Bacterial artificial chromosome fingerprint arrays for the differentiation of transcriptomic differences in mycobacteria

Li, Alice H.; Lam, Wan L.; Stokes, Richard W.

doi:10.1016/j.mimet.2008.07.008

Cited by 3 publications

(4 citation statements)

References 25 publications

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“…Previously, our laboratory reported the use of a bacterial artificial chromosome (BAC) based approach, BAC fingerprint array (BACFA), to analyze expression differences between the two highly related Mtb strains, H37Ra and H37Rv [27]. The data presented here are complementary to those obtained using BACFA analysis in that both data sets suggest a scenario where H37Rv is better equipped to adapt to intracellular environments.…”

Section: Discussionmentioning

confidence: 84%

“…Bacteria were recovered by centrifugation, before Trizol extraction. DNase treatment and purification of extracted RNA was performed as previously described [27].…”

Section: Methodsmentioning

confidence: 99%

“…For our specific purposes, we chose the bone-marrow-derived macrophage for their reproducibility and similarities to the alveolar macrophage [26], a presumed natural niche for Mtb upon infecting the host lung. Previously, our laboratory had used an alternative genome analytical method, bacterial artificial chromosome fingerprint arrays (BACFA), to examine intracellular transcriptomes of Mtb [27]. Using BACFA, we were able to identify an operon of genes ( frdABCD ) encoding fumarate reductase, which appeared to be important for intracellular survival of the tubercle bacterium.…”

Section: Introductionmentioning

confidence: 99%

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Contrasting Transcriptional Responses of a Virulent and an Attenuated Strain of Mycobacterium tuberculosis Infecting Macrophages

Waddell

Hinds

et al. 2010

PLoS ONE

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BackgroundH37Rv and H37Ra are well-described laboratory strains of Mycobacterium tuberculosis derived from the same parental strain, H37, that show dramatically different pathogenic phenotypes.Methodology/Principal FindingsIn this study, the transcriptomes of the two strains during axenic growth in broth and during intracellular growth within murine bone-marrow macrophages were compared by whole genome expression profiling. We identified and compared adaptations of either strain upon encountering an intracellular environment, and also contrasted the transcriptomes of the two strains while inside macrophages. In the former comparison, both strains induced genes that would facilitate intracellular survival including those involved in mycobactin synthesis and fatty acid metabolism. However, this response was stronger and more extensive for H37Rv than for H37Ra. This was manifested as the differential expression of a greater number of genes and an increased magnitude of expression for these genes in H37Rv. In comparing intracellular transcriptional signatures, fifty genes were found to be differentially expressed between the strains. Of these fifty, twelve were under control of the PhoPR regulon. Further differences between strains included genes whose products were members of the ESAT-6 family of proteins, or were associated with their secretion.Conclusions/SignificanceAlong with the recent identification of single nucleotide polymorphisms in H37Ra when compared to H37Rv, our demonstration of differential expression of PhoP-regulated and ESX-1 region-related genes during macrophage infection further highlights the significance of these genes in the attenuation of H37Ra.

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Section: Discussionmentioning

confidence: 84%

“…Bacteria were recovered by centrifugation, before Trizol extraction. DNase treatment and purification of extracted RNA was performed as previously described [27].…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Contrasting Transcriptional Responses of a Virulent and an Attenuated Strain of Mycobacterium tuberculosis Infecting Macrophages

Waddell

Hinds

et al. 2010

PLoS ONE

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“…These include in vivo - induced antigen technology, subtractive hybridization, in vivo expression technology etc. [3-5]. …”

Section: Introductionmentioning

confidence: 99%

Analysis of intracellular expressed proteins of Mycobacterium tuberculosis clinical isolates

et al. 2012

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BackgroundTuberculosis (TB) is the most threatening infectious disease globally. Although progress has been made to reduce global incidence of TB, emergence of multidrug resistant (MDR) TB threatens to undermine these advances. To combat the disease, novel intervention strategies effective against drug resistant and sensitive subpopulations of M. tuberculosis are urgently required as adducts in the present treatment regimen. Using THP-1 cells we have analyzed and compared the global protein expression profile of broth-cultured and intraphagosomally grown drug resistant and sensitive M.tuberculosis clinical isolates.ResultsOn comparing the two dimensional (2-DE) gels, many proteins were found to be upregulated/expressed during intracellular state which were identified by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). Four proteins (adenosylhomocysteinase, aspartate carbomyltransferase, putatitive thiosulfate sulfurtransferase and universal stress protein) were present in both intracellular MDR and sensitive isolates and three of these belonged to intermediary metabolism and respiration category. Two proteins (alanine dehydrogenase and adenosine kinase) of intracellular MDR isolate and two (glucose-6-phosphate isomerase and ATP synthase epsilon chain) of intracellular sensitive isolate belonged to intermediary metabolism and respiration category. One protein (Peroxidase/Catalase) of intracellular MDR and three (HSPX, 14 kDa antigen and 10 kDa chaperonin) of sensitive isolate belonged to virulence, detoxification and adaptation category. ESAT-6 of intracellular MDR belonged to cell wall and cell processes category. Two proteins (Antigen 85-C and Antigen 85-A) of intracellular sensitive isolate were involved in lipid metabolism while probable peptidyl-prolyl cis-trans isomerase A was involved in information pathways. Four (Rv0635, Rv1827, Rv0036c and Rv2032) of intracellular MDR and two proteins (Rv2896c and Rv2558c) of sensitive isolate were hypothetical proteins which were functionally characterized using bioinformatic tools. Bioinformatic findings revealed that the proteins encoded by Rv0036, Rv2032c, Rv0635, Rv1827 and Rv2896c genes are involved in cellular metabolism and help in intracellular survival.ConclusionsMass spectrometry and bioinformatic analysis of both MDR and sensitive isolates of M. tuberculosis during intraphagosomal growth showed that majority of commonly upregulated/expressed proteins belonged to the cellular metabolism and respiration category. Inhibitors of the metabolic enzymes/intermediate can therefore serve as suitable drug targets against drug-resistant and sensitive subpopulations of M. tuberculosis.

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Adjusting to a new home: Mycobacterium tuberculosis gene expression in response to an intracellular lifestyle

Stokes

Waddell

2009

Future Microbiol.

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Mycobacterium tuberculosis remains the most significant single species of bacteria causing disease in mankind. The ability of M. tuberculosis to survive and replicate within host macrophages is a pivotal step in its pathogenesis. Understanding the microenvironments that M. tuberculosis encounters within the macrophage and the adaptations that the bacterium undergoes to facilitate its survival will lead to insights into possible therapeutic targets for improved treatment of tuberculosis. This is urgently needed with the emergence of multi- and extensively drug resistant strains of M. tuberculosis. Significant advances have been made in understanding the macrophage response on encountering M. tuberculosis. Complementary information is also accumulating regarding the counter responses of M. tuberculosis during the various stages of its interactions with the host. As such, a picture is emerging delineating the gene expression of intracellular M. tuberculosis at different stages of the interaction with macrophages.

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Bacterial artificial chromosome fingerprint arrays for the differentiation of transcriptomic differences in mycobacteria

Cited by 3 publications

References 25 publications

Contrasting Transcriptional Responses of a Virulent and an Attenuated Strain of Mycobacterium tuberculosis Infecting Macrophages

Contrasting Transcriptional Responses of a Virulent and an Attenuated Strain of Mycobacterium tuberculosis Infecting Macrophages

Analysis of intracellular expressed proteins of Mycobacterium tuberculosis clinical isolates

Adjusting to a new home: Mycobacterium tuberculosis gene expression in response to an intracellular lifestyle

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