Bacterial Methylation and Role in Understanding Disease

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1 Bacterial Methylation and Role in Understanding Disease Michael Jennings Pacific Biosciences, West Coast UGM, Stanford, December 3, 2015

2 Host adapted bacterial pathogens Neisseria meningitis, septicemia gonorrhea Haemophilus respiratory tract, otitis media Helicobacter pylori gastritis, cancer Streptococcus pneumoniae Pneumonia, meningitis, septicemia These bacteria express on their surface virulence factors that are essential for successful colonization of the host.

3 Surface expressed virulence factors. From: Rosenstein et al., NEJM (18):

5 Phase variation Phase variation is the reversible, high frequency switching of bacterial surface antigens.

6 Phase variation via simple tandem repeats P Repeats in coding sequence Homopolymers eg poly G or poly C Tetranucleotides eg GATC Heptanucletidies eg CAAACAA All changes are reversible

7 Phase variation via simple tandem repeats P Repeats in coding sequence Alter expression by frame shift mutations. All changes are reversible

8 Phase variation - a powerful system for generating phenotypic diversity 3 genes expressing different phase variable virulence factors Independent switching of these factors can give rise to 8 different phenotypes (2 3 =8) from a single strain. The genome of N. meningitidis strain MC58 is reported to have >50 phase variable genes - a potential repertoire of 5 x phenotypes (2 50 = 5 x ) from a single strain!

Avogadro and the meningococcus Considering both phase- (5 x 10 14

surfaces that can be made by a single organism = 5 x 10 21!

9 Avogadro and the meningococcus Considering both phase- (5 x ) and antigenic variation (pili only ~10 7 ), the repertoire of surfaces that can be made by a single organism = 5 x 10 21! As this is a conservative estimate, Avogadro s number is not far away..

11 Many species containing phase variable Type III restric5on modifica5on systems

12 Restriction-Modification Systems 2005, 8: Composed of pairs of opposing enzyme activities - Endonuclease (Res) - Methyltransferase (Mod)

13 Many phase variable type III restric5on modifica5on systems that have lost Res func5on

14 This situa5on has evolved independently at least 11 5mes selec5ve advantage?

15 Phase variable R-M systems Many genes involved in virulence are phase-variable. Methylation of DNA can control gene expression and is involved in phase variation mechanisms of specific genes. eg. Dam in E. coli. Could the phase-variable methyltransferase play a role in pathogenicity by mediating phase variation of multiple genes?

16 Phase variable H. influenzae type III mod gene

Nontypeable Haemophilus influenzae (NTHi) Causes upper and lower respiratory tract infections pneumonia, sinusitis, COPD exacerbations and otitis

17 Nontypeable Haemophilus influenzae (NTHi) Causes upper and lower respiratory tract infections pneumonia, sinusitis, COPD exacerbations and otitis media. Otits media - middle ear infection that occurs when bacteria colonising the nasopharynx enter the middle ear space via the Eustachian tube.

18 Nontypeable H. influenzae moda allele distribution Nature Communications : 7828

19 Microarray expression profiling moda2 strain 723 Repeats ON mod Random switching ~1/100 Repeats OFF mod X MOD expressed Methylation of target sites MOD not expressed No methylation of target sites Microarray 10 genes upregulated 26 genes upregulated Many genes required for anaerobic growth

20 Phase variation of a single gene vs the phase variable regulon - Phasevarion Repeats (-/+) Repeats (-/+) Mod PNAS :

21 Nontypeable H. influenzae moda2 in an OM model Nature Communications : 7828

Chinchillas challenged with 723 moda2 ON vs OFF Days: +2 +4 +7 +10 +14 +18 Epitympanic tap and nasopharyngeal lavage to determine the load of NTHI +22

22 Chinchillas challenged with 723 moda2 ON vs OFF Days: Epitympanic tap and nasopharyngeal lavage to determine the load of NTHI +22 Challenge chinchillas intranasally and transbullarly COHORTS: 1) NTHI 723 moda2 (ON) 2) NTHI 723 moda2 (OFF) Use histology to assess middle ear pathology

23 Nature Communications : 7828 Chinchillas challenged with 723 moda2 ON vs OFF DAY OFF 22ON Death ON OFF No Data

24 Chinchillas challenged with 723 moda2 ON vs OFF The observation that ModA2 OFF challenge switches to ON over time supports the hypothesis of niche adaptation in the middle ear.

25 Nontypeable H. influenzae moda allele distribution All other NTHi moda alleles tested display gene regulation and virulence phenotypes. Nature Communications : 7828

26 Vaccine-candidate Westerns using OMPs Nature Communications : 7828

27 Phasevarions based on Type III R-M methyltransferases are present in many host adapted Gram negative bacterial pathogens. Nature Microbiology Reviews :

28 Phasevarion mediated gene switching confirmed in 4 species Nature Microbiology Reviews :

29 Evidence for methylation?

30 Until recently only a few Mod target sites had been defined. This has limited studies on the mechanism of gene regulation at the promoter level.

31 Principle of detecting modified DNA bases during PacBio SMRT sequencing.

32 SMRT methylome analysis was performed by our collaborators at Pacific Biosciences

33 Methylome analysis - ModA12 Methyltransferase Specificity Modified Base Nm moda12 ON Nm moda12 KAN 5 -GATC-3 3 -CTAG-5 m6a 5 -GAAGG-3 3 -CTTCC-5 m6a 5 -ACACC-3 3 -TGTGG-5 m6a PACIFIC BIOSCIENCES CONFIDENTIAL 33

34 Nontypeable H. influenzae moda allele distribution Nature Communications : 7828

35 Haemophilus methylomes Nature Communications : 7828

36 Haemophilus methylomes Nature Communications : 7828

37 How do changes in methyla5on regulate gene expression?

38 Mapping methyla=on sites to promoter regions

39 What about Gram-positive bacterial pathogens?

40 Transparent and Streptococcus opaque colonies of strains TIGR4 pneumoniae (A) and L82016 (B) growing on TSA supplemented with cysteine. Gram-positive bacterial pathogen. Causes meningitis, sepsis, pneumonia and otitis media. Major morbidity and mortality worldwide.

41 Transparent and opaque colonies of strains TIGR4 (A) and L82016 (B) growing on TSA supplemented with cysteine. Streptococcus pneumoniae Briles D E et al. Infect. Immun. 2005;73:

42 Are DNA methylation changes driving this switching process in the pneumococcus?

43 Nature Communications : 5055

44 Principle of detecting modified DNA bases during PacBio SMRT sequencing.

45 Nature Communications : 5055

46 Type I RM system and regulation of gene expression Each of the 6 alternate methylation patterns results in a distinct gene expression profile (RNA seq). Some of these genes included well characterized virulence factors such as capsule.

47 Nature Communications : 5055

48 SpnIII system and a role in virulence? Mice were challenged with wild type D39 via intranasal (carriage model) or intravenous (invasive model) and relative fitness of SpnIII alleles was examined.

49 In vivo selection for switching in the mouse model NP Inoculum, black Day 1, grey Day 3, white Day 7, striped Nature Communications : 5055

50 In vivo selection for switching in the mouse model BLOOD Inoculum, black 4 hours, grey 30 hours, white Nature Communications : 5055

51 A phase variable regulon - Phasevarion Multiple genes switched in a coordinated fashion resulting in differentiation into two (on/off Type III) or 6 (6-way recombination Type I) distinct cell types via methylation changes. Distinct phenotypes in virulence model systems. Major implications for host/pathogen interactions and vaccine development. SMRT methylome analysis has facilitated rapid determination of target sites - confirming methylation and enabling promoter level studies.

Acknowledgements Ins$tute for Glycomics, Griffith University The University of Adelaide Freda Jen, John Atack, Kate Seib, Jayde Prof James Paton

Monash University (formerly GU) Yogitha Srikhanta Pacific Biosciences MaQhew Boitano Tyson Clark, Jonas Korlach Leicester University Marco Oggioni

52 Acknowledgements Ins$tute for Glycomics, Griffith University The University of Adelaide Freda Jen, John Atack, Kate Seib, Jayde Prof James Paton Gawthorne The Research Ins$tute at Na$onwide Children's Hospital Jennifer Edwards, Lauren Bakaletz and team The University of Iowa, Michael Apicella Monash University (formerly GU) Yogitha Srikhanta Pacific Biosciences MaQhew Boitano Tyson Clark, Jonas Korlach Leicester University Marco Oggioni Funding: Australian Research Council, Na5onal Health & Medical Research Council (Australia); NIH, Helen C LeviQ Visi5ng Professorship (University of Iowa).

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