Chlamydia trachomatis is obligate intracellular bacterial pathogen that remains a significant public health burden worldwide. A critical early event during infection is chlamydial entry into non-phagocytic host epithelial cells. Like other Gram-negative bacteria, C.trachomatis uses a type III secretion system (T3SS) to deliver virulence effector proteins into host cells. These effectors trigger bacterial uptake and promote bacterial survival and replication within the host cell. In this review, we highlight recent cryo-electron tomography that has provided striking insights into the initial interactions between Chlamydia and its host. We describe the polarised structure of extracellular C.trachomatis elementary bodies (EBs), and the supramolecular organisation of T3SS complexes on the EB surface, in addition to the changes in host and pathogen architecture that accompany bacterial internalisation and EB encapsulation into early intracellular vacuoles. Finally, we consider the implications for further understanding the mechanism of C.trachomatis entry and how this might relate to those of other bacteria and viruses.
BACKGROUND: In recent years there has been interest in websites as a means of increasing access to free chlamydia tests through the National Chlamydia Screening Programme (NCSP) in England. We aimed to describe and evaluate online access to chlamydia testing within the NCSP. METHODS: We analysed NCSP chlamydia testing data (2006–2010) for 15–24 year olds from the 71/95 programme areas in England where site codes were available to identify tests ordered through the internet. The characteristics of people using online testing services in 2010 were compared with those testing in general practice (GP) or community sexual and reproductive health (SRH) services. We evaluated 58 websites offering free chlamydia tests through the NCSP, and 32 offering kits on a commercial basis for signposting to clinical service and health promotion advice offered. RESULTS: Between 2006 and 2010, 5% of all tests in the included programme areas were accessed through the internet. The number of internet tests increased from 18 (<1% of all tests) in 2006 to 59,750 in 2010 (6% of all NCSP tests). In 2010 the proportion of NCSP tests accessed online by programme area ranged from <1% to 38%. The proportion of tests with a positive result on the internet was higher than tests from general practice and comparable to those from community SRH services (internet 7.6%; GP 5.6%; Community SRH 8.2%). A higher proportion of people accessing online testing were male, aged 20--24 and reported >1 sexual partner in the past year. Provision of sexual health information and appropriate signposting for those in need of clinical services varied between websites. Service provision within the NCSP was fragmented with multiple providers serving specific geographical catchment areas. CONCLUSION: Internet testing reaches a population with a relatively high risk of chlamydia infection and appears acceptable to young men, a group that has been difficult to engage with chlamydia testing. In order to maximise the potential benefit of these services, websites should be consistent with national guidelines and adhere to minimum standards for signposting to clinical care and health promotion information. The current system with multiple providers servicing geographically specific catchment areas is contrary to the geographically unrestricted nature of the internet and potentially confusing for clients.
Chlamydia trachomatis is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive Chlamydia infections. Despite its importance for Chlamydia biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated C. trachomatis inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells' antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the C. trachomatis infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that C. trachomatis hijacks retrograde pathways for effective infection.
The peptidoglycan (PG) cell wall is a peptide cross-linked glycan polymer essential for bacterial division and maintenance of cell shape and hydrostatic pressure. Bacteria in the Chlamydiales were long thought to lack PG until recent advances in PG labeling technologies revealed the presence of this critical cell wall component in Chlamydia trachomatis. In this study, we utilize bio-orthogonal D-amino acid dipeptide probes combined with super-resolution microscopy to demonstrate that four pathogenic Chlamydiae species each possess a ≤ 140 nm wide PG ring limited to the division plane during the replicative phase of their developmental cycles. Assembly of this PG ring is rapid, processive, and linked to the bacterial actin-like protein, MreB. Both MreB polymerization and PG biosynthesis occur only in the intracellular form of pathogenic Chlamydia and are required for cell enlargement, division, and transition between the microbe’s developmental forms. Our kinetic, molecular, and biochemical analyses suggest that the development of this limited, transient, PG ring structure is the result of pathoadaptation by Chlamydia to an intracellular niche within its vertebrate host.
Weak intracellular penetration of antibiotics makes some infections difficult to treat. The Trojan horse strategy for targeted drug delivery is among the interesting routes being explored to overcome this therapeutic difficulty. Chlamydia trachomatis, as an obligate intracellular human pathogen, is responsible for both trachoma and sexually transmitted diseases. Chlamydia develops in a vacuole and is therefore protected by four membranes (plasma membrane, bacterial inclusion membrane, and bacterial membranes). In this work, the iron-transport protein, human serum-transferrin, was used as a Trojan horse for antibiotic delivery into the bacterial vacuole. Amoxicillin was grafted onto transferrin. The transferrin-amoxicillin construct was characterized by mass spectrometry and absorption spectroscopy. Its affinity for transferrin receptor 1, determined by fluorescence emission titration [KaffTf-amox = (1.3 ± 1.0) x 108], is very close to that of transferrin [4.3 x 108]. Transmission electron and confocal microscopies showed a co-localization of transferrin with the bacteria in the vacuole and were also used to evaluate the antibiotic capability of the construct. It is significantly more effective than amoxicillin alone. These promising results demonstrate targeted delivery of amoxicillin to suppress Chlamydia and are of interest for Chlamydiaceae and maybe other intracellular bacteria therapies.
The search for new nontraditional targets is a high priority in antibiotic design today. Bacterial membrane energetics based on sodium ion circulation offers potential alternative targets. The present work identifies the Na(+)-translocating NADH:ubiquinone oxidoreductase (Na(+)-NQR), a key respiratory enzyme in many microbial pathogens, as indispensible for the Chlamydia trachomatis infectious process. Infection by Chlamydia trachomatis significantly increased first H(+) and then Na(+) levels within the host mammalian cell. A newly designed furanone Na(+)-NQR inhibitor, PEG-2S, blocked the changes in both H(+) and Na(+) levels induced by Chlamydia trachomatis infection. It also inhibited intracellular proliferation of Chlamydia trachomatis with a half-minimal inhibitory concentration in the submicromolar range but did not affect the viability of mammalian cells or bacterial species representing benign intestinal microflora. At low nanomolar concentrations (IC50 value = 1.76 nmol/L), PEG-2S inhibited the Na(+)-NQR activity in sub-bacterial membrane vesicles isolated from Vibrio cholerae. Taken together, these results show, for the first time, that Na(+)-NQR is critical for the bacterial infectious process and is susceptible to a precisely targeted bactericidal compound in situ. The obtained data have immediate relevance for many different diseases caused by pathogenic bacteria that rely on Na(+)-NQR activity for growth, including sexually transmitted, pulmonary, oral, gum, and ocular infections.
Infectious diseases caused by chlamydia or schistosomes are a major health problem worldwide, and particularly so in developing countries. The lack of appropriate vaccines renders the search for potent natural products against these disease-causing agents an urgent endeavor. Sponge-associated actinomycetes represent a rich reservoir for natural products. Among them, members of the genus Streptomyces are capable of synthesizing an impressive array of diverse natural products with a wide variety of biological activities. The naphthacene glycoside SF2446A2 was isolated from the calcium alginate beads culture of Streptomyces sp. strain RV15 that had originally been obtained from the Mediterranean sponge Dysidea tupha. Its structure was identified by spectroscopic analysis and MS and comparison with the literature data. SF2446A2 showed inhibitory activity against Chlamydia trachomatis and was able to inhibit the primary infection in a dose-dependent manner, as well as progeny formation. Moreover, it caused disruptive effects on the surface area of Schistosoma mansoni and affected the gonads by impairing oogenesis and spermatogenesis. Our current study demonstrates that sponge-associated actinomycetes are capable of providing compounds with new pharmacological activities and with relevance to drug discovery.The Journal of Antibiotics advance online publication, 20 May 2015; doi:10.1038/ja.2015.54.
Chlamydia trachomatis is an obligate intracellular bacterial pathogen of medical importance. C. trachomatis develops inside a membranous vacuole in the cytosol of epithelial cells but manipulates the host cell in numerous ways. One prominent effect of chlamydial infection is the inhibition of apoptosis in the host cell, but molecular aspects of this inhibition are unclear. Tumour necrosis factor (TNF) is a cytokine with important roles in immunity, which is produced by immune cells in chlamydial infection and which can have pro-apoptotic and non-apoptotic signalling activity. We here analysed the signalling through TNF in cells infected with C. trachomatis. The pro-apoptotic signal of TNF involves the activation of caspase-8 and is controlled by inhibitor of apoptosis proteins (IAPs). We found that in C. trachomatis-infected cells, TNF-induced apoptosis was blocked upstream of caspase-8 activation even when IAPs were inhibited or the inhibitor of caspase-8-activation, cFLIP, was targeted by RNAi. However, when caspase-8 was directly activated by experimental over-expression of its upstream adapter FADD, C. trachomatis was unable to inhibit apoptosis. Non-apoptotic TNF-signalling, in particular the activation of NF-κB, initiates at the plasma membrane while the activation of caspase-8 and pro-apoptotic signalling occur subsequently to internalisation of TNF receptor and the formation of a cytosolic signalling complex. In C. trachomatis infected cells, NF-κB-activation through TNF was unaffected while the internalisation of the TNF-TNF-receptor complex was blocked, explaining the lack of caspase-8-activation. These results identify a dichotomy of TNF signalling in C. trachomatis-infected cells: apoptosis is blocked at the internalisation of the TNF receptor but non-apoptotic signalling through this receptor remains intact, permitting a response to this cytokine at sites of infection.
Chlamydiae are obligate intracellular bacteria with two distinct morphological stages, the infectious elementary bodies (EBs) and non-infectious reticulate bodies (RBs). Here we describe a rapid and straightforward protocol for the purification of EBs and RBs involving special density gradients. It has been successfully applied to three chlamydial species.
The lifestyle of Chlamydiae is unique: the bacteria alternate between two morphologically distinct forms, an infectious non-replicative elementary body (EB), and a replicative, non-infectious reticulate body (RB). This review focuses on recent advances in understanding the structure and function of the infectious form of the best-studied member of the phylum, the human pathogen Chlamydia trachomatis. Once considered as an inert particle of little functional capacity, the EB is now perceived as a sophisticated entity that encounters at least three different environments during each infectious cycle. We review current knowledge on its composition and morphology, and emerging metabolic activities. These features confer resistance to the extracellular environment, the ability to penetrate a host cell and ultimately enable the EB to establish a niche enabling bacterial survival and growth. The bacterial and host molecules involved in these processes are beginning to emerge.