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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.

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.

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.

Bacterial cell division predominantly occurs by a highly conserved process, termed binary fission, that requires the bacterial homologue of tubulin, FtsZ. Other mechanisms of bacterial cell division that are independent of FtsZ are rare. Although the obligate intracellular human pathogen Chlamydia trachomatis, the leading bacterial cause of sexually transmitted infections and trachoma, lacks FtsZ, it has been assumed to divide by binary fission. We show here that Chlamydia divides by a polarized cell division process similar to the budding process of a subset of the Planctomycetes that also lack FtsZ. Prior to cell division, the major outer-membrane protein of Chlamydia is restricted to one pole of the cell, and the nascent daughter cell emerges from this pole by an asymmetric expansion of the membrane. Components of the chlamydial cell division machinery accumulate at the site of polar growth prior to the initiation of asymmetric membrane expansion and inhibitors that disrupt the polarity of C. trachomatis prevent cell division. The polarized cell division of C. trachomatis is the result of the unipolar growth and FtsZ-independent fission of this coccoid organism. This mechanism of cell division has not been documented in other human bacterial pathogens suggesting the potential for developing Chlamydia-specific therapeutic treatments., Author Summary The obligate intracellular human pathogen Chlamydia trachomatis is the leading bacterial cause of sexually transmitted infections. C. trachomatis does not express FtsZ, which is necessary for the highly conserved process of binary fission that most bacteria utilize to divide. Nonetheless, it has been assumed that this bacterial pathogen divides by binary fission. We show here that C. trachomatis divides by a polarized cell division process that is similar to the budding process of some other bacteria that lack FtsZ, such as the Planctomycetes. This novel mode of cell division has not been documented in other human bacterial pathogens suggesting the potential for developing Chlamydia-specific therapeutic treatments.

Summary 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.

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.

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.

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 β-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.

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.

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 novel series of N-benzyl substituted 3'-(R)- 3'-aminomethyl-3'-hydroxy spectinomycins was developed based on 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 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 leads 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. In addition to improved antibacterial potency, compounds also demonstrated no mammalian cytotoxicity, improved microsomal stability, and favorable pharmacokinetic properties in rats. The lead compound from the series, compound 1, exhibited excellent chemical stability, which was superior to spectinomycin and had no significant interaction with a panel of human receptors and drug metabolism enzymes suggesting low potential for adverse reactions or drug-drug interactions in vivo. Compound 1 was active in vitro against a panel of penicillin, macrolide, and cephalosporin resistant S. pneumoniae clinical isolates and cured mice of fatal pneumococcal pneumonia and sepsis at a dose of 5 mg/kg. Together, these studies indicate N-benzyl aminomethyl spectinomycins possess suitable properties for further development as novel antibacterial agents to treat drug resistant respiratory tract and sexually transmitted bacterial infections.

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.

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.

Bacterial cell division predominantly occurs by a highly conserved process, termed binary fission, that requires the bacterial homologue of tubulin, FtsZ. Other mechanisms of bacterial cell division that are independent of FtsZ are rare. Although the obligate intracellular human pathogen Chlamydia trachomatis, the leading bacterial cause of sexually transmitted infections and trachoma, lacks FtsZ, it has been assumed to divide by binary fission. We show here that Chlamydia divides by a polarized cell division process similar to the budding process of a subset of the Planctomycetes that also lack FtsZ. Prior to cell division, the major outer-membrane protein of Chlamydia is restricted to one pole of the cell, and the nascent daughter cell emerges from this pole by an asymmetric expansion of the membrane. Components of the chlamydial cell division machinery accumulate at the site of polar growth prior to the initiation of asymmetric membrane expansion and inhibitors that disrupt the polarity of C. trachomatis prevent cell division. The polarized cell division of C. trachomatis is the result of the unipolar growth and FtsZ-independent fission of this coccoid organism. This mechanism of cell division has not been documented in other human bacterial pathogens suggesting the potential for developing Chlamydia-specific therapeutic treatments.

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.

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.

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.