Engineering of obligate intracellular bacteria: progress, challenges and paradigms

McClure, Erin E.; Chávez, Adela S. Oliva; Shaw, Dana K.; Carlyon, Jason A.; Ganta, Roman R.; Noh, Susan M.; Wood, Douglas; Bavoil, Patrik M.; Brayton, Kelly A.; Martínez, Juan José; McBride, Jere W.; Valdivia, Raphael H.; Munderloh, Ulrike G.; Pedra, Joao H. F.

doi:10.1038/nrmicro.2017.59

Cited by 126 publications

(120 citation statements)

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“…Development of the mariner transposase Himar1, which can function in many organisms Pelicic et al, 2000;Ashour and Hondalus, 2003) and the development of hyperactive Himar1 mutants have effectively diminished this last obstacle. Himar transposition provides an ideal system for global genome functional analysis of the AT-rich organism like Ehrlichia species (Munderloh et al, 2012;Cheng et al, 2013;McClure et al, 2017), since it recognizes AT sites and inserts at a single site per genome . In the present study, we constructed HF strain Himar1 transposon mutant library, and cloned selected mutants and characterized cloned mutant pathogenicity in laboratory mice.…”

Section: Introductionmentioning

confidence: 99%

Discovery of in vivo Virulence Genes of Obligatory Intracellular Bacteria by Random Mutagenesis

Bekebrede

Lin

Teymournejad

et al. 2020

Front. Cell. Infect. Microbiol.

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Ehrlichia spp. are emerging tick-borne obligatory intracellular bacteria that cause febrile and sometimes fatal diseases with abnormal blood cell counts and signs of hepatitis. Ehrlichia HF strain provides an excellent mouse disease model of fatal human ehrlichiosis. We recently obtained and established stable culture of Ehrlichia HF strain in DH82 canine macrophage cell line, and obtained its whole genome sequence and annotation. To identify genes required for in vivo virulence of Ehrlichia, we constructed random insertional HF strain mutants by using Himar1 transposon-based mutagenesis procedure. Of total 158 insertional mutants isolated via antibiotic selection in DH82 cells, 74 insertions were in the coding regions of 55 distinct protein-coding genes, including TRP120 and multicopy genes, such as p28/omp-1, virB2, and virB6. Among 84 insertions mapped within the non-coding regions, seven are located in the putative promoter region since they were within 50 bp upstream of the seven distinct genes. Using limited dilution methods, nine stable clonal mutants that had no apparent defect for multiplication in DH82 cells, were obtained. Mouse virulence of seven mutant clones was similar to that of wild-type HF strain, whereas two mutant clones showed significantly retarded growth in blood, livers, and spleens, and the mice inoculated with them lived longer than mice inoculated with wild-type. The two clones contained mutations in genes encoding a conserved hypothetical protein and a staphylococcal superantigen-like domain protein, respectively, and both genes are conserved among Ehrlichia spp., but lack homology to other bacterial genes. Inflammatory cytokine mRNA levels in the liver of mice infected with the two mutants were significantly diminished than those infected with HF strain wild-type, except IL-1β and IL-12 p40 in one clone. Thus, we identified two Ehrlichia virulence genes responsible for in vivo infection, but not for infection and growth in macrophages.Keywords: Ehrlichia HF strain, Himar1 transposon mutagenesis, mouse virulence, inflammatory cytokines, staphylococcal superantigen-like domain, obligatory intracellular bacteria IMPORTANCE Ehrlichiosis, a sometimes deadly febrile disease in man and animal, is caused by infection with Gram-negative obligatory intracellular bacteria, Ehrlichia. Ehrlichia species lack typical pathogen-associate molecular patterns (PAMPs), such as lipopolysaccharide, peptidoglycan, pili, and flagella, yet they induce acute and/or chronic inflammatory cytokines in infected animals.Bekebrede et al. In vivo Virulence Genes of EhrlichiaStudies to identify virulence factors and PAMPs of Ehrlichia species are hampered by the limitation to applicable genetic tools and small laboratory animal models. Mouse infection with Ehrlichia HF strain provides an excellent model for fatal human ehrlichiosis, as the HF strain is highly virulent in laboratory mice. However, due to inability to culture the HF stain, the use of this model has been limited. As we have succeeded in culturing and who...

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Section: Introductionmentioning

confidence: 99%

Discovery of in vivo Virulence Genes of Obligatory Intracellular Bacteria by Random Mutagenesis

Bekebrede

Lin

Teymournejad

et al. 2020

Front. Cell. Infect. Microbiol.

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“…These techniques can only diagnose bacterial infections when there has been significant systemic tissue damage,which poses challenges in treatment owing to the high bacterial burden despite the availability of antibiotics.T oc omplicate matters,s ome bacteria are able to survive inside the host cells,w hich render the available treatments ineffective,l eading to recurrent or chronic infections. [5] These intractable infections are difficult to treat owing to the challenge in detection and high resistance towards traditional antimicrobial therapies.T ot his end, the development of new strategies for detection and elimination of intracellular bacteria is highly attractive,w hich will have as ignificant impact in the treatment against bacterial infections.…”

Section: Introductionmentioning

confidence: 99%

An AIEgen‐Peptide Conjugate as a Phototheranostic Agent for Phagosome‐Entrapped Bacteria

Kenry

et al. 2019

Angew Chem Int Ed

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The detection and elimination of intracellular bacteria remain am ajor challenge.I nt his work, we report an aggregation-induced emission (AIE) bioprobe that can detect bacterial infection and kill bacteria surviving inside macrophages through adynamic process,notably specific molecular tailoring of the probe by caspase-1 activation in infected macrophages and accumulation of the residue on phagosomes containing bacteria, leading to light-up fluorescent signals. Moreover,t he AIEgen can serve as ap hotosensitizer for generation of reactive oxygen species (ROS);a nd the average ROSi ndicator fluorescent signal intensity per unit area in the bacterial phagosomes is approximately 2.7-fold higher than that in the cytoplasm. This,inturn, induces bacteria killing with high efficiency and minimal cytotoxicity towards macrophages. We envision that this specific light-up bioprobe may provide an ew approach for selective and sensitive detection and eradication of intracellular bacterial infections.

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“…The genomic simplicity of these organisms combined with their intimate relationship with their cellular hosts makes them attractive systems for studying bacterial physiology, host–pathogen biology, and questions about how bacteria sense and respond to their environment. Obligate intracellular bacteria are difficult to propagate in the laboratory, however, and are mostly genetically intractable 9 , thus our understanding of their biology lags behind that of free-living counterparts.…”

Section: Introductionmentioning

confidence: 99%

The obligate intracellular bacterium Orientia tsutsugamushi differentiates into a developmentally distinct extracellular state

Atwal

Wongsantichon

Giengkam

et al. 2020

Preprint

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Developmental 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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Engineering of obligate intracellular bacteria: progress, challenges and paradigms

Cited by 126 publications

References 112 publications

Discovery of in vivo Virulence Genes of Obligatory Intracellular Bacteria by Random Mutagenesis

Discovery of in vivo Virulence Genes of Obligatory Intracellular Bacteria by Random Mutagenesis

An AIEgen‐Peptide Conjugate as a Phototheranostic Agent for Phagosome‐Entrapped Bacteria

The obligate intracellular bacterium Orientia tsutsugamushi differentiates into a developmentally distinct extracellular state

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