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.

IL-1-conferred gene expression pattern in ERα+ BCa and AR+ PCa cells is intrinsic to ERα- BCa and AR- PCa cells and promotes cell survival.

BACKGROUND: Breast (BCa) and prostate (PCa) cancers are hormone receptor (HR)-driven cancers. Thus, BCa and PCa patients are given therapies that reduce hormone levels or directly block HR activity; but most patients eventually develop treatment resistance. We have previously reported that interleukin-1 (IL-1) inflammatory cytokine downregulates ERα and AR mRNA in HR-positive (HR+) BCa and PCa cell lines, yet the cells can remain viable. Additionally, we identified pro-survival proteins and processes upregulated by IL-1 in HR+ BCa and PCa cells, that are basally high in HR- BCa and PCa cells. Therefore, we hypothesize that IL-1 confers a conserved gene expression pattern in HR+ BCa and PCa cells that mimics conserved basal gene expression patterns in HR- BCa and PCa cells to promote HR-independent survival and tumorigenicity. METHODS: We performed RNA sequencing (RNA-seq) for HR+ BCa and PCa cell lines exposed to IL-1 and for untreated HR- BCa and PCa cell lines. We confirmed expression patterns of select genes by RT-qPCR and used siRNA and/or drug inhibition to silence select genes in the BCa and PCa cell lines. Finally, we performed Ingenuity Pathway Analysis (IPA) and used the gene ontology web-based tool, GOrilla, to identify signaling pathways encoded by our RNA-seq data set. RESULTS: We identified 350 genes in common between BCa and PCa cells that are induced or repressed by IL-1 in HR+ cells that are, respectively, basally high or low in HR- cells. Among these genes, we identified Sequestome-1 (SQSTM1/p62) and SRY (Sex-Determining Region Y)-Box 9 (SOX9) to be essential for survival of HR- BCa and PCa cell lines. Analysis of publicly available data indicates that p62 and SOX9 expression are elevated in HR-independent BCa and PCa sublines generated in vitro, suggesting that p62 and SOX9 have a role in acquired hormone receptor independence and treatment resistance. We also assessed HR- cell line viability in response to the p62-targeting drug, verteporfin, and found that verteporfin is cytotoxic for HR- cell lines. CONCLUSIONS: Our 350 gene set can be used to identify novel therapeutic targets and/or biomarkers conserved among acquired (e.g. due to inflammation) or intrinsic HR-independent BCa and PCa.

A Loss-of-Function Mutation in the Integrin Alpha L (Itgal) Gene Contributes to Susceptibility to Salmonella enterica Serovar Typhimurium Infection in Collaborative Cross Strain CC042.

Salmonella is an intracellular bacterium found in the gastrointestinal tract of mammalian, avian, and reptilian hosts. Mouse models have been extensively used to model in vivo distinct aspects of human Salmonella infections and have led to the identification of several host susceptibility genes. We have investigated the susceptibility of Collaborative Cross strains to intravenous infection with Salmonella enterica serovar Typhimurium as a model of human systemic invasive infection. In this model, strain CC042/GeniUnc (CC042) mice displayed extreme susceptibility with very high bacterial loads and mortality. CC042 mice showed lower spleen weights and decreased splenocyte numbers before and after infection, affecting mostly CD8+ T cells, B cells, and all myeloid cell populations, compared with control C57BL/6J mice. CC042 mice also had lower thymus weights with a reduced total number of thymocytes and double-negative and double-positive (CD4+, CD8+) thymocytes compared to C57BL/6J mice. Analysis of bone marrow-resident hematopoietic progenitors showed a strong bias against lymphoid-primed multipotent progenitors. An F2 cross between CC042 and C57BL/6N mice identified two loci on chromosome 7 (Stsl6 and Stsl7) associated with differences in bacterial loads. In the Stsl7 region, CC042 carried a loss-of-function variant, unique to this strain, in the integrin alpha L (Itgal) gene, the causative role of which was confirmed by a quantitative complementation test. Notably, Itgal loss of function increased the susceptibility to S. Typhimurium in a (C57BL/6J × CC042)F1 mouse background but not in a C57BL/6J mouse inbred background. These results further emphasize the utility of the Collaborative Cross to identify new host genetic variants controlling susceptibility to infections and improve our understanding of the function of the Itgal gene.

A balanced approach to falls prevention: Application in the real world.

BACKGROUND: Falls place a heavy burden on the health system, impacting on an individual's quality of life, often resulting in a fear of falling, reduction in independence and at times admission to residential care. This study aimed to determine health professionals' confidence in discussing falls prevention strategies, topics discussed and the barriers and enablers to falls prevention discussions with clients aged 60 years and over. METHOD: A cross-sectional self-complete online survey was undertaken with a sample of health professionals (n = 191) who had engaged in the services of the Stay On Your Feet® programs delivered by the Injury Matters in Western Australia (WA). RESULTS: The majority of participants were physiotherapists (25.7%), registered nurses (17.8%) and occupational therapists (11%) located in metropolitan (56%) and regional (44%) WA. Most health professionals (80.2%) were "highly" confident discussing falls prevention strategies. Discussion of falls prevention included the benefits of strength and balance exercises (83%), eating a healthy diet (78.7%), regular eyesight checks (64.5%), reviewing medications (54.8%) and exposure to sunlight (50.3%). The main enablers to falls prevention were knowledge (89.7%), skills to identify (77.7%) and implement (66.3%) falls prevention strategies, and access to printed resources (74.9%), while the main barrier was appointment times (14.6%). CONCLUSION: Health professionals' indicated that they are confident in discussing falls prevention strategies, and although a range of falls prevention strategies were discussed, limited attention was directed at the pharmacists' review of medications, eyesight checks and increasing vitamin D levels. SO WHAT?: Health professionals are discussing falls prevention strategies with their clients. A more multifaceted approach should be encouraged with an emphasis on all prevention strategies.

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.

Trends in antibiotic susceptibility of enteric fever isolates in East London.

BACKGROUND: The study sought evidence for changes in the proportions of antibiotic resistant strains among isolates of Salmonella enterica serovar Typhi (S. typhi) and Salmonella enterica serovar Paratyphi (S. paratyphi) between 2005 and 2012. METHODS: Blood culture isolates of S. typhi and S. paratyphi from patients attending Newham and The Royal London Hospitals were included in the study. The organisms were cultured on selective media and identified by Maldi-ToF, API 20E and serology. Minimum inhibitory concentrations (MICs) of augmentin, chloramphenicol, co-trimoxazole, ceftriaxone, ciprofloxacin and azithromycin were determined by E tests for 194 isolates. RESULTS: Median MICs of ciprofloxacin and ceftriaxone were stable at 0.5 mg/L and 0.125 mg/L, respectively. Chloramphenicol, azithromycin, co-trimoxazole and augmentin median MICs were 4 mg/L, 8 mg/L, 0.064 mg/L and 0.5 mg/L, respectively. MIC90 values were lower than the resistant breakpoint for ceftriaxone, azithromycin and augmentin, but were >256 mg/L for chloramphenicol, 32 mg/L for co-trimoxazole and 1 mg/L for ciprofloxacin. CONCLUSIONS: Antibiotic resistance remained stable for enteric fever isolates between 2005 and 2012. The isolates remained susceptible to augmentin, ceftriaxone and azithromycin over this period, but the MIC90 was greater than the resistant breakpoint for chloramphenicol, cotrimoxazole and ciprofloxacin. The implications for clinical practice are that isolates of S. typhi and S. paratyphi from East London remain sensitive to ceftriaxone and azithromycin.

East London experience with enteric fever 2007-2012.

PURPOSE: The clinical presentation and epidemiology for patients with enteric fever at two hospitals in East London during 2007-2012 is described with the aim to identify preventive opportunities and to reduce the cost of treatment. METHODS: A retrospective analysis of case notes from patients admitted with enteric fever during 2007 to 2012 with a microbiologically confirmed diagnosis was undertaken. Details on clinical presentation, travel history, demographic data, laboratory parameters, treatment, patient outcome and vaccination status were collected. RESULTS: Clinical case notes were available for 98/129 (76%) patients including 69 Salmonella enterica serovar Typhi (S. Typhi) and 29 Salmonella enterica serovar Paratyphi (S. Paratyphi). Thirty-four patients (35%) were discharged from emergency medicine without a diagnosis of enteric fever and then readmitted after positive blood cultures. Seventy-one of the 98 patients (72%) were UK residents who had travelled abroad, 23 (23%) were foreign visitors/new entrants to the UK and four (4%) had not travelled abroad. Enteric fever was not considered in the initial differential diagnosis for 48/98 (49%) cases. The median length of hospital stay was 7 days (range 0-57 days). The total cost of bed days for managing enteric fever was £454,000 in the two hospitals (mean £75,666/year). Median time to clinical resolution was five days (range 1-20). Seven of 98 (7%) patients were readmitted with relapsed or continued infection. Six of the 71 (8%) patients had received typhoid vaccination, 34 (48%) patients had not received vaccination, and for 31 cases (44%) vaccination status was unknown. CONCLUSIONS: Further interventions regarding education and vaccination of travellers and recognition of the condition by emergency medicine clinicians in travellers to South Asia is required.