Distinguishing the effects of Isoniazid and Rifampin on by murplelake77


									                                      Distinguishing the effects of Isoniazid and Rifampin on Mycobacterium tuberculosis: A
                                                         comparison of in vitro and in vivo proteomics
                                                                   Nicole A. Kruh1, JoLynn Troudt1, Angelo Izzo1, Jessica Prenni2  and Karen Dobos1
                                                             Department of Microbiology, Immunology and Pathology1; Proteomics and Metabolomics Facility2,
                                                                                      Colorado State University, Fort Collins, CO

                                      Abstract                                                                        In vitro Scaffold Results & Spectral Counting                                                                                  Multiple Reaction Monitoring (MRM)
For nearly half a century, a combinatorial chemotherapy approach has been taken for                                                                                                                                                 Initial proteomic data was collected as full-scan MS/MS spectra in a data-dependent
treating Mycobacterium tuberculosis (Mtb).  While the treatment of pan-sensitive strains is a          The program Scaffold was used to compile all database search results. This program allows us
well-established six-month regimen, the treatment of multi-drug resistant tuberculosis                 to view all of the proteins identified in each sample, as well as to make direct comparisons                                 manner, which gives information about the most abundant or most ionizable tryptic
(MDR-TB) is a challenge to both clinicians and researchers.   MDR-TB is defined as                     between each growth condition. Below is a screenshot of the Scaffold result file from the in vitro                           fragments in each sample. In order to get more quantitative information and to identify
resistance to the two first-line drugs isoniazid (INH) and rifampin (RIF), and is linked               experiment; highlighted are the statistically significant differentially expressed proteins found                            less prominent proteins (which may be obscured by more abundant ions), we will be
to prolonged drug treatment, increased chance of surgery and a high rate of
                                                                                                       upon exposure of in vitro grown Mtb to drug. This includes the acyl carrier protein, AcpM,                                   using MRM. This technique is highly specific, allowing us to monitor and collect data for
mortality.     In order to effectively develop new drugs, it is important to fully                                                                                                                                                  targeted peptides from the protein of interest.
understand the mechanism of action of the current drugs, the methods by which                          which has the best ANOVA score.
resistance develops, and the overall response within the bacterium.   The majority of
evidence related to changes in protein expression in response to drug treatment is
                                                                                                       Spectral counting is a method of label-free relative quantitation in complex samples, such as
                                                                                                       WCL. In general, if a protein is more abundant, spectra count, peptide count and sequence
                                                                                                                                                                                                                                                                                   MRM Design
correlated from microarray data.     While the fluctuation in mRNA levels is often                                                                                                                                                  Designing a MRM experiment is not trivial – one or more tryptic peptides per protein
indicative of protein expression, studies have shown that it is not always an accurate                 coverage will all increase; thus, this data is normalized to determine the quantities of protein
representation of the proteome.  For this reason, we have taken a proteomic approach                   relative to the population (1, 2).                                                                                           must be chosen for monitoring based on various rules. Shown below is an MRM design
to discerning changes within the bacteria upon drug exposure.  Utilizing Mtb H37Rv,                                                                                           Each lane represents the data from three
                                                                                                                                                                                                                                    for the AcpM protein. AcpM peptide and fragment ion pairs (transitions) were chosen
INH, RIF and the combination of INH/RIF, was introduced in GAS media and                                                                                                      technical replicates of one biological                from a combination of experimental and purified protein MS/MS data.
samples were grown as (biological) triplicates. Each sample was analyzed in triplicate                                                                                        replicate
on a linear ion trap mass spectrometer directly coupled with nanoscale reverse phase                                                                                                                                                For optimal results, MRM peptide candidates must abide by several rules:
chromatography (nLC-LTQ).  The raw data files were searched against the TB protein                                                                                                                                                           •  Size: 7-30 amino acids in length
                                                                                                                                  Total Proteins
sequence database using both the Sequest and Mascot database search engines. The                                                                                 ANOVA
resulting analysis files were compiled in Scaffold.     Initial quantitation by spectral
                                                                                                                                    Identified                    test                                                                       •  Ion charge of 2+
counting was performed to identify the dominant proteins expressed under each                                                                                                                                                                •  Peptides must be unmodified (ex: Met can be oxidized)
condition, as well as to distinguish the individual versus combined effects of each
drug.  Absolute quantitation on specific proteins of interest, including members of the
                                                                                                                                                                                                                                             •  Peptides which tend to digest incompletely should be avoided
FAS-II pathway using multiple reaction monitoring (MRM) analysis on a triple                                                                                                                                                                 •  Peptide must be unique to the protein of interest
quadrapole mass spectrometer is underway. In addition, an in vivo translation of the
experiment – involving H37Rv infected guinea pigs, is being completed to ensure that                                                                                                                                                Analysis of the MS/MS data revealed 3 AcpM peptides in the recombinant protein digest
the in vitro model correlates with the infection model and that the drug effect is                                                                                                                                                  and 5 peptides in the WCL digest. After applying these rules to the 5 peptides found in
                                                                                                                                                                                                                                    the AcpM analysis – two ideal peptides remained (Table below).

                         Experimental Design                                                                                                                                                                                           Protein   Peptide             Observed mass
                                                                                                                                                                                                                                                                                                  Actual mass
                                                                                                                                                                                                                                                                                                                      Charge   Ion 1               Ion 2

                                                                                                                                                                                                                                       AcpM      IPDEDLAGLR          549.38 – 550.32    549.85    1096.74 – 1098.63    2+      Y8      888.26      Y9      985.3
    H37Rv In vitro growth conditions                   H37Rv In vivo growth conditions                                                                                                                                                           TVGDVVAYIQK         597.81 – 598.44    598.13    1193.61 -1194.86     2+      Y9      992.32      Y6      721.28

                                                                                                                                                                                                                                       Above: Table summarizing the two AcpM
                                                                                                                          Venn diagram showing the unique and                                                                          peptides and their properties – including two
                  x3               x3                            x5                          x5                                                                                                                                        transition ions chosen for monitoring.
                                                                                                                          shared proteins in this analysis. Of the 47
     No Drug (control) Rifampin                       No Drug (control)          INH/RIF                                  shared proteins, AcpM represents one of
                       0.125 µg/mL                                                                                        the differentially expressed proteins.                                                                       To the right, the MS/MS spectra for the
                                                                                                                                                                                                                                       peptides: IPDEDLAGLR – from which
                                                                                                                                                                                                                                       transition ions (purple boxes) were identified
                                                                       x5                    x5
                    x3                x3                   Isoniazid             Rifampin                           Biological samples in triplicate
                                                                                                                                                                                 The FAS-II Pathway                                    (Y8 & Y9) and TVGDVVAYIQK (Y6 & Y9).
        Isoniazid        Isoniazid/
        0.5 µg/mL        Rifampin                                                                               AcpM                                                                                                                   Below: The preliminary MRM data for both
                                               Aerosol            Begin Drug                 Harvest                                               INH
                                                                                                                                                                                                                                       AcpM peptides #1 and #2, showing a resolved
                                               Infection          Treatments                  Lungs                                                INH/RIF                                                                             peak eluting at approximately 46.5 and 48
                                                                                                                                                   No drug                                                                             minutes respectively.
                 •  Centrifuge cells          Day 0                    Day14                  Day 28                                               RIF                                                         Rv0636
                 •  Delipidate cells
                 •  Sonicate to prepare WCL                       •  Homogenize lungs
                                                                  •  Collagenase/DNase treatment
                                                                                                                                                                                                                                                            Peptide # 1                  Peptide # 2
                                                                  •  Delipidate cells
                                                                                                                                                                                                                                                            Y9 Transition                Y9 Transition
                                                                  •  Sonicate to prepare WCL

                                                                                                           The chart above compares AcpM spectral counts
                                                                                                           in each biological replicate, clearly showing that                                                                                                                                                                  Above: Preliminary MRM data from
                                                                                                           upon treatment with RIF the amount of AcpM              During long chain fatty acid synthesis, AcpM serves as a                                                                                                    Rv0636 dehydrase protein – not seen
                         Sample preparation                                                                slighty increases. The increase is much more
                                                                                                           dramatic after exposure to INH. Unexpectedly,
                                                                                                                                                                   carrier protein. It presents the growing acyl chain to the
                                                                                                                                                                   various FAS-II proteins – allowing the acyl chain to undergo
                                                                                                                                                                                                                                                            Peptide # 1
                                                                                                                                                                                                                                                            Y8 Transition
                                                                                                                                                                                                                                                                                        Peptide # 2
                                                                                                                                                                                                                                                                                        Y6 Transition
                                                                                                                                                                                                                                                                                                                               during normal LTQ analysis. Peptide and
                                                                                                                                                                                                                                                                                                                               transitions were optimized on
                                                                                                                                                                                                                                                                                                                               recombinant protein (data not shown).
                                                                                                           after subjecting the bacteria to both drugs, as in      keto-reduction, dehydration, enoyl-reduction and finally a
                                                                                                           standard anti-mycobacterial chemotherapy, the           condersation which adds an additonal 2 carbons to the chain.
                                                      50 – 100 ng peptides/injection
                                                                                                           effect is synergistic – drastically increasing the      These acyl chains serve as the precursors to mycolic acids – a   All of the initial MS work was performed on an LTQ mass spectrometer, including global
                                                                                                           levels of AcpM.                                         vital component to the mycobacterial cell wall.
                             Digest with                   3 technical replicates                                                                                                                                                   analysis of in vitro WCL and in vivo lung homogenates. Traditionally MRM experiments are
           10 µg WCL           trypsin                                                                                                                                                                                              completed on a triple quadrupole mass spectrometer (QQQ), our preliminary MRM runs
                                                                                                                 AcpM, The FAS-II Pathway & In vivo Results                                                                         were executed on a Waters Xevo TQ. The use of MRM on either instrument will yield a
                                                                                                                                                                                                                                    higher sensitivity than the LTQ – allowing us to check for the presence of lower
                                                                                                       The acyl carrier protein, AcpM, is a key component in the fatty acid synthesis, type II                                      abundance proteins in all in vivo samples. Also, with the use of isotopically-labeled peptide
                                                       Reverse phase separation on a                   pathway (FAS-II)(shown above, right). Genes involved in the FAS-II pathway are found                                         standards, absolute quantitation can be achieved.
                                                        Zorbax 300SB-C18 column
                                                                                                       primarily in three operons: 1 - the kas operon, which is comprised of five genes, including
                                                          over a 90-minute linear
                                                           acetonitrile gradient
                                                                                                       acpM and two -ketoacyl-ACP-synthases, kasA and kasB (3); 2 - The mabA operon, which                                                                                                  Future
           Data-dependent acquisition on                                                               contains the the two genes mabA and inhA, which encode the oxoacyl-ACP- reductase and                                        Similar to microarray analysis, mass spectrometry allows us to compare different bacterial
                 Nanospray-LTQ                                                                         enoyl-ACP-reductase respectively (4); 3 - The last and most recently characterized operon                                    states – i.e. drug exposed vs untreated. In the future, we would like to use the MRM
                                                                                                       encodes the three dehydrase proteins, Rv0635-Rv0637 (5). There is extensive evidence                                         technique to quantitate various proteins in the FAS-II pathway, elucidating the changes
                                                                                                       implicating the inhibitory effects of INH on the FAS-II pathway. Previous microarray and                                     which occur on the protein level. Additionally, establishment of MRM protocols will
                                 Data Analysis                                                         real time PCR experiments have shown a similar upregulation of the kas operon in response                                    allow us to perform quantitation on virtually any given protein set. Unlike mRNA needed
                                                                                                       to treatment with INH (3). This observation gives validation to our experiment because we                                    for microarrays, protein is much more stable, making these types of experiments feasable.
                                   Data                                                                were able to reproduce this expected response at the protein level and also because the
                                  Mining        Mascot                                                 protein KasA is thought to be a target of INH. While we did not detect KasA in vitro, its
       Raw LTQ Data
                                                                                                       regulation should follow that of AcpM as they are integrated together in this operon.
       (MS/MS Spectra)                                                                                                                                                                                                              I would like to thank the entire Dobos lab, as well as the Tonge lab at Stony Brook University for providing purified
                                              SEQUEST                                                                                      In preliminary in vivo studies, 6 H37Rv infected guinea pig lung                         recombinant FAS-II proteins and expression plasmids.
                                                                                                                                           homogenates were analyzed. The AcpM protein was found to                                 This work was supported by NIH Contract DHH5266200400091c and Gates Foundation Contract # 42775.
                                                                               Statistical                                                 be upregulated in long term infection (90 days; data to the left).
                                                                               Analysis                                                    Variation in spectral counts is attributed to the complexity of                                                                               References
                X! Tandem                                                                                                                  the protein samples – containing both bacteria and host                                  (1) Liu H, Sadygov RG, Yates JR 3rd.; Anal Chem. 2004 Jul 15;76(14):4193-201.
                                                                                                                                           proteins. We hypothesize that in drug treated guinea pigs,                               (2) htp://proteomics.btk.fi/uploads/media/Talk_3__Introduction_Spectral_Counting_03.pd
                                                                                                                                           AcpM and other FAS-II pathway components will be                                         (3) Betts JC, McLaren A, Lennon MG, Kelly FM, Lukey PT, Blakemore SJ, Duncan K.; Antimicrob Agents
                                                                                                                                           upregulated in earlier infection time points (15 and 30 day) as                          Chemother. 2003 Sep;47(9):2903-13.
                                                                                                                                                                                                                                    (4) Marrakchi H, Ducasse S, Labesse G, Montrozier H, Margeat E, Emorine L, Charpentier X, Daffé M, Quémard A.;
                                                                                                        Spectral counts for 6 different 90 seen in the in vitro experiments. In order to quantitatively                             Microbiology. 2002 Apr;148 (Pt 4):951-60.
    Data was searched against a concatenated Mtb + IPI Mouse database containing                        day infected guinea pig lung
                               222,498 sequence entries
                                                                                                                                           compare these samples, we have chosen to use a directed,                                 (5) Sacco E, Covarrubias AS, O'Hare HM, Carroll P, Eynard N, Jones TA, Parish T, Daffé M, Bäckbro K, Quémard
                                                                                                                                           quantitative mass spectrometry technique – MRM.                                          A.; Proc Natl Acad Sci U S A. 2007 Sep 11;104(37):14628-33. Epub 2007 Sep 5.

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