Bringing genetics to heretofore intractable obligate intracellular bacterial pathogens: Chlamydia and beyond

Ölander, Magnus; Sixt, Barbara S.

doi:10.1371/journal.ppat.1010669

Cited by 4 publications

(4 citation statements)

References 80 publications

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“…Better methods to determine the transformation efficiency in Chlamydia will have to be developed in the future. Furthermore, although clonal purification is a crucial step for the use of transformed bacteria in experiments ( 15 ), we omitted this step for initial confirmation because plaque formation is not visible for C. suis and limiting dilution is very challenging with incomplete success ( 26 , 51 ). To ensure a mostly clonal population of transformants for sequencing, we reduced the number of untransformed Chlamydia by growing transformed cultures multiple passages in the presence of antibiotic selection prior to sample collection.…”

Section: Discussionmentioning

confidence: 99%

“…In future studies, we would like to investigate the rrn-nqrF plasticity zone in further detail, particularly if complete vector integration is limited to this region and the role of individual genes/operons such as the rrn, inv , and nqrF during the integration process. With all the new genetic tools available ( 14 , 15 ), it would be interesting to investigate whether rrn-nqrF is equally transformable in other species. The recently published plasmid pBBR1MCS-4 could play a key role in exploring these complex dynamics considering that this broad host-range vector from Bordetella pertussis yielded similar results with complete vector integration in the incA region, located outside of the rrn-nqrF zone, and retention of free vector in the transformants ( 35 ).…”

Section: Discussionmentioning

confidence: 99%

“…Despite the clinical importance of Chlamydia , genetic manipulation tools to achieve a better understanding of chlamydial pathogenesis have only recently expanded ( 14 , 15 ). To date, the genetic toolbox for Chlamydia ranges from chemical mutagenesis to gene disruption by fluorescence-reported allelic exchange mutagenesis (FRAEM) ( 16 ) or TargeTron ( 17 ), and was recently enhanced by the implementation of CRISPRi technology ( 18 ).…”

Section: Introductionmentioning

confidence: 99%

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Chlamydia suisdisplays high transformation capacity with complete cloning vector integration into the chromosomalrrn-nqrFplasticity zone

Marti,

Biggel,

Shima

et al. 2023

Microbiol Spectr

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Chlamydia , comprising several human and zoonotic pathogens, is a genus of the conserved bacterial phylum Chlamydiota. Their obligate intracellular niche serves as a barrier for natural genetic exchange via horizontal gene transfer (HGT), and further limits the development and application of genetic tools. To date, the only example for recent inter-phylum HGT among the Chlamydiota is tetracycline resistance in the potentially zoonotic species Chlamydia suis , a close phylogenetic relative of human C. trachomatis , which causes bacterial sexually transmitted infections and ocular trachoma. Tetracycline resistance in porcine C. suis strains has been described worldwide and is always part of a genomic island dividing invasin ( inv ), located within a chromosomal region between the rRNA operon ( rrn ) and the nqrF reductase. Here, we aimed to expand the still modest number of available genetic manipulation systems for Chlamydia by generating allele-replacement and integration vectors for C. suis . These vectors comprised homologous C. suis sequences of the chromosomal region of interest, an E. coli origin of replication ( ori ) and selection markers but lacked the native chlamydial plasmids or its ori . We first recovered allele-replacement mutants using a vector that targets the tryptophan ( trp ) operon of C. suis . The vector was further successfully maintained as a free plasmid in C. trachomatis without allele replacement, suggesting complex plasmid dynamics in the absence of a chlamydial ori . Moreover, we showed that the hypervariable rrn-nqrF intergenic region of C. suis is highly susceptible to transformation, resulting in complete vector integration upstream of nqrF without interruption of the targeted inv gene. IMPORTANCE The obligate intracellular Chlamydia genus contains many pathogens with a negative impact on global health and economy. Despite recent progress, there is still a lack of genetic tools limiting our understanding of these complex bacteria. This study provides new insights into genetic manipulation of Chlamydia with the opportunistic porcine pathogen Chlamydia suis , the only chlamydial species naturally harboring an antibiotic resistance gene, originally obtained by horizontal gene transfer. C. suis is transmissible to humans, posing a potential public health concern. We report that C. suis can take up vectors that lack the native plasmid, a requirement for most chlamydial transformation systems described to date. Additionally, we show that C. trachomatis , the most common cause for bacterial sexually transmitted infections and infectious blindness worldwide, can be transformed with C. suis vectors. Finally, the chromosomal region that harbors the resistance gene of C. suis is highly susceptible to complete vector integration.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Chlamydia suisdisplays high transformation capacity with complete cloning vector integration into the chromosomalrrn-nqrFplasticity zone

Marti,

Biggel,

Shima

et al. 2023

Microbiol Spectr

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“…While there are inherent difficulties in the genetic manipulation of obligate intracellular bacteria such as Chlamydia, it is noteworthy that ongoing refinement of novel genetic tools continues at a rapid pace. These future improvements presently include the potential development of counterselectable markers to replace genes at desired sites in the chlamydial chromosome (now possible with the obligate intracellular bacterial pathogen Coxiella burnetii ) [ 91 ], saturation mutagenesis enabling transposon-insertion sequencing [ 92 ], and the creation of synthetic riboswitches to control gene expression in Chlamydia [ 93 , 94 ].…”

Section: Potential For Use As a Bioweaponmentioning

confidence: 99%

Psittacosis: An Underappreciated and Often Undiagnosed Disease

Dembek,

Mothershead,

Owens

et al. 2023

Pathogens

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The bacterial agent Chlamydia psittaci, and the resulting disease of psittacosis, is a little-known and underappreciated infectious disease by healthcare practitioners and in public health in general. C. psittaci infections can cause significant psittacosis outbreaks with pandemic potential, with person-to-person transmission being documented in the last decade. In this publication, we review the pathogen and its disease, as well as examine the potential for genetic manipulation in this organism to create a more deadly pathogen. Recent disease surveys indicate that currently, the highest incidences of human disease exist in Australia, Germany and the UK. We recommend the universal public health reporting of C. psittaci and psittacosis disease and increasing the promotion of public health awareness.

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Development of a suite of Proteus mirabilis -derived urea-inducible promoters

Fitzgerald,

Scavone,

Moolaveesala

et al. 2024

Appl Environ Microbiol

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Catheter-associated urinary tract infections (CAUTIs) are a significant burden on healthcare systems, accounting for up to 40% of hospital-acquired infections globally. A prevalent CAUTI pathogen, Proteus mirabilis, is an understudied Gram-negative bacterium. One sequela of P. mirabilis CAUTI is the production of urinary stones, which complicates treatment and clearing of the infection. Stone formation is induced by the activity of urease, a nickel-metalloenzyme that is regulated by UreR in a urea-dependent manner. As urea is abundant in the urinary tract, urease genes are highly expressed during experimental UTI. We sought to leverage the urease promoter to create an expression system that would enable urea-inducible expression of genes during in vitro experiments as well as during experimental UTI. During preliminary studies, we observed unexpectedly high levels of basal expression of the urease promoter. This was somewhat dependent on the presence of regulator UreR. To further develop this expression system, we generated a series of reporter constructs to assess the impact of specific promoter elements on promoter activity in the presence and absence of urea. Elements of interest included known regulatory binding sites, alternative translational start sites, and single-nucleotide polymorphisms identified through comparative genomics. This work describes a suite of urea-inducible promoters, constructed during this study, that exhibit a variety of expression dynamics, providing a customizable platform for gene expression. IMPORTANCE Urea is an inexpensive molecule that can easily be supplied during in vitro experiments. A urea-inducible promoter would also be activated by environments where urea naturally occurs, such as in the urinary tract. Thus, the development of a urea-inducible system for selective gene expression is of great interest to the field of uropathogenesis as it would enable selective gene induction during experimental urinary tract infection. This expression system would also have important applications for recombinant protein production in biotech and manufacturing.

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Bringing genetics to heretofore intractable obligate intracellular bacterial pathogens: Chlamydia and beyond

Cited by 4 publications

References 80 publications

Chlamydia suisdisplays high transformation capacity with complete cloning vector integration into the chromosomalrrn-nqrFplasticity zone

Chlamydia suisdisplays high transformation capacity with complete cloning vector integration into the chromosomalrrn-nqrFplasticity zone

Psittacosis: An Underappreciated and Often Undiagnosed Disease

Development of a suite of Proteus mirabilis -derived urea-inducible promoters

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