Defining the mechanism of Yersinia entomophaga MH96 exoprotein release : a thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy

There is an increasing need in New Zealand for the development of new solutions and improvement of currently available biological control agents of pasture pests. In the last two decades, the entomopathogen Yersinia entomophaga was isolated, characterised, and defined as an economically viable agent for use in biopesticides against pasture pests, such as the New Zealand grass grub Costelytra giveni, the black beetle Hetreronychus arator, and other insects from the orders Coleoptera and Lepidoptera. Y. entomophaga produces an ABC toxin complex, called the Yen-TC, which is its main orally active virulence factor. Although previous studies have revealed how the toxin is affecting the host, the mechanism for Yen-TC production and cellular release have yet to be fully elucidated. Furthermore, the roles of other virulence factors of Y. entomophaga and their effects and mechanisms of cellular release are yet to be determined. In silico analysis of the draft genome sequence revealed several gene clusters encoding for virulence factors such as Rhs with predicted function in the type 6 secretion system, YenT as a possible heat-stable enterotoxin, and PirAB, a haemocoelic-active toxin. This study aimed to identify regulators of exoproteome release in Y. entomophaga MH96. The Yersinia entomophaga region of exoproteome release (YeRER) was identified by an exoproteome screening assay developed in this study. The YeRER is comprised of the transcriptional regulator RoeA, a regulatory ncRNA, and the Yersinia lysis cassette (YLC). Transcriptomics, mutagenesis, and trans complementation experiments were used to elucidates the importance of YeRER in Y. entomophaga protein release and changes in cell morphology. The global regulator, RoeA, strictly down-regulates the YLC expression which is involved in protein release and vesicle formation. Expression levels of the encoded secretion systems in MH96, T1SS, T3SS, T3SS2, and T6SS are not under the control of YeRER or quorum sensing (QS), which are involved in protein secretion. The T2SS expression is increased in roeA and QS mutants, which in turn showed reduced global exoproteome concentration. It is unlikely that the MH96 secretion systems are involved in MH96 exoproteome production in vitro. While underlying mechanisms have yet to be investigated, this study strongly suggests that exoproteins such as the Yen-TC are secreted by membrane vesicles which are induced by activation of a holin-endolysin complex

Identification of genes involved in exoprotein release using a high-throughput exoproteome screening assay in Yersinia entomophaga

Bacterial protein secretion is crucial to the maintenance of viability and pathogenicity. Although many bacterial secretion systems have been identified, the underlying mechanisms regulating their expression are less well explored. Yersinia entomophaga MH96, an entomopathogenic bacterium, releases an abundance of proteins including the Yen-Tc into the growth medium when cultured in Luria Bertani broth at ≤ 25˚C. Through the development of a high-throughput exoproteome screening assay (HESA), genes involved in MH96 exoprotein production were identified. Of 4,080 screened transposon mutants, 34 mutants exhibited a decreased exoprotein release, and one mutation located in the intergenic region of the Yen-Tc operon displayed an elevated exoprotein release relative to the wild-type strain MH96. DNA sequencing revealed several transposon insertions clustered in gene regions associated with lipopolysaccharide (LPSI and LPSII), and N-acyl-homoserine lactone synthesis (quorum sensing). Twelve transposon insertions were located within transcriptional regulators or intergenic regions. The HESA will have broad applicability for identifying genes associated with exoproteome production in a range of microorganisms

Identification of a BRCA2-Specific modifier locus at 6p24 related to breast cancer risk

Common genetic variants contribute to the observed variation in breast cancer risk for BRCA2 mutation carriers; those known to date have all been found through population-based genome-wide association studies (GWAS). To comprehensively identify breast cancer risk modifying loci for BRCA2 mutation carriers, we conducted a deep replication of an ongoing GWAS discovery study. Using the ranked P-values of the breast cancer associations with the imputed genotype of 1.4 M SNPs, 19,029 SNPs were selected and designed for inclusion on a custom Illumina array that included a total of 211,155 SNPs as part of a multi-consortial project. DNA samples from 3,881 breast cancer affected and 4,330 unaffected BRCA2 mutation carriers from 47 studies belonging to the Consortium of Investigators of Modifiers of BRCA1/2 were genotyped and available for analysis. We replicated previously reported breast cancer susceptibility alleles in these BRCA2 mutation carriers and for several regions (including FGFR2, MAP3K1, CDKN2A/B, and PTHLH) identified SNPs that have stronger evidence of association than those previously published. We also identified a novel susceptibility allele at 6p24 that was inversely associated with risk in BRCA2 mutation carriers (rs9348512; per allele HR = 0.85, 95% CI 0.80-0.90, P = 3.9×10−8). This SNP was not associated with breast cancer risk either in the general population or in BRCA1 mutation carriers. The locus lies within a region containing TFAP2A, which encodes a transcriptional activation protein that interacts with several tumor suppressor genes. This report identifies the first breast cancer risk locus specific to a BRCA2 mutation background. This comprehensive update of novel and previously reported breast cancer susceptibility loci contributes to the establishment of a panel of SNPs that modify breast cancer risk in BRCA2 mutation carriers. This panel may have clinical utility for women with BRCA2 mutations weighing options for medical prevention of breast cancer

Appendices to Defining the mechanism of Yersinia entomophaga MH96 exoprotein release (PhD thesis)

Table S3: Transcriptomics QS mutant KTMS23 compared to wild type strain Y. entomophaga MH96Table S6:Transcriptomics of roeA mutant KTMS12Table S8: Transcriptomics of fermenter of Y. entomophaga MH96 -Cluster 1.Table S9: Transcriptomics of fermenter of Y. entomophaga MH96 – Cluster 4Table S10: Transcriptomics of fermenter of Y. entomophaga MH96 – Cluster 5 and Cluster 7Table S11: Comparison of Down-regulated genes in KTMS12, KTMS23 with genes of Cluster 5 and 7 from the fermenter, derived from transcriptome dataTable S12: Transcriptomics of fermenter of Y. entomophaga MH96 – Cluster 2 Table S13: Transcriptomics of fermenter of Y. entomophaga MH96 – Cluster 3</div

Identification of a BRCA2-specific modifier locus at 6p24 related to breast cancer risk.

Common genetic variants contribute to the observed variation in breast cancer risk for BRCA2 mutation carriers; those known to date have all been found through population-based genome-wide association studies (GWAS). To comprehensively identify breast cancer risk modifying loci for BRCA2 mutation carriers, we conducted a deep replication of an ongoing GWAS discovery study. Using the ranked P-values of the breast cancer associations with the imputed genotype of 1.4 M SNPs, 19,029 SNPs were selected and designed for inclusion on a custom Illumina array that included a total of 211,155 SNPs as part of a multi-consortial project. DNA samples from 3,881 breast cancer affected and 4,330 unaffected BRCA2 mutation carriers from 47 studies belonging to the Consortium of Investigators of Modifiers of BRCA1/2 were genotyped and available for analysis. We replicated previously reported breast cancer susceptibility alleles in these BRCA2 mutation carriers and for several regions (including FGFR2, MAP3K1, CDKN2A/B, and PTHLH) identified SNPs that have stronger evidence of association than those previously published. We also identified a novel susceptibility allele at 6p24 that was inversely associated with risk in BRCA2 mutation carriers (rs9348512; per allele HR = 0.85, 95% CI 0.80–0.90, P = 3.961028). This SNP was not associated with breast cancer risk either in the general population or in BRCA1 mutation carriers. The locus lies within a region containing TFAP2A, which encodes a transcriptional activation protein that interacts with several tumor suppressor genes. This report identifies the first breast cancer risk locus specific to a BRCA2 mutation background. This comprehensive update of novel and previously reported breast cancer susceptibility loci contributes to the establishment of a panel of SNPs that modify breast cancer risk in BRCA2 mutation carriers. This panel may have clinical utility for women with BRCA2 mutations weighing options for medical prevention of breast cancer

Multiple independent variants at the TERT locus are associated with telomere length and risks of breast and ovarian cancer

TERT-locus single nucleotide polymorphisms (SNPs) and leucocyte telomere measures are reportedly associated with risks of multiple cancers. Using the iCOGs chip, we analysed ~480 TERT-locus SNPs in breast (n=103,991), ovarian (n=39,774) and BRCA1 mutation carrier (11,705) cancer cases and controls. 53,724 participants have leucocyte telomere measures. Most associations cluster into three independent peaks. Peak 1 SNP rs2736108 minor allele associates with longer telomeres (P=5.8×10−7), reduced estrogen receptor negative (ER-negative) (P=1.0×10−8) and BRCA1 mutation carrier (P=1.1×10−5) breast cancer risks, and altered promoter-assay signal. Peak 2 SNP rs7705526 minor allele associates with longer telomeres (P=2.3×10−14), increased low malignant potential ovarian cancer risk (P=1.3×10−15) and increased promoter activity. Peak 3 SNPs rs10069690 and rs2242652 minor alleles increase ER-negative (P=1.2×10−12) and BRCA1 mutation carrier (P=1.6×10−14) breast and invasive ovarian (P=1.3×10−11) cancer risks, but not via altered telomere length. The cancer-risk alleles of rs2242652 and rs10069690 respectively increase silencing and generate a truncated TERT splice-variant

Multiple independent variants at the TERT locus are associated with telomere length and risks of breast and ovarian cancer

<p>TERT-locus SNPs and leukocyte telomere measures are reportedly associated with risks of multiple cancers. Using the Illumina custom genotyping array iCOG, we analyzed similar to 480 SNPs at the TERT locus in breast (n = 103,991), ovarian (n = 39,774) and BRCA1 mutation carrier (n = 11,705) cancer cases and controls. Leukocyte telomere measurements were also available for 53,724 participants. Most associations cluster into three independent peaks. The minor allele at the peak 1 SNP rs2736108 associates with longer telomeres (P = 5.8 x 10(-7)), lower risks for estrogen receptor (ER)-negative (P = 1.0 x 10(-8)) and BRCA1 mutation carrier (P = 1.1 x 10(-5)) breast cancers and altered promoter assay signal. The minor allele at the peak 2 SNP rs7705526 associates with longer telomeres (P = 2.3 x 10(-14)), higher risk of low-malignant-potential ovarian cancer (P = 1.3 x 10(-15)) and greater promoter activity. The minor alleles at the peak 3 SNPs rs10069690 and rs2242652 increase ER-negative (P = 1.2 x 10(-12)) and BRCA1 mutation carrier (P = 1.6 x 10-14) breast and invasive ovarian (P = 1.3 x 10(-11)) cancer risks but not via altered telomere length. The cancer risk alleles of rs2242652 and rs10069690, respectively, increase silencing and generate a truncated TERT splice variant.</p>

Associations between SNPs in the region surrounding rs9348512 on chromosome 6 and breast cancer risk.

<p>Results based on imputed and observed genotypes. The blue spikes indicate the recombination rate at each position. Genotyped SNPs are represented by diamonds and imputed SNPs are represented by squares. Color saturation indicates the degree of correlation with the SNP rs9348512.</p