scholarly journals A metabolic dependency for host isoprenoids in the obligate intracellular pathogenRickettsia parkeriunderlies a sensitivity for the statin class of host-targeted therapeutics

2019 ◽  
Author(s):  
Vida Ahyong ◽  
Charles A. Berdan ◽  
Daniel K. Nomura ◽  
Matthew D. Welch
Keyword(s):  
Cell Wall ◽  
Host Cell ◽  
Bacterial Growth ◽  
Spotted Fever ◽  
Intracellular Pathogens ◽  
Biosynthetic Pathways ◽  
Targeted Therapeutics ◽  
Rickettsia Parkeri ◽  
Obligate Intracellular ◽  
Intracellular Parasites

AbstractGram-negative bacteria in the order Rickettsiales are obligate intracellular parasites that cause human diseases such typhus and spotted fever. They have evolved a dependence on essential nutrients and metabolites from the host cell as a consequence of extensive genome streamlining. However, it remains largely unknown which nutrients they require and whether their metabolic dependency can be exploited therapeutically. Here, we describe a genetic rewiring of bacterial isoprenoid biosynthetic pathways in the Rickettsiales that has resulted from reductive genome evolution. We further investigated whether the spotted fever groupRickettsiaspeciesRickettsia parkeriscavenges isoprenoid precursors directly from the host. Using targeted mass spectrometry in uninfected and infected cells, we found decreases in host isoprenoid products and concomitant increases in bacterial isoprenoid metabolites. Additionally, we report that bacterial growth is prohibited by inhibition of the host isoprenoid pathway with the statins class of drugs. We show that growth inhibition correlates with changes in bacterial size and shape that mimic those caused by antibiotics that inhibit peptidoglycan biosynthesis, suggesting statins inhibit cell wall synthesis. Altogether, our results describe an Achilles’ heel of obligate intracellular pathogens that can be exploited with host-targeted therapeutics that interfere with metabolic pathways required for bacterial growth.ImportanceObligate intracellular parasites, which include viruses as well as certain bacteria and eukaryotes, extract essential nutrients and metabolites from their host cell. As a result, these pathogens have often lost essential biosynthetic pathways and are metabolically dependent on the host. In this study, we describe a metabolic dependency of the bacterial pathogenRickettsia parkerion host isoprenoid molecules that are used in the biosynthesis of downstream products including cholesterol, steroid hormones, and heme. Bacteria make products from isoprenoids such as an essential lipid carrier for making the bacterial cell wall. We show that bacterial metabolic dependency can represent an Achilles’ heel, and that inhibiting host isoprenoid biosynthesis with the FDA-approved statin class of drugs inhibits bacterial growth by interfering with the integrity of the cell wall. This work highlights a potential to treat infections by obligate intracellular pathogens through inhibition of host biosynthetic pathways that are susceptible to parasitism.

scholarly journals A Metabolic Dependency for Host Isoprenoids in the Obligate Intracellular Pathogen Rickettsia parkeri Underlies a Sensitivity to the Statin Class of Host-Targeted Therapeutics

mSphere ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Vida Ahyong ◽  
Charles A. Berdan ◽  
Thomas P. Burke ◽  
Daniel K. Nomura ◽  
Matthew D. Welch
Keyword(s):  
Cell Wall ◽  
Host Cell ◽  
Bacterial Growth ◽  
Spotted Fever ◽  
Intracellular Pathogens ◽  
Biosynthetic Pathways ◽  
Targeted Therapeutics ◽  
Rickettsia Parkeri ◽  
Content Type ◽  
Obligate Intracellular

ABSTRACT Gram-negative bacteria in the order Rickettsiales have an obligate intracellular growth requirement, and some species cause human diseases such as typhus and spotted fever. The bacteria have evolved a dependence on essential nutrients and metabolites from the host cell as a consequence of extensive genome reduction. However, it remains largely unknown which nutrients they acquire and whether their metabolic dependency can be exploited therapeutically. Here, we describe a genetic rewiring of bacterial isoprenoid biosynthetic pathways in the Rickettsiales that has resulted from reductive genome evolution. Furthermore, we investigated whether the spotted fever group Rickettsia species Rickettsia parkeri scavenges isoprenoid precursors directly from the host. Using targeted mass spectrometry, we found that infection caused decreases in host isoprenoid products and concomitant increases in bacterial isoprenoid metabolites. Additionally, we report that treatment of infected cells with statins, which inhibit host isoprenoid synthesis, prohibited bacterial growth. We show that growth inhibition correlates with changes in bacterial size and shape that mimic those caused by antibiotics that inhibit peptidoglycan biosynthesis, suggesting that statins lead to an inhibition of cell wall synthesis. Altogether, our results describe a potential Achilles’ heel of obligate intracellular pathogens that can potentially be exploited with host-targeted therapeutics that interfere with metabolic pathways required for bacterial growth. IMPORTANCE Obligate intracellular pathogens, which include viruses as well as certain bacteria and eukaryotes, are a subset of infectious microbes that are metabolically dependent on and unable to grow outside an infected host cell because they have lost or lack essential biosynthetic pathways. In this study, we describe a metabolic dependency of the bacterial pathogen Rickettsia parkeri on host isoprenoid molecules that are used in the biosynthesis of downstream products, including cholesterol, steroid hormones, and heme. Bacteria make products from isoprenoids, such as an essential lipid carrier for making the bacterial cell wall. We show that bacterial metabolic dependency can represent a potential Achilles’ heel and that inhibiting host isoprenoid biosynthesis with the FDA-approved statin class of drugs inhibits bacterial growth by interfering with the integrity of the cell wall. This work supports the potential to treat infections by obligate intracellular pathogens through inhibition of host biosynthetic pathways that are susceptible to parasitism.

scholarly journals A streamlined method for transposon mutagenesis ofRickettsia parkeriyields numerous mutations that impact infection

2018 ◽  
Author(s):  
Rebecca L. Lamason ◽  
Natasha M. Kafai ◽  
Matthew D. Welch
Keyword(s):  
Host Cell ◽  
Virulence Factors ◽  
Vascular Endothelial Cells ◽  
Spotted Fever ◽  
Transposon Mutagenesis ◽  
Host Cells ◽  
Spotted Fever Group ◽  
Loss Of Function ◽  
Rickettsia Parkeri ◽  
Obligate Intracellular

AbstractThe rickettsiae are obligate intracellular alphaproteobacteria that exhibit a complex infectious life cycle in both arthropod and mammalian hosts. As obligate intracellular bacteria,Rickettsiaare highly adapted to living inside a variety of host cells, including vascular endothelial cells during mammalian infection. Although it is assumed that the rickettsiae produce numerous virulence factors that usurp or disrupt various host cell pathways, they have been challenging to genetically manipulate to identify the key bacterial factors that contribute to infection. Motivated to overcome this challenge, we sought to expand the repertoire of available rickettsial loss-of-function mutants, using an improvedmariner-based transposon mutagenesis scheme. Here, we present the isolation of over 100 transposon mutants in the spotted fever group speciesRickettsia parkeri. These mutants targeted genes implicated in a variety of pathways, including bacterial replication and metabolism, hypothetical proteins, the type IV secretion system, as well as factors with previously established roles in host cell interactions and pathogenesis. Given the need to identify critical virulence factors, forward genetic screens such as this will provide an excellent platform to more directly investigate rickettsial biology and pathogenesis.

scholarly journals Identification and Characterization of the Chlamydia trachomatis L2 S-Adenosylmethionine Transporter

mBio ◽  
2011 ◽  
Vol 2 (3) ◽  
Author(s):  
Rachel Binet ◽  
Reinaldo E. Fernandez ◽  
Derek J. Fisher ◽  
Anthony T. Maurelli
Keyword(s):  
Chlamydia Trachomatis ◽  
Host Cell ◽  
Genomic Library ◽  
Dual Function ◽  
Enzymatic Reactions ◽  
Content Type ◽  
Methyl Group ◽  
E Coli ◽  
Obligate Intracellular ◽  
Intracellular Parasites

ABSTRACTMethylation is essential to the physiology of all cells, including the obligate intracellular bacteriumChlamydia. Nevertheless, the methylation cycle is under strong reductive evolutionary pressure inChlamydia. OnlyParachlamydia acanthamoebaeandWaddlia chondrophilagenome sequences harbor homologs tometK, encoding theS-adenosylmethionine (SAM) synthetase required for synthesis of SAM, and tosahH, which encodes theS-adenosylhomocysteine (SAH) hydrolase required for detoxification of SAH formed after the transfer of the methyl group from SAM to the methylation substrate. Transformation of a conditional-lethal ΔmetKmutant ofEscherichia coliwith a genomic library ofChlamydia trachomatisL2 identified CTL843 as a putative SAM transporter based on its ability to allow the mutant to survivemetKdeficiency only in the presence of extracellular SAM. CTL843 belongs to the drug/metabolite superfamily of transporters and allowedE. colito transportS-adenosyl-l-[methyl-14C]methionine with an apparentKmof 5.9 µM and aVmaxof 32 pmol min−1 mg−1. Moreover, CTL843 conferred a growth advantage to a Δpfs E. colimutant that lost the ability to detoxify SAH, while competition and back-transport experiments further implied that SAH was an additional substrate for CTL843. We propose that CTL843 acts as a SAM/SAH transporter (SAMHT) serving a dual function by allowingChlamydiato acquire SAM from the host cell and excrete the toxic by-product SAH. The demonstration of a functional SAMHT provides further insight into the reductive evolution associated with the obligate intracellular lifestyle ofChlamydiaand identifies an excellent chemotherapeutic target.IMPORTANCEObligate intracellular parasites likeChlamydiahave followed a reductive evolutionary path that has made them almost totally dependent on their host cell for nutrients. In this work, we identify a unique transporter of a metabolite essential for all methylation reactions that potentially bypasses the need for two enzymatic reactions inChlamydia. The transporter, CTL843, allowsChlamydia trachomatisL2 to stealS-adenosylmethionine (SAM) from the eukaryotic host cytosol and to likely remove the toxicS-adenosylhomocysteine (SAH) formed when SAM loses its methyl group, acting as a SAM/SAH transporter (SAMHT). In addition to reflecting the adaptation ofChlamydiato an obligate intracellular lifestyle, the specific and central roles of SAMHT inChlamydiametabolism provide a target for the development of therapeutic agents for the treatment of chlamydial infections.

scholarly journals Loss of a doublecortin (DCX) domain containing protein causes structural defects in a tubulin-based organelle ofToxoplasma gondiiand impairs host cell invasion

2016 ◽  
Author(s):  
Eiji Nagayasu ◽  
Yu-chen Hwang ◽  
Jun Liu ◽  
John M. Murray ◽  
Ke Hu
Keyword(s):  
Host Cell ◽  
Cell Invasion ◽  
Polymer Structure ◽  
Structural Defects ◽  
Host Cell Invasion ◽  
Obligate Intracellular ◽  
Intracellular Parasites ◽  
Apical Complex ◽  
Cytoskeletal Elements ◽  
Special Arrangement

AbstractThe ~6000 species in phylum Apicomplexa are single-celled obligate intracellular parasites. Their defining characteristic is the “apical complex”, membranous and cytoskeletal elements at the apical end of the cell that participate in host-cell invasion. The apical complex ofToxoplasma gondiiand some other apicomplexans includes a cone-shaped assembly, the “conoid”, which (inT. gondii) comprises 14 spirally arranged fibers that are non-tubular polymers of tubulin. The tubulin dimers used for the conoid fibers make canonical microtubules elsewhere in the same cell, suggesting that their special arrangement in the conoid fibers is dictated by non-tubulin proteins. One candidate for this role is TgDCX, which has a doublecortin (DCX) domain and a TPPP/P25-alpha domain, known modulators of tubulin polymer structure. Loss of TgDCX radically disrupts the structure of the conoid, severely impairs host cell invasion, and slows growth. The defects of TgDCX-null parasites are corrected by re-introduction of a TgDCX coding sequence.

scholarly journals Genome Sequence of Thermofilum pendens Reveals an Exceptional Loss of Biosynthetic Pathways without Genome Reduction

2008 ◽  
Vol 190 (8) ◽  
pp. 2957-2965 ◽  
Author(s):  
Iain Anderson ◽  
Jason Rodriguez ◽  
Dwi Susanti ◽  
Iris Porat ◽  
Claudia Reich ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Housekeeping Genes ◽  
Biosynthetic Pathways ◽  
Phosphotransferase System ◽  
Obligate Intracellular ◽  
Intracellular Parasites ◽  
Associated Proteins ◽  
Short Palindromic Repeat ◽  
Thermoproteus Tenax ◽  
First Time

ABSTRACT We report the complete genome of Thermofilum pendens, a deeply branching, hyperthermophilic member of the order Thermoproteales in the archaeal kingdom Crenarchaeota. T. pendens is a sulfur-dependent, anaerobic heterotroph isolated from a solfatara in Iceland. It is an extracellular commensal, requiring an extract of Thermoproteus tenax for growth, and the genome sequence reveals that biosynthetic pathways for purines, most amino acids, and most cofactors are absent. In fact, T. pendens has fewer biosynthetic enzymes than obligate intracellular parasites, although it does not display other features that are common among obligate parasites and thus does not appear to be in the process of becoming a parasite. It appears that T. pendens has adapted to life in an environment rich in nutrients. T. pendens was known previously to utilize peptides as an energy source, but the genome revealed a substantial ability to grow on carbohydrates. T. pendens is the first crenarchaeote and only the second archaeon found to have a transporter of the phosphotransferase system. In addition to fermentation, T. pendens may obtain energy from sulfur reduction with hydrogen and formate as electron donors. It may also be capable of sulfur-independent growth on formate with formate hydrogen lyase. Additional novel features are the presence of a monomethylamine:corrinoid methyltransferase, the first time that this enzyme has been found outside the Methanosarcinales, and the presence of a presenilin-related protein. The predicted highly expressed proteins do not include proteins encoded by housekeeping genes and instead include ABC transporters for carbohydrates and peptides and clustered regularly interspaced short palindromic repeat-associated proteins.

scholarly journals Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction with Calcium-Dependent Protein Kinase 1

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Matthew A. Child ◽  
Megan Garland ◽  
Ian Foe ◽  
Peter Madzelan ◽  
Moritz Treeck ◽  
...  
Keyword(s):  
Host Cell ◽  
Kinase Activity ◽  
Effector Proteins ◽  
Apicomplexan Parasites ◽  
Obligate Intracellular ◽  
Calcium Dependent ◽  
Intracellular Parasites ◽  
Dependent Protein ◽  
Protective Environment ◽  
Calcium Dependent Protein Kinases

ABSTRACT Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson’s disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondii. IMPORTANCE Apicomplexan parasites such as Toxoplasma and Plasmodium are obligate intracellular parasites that require the protective environment of a host cell in order to replicate and survive within a host organism. These parasites secrete effector proteins from specialized apical organelles to select and invade a chosen host cell. The secretion of these organelles is a tightly regulated process coordinated by endogenous small molecules and calcium-dependent protein kinases. We previously identified the Toxoplasma orthologue of the highly conserved protein DJ-1 as a regulator of microneme secretion, but the molecular basis for this was not known. We have now identified the molecular mechanism for how TgDJ-1 regulates microneme secretion. TgDJ-1 interacts with the kinase responsible for the secretion of these organelles (calcium-dependent kinase 1) and synergizes with calcium to potentiate kinase activity. This interaction is direct, phosphodependent, and necessary for the normal secretion of these important organelles. IMPORTANCE Apicomplexan parasites such as Toxoplasma and Plasmodium are obligate intracellular parasites that require the protective environment of a host cell in order to replicate and survive within a host organism. These parasites secrete effector proteins from specialized apical organelles to select and invade a chosen host cell. The secretion of these organelles is a tightly regulated process coordinated by endogenous small molecules and calcium-dependent protein kinases. We previously identified the Toxoplasma orthologue of the highly conserved protein DJ-1 as a regulator of microneme secretion, but the molecular basis for this was not known. We have now identified the molecular mechanism for how TgDJ-1 regulates microneme secretion. TgDJ-1 interacts with the kinase responsible for the secretion of these organelles (calcium-dependent kinase 1) and synergizes with calcium to potentiate kinase activity. This interaction is direct, phosphodependent, and necessary for the normal secretion of these important organelles.

scholarly journals Untargeted Metabolomics Uncovers the Essential Lysine Transporter in Toxoplasma gondii

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 476
Author(s):  
Joachim Kloehn ◽  
Matteo Lunghi ◽  
Emmanuel Varesio ◽  
David Dubois ◽  
Dominique Soldati-Favre
Keyword(s):  
Amino Acids ◽  
Toxoplasma Gondii ◽  
Rna Degradation ◽  
Major Facilitator Superfamily ◽  
Untargeted Metabolomics ◽  
Apicomplexan Parasites ◽  
Obligate Intracellular ◽  
Intracellular Parasites ◽  
Major Facilitator ◽  
Plasma Membrane Transporter

Apicomplexan parasites are responsible for devastating diseases, including malaria, toxoplasmosis, and cryptosporidiosis. Current treatments are limited by emerging resistance to, as well as the high cost and toxicity of existing drugs. As obligate intracellular parasites, apicomplexans rely on the uptake of many essential metabolites from their host. Toxoplasma gondii, the causative agent of toxoplasmosis, is auxotrophic for several metabolites, including sugars (e.g., myo-inositol), amino acids (e.g., tyrosine), lipidic compounds and lipid precursors (cholesterol, choline), vitamins, cofactors (thiamine) and others. To date, only few apicomplexan metabolite transporters have been characterized and assigned a substrate. Here, we set out to investigate whether untargeted metabolomics can be used to identify the substrate of an uncharacterized transporter. Based on existing genome- and proteome-wide datasets, we have identified an essential plasma membrane transporter of the major facilitator superfamily in T. gondii—previously termed TgApiAT6-1. Using an inducible system based on RNA degradation, TgApiAT6-1 was depleted, and the mutant parasite’s metabolome was compared to that of non-depleted parasites. The most significantly reduced metabolite in parasites depleted in TgApiAT6-1 was identified as the amino acid lysine, for which T. gondii is predicted to be auxotrophic. Using stable isotope-labeled amino acids, we confirmed that TgApiAT6-1 is required for efficient lysine uptake. Our findings highlight untargeted metabolomics as a powerful tool to identify the substrate of orphan transporters.

scholarly journals Full-Length Transcriptome of Thalassiosira weissflogii as a Reference Resource and Mining of Chitin-Related Genes

Marine Drugs ◽  
2021 ◽  
Vol 19 (7) ◽  
pp. 392
Author(s):  
Haomiao Cheng ◽  
Chris Bowler ◽  
Xiaohui Xing ◽  
Vincent Bulone ◽  
Zhanru Shao ◽  
...  
Keyword(s):  
Cell Wall ◽  
Full Length ◽  
Biosynthetic Pathways ◽  
Glycosidic Linkage ◽  
Thalassiosira Weissflogii ◽  
Ecological Value ◽  
Pacbio Sequencing ◽  
Non Coding Rnas ◽  
Chitin And Chitosan ◽  
Simple Sequence

β-Chitin produced by diatoms is expected to have significant economic and ecological value due to its structure, which consists of parallel chains of chitin, its properties and the high abundance of diatoms. Nevertheless, few studies have functionally characterised chitin-related genes in diatoms owing to the lack of omics-based information. In this study, we first compared the chitin content of three representative Thalassiosira species. Cell wall glycosidic linkage analysis and chitin/chitosan staining assays showed that Thalassiosira weissflogii was an appropriate candidate chitin producer. A full-length (FL) transcriptome of T. weissflogii was obtained via PacBio sequencing. In total, the FL transcriptome comprised 23,362 annotated unigenes, 710 long non-coding RNAs (lncRNAs), 363 transcription factors (TFs), 3113 alternative splicing (AS) events and 3295 simple sequence repeats (SSRs). More specifically, 234 genes related to chitin metabolism were identified and the complete biosynthetic pathways of chitin and chitosan were explored. The information presented here will facilitate T. weissflogii molecular research and the exploitation of β-chitin-derived high-value enzymes and products.

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