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.

Multiplex Imaging of Polymicrobial Communities-Murine Models to Study Oral Microbiome Interactions.

Similar to other mucosal surfaces of the body, the oral cavity hosts a diverse microbial flora that live in polymicrobial biofilm communities. It is the ecology of these communities that are the primary determinants of oral health (symbiosis) or disease (dysbiosis). As such, both symbiosis and dysbiosis are inherently polymicrobial phenomena. In an effort to facilitate studies of polymicrobial communities within rodent models, we developed a suite of synthetic luciferases suitable for multiplexed in situ analyses of microbial ecology and specific gene expression. Using this approach, it is feasible to noninvasively measure multiple luciferase signals in vivo with both spatial and temporal resolution. In the following chapter, we describe the relevant details and protocols used to establish a biophotonic imaging platform for the study of experimental polymicrobial oral biofilms and abscesses in mice. The protocols described here are specifically tailored for use with oral streptococci, but the general strategies are adaptable for a wide range of polymicrobial infection studies using other species.

Chlamydia trachomatis utilizes the mammalian CLA1 lipid transporter to acquire host phosphatidylcholine essential for growth.

Phosphatidylcholine is a constituent of Chlamydia trachomatis membranes that must be acquired from its mammalian host to support bacterial proliferation. The CLA1 (SR-B1) receptor is a bi-directional phosphatidylcholine/cholesterol transporter that is recruited to the inclusion of Chlamydia-infected cells along with ABCA1. C. trachomatis growth was inhibited in a dose-dependent manner by BLT-1, a selective inhibitor of CLA1 function. Expression of a BLT-1-insensitive CLA1(C384S) mutant ameliorated the effect of the drug on chlamydial growth. CLA1 knockdown using shRNAs corroborated an important role for CLA1 in the growth of C. trachomatis. Trafficking of a fluorescent phosphatidylcholine analogue to Chlamydia was blocked by the inhibition of CLA1 or ABCA1 function, indicating a critical role for these transporters in phosphatidylcholine acquisition by this organism. Our analyses using a dual-labelled fluorescent phosphatidylcholine analogue and mass spectrometry showed that the phosphatidylcholine associated with isolated Chlamydia was unmodified host phosphatidylcholine. These results indicate that C. trachomatis co-opts host phospholipid transporters normally used to assemble lipoproteins to acquire host phosphatidylcholine essential for growth.

Identification and Partial Characterization of Potential FtsL and FtsQ Homologs of Chlamydia.

Chlamydia is amongst the rare bacteria that lack the critical cell division protein FtsZ. By annotation, Chlamydia also lacks several other essential cell division proteins including the FtsLBQ complex that links the early (e.g., FtsZ) and late (e.g., FtsI/Pbp3) components of the division machinery. Here, we report chlamydial FtsL and FtsQ homologs. Ct271 aligned well with Escherichia coli FtsL and shared sequence homology with it, including a predicted leucine-zipper like motif. Based on in silico modeling, we show that Ct764 has structural homology to FtsQ in spite of little sequence similarity. Importantly, ct271/ftsL and ct764/ftsQ are present within all sequenced chlamydial genomes and are expressed during the replicative phase of the chlamydial developmental cycle, two key characteristics for a chlamydial cell division gene. GFP-Ct764 localized to the division septum of dividing transformed chlamydiae, and, importantly, over-expression inhibited chlamydial development. Using a bacterial two-hybrid approach, we show that Ct764 interacted with other components of the chlamydial division apparatus. However, Ct764 was not capable of complementing an E. coli FtsQ depletion strain in spite of its ability to interact with many of the same division proteins as E. coli FtsQ, suggesting that chlamydial FtsQ may function differently. We previously proposed that Chlamydia uses MreB and other rod-shape determining proteins as an alternative system for organizing the division site and its apparatus. Chlamydial FtsL and FtsQ homologs expand the number of identified chlamydial cell division proteins and suggest that Chlamydia has likely kept the late components of the division machinery while substituting the Mre system for the early components.

Aminomethyl spectinomycins as therapeutics for drug-resistant respiratory tract and sexually transmitted bacterial infections.

The antibiotic spectinomycin is a potent inhibitor of bacterial protein synthesis with a unique mechanism of action and an excellent safety index, but it lacks antibacterial activity against most clinically important pathogens. A series of N-benzyl-substituted 3'-(R)-3'-aminomethyl-3'-hydroxy spectinomycins was developed on the basis of a computational analysis of the aminomethyl spectinomycin binding site and structure-guided synthesis. These compounds had ribosomal inhibition values comparable to spectinomycin but showed increased potency against the common respiratory tract pathogens Streptococcus pneumoniae, Haemophilus influenzae, Legionella pneumophila, and Moraxella catarrhalis, as well as the sexually transmitted bacteria Neisseria gonorrhoeae and Chlamydia trachomatis. Non-ribosome-binding 3'-(S) isomers of the lead compounds demonstrated weak inhibitory activity in in vitro protein translation assays and poor antibacterial activity, indicating that the antibacterial activity of the series remains on target against the ribosome. Compounds also demonstrated no mammalian cytotoxicity, improved microsomal stability, and favorable pharmacokinetic properties in rats. The lead compound from the series exhibited excellent chemical stability superior to spectinomycin; no interaction with a panel of human receptors and drug metabolism enzymes, suggesting low potential for adverse reactions or drug-drug interactions in vivo; activity in vitro against a panel of penicillin-, macrolide-, and cephalosporin-resistant S. pneumoniae clinical isolates; and the ability to cure mice of fatal pneumococcal pneumonia and sepsis at a dose of 5 mg/kg. Together, these studies indicate that N-benzyl aminomethyl spectinomycins are suitable for further development to treat drug-resistant respiratory tract and sexually transmitted bacterial infections.

Type II fatty acid synthesis is essential for the replication of Chlamydia trachomatis.

The major phospholipid classes of the obligate intracellular bacterial parasite Chlamydia trachomatis are the same as its eukaryotic host except that they also contain chlamydia-made branched-chain fatty acids in the 2-position. Genomic analysis predicts that C. trachomatis is capable of type II fatty acid synthesis (FASII). AFN-1252 was deployed as a chemical tool to specifically inhibit the enoyl-acyl carrier protein reductase (FabI) of C. trachomatis to determine whether chlamydial FASII is essential for replication within the host. The C. trachomatis FabI (CtFabI) is a homotetramer and exhibited typical FabI kinetics, and its expression complemented an Escherichia coli fabI(Ts) strain. AFN-1252 inhibited CtFabI by binding to the FabI·NADH complex with an IC50 of 0.9 µM at saturating substrate concentration. The x-ray crystal structure of the CtFabI·NADH·AFN-1252 ternary complex revealed the specific interactions between the drug, protein, and cofactor within the substrate binding site. AFN-1252 treatment of C. trachomatis-infected HeLa cells at any point in the infectious cycle caused a decrease in infectious titers that correlated with a decrease in branched-chain fatty acid biosynthesis. AFN-1252 treatment at the time of infection prevented the first cell division of C. trachomatis, although the cell morphology suggested differentiation into a metabolically active reticulate body. These results demonstrate that FASII activity is essential for C. trachomatis proliferation within its eukaryotic host and validate CtFabI as a therapeutic target against C. trachomatis.

Morphologic and molecular evaluation of Chlamydia trachomatis growth in human endocervix reveals distinct growth patterns.

In vitro models of Chlamydia trachomatis growth have long been studied to predict growth in vivo. Alternative or persistent growth modes in vitro have been shown to occur under the influence of numerous stressors but have not been studied in vivo. Here, we report the development of methods for sampling human infections from the endocervix in a manner that permits a multifaceted analysis of the bacteria, host and the endocervical environment. Our approach permits evaluating total bacterial load, transcriptional patterns, morphology by immunofluorescence and electron microscopy, and levels of cytokines and nutrients in the infection microenvironment. By applying this approach to two pilot patients with disparate infections, we have determined that their contrasting growth patterns correlate with strikingly distinct transcriptional biomarkers, and are associated with differences in local levels of IFNγ. Our multifaceted approach will be useful to dissect infections in the human host and be useful in identifying patients at risk for chronic disease. Importantly, the molecular and morphological analyses described here indicate that persistent growth forms can be isolated from the human endocervix when the infection microenvironment resembles the in vitro model of IFNγ-induced persistence.

Host HDL biogenesis machinery is recruited to the inclusion of Chlamydia trachomatis-infected cells and regulates chlamydial growth.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that is the most common cause of sexually transmitted bacterial infections and is the etiological agent of trachoma, the leading cause of preventable blindness. The organism infects epithelial cells of the genital tract and eyelid resulting in a damaging inflammatory response. Chlamydia trachomatis grows within a vacuole termed the inclusion, and its growth depends on numerous host factors, including lipids. Although a variety of mechanisms are involved in the acquisition of host cell cholesterol and glycosphingolipids by C. trachomatis, none of the previously documented pathways for lipid acquisition are absolutely required for growth. Here we demonstrate that multiple components of the host high-density lipoprotein (HDL) biogenesis machinery including the lipid effluxers, ABCA1 and CLA 1, and their extracellular lipid acceptor, apoA-1, are recruited to the inclusion of C. trachomatis-infected cells. Furthermore, the apoA-1 that accumulates within the inclusion colocalizes with pools of phosphatidylcholine. Knockdown of ABCA1, which mediates the cellular efflux of cholesterol and phospholipids to initiate the formation of HDL in the serum, prevents the growth of C. trachomatis in infected HeLa cells. In addition, drugs that inhibit the lipid transport activities of ABCA1 and CLA 1 also inhibit the recruitment of phospholipids to the inclusion and prevent chlamydial growth.These results strongly suggest that C. trachomatis co-opts the host cell lipid transport system involved in the formation of HDL to acquire lipids, such as phosphatidylcholine, that are necessary for growth.

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.

Toll-like receptor 2 activation by Chlamydia trachomatis is plasmid dependent, and plasmid-responsive chromosomal loci are coordinately regulated in response to glucose limitation by C. trachomatis but not by C. muridarum.

We previously demonstrated that plasmid-deficient Chlamydia muridarum retains the ability to infect the murine genital tract but does not elicit oviduct pathology because it fails to activate Toll-like receptor 2 (TLR2). We derived a plasmid-cured derivative of the human genital isolate Chlamydia trachomatis D/UW-3/Cx, strain CTD153, which also fails to activate TLR2, indicating this virulence phenotype is associated with plasmid loss in both C. trachomatis and C. muridarum. As observed with plasmid-deficient C. muridarum, CTD153 displayed impaired accumulation of glycogen within inclusions. Transcriptional profiling of the plasmid-deficient strains by using custom microarrays identified a conserved group of chromosomal loci, the expression of which was similarly controlled in plasmid-deficient C. muridarum strains CM972 and CM3.1 and plasmid-deficient C. trachomatis CTD153. However, although expression of glycogen synthase, encoded by glgA, was greatly reduced in CTD153, it was unaltered in plasmid-deficient C. muridarum strains. Thus, additional plasmid-associated factors are required for glycogen accumulation by this chlamydial species. Furthermore, in C. trachomatis, glgA and other plasmid-responsive chromosomal loci (PRCLs) were transcriptionally responsive to glucose limitation, indicating that additional regulatory elements may be involved in the coordinated expression of these candidate virulence effectors. Glucose-limited C. trachomatis displayed reduced TLR2 stimulation in an in vitro assay. During human chlamydial infection, glucose limitation may decrease chlamydial virulence through its effects on plasmid-responsive chromosomal genes.

Developmental expression of non-coding RNAs in Chlamydia trachomatis during normal and persistent growth.

Chlamydia trachomatis is an obligate intracellular bacterium that exhibits a unique biphasic developmental cycle that can be disrupted by growth in the presence of IFN-γ and ß-lactams, giving rise to an abnormal growth state termed persistence. Here we have examined the expression of a family of non-coding RNAs (ncRNAs) that are differentially expressed during the developmental cycle and the induction of persistence and reactivation. ncRNAs were initially identified using an intergenic tiling microarray and were confirmed by northern blotting. ncRNAs were mapped, characterized and compared with the previously described chlamydial ncRNAs. The 5'- and 3'-ends of the ncRNAs were determined using an RNA circularization procedure. Promoter predictions indicated that all ncRNAs were expressed from σ(66) promoters and eight ncRNAs contained non-templated 3'-poly-A or poly-AG additions. Expression of ncRNAs was studied by northern blotting during (i) the normal developmental cycle, (ii) IFN-γ-induced persistence and (iii) carbenicillin-induced persistence. Differential temporal expression during the developmental cycle was seen for all ncRNAs and distinct differences in expression were seen during IFN-γ and carbenicillin-induced persistence and reactivation. A heterologous co-expression system was used to demonstrate that one of the identified ncRNAs regulated the expression of FtsI by inducing degradation of ftsI mRNA.

Global transcriptional upregulation in the absence of increased translation in Chlamydia during IFNgamma-mediated host cell tryptophan starvation.

The developmentally regulated intracellular pathogen Chlamydia pneumoniae is a natural tryptophan auxotroph. These organisms survive tryptophan starvation induced by host cell activation with IFNgamma by blocking maturation to the infectious form. In most bacteria, the stringent response is induced during amino acid starvation to promote survival. However, the response of obligate intracellular pathogens, which are predicted to lack stringent responses to amino acid starvation, is poorly characterized. Chlamydial transcription and translation were analysed during IFNgamma-mediated tryptophan starvation using genomic normalization methods, and the data revealed the novel findings that: (i) global chlamydial transcription was upregulated; and (ii) protein synthesis was dramatically reduced. These results indicate a dysregulation of developmental gene expression and an uncoupling of transcription from translation. These observations represent an alternative survival strategy for host-adapted obligate intracellular bacterial pathogens that have lost the genes for stringent control during reductive evolution.

The Chlamydia pneumoniae type III secretion-related lcrH gene clusters are developmentally expressed operons.

Two chlamydial homologues of the Yersinia lcrH chaperone for type III secretion system structural components are present within separate gene clusters. Quantitative transcriptional analyses demonstrated that each cluster is differentially regulated and expressed as an operon using major sigma factor elements, suggesting the presence of more elaborate developmental regulation mechanisms in chlamydiae.

The chlamydial developmental cycle.

Intracellular parasitism by bacterial pathogens is a complex, multi-factorial process that has been exploited successfully by a wide variety of organisms. Members of the Order Chlamydiales are obligate intracellular bacteria that are transmitted as metabolically inactive particles and must differentiate, replicate, and re-differentiate within the host cell to carry out their life cycle. Understanding the developmental cycle has been greatly advanced by the availability of complete genome sequences, DNA microarrays, and advanced cell biology techniques. Measuring transcriptional changes throughout the cycle has allowed investigators to determine the nature of the temporal gene expression changes required for bacterial growth and development.