Understanding the regulation of cytochrome P450s: core enzymes involved in Mycobacteria metabolism

Introduction• Mycobacterium is a genus of genetically similar bacteria including various human & animal

pathogens: M. tuberculosis; the cause of tuberculosis (TB) in humans, M. bovis; the cause of TB incattle; and others such as M. avium; causing opportunistic infections in immunocompromised people.

• In 2014 alone, there were 9.6 million M. tuberculosis infections worldwide, leading to over 1.5 milliondeaths, the biggest cause of death by infectious disease worldwide1.

• M. bovis has been estimated to have cost the British government an estimated £500 million for thepast decade due to diagnostics, surveillance and culling of infected cattle. With this figure set to riseto over £1 billion in the next decade if improvements are not made in vaccine discovery/diagnostics2.

• Extensive research has been conducted on Mycobacterium to discover new drug targets & vaccinecandidates, but many have fallen short in their promise as new and effective drugs or vaccines.

• One particular area that shows promise is targeting cytochrome P450s (CYPs), enzymes involved incore metabolic pathways in some bacteria such as the Actinomycetales group.

• M. tuberculosis and M. bovis differ in their number of CYPs, with 20 & 17 respectively, whereas otherbacteria have none, such as E. coli and others such as Streptomyces avermitilis have 33 CYPs.

• Anti-fungal drugs such as azoles have been identified to inhibit Mycobacterial CYPs but their use aseffective anti-tuberculosis drugs have yet to be utilised3,4.

• CYPs have been identified as potential targets, but their regulation in Mycobacterium is widelyunknown, with 8 CYP genes within close proximity to TetR family of transcriptional regulators (TFTRs)genes, a group of transcriptional regulators in high abundance in Mycobacterium genomes, but thefunction of all TFTRs in M. tuberculosis is unknown5.

• A range of bioinformatic and molecular techniques is discussed in this poster and how they will beused to further understand the role of TFTRs in CYP regulation.

Methods

Ligation-Independent Cloning (LIC):

pNIC28-Bsa4 digestion

Plasmid prep. from E. coli DH5α

Identifying TFTRs by bioinformatics:M. tuberculosis H37Rv

Genome Sequence

IdentifyTFTRs

TetR Clones• BL21 (DE3) pLysS• Rosetta 2 (DE3) pLysS

Confirmation of expressionSDS-PAGE

Protein expression and further studies:

Deletion mutant generation

• TFTR mutants• CYP mutants

Electrophoretic mobility shift assays (EMSAs)• Shift in protein bands

Ligand binding• Activator of TFTR Purified TFTR

Determine Function

RNA-Seq• Deletion mutant• Up/down regulated TFTR

Identify TFTR homologues in: • M. bovis • M. smegmatis• And other Mycobacteria

BLAST

MEME& FIMO

software

Predicted binding motifs

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CTetR CYPIdentify TFTRs close to CYPs

pNIC28-Bsa47284 bp

Cut pNIC28-Bsa45359 bp

pNIC28-Bsa4:TetR~5965 bp

TetR gene

Digestion

PCR of TFTR

Predicted motif occurrences in genome: informs selection

of candidates>M. tuberculosis

>M. bovis

>M. smegmatis

Save the world

Conclusions• TFTRs are in high abundance in the M.

tuberculosis genome, with 11 within closeproximity to CYP genes (Figure 1).

• Three TFTRs (Rv0135c, Rv0775 andRv1255c) chosen based on bioinformaticanalyses and their close proximity & likelyregulation of CYP genes (Figure 2).

• CYP-associated TFTRs have homologuesin M. bovis, except Rv1255c in M. boviswhere a region of difference is present(Figure 2C).

• Predicted binding motifs of the threechosen CYP-associated TFTRs (Rv0135c,Rv0775 & Rv1255c) identified (Figure 3).

• All three TFTRs were successfully clonedinto pNIC28-Bsa4 vector using ligationindependent cloning (Figure 4). Withsequencing results showing 100%sequence identity & coverage compared toM. tuberculosis TFTR gene sequences.

• Expression of three CYP-associatedTFTRs confirmed in Rosetta 2 (DE3) pLysSor in BL21 (DE3) pLysS (Figure 5). Likelythat addition of numerous rare codons inRosetta 2 strain helps with expression.

• Currently, expression of CYP-associatedTFTR is being pursued and optimised forefficient expression of Rv0775, withpurification being conducted.

Ashley D. Otter1 and Sharon L. Kendall1

1 Royal Veterinary College, Dept. of Pathology and Pathogen Biology, Camden, London.

Corresponding author: Ashley D. Otter – aotter@rvc.ac.uk

Figure 1: BLAST Rings of Mycobacterium genomes with TFTRs annotated asred notches and CYPs as blue notches. Arrows represent those TFTRsassociated with a TFTR.

BLAST Ring comparison with annotated TFTRs and CYPs

E-value

8.7e-038

6.2e-033

4.8e-009

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Predicted motif from MEME

Figure 3: Predicted binding motifs of 3 chosen TFTRs. MEMEwas used to identify possible binding motifs with an E-value.

Predicted binding motifs of CYP related TFTRs

Figure 4: Agarose gel of PCR fragments from all 3 TFTR DH5α clones. + represents amplification of TFTRfrom M. tuberculosis gDNA using standard primers, - represents amplification of empty pNIC28-Bsa4vector (SacB gene) using pLIC primers.

PCR confirmation of cloning selected CYP related TFTRs into pNIC28-Bsa4

Rv0135c

A)

B)

Rv0775

Rv1255c

C)

Figure 2: BLAST homology comparisons (grey sections) for 12 kb regions nearchosen TFTRs. Red represents TFTRs and green represent CYPs.

M. tuberculosis

M. bovis

M. tuberculosis

M. bovis

M. tuberculosis

M. bovis

Gene organisation of selected CYP related TFTRs

Future work• Verification of binding motifs by performing electrophoretic mobility shift

assays (EMSAs).• Confirmation of expression by Western Blot using anti-his tag antibody.• Generation of TFTR deletion mutants in Mycobacterium species e.g. M.

tuberculosis and M. bovis. Other deletion work will potentially includedeletion of named CYPs associated with TFTRs.

• From deletion mutants: study expression pattern changes using GFP/RFPreporter gene constructs and RNA-Seq/Microarrays.

• Phenotypic analyses in deletion mutants including drug sensitivity, asrecent publications have shown that Rv1256c can be inhibited by anti-fungal Azole drugs, drugs already approved for use.

• From this work, it is hoped CYPs & TFTR regulators of M. tuberculosis willbe further understood and facilitate in the discovery of future drug targetsand to further understand mycobacterial metabolism.

References1) WorldHealthOrganization(2012).Globaltuberculosisreport2012.Geneva,Switzerland.

http://www.who.int/tb/publications/global_report/2) DepartmentforEnvironmentFoodandRuralAffairs(2014).TheStrategyforachievingOfficiallyBovineTuberculosisFreestatusfor

England.London,UK.www.defra.gov.uk3) Munro,aW.,McLean,K.J.,Marshall,K.R.,Warman,aJ.,Lewis,G.,Roitel,O.,Leys,D.(2003).CytochromesP450:noveldrugtargetsin

thewaragainstmultidrug-resistantMycobacteriumtuberculosis.BiochemicalSocietyTransactions,31:625–630.4) McLeanKJ,MarshallKR,RichmondA,HunterIS,FowlerK,Kieser T,Gurcha SS,Besra GS,MunroAW(2002).Azoleantifungalsarepotent

inhibitorsofcytochromeP450mono-oxygenases andbacterialgrowthinmycobacteriaandstreptomycetes.Microbiology.148:2937-49.5) Balhana,R.J.C.,Singla,A.,Sikder,M.H.,Withers,M.,&Kendall, S.L.(2015).GlobalanalysesofTetR familytranscriptional

regulatorsinmycobacteriaindicatesconservationacrossspeciesanddiversityinregulatedfunctions.BMCGenomics,16(1),479.

SDS-PAGE of CYP-associated TFTRsWCL Sol. Ins. WCL Sol. Ins.

Rosetta 2 (DE3)

25 kDA

BL21 (DE3)

Rv1

255cWCL Sol. Ins.WCL Sol. Ins.

BL21 (DE3) Rosetta 2 (DE3)

Rv0

775

25 kDA

WCL Sol. Ins. WCL Sol. Ins.

BL21 (DE3) Rosetta 2 (DE3)

Rv0

135c

Figure 5: SDS-Page of each CYP-associated TFTR. WCL – Whole cell lysate, Sol. – soluble fraction, Ins. – insoluble fraction.

ScanQRcodeforelectronicversion:

ortakeacopyfrombelow

25 kDA