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Fortune, Sarah

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Fortune

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Sarah

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Fortune, Sarah
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    IFN-γ-independent immune markers of Mycobacterium tuberculosis exposure
    (Springer Science and Business Media LLC, 2019-05-20) Lu, Lenette; Smith, Malisa; Yu, Krystle K. Q.; Luedemann, Corinne; Suscovich, Todd; Grace, Patricia; Cain, Adam; Yu, Wen-Han; McKitrick, Tanya; Lauffenburger, Douglas; Cummings, Richard; Mayanja-Kizza, Harriet; Hawn, Thomas R.; Boom, W. Henry; Stein, Catherine M.; Fortune, Sarah; Seshadri, Chetan; Alter, Galit
    Exposure to Mycobacterium tuberculosis (Mtb) results in heterogeneous clinical outcomes including primary progressive tuberculosis and latent Mtb infection (LTBI). Mtb infection is identified using the tuberculin skin test and interferon-γ (IFN-γ) release assay IGRA, and a positive result may prompt chemoprophylaxis to prevent progression to tuberculosis. In the present study, we report on a cohort of Ugandan individuals who were household contacts of patients with TB. These individuals were highly exposed to Mtb but tested negative by IFN-γ release assay and tuberculin skin test, ‘resisting’ development of classic LTBI. We show that ‘resisters’ possess IgM, class-switched IgG antibody responses and non-IFN-γ T cell responses to the Mtb-specific proteins ESAT6 and CFP10, immunologic evidence of exposure to Mtb. Compared to subjects with classic LTBI, ‘resisters’ display enhanced antibody avidity and distinct Mtb-specific IgG Fc profiles. These data reveal a distinctive adaptive immune profile among Mtb-exposed subjects, supporting an expanded definition of the host response to Mtb exposure, with implications for public health and the design of clinical trials.
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    The Importance of First Impressions: Early Events in Mycobacterium tuberculosis Infection Influence Outcome
    (American Society for Microbiology, 2016) Cadena, Anthony M.; Flynn, JoAnne L.; Fortune, Sarah
    ABSTRACT Tuberculosis remains a major health threat in much of the world. New vaccines against Mycobacterium tuberculosis are essential for preventing infection, disease, and transmission. However, the host immune responses that need to be induced by an effective vaccine remain unclear. Increasingly, it has become clear that early events in infection are of major importance in the eventual outcome of the infection. Studying such events in humans is challenging, as they occur within the lung and thoracic lymph nodes, and any clinical signs of early infection are relatively nonspecific. Nonetheless, clinical studies and animal models of tuberculosis have provided new insights into the local events that occur in the first few weeks of tuberculosis. Development of an effective vaccine requires a clear understanding of the successful (and detrimental) early host responses against M. tuberculosis, with the goal to improve upon natural immune responses and prevent infection or disease.
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    Small RNA profiling in Mycobacterium tuberculosis identifies MrsI as necessary for an anticipatory iron sparing response
    (National Academy of Sciences, 2018) Gerrick, Elias R.; Barbier, Thibault; Chase, Michael; Xu, Raylin; François, Josie; Lin, Vincent; Szucs, Matthew J.; Rock, Jeremy M.; Ahmad, Rushdy; Tjaden, Brian; Livny, Jonathan; Fortune, Sarah
    One key to the success of Mycobacterium tuberculosis as a pathogen is its ability to reside in the hostile environment of the human macrophage. Bacteria adapt to stress through a variety of mechanisms, including the use of small regulatory RNAs (sRNAs), which posttranscriptionally regulate bacterial gene expression. However, very little is currently known about mycobacterial sRNA-mediated riboregulation. To date, mycobacterial sRNA discovery has been performed primarily in log-phase growth, and no direct interaction between any mycobacterial sRNA and its targets has been validated. Here, we performed large-scale sRNA discovery and expression profiling in M. tuberculosis during exposure to five pathogenically relevant stresses. From these data, we identified a subset of sRNAs that are highly induced in multiple stress conditions. We focused on one of these sRNAs, ncRv11846, here renamed mycobacterial regulatory sRNA in iron (MrsI). We characterized the regulon of MrsI and showed in mycobacteria that it regulates one of its targets, bfrA, through a direct binding interaction. MrsI mediates an iron-sparing response that is required for optimal survival of M. tuberculosis under iron-limiting conditions. However, MrsI is induced by multiple host-like stressors, which appear to trigger MrsI as part of an anticipatory response to impending iron deprivation in the macrophage environment.
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    The Capacity of Mycobacterium tuberculosis To Survive Iron Starvation Might Enable It To Persist in Iron-Deprived Microenvironments of Human Granulomas
    (American Society for Microbiology, 2017) Kurthkoti, Krishna; Amin, Hamel; Marakalala, Mohlopheni; Ghanny, Saleena; Subbian, Selvakumar; Sakatos, Alexandra; Livny, Jonathan; Fortune, Sarah; Berney, Michael; Rodriguez, G. Marcela
    ABSTRACT This study was conducted to investigate the role of iron deprivation in the persistence of Mycobacterium tuberculosis. We present evidence of iron restriction in human necrotic granulomas and demonstrate that under iron starvation M. tuberculosis persists, refractive to antibiotics and capable of restarting replication when iron is made available. Transcriptomics and metabolomic analyses indicated that the persistence of M. tuberculosis under iron starvation is dependent on strict control of endogenous Fe utilization and is associated with upregulation of pathogenicity and intrinsic antibiotic resistance determinants. M. tuberculosis mutants compromised in their ability to survive Fe starvation were identified. The findings of this study advance the understanding of the physiological settings that may underpin the chronicity of human tuberculosis (TB) and are relevant to the design of effective antitubercular therapies.
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    Seq-Well: A Portable, Low-Cost Platform for High-Throughput Single-Cell RNA-Seq of Low-Input Samples
    (2017) Gierahn, Todd M.; Wadsworth, Marc H.; Hughes, Travis; Bryson, Bryan; Butler, Andrew; Satija, Rahul; Fortune, Sarah; Love, J. Christopher; Shalek, Alex K.
    Single-cell RNA-Seq can precisely resolve cellular states but application to sparse samples is challenging. Here, we present Seq-Well, a portable, low-cost platform for massively-parallel single-cell RNA-Seq. Barcoded mRNA capture beads and single cells are sealed in an array of subnanoliter wells using a semi-permeable membrane, enabling efficient cell lysis and transcript capture. We characterize Seq-Well using species-mixing experiments and PBMCs, and profile thousands of primary human macrophages exposed to tuberculosis.
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    Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform
    (2016) Rock, Jeremy; Hopkins, Forrest; Chavez, Alejandro; Diallo, Marieme; Chase, Michael; Gerrick, Elias; Pritchard, Justin R.; Church, George; Rubin, Eric; Sassetti, Christopher M.; Schnappinger, Dirk; Fortune, Sarah
    Development of new drug regimens that allow rapid, sterilizing treatment of tuberculosis has been limited by the complexity and time required for genetic manipulations in Mycobacterium tuberculosis. CRISPR interference (CRISPRi) promises to be a robust, easily engineered, and scalable platform for regulated gene silencing. However, in M. tuberculosis, the existing Streptococcus pyogenes Cas9-based CRISPRi system is of limited utility because of relatively poor knockdown efficiency and proteotoxicity. To address these limitations, we screened eleven diverse Cas9 orthologues and identified four that are broadly functional for targeted gene knockdown in mycobacteria. The most efficacious of these proteins, the CRISPR1 Cas9 from Streptococcus thermophilus (dCas9Sth1), typically achieves 20–100 fold knockdown of endogenous gene expression with minimal proteotoxicity. In contrast to other CRISPRi systems, dCas9Sth1-mediated gene knockdown is robust when targeted far from the transcriptional start site, thereby allowing high-resolution dissection of gene function in the context of bacterial operons. We demonstrate the utility of this system by addressing persistent controversies regarding drug synergies in the mycobacterial folate biosynthesis pathway. We anticipate that the dCas9Sth1 CRISPRi system will have broad utility for functional genomics, genetic interaction mapping, and drug-target profiling in M. tuberculosis.
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    The ESX System in Bacillus subtilis Mediates Protein Secretion
    (Public Library of Science, 2014) Huppert, Laura Ann; Ramsdell, Talia L.; Chase, Michael; Sarracino, David A.; Fortune, Sarah; Burton, Briana
    Esat-6 protein secretion systems (ESX or Ess) are required for the virulence of several human pathogens, most notably Mycobacterium tuberculosis and Staphylococcus aureus. These secretion systems are defined by a conserved FtsK/SpoIIIE family ATPase and one or more WXG100 family secreted substrates. Gene clusters coding for ESX systems have been identified amongst many organisms including the highly tractable model system, Bacillus subtilis. In this study, we demonstrate that the B. subtilis yuk/yue locus codes for a nonessential ESX secretion system. We develop a functional secretion assay to demonstrate that each of the locus gene products is specifically required for secretion of the WXG100 virulence factor homolog, YukE. We then employ an unbiased approach to search for additional secreted substrates. By quantitative profiling of culture supernatants, we find that YukE may be the sole substrate that depends on the FtsK/SpoIIIE family ATPase for secretion. We discuss potential functional implications for secretion of a unique substrate.
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    Sterilization of granulomas is common in both active and latent tuberculosis despite extensive within-host variability in bacterial killing
    (2013) Lin, Philana Ling; Ford, Christopher B.; Coleman, M. Teresa; Myers, Amy J.; Gawande, Richa; Ioerger, Thomas; Sacchettini, James; Fortune, Sarah; Flynn, JoAnne L.
    Over 30% of the world’s population is infected with Mycobacterium tuberculosis (Mtb), yet only ~5–10% will develop clinical disease1. Despite considerable effort, we understand little about what distinguishes individuals who progress to active tuberculosis (TB) from those who remain latent for decades. The variable course of disease is recapitulated in cynomolgus macaques infected with Mtb2. Active disease in macaques is defined by clinical, microbiologic and immunologic signs and occurs in ~45% of animals, while the remaining are clinically asymptomatic2,3. Here, we use barcoded Mtb isolates and quantitative measures of culturable and cumulative bacterial burden to show that most lesions are likely founded by a single bacterium and reach similar maximum burdens. Despite common origins, the fate of individual lesions varies substantially within the same host. Strikingly, in active disease, the host sterilizes some lesions even while others progress. Our data suggest that lesional heterogeneity arises, in part, through differential killing of bacteria after the onset of adaptive immunity. Thus, individual lesions follow diverse and overlapping trajectories, suggesting critical responses occur at a lesional level to ultimately determine the clinical outcome of infection. Defining the local factors that dictate outcome will be important in developing effective interventions to prevent active TB.
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    Mycobacterium tuberculosis mutation rate estimates from different lineages predict substantial differences in the emergence of drug resistant tuberculosis
    (2013) Ford, Christopher Burton; Shah, Rupal R.; Maeda, Midori Kato; Gagneux, Sebastien; Murray, Megan; Cohen, Ted; Johnston, James C.; Gardy, Jennifer; Lipsitch, Marc; Fortune, Sarah
    A critical question in tuberculosis control is why some strains of Mycobacterium tuberculosis are preferentially associated with multiple drug resistances. We demonstrate that M. tuberculosis strains from Lineage 2 (East Asian lineage and Beijing sublineage) acquire drug resistances in vitro more rapidly than M. tuberculosis strains from Lineage 4 (Euro-American lineage) and that this higher rate can be attributed to a higher mutation rate. Moreover, the in vitro mutation rate correlates well with the bacterial mutation rate in humans as determined by whole genome sequencing of clinical isolates. Finally, using a stochastic mathematical model, we demonstrate that the observed differences in mutation rate predict a substantially higher probability that patients infected with a drug susceptible Lineage 2 strain will harbor multidrug resistant bacteria at the time of diagnosis. These data suggest that interventions to prevent the emergence of drug resistant tuberculosis should target bacterial as well as treatment-related risk factors.
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    DNA Methylation Impacts Gene Expression and Ensures Hypoxic Survival of Mycobacterium tuberculosis
    (Public Library of Science, 2013) Shell, Scarlet S.; Prestwich, Erin G.; Baek, Seung-Hun; Shah, Rupal R.; Sassetti, Christopher M.; Dedon, Peter C.; Fortune, Sarah
    DNA methylation regulates gene expression in many organisms. In eukaryotes, DNA methylation is associated with gene repression, while it exerts both activating and repressive effects in the Proteobacteria through largely locus-specific mechanisms. Here, we identify a critical DNA methyltransferase in M. tuberculosis, which we term MamA. MamA creates N6-methyladenine in a six base pair recognition sequence present in approximately 2,000 copies on each strand of the genome. Loss of MamA reduces the expression of a number of genes. Each has a MamA site located at a conserved position relative to the sigma factor −10 binding site and transcriptional start site, suggesting that MamA modulates their expression through a shared, not locus-specific, mechanism. While strains lacking MamA grow normally in vitro, they are attenuated in hypoxic conditions, suggesting that methylation promotes survival in discrete host microenvironments. Interestingly, we demonstrate strikingly different patterns of DNA methyltransferase activity in different lineages of M. tuberculosis, which have been associated with preferences for distinct host environments and different disease courses in humans. Thus, MamA is the major functional adenine methyltransferase in M. tuberculosis strains of the Euro-American lineage while strains of the Beijing lineage harbor a point mutation that largely inactivates MamA but possess a second functional DNA methyltransferase. Our results indicate that MamA influences gene expression in M. tuberculosis and plays an important but strain-specific role in fitness during hypoxia.