Stratification of Fusobacterium nucleatum by local health status in the oral cavity defines its subspecies disease association.

The ubiquitous inflammophilic oral pathobiont Fusobacterium nucleatum (Fn) is widely recognized for its strong association with inflammatory dysbiotic diseases and cancer. Fn is subdivided into four subspecies, which are historically considered functionally interchangeable in the oral cavity. To test this assumption, we analyzed patient-matched dental plaque and odontogenic abscess clinical specimens and examined whether an inflammatory environment selects for/against particular Fn subspecies. Dental plaque harbored a greater diversity of fusobacteria, with Fn. polymorphum dominating, whereas odontogenic abscesses were exceptionally biased for the largely uncharacterized organism Fn. animalis. Comparative genomic analyses revealed significant genotypic distinctions among Fn subspecies that correlate with their preferred ecological niches and support a taxonomic reassignment of each as a distinct Fusobacterium species. Despite originating as a low-abundance organism in dental plaque, Fn. animalis typically outcompetes other oral fusobacteria within the inflammatory abscess environment, which may explain its prevalence in other oral and extraoral diseases.

The prevalence of Fusobacterium nucleatum subspecies in the oral cavity stratifies by local health status.

The ubiquitous inflammophilic pathobiont Fusobacterium nucleatum is widely recognized for its strong association with a variety of human dysbiotic diseases such as periodontitis and oral/extraoral abscesses, as well as multiple types of cancer. F. nucleatum is currently subdivided into four subspecies: F. nucleatum subspecies nucleatum (Fn. nucleatum), animalis (Fn. animalis), polymorphum (Fn. polymorphum), and vincentii/fusiforme (Fn. vincentii). Although these subspecies have been historically considered as functionally interchangeable in the oral cavity, direct clinical evidence is largely lacking for this assertion. Consequently, we assembled a collection of oral clinical specimens to determine whether F. nucleatum subspecies prevalence in the oral cavity stratifies by local oral health status. Patient-matched clinical specimens of both disease-free dental plaque and odontogenic abscess were analyzed with newly developed culture-dependent and culture-independent approaches using 44 and 60 oral biofilm/tooth abscess paired specimens, respectively. Most oral cavities were found to simultaneously harbor multiple F. nucleatum subspecies, with a greater diversity present within dental plaque compared to abscesses. In dental plaque, Fn. polymorphum is clearly the dominant organism, but this changes dramatically within odontogenic abscesses where Fn. animalis is heavily favored over all other fusobacteria. Surprisingly, the most commonly studied F. nucleatum subspecies, Fn. nucleatum, is only a minor constituent in the oral cavity. To gain further insights into the genetic basis for these phenotypes, we subsequently performed pangenome, phylogenetic, and functional enrichment analyses of oral fusobacterial genomes using the Anvi'o platform, which revealed significant genotypic distinctions among F. nucleatum subspecies. Accordingly, our results strongly support a taxonomic reassignment of each F. nucleatum subspecies into distinct Fusobacterium species. Of these, Fn. animalis should be considered as the most clinically relevant at sites of active inflammation, despite being among the least characterized oral fusobacteria.

Development of the First Tractable Genetic System for Parvimonas micra, a Ubiquitous Pathobiont in Human Dysbiotic Disease.

Parvimonas micra is a Gram-positive obligate anaerobe and a typical member of the human microbiome. P. micra is among the most highly enriched species at numerous sites of mucosal dysbiotic disease and is closely associated with the development of multiple types of malignant tumors. Despite its strong association with disease, surprisingly little is known about P. micra pathobiology, which is directly attributable to its longstanding genetic intractability. To address this problem, we directly isolated a collection of P. micra strains from odontogenic abscess clinical specimens and then screened these isolates for natural competence. Amazingly, all of the P. micra clinical isolates exhibited various levels of natural competence, including the reference strain ATCC 33270. By exploiting this ability, we were able to employ cloning-independent methodologies to engineer and complement a variety of targeted chromosomal genetic mutations directly within low-passage-number clinical isolates. To develop a tractable genetic system for P. micra, we first adapted renilla-based bioluminescence for highly sensitive reporter studies. This reporter system was then applied for the development of the novel Theo+ theophylline-inducible riboswitch for tunable gene expression studies over a broad dynamic range. Finally, we demonstrate the feasibility of generating mariner-based transposon sequencing (Tn-seq) libraries for forward genetic screening in P. micra. With the availability of a highly efficient transformation protocol and the current suite of genetic tools, P. micra should now be considered a fully genetically tractable organism suitable for molecular genetic research. The methods presented here provide a clear path to investigate the understudied role of P. micra in polymicrobial infections and tumorigenesis. IMPORTANCE Parvimonas micra is among the most highly enriched species at numerous sites of mucosal dysbiotic disease and is closely associated with numerous cancers. Despite this, little is known about P. micra pathobiology, which is directly attributable to its longstanding genetic intractability. In this study, we provide the first report of P. micra natural competence and describe the only tractable genetic system for this species. The methods presented here will allow for the detailed study of P. micra and its role in infection and tumorigenesis.

Plasmid-cured Chlamydia caviae activates TLR2-dependent signaling and retains virulence in the guinea pig model of genital tract infection.

Loss of the conserved "cryptic" plasmid from C. trachomatis and C. muridarum is pleiotropic, resulting in reduced innate inflammatory activation via TLR2, glycogen accumulation and infectivity. The more genetically distant C. caviae GPIC is a natural pathogen of guinea pigs and induces upper genital tract pathology when inoculated intravaginally, modeling human disease. To examine the contribution of pCpGP1 to C. caviae pathogenesis, a cured derivative of GPIC, strain CC13, was derived and evaluated in vitro and in vivo. Transcriptional profiling of CC13 revealed only partial conservation of previously identified plasmid-responsive chromosomal loci (PRCL) in C. caviae. However, 2-deoxyglucose (2DG) treatment of GPIC and CC13 resulted in reduced transcription of all identified PRCL, including glgA, indicating the presence of a plasmid-independent glucose response in this species. In contrast to plasmid-cured C. muridarum and C. trachomatis, plasmid-cured C. caviae strain CC13 signaled via TLR2 in vitro and elicited cytokine production in vivo similar to wild-type C. caviae. Furthermore, inflammatory pathology induced by infection of guinea pigs with CC13 was similar to that induced by GPIC, although we observed more rapid resolution of CC13 infection in estrogen-treated guinea pigs. These data indicate that either the plasmid is not involved in expression or regulation of virulence in C. caviae or that redundant effectors prevent these phenotypic changes from being observed in C. caviae plasmid-cured strains.

Inhibition of indoleamine 2,3-dioxygenase activity by levo-1-methyl tryptophan blocks gamma interferon-induced Chlamydia trachomatis persistence in human epithelial cells.

Gamma interferon (IFN-γ) induces expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO1) in human epithelial cells, the permissive cells for the obligate intracellular bacterium Chlamydia trachomatis. IDO1 depletes tryptophan by catabolizing it to kynurenine with consequences for C. trachomatis, which is a tryptophan auxotroph. In vitro studies reveal that tryptophan depletion can result in the formation of persistent (viable but noncultivable) chlamydial forms. Here, we tested the effects of the IDO1 inhibitor, levo-1-methyl-tryptophan (L-1MT), on IFN-γ-induced C. trachomatis persistence. We found that addition of 0.2 mM L-1MT to IFN-γ-exposed infected HeLa cell cultures restricted IDO1 activity at the mid-stage (20 h postinfection [hpi]) of the chlamydial developmental cycle. This delayed tryptophan depletion until the late stage (38 hpi) of the cycle. Parallel morphological and gene expression studies indicated a consequence of the delay was a block in the induction of C. trachomatis persistence by IFN-γ. Furthermore, L-1MT addition allowed C. trachomatis to undergo secondary differentiation, albeit with limited productive multiplication of the bacterium. IFN-γ-induced persistent infections in epithelial cells have been previously reported to be more resistant to doxycycline than normal productive infections in vitro. Pertinent to this observation, we found that L-1MT significantly improved the efficacy of doxycycline in clearing persistent C. trachomatis forms. It has been postulated that persistent forms of C. trachomatis may contribute to chronic chlamydial disease. Our findings suggest that IDO1 inhibitors such as L-1MT might provide a novel means to investigate, and potentially target, persistent chlamydial forms, particularly in conjunction with conventional therapeutics.