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 tw… Show more
“…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 )].…”
Section: Introductionmentioning
confidence: 99%
“…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.…”
Chlamydia trachomatis is a strict intracellular human pathogen. It is the main bacterial cause of sexually transmitted infections and the etiologic agent of trachoma, which is the leading cause of preventable blindness. Despite over 100 years since C. trachomatis was first identified, there is still no vaccine. However in recent years, the advancement of genetic manipulation approaches for C. trachomatis has increased our understanding of the molecular pathogenesis of C. trachomatis and progress towards a vaccine. In this mini-review, we aimed to outline the factors related to the developmental cycle phase and specific pathogenesis activity of C. trachomatis in order to focus priorities for future genetic approaches. We highlight the factors known to be critical for developmental cycle stages, gene expression regulatory factors, type III secretion system and their effectors, and individual virulence factors with known impacts.
“…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 )].…”
Section: Introductionmentioning
confidence: 99%
“…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.…”
Chlamydia trachomatis is a strict intracellular human pathogen. It is the main bacterial cause of sexually transmitted infections and the etiologic agent of trachoma, which is the leading cause of preventable blindness. Despite over 100 years since C. trachomatis was first identified, there is still no vaccine. However in recent years, the advancement of genetic manipulation approaches for C. trachomatis has increased our understanding of the molecular pathogenesis of C. trachomatis and progress towards a vaccine. In this mini-review, we aimed to outline the factors related to the developmental cycle phase and specific pathogenesis activity of C. trachomatis in order to focus priorities for future genetic approaches. We highlight the factors known to be critical for developmental cycle stages, gene expression regulatory factors, type III secretion system and their effectors, and individual virulence factors with known impacts.
“…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.…”
Pathogenic species within theRickettsiagenus are transmitted to humans through arthropod vectors and cause a spectrum of diseases ranging from mild to life-threatening. Despite rickettsiae posing an emerging global health risk, the genetic requirements of their infectious life cycles remain poorly understood. A major hurdle toward building this understanding has been the lack of efficient tools for genetic manipulation, owing to the technical difficulties associated with their obligate intracellular nature. To this end, we implemented the Tet-On system to enable conditional gene expression inRickettsia parkeri. Using Tet-On, we show inducible expression of antibiotic resistance and a fluorescent reporter. We further used this inducible promoter to screen the ability ofR. parkerito express four variants of the catalytically dead Cas9 (dCas9). We demonstrate that all four dCas9 variants can be expressed inR. parkeriand used for CRISPR interference (CRISPRi)-mediated targeted gene knockdown. We show targeted knockdown of an antibiotic resistance gene as well as the endogenous virulence factorsca2. Altogether, we have developed systems for inducible gene expression and CRISPRi-mediated gene knockdown for the first time in rickettsiae, laying the groundwork for more scalable, targeted mechanistic investigations into their infectious life cycles.
“…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 ).…”
Chlamydia
, an obligate intracellular bacterial pathogen, has a unique developmental cycle involving the differentiation of invading elementary bodies (EBs) to noninfectious reticulate bodies (RBs), replication of RBs, and redifferentiation of RBs into progeny EBs. Progression of this cycle is regulated by three sigma factors, which direct the RNA polymerase to their respective target gene promoters. We hypothesized that the
Chlamydia-
specific transcriptional regulator GrgA, previously shown to activate σ66 and σ28, plays an essential role in chlamydial development and growth. To test this hypothesis, we applied a novel genetic tool known as dependence on plasmid-mediated expression to create
Chlamydia trachomatis
with conditional GrgA deficiency. We show that GrgA-deficient
C. trachomatis
RBs have a growth rate that is approximately half of the normal rate and fail to transition into progeny EBs. In addition, GrgA-deficient
C. trachomatis
fails to maintain its virulence plasmid. Results of RNA-Seq analysis indicate that GrgA promotes RB growth by optimizing tRNA synthesis and expression of nutrient-acquisition genes, while it enables RB-to-EB conversion by facilitating the expression of a histone and outer membrane proteins required for EB morphogenesis. GrgA also regulates numerous other late genes required for host cell exit and subsequent EB invasion into host cells. Importantly, GrgA stimulates the expression of σ54, the third and last sigma factor, and its activator, AtoC, and thereby indirectly upregulating the expression of σ54-dependent genes. In conclusion, our work demonstrates that GrgA is a master transcriptional regulator in
Chlamydia
and plays multiple essential roles in chlamydial pathogenicity.
IMPORTANCE
Hallmarks of the developmental cycle of the obligate intracellular pathogenic bacterium
Chlamydia
are the primary differentiation of the infectious elementary body (EB) into the proliferative reticulate body (RB) and the secondary differentiation of RBs back into EBs. The mechanisms regulating these transitions remain unclear. In this report, we developed an effective novel strategy termed dependence on plasmid-mediated expression (DOPE) that allows for the knockdown of essential genes in
Chlamydia
. We demonstrate that GrgA, a
Chlamydia
-specific transcription factor, is essential for the secondary differentiation and optimal growth of RBs. We also show that GrgA, a chromosome-encoded regulatory protein, controls the maintenance of the chlamydial virulence plasmid. Transcriptomic analysis further indicates that GrgA functions as a critical regulator of all three sigma factors that recognize different promoter sets at developmental stages. The DOPE strategy outlined here should provide a valuable tool for future studies examining chlamydial growth, development, and pathogenicity.
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