Mycobacterium tuberculosis strains disrupted in mce3 and mce4 operons are attenuated in mice

Senaratne, Ryan H.; Sidders, Ben; Sequeira, Patrícia Carvalho de; Saunders, G. F. T.; Dunphy, Kathleen Y.; Marjanovic, Olivera; Reader, J. Rachel; Lima, Patricia; Chan, Stephen; Kendall, Sharon L.; McFadden, Johnjoe; Riley, Lee W.

doi:10.1099/jmm.0.47454-0

Cited by 97 publications

(81 citation statements)

References 16 publications

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“…Hybridization of the loop to the mce4A mRNA will create a temporary siRNAmRNA duplex, which induces the bacterial RNA Science Publications AJBB interference machinery () to cleave the target mRNA and release the fragments (Van Der Oost and Brouns, 2009;Wiedenheft et al, 2012). Because the mycobacterium utilizes the product of mce4A for survival on cholesterol for carbon and energy source (Xu et al, 2007;Senaratne et al, 2008;Miner et al, 2009;Rathor et al, 2013), degradation of the mce4A mRNA will lead to its reduced survival. This study tested the ability of the mce4A siRNA to attenuate its target mce4A mRNA in macrophages infected with recombinant E.coli-4A and M. smegmatis.…”

Section: Discussionmentioning

confidence: 99%

A Short Interfering Rna Molecular Beacon for the Attenuation of Mycobacterial Infection

George¹

2014

American Journal of Biochemistry and Biotechnology

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The ability of the pathogen Mycobacterium Tuberculosis (MTB) to invade and survive within macrophages of granulomas is attributed to the product of the Mammalian Cell Entry (MCE) operon whose gene, mce4A, encodes a cholesterol transporter that transports host lipids into the bacterium that allows the bacterium to survive during chronic infection. Here, we proposed and tested the hypothesis that a mce4A siRNA molecular beacon can be used to attenuate mycobacterial infection in macrophages. Mce4A gene was cloned and expressed in E. coli (E. coli-4A) and differentiated U937 cells were transduced with piLenti-siRNA-GFP phage expressing the mce4A siRNA for 24 h. This was followed by infection with either E. coli-4A or M. smegmatis for 3 h followed by incubation for 0, 3, 6, 24 and 48 h. The cells were lysed and the lysates were plated on LB agar plates containing ampicillin (100 µg mL −1 ) or on 7H11 media and incubated at 37°C overnight. Our results showed that the siRNA treatment attenuated E.coli-4A infection in macrophages at 3, 6, 24 and 48 h by 0, 77, 59.6 and 99.7%, respectively. Our results also showed that the siRNA treatment attenuated M. smegmatis infection in macrophages at 3, 6, 24 and 48 h. by 94.8, 70.3, 98.9 and 93.4%, respectively. In conclusion, a mce4A siRNA molecular beacon was successfully delivered and stably expressed in macrophages which attenuated E. coli expressing mce4A (E. coli-4A) and M. smegmatis infection in macrophages.

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Section: Discussionmentioning

confidence: 99%

A Short Interfering Rna Molecular Beacon for the Attenuation of Mycobacterial Infection

George¹

2014

American Journal of Biochemistry and Biotechnology

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“…It is noteworthy that most recently, a fifth Mce operon was identified in M. canettii strain STB-J of the early branching tubercle bacilli (35). Certain mce operons of M. tuberculosis have been associated with a role in pathogenicity (41,42); in particular, the Mce4 operon seems to be required for cholesterol uptake and enhanced persistence of M. tuberculosis (43,44).…”

Section: Mycobacterial Genomicsmentioning

confidence: 99%

Mycobacterial Pathogenomics and Evolution

et al. 2014

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Most mycobacterial species are harmless saprophytes, often found in aquatic environments. A few species seem to have evolved from this pool of environmental mycobacteria into major human pathogens, such as Mycobacterium tuberculosis, the agent of tuberculosis, Mycobacterium leprae, the leprosy bacillus, and Mycobacterium ulcerans, the agent of Buruli ulcer. While the pathogenicity of M. ulcerans relates to the acquisition of a large plasmid encoding a polyketide-derived toxin, the molecular mechanisms by which M. leprae or M. tuberculosis have evolved to cause disease are complex and involve the interaction between the pathogen and the host.Here we focus on M. tuberculosis and closely related mycobacteria and discuss insights gained from recent genomic and functional studies. Comparison of M. tuberculosis genome data with sequences from nontuberculous mycobacteria, such as Mycobacterium marinum or Mycobacterium kansasii, provides a perception of the more distant evolution of M. tuberculosis, while the recently accomplished genome sequences of multiple tubercle bacilli with smooth colony morphology, named Mycobacterium canettii, have allowed the ancestral gene pool of tubercle bacilli to be estimated. The resulting findings are instrumental for our understanding of the pathogenomic evolution of tuberculosis-causing mycobacteria. Comparison of virulent and attenuated members of the M. tuberculosis complex has further contributed to identification of a specific secretion pathway, named ESX or Type VII secretion. The molecular machines involved are key elements for mycobacterial pathogenicity, strongly influencing the ability of M. tuberculosis to cope with the immune defense mounted by the host.

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“…Previous studies suggest that latex beads coated with Mce1A, Mce3A, and Mce3E are internalized by nonphagocytic HeLa cells (18,19). Additional studies have demonstrated that the M. tuberculosis strains with mce operon disruption were attenuated in mice (20,21). Among the four mce operons, the mce3 operon is located in the region of difference 15 of the M. tuberculosis genome, which is of special interest because it is absent in the vaccine strain of Mycobacterium bovis bacillus Calmette-Guérin (BCG) (22).…”

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confidence: 99%

Mycobacterium tuberculosis Mce3E Suppresses Host Innate Immune Responses by Targeting ERK1/2 Signaling

Chai

Zhang

et al. 2015

The Journal of Immunology

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Crucial to the pathogenesis of the tuberculosis (TB)-causing pathogen Mycobacterium tuberculosis is its ability to subvert host immune defenses to promote its intracellular survival. The mammalian cell entry protein 3E (Mce3E), located in the region of difference 15 of the M. tuberculosis genome and absent in Mycobacterium bovis bacillus Calmette-Guérin, has an essential role in facilitating the internalization of mammalian cells by mycobacteria. However, relatively little is known about the role of Mce3E in modulation of host innate immune responses. In this study, we demonstrate that Mce3E inhibits the activation of the ERK1/2 signaling pathway, leading to the suppression of Tnf and Il6 expression, and the promotion of mycobacterial survival within macrophages. Mce3E interacts and colocalizes with ERK1/2 at the endoplasmic reticulum in a DEF motif (an ERK-docking motif)–dependent manner, relocates ERK1/2 from cytoplasm to the endoplasmic reticulum, and finally reduces the association of ERK1/2 with MEK1 and blocks the nuclear translocation of phospho-ERK1/2. A DEF motif mutant form of Mce3E (F294A) loses its ability to suppress Tnf and Il6 expression and to promote intracellular survival of mycobacteria. Inhibition of the ERK1/2 pathway in macrophages using U0126, a specific inhibitor of the ERK pathway, also leads to the suppressed Tnf and Il6 expression and the enhanced intracellular survival of mycobacteria. Taken together, these results suggest that M. tuberculosis Mce3E exploits the ERK1/2 signaling pathway to suppress host innate immune responses, providing a potential Mce3E–ERK1/2 interface–based drug target against M. tuberculosis.

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Mycobacterium tuberculosis strains disrupted in mce3 and mce4 operons are attenuated in mice

Cited by 97 publications

References 16 publications

A Short Interfering Rna Molecular Beacon for the Attenuation of Mycobacterial Infection

A Short Interfering Rna Molecular Beacon for the Attenuation of Mycobacterial Infection

Mycobacterial Pathogenomics and Evolution

Mycobacterium tuberculosis Mce3E Suppresses Host Innate Immune Responses by Targeting ERK1/2 Signaling

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