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High-resolution imaging of protein secretion at the single-cell level using plasmon-enhanced FluoroDOT assay

Seth, Anushree; Mittal, Ekansh; Luan, Jingyi; Kolla, Samhitha; Mazer, Monty B; Joshi, Hemant; Gupta, Rohit; Rathi, Priya; Wang, Zheyu; Morrissey, Jeremiah J; Ernst, Joel D; Portal-Celhay, Cynthia; Morley, Sharon Celeste; Philips, Jennifer A; Singamaneni, Srikanth
Secreted proteins mediate essential physiological processes. With conventional assays, it is challenging to map the spatial distribution of proteins secreted by single cells, to study cell-to-cell heterogeneity in secretion, or to detect proteins of low abundance or incipient secretion. Here, we introduce the "FluoroDOT assay," which uses an ultrabright nanoparticle plasmonic-fluor that enables high-resolution imaging of protein secretion. We find that plasmonic-fluors are 16,000-fold brighter, with nearly 30-fold higher signal-to-noise compared with conventional fluorescence labels. We demonstrate high-resolution imaging of different secreted cytokines in the single-plexed and spectrally multiplexed FluoroDOT assay that revealed cellular heterogeneity in secretion of multiple proteins simultaneously. Using diverse biochemical stimuli, including Mycobacterium tuberculosis infection, and a variety of immune cells such as macrophages, dendritic cells (DCs), and DC-T cell co-culture, we demonstrate that the assay is versatile, facile, and widely adaptable for enhancing biological understanding of spatial and temporal dynamics of single-cell secretome.
PMID: 36046626
ISSN: 2667-2375
CID: 5337762

Consequence of enhanced LC3-trafficking for a live, attenuated M. tuberculosis vaccine

Koster, Stefan; Klevorn, Thais; Papavinasasundaram, Kadamba; Sassetti, Christopher M; Portal-Celhay, Cynthia; Philips, Jennifer A
Development of a new vaccine against tuberculosis is urgently needed. Recent work has demonstrated that two related LC3-associated trafficking pathways, autophagy and LC3-associated phagocytosis (LAP), enhance antigen presentation and might play a role in vaccine efficacy. Mycobacterium tuberculosis inhibits both LC3-trafficking pathways. Moreover, the vaccine strain, BCG, induces even less LC3-trafficking than M. tuberculosis, which may help explain its limited efficacy. To determine whether enhanced LC3-trafficking can improve efficacy of a live, attenuated M. tuberculosis vaccine, we took advantage of our recent finding that the bacterial virulence factor CpsA inhibits LAP. When we deleted cpsA in the mc26206 vaccine strain, it dramatically increased LC3-trafficking. We compared the protective efficacy of the strain lacking cpsA to the parent strain and to BCG in mice challenged with M. tuberculosis. We found that the strain lacking cpsA generated modestly enhanced protection in the spleen, but overall did not outperform BCG.
PMID: 29343411
ISSN: 1873-2518
CID: 2978852

Mycobacterium tuberculosis EsxH inhibits ESCRT-dependent CD4+ T-cell activation

Portal-Celhay, Cynthia; Tufariello, JoAnn M; Srivastava, Smita; Zahra, Aleena; Klevorn, Thais; Grace, Patricia S; Mehra, Alka; Park, Heidi S; Ernst, Joel D; Jacobs, William R Jr; Philips, Jennifer A
Mycobacterium tuberculosis (Mtb) establishes a persistent infection, despite inducing antigen-specific T-cell responses. Although T cells arrive at the site of infection, they do not provide sterilizing immunity. The molecular basis of how Mtb impairs T-cell function is not clear. Mtb has been reported to block major histocompatibility complex class II (MHC-II) antigen presentation; however, no bacterial effector or host-cell target mediating this effect has been identified. We recently found that Mtb EsxH, which is secreted by the Esx-3 type VII secretion system, directly inhibits the endosomal sorting complex required for transport (ESCRT) machinery. Here, we showed that ESCRT is required for optimal antigen processing; correspondingly, overexpression and loss-of-function studies demonstrated that EsxH inhibited the ability of macrophages and dendritic cells to activate Mtb antigen-specific CD4+ T cells. Compared with the wild-type strain, the esxH-deficient strain induced fivefold more antigen-specific CD4+ T-cell proliferation in the mediastinal lymph nodes of mice. We also found that EsxH undermined the ability of effector CD4+ T cells to recognize infected macrophages and clear Mtb. These results provide a molecular explanation for how Mtb impairs the adaptive immune response.
PMID: 27918526
ISSN: 2058-5276
CID: 2353402

The role of metal-dependent regulation of ESX-3 secretion on the intracellular survival of Mycobacterium tuberculosis

Tinaztepe, Emir; Wei, Jun-Rong; Raynowska, Jenelle; Portal-Celhay, Cynthia; Thompson, Victor; Philips, Jennifer A
More people die every year from Mycobacterium tuberculosis (Mtb) than any other bacterial pathogen. Type VII secretion systems (T7SS) are used by both environmental and pathogenic mycobacteria to secrete proteins across their complex cell envelope. In the non-pathogen, Mycobacterium smegmatis (Msmeg), the ESX-1 T7SS plays a role in conjugation, and the ESX-3 T7SS is involved in metal homeostasis. In Mtb, these secretion systems have taken on roles in virulence, and they also are targets of the host immune response. ESX-3 secretes a heterodimer composed of EsxG (TB9.8) and EsxH (TB10.4), which impairs phagosome maturation in macrophages and is essential for virulence in mice. Given the importance of EsxG and EsxH during infection, we examined their regulation. In Mtb, the secretion of EsxG and EsxH was regulated in response to iron and zinc, in accordance with the previously described transcriptional response of the esx-3 locus to these metals. While iron regulated esx-3 expression in both Mtb and Msmeg, there is a significant difference in the dynamics of this regulation. In Msmeg the esx-3 locus behaved like other iron-regulated genes such as mbtB In Mtb both iron and zinc modestly repressed esx-3 expression. Diminished secretion of EsxG and EsxH in response to these metals altered the interaction of Mtb with macrophages, leading to impaired intracellular Mtb survival. Our findings detail the regulatory differences of esx-3 in Mtb and Msmeg and demonstrate the importance of metal-dependent regulation of ESX-3 for virulence in Mtb.
PMID: 27245412
ISSN: 1098-5522
CID: 2125092

Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism

Ouimet, Mireille; Koster, Stefan; Sakowski, Erik; Ramkhelawon, Bhama; van Solingen, Coen; Oldebeken, Scott; Karunakaran, Denuja; Portal-Celhay, Cynthia; Sheedy, Frederick J; Ray, Tathagat Dutta; Cecchini, Katharine; Zamore, Philip D; Rayner, Katey J; Marcel, Yves L; Philips, Jennifer A; Moore, Kathryn J
Mycobacterium tuberculosis (Mtb) survives in macrophages by evading delivery to the lysosome and promoting the accumulation of lipid bodies, which serve as a bacterial source of nutrients. We found that by inducing the microRNA (miRNA) miR-33 and its passenger strand miR-33*, Mtb inhibited integrated pathways involved in autophagy, lysosomal function and fatty acid oxidation to support bacterial replication. Silencing of miR-33 and miR-33* by genetic or pharmacological means promoted autophagy flux through derepression of key autophagy effectors (such as ATG5, ATG12, LC3B and LAMP1) and AMPK-dependent activation of the transcription factors FOXO3 and TFEB, which enhanced lipid catabolism and Mtb xenophagy. These data define a mammalian miRNA circuit used by Mtb to coordinately inhibit autophagy and reprogram host lipid metabolism to enable intracellular survival and persistence in the host.
PMID: 27089382
ISSN: 1529-2916
CID: 2079882

Ca2+ Signaling but Not Store-Operated Ca2+ Entry Is Required for the Function of Macrophages and Dendritic Cells

Vaeth, Martin; Zee, Isabelle; Concepcion, Axel R; Maus, Mate; Shaw, Patrick; Portal-Celhay, Cynthia; Zahra, Aleena; Kozhaya, Lina; Weidinger, Carl; Philips, Jennifer; Unutmaz, Derya; Feske, Stefan
Store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels is essential for immunity to infection. CRAC channels are formed by ORAI1 proteins in the plasma membrane and activated by stromal interaction molecule (STIM)1 and STIM2 in the endoplasmic reticulum. Mutations in ORAI1 and STIM1 genes that abolish SOCE cause severe immunodeficiency with recurrent infections due to impaired T cell function. SOCE has also been observed in cells of the innate immune system such as macrophages and dendritic cells (DCs) and may provide Ca2+ signals required for their function. The specific role of SOCE in macrophage and DC function, as well as its contribution to innate immunity, however, is not well defined. We found that nonselective inhibition of Ca2+ signaling strongly impairs many effector functions of bone marrow-derived macrophages and bone marrow-derived DCs, including phagocytosis, inflammasome activation, and priming of T cells. Surprisingly, however, macrophages and DCs from mice with conditional deletion of Stim1 and Stim2 genes, and therefore complete inhibition of SOCE, showed no major functional defects. Their differentiation, FcR-dependent and -independent phagocytosis, phagolysosome fusion, cytokine production, NLRP3 inflammasome activation, and their ability to present Ags to activate T cells were preserved. Our findings demonstrate that STIM1, STIM2, and SOCE are dispensable for many critical effector functions of macrophages and DCs, which has important implications for CRAC channel inhibition as a therapeutic strategy to suppress pathogenic T cells while not interfering with myeloid cell functions required for innate immunity.
PMID: 26109647
ISSN: 1550-6606
CID: 1640972


Chapter by: Maslow, Melanie Jane; Portal-Celhay, Cynthia
in: Mandell, Douglas, and Bennett's principles and practice of infectious diseases by Bennett, John E; Dolin, Raphael; Blaser, Martin J [Eds]
Philadelphia, PA : Elsevier/Saunders, 2015
pp. 339-349.e3
ISBN: 9780323263733
CID: 1685642

Effect of Caenorhabditis elegans age and genotype on horizontal gene transfer in intestinal bacteria

Portal-Celhay, Cynthia; Nehrke, Keith; Blaser, Martin J
Horizontal gene transfer (HGT) between bacteria occurs in the intestinal tract of their animal hosts and facilitates both virulence and antibiotic resistance. A model in which both the pathogen and the host are genetically tractable facilitates developing insight into mechanistic processes enabling or restricting the transfer of antibiotic resistance genes. Here we develop an in vivo experimental system to study HGT in bacteria using Caenorhabditis elegans as a model host. Using a thermosensitive conjugative system, we provide evidence that conjugation between two Escherichia coli strains can take place in the intestinal lumen of N2 wild-type worms at a rate of 10(-3) and 10(-2) per donor. We also show that C. elegans age and genotype are important determinants of the frequency of conjugation. Whereas approximately 1 transconjugant for every 100 donor cells could be recovered from the intestine of N2 C. elegans, for the age-1 and tol-1 mutants, the detected rate of transconjugation (10(-3) and 10(-4) per donor cell, respectively) was significantly lower. This work demonstrates that increased recombination among lumenal microbial populations is a phenotype associated with host aging, and the model provides a framework to study the dynamics of bacterial horizontal gene transfer within the intestinal environment.-Portal-Celhay, C., Nehrke, K., Blaser, M. J. Effect of Caenorhabditis elegans age and genotype on horizontal gene transfer in intestinal bacteria.
PMID: 23085995
ISSN: 0892-6638
CID: 227052

Control of intestinal bacterial proliferation in regulation of lifespan in Caenorhabditis elegans

Portal-Celhay, C; Bradley, ER; Blaser, MJ
ABSTRACT: BACKGROUND: A powerful approach to understanding complex processes such as aging is to use model organisms amenable to genetic manipulation, and to seek relevant phenotypes to measure. Caenorhabditis elegans is particularly suited to studies of aging, since numerous single-gene mutations have been identified that affect its lifespan; it possesses an innate immune system employing evolutionarily conserved signaling pathways affecting longevity. As worms age, bacteria accumulate in the intestinal tract. However, quantitative relationships between worm genotype, lifespan, and intestinal lumen bacterial load have not been examined. We hypothesized that gut immunity is less efficient in older animals, leading to enhanced bacterial accumulation, reducing longevity. To address this question, we evaluated the ability of worms to control bacterial accumulation as a functional marker of intestinal immunity. RESULTS: We show that as adult worms age, several C. elegans genotypes show diminished capacity to control intestinal bacterial accumulation. We provide evidence that intestinal bacterial load, regulated by gut immunity, is an important causative factor of lifespan determination; the effects are specified by bacterial strain, worm genotype, and biologic age, all acting in concert. CONCLUSIONS: In total, these studies focus attention on the worm intestine as a locus that influences longevity in the presence of an accumulating bacterial population. Further studies defining the interplay between bacterial species and host immunity in C. elegans may provide insights into the general mechanisms of aging and age-related diseases.
PMID: 22452899
ISSN: 1471-2180
CID: 165584

Competition and Resilience between Founder and Introduced Bacteria in the Caenorhabditis elegans Gut

Portal-Celhay, Cynthia; Blaser, Martin J
The microbial communities that reside within the intestinal tract in vertebrates are complex and dynamic. In this report, we establish the utility of Caenorhabditis elegans as a model system for identifying the factors that contribute to bacterial persistence and for host control of gut luminal populations. We found that for N2 worms grown on mixed lawns of bacteria, Salmonella enterica serovar Typhimurium substantially outcompeted Escherichia coli, even when E. coli was initially present at 100-fold-higher concentrations. To address whether innate immunity affects the competition, the daf-2 and daf-16 mutants were studied; their total gut bacterial levels reflect overall capacity for colonization, but Salmonella outcompeted E. coli to an extent similar to wild-type worms. To address the role of virulence properties, Salmonella Deltaspi-1 Deltaspi-2 was used to compete with E. coli. The net differential was significantly less than that for wild-type Salmonella; thus, spi-1 spi-2 encodes C. elegans colonization factors. An E. coli strain with repeated in vivo passage had an enhanced ability to compete against an in vitro-passed E. coli strain and against Salmonella. Our data provide evidence of active competition for colonization niches in the C. elegans gut, as determined by bacterial factors and subject to in vivo selection.
PMID: 22184417
ISSN: 0019-9567
CID: 160245