The genome of Chlamydophila caviae (formerly Chlamydia psittaci, GPIC isolate) (1 173 390 nt with a plasmid of 7966 nt) was determined, representing the fourth species with a complete genome sequence from the Chlamydiaceae family of obligate intracellular bacterial pathogens. Of 1009 annotated genes, 798 were conserved in all three other completed Chlamydiaceae genomes. The C.caviae genome contains 68 genes that lack orthologs in any other completed chlamydial genomes, including tryptophan and thiamine biosynthesis determinants and a ribose-phosphate pyrophosphokinase, the product of the prsA gene. Notable amongst these was a novel member of the virulence-associated invasin/intimin family (IIF) of Gram-negative bacteria. Intriguingly, two authentic frameshift mutations in the ORF indicate that this gene is not functional. Many of the unique genes are found in the replication termination region (RTR or plasticity zone), an area of frequent symmetrical inversion events around the replication terminus shown to be a hotspot for genome variation in previous genome sequencing studies. In C.caviae, the RTR includes several loci of particular interest including a large toxin gene and evidence of ancestral insertion(s) of a bacteriophage. This toxin gene, not present in Chlamydia pneumoniae, is a member of the YopT effector family of type III-secreted cysteine proteases. One gene cluster (guaBA-add) in the RTR is much more similar to orthologs in Chlamydia muridarum than those in the phylogenetically closest species C.pneumoniae, suggesting the possibility of horizontal transfer of genes between the rodent-associated Chlamydiae. With most genes observed in the other chlamydial genomes represented, C.caviae provides a good model for the Chlamydiaceae and a point of comparison against the human atherosclerosis-associated C.pneumoniae. This crucial addition to the set of completed Chlamydiaceae genome sequences is enabling dissection of the roles played by niche-specific genes in these important bacterial pathogens.

Infections caused by the obligate intracellular pathogen Chlamydia trachomatis have a marked impact on human health. C. trachomatis serovariants are the leading cause of bacterial sexually transmitted disease and infectious preventable blindness. Despite decades of effort, there is no practical vaccine against C. trachomatis diseases. Here we report that all C. trachomatis reference serotypes responsible for sexually transmitted disease and blinding trachoma synthesize a highly conserved surface-exposed antigen termed polymorphic membrane protein D (PmpD). We show that Ab specific to PmpD are neutralizing in vitro. We also present evidence that Ab against serovariable-neutralizing targets, such as the major outer membrane protein, block PmpD neutralization. This finding suggests that a decoy-like immune evasion strategy may be active in vivo whereby immunodominant type-specific surface antigens block the neutralizing ability of species-common PmpD Ab. Collectively, these results show that PmpD is a previously uncharacterized C. trachomatis species-common pan-neutralizing target. Moreover, a vaccine protocol using recombinant PmpD to elicit neutralizing Ab in the absence of immunodominant type-specific Ab might be highly efficacious and surpass the level of protection achieved through natural immunity.Chlamydia ͉ autotransporter ͉ neutralizing target ͉ surface antigen ͉ vaccine

SummaryFour genes of Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC), whose predicted products are highly homologous to structural and regulatory components of a contact-dependent or type III secretion apparatus, were isolated. Related to genes present in several animal and plant bacterial pathogens, these genes may represent a section of a previously undetected chromosomal virulence locus analogous to several recently described virulenceassociated type III secretion loci. The existence of contact-dependent secretion in Chlamydia strongly suggests that these bacteria use pathogenic mechanisms that are similar to those of other intracellular bacterial pathogens. Unlike other intracellular bacteria, however, chlamydiae are metabolically inactive extracellularly and only become capable of global protein synthesis several hours after infection. This implies that chlamydial contact-dependent secretion is only active from within, uniquely after the bacteria have been internalized by eukaryotic cells. The possible role(s) of this pathway in chlamydial pathogenesis are discussed.

Secondary caries remains the main reason for dental restoration failure. Replacement of failed restorations accounts for 50-70% of all restorations performed. Antibacterial adhesives could inhibit biofilm acids at tooth-restoration margins, and calcium phosphate (CaP) ions could remineralize tooth lesions. The objectives of this study were to: (1) incorporate nanoparticles of silver (NAg), quaternary ammonium dimethacrylate (QADM), and nanoparticles of amorphous calcium phosphate (NACP) into bonding agent; and (2) investigate their effects on dentin bonding and microcosm biofilms. An experimental primer was made with pyromellitic glycerol dimethacrylate (PMGDM) and 2-hydroxyethyl methacrylate (HEMA). An adhesive was made with bisphenol-A-glycerolate dimethacrylate (BisGMA) and triethylene glycol dimethacrylate (TEGDMA). NAg was incorporated into primer at 0.1wt%. The adhesive contained 0.1% NAg and 10% QADM, and 0-40% NACP. Incorporating NAg into primer and NAg-QADM-NACP into adhesive did not adversely affect dentin bond strength (p>0.1). SEM showed numerous resin tags, and TEM revealed NAg and NACP in dentinal tubules. Viability of human saliva microcosm biofilms on primer/adhesive/composite disks was substantially reduced via NAg and QADM. Metabolic activity, lactic acid, and colony-forming units of biofilms were much lower on the new bonding agents than control (p<0.05). In conclusion, novel dental bonding agents containing NAg, QADM and NACP were developed with the potential to kill residual bacteria in the tooth cavity and inhibit the invading bacteria along tooth-restoration margins, with NACP to remineralize tooth lesions. The novel method of combining antibacterial agents (NAg and QADM) with remineralizing agent (NACP) may have wide applicability to other adhesives for caries inhibition.

Although endosomal compartments have been suggested to play a role in unconventional protein secretion, there is scarce experimental evidence for such involvement. Here we report that recycling endosomes are essential for externalization of cytoplasmic secretory protein tissue transglutaminase (tTG). The de novo synthesized cytoplasmic tTG does not follow the classical ER/Golgi-dependent secretion pathway, but is targeted to perinuclear recycling endosomes, and is delivered inside these vesicles prior to externalization. On its route to the cell surface tTG interacts with internalized β1 integrins inside the recycling endosomes and is secreted as a complex with recycled β1 integrins. Inactivation of recycling endosomes, blocking endosome fusion with the plasma membrane, or downregulation of Rab11 GTPase that controls outbound trafficking of perinuclear recycling endosomes, all abrogate tTG secretion. The initial recruitment of cytoplasmic tTG to recycling endosomes and subsequent externalization depend on its binding to phosphoinositides on endosomal membranes. These findings begin to unravel the unconventional mechanism of tTG secretion which utilizes the long loop of endosomal recycling pathway and indicate involvement of endosomal trafficking in non-classical protein secretion.

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Investigations conducted on feral African Sacred Ibises ( Threskiornis aethiopicus ) in western France led to the isolation of a strain with chlamydial genetic determinants. Ultrastructural analysis, comparative sequence analysis of the 16S rRNA gene, ompA, and of a concatenate of 31 highly conserved genes, as well as determination of the whole genome sequence confirmed the relatedness of the new isolate to members of the Chlamydiaceae, while, at the same time demonstrating a unique position outside the currently recognized species of this family. We propose to name this new chlamydial species Chlamydia ibidis .

Late C. trachomatis inclusions express each member of the surface-exposed polymorphic membrane protein family (Pmp subtypes A through I) with a reproducible distribution of fully-on, fully-off and intermediate phenotypes. This observation is consistent with observed variable Pmp antibody profiles in C. trachomatis-infected patients and has led to the hypothesis that the pmp gene family forms the basis of a phase variation-like mechanism of antigenic variation. Here we investigate and compare the developmental expression of each of the nine pmp genes under conditions of optimal in vitro growth with that under conditions that promote prolonged survival of chlamydiae when exposed to penicillin-induced stress. We demonstrate that the pmp gene family includes distinct transcriptional units that are differentially expressed along development and differentially responsive to stress. In particular, our results indicate that expression of pmpA, pmpD and pmpI is uniquely unaffected by stress, suggesting that the PmpA, PmpD and PmpI proteins play a critical role in the pathogenesis of C. trachomatis.