BIOL368/F14:Nicole Anguiano Week 11

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Finding a Journal Club Article/Microarray Dataset

  1. An Epigenetic Antimalarial Resistance Mechanism Involving Parasite Genes Linked to Nutrient Uptake. Sharma P, Wollenberg K, Sellers M, Zainabadi K, Galinsky K, Moss E, Nguitragool W, Neafsey D, Desai SA. , Europe PMC 23720749 (ArrayExpress)
  2. Identification of a subtelomeric gene family expressed during the asexual-sexual stage transition in Plasmodium falciparum. Eksi S, Haile Y, Furuya T, Ma L, Su X, Williamson KC. , Europe PMC 15996767 (ArrayExpress)
  3. The Mycobacterium tuberculosis sigma factor sigmaB is required for full response to cell envelope stress and hypoxia in vitro, but it is dispensable for in vivo growth. Fontán PA, Voskuil MI, Gomez M, Tan D, Pardini M, Manganelli R, Fattorini L, Schoolnik GK, Smith I. (ArrayExpress)
    • Third paper is approved. Kam D. Dahlquist 18:25, 5 November 2014 (EST).
    • Note that you have linked to the European version of PubMedCentral and that you need to go to the publisher site for the real full text version of the article.
  4. Phenylalanine-rich peptides potently bind ESAT6, a virulence determinant of Mycobacterium tuberculosis, and concurrently affect the pathogen's growth. Kumar K, Tharad M, Ganapathy S, Ram G, Narayan A, Khan JA, Pratap R, Ghosh A, Samuchiwal SK, Kumar S, Bhalla K, Gupta D, Natarajan K, Singh Y, Ranganathan A.(ArrayExpress)
  5. Nitrogen stress response and stringent response are coupled in Escherichia coli. Brown DR, Barton G, Pan Z, Buck M, Wigneshweraraj S. Nat Commun 5:4115 (2014), Europe PMC 24947454 (ArrayExpress)

Approved Article Information

Fontán P. A., Voskuil M. I., Gomez M., Tan D., Pardini M., Manganelli R., Fattorini L., Schoolnik G. K., Smith I. (2009 July 10). The Mycobacterium tuberculosis sigma factor sigmaB is required for full response to cell envelope stress and hypoxia in vitro, but it is dispensable for in vivo growth. J. Bacteriol, 191, 5628-5633. doi:10.1128/JB.00510-09

Preparation for Journal Club 3

  • Link to Article. For full citation, see "Approved Article Information" above.

Vocabulary

  1. Hypoxic: "Partial lack of oxygen; Pertaining to or suffering from hypoxia" (Biology Online)
  2. Concomitant: "Occurring during the same time period; usually refers to secondary symptoms that occur with a main symptom" (Medline Plus)
  3. Siderophores: "Relatively low molecular weight, ferric ion specific chelating agents elaborated by bacteria and fungi growing under low iron stress;" "the role of these compounds is to scavenge iron from the environment and to make the mineral, which is almost always essential, available to the microbial cell" (Neilands, J. B. (1995 Nov 10). Siderophores: Structure and Function of Microbial Iron Transport Compounds. J. Bacteriol, 270: 26723-26726. doi: 10.1074/jbc.270.45.26723)
  4. Cassette mutagenesis: "The production of mutants within a region (often bounded by unique restriction sites) by the use of synthetic oligonucleotides that fill the gap with mutants designed into the synthetic genetic material" (Biology Online)
  5. Specificity: "The ability of the immune response to interact with individual antigens." (Biology Online)
  6. Structural Gene: "A gene that encodes a protein with a chief function as part of a physical structure within a cell. This is in contrast to genes that encode proteins with a function that is enzymatic or regulatory." (Genetics Home Reference)
  7. Regulon: "group of operons that are transcriptionally co-regulated by the same regulatory machinery, consisting of trans regulators (transcription factors or simply TFs) and cis regulatory binding elements in the promoters of the operons they regulate;" "contains operons regulated by one same transcription factor" ([http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0029496 Zhang H., Yin Y., Olman V., Xu Y. (2012 Jan 03). Genomic Arrangement of Regulons in Bacterial Genomes. PLOS ONE. doi: 10.1371/journal.pone.0029496
  8. Multiplicity of infection (MOI): "a frequently used term in virology which refers to the number of virions that are added per cell during infection" (Virology Blog)
  9. Optical Density (OD): "In microbiology, the values can be used to measure turbidity, which in turn is used to estimate the number of bacteria (or concentration of molecules like proteins) in a solution" (Biology Online)
  10. Physiological: "Of, or pertaining to physiology or normal functioning of an organism." (Biology Online)

Outline

Introduction

  • A σ factor is a component of RNA polymerase that allows it to bind to a promoter.
    • Mycobacterium tuberculosis has 13 σ factors, meaning it can more easily adapt to stressful conditions.
  • σA, the primary σ factor, and σB have a similar amino acid sequence.
  • The structural gene of σB, sigB, is positively regulated by extracytoplasmic function (ECF) sigma factors σE, σH, and σL.
    • These three factors transcribe sigB at the same transcription start site.
    • RNA polymerase containing σF transcribe sigB at a different transcription start site, but overexpression of σF made no difference in sigB expression.
  • In Mycobacterium tuberculosis, σB regulates cell envelope response, σH regulates response to oxidative stress and heat shock.
    • σB is a component of both regulons.
    • σB is potentially is a key component of the stress response.
  • To study how much of the stress response of σE and σH is transmitted through σB, the σB regulon corresponding to the cell envelope and oxidative stress in vitro was analyzed.
    • There were two separate ways in which this was studied in a M. tuberculosis sigB mutant.
      1. The σB regulon activated by cell envelope and oxidative stress in vitro was analyzed in order to determine the extent to which the response is transmitted through σB.
      2. The growth of the mutant strain in THP-1 macrophage-like cells and in vivo in mouse and guinea pig models of infection was also evaluated.

Materials and Methods

M. tuberculosis infection, incubation, and isolation
  • The M. tuberculosis strain that was used as the sigB mutant had a kanamycin cassette inserted.
  • THP-1 monocytic cells were infected, and 105</sub> were differentiated with 40nM phorbol myristate acetate in 96-well plagtes at a MOI of 0.05 bacterium per macrophage.
    • Triplicate wells were treated with 0.05% sodium dodecyl sulfate (SDS). CFU was determined by plating serial dilutions, and colonies were counted after 2-3 weeks of incubation at 37°C.
  • BALB/c mice were infected intravenously with one of the three M. tuberculosis strains: the sigB mutant, the complemented mutant strain, or the wild-type parental strain.
    • Three mice were killed at determined times (not stated in the paper), and their lungs, spleens, and livers were harvested for analysis of bacterial survival. CFU was determined the same way as above.
  • Hartley strain guinea pigs between 250-300g were infected through the air.
    • Five were killed at days 1, 21, and 56 after infection, and the lungs were analyzed. CFU was determined the same way as above.
  • In vitro, DNA samples were cultivated in roller bottles until the early phase of exponential growth. Aliquots of 40mL were treated with 0.05% SDS or 5mM diamide.
  • To isolate the RNA for the in vitro cultures, bacterial cell pellets were centrifuged in a microcentrifuge tube containing 0.5mL ziconia beads, and disrupted by two 1-minute pulses in a BeadBeater.
    • RNA from the samples were purified using RNeasy columns, and kept at -80°C.
Microarray Analysis
  • The control and no treatment sigB mutants, as well as the control and no treatment wild type strains, were marked with Cy3 dye. The sigB mutants treated with 0.05% SDS or 5mM diamide, as well as the wild type strains treated with 0.05% SDS or 5 mM diamide, were marked with Cy5 dye.
  • The microarrays were scanned using a GenePix 4000A imaging device by Axon Instruments. The dye intensity was quantified using ScanAlyze.
  • Gene specific spots outside of those whose induction ratio was in the top or bottom 5% were used to normalize the intensities of the dye.
    • Large percentage fluctuations were eliminated by adjusting low intensity spots to a minimum noise value.
      • The noise value was determined by calculating the average intensity for the bottom 20% of the spots. Every value below the noise value was raised to it.
  • The ratios of red/green fluorescence were calculated from three biological replicates and two microarrays for each biological replicate.
  • To determined whether a gene was regulated, A false discovery rate of <2% (determined by the Significance Analysis of Microarray program) and a regulation of at least 1.8-fold were used.
  • There is no reference in the data to the log transformation, the normalization of log ratios for each slide, nor the normalization of log ratios across slides.
Hypoxic Treatment and Zone Diffusion
  • Cultures of the strains were isolated with methylene blue at 37°C with rotation for 4 days, at which point the blue color disappeared, indicating that the oxygen had been entirely used up.
    • Three tubes at each time point were opened to determined the CFU/ml at each culture.
  • The strains were grown to exponential phase, then 1ml was played, and paper disks containing 20μl of 10% SDS were placed on the agar.
    • Diameters of zones of inhibition were measured after 10 days of incubation at 37°C.

Results

  • The sigB mutant strain was created by inserting a kanamycin cassette in the sigB gene.
    • A complemented strain was created by placing the sigB gene back in the mutant DNA using a plasmid.
  • The survival of the sigB mutant and the wild type, as well as the sigB and the complemented strain, were compared under different conditions.
  • Figure 1 shows that the sigB was more sensitive to SDS than the complemented or wind-type strains.
    • The sigB mutant strain had an average colony diameter of over 3.5cm, whereas the wild type strain had an average colony diameter of slightly over 2.5cm, and the complemented strain had an average colony diameter of about 3cm.
  • Treatment of broth cultures of the three strains at high temperatures (45°C) for short periods (≥24 hours) caused a decrease in survivial in the sigB only in standing instead of rolling cultures.
    • It was hypothesized that decreased survival was due not to the heat, but to the lower amount of oxygen, so that hypothesis was tested.
      • Figure 2, which shows the effects of hypoxia treatment on the grown of the three tested strains, shows that this hypothesis was correct.
        • a) A in figure 2 shows the means and standard deviations for the three biological replicates represented as optical densities (ODs). The sigB mutant is shown as a square, the complemented strain is shown as a triangle, and the wild type is shown as a diamond. Though it is difficult to see the symbols, the square is on the bottom of the three at the third week, and the diamond is on the top.
        • b) B in figure 2 shows the means and standard deviations for the three biological replicates represented in CFU/ml. Again, he sigB mutant is shown as a square, the complemented strain is shown as a triangle, and the wild type is shown as a diamond. It is easily apparent in this second figure that the sigB mutant has much lower survival than the other two strains, with a 3 log order lower survival than the other two strains.
  • Due to σE (cell envelope response) and σH (hypoxia and heat shock response) have responses that converge in the activation of σB, it was hypothesized that analysis under the two stress conditions they correspond to may show what genes σB targets that are relevant for pathogenicity.
    • The wild type and sigB mutant strains were compared under those conditions.
  • Table 1 shows the distribution of functional categories of genes who are regulated by σB in the wild type strain when it is under cell envelope or oxidative stress. The vast majority are hypothetical proteins, but of those with known function, 20 are cell wall-associated genes in the case of cell envelope stress, and 12 are intermediate metabolism genes in the case of oxidative stress.
  • In another hypoxic treatment, only eight genes of the mutant strain were downregulated in comparison to the levels of regulation in the wild-type strain, but 28 genes were upregulated (particularly sigE and other genes in the σE regulon).
    • Expression of sigE in the mutant strain may be caused by the absence of σB allowing for σE or other transcription factors promoting sigE.
    • The absence of σB may also cause a stress that causes sigE to be promoted.
  • With the treatment of SDS, 72 genes were downregulated in the sigB mutant strain.
    • Overlap in σE and σB was expected, but they had only one gene, Rv0465c (a putative transcriptional regulator), in common.
    • Most genes downregulated related to the cell envelope stress response or transcriptional regulators.
      • In the wild type strain, ideR was upregulated with regulation dependent on σB.
      • ideR was downregulated in the mutant strain, which caused the upregulation of genes from the mbt cluster.
        • The mbt cluster encodes enzymes for the biosynthesis of siderophores and is usually repressed by ideR.
  • Under oxidative stress caused by diamide, analysis of the sigB mutant showed that there were 40 genes indicated to be part of σB regulation.
    • Overlap was expected between the σB and σH regulons, but only 2 genes encoding heat shock proteins, Rv0251c and Rv0384c, were seen to overlap.
      • Both of these genes were previously observed to be a part of σH regulation.
    • furA (Rv1909), a peroxidase, was observed to be under the control of σB.
      • Previous research by Mulder et al. showed an increase in expression of catalase and peroxidase mRNA when σB was overexpressed in Mycobacterium bovis.
      • The DNA microarray analysis indicates that σB regulates furA and katG (katG is in the same transcriptional unit of furA) in M. tuberculosis.
    • The regulation of ideR (Rv2711), PPE19 (Rv1361c), and hsp20 (Rv0251c) was confirmed by RT-PCR.
      • Figure 3 shows the expression of σB regulated genes in M. tuberculosis.
        • a) A shows the expression of three genes found to be regulated by σB in the DNA microarrays, confirmed by RT-PCR. The black bars indicate the wild type, and the white bars indicate the mutant strain. The bars indicate the expression after it has been normalized to the expression of sigA. The expression of Rv1361c (PPE19) in nonstressed broth cultures shows a significantly lower amount of expression in the mutant strain than the wild type. The expression of ideR in SDS-treated broth cultures also shows much less expression in the mutant than the wild type. The expression of Rv1361c (hsp20) in diamide-treated broth cultures shows less expression in the mutant strain than the wild type strain.
        • b) B shows the alignment of the sequences of the promoter regions of σB regulated genes, with the proposed -35 and -10 consensus regions at the bottom. The promoter sequences in the consensus region are almost identical among the genes.
      • σB's effect on ideR has been reported in previous studies that showed the effects of σB overproduction.
  • Figure 4 shows the growth of M. tuberculosis in mactophages, mice, and guinea pigs.
    • a) shows infection in the THP-1 macrophage-like cells. No difference can be seen between the mutant, complemented, and wild type strains.
    • b) shows infection in BALB/c mice infected intravenously. No difference can be seen between the mutant, complemented, and wild type strains.
    • c) shows infection in aerosol-infected guinea pigs. No difference can be seen between the mutant, complemented, and wild type strains.
    • As it was suspected that differences between the strains became apparent in low oxygen environments, they decided to grow the mutants and wild type in guinea pigs, who have a low-oxygen environment in their lungs.
      • Even in the guinea pigs, no differences were seen in the growth of the wild type or mutant strains (Fig. 4).

Discussion

  • Main Point: A sigB mutant is sensitive to SDS stress and hypoxic treatment in vivo, but the mutation does not affect survival in vitro.
  • σB is a central regulator in M. tuberculosis stress response since sigB, its structural gene, is positively regulated by three other sigma factors.
  • Using DNA microarrays, two sets of genes under σB regulation were found depending on whether the bacteria was exposed to cell wall or oxidative stress.
    • Other regulatory mechanisms are activated in response to a particular stress.
    • Rv1130, RV1131, and hsp20 are under σB, but are also regulated by σE, MprAB, and PhoP.
  • There was only a small overlap between the σB regulon and the regulons of σH (which responds to heat stress and oxidative stress) and σE (which responds to cell envelope stress).
    • This may be due to expression of sigB in sigH and sigE mutants tested in other experiments.
    • It could also be do to the differences in the kind of DNA microarray used (oligonucleotide array in this experiments and amplicon arrays in others).
  • Between a different report and the current, there was only a small overlap in the genes of the σB regulon, which is likely due to the different methodologies used.
    • In the other report, the wild type was compared to a mutant overexpressing σB, and not a sigB mutant.
    • Evaluation of the regulon under stress likely gives a better representation of the conditions in which the sigma factor activates.
  • The lack of σB in vivo could be due to redundancy of sigma factors and other regulators.
    • However, the differences between in vivo and in vitro may indicate a poor representation of virulence in infection by the animal models available.
    • Similar differences have been seen in other regulators (such as dosR), which seem indispensable in vitro, but not as important in vivo under certain conditions.
      • dosR seems required in some in vivo environments.

Presentation Link

Journal Club 3 Presentation

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Nicole Anguiano
BIOL 368, Fall 2014

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