264 research outputs found
Contact-dependent growth inhibition (CDI) systems deploy a large family of polymorphic ionophoric toxins for inter-bacterial competition
No full textContact-dependent growth inhibition (CDI) is a widespread form of inter-bacterial competition mediated by CdiA effector proteins. CdiA is presented on the inhibitor cell surface and delivers its toxic C-terminal region (CdiA-CT) into neighboring bacteria upon contact. Inhibitor cells also produce CdiI immunity proteins, which neutralize CdiA-CT toxins to prevent auto-inhibition. Here, we describe a diverse group of CDI ionophore toxins that dissipate the transmembrane potential in target bacteria. These CdiA-CT toxins are composed of two distinct domains based on AlphaFold2 modeling. The C-terminal ionophore domains are all predicted to form five-helix bundles capable of spanning the cell membrane. The N-terminal "entry" domains are variable in structure and appear to hijack different integral membrane proteins to promote toxin assembly into the lipid bilayer. The CDI ionophores deployed by E. coli isolates partition into six major groups based on their entry domain structures. Comparative sequence analyses led to the identification of receptor proteins for ionophore toxins from groups 1 & 3 (AcrB), group 2 (SecY) and groups 4 (YciB). Using forward genetic approaches, we identify novel receptors for the group 5 and 6 ionophores. Group 5 exploits homologous putrescine import proteins encoded by puuP and plaP, and group 6 toxins recognize di/tripeptide transporters encoded by paralogous dtpA and dtpB genes. Finally, we find that the ionophore domains exhibit significant intra-group sequence variation, particularly at positions that are predicted to interact with CdiI. Accordingly, the corresponding immunity proteins are also highly polymorphic, typically sharing only ~30% sequence identity with members of the same group. Competition experiments confirm that the immunity proteins are specific for their cognate ionophores and provide no protection against other toxins from the same group. The specificity of this protein interaction network provides a mechanism for self/nonself discrimination between E. coli isolates
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Why WhiB1: Manipulation of Clp System to Determine Role of WhiB1 in Regulating Mycobacterial Cell Division
No full textMycobacterium tuberculosis (Mtb) is the causal agent of tuberculosis (TB), a major public health issue in the world. In the 2015 WHO report, there were approximately 10.4 million new cases of TB worldwide and 1.4 million TB deaths. Moreover, the rising number of multi-drug-resistant (MDR) and extensively drug-resistant (XDR) TB cases highlights the urgent demand for new TB drugs. In Mtb, the Clp system is essential and provides a promising target for new antibiotics. In this thesis, we identified the preferred cleavage sites of ClpP1P2 and synthesized new peptide boronate inhibitors that are specific to mycobacteria. Several compounds inhibited ClpP1P2 activity in vitro and growth of both Mtb and Mycobacterium smegmatis (Msm) by specifically targeting ClpP1P2 proteolytic activity.
As more compounds inhibiting the Clp system are identified in mycobacteria, there is a growing need to understand the precise function of the mycobacterial Clp system. We hypothesized that Clp essentiality stems from its role as a proteolytic regulator of substrates that become toxic when accumulated. To determine the substrates of mycobacterial Clp, we conducted quantitative proteomics to identify proteins that accumulate upon depletion of the Clp protease. One identified substrate was WhiB1, an essential transcriptional regulator whose function was largely unknown. To further understand WhiB1 as a Clp substrate, we utilized peptide boronates and genetic depletions of Clp components. Blocking the degradation sequence of WhiB1 resulted in toxic accumulation. We found that the transcriptional regulatory function of WhiB1 was responsible for the toxicity of WhiB1 accumulation. RNAseq identified essential genes involved in mycobacterial cell division that were repressed by WhiB1, namely sepF, ftsZ, and mtrAB. WhiB1 accumulation decreased levels of FtsZ in cells and caused elongation, branching, and loss of FtsZ ring formation. Taken together, we conclude that Clp essentiality is in part due to its role in degrading WhiB1, a cell division regulator.Biology, Molecular and CellularClp, Mycobacteria, WhiB
PLoS Pathog
No full textMycobacterium tuberculosis (Mtb) remains a major public health problem, with an effective vaccine continuing to prove elusive. Progress in vaccination strategies has been hampered by a lack of appreciation of the bacterium's response to dynamic changes in the host immune environment. Here, we utilize reporter Mtb strains that respond to specific host immune stresses such as hypoxia and nitric oxide (hspX'::GFP), and phagosomal maturation (rv2390c'::GFP), to investigate vaccine-induced alterations in the environmental niche during experimental murine infections. While vaccination undoubtedly decreased bacterial burden, we found that it also appeared to accelerate Mtb's adoption of a phenotype better equipped to survive in its host. We subsequently utilized a novel replication reporter strain of Mtb to demonstrate that, in addition to these alterations in host stress response, there is a decreased percentage of actively replicating Mtb in vaccinated hosts. This observation was supported by the differential sensitivity of recovered bacteria to the front-line drug isoniazid. Our study documents the natural history of the impact that vaccination has on Mtb's physiology and replication and highlights the value of reporter Mtb strains for probing heterogeneous Mtb populations in the context of a complex, whole animal model.20141DP2LM011952-01/DP/NCCDPHP CDC HHS/United StatesAI067027/AI/NIAID NIH HHS/United StatesDP2 LM011952/LM/NLM NIH HHS/United StatesHL055936/HL/NHLBI NIH HHS/United StatesR01 AI067027/AI/NIAID NIH HHS/United StatesR01 HL055936/HL/NHLBI NIH HHS/United States25233380PMC41695031018
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Elucidation of an essential genetic pathway under antibiotic selection in Mycobacterium tuberculosis
No full textTuberculosis remains the world’s deadliest infectious disease caused by a single agent. Although tuberculosis is curable, treatment success is limited by our narrow understanding of genetic factors allowing its causative agent, Mycobacterium tuberculosis (Mtb), to evade antibiotic clearance. Large-scale sequencing of clinical Mtb populations revealed ongoing selection on genetic variants that could confer fitness advantages in the presence of drug pressure. This unbiased approach allowed identification of genes with no previous link to drug resistance, including two essential genes dnaA and resR. Although initially investigated independently, DnaA and ResR share a common binding site at the Rv0010c-Rv0011c intergenic region (IGR) and this IGR itself is one of the highly mutated non-coding regions on Mtb genome. Clinical IGR variants overlap with DnaA and ResR binding sites and phenocopy dnaA and resR variants, revealing a genetic pathway under selection. Yet this genetic pathway and the function of Rv0010c-Rv0011c IGR remains uncharacterized.
Isogenic variants in the Rv0010c-Rv0011c IGR phenocopy dnaA and resR variants, showing similar increases in cell length, antibiotic resilience, and low-level isoniazid resistance. We found that DnaA and ResR bind at neighboring sites in the most conserved regions of this IGR, which paradoxically are where more recent clinical mutations accumulate. Knockout of the entire the Rv0010c-Rv0011c operon, including its 155bp IGR, resulted in shorter cells with increased sensitivity to isoniazid. This defect can only be complemented with the entire operon, though this complementation does not require translation of the two coding genes. Complementation with the intact operon carrying clinically relevant IGR variants recapitulates isogenic variant phenotypes. Meanwhile, complementation with the intact operon carrying DnaA or ResR binding site deletions failed, highlighting the requirement of protein binding in its downstream function.
To understand the functional consequence of protein binding, we used biochemical approaches and found that DnaA and ResR bind cooperatively at this IGR. Clinical IGR variants increase the binding affinity of two proteins and binding site deletions reduce their affinity. Using transcriptomics, we identified genes that are differentially expressed in strains with clinical IGR variants versus strains with binding site deletions to pinpoint transcriptional changes correlating with divergent phenotypes in these strains. These genes included whiB2 and its regulon of division related genes. Notably, the promoter of whiB2 is a known direct target of ResR and is also highly mutated in clinical Mtb populations. We propose a model where clinical variations sequester ResR through its interaction with DnaA at the Rv0010c-Rv0011c IGR. This sequestration reduces ResR’s ability to activate division related genes and alter division dynamics, resulting in morphology and drug phenotypes.
Together, the data in this dissertation provide functional insight into an essential and previously uncharacterized genetic pathway under selection in clinical Mtb populations. We propose that clinical mutations in this pathway alter dynamics of cell cycle events and contribute to changes in Mtb morphology and drug response. Understanding non-canonical drug determinants is critical to elucidate other mechanisms Mtb use to evade antibiotics killing and we hope to inspire future studies on intergenic regions and unknown genetic pathways to better understand Mtb biology and improve treatment design.Medical SciencesMedical Science
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Improving and understanding macrophage restriction of Mycobacterium tuberculosis infection
No full textTuberculosis, the disease caused by Mycobacterium tuberculosis (Mtb), is a leading cause of death due to infectious disease. New biomedical interventions are necessary to meet World Health Organization goals for tuberculosis control. However, the creation of these interventions is constrained by insufficient knowledge of how the human immune system fights infection with Mtb, which limits our tools for improving immunity. Macrophages are a protective replicative niche for Mtb but can kill the infecting bacterium when appropriately activated. We sought to identify compounds that best activate macrophage restriction, and elucidate the resulting mechanism of Mtb control.
A host-side CRISPR screen during mycobacterial infection identified macrophage genes for which genetic and pharmacologic targeting each increased macrophage survival and Mtb restriction. In a complementary approach, we systematically compared bacterial restriction by human macrophages after treatment with 26 activators, including compounds currently in clinical trials for tuberculosis. Both approaches yielded pharmacological inducers of Mtb restriction that were robust across macrophage contexts.
To understand mechanisms of macrophage restriction of Mtb infection we studied the compound all-trans-retinoic acid (ATRA), an active metabolite of vitamin A which most effectively increased Mtb control. By comparing ATRA to closely related yet non-restrictive compounds, we found that bacterial clearance was transcriptionally and functionally associated with macrophage cholesterol efflux; this limitation of macrophage cholesterol was necessary for restriction of Mtb. To determine how cholesterol efflux affected bacterial control, we performed the first Mtb CRISPR interference screen in an infection model, identifying Mtb genes specifically required to survive in ATRA-activated macrophages. These data showed that ATRA treatment starves Mtb of cholesterol and the downstream metabolite propionyl-CoA. Supplementation with sources of propionyl-CoA abrogated the restrictive effect of ATRA, and this effect was dependent on pathways that integrate propionate into central carbon metabolism.
The data in this dissertation demonstrate that host genetic screening and systematic comparative analysis can each identify effective activators of Mtb restriction by macrophages, enabling future therapeutic development. They also show that targeting the coupled metabolism of Mtb and the macrophage improves control of infection, and that it is possible to genetically map the mode of bacterial death using CRISPR interference.Medical SciencesMedical Science
Glia in Health and Disease
No full textThis eBook is a collection of articles from a Frontiers Research Topic. Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office: frontiersin.org/about/contac
Motility of urease-deficient derivatives of Helicobacter pylori
No full textThis article cites 29 articles, 19 of which can be accessed fre
Glia in Health and Disease
No full textThis eBook is a collection of articles from a Frontiers Research Topic. Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office: frontiersin.org/about/contac
Public Choice for Influencers Versus Celebrities: Analysis According to Edgar Morin
No full textThis article examines why the public pays more attention to influencers today than to celebrities. It aims to integrate the author\u27s ideas into the academic field of influencer research and mass culture. We will analyze the psychological differences that motivate the public\u27s adherence to influencers compared to celebrities, drawing on Edgar Morin\u27s theory of mass culture. We will conclude that these differences manifest primarily in three aspects
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