Tick-borne relapsing fever (TBRF), characterized by recurring febrile episodes, is globally distributed and among the most common bacterial infections in some African countries. Despite the public health concern that this disease represents, little is known regarding the virulence determinants required by TBRF Borrelia during infection. Because the chromosomes of TBRF Borrelia show extensive colinearity with those of Lyme disease (LD) Borrelia , the exceptions represent unique genes encoding proteins that are potentially essential to the disparate enzootic cycles of these two groups of spirochetes. One such exception is a gene encoding an HtrA family protease, BtpA, that is present in TBRF Borrelia , but not in LD spirochetes. Previous work suggested that btpA orthologs may be important for resistance to stresses faced during mammalian infection. Herein, proteomic analyses of the TBRF spirochete, Borrelia turicatae , demonstrated that BtpA, as well as proteins encoded by adjacent genes in the B. turicatae genome, were produced in response to culture at mammalian body temperature, suggesting a role in mammalian infection. Further, transcriptional analyses revealed that btpA was expressed with the genes immediately upstream and downstream as part of an operon. To directly assess if btpA is involved in resistance to environmental stresses, btpA deletion mutants were generated. btpA mutants demonstrated no growth defect in response to heat shock, but were more sensitive to oxidative stress produced by t- butyl peroxide compared to wild-type B. turicatae . Finally, btpA mutants were fully infectious in a murine relapsing fever (RF) infection model. These results indicate that BtpA is either not required for mammalian infection, or that compensatory mechanisms exist in TBRF spirochetes to combat environmental stresses encountered during mammalian infection in the absence of BtpA.

Relapsing fever (RF), caused by spirochetes of the genus Borrelia, is a globally distributed, vector-borne disease with high prevalence in developing countries. To date, signaling pathways required for infection and virulence of RF Borrelia spirochetes are unknown. Cyclic di-AMP (c-di-AMP), synthesized by diadenylate cyclases (DACs), is a second messenger predominantly found in Gram-positive organisms that is linked to virulence and essential physiological processes. Although Borrelia is Gram-negative, it encodes one DAC (CdaA), and its importance remains undefined. To investigate the contribution of c-di-AMP signaling in the RF bacterium, Borrelia turicatae, a cdaA mutant was generated. The mutant was significantly attenuated during murine infection, and genetic complementation reversed this phenotype. Because c-di-AMP is essential for viability in many bacteria, whole genome sequencing was performed on cdaA mutants, and single nucleotide polymorphisms identified potential suppressor mutations. Additionally, conditional mutation of cdaA confirmed that CdaA is important for normal growth and physiology. Interestingly, mutation of cdaA did not affect expression of homologs of virulence regulators whose levels are impacted by c-di-AMP signaling in the Lyme disease bacterium, Borrelia burgdorferi. Finally, the cdaA mutant had a significant growth defect when grown with salts, at decreased osmolarity, and without pyruvate. While the salt treatment phenotype was not reversed by genetic complementation, possibly due to suppressor mutations, growth defects at decreased osmolarity and in media lacking pyruvate could be attributed directly to cdaA inactivation. Overall, these results indicate CdaA is critical for B. turicatae pathogenesis and link c-di-AMP to osmoregulation and central metabolism in RF spirochetes.

Background: Studies showed that folate and related single nucleotide polymorphisms (SNPs) could predict prostate cancer (PCa) risk. However, little is known about the interactions of folate-related SNPs associated with PCa aggressiveness. The study’s objective is to evaluate SNP–SNP interactions among the DHFR 19-bp polymorphism and 10 SNPs in folate metabolism and the one-carbon metabolism pathway associated with PCa aggressiveness. Methods: We evaluated 1294 PCa patients, including 690 European Americans (EAs) and 604 African Americans (AAs). Both individual SNP effects and pairwise SNP–SNP interactions were analyzed. Results: None of the 11 individual polymorphisms were significant for EAs and AAs. Three SNP–SNP interaction pairs can predict PCa aggressiveness with a medium to large effect size. For the EA PCa patients, the interaction between rs1801133 (MTHFR) and rs2236225 (MTHFD1), and rs1801131 (MTHFR) and rs7587117 (SLC4A5) were significantly associated with aggressive PCa. For the AA PCa patients, the interaction of DHFR-19bp polymorphism and rs4652 (LGALS3) was significantly associated with aggressive PCa. Conclusions: These SNP–SNP interactions in the folate metabolism-related genes have a larger impact than SNP individual effects on tumor aggressiveness for EA and AA PCa patients. These findings can provide valuable information for potential biological mechanisms of PCa aggressiveness.

Introduction: Prostate Cancer (PCa) disproportionately affects African Americans, as AA men are more commonly diagnosed with aggressive PCa and twice as likely to have mortality from the disease compared to European Americans (EA). The one- carbon metabolism pathway has been extensively studied for cancer development, yet little research has been reported on its association with aggressiveness of cancer at diagnosis. The purpose of our study is to determine whether there is difference by race for the impact of a DHFR 19bp polymorphism together with multiple single nucleotide polymorphisms (SNPs) in one-carbon metabolism genes on a cohort of men diagnosed with either low-aggressive or high-aggressive PCa. Methods: DNA samples from a population-based study of 1,498 PCa subjects in the North Carolina- Louisiana Prostate Cancer Project were analyzed. The DHFR 19bp polymorphism was targeted by utilizing two TaqMan TAMRA probes, one with a FAM fluorescent probe to identify the insertion allele and one with a VIC fluorescent probe to identify the deletion allele. Six SNPs were genotyped via TaqMan SNP genotyping assays by Applied Biosystems. Chi-Square analyses were performed to examine differences in genotype between race. A multivariable logistic regression model adjusted for confounders was utilized to estimate the association of the 19bp DHFR polymorphism and one-carbon metabolism SNPs with PCa aggressiveness. Results: A total of 152 participants were excluded due to insufficient DNA samples, resulting in a sample of 1,346 participants. The analysis consisted of 993 low-aggressive and 345 high aggressive PCa cases. Chi-square analyses revealed significant frequency differences between AAs and EAs for the 19bp DHFR deletion polymorphism (31.2% vs 17.6%), as well as MTHFR rs1801131, MTHFR rs1801133, MTR rs1805087, MTHFD1 rs2236225, and MTHFD2 rs7587117 (P values < 0.0001). After adjusting for confounders, the cohort was stratified based on DHFR polymorphism status. Among subjects with the 19bp DHFR double deletion, individuals with heterozygous MTR rs1805087 (AG) were significantly less likely to have aggressive PCa (OR = 0.27 [0.10, 0.74]) when compared to individuals with the GG genotype. Among the group that had at least one copy of wild-type DHFR, heterozygous individuals (GA) at MTHFD1 rs2236225 had a significantly lower chance of aggressive PCa diagnosis (OR = 0.60 [0.38, 0.95]) compared to individuals with the AA genotype. Conclusion: Substantial differences exist between AAs and EAs in regard to polymorphisms within the one-carbon metabolism pathway. There appears to be significant interaction among genes involved that can be attributable to the racial distribution of genetic polymorphisms. Studies examining the association between folate metabolism and PCa should consider the modulation effect of gene-gene interaction when analyzing the polymorphisms collectively. Citation Format: C. Tyler Ratliff, Lora J. Rogers, L. Joseph Su. Racial differences in the association of one-carbon metabolism polymorphisms and prostate cancer aggressiveness [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-161.