Review Article | Published:

Engineering of obligate intracellular bacteria: progress, challenges and paradigms

Nature Reviews Microbiology volume 15, pages 544558 (2017) | Download Citation

Abstract

It is estimated that approximately one billion people are at risk of infection with obligate intracellular bacteria, but little is known about the underlying mechanisms that govern their life cycles. The difficulty in studying Chlamydia spp., Coxiella spp., Rickettsia spp., Anaplasma spp., Ehrlichia spp. and Orientia spp. is, in part, due to their genetic intractability. Recently, genetic tools have been developed; however, optimizing the genomic manipulation of obligate intracellular bacteria remains challenging. In this Review, we describe the progress in, as well as the constraints that hinder, the systematic development of a genetic toolbox for obligate intracellular bacteria. We highlight how the use of genetically manipulated pathogens has facilitated a better understanding of microbial pathogenesis and immunity, and how the engineering of obligate intracellular bacteria could enable the discovery of novel signalling circuits in host–pathogen interactions.

Key points

  • Extracellular bacteria are free-living organisms, whereas facultative intracellular bacteria replicate either inside eukaryotic host cells or in an environmental niche.

  • Obligate intracellular bacteria, which include Chlamydia spp., Anaplasma spp., Ehrlichia spp., Rickettsia spp., Orientia spp. and Coxiella spp., replicate exclusively inside of eukaryotic host cells.

  • Genetic tools for the manipulation of obligate intracellular bacteria have historically been limited; however, there has been considerable recent progress in refining these methods. Such tools include transformation strategies, shuttle vectors, random and targeted mutagenesis through allelic exchange, and mobile group II introns.

  • Novel bacterial molecules that shed light on both microbial pathogenesis mechanisms and host cell biology have been characterized by applying genetic tools to study Chlamydia trachomatis serovar L2 and Rickettsia parkeri.

  • Vaccines against obligate intracellular bacterial infections are lacking. Refining genetic tools would enable the characterization of virulence factors and the development of vaccine candidates.

  • Key questions in bacterial pathogenesis and physiology are primed for investigation once all obligate intracellular bacteria can be genetically manipulated on a routine basis.

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Acknowledgements

The authors apologize to those colleagues whose work could not be cited owing to the broad scope of the Review and space limitations. Work in the authors' laboratories was supported by the US National Institutes of Health (Institutional Training Grant T32AI007540 to E.E.M.; R01 AI072683, R56 AI123346 and R21 AI122014 to J.A.C.; R01 AI100759 to R.H.V.; U19 AI084044 to P.M.B.; R01 AI070908 to R.R.G.; R01 AI020384 and R21 AI103272 to D.O.W.; R01 AI042792 to U.G.M. and K.A.B.; R01 AI072606 and R21 AI111086 to J.J.M.; R01 AI106859 and R21 AI115449 to J.W.M.; and R01 AI093653 and R01AI116523 to J.H.F.P.), the US Department of Agriculture (USDA-ARS 2090-32000-038-00D to S.M.N.), the Center of Excellence for Vector-Borne Diseases at Kansas State University (to R.R.G.) and the University of Maryland School of Medicine (to J.H.F.P.). The content of this Review is solely the responsibility of the authors and does not necessarily represent the official views of the US National Institute of Allergy and Infectious Diseases or the US National Institutes of Health.

Author information

Author notes

    • Ulrike G. Munderloh
    •  & Joao H. F. Pedra

    These authors contributed equally to this work.

Affiliations

  1. Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.

    • Erin E. McClure
    • , Adela S. Oliva Chávez
    • , Dana K. Shaw
    •  & Joao H. F. Pedra
  2. Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298, USA.

    • Jason A. Carlyon
  3. Center of Excellence for Vector-Borne Diseases, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas 66506, USA.

    • Roman R. Ganta
  4. Animal Disease Research Unit, Agricultural Research Service, U.S. Department of Agriculture and the Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington 99164, USA.

    • Susan M. Noh
  5. Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, Alabama 36688, USA.

    • David O. Wood
  6. Department of Microbial Pathogenesis, University of Maryland School of Dentistry, Baltimore, Maryland 21201, USA.

    • Patrik M. Bavoil
  7. Department of Veterinary Microbiology and Pathology and the Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington, 99164, USA.

    • Kelly A. Brayton
  8. Vector Borne Disease Laboratories, Department of Pathobiological Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, Louisiana 70803, USA.

    • Juan J. Martinez
  9. Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555, USA.

    • Jere W. McBride
  10. Department for Molecular Genetics and Microbiology, Duke University, Durham, North Carolina 27710, USA.

    • Raphael H. Valdivia
  11. Department of Entomology, University of Minnesota, St. Paul, Minnesota 55108, USA.

    • Ulrike G. Munderloh

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Correspondence to Joao H. F. Pedra.

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    Disease pathogenesis and immune response against selected obligates.

Glossary

Transformation

A technique that induces bacteria to take up exogenous DNA molecules, usually through chemical or electrical methods.

Polyamidoamine dendrimers

(PAMAM dendrimers). Highly branched polymers that can be used to deliver small molecules or DNA to cells.

Allelic exchange

A common method that is used to knock in, knock out or otherwise mutagenize a DNA segment that relies on homologous recombination between the wild-type gene and an exogenous DNA construct.

Fluorescence-reported allelic exchange mutagenesis

(FRAEM). A method for allelic exchange in Chlamydia trachomatis serovar L2 that can be monitored byobserving fluorescent chlamydial inclusions.

Dot/Icm type IV secretion system

A set of bacterial proteins that inject effector molecules into the eukaryotic host cytosol to remodel the intracellular niche.

Mariner transposon

An abundant class II transposable element first discovered in Drosophila spp. that integrates into a wide range of genomes.

Ethyl methanesulfonate mutagenesis

(EMS mutagenesis). A technique in which a DNA-alkylating agent (EMS) is applied to a population of cells to create a library of strains that contain random mutations.

Mobile group II introns

Mobile bacterial ribozymes that self-splice, reverse transcribe the spliced RNA into DNA, and then integrate the DNA into the bacterial chromosome.

Necrosis

A type of inflammatory cell death that occurs spontaneously after damage to a cell.

Apoptosis

A mode of non-inflammatory programmed cell death.

Stimulator of interferon genes

(STING). An endoplasmic reticulum-associated cytosolic intracellular pattern recognition molecule that senses cyclic dinucleotides and induces the production of type I interferons.

Vinculin

A mammalian cytoskeletal protein that anchors the cell membrane to the actin cytoskeleton.

Granulocytic anaplasmosis

A mild-to-severe tick-borne infectious disease caused by Anaplasma phagocytophilum, which infects neutrophils and myeloid cells, that is characterized by fever, thrombocytopenia, leukopenia and liver damage.

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DOI

https://doi.org/10.1038/nrmicro.2017.59

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