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 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 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 Toxoplasma gondii Development of Its Replicative Niche: in Its Host Cell and Beyond

2014 ◽  
Vol 13 (8) ◽  
pp. 965-976 ◽  
Author(s):  
Ira J. Blader ◽  
Anita A. Koshy
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Host Cells ◽  
Content Type ◽  
Obligate Intracellular ◽  
Cellular Processes ◽  
High Prevalence ◽  
Life Threatening ◽  
Cell Processes ◽  
Cellular Pathways

ABSTRACTIntracellular pathogens can replicate efficiently only after they manipulate and modify their host cells to create an environment conducive to replication. While diverse cellular pathways are targeted by different pathogens, metabolism, membrane and cytoskeletal architecture formation, and cell death are the three primary cellular processes that are modified by infections.Toxoplasma gondiiis an obligate intracellular protozoan that infects ∼30% of the world's population and causes severe and life-threatening disease in developing fetuses, in immune-comprised patients, and in certain otherwise healthy individuals who are primarily found in South America. The high prevalence ofToxoplasmain humans is in large part a result of its ability to modulate these three host cell processes. Here, we highlight recent work defining the mechanisms by whichToxoplasmainteracts with these processes. In addition, we hypothesize why some processes are modified not only in the infected host cell but also in neighboring uninfected cells.

scholarly journals EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Dipu Mohan Kumar ◽  
Mingqun Lin ◽  
Qingming Xiong ◽  
Mathew James Webber ◽  
Comert Kural ◽  
...  
Keyword(s):  
Cell Surface ◽  
Host Cell ◽  
Actin Polymerization ◽  
Ehrlichia Chaffeensis ◽  
Content Type ◽  
Hnrnp K ◽  
Obligate Intracellular ◽  
Human Proteins ◽  
Coated Bead ◽  
Human Monocytic Ehrlichiosis

ABSTRACTObligate intracellular bacteria, such asEhrlichia chaffeensis, perish unless they can enter eukaryotic cells.E. chaffeensisis the etiological agent of human monocytic ehrlichiosis, an emerging infectious disease. To infect cells,Ehrlichiauses theCterminus of the outer membrane invasinentry-triggeringprotein (EtpE) ofEhrlichia(EtpE-C), which directly binds the mammalian cell surface glycosylphosphatidyl inositol-anchored protein, DNase X. How this binding drivesEhrlichiaentry is unknown. Here, using affinity pulldown of host cell lysates with recombinant EtpE-C (rEtpE-C), we identified two new human proteins that interact with EtpE-C: CD147 and heterogeneous nuclear ribonucleoprotein K (hnRNP-K). The interaction of CD147 with rEtpE-C was validated by far-Western blotting and coimmunoprecipitation of native EtpE with endogenous CD147. CD147 was ubiquitous on the cell surface and also present around foci of rEtpE-C-coated-bead entry. Functional neutralization of surface-exposed CD147 with a specific antibody inhibitedEhrlichiainternalization and infection but not binding. Downregulation of CD147 by short hairpin RNA (shRNA) impairedE. chaffeensisinfection. Functional ablation of cytoplasmic hnRNP-K by a nanoscale intracellular antibody markedly attenuated bacterial entry and infection but not binding. EtpE-C also interacted with neuronal Wiskott-Aldrich syndrome protein (N-WASP), which is activated by hnRNP-K. Wiskostatin, which inhibits N-WASP activation, and cytochalasin D, which inhibits actin polymerization, inhibitedEhrlichiaentry. Upon incubation with host cell lysate, EtpE-C but not an EtpE N-terminal fragment stimulatedin vitroactin polymerization in an N-WASP- and DNase X-dependent manner. Time-lapse video images revealed N-WASP recruitment at EtpE-C-coated bead entry foci. Thus, EtpE-C binding to DNase X drivesEhrlichiaentry by engaging CD147 and hnRNP-K and activating N-WASP-dependent actin polymerization.IMPORTANCEEhrlichia chaffeensis, an obligate intracellular bacterium, causes a blood-borne disease called human monocytic ehrlichiosis, one of the most prevalent life-threatening emerging tick-transmitted infectious diseases in the United States. The survival ofEhrlichiabacteria, and hence, their ability to cause disease, depends on their specific mode of entry into eukaryotic host cells. Understanding the mechanism by whichE. chaffeensisenters cells will create new opportunities for developing effective therapies to prevent bacterial entry and disease in humans. Our findings reveal a novel cellular signaling pathway triggered by an ehrlichial surface protein called EtpE to induce its infectious entry. The results are also important from the viewpoint of human cell physiology because three EtpE-interacting human proteins, DNase X, CD147, and hnRNP-K, are hitherto unknown partners that drive the uptake of small particles, including bacteria, into human cells.

scholarly journals Impact of Active Metabolism onChlamydia trachomatisElementary Body Transcript Profile and Infectivity

2018 ◽  
Vol 200 (14) ◽  
Author(s):  
Scott Grieshaber ◽  
Nicole Grieshaber ◽  
Hong Yang ◽  
Briana Baxter ◽  
Ted Hackstadt ◽  
...  
Keyword(s):  
United States ◽  
Host Cell ◽  
Terminal Differentiation ◽  
The United States ◽  
Target Cells ◽  
Developmental Cycle ◽  
Content Type ◽  
Obligate Intracellular ◽  
Cell Form ◽  
Mucosal Surfaces

ABSTRACTBacteria of the genusChlamydiainclude the significant human pathogensChlamydia trachomatisandC. pneumoniae. All chlamydiae are obligate intracellular parasites that depend on infection of a host cell and transition through a biphasic developmental cycle. Following host cell invasion by the infectious elementary body (EB), the pathogen transitions to the replicative but noninfectious reticulate body (RB). Differentiation of the RB back to the EB is essential to generate infectious progeny. While the EB form has historically been regarded as metabolically inert, maintenance of infectivity during incubation with specific nutrients has revealed active maintenance of the infectious phenotype. Using transcriptome sequencing, we show that the transcriptome of extracellular EBs incubated under metabolically stimulating conditions does not cluster with germinating EBs but rather with the transcriptome of EBs isolated directly from infected cells. In addition, the transcriptional profile of the extracellular metabolizing EBs more closely resembled that of EB production than germination. Maintenance of infectivity of extracellular EBs was achieved by metabolizing chemically diverse compounds, including glucose 6-phosphate, ATP, and amino acids, all of which can be found in extracellular environments, including mucosal secretions. We further show that the EB cell type actively maintains infectivity in the inclusion after terminal differentiation. Overall, these findings contribute to the emerging understanding that the EB cell form is actively maintained through metabolic processes after terminal differentiation to facilitate prolonged infectivity within the inclusion and under host cell free conditions, for example, following deposition at mucosal surfaces.IMPORTANCEChlamydiae are obligate intracellular Gram-negative bacteria that are responsible for a wide range of diseases in both animal and human hosts. According to the Centers for Disease Control and Prevention,C. trachomatisis the most frequently reported sexually transmitted infection in the United States, costing the American health care system nearly $2.4 billion annually. Every year, there are over 4 million new cases ofChlamydiainfections in the United States and an estimated 100 million cases worldwide. To cause disease,Chlamydiamust successfully complete its complex biphasic developmental cycle, alternating between an infectious cell form (EB) specialized for initiating entry into target cells and a replicative form (RB) specialized for creating and maintaining the intracellular replication niche. The EB cell form has historically been considered metabolically quiescent, a passive entity simply waiting for contact with a host cell to initiate the next round of infection. Recent studies and data presented here demonstrate that the EB maintains its infectious phenotype by actively metabolizing a variety of nutrients. Therefore, the EB appears to have an active role in chlamydial biology, possibly within multiple environments, such as mucosal surfaces, fomites, and inside the host cell after formation.

scholarly journals Glycoside Hydrolase Genes Are Required for Virulence ofAgrobacterium tumefaciensonBryophyllum daigremontianaand Tomato

2019 ◽  
Vol 85 (15) ◽  
Author(s):  
Stephanie L. Mathews ◽  
Haylea Hannah ◽  
Hillary Samagaio ◽  
Camille Martin ◽  
Eleanor Rodriguez-Rassi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Host Cell ◽  
Plant Cell ◽  
Crown Gall ◽  
Plant Cell Wall ◽  
Tumor Formation ◽  
Glycoside Hydrolases ◽  
Plant Host ◽  
Content Type ◽  
Limited Digestion

ABSTRACTAgrobacterium tumefaciensis a rhizosphere bacterium that can infect wound sites on plants. The bacterium transfers a segment of DNA (T-DNA) from the Ti plasmid to the plant host cell via a type IV secretion system where the DNA becomes integrated into the host cell chromosomes. The expression of T-DNA in the plant results in tumor formation. Although the binding of the bacteria to plant surfaces has been studied previously, there is little work on possible interactions of the bacteria with the plant cell wall. Seven of the 48 genes encoding putative glycoside hydrolases (Atu2295,Atu2371,Atu3104,Atu3129,Atu4560,Atu4561, andAtu4665) in the genome ofA. tumefaciensC58 were found to play a role in virulence on tomato andBryophyllum daigremontiana. Two of these genes (pglAandpglB;Atu3129andAtu4560) encode enzymes capable of digesting polygalacturonic acid and, thus, may play a role in the digestion of pectin. One gene (arfA;Atu3104) encodes an arabinosylfuranosidase, which could remove arabinose from the ends of polysaccharide chains. Two genes (bglAandbglB;Atu2295andAtu4561) encode proteins with β-glycosidase activity and could digest a variety of plant cell wall oligosaccharides and polysaccharides. One gene (xynA;Atu2371) encodes a putative xylanase, which may play a role in the digestion of xylan. Another gene (melA;Atu4665) encodes a protein with α-galactosidase activity and may be involved in the breakdown of arabinogalactans. Limited digestion of the plant cell wall byA. tumefaciensmay be involved in tumor formation on tomato andB. daigremontiana.IMPORTANCEA. tumefaciensis used in the construction of genetically engineered plants, as it is able to transfer DNA to plant hosts. Knowledge of the mechanisms of DNA transfer and the genes required will aid in the understanding of this process. Manipulation of glycoside hydrolases may increase transformation and widen the host range of the bacterium.A. tumefaciensalso causes disease (crown gall tumors) on a variety of plants, including stone fruit trees, grapes, and grafted ornamentals such as roses. It is possible that compounds that inhibit glycoside hydrolases could be used to control crown gall disease caused byA. tumefaciens.

scholarly journals Cyclic di-AMP Is Critical for Listeria monocytogenes Growth, Cell Wall Homeostasis, and Establishment of Infection

mBio ◽  
2013 ◽  
Vol 4 (3) ◽  
Author(s):  
Chelsea E. Witte ◽  
Aaron T. Whiteley ◽  
Thomas P. Burke ◽  
John-Demian Sauer ◽  
Daniel A. Portnoy ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Bacterial Growth ◽  
Genetic Screen ◽  
Content Type ◽  
Forward Genetic Screen ◽  
Innate Immune Signaling ◽  
Food Borne Illness ◽  
Multidrug Resistance Transporters

ABSTRACT Listeria monocytogenes infection leads to robust induction of an innate immune signaling pathway referred to as the cytosolic surveillance pathway (CSP), characterized by expression of beta interferon (IFN-β) and coregulated genes. We previously identified the IFN-β stimulatory ligand as secreted cyclic di-AMP. Synthesis of c-di-AMP in L. monocytogenes is catalyzed by the diadenylate cyclase DacA, and multidrug resistance transporters are necessary for secretion. To identify additional bacterial factors involved in L. monocytogenes detection by the CSP, we performed a forward genetic screen for mutants that induced altered levels of IFN-β. One mutant that stimulated elevated levels of IFN-β harbored a transposon insertion in the gene lmo0052. Lmo0052, renamed here PdeA, has homology to a cyclic di-AMP phosphodiesterase, GdpP (formerly YybT), of Bacillus subtilis and is able to degrade c-di-AMP to the linear dinucleotide pApA. Reduction of c-di-AMP levels by conditional depletion of the di-adenylate cyclase DacA or overexpression of PdeA led to marked decreases in growth rates, both in vitro and in macrophages. Additionally, mutants with altered levels of c-di-AMP had different susceptibilities to peptidoglycan-targeting antibiotics, suggesting that the molecule may be involved in regulating cell wall homeostasis. During intracellular infection, increases in c-di-AMP production led to hyperactivation of the CSP. Conditional depletion of dacA also led to increased IFN-β expression and a concomitant increase in host cell pyroptosis, a result of increased bacteriolysis and subsequent bacterial DNA release. These data suggest that c-di-AMP coordinates bacterial growth, cell wall stability, and responses to stress and plays a crucial role in the establishment of bacterial infection. IMPORTANCE Listeria monocytogenes is a Gram-positive intracellular pathogen and the causative agent of the food-borne illness listeriosis. Upon infection, L. monocytogenes stimulates expression of IFN-β and coregulated genes dependent upon host detection of a secreted bacterial signaling nucleotide, c-di-AMP. Using a forward genetic screen for mutants that induced high levels of host IFN-β expression, we identified a c-di-AMP phosphodiesterase, PdeA, that degrades c-di-AMP. Here we characterize L. monocytogenes mutants that express enhanced or diminished levels of c-di-AMP. Decreased c-di-AMP levels by conditional depletion of the diadenylate cyclase (DacA) or overexpression of PdeA attenuated bacterial growth and led to bacteriolysis, suggesting that its production is essential for viability and may regulate cell wall metabolism. Mutants lacking PdeA had a distinct transcriptional profile, which may provide insight into additional roles for the molecule. This work demonstrates that c-di-AMP is a critical signaling molecule required for bacterial replication, cell wall stability, and pathogenicity.

scholarly journals Susceptibility of Inbred Mice to Rickettsia parkeri

2012 ◽  
Vol 80 (5) ◽  
pp. 1846-1852 ◽  
Author(s):  
Britton J. Grasperge ◽  
Kathryn E. Reif ◽  
Timothy D. Morgan ◽  
Piyanate Sunyakumthorn ◽  
Joseph Bynog ◽  
...  
Keyword(s):  
Inbred Mice ◽  
Spotted Fever ◽  
Inbred Mouse ◽  
Transmission Model ◽  
Mouse Strains ◽  
Spotted Fever Group ◽  
Limited Information ◽  
Rickettsia Parkeri ◽  
Content Type ◽  
Boutonneuse Fever

ABSTRACTRickettsia parkeri, a member of the spotted fever groupRickettsia, is the causative agent of American boutonneuse fever in humans. Despite the increased recognition of human cases, limited information is available regarding the infection of invertebrate and vertebrate hosts for this emerging tick-borne disease. Toward the development of a viable transmission model and to further characterize the pathology associated withR. parkeriinfection, inbred mouse strains (A/J, BALB/c, C3H/HeJ, and C3H/HeN) were intravenously and intradermally inoculated with 105low-passage-numberR. parkeri(Portsmouth strain), and infection, gross pathology, and histopathology were scored. Additionally, a quantitative real-time PCR (qPCR) was performed to estimate rickettsial load in heart, lung, spleen, and liver tissues of infected mice at 19 days postinoculation. Of the A/J, BALB/c, and C3H/HeN mice, none displayed universal pathology consistent with sustained infection. Compared to age-matched control mice, the intravenously inoculated C3H/HeJ mice exhibited marked facial edema and marked splenomegaly upon gross examination, while the intradermally inoculated mice developed characteristic eschar-like lesions. The C3H/HeJ mice also exhibited the greatest concentrations of rickettsial DNA from heart, lung, liver, and spleen samples when examined by qPCR. The similarity of the pathology of human disease and sustained infection suggests that the C3H/HeJ strain of mice is a promising candidate for subsequent experiments to examine the tick transmission, dissemination, and pathology ofR. parkeririckettsiosis.

scholarly journals The Rickettsial Ankyrin Repeat Protein 2 Is a Type IV Secreted Effector That Associates with the Endoplasmic Reticulum

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Stephanie S. Lehman ◽  
Nicholas F. Noriea ◽  
Karin Aistleitner ◽  
Tina R. Clark ◽  
Cheryl A. Dooley ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Host Cell ◽  
Spotted Fever ◽  
Rocky Mountain ◽  
Ankyrin Repeat ◽  
Avirulent Strain ◽  
Rocky Mountain Spotted Fever ◽  
Content Type ◽  
Type Iv ◽  
Plaque Phenotype

ABSTRACTStrains ofRickettsia rickettsii, the tick-borne agent of Rocky Mountain spotted fever, vary considerably in virulence. Genomic comparisons ofR. rickettsiistrains have identified a relatively small number of genes divergent in an avirulent strain. Among these is one annotated asRickettsiaankyrin repeat protein 2 (RARP-2). Homologs of RARP-2 are present in all strains ofR. rickettsii, but the protein in the avirulent strain Iowa contains a large internal deletion relative to the virulent Sheila Smith strain. RARP-2 is secreted in a type IV secretion system-dependent manner and exposed to the host cell cytosol. RARP-2 of Sheila Smith colocalizes with multilamellar membranous structures bearing markers of the endoplasmic reticulum (ER), whereas the Iowa protein shows no colocalization with host cell organelles and evidence of proteolytic degradation is detected. Overexpression of Sheila Smith RARP-2 inR. rickettsiiIowa converts this avirulent strain’s typically nonlytic or opaque plaque type to a lytic plaque phenotype similar to that of the virulent Sheila Smith strain. Mutation of a predicted proteolytic active site of Sheila Smith RARP-2 abolished the lytic plaque phenotype but did not eliminate association with host membrane. RARP-2 is thus a type IV secreted effector and released from the rickettsiae into the host cytosol to modulate host processes during infection. Overexpression of Sheila Smith RARP-2 did not, however, restore the virulence of the Iowa strain in a guinea pig model, likely due to the multifactorial nature of rickettsial virulence.IMPORTANCEMembers of the genusRickettsiaare obligate intracellular bacteria that exhibit a range of virulence from harmless endosymbionts of arthropods to the etiologic agents of severe disease. Despite the growing number of available genomes, little is known regarding virulence determinants of rickettsiae. Here, we have characterized an ankyrin repeat-containing protein, RARP-2, which differs between a highly virulent and an avirulent strain ofR. rickettsii, the agent of Rocky Mountain spotted fever. RARP-2 is secreted by a type IV secretion system into the cytosol of the host cell, where it interacts with and manipulates the structure of the endoplasmic reticulum. RARP-2 from the avirulent strain is truncated by the loss of seven of 10 ankyrin repeat units but, although secreted, fails to alter ER structure. Recognition of those rickettsial factors associated with virulence will facilitate understanding of regional and strain-specific variation in severity of disease.

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.

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