tb) is its ability to enter a dormant state within its human host. Since cell division is intimately linked to metabolic shut down, understanding the mechanism of septum formation and its integration with other events in the division pathway is likely to offer clues to the molecular basis of dormancy. The M. tb genome lacks obvious homologues of several conserved cell division proteins, and this study was aimed at identifying and functionally characterising mycobacterial homologues of the E. coli septum site specification protein MinD (Ec MinD). Sequence homology based analyses suggested that the genomes of both M. tb and the saprophyte Mycobacterium smegmatis (M. smegmatis) encode two putative Ec MinD homologues - Rv1708/MSMEG_3743 and Rv3660c/ MSMEG_6171. Of these, Rv1708/MSMEG_3743 were found to be the true homologues, through complementation of the E. coli ∆minDE mutant HL1, overexpression studies, and structural comparisons. Rv1708 and MSMEG_3743 fully complemented the mini-cell phenotype of HL1, and over-expression of MSMEG_3743 in M. smegmatis led to cell elongation and a drastic decrease in c.f.u. counts, indicating its essentiality in cell-division. MSMEG_3743 displayed ATPase activity, consistent with its containing a conserved Walker A motif. Interaction of Rv1708 with the chromosome associated proteins ScpA and ParB, implied a link between its septum formation role, and chromosome segregation. Comparative structural analyses showed Rv1708 to be closer in similarity to Ec MinD than Rv3660c. In summary we identify Rv1708 and MSMEG_3743 to be homologues of Ec MinD, adding a critical missing piece to the mycobacterial cell division puzzle.
Asunto(s)
Proteínas de Escherichia coli , Mycobacterium tuberculosis , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , División Celular/genética , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMEN
Macrophages are the primary hosts for Mycobacterium tuberculosis (M. tb), an intracellular pathogen, and the causative organism of tuberculosis (TB) in humans. While M. tb has the ability to enter and survive in host macrophages, the precise mechanism of its internalization, and factors that control this essential process are poorly defined. We have previously demonstrated that perturbations in levels of cholesterol and sphingolipids in macrophages lead to significant reduction in the entry of Mycobacterium smegmatis (M. smegmatis), a surrogate model for mycobacterial internalization, signifying a role for these plasma membrane lipids in interactions at the host-pathogen interface. In this work, we investigated the role of the host actin cytoskeleton, a critical protein framework underlying the plasma membrane, in the entry of M. smegmatis into human macrophages. Our results show that cytochalasin D mediated destabilization of the actin cytoskeleton of host macrophages results in a dose-dependent reduction in the entry of mycobacteria. Notably, the internalization of Escherichia coli remained invariant upon actin destabilization of host cells, implying a specific involvement of the actin cytoskeleton in mycobacterial infection. By monitoring the F-actin content of macrophages utilizing a quantitative confocal microscopy-based technique, we observed a close correlation between the entry of mycobacteria into host macrophages with cellular F-actin content. Our results constitute the first quantitative analysis of the role of the actin cytoskeleton of human macrophages in the entry of mycobacteria, and highlight actin-mediated mycobacterial entry as a potential target for future anti-TB therapeutics.
Asunto(s)
Actinas , Mycobacterium tuberculosis , Humanos , Actinas/metabolismo , Citocalasina D/farmacología , Citocalasina D/metabolismo , Citoesqueleto de Actina/metabolismo , Macrófagos/metabolismo , Mycobacterium tuberculosis/metabolismo , Colesterol/metabolismo , EsfingolípidosRESUMEN
We have investigated the physiological role of the autophagy receptor Optineurin/Optn in endoplasmic reticulum (ER) stress response using cellular and animal models. In comparison to their normal counterparts, Optn-deficient mouse embryonic fibroblasts showed significantly higher cell death and caspase-3 activation upon treatment with tunicamycin and thapsigargin, inducers of ER stress. The transcript levels of some of the genes regulated by the IRE1-XBP1 and PERK-ATF4 pathways were upregulated in Optn-deficient cells, in comparison with normal cells, upon treatment with tunicamycin, and also in the brain cortex and liver of tunicamycin treated Optn-deficient mice. Also, the basal levels of IRE1α and PERK were higher in Optn-deficient cells. These results suggest that Optn modulates ER stress-induced signaling pathways and provides protection from ER stress-induced cell death.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Estrés del Retículo Endoplásmico/fisiología , Proteínas de Transporte de Membrana/fisiología , Animales , Autofagia/fisiología , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/genética , Muerte Celular/fisiología , Células Cultivadas , Estrés del Retículo Endoplásmico/genética , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Fibroblastos/citología , Fibroblastos/fisiología , Expresión Génica , Proteínas de Transporte de Membrana/deficiencia , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/fisiología , Respuesta de Proteína Desplegada/genética , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
In a Mycobacterium smegmatis mutant library screen, transposon mutants with insertions in fhaA, dprE2, rpsT, and parA displayed hypersusceptibility to antibiotics, including the ß-lactams meropenem, ampicillin, amoxicillin, and cefotaxime. Sub-MIC levels of octoclothepin, a psychotic drug inhibiting ParA, phenocopied the parA insertion and enhanced the bactericidal activity of meropenem against Mycobacterium tuberculosis in combination with clavulanate. Our study identifies novel factors associated with antibiotic resistance, with implications in repurposing ß-lactams for tuberculosis treatment.
Asunto(s)
Antibacterianos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/patogenicidad , Tuberculosis/tratamiento farmacológico , beta-Lactamas/metabolismo , Ácido Clavulánico/farmacología , Dibenzotiepinas/farmacología , Farmacorresistencia Microbiana/genética , Meropenem , Pruebas de Sensibilidad Microbiana , Tienamicinas/farmacología , Tuberculosis/microbiología , beta-Lactamasas/genéticaRESUMEN
The phenomenal success of Mycobacterium tuberculosis (M.tb) as a pathogen is primarily based on its ability to modulate host immune responses. The genome of M.tb encodes multiple immunomodulatory proteins, including several members of the multigenic PE_PPE family of which the PE_PGRS proteins are a subset. Curiously, 56 of the 61 PE_PGRS proteins contain multiple copies of the glycine-rich sequence motif GGXGXD/NXUX, a nonapeptide sequence predicted to bind Ca(2+), but the functional significance of these motifs remains a mystery. Here we provide evidence via isothermal titration calorimetry, (45)Ca blotting, fluorescence, and circular dichroism spectroscopy that Ca(2+) binds to the PE_PGRS proteins, PE_PGRS33 (Rv1818c) (10 motifs) and PE_PGRS61 (Rv3653) (one motif). Ca(2+) was observed not to bind to PE_PGRS8 (Rv0742), which lacks nonapeptide motifs. Using recombinant Mycobacterium smegmatis strains expressing Rv1818c and Rv3653 and the THP-1 macrophage model of infection, we show that the two proteins mediate Ca(2+)-dependent upregulation of the anti-inflammatory cytokine IL-10, events critical to the pathogenesis of M.tb. Both Rv1818c and Rv3653 interact with TLR2 in a Ca(2+)-dependent manner, providing a novel mechanistic basis for their immunomodulatory effects. Mutations in the nonapeptide motif of Rv3653 led to compromised Ca(2+) binding, validating the functional criticality of this motif. This study demonstrates for the first time not only their Ca(2+) binding properties but also an essential role for Ca(2+) in the functioning of the M.tb PE_PGRS proteins, opening up the possibility of developing novel anti-tuberculosis therapeutics that inhibit Ca(2+)-PE_PGRS binding.
Asunto(s)
Proteínas Bacterianas/química , Calcio/química , Interacciones Huésped-Patógeno , Mycobacterium tuberculosis/química , Dicroismo Circular , Espectrometría de FluorescenciaRESUMEN
The hallmark feature of Mycobacterium tuberculosis (M.tb) the causative agent of human tuberculosis, is its complex lipid rich cell wall comprised primarily of mycolic acids, long chain fatty acids that play a key role in structural stability and permeability of the cell wall. In addition, they are involved in inhibiting phagosome-lysosome fusion and aid in granuloma formation during the pathogenic process. M.tb DesA1 is an essential acyl-acyl carrier protein desaturase predicted to catalyze the introduction of position specific double bonds during the biosynthesis of mycolic acids. This protein is one among three annotated desaturases (DesA1-3) in the M.tb genome but is unique in containing a ßγ-crystallin Greek key signature motif, a well-characterized fold known to mediate Ca2+ binding in both prokaryotic and eukaryotic organisms. Using Isothermal Titration Calorimetry and 45CaCl2 overlay, we demonstrate that Ca2+ binds to DesA1. Spectroscopic measurements suggested that this binding induces changes in protein conformation but does not lead to significant alterations in the secondary structure of the protein, a feature common to several ßγ-crystallins. An M. smegmatis strain over-expressing M.tb desA1 showed a Ca2+ dependent variation in surface phenotype, revealing a functional role for Ca2+in DesA1 activity. This study represents the first identification of a Ca2+ binding ßγ-crystallin in M.tb, emphasizing the implicit role of Ca2+ in the pathogenesis of M.tb.
Asunto(s)
Antígenos Bacterianos/química , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Calcio/metabolismo , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Calorimetría , Dicroismo Circular , Ácido Graso Desaturasas/química , Ácido Graso Desaturasas/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/química , Conformación Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de FluorescenciaRESUMEN
Toll-like receptor (TLR)-mediated interactions of Mycobacterium tuberculosis (M. tb) with macrophages are major determinant in the outcome of innate immune defence and subsequent adaptive immune responses. Here we report a novel interaction of the M. tb protein pair PE9 (Rv1088)-PE10 (Rv1089) with the macrophage TLR4 leading to apoptosis and modulation of cytokine levels. We demonstrate that the two proteins physically interact, and that PE9 is required for the cell wall localization of PE10 in Mycobacterium smegmatis. Interaction of the PE9-PE10 complex with TLR4 in THP-1 macrophages was associated with increased levels of phospho-IRF-3, which correlated with an increase in transcript levels of its target gene interferon-ß. THP-1 macrophages treated with PE9-PE10 complex showed multiple hallmarks of apoptosis and modulation of interleukin (IL)-1b and IL-10 levels. All of these effects were abrogated when cells were treated either with an antibody to PE10 or an anti-TLR4 antibody, indicating that the complex specifically interacts with TLR4 through PE10, establishing this protein pair as a TLR4 ligand. This novel observation of two proline-glutamate (PE) proteins forming functional heterodimers represents a considerable expansion of the PE_PPE repertoire in the context of receptor engagement and the concomitant modulation of host responses by this unique class of proteins.
Asunto(s)
Apoptosis , Proteínas Bacterianas/metabolismo , Macrófagos/fisiología , Mycobacterium tuberculosis/inmunología , Multimerización de Proteína , Receptor Toll-Like 4/agonistas , Línea Celular , Humanos , Mycobacterium smegmatis/inmunologíaRESUMEN
The extraordinary success of Mycobacterium tuberculosis (M. tb) has been attributed to its ability to modulate host immune responses, and its genome encodes multiple immunomodulatory factors, including several proteins of the multigenic PE_PPE family. To understand its role in M. tb pathophysiology we have characterised the PPE50 (Rv3135)-PPE51 (Rv3136) gene cluster, one of nine PPE-PPE clusters in the genome. We demonstrate here that this cluster is operonic, and that PPE50 and PPE51 interact - the first demonstration of PPE-PPE interaction. THP-1 macrophages infected with recombinant Mycobacterium smegmatis strains expressing PPE50 and PPE51 showed lower intracellular viability than the control, which correlated with an increase in transcript levels of iNOS2. Infected macrophages also exhibited an upregulation in levels of IL-10, indicating an immunomodulatory role for these proteins. Using pull-downs and signalling assays, we identified TLR1 to be the cognate receptor for PPE50 - all the phenotypes observed on infection of THP-1 macrophages were reversed on pre-treatment with an anti-TLR1 antibody, validating the functional outcome of PPE50-TLR1 interaction. Our data reveals a TLR1 dependent role for the PPE50-PPE51 cluster in promoting bacillary persistence, via CFU reduction and concomitant upregulation of the anti-inflammatory response - a two-pronged strategy to circumvent host immune surveillance.
Asunto(s)
Mycobacterium tuberculosis , Proteínas Bacterianas/metabolismo , Receptor Toll-Like 1/genética , Receptor Toll-Like 1/metabolismo , Mycobacterium smegmatis/genética , Familia de MultigenesRESUMEN
Autophagy has emerged as a critical innate immune mechanism for host elimination of intracellular pathogens, however, the role of the autophagy receptor Optineurin during mycobacterial infection is not fully understood. To address this lacuna, we infected bone marrow-derived macrophages (BMDMs) derived from Optn+/+ and Optn-/- mice with Mycobacterium smegmatis, and observed the infection outcome at sequential time points. While low multiplicity of infection (MOI) did not show any significant difference between BMDMs from the two groups, at high MOI Optn-/- mice-derived BMDMs showed significantly lower colony forming unit counts, as well as lower cell counts at 12 h and 24 h post-infection. Quantification of cell numbers and nuclear morphologies at various time points post-infection indicated a markedly higher cell death in the Optineurin-deficient macrophages. Optineurin-deficient BMDMs showed significantly lower levels of the autophagosomal protein LC3-II upon infection, indicating a potential role for Optineurin in regulating autophagy during mycobacterial infection. Moreover, when stimulated by bacterial LPS, Optineurin deficient macrophages, showed altered levels of the inflammatory cytokine pro-IL-1ß. These observations taken together suggest a novel regulatory role for Optineurin during mycobacterial infection. Its deficiency leads to an impairment in macrophage responses, directly impacting the pathophysiology of infection.
RESUMEN
Mycobacterium tuberculosis (M. tb) has a complex cell wall, composed largely of mycolic acids, that are crucial to its structural maintenance. The M. tb desaturase A1 (DesA1) is an essential Ca2+-binding protein that catalyses a key step in mycolic acid biosynthesis. To investigate the structural and functional significance of Ca2+ binding, we introduced mutations at key residues in its Ca2+-binding ßγ-crystallin motif to generate DesA1F303A, E304Q, and F303A-E304Q. Complementation of a conditional ΔdesA1 strain of Mycobacterium smegmatis, with the Ca2+ non-binders F303A or F303A-E304Q, failed to rescue its growth phenotype; these complements also exhibited enhanced cell wall permeability. Our findings highlight the criticality of Ca2+ in DesA1 function, and its implicit role in the maintenance of mycobacterial cellular integrity.
Asunto(s)
Proteínas Bacterianas , Calcio , Pared Celular , Mycobacterium tuberculosis , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/genética , Calcio/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Pared Celular/metabolismo , Pared Celular/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/genética , Mutación , Unión Proteica , Ácidos Micólicos/metabolismoRESUMEN
The rabbit model of tuberculosis is attractive because of its pathophysiologic resemblance to the disease in humans. Rabbits are naturally resistant to infection but may manifest cavitary lung lesions. We describe here a novel approach that utilizes presensitization and bronchoscopic inoculation to reliably produce cavities in the rabbit model. With a fixed inoculum of bacilli, we were able to reproducibly generate cavities by using Mycobacterium bovis Ravenel, M. bovis AF2122, M. bovis BCG, M. tuberculosis H37Rv, M. tuberculosis CDC1551, and the M. tuberculosis CDC1551 DeltasigC mutant. M. bovis infections generated cavitary CFU counts of 10(6) to 10(9) bacilli, while non-M. bovis species and BCG yielded CFU counts that ranged from 10(4) to 10(8) bacilli. Extrapulmonary dissemination was almost exclusively noted among rabbits infected with M. bovis Ravenel and AF2122. Though all of the species yielded secondary lesions at intrapulmonary sites, M. bovis infections led to the most apparent gross pathology. Using the M. tuberculosis icl and dosR gene expression patterns as molecular sentinels, we demonstrated that both the cavity wall and cavity lumen are microenvironments associated with hypoxia, upregulation of the bacterial dormancy program, and the use of host lipids for bacterial catabolism. This unique cavitary model provides a reliable animal model to study cavity pathogenesis and extrapulmonary dissemination.
Asunto(s)
Mycobacterium bovis/fisiología , Mycobacterium tuberculosis/fisiología , Tuberculosis Pulmonar/microbiología , Tuberculosis Pulmonar/patología , Animales , Broncoscopía , Modelos Animales de Enfermedad , Regulación Bacteriana de la Expresión Génica/fisiología , Pulmón/patología , Mycobacterium tuberculosis/genética , Conejos , Reproducibilidad de los ResultadosRESUMEN
Mycobacteria are intracellular pathogens that can invade and survive within host macrophages. Mycobacterial infections remain a major cause of mortality and morbidity worldwide, with serious concerns of emergence of multi and extensively drug-resistant tuberculosis. While significant advances have been made in identifying mycobacterial virulence determinants, the detailed molecular mechanism of internalization of mycobacteria into host cells remains poorly understood. Although several studies have highlighted the crucial role of sphingolipids in mycobacterial growth, persistence and establishment of infection, the role of sphingolipids in the entry of mycobacteria into host cells is not known. In this work, we explored the role of host membrane sphingolipids in the entry of Mycobacterium smegmatis into J774A.1 macrophages. Our results show that metabolic depletion of sphingolipids in host macrophages results in a significant reduction in the entry of M. smegmatis. Importantly, the entry of Escherichia coli into host macrophages under similar conditions remained invariant, implying the specificity of the requirement of sphingolipids in mycobacterial entry. To the best of our knowledge, our results constitute the first report demonstrating the role of host macrophage sphingolipids in the entry of mycobacteria. Our results could help in the development of novel therapeutic strategies targeting sphingolipid-mediated entry of mycobacteria into host cells.
Asunto(s)
Mycobacterium smegmatis/fisiología , Esfingolípidos/metabolismo , Línea Celular , Membrana Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Fumonisinas/farmacología , Humanos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Microscopía Confocal , Esfingolípidos/químicaRESUMEN
FhaA is a forkhead-associated domain-containing protein, the depletion of which leads to accumulation of peptidoglycan (PG) precursors at the septum and poles in Mycobacterium smegmatis (M. smegmatis), by a mechanism undefined thus far. To elucidate its function, we constructed an fhaA (MSMEG_0035) knockout (ΔfhaA) strain in M. smegmatis and demonstrated that this gene is dispensable for in vitro growth. The mutant showed a short cell length phenotype due to a probable defect in cell elongation/cell wall synthesis, which was reversed by complementation with both M. smegmatis and Mycobacterium tuberculosis (M. tb) fhaA (Rv0020c), confirming their association with the observed phenotype. The identification of penicillin binding protein A (PbpA), a PG biosynthesis enzyme as an interacting partner for mycobacterial FhaA, provided a hint into the functioning of FhaA. A drastic reduction in the levels of ectopically expressed PbpA in the ΔfhaA mutant vs wild-type M. smegmatis suggested that FhaA interacts with and stabilises PbpA. In addition, the fhaA deletion mutant was sensitive to multiple classes of antibiotics pointing to a general permeability defect. Our findings uncover a role for FhaA in PG biosynthesis and suggest its involvement in the maintenance of mycobacterial cell envelope integrity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Ciclo Celular/metabolismo , División Celular , Mycobacterium smegmatis/fisiología , Mycobacterium tuberculosis/fisiología , Proteínas Bacterianas/genética , Proteínas de Ciclo Celular/genética , Pared Celular/metabolismo , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteínas de Unión a las Penicilinas/metabolismo , Peptidoglicano/metabolismo , Unión Proteica , Mapeo de Interacción de ProteínasRESUMEN
As a successful pathogen, Mycobacterium tuberculosis has effectively infected one-third of the world's population. Despite the existence of compound libraries developed by recent advances in combinatorial chemistry, few compounds have been screened against M. tuberculosis. The use of a regulable promoter to control the level of expression of a drug target in living organisms has been shown to be advantageous compared with targetless whole-cell-based or in vitro biochemical screening approaches towards antibiotic discovery. In this study, we demonstrate that the acetamidase promoter from Mycobacterium smegmatis responds in a dose-dependent manner to different concentrations of its inducer acetamide. Using this promoter to regulate expression of a zeocin resistance gene in M. smegmatis, we show that the test strain exhibits increased sensitivity to zeocin at a low concentration of acetamide compared with a fully resistant phenotype at high doses of the inducer. This model system has indicated the feasibility of using a regulable promoter in designing a whole-cell-based high throughput screen for specific inhibitors against potential drug targets of M. tuberculosis.
Asunto(s)
Amidohidrolasas/genética , Genes Bacterianos/efectos de los fármacos , Mycobacterium/genética , Regiones Promotoras Genéticas , Secuencia de Bases , Bleomicina/farmacología , Cartilla de ADN , Farmacorresistencia Microbiana , Mycobacterium/efectos de los fármacos , Mycobacterium/enzimologíaRESUMEN
Despite the known association of several genes to clinical Isoniazid (INH) resistance, its molecular basis remains unknown in ~16% of clinical isolates of Mycobacterium tuberculosis (M. tb). While screening a set of Mycobacterium smegmatis (M. smegmatis) transposon mutants with altered colony morphology for differential susceptibility to INH, we found six resistant mutants and mapped their transposon insertion sites. The disrupted genes in six INH resistant mutants were homologs of M. tb ctaE, rplY, tatA, csd and tatB with one insertion mapping to the promoter region of M. smegmatis ctaE. MIC measurements indicated a wide spectrum of INH resistance in these mutants, with complementation analyses of four selected mutants with the cognate M. smegmatis genes and their M. tb homologs confirming the association of the disrupted genes with INH resistance. Our discovery of novel genes associated with INH resistance could lead to the identification of novel INH resistance mechanisms and possibly new diagnostic modalities as well.
Asunto(s)
Antibacterianos/farmacología , ADN Bacteriano/genética , Farmacorresistencia Bacteriana/genética , Sitios Genéticos , Isoniazida/farmacología , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Elementos Transponibles de ADN , Genotipo , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/crecimiento & desarrollo , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/crecimiento & desarrollo , Fenotipo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The major virulence trait of Mycobacterium tuberculosis is its ability to enter a latent state in the face of robust host immunity. Clues to the molecular basis of latency can emerge from understanding the mechanism of cell division, beginning with identification of proteins involved in this process. Using complementation of Escherichia coli mutants, we functionally annotated M. tuberculosis and Mycobacterium smegmatis homologs of divisome proteins FtsW and AmiC. Our results demonstrate that E. coli can be used as a surrogate model to discover mycobacterial cell division genes, and should prove invaluable in delineating the mechanisms of this fundamental process in mycobacteria.
Asunto(s)
División Celular , Genes Bacterianos , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Escherichia coli/genética , Eliminación de Gen , Prueba de Complementación Genética/métodos , Genética Microbiana/métodos , Mycobacterium smegmatis/fisiología , Mycobacterium tuberculosis/fisiologíaRESUMEN
Mycobacteria are intracellular pathogens that can invade and survive within host macrophages, and are a major cause of mortality and morbidity worldwide. The molecular mechanism involved in the internalization of mycobacteria is poorly understood. In this work, we have explored the role of host membrane cholesterol in the entry of the avirulent surrogate mycobacterial strain Mycobacterium smegmatis into THP-1 macrophages. Our results show that depletion of host membrane cholesterol using methyl-ß-cyclodextrin results in a significant reduction in the entry of M. smegmatis into host cells. More importantly, we show that the inhibition in the ability of M. smegmatis to enter host macrophages could be reversed upon replenishment of membrane cholesterol. To the best of our knowledge, these results constitute the first report showing that membrane cholesterol replenishment can reverse the inhibition in the entry of mycobacteria into host cells. In addition, we demonstrate that cholesterol complexation using amphotericin B (without physical depletion) is sufficient to inhibit mycobacterial entry. Importantly, we observed a significant reduction in mycobacterial entry upon enrichment of host membrane cholesterol. Taken together, our results demonstrate, for the first time, that an optimum host plasma membrane cholesterol is necessary for the entry of mycobacteria. These results assume relevance in the context of developing novel therapeutic strategies targeting cholesterol-mediated mycobacterial host cell entry.
Asunto(s)
Colesterol/metabolismo , Mycobacterium smegmatis/fisiología , Anfotericina B/farmacología , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos/microbiología , Microscopía Confocal , Mycobacterium smegmatis/efectos de los fármacos , Fagocitosis/efectos de los fármacos , beta-Ciclodextrinas/farmacologíaRESUMEN
Cell envelope associated components of Mycobacterium tuberculosis (M.tb) have been implicated in stress response, immune modulation and in vivo survival of the pathogen. Although many such factors have been identified, there is a large disparity between the number of genes predicted to be involved in functions linked to the envelope and those described in the literature. To identify and characterise novel stress related factors associated with the mycobacterial cell envelope, we isolated colony morphotype mutants of Mycobacterium smegmatis (M. smegmatis), based on the hypothesis that mutants with unusual colony morphology may have defects in the biosynthesis of cell envelope components. On testing their susceptibility to stress conditions relevant to M.tb physiology, multiple mutants were found to be sensitive to Isoniazid, Diamide and H2O2, indicative of altered permeability due to changes in cell envelope composition. Two mutants showed defects in biofilm formation implying possible roles for the target genes in antibiotic tolerance and/or virulence. These assays identified novel stress associated roles for several mycobacterial genes including sahH, tatB and aceE. Complementation analysis of selected mutants with the M. smegmatis genes and their M.tb homologues showed phenotypic restoration, validating their link to the observed phenotypes. A mutant carrying an insertion in fhaA encoding a forkhead associated domain containing protein, showed reduced survival in THP-1 macrophages, providing in vivo validation to this screen. Taken together, these results suggest that the M.tb homologues of a majority of the identified genes may play significant roles in the pathogenesis of tuberculosis.
Asunto(s)
Genes Bacterianos/genética , Mutagénesis/genética , Mycobacterium smegmatis/genética , Estrés Fisiológico/genética , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Pared Celular/efectos de los fármacos , Pared Celular/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/genética , Peróxido de Hidrógeno/farmacología , Macrófagos/efectos de los fármacos , Mutación/genética , Mycobacterium smegmatis/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacosRESUMEN
The ability to metabolize aromatic beta-glucosides such as salicin and arbutin varies among members of the Enterobacteriaceae. The ability of Escherichia coli to degrade salicin and arbutin appears to be cryptic, subject to activation of the bgl genes, whereas many members of the Klebsiella genus can metabolize these sugars. We have examined the genetic basis for beta-glucoside utilization in Klebsiella aerogenes. The Klebsiella equivalents of bglG, bglB and bglR have been cloned using the genome sequence database of Klebsiella pneumoniae. Nucleotide sequencing shows that the K. aerogenes bgl genes show substantial similarities to the E. coli counterparts. The K. aerogenes bgl genes in multiple copies can also complement E. coli mutants deficient in bglG encoding the antiterminator and bglB encoding the phospho-beta-glucosidase, suggesting that they are functional homologues. The regulatory region bglR of K. aerogenes shows a high degree of similarity of the sequences involved in BglG-mediated regulation. Interestingly, the regions corresponding to the negative elements present in the E. coli regulatory region show substantial divergence in K. aerogenes. The possible evolutionary implications of the results are discussed.
Asunto(s)
Proteínas Bacterianas/genética , Enterobacter aerogenes/genética , Escherichia coli/genética , Glucósidos/metabolismo , Proteínas de Unión al ARN/genética , Secuencia de Bases , Secuencia Conservada , Enterobacter aerogenes/enzimología , Dosificación de Gen , Silenciador del Gen , Genes Bacterianos , Variación Genética , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/genética , Datos de Secuencia Molecular , Operón , Fenotipo , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/genética , beta-Glucosidasa/metabolismoRESUMEN
The pathogenesis of Mycobacterium tuberculosis involves the coordinate action of multiple bacillary components that modulate host immune responses to ensure its survival. One such group of factors is the multigenic PE_PPE protein family, several members of which have been implicated in host immune evasion. Here we investigate the function of the PE-PPE gene pair PE35 (Rv3872)-PPE68 (Rv3873), located in the region of difference 1, encoding a specialized mycobacterial secretion system that is deleted in all vaccine strains of Mycobacterium bovis BCG. We report that this gene pair is co-operonic in M. tuberculosis, and demonstrate that its gene products interact with each other. Stimulation of THP-1 macrophages with recombinant PE35 and PPE68, singly or in combination, led to a dose-dependent increase in levels of the anti-inflammatory cytokine interleukin (IL)-10 and the chemokine monocyte chemoattractant protein-1, and caused a reciprocal decrease in levels of the proinflammatory cytokine IL-12. PE35/PPE68-stimulated production of IL-10 and monocyte chemoattractant protein-1 was observed to be dependent on toll-like receptor 2, as receptor blockade caused a significant reduction in their levels. Pharmacological inhibition indicated that this induction involved activation of the mitogen-activated protein kinase signalling axis. In a transwell migration assay, culture supernatants from PE35/PPE68-treated THP-1 cells were observed to stimulate the migration of monocytes. Our findings suggest that the PE35-PPE68 gene pair plays an important immunomodulatory role in regulating the pathophysiology of M. tuberculosis. STRUCTURED DIGITAL ABSTRACT: TLR2 physically interacts with PPE68 by anti bait coimmunoprecipitation (View interaction) PE35 binds to PPE68 by pull down (View interaction) PE35 physically interacts with PPE68 by anti tag coimmunoprecipitation (View interaction) TLR2 physically interacts with PE35 by anti bait coimmunoprecipitation (View interaction) PPE68 and PE35 physically interact by dihydrofolate reductase reconstruction (View interaction).