Developmental differentiation in a cytoplasm-dwelling obligate intracellular bacterium

AbstractDevelopmental differentiation has been described for several vacuole-dwelling obligate intracellular bacteria but never for an obligate intracellular bacterium that resides in the cytoplasm. Here, we show that the cytoplasm-dwelling obligate intracellular bacterium Orientia tsutsugamushi (Ot) exists in five distinct subpopulations. We show that Ot differentiates into a distinct, metabolically inactive, extracellular state upon budding from the surface of host cells and that this stage is preceded by a surface-associated maturation stage. We identify proteins that are differentially expressed in intracellular, replicative bacteria and extracellular, metabolically inactive bacteria. Metabolic activity resumes rapidly upon entry into the cytoplasm and is triggered by the host cell reducing environment. This example of developmental differentiation in a species of Rickettsiaceae provides a new model system for studying synchronized differentiation in a bacterium that has a minimal genome and where the interactions between bacterium and host cell are more direct than they are for bacteria separated from the eukaryote cell host by a vacuolar membrane.Author SummaryScrub typhus is a life-threatening human infection that is caused by the bacterium Orientia tsutsugamushi and spread by mites. Although the disease is estimated to affect at least one million people annually and is often fatal, the infectious agent is much less well understood than many other pathogens. O. tsutsugamushi is an intracellular bacterium that can only grow and divide within eukaryotic cells. During infection, it is found primarily in the cells that make up the lining of blood vessels and in certain immune cell types. O. tsutsugamushi bacteria can remain inside a single infected cell for seven days or more before budding out. The ways in which the bacterium itself changes during the course of an intracellular infection cycle have not been studied. In the current work, we used a range of techniques to show that O. tsutsugamushi differentiates into five distinct subpopulations, and that these are associated with measurable differences in metabolic activity, replication and infectivity. This work opens new avenues of research into the regulation and mechanisms of differentiation of O. tsutsugamushi, which could lead to improved diagnosis and treatments. It also provides a new model system for studying fundamental questions about bacterial development.

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Coxiella burnetiiType 4B Secretion System-dependent manipulation of endolysosomal maturation is required for bacterial growth

10.1101/645382 ◽

2019 ◽

Author(s):

Dhritiman Samanta ◽

Tatiana M. Clemente ◽

Stacey D. Gilk

Keyword(s):

Host Cell ◽

Secretion System ◽

Intracellular Bacterium ◽

Host Cells ◽

Acidic Ph ◽

Obligate Intracellular Bacterium ◽

Cell Infection ◽

Obligate Intracellular ◽

Endosomal Acidification ◽

Infected Cells

AbstractUpon host cell infection, the obligate intracellular bacteriumC. burnetiiresides and multiplies within theCoxiella–ContainingVacuole (CCV). The nascent CCV progresses through the endosomal maturation pathway into a phagolysosome, acquiring lysosomal markers as well as acidic pH and active proteases and hydrolases. Approximately 24-48 hours post infection, heterotypic fusion between the CCV and host endosomes/lysosomes leads to CCV expansion and subsequent bacterial replication in the mature CCV. Initial CCV acidification is required to activateC. burnetiimetabolism and the Type 4B Secretion System (T4BSS), which secretes effector proteins required for CCV maturation. However, we recently found that the mature CCV is less acidic (pH~5.2) than lysosomes (pH~4.8). Further, CCV acidification to pH~4.8 causesC. burnetiilysis, suggestingC. burnetiiactively regulates CCV pH. Because heterotypic fusion with host endosomes/lysosomes may influence CCV pH, we investigated endosomal maturation in cells infected with wildtype (WT) or T4BSS mutant (ΔdotA)C. burnetii. We observed significantly fewer LAMP1-positive lysosomes, along with less acidic “mature” endosomes (pH~5.8), in WT-infected cells, compared to mock or ΔdotA-infected cells. Further, while endosomes progressively acidified from the periphery (pH~5.5) to the perinuclear area (pH~4.7) in both mock and ΔdotA-infected cells, endosomes did not acidify beyond pH~5.2 in WT-infected cells, indicating that theC. burnetiiT4BSS inhibits endosomal maturation. Finally, increasing the number of acidic lysosomes by overexpressing the transcription factor EB inhibitedC. burnetiigrowth, indicating lysosomes are detrimental toC. burnetii. Overall, our data suggest thatC. burnetiiregulates CCV pH, possibly by reducing the number of host lysosomes available for heterotypic fusion.Author summaryThe obligate intracellular bacteriumCoxiella burnetiicauses human Q fever, which manifests as a flu-like illness but can develop into a life-threatening and difficult to treat endocarditis.C. burnetii,in contrast to many other intracellular bacteria, thrives within a lysosome-like vacuole in host cells. However, we previously found that theC. burnetiivacuole is not as acidic as lysosomes and increased acidification kills the bacteria, suggesting thatC. burnetiiregulates the pH of its vacuole. Here, we discovered thatC. burnetiiblocks endosomal maturation and acidification during host cell infection, resulting in fewer lysosomes in the host cell. Moreover, increasing lysosomes in the host cells blockedC. burnetiigrowth. Together, our study suggests thatC. burnetiiregulates vacuole acidity and blocks endosomal acidification in order to produce a permissive intracellular niche.

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Exploitation of the Endocytic Pathway by Orientia tsutsugamushi in Nonprofessional Phagocytes

Infection and Immunity ◽

10.1128/iai.01620-05 ◽

2006 ◽

Vol 74 (7) ◽

pp. 4246-4253 ◽

Cited By ~ 33

Author(s):

Hyuk Chu ◽

Jung-Hee Lee ◽

Seung-Hoon Han ◽

Se-Yoon Kim ◽

Nam-Hyuk Cho ◽

...

Keyword(s):

Host Cell ◽

Causative Agent ◽

Scrub Typhus ◽

Adaptor Protein ◽

Endocytic Pathway ◽

Intracellular Bacterium ◽

Orientia Tsutsugamushi ◽

Obligate Intracellular Bacterium ◽

Obligate Intracellular ◽

Endocytic Vesicles

ABSTRACT Orientia tsutsugamushi, a causative agent of scrub typhus, is an obligate intracellular bacterium that requires the exploitation of the endocytic pathway in the host cell. We observed the localization of O. tsutsugamushi with clathrin or adaptor protein 2 within 30 min after the infection of nonprofessional phagocytes. We have further confirmed that the infectivity of O. tsutsugamushi is significantly reduced by drugs that block clathrin-mediated endocytosis but not by filipin III, an inhibitor that blocks caveola-mediated endocytosis. In the present study, with a confocal microscope, O. tsutsugamushi was sequentially colocalized with the early and late endosomal markers EEA1 and LAMP2, respectively, within 1 h after infection. The colocalization of O. tsutsugamushi organisms with EEA1 and LAMP2 gradually disappeared until 2 h postinfection, and then free O. tsutsugamushi organisms were found in the cytoplasm. When the acidification of endocytic vesicles was blocked by treating the cells with NH4Cl or bafilomycin A, the escape of O. tsutsugamushi organisms from the endocytic pathway was severely impaired, and the infectivity of O. tsutsugamushi was drastically reduced. To our knowledge, this is the first report that the invasion of O. tsutsugamushi is dependent on the clathrin-dependent endocytic pathway and the acidification process of the endocytic vesicles in nonprofessional phagocytes.

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The Whole-genome Sequencing of the Obligate Intracellular Bacterium Orientia tsutsugamushi Revealed Massive Gene Amplification During Reductive Genome Evolution

DNA Research ◽

10.1093/dnares/dsn011 ◽

2008 ◽

Vol 15 (4) ◽

pp. 185-199 ◽

Cited By ~ 113

Author(s):

K. Nakayama ◽

A. Yamashita ◽

K. Kurokawa ◽

T. Morimoto ◽

M. Ogawa ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Evolution ◽

Gene Amplification ◽

Genome Sequencing ◽

Intracellular Bacterium ◽

Whole Genome ◽

Orientia Tsutsugamushi ◽

Obligate Intracellular Bacterium ◽

Obligate Intracellular

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Extrusions promote engulfment and Chlamydia survival within macrophages

10.1101/041079 ◽

2016 ◽

Cited By ~ 1

Author(s):

Meghan Zuck ◽

Tisha C Ellis ◽

Anthony Venida ◽

Kevin Hybiske

Keyword(s):

Immune Responses ◽

Host Cell ◽

Membrane Structure ◽

Functional Role ◽

Host Cells ◽

Obligate Intracellular Bacterium ◽

Obligate Intracellular ◽

Primary Bone ◽

New Hosts ◽

Primary Bone Marrow

All obligate intracellular pathogens must exit their host cells in order to propagate and survive as a species; the precise strategies they use have a direct impact on their ability to disseminate within a host, transmit to new hosts, and engage or avoid immune responses. The obligate intracellular bacterium Chlamydia trachomatis exits the host cell by two distinct exit strategies, lysis and extrusion. Despite being equally active pathways, lysis and extrusion differ greatly in their mechanisms. The defining characteristics of extrusions, and advantages gained by Chlamydia within this unique double-membrane structure are not well understood. Here, we present data that defines extrusions as being largely devoid of host organelles, comprised mostly of Chlamydia elementary bodies, and containing phosphatidylserine on the outer surface of the extrusion membrane. Towards defining a functional role for extrusions in Chlamydia pathogenesis, we demonstrate that extrusions confer significant infectious advantages for Chlamydia by serving as transient, intracellular-like niches for extracellular Chlamydia, as compared to Chlamydia that would otherwise exit by lysing the host cell. In addition to enhanced survival outside of the host cell, we report the key discovery that chlamydial extrusions can be engulfed by primary bone marrow-derived macrophages, after which they provide a protective microenvironment for Chlamydia. Extrusion-derived Chlamydia were able to stave off macrophage based killing beyond 8 h, and culminated in the release of infectious EB from the macrophage. Based on these findings, we propose a model in which a major outcome of Chlamydia exiting epithelial cells inside extrusions is to hijack macrophages as vehicles for dissemination within the host.

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Orientia tsutsugamushi Strain Ikeda Ankyrin Repeat-Containing Proteins Recruit SCF1 Ubiquitin Ligase Machinery via Poxvirus-Like F-Box Motifs

Journal of Bacteriology ◽

10.1128/jb.00276-15 ◽

2015 ◽

Vol 197 (19) ◽

pp. 3097-3109 ◽

Cited By ~ 13

Author(s):

Andrea R. Beyer ◽

Lauren VieBrock ◽

Kyle G. Rodino ◽

Daniel P. Miller ◽

Brittney K. Tegels ◽

...

Keyword(s):

Host Cell ◽

Ubiquitin Ligase ◽

Scrub Typhus ◽

Ankyrin Repeat ◽

Intracellular Bacterium ◽

Host Cells ◽

Orientia Tsutsugamushi ◽

Ligand Interaction ◽

Content Type ◽

Cell Functions

ABSTRACTA rising theme among intracellular microbes is the delivery of ankyrin repeat-containing effectors (Anks) that interact with target proteins to co-opt host cell functions.Orientia tsutsugamushi, an obligate intracellular bacterium and the etiologic agent of scrub typhus, encodes one of the largest Ank repertoires of any sequenced microorganism. They have been previously identified as type 1 secretion system substrates. Here,in silicoand manual sequence analyses revealed that a large proportion ofO. tsutsugamushistrain Ikeda Anks bear a eukaryotic/poxvirus-like F-box motif, which is known to recruit host cell SCF1 ubiquitin ligase machinery. We assessed the Anks for the ability to serve as F-box proteins. Coimmunoprecipitation assays demonstrated that F-box-containing Anks interact with overexpressed and/or endogenous SCF1 components. When coexpressed with FLAG-Ank4_01 or FLAG-Ank9, a glutathioneS-transferase (GST)-tagged version of the SCF1 component SKP1 localized to subcellular sites of FLAG-Ank accumulation. The abilities of recombinant Anks to interact and colocalize with SKP1 were F-box dependent. GST-SKP1 precipitatedO. tsutsugamushi-derived Ank9 from infected host cells, verifying both that the pathogen expresses Ank9 during infection and the protein's capability to bind SKP1. AligningO. tsutsugamushi, poxviral, and eukaryotic F-box sequences delineated three F-box residues that are highly conserved and likely to be functionally important. Substitution of these residues ablated the ability of GFP-Ank9 to interact with GST-SKP1. These results demonstrate thatO. tsutsugamushistrain Ikeda Anks can co-opt host cell polyubiquitination machinery, provide the first evidence that anO. tsutsugamushiAnk does so during infection, and advance overall understanding of microbial F-box proteins.IMPORTANCEAnkyrin repeat-containing proteins (Anks) are important virulence factors of intracellular bacteria that mediate protein-protein interactions with host cell targets.Orientia tsutsugamushi, which causes a debilitating infection called scrub typhus in one of the most densely populated regions of the world, encodes one of the largest Ank armamentariums of any sequenced bacterium. This study demonstrates thatO. tsutsugamushistrain Ikeda Anks also bear F-box motifs that interact with host cell polyubiquitination machinery. By proving that anOrientia-derived Ank interacts with SKP1 in infected cells, this evidences the first bona fideOrientiaeffector and the first example of an endogenous F-box-containing Ank–mammalian-host ligand interaction for any intracellular bacterium. Also, importantly, this work identifies key residues that are essential for microbial F-box function.

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

10.1101/277160 ◽

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.

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Host range of obligate intracellular bacterium Parachlamydia acanthamoebae

Microbiology and Immunology ◽

10.1111/j.1348-0421.2010.00265.x ◽

2010 ◽

Vol 54 (11) ◽

pp. 707-713 ◽

Cited By ~ 11

Author(s):

Yasuhiro Hayashi ◽

Shinji Nakamura ◽

Junji Matsuo ◽

Tatsuya Fukumoto ◽

Mitsutaka Yoshida ◽

...

Keyword(s):

Host Range ◽

Intracellular Bacterium ◽

Obligate Intracellular Bacterium ◽

Obligate Intracellular

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

Eukaryotic Cell ◽

10.1128/ec.00081-14 ◽

2014 ◽

Vol 13 (8) ◽

pp. 965-976 ◽

Cited By ~ 32

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.

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Invasion of Toxoplasma gondii occurs by active penetration of the host cell

Journal of Cell Science ◽

10.1242/jcs.108.6.2457 ◽

1995 ◽

Vol 108 (6) ◽

pp. 2457-2464 ◽

Cited By ~ 4

Author(s):

J.H. Morisaki ◽

J.E. Heuser ◽

L.D. Sibley

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Tyrosine Phosphorylation ◽

Host Cells ◽

The Novel ◽

Host Proteins ◽

Host Cell Membrane ◽

Membrane Ruffling ◽

Obligate Intracellular ◽

Obligate Intracellular Parasite

Toxoplasma gondii is an obligate intracellular parasite that infects a wide variety of vertebrate cells including macrophages. We have used a combination of video microscopy and fluorescence localization to examine the entry of Toxoplasma into macrophages and nonphagocytic host cells. Toxoplasma actively invaded host cells without inducing host cell membrane ruffling, actin microfilament reorganization, or tyrosine phosphorylation of host proteins. Invasion occurred rapidly and within 25–40 seconds the parasite penetrated into a tight-fitting vacuole formed by invagination of the plasma membrane. In contrast, during phagocytosis of Toxoplasma, extensive membrane ruffling captured the parasite in a loose-fitting phagosome that formed over a period of 2–4 minutes. Phagocytosis involved both reorganization of the host cytoskeleton and tyrosine phosphorylation of host proteins. In some cases, parasites that were first internalized by phagocytosis, were able to escape from the phagosome by a process analogous to invasion. These studies reveal that active penetration of the host cell by Toxoplasma is fundamentally different from phagocytosis or induced endocytic uptake. The novel ability to penetrate the host cell likely contributes to the capability of Toxoplasma-containing vacuoles to avoid endocytic processing.

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