Recent advances in genetic systems in obligate intracellular human-pathogenic bacteria

Abstract

The ability to genetically manipulate a pathogen is fundamental to discovering factors governing host–pathogen interactions at the molecular level and is critical for devising treatment and prevention strategies. While the genetic “toolbox” for many important bacterial pathogens is extensive, approaches for modifying obligate intracellular bacterial pathogens were classically limited due in part to the uniqueness of their obligatory lifestyles. Many researchers have confronted these challenges over the past two and a half decades leading to the development of multiple approaches to construct plasmid-bearing recombinant strains and chromosomal gene inactivation and deletion mutants, along with gene-silencing methods enabling the study of essential genes. This review will highlight seminal genetic achievements and recent developments (past 5 years) for Anaplasma spp., Rickettsia spp., Chlamydia spp., and Coxiella burnetii including progress being made for the still intractable Orientia tsutsugamushi. Alongside commentary of the strengths and weaknesses of the various approaches, future research directions will be discussed to include methods for C. burnetii that should have utility in the other obligate intracellular bacteria. Collectively, the future appears bright for unraveling the molecular pathogenic mechanisms of these significant pathogens.

Molecular pathogenesis of Chlamydia trachomatis

“…The intracellular nature and unusual bi-phasic developmental cycle has, until recently, hampered progress on biological investigations and genetic manipulation of Chlamydia . Recently, genetic methods have rapidly progressed and accordingly our understanding of pathogenic mechanisms has increased ( Fisher and Beare, 2023 ). However, each genetic approach has their advantages and limitations [reviewed in, ( Banerjee and Nelson, 2021 ; Wan et al., 2023 )].…”

“…As such, specialised techniques were developed to better understand this clinically significant pathogen. Whole genome sequencing ( Stephens et al., 1998 ), allelic exchange mutagenesis ( Mueller et al., 2016 ), CRISPRi knockdown ( Ouellette, 2018 ; Ouellette et al., 2021 ) and intron insertion gene inactivation ( O'Neill et al., 2021 ) assist in associating genes with chlamydial phenotypic traits, bypassing impractical traditional approaches in genome studies [reviewed in ( Read and Massey, 2014 ; Fisher and Beare, 2023 ; Luu et al., 2023 )]. Nonetheless, many of these genetic manipulation approaches for Chlamydia are still relatively time consuming and requires expert culture skills, hence we set out to review what is known about potential virulence factors to guide priorities for future genetic experiments.…”

An expanded genetic toolkit for inducible expression and targeted gene silencing inRickettsia parkeri

“…For instance, by varying the timing and dosage of induction, this tool will allow us to study the kinetic requirements of a given virulence gene during the infectious life cycle. While conditional expression systems have been developed in C. burnetii and C. trachomatis , many obligate intracellular bacteria still lack them 42 . Here, we have adapted the Tet-On system for use in R. parkeri , enabling conditional gene expression for the first time in a Rickettsia species.…”

Requirement of GrgA for Chlamydia infectious progeny production, optimal growth, and efficient plasmid maintenance

“…Despite recent advancements in the genetic manipulation of Chlamydia ( 20 , 21 ), the tools available to study essential genes in this obligate intracellular bacterium remain limited. We attempted but failed to disrupt grgA through group II intron (Targetron) insertional mutagenesis ( 22 ).…”