Genome-wide screen identifies host loci that modulate Mycobacterium tuberculosis fitness in immunodivergent mice.

Genetic differences among mammalian hosts and among strains of Mycobacterium tuberculosis (Mtb) are well-established determinants of tuberculosis (TB) patient outcomes. The advent of recombinant inbred mouse panels and next-generation transposon mutagenesis and sequencing approaches has enabled dissection of complex host-pathogen interactions. To identify host and pathogen genetic determinants of Mtb pathogenesis, we infected members of the highly diverse BXD family of strains with a comprehensive library of Mtb transposon mutants (TnSeq). Members of the BXD family segregate for Mtb-resistant C57BL/6J (B6 or B) and Mtb-susceptible DBA/2J (D2 or D) haplotypes. The survival of each bacterial mutant was quantified within each BXD host, and we identified those bacterial genes that were differentially required for Mtb fitness across BXD genotypes. Mutants that varied in survival among the host family of strains were leveraged as reporters of "endophenotypes," each bacterial fitness profile directly probing specific components of the infection microenvironment. We conducted quantitative trait loci (QTL) mapping of these bacterial fitness endophenotypes and identified 140 host-pathogen QTL (hpQTL). We located a QTL hotspot on chromosome 6 (75.97-88.58 Mb) associated with the genetic requirement of multiple Mtb genes: Rv0127 (mak), Rv0359 (rip2), Rv0955 (perM), and Rv3849 (espR). Together, this screen reinforces the utility of bacterial mutant libraries as precise reporters of the host immunological microenvironment during infection and highlights specific host-pathogen genetic interactions for further investigation. To enable downstream follow-up for both bacterial and mammalian genetic research communities, all bacterial fitness profiles have been deposited into GeneNetwork.org and added into the comprehensive collection of TnSeq libraries in MtbTnDB.

Genome-wide screen identifies host loci that modulate M. tuberculosis fitness in immunodivergent mice.

Genetic differences among mammalian hosts and Mycobacterium tuberculosis ( Mtb ) strains determine diverse tuberculosis (TB) patient outcomes. The advent of recombinant inbred mouse panels and next-generation transposon mutagenesis and sequencing approaches has enabled dissection of complex host- pathogen interactions. To identify host and pathogen genetic determinants of Mtb pathogenesis, we infected members of the BXD family of mouse strains with a comprehensive library of Mtb transposon mutants (TnSeq). Members of the BXD family segregate for Mtb -resistant C57BL/6J (B6 or B ) and Mtb -susceptible DBA/2J (D2 or D ) haplotypes. The survival of each bacterial mutant was quantified within each BXD host, and we identified those bacterial genes that were differentially required for Mtb fitness across BXD genotypes. Mutants that varied in survival among the host family of strains were leveraged as reporters for "endophenotypes", each bacterial fitness profile directly probing specific components of the infection microenvironment. We conducted QTL mapping of these bacterial fitness endophenotypes and identified 140 h ost- p athogen quantitative trait loci ( hp QTL). We identified a QTL hotspot on chromosome 6 (75.97-88.58 Mb) associated with the genetic requirement of multiple Mtb genes; Rv0127 ( mak ), Rv0359 ( rip2 ), Rv0955 ( perM ), and Rv3849 ( espR ). Together, this screen reinforces the utility of bacterial mutant libraries as precise reporters of the host immunological microenvironment during infection and highlights specific host-pathogen genetic interactions for further investigation. To enable downstream follow-up for both bacterial and mammalian genetic research communities, all bacterial fitness profiles have been deposited into GeneNetwork.org and added into the comprehensive collection of TnSeq libraries in MtbTnDB.

Differential Requirement for IRGM Proteins during Tuberculosis Infection in Mice.

Mycobacterium tuberculosis (Mtb) is a bacterium that exclusively resides in human hosts and remains a dominant cause of morbidity and mortality among infectious diseases worldwide. Host protection against Mtb infection is dependent on the function of immunity-related GTPase clade M (IRGM) proteins. Polymorphisms in human IRGM associate with altered susceptibility to mycobacterial disease, and human IRGM promotes the delivery of Mtb into degradative autolysosomes. Among the three murine IRGM orthologs, Irgm1 has been singled out as essential for host protection during Mtb infections in cultured macrophages and in vivo. However, whether the paralogous murine Irgm genes, Irgm2 and Irgm3, play roles in host defense against Mtb or exhibit functional relationships with Irgm1 during Mtb infection remains undetermined. Here, we report that Irgm1-/- mice are indeed acutely susceptible to aerosol infection with Mtb, yet the additional deletion of the paralogous Irgm3 gene restores protective immunity to Mtb infections in Irgm1-deficient animals. Mice lacking all three Irgm genes (panIrgm-/-) are characterized by shifted lung cytokine profiles at 5 and 24 weeks postinfection, but control disease until the very late stages of the infection, when panIrgm-/- mice display increased mortality compared to wild-type mice. Collectively, our data demonstrate that disruptions in the balance between Irgm isoforms is more detrimental to the Mtb-infected host than total loss of Irgm-mediated host defense, a concept that also needs to be considered in the context of human Mtb susceptibility linked to IRGM polymorphisms.

Host-pathogen genetic interactions underlie tuberculosis susceptibility in genetically diverse mice.

The outcome of an encounter with Mycobacterium tuberculosis (Mtb) depends on the pathogen's ability to adapt to the variable immune pressures exerted by the host. Understanding this interplay has proven difficult, largely because experimentally tractable animal models do not recapitulate the heterogeneity of tuberculosis disease. We leveraged the genetically diverse Collaborative Cross (CC) mouse panel in conjunction with a library of Mtb mutants to create a resource for associating bacterial genetic requirements with host genetics and immunity. We report that CC strains vary dramatically in their susceptibility to infection and produce qualitatively distinct immune states. Global analysis of Mtb transposon mutant fitness (TnSeq) across the CC panel revealed that many virulence pathways are only required in specific host microenvironments, identifying a large fraction of the pathogen's genome that has been maintained to ensure fitness in a diverse population. Both immunological and bacterial traits can be associated with genetic variants distributed across the mouse genome, making the CC a unique population for identifying specific host-pathogen genetic interactions that influence pathogenesis.

Identification of an IL-1-induced gene expression pattern in AR+ PCa cells that mimics the molecular phenotype of AR- PCa cells.

BACKGROUND: In immunosurveillance, bone-derived immune cells infiltrate the tumor and secrete inflammatory cytokines to destroy cancer cells. However, cancer cells have evolved mechanisms to usurp inflammatory cytokines to promote tumor progression. In particular, the inflammatory cytokine, interleukin-1 (IL-1), is elevated in prostate cancer (PCa) patient tissue and serum, and promotes PCa bone metastasis. IL-1 also represses androgen receptor (AR) accumulation and activity in PCa cells, yet the cells remain viable and tumorigenic; suggesting that IL-1 may also contribute to AR-targeted therapy resistance. Furthermore, IL-1 and AR protein levels negatively correlate in PCa tumor cells. Taken together, we hypothesize that IL-1 reprograms AR positive (AR+ ) PCa cells into AR negative (AR- ) PCa cells that co-opt IL-1 signaling to ensure AR-independent survival and tumor progression in the inflammatory tumor microenvironment. METHODS: LNCaP and PC3 PCa cells were treated with IL-1ß or HS-5 bone marrow stromal cell (BMSC) conditioned medium and analyzed by RNA sequencing and RT-QPCR. To verify genes identified by RNA sequencing, LNCaP, MDA-PCa-2b, PC3, and DU145 PCa cell lines were treated with the IL-1 family members, IL-1α or IL-1ß, or exposed to HS-5 BMSC in the presence or absence of Interleukin-1 Receptor Antagonist (IL-1RA). Treated cells were analyzed by western blot and/or RT-QPCR. RESULTS: Comparative analysis of sequencing data from the AR+ LNCaP PCa cell line versus the AR- PC3 PCa cell line reveals an IL-1-conferred gene suite in LNCaP cells that is constitutive in PC3 cells. Bioinformatics analysis of the IL-1 regulated gene suite revealed that inflammatory and immune response pathways are primarily elicited; likely facilitating PCa cell survival and tumorigenicity in an inflammatory tumor microenvironment. CONCLUSIONS: Our data supports that IL-1 reprograms AR+ PCa cells to mimic AR- PCa gene expression patterns that favor AR-targeted treatment resistance and cell survival.