Affiliation:
1. Department of Microbiology & Molecular Genetics, The University of Texas Health Science Center, Houston, Texas, USA
Abstract
ABSTRACT
Gene inactivation by creating in-frame deletion mutations in
Fusobacterium nucleatum
is time consuming, and most fusobacterial strains are genetically intractable. Addressing these problems, we introduced a riboswitch-based inducible CRISPR interference (CRISPRi) system. This system employs the nuclease-inactive
Streptococcus pyogenes
Cas9 protein (dCas9), specifically guided to the gene of interest by a constantly expressed single-guide RNA (sgRNA). Mechanistically, this dCas9-sgRNA complex serves as an insurmountable roadblock for RNA polymerase, thus repressing the target gene transcription. Leveraging this system, we first examined two non-essential genes,
ftsX
and
radD
, which are pivotal for fusobacterial cytokinesis and coaggregation. Upon adding the inducer, theophylline,
ftsX
suppression caused filamentous cell formation akin to chromosomal
ftsX
deletion, while targeting
radD
significantly reduced RadD protein levels, abolishing RadD-mediated coaggregation. The system was then extended to probe essential genes
bamA
and
ftsZ
, which are vital for outer membrane biogenesis and cell division. Impressively,
bamA
suppression disrupted membrane integrity and bacterial separation, stalling growth, while
ftsZ
targeting yielded elongated cells in broth with compromised agar growth. Further studies on
F. nucleatum
clinical strain CTI-2 and
Fusobacterium periodonticum
revealed reduced indole synthesis when targeting
tnaA
. Moreover, silencing
clpB
in
F. periodonticum
decreased ClpB, increasing thermal sensitivity. In summary, our CRISPRi system streamlines gene inactivation across various fusobacterial strains.
IMPORTANCE
How can we effectively investigate the gene functions in
Fusobacterium nucleatum
, given the dual challenges of gene inactivation and the inherent genetic resistance of many strains? Traditional methods have been cumbersome and often inadequate. Addressing this, our work introduces a novel inducible CRISPR interference (CRISPRi) system in which dCas9 expression is controlled at the translation level by a theophylline-responsive riboswitch unit, and single-guide RNA expression is driven by the robust, constitutive
rpsJ
promoter. This approach simplifies gene inactivation in the model organism (ATCC 23726) and extends its application to previously considered genetically intractable strains like CTI-2 and
Fusobacterium periodonticum
. With CRISPRi’s potential, it is a pivotal tool for in-depth genetic studies into fusobacterial pathogenesis, potentially unlocking targeted therapeutic strategies.
Funder
HHS | NIH | National Institute of Dental and Craniofacial Research
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology