Molecular Regulatory Mechanisms Drive Emergent Pathogenetic Properties of Neisseria gonorrhoeae.

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection (STI) gonorrhea, with an estimated 87 million annual cases worldwide. N. gonorrhoeae predominantly colonizes the male and female genital tract (FGT). In the FGT, N. gonorrhoeae confronts fluctuating levels of nutrients and oxidative and non-oxidative antimicrobial defenses of the immune system, as well as the resident microbiome. One mechanism utilized by N. gonorrhoeae to adapt to this dynamic FGT niche is to modulate gene expression primarily through DNA-binding transcriptional regulators. Here, we describe the major N. gonorrhoeae transcriptional regulators, genes under their control, and how these regulatory processes lead to pathogenic properties of N. gonorrhoeae during natural infection. We also discuss the current knowledge of the structure, function, and diversity of the FGT microbiome and its influence on gonococcal survival and transcriptional responses orchestrated by its DNA-binding regulators. We conclude with recent multi-omics data and modeling tools and their application to FGT microbiome dynamics. Understanding the strategies utilized by N. gonorrhoeae to regulate gene expression and their impact on the emergent characteristics of this pathogen during infection has the potential to identify new effective strategies to both treat and prevent gonorrhea.

Microbial Lipid A Remodeling Controls Cross-Presentation Efficiency and CD8 T Cell Priming by Modulating Dendritic Cell Function.

The majority of Gram-negative bacteria elicit a potent immune response via recognition of lipid A expressed on the outer bacterial membrane by the host immune receptor Toll-like receptor 4 (TLR4). However, some Gram-negative bacteria evade detection by TLR4 or alter the outcome of TLR4 signaling by modification of lipid A species. Although the role of lipid A modifications on host innate immunity has been examined in some detail, it is currently unclear how lipid A remodeling influences host adaptive immunity. One prototypic Gram-negative bacterium that modifies its lipid A structure is Porphyromonas gingivalis, an anaerobic pathobiont that colonizes the human periodontium and induces chronic low-grade inflammation that is associated with periodontal disease as well as a number of systemic inflammatory disorders. P. gingivalis produces dephosphorylated and deacylated lipid A structures displaying altered activities at TLR4. Here, we explored the functional role of P. gingivalis lipid A modifications on TLR4-dependent innate and adaptive immune responses in mouse bone marrow-derived dendritic cells (BMDCs). We discovered that lipid A 4'-phosphate removal is required for P. gingivalis to evade BMDC-dependent proinflammatory cytokine responses and markedly limits the bacterium's capacity to induce beta interferon (IFN-ß) production. In addition, lipid A 4'-phosphatase activity prevents canonical bacterium-induced delay in antigen degradation, which leads to inefficient antigen cross-presentation and a failure to cross-prime CD8 T cells specific for a P. gingivalis-associated antigen. We propose that lipid A modifications produced by this bacterium alter host TLR4-dependent adaptive immunity to establish chronic infections associated with a number of systemic inflammatory disorders.

Global Network Analysis of Neisseria gonorrhoeae Identifies Coordination between Pathways, Processes, and Regulators Expressed during Human Infection.

Neisseria gonorrhoeae is a Gram-negative diplococcus that is responsible for the sexually transmitted infection gonorrhea, a high-morbidity disease in the United States and worldwide. Over the past several years, N. gonorrhoeae strains resistant to antibiotics used to treat this infection have begun to emerge across the globe. Thus, new treatment strategies are needed to combat this organism. Here, we utilized N. gonorrhoeae transcriptomic data sets, including those obtained from natural infection of the human genital tract, to infer the first global gene coexpression network of this pathogen. Interrogation of this network revealed genes central to the network that are likely critical for gonococcal growth, metabolism, and virulence, including genes encoding hypothetical proteins expressed during mucosal infection. In addition, network analysis revealed overlap in the response of N. gonorrhoeae to incubation with neutrophils and exposure to hydrogen peroxide stress in vitro Network analysis also identified new targets of the gonococcal global regulatory protein Fur, while examination of the network neighborhood of genes allowed us to assign additional putative categories to several proteins. Collectively, the characterization of the first gene coexpression network for N. gonorrhoeae described here has revealed new regulatory pathways and new categories for proteins and has shown how processes important to gonococcal infection in both men and women are linked. This information fills a critical gap in our understanding of virulence strategies of this obligate human pathogen and will aid in the development of new treatment strategies for gonorrhea.IMPORTANCE Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection (STI) gonorrhea, a disease with high morbidity worldwide with an estimated 87 million cases annually. Current therapeutic and pharmacologic approaches to treat gonorrhea have been compromised by increased antibiotic resistance worldwide, including to the most recent FDA-approved antibiotic. New treatment strategies are urgently needed to combat this organism. In this study, we used network analysis to interrogate and define the coordination of pathways and processes in N. gonorrhoeae An analysis of the gonococcal network was also used to assign categories to genes and to expand our understanding of regulatory strategies. Network analysis provides important insights into pathogenic mechanisms of this organism that will guide the design of new strategies for disease treatment.

Comparison of Prevalence and Types of Mutations in Lung Cancers Among Black and White Populations.

IMPORTANCE: Lung cancer is the leading cause of cancer death in the United States in all ethnic and racial groups. The overall death rate from lung cancer is higher in black patients than in white patients. OBJECTIVE: To compare the prevalence and types of somatic alterations between lung cancers from black patients and white patients. Differences in mutational frequencies could illuminate differences in prognosis and lead to the reduction of outcome disparities by more precisely targeting patients' treatment. DESIGN, SETTING, AND PARTICIPANTS: Tumor specimens were collected from Baptist Cancer Center (Memphis, Tennessee) over the course of 9 years (January 2004-December 2012). Genomic analysis by massively parallel sequencing of 504 cancer genes was performed at Dana-Farber Cancer Institute (Boston, Massachusetts). Overall, 509 lung cancer tumors specimens (319 adenocarcinomas; 142 squamous cell carcinomas) were profiled from 245 black patients and 264 white patients. MAIN OUTCOMES AND MEASURES: The frequencies of genomic alterations were compared between tumors from black and white populations. RESULTS: Overall, 509 lung cancers were collected and analyzed (273 women [129 black patients; 144 white patients] and 236 men [116 black patients; 120 white patients]). Using 313 adenocarcinomas and 138 squamous cell carcinomas with genetically supported ancestry, overall mutational frequencies and copy number changes were not significantly different between black and white populations in either tumor type after correcting for multiple hypothesis testing. Furthermore, specific activating alterations in members of the receptor tyrosine kinase/Ras/Raf pathway including EGFR and KRAS were not significantly different between populations in lung adenocarcinoma. CONCLUSIONS AND RELEVANCE: These results demonstrate that lung cancers from black patients are similar to cancers from white patients with respect to clinically actionable genomic alterations and suggest that clinical trials of targeted therapies could significantly benefit patients in both groups.

Integration of gene mutations in risk prognostication for patients receiving first-line immunochemotherapy for follicular lymphoma: a retrospective analysis of a prospective clinical trial and validation in a population-based registry.

BACKGROUND: Follicular lymphoma is a clinically and genetically heterogeneous disease, but the prognostic value of somatic mutations has not been systematically assessed. We aimed to improve risk stratification of patients receiving first-line immunochemotherapy by integrating gene mutations into a prognostic model. METHODS: We did DNA deep sequencing to retrospectively analyse the mutation status of 74 genes in 151 follicular lymphoma biopsy specimens that were obtained from patients within 1 year before beginning immunochemotherapy consisting of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). These patients were recruited between May 4, 2000, and Oct 20, 2010, as part of a phase 3 trial (GLSG2000). Eligible patients had symptomatic, advanced stage follicular lymphoma and were previously untreated. The primary endpoints were failure-free survival (defined as less than a partial remission at the end of induction, relapse, progression, or death) and overall survival calculated from date of treatment initiation. Median follow-up was 7·7 years (IQR 5·5-9·3). Mutations and clinical factors were incorporated into a risk model for failure-free survival using multivariable L1-penalised Cox regression. We validated the risk model in an independent population-based cohort of 107 patients with symptomatic follicular lymphoma considered ineligible for curative irradiation. Pretreatment biopsies were taken between Feb 24, 2004, and Nov 24, 2009, within 1 year before beginning first-line immunochemotherapy consisting of rituximab, cyclophosphamide, vincristine, and prednisone (R-CVP). Median follow-up was 6·7 years (IQR 5·7-7·6). FINDINGS: We established a clinicogenetic risk model (termed m7-FLIPI) that included the mutation status of seven genes (EZH2, ARID1A, MEF2B, EP300, FOXO1, CREBBP, and CARD11), the Follicular Lymphoma International Prognostic Index (FLIPI), and Eastern Cooperative Oncology Group (ECOG) performance status. In the training cohort, m7-FLIPI defined a high-risk group (28%, 43/151) with 5-year failure-free survival of 38·29% (95% CI 25·31-57·95) versus 77·21% (95% CI 69·21-86·14) for the low-risk group (hazard ratio [HR] 4·14, 95% CI 2·47-6·93; p<0·0001; bootstrap-corrected HR 2·02), and outperformed a prognostic model of only gene mutations (HR 3·76, 95% CI 2·10-6·74; p<0·0001; bootstrap-corrected HR 1·57). The positive predictive value and negative predictive value for 5-year failure-free survival were 64% and 78%, respectively, with a C-index of 0·80 (95% CI 0·71-0·89). In the validation cohort, m7-FLIPI again defined a high-risk group (22%, 24/107) with 5-year failure-free survival of 25·00% (95% CI 12·50-49·99) versus 68·24% (58·84-79·15) in the low-risk group (HR 3·58, 95% CI 2·00-6·42; p<0.0001). The positive predictive value for 5-year failure-free survival was 72% and 68% for negative predictive value, with a C-index of 0·79 (95% CI 0·69-0·89). In the validation cohort, risk stratification by m7-FLIPI outperformed FLIPI alone (HR 2·18, 95% CI 1·21-3·92), and FLIPI combined with ECOG performance status (HR 2·03, 95% CI 1·12-3·67). INTERPRETATION: Integration of the mutational status of seven genes with clinical risk factors improves prognostication for patients with follicular lymphoma receiving first-line immunochemotherapy and is a promising approach to identify the subset at highest risk of treatment failure. FUNDING: Deutsche Krebshilfe, Terry Fox Research Institute.

Exome sequencing identifies BRAF mutations in papillary craniopharyngiomas.

Craniopharyngiomas are epithelial tumors that typically arise in the suprasellar region of the brain. Patients experience substantial clinical sequelae from both extension of the tumors and therapeutic interventions that damage the optic chiasm, the pituitary stalk and the hypothalamic area. Using whole-exome sequencing, we identified mutations in CTNNB1 (ß-catenin) in nearly all adamantinomatous craniopharyngiomas examined (11/12, 92%) and recurrent mutations in BRAF (resulting in p.Val600Glu) in all papillary craniopharyngiomas (3/3, 100%). Targeted genotyping revealed BRAF p.Val600Glu in 95% of papillary craniopharyngiomas (36 of 39 tumors) and mutation of CTNNB1 in 96% of adamantinomatous craniopharyngiomas (51 of 53 tumors). The CTNNB1 and BRAF mutations were clonal in each tumor subtype, and we detected no other recurrent mutations or genomic aberrations in either subtype. Adamantinomatous and papillary craniopharyngiomas harbor mutations that are mutually exclusive and clonal. These findings have important implications for the diagnosis and treatment of these neoplasms.

Genomic sequencing of meningiomas identifies oncogenic SMO and AKT1 mutations.

Meningiomas are the most common primary nervous system tumor. The tumor suppressor NF2 is disrupted in approximately half of all meningiomas, but the complete spectrum of genetic changes remains undefined. We performed whole-genome or whole-exome sequencing on 17 meningiomas and focused sequencing on an additional 48 tumors to identify and validate somatic genetic alterations. Most meningiomas had simple genomes, with fewer mutations, rearrangements and copy-number alterations than reported in other tumors in adults. However, several meningiomas harbored more complex patterns of copy-number changes and rearrangements, including one tumor with chromothripsis. We confirmed focal NF2 inactivation in 43% of tumors and found alterations in epigenetic modifiers in an additional 8% of tumors. A subset of meningiomas lacking NF2 alterations harbored recurrent oncogenic mutations in AKT1 (p.Glu17Lys) and SMO (p.Trp535Leu) and exhibited immunohistochemical evidence of activation of these pathways. These mutations were present in therapeutically challenging tumors of the skull base and higher grade. These results begin to define the spectrum of genetic alterations in meningiomas and identify potential therapeutic targets.