Genomics of "chlamydia"-related bacteria

AbstractThe Chlamydiales order is an important bacterial phylum that comprises some of the most successful human pathogens such as Chlamydia trachomatis, the leading infectious cause of blindness worldwide. Since some years, several new bacteria related to Chlamydia have been discovered in clinical or environmental samples and might represent emerging pathogens. The genome sequencing of classical Chlamydia has brought invaluable information on these obligate intracellular bacteria otherwise difficult to study due to the lack of tools to perform basic genetic manipulation. The recent emergence of high-throughput sequencing technologies yielding millions of reads in a short time lowered the costs of genome sequencing and thus represented a unique opportunity to study Chlamydia-re\ated bacteria. Based on the sequencing and the analysis of Chlamydiales genomes, this thesis provides significant insights into the genetic determinants of the intracellular lifestyle, the pathogenicity, the metabolism and the evolution of Chlamydia-related bacteria. A first approach showed the efficacy of rapid sequencing coupled to proteomics to identify immunogenic proteins. This method, particularly useful for an emerging pathogen such as Parachlamydia acanthamoebae, enabled us to discover good candidates for the development of diagnostic tools that would permit to evaluate at larger scale the role of this bacterium in disease. Second, the complete genome of Waddlia chondrophila, a potential agent of miscarriage, encodes numerous virulence factors to manipulate its host cell and resist to environmental stresses. The reconstruction of metabolic pathways showed that the bacterium possesses extensive capabilities compared to related organisms. However, it is still incapable of synthesizing some essential components and thus has to import them from its host. Third, the genome comparison of Protochlamydia naegleriophila to its closest known relative Protochlamydia amoebophila revealed a particular evolutionary dynamic with the occurrence of an unexpected genome rearrangement. Fourth, a phylogenetic analysis of P. acanthamoebae and Legionella drancourtii identified several genes probably exchanged by horizontal gene transfer with other intracellular bacteria that might occur within their amoebal host. These genes often encode mechanisms for resistance to metal or toxic compounds. As a whole, the analysis of the different genomes enabled us to highlight a large diversity in size, GC percentage, repeat content as well as plasmid organization. The abundant genomic data obtained during this thesis have a wide impact since they provide the necessary bases for detailed investigations on countless aspects of the biology and the evolution of Chlamydia-related bacteria, whether in wet lab or by bioinformatical analyses.RésuméL'ordre des Chlamydiales est un important phylum bactérien qui comprend de nombreuses espèces pathogènes pour l'homme et les animaux, dont Chlamydia trachomatis, responsable du trachome, la cause majeure de cécité d'origine infectieuse à travers le monde. Durant ces dernières décennies, de nombreuses bactéries apparentées aux Chlamydia ont été découvertes dans des échantillons environnementaux ou cliniques mais leur éventuel rôle pathogène dans le développement de maladies reste peu connu. Ces bactéries sont des intracellulaires obligatoires car elles ont besoin d'une cellule hôte pour se multiplier, ce qui rend leur étude particulièrement difficile. Le développement de nouvelles technologies permettant de séquencer le génome d'un organisme rapidement et à moindre coût ainsi que l'essor des méthodes d'analyse s'y rapportant représentent une opportunité exceptionnelle d'étudier ces organismes. Dans ce contexte, cette thèse démontre l'utilité de la génomique pour développer de nouveaux outils diagnostiques ainsi que pour étudier le métabolisme de ces bactéries, leurs facteurs de virulence et leur évolution.Ainsi, une première approche a illustré l'utilité d'un séquençage rapide pour obtenir les informations nécessaires à l'identification de protéines qui sont reconnues par des anticorps humains ou animaux. Cette méthode, particulièrement utile pour un pathogène émergent tel que Parachlamydia acanthamoebae, a permis de découvrir de bons candidats pour le développement d'un outil diagnostique qui permettrait d'évaluer à plus large échelle le rôle de cette bactérie notamment dans la pneumonie. L'analyse du contenu génique de Waddlia chondrophila, un autre germe qui pourrait être impliqué dans les avortements et tes fausses-couches, a en outre mis en évidence la présence de nombreux facteurs connus qui lui permettent de manipuler son hôte. Cette bactérie possède de plus grandes capacités métaboliques que les autres Chlamydia, mais elle est incapable de synthétiser certains composants et doit donc les importer de son hôte pour subvenir à ses besoins. La comparaison du génome de Protochlamydia naegleriophila à son plus proche parent, Protochlamydia amoebophila, a dévoilé une évolution dynamique particulière avec l'occurrence d'un réarrangement majeur inattendu après la séparation de ces deux espèces. En outre, ces études ont montré l'occurrence de plusieurs transferts de gène avec d'autres organismes plus éloignés, notamment d'autres intracellulaires d'amibes, souvent pour l'acquisition de mécanismes de résistances à des composés toxiques. Les données génomiques acquises durant ce travail posent les fondements nécessaires a de nombreuses analyses qui permettront progressivement de mieux comprendre de nombreux aspects de ces bactéries fascinantes.

Study of Staphylococcus aureus pathogenic genes by transfer and expression in the less virulent organism Streptococcus gordonii.

Because Staphylococcus aureus strains contain multiple virulence factors, studying their pathogenic role by single-gene inactivation generated equivocal results. To circumvent this problem, we have expressed specific S. aureus genes in the less virulent organism Streptococcus gordonii and tested the recombinants for a gain of function both in vitro and in vivo. Clumping factor A (ClfA) and coagulase were investigated. Both gene products were expressed functionally and with similar kinetics during growth by streptococci and staphylococci. ClfA-positive S. gordonii was more adherent to platelet-fibrin clots mimicking cardiac vegetations in vitro and more infective in rats with experimental endocarditis (P < 0.05). Moreover, deleting clfA from clfA-positive streptococcal transformants restored both the low in vitro adherence and the low in vivo infectivity of the parent. Coagulase-positive transformants, on the other hand, were neither more adherent nor more infective than the parent. Furthermore, coagulase did not increase the pathogenicity of clfA-positive streptococci when both clfA and coa genes were simultaneously expressed in an artificial minioperon in streptococci. These results definitively attribute a role for ClfA, but not coagulase, in S. aureus endovascular infections. This gain-of-function strategy might help solve the role of individual factors in the complex the S. aureus-host relationship.

Endothelial nitric oxide synthase gene transfer restores endothelium-dependent relaxations and attenuates lesion formation in carotid arteries in apolipoprotein E-deficient mice.

Nitric oxide (NO) and monocyte chemoattractant protein-1 (MCP-1) exert partly opposing effects in vascular biology. NO plays pleiotropic vasoprotective roles including vasodilation and inhibition of platelet aggregation, smooth muscle cell proliferation, and endothelial monocyte adhesion, the last effect being mediated by MCP-1 downregulation. Early stages of arteriosclerosis are associated with reduced NO bioactivity and enhanced MCP-1 expression. We have evaluated adenovirus-mediated gene transfer of human endothelial NO synthase (eNOS) and of a N-terminal deletion (8ND) mutant of the MCP-1 gene that acts as a MCP-1 inhibitor in arteriosclerosis-prone, apolipoprotein E-deficient (ApoE(-/-)) mice. Endothelium-dependent relaxations were impaired in carotid arteries instilled with a noncoding adenoviral vector but were restored by eNOS gene transfer (p < 0.01). A perivascular collar was placed around the common carotid artery to accelerate lesion formation. eNOS gene transfer reduced lesion surface areas, intima/media ratios, and macrophage contents in the media at 5-week follow-up (p < 0.05). In contrast, 8ND-MCP-1 gene transfer did not prevent lesion formation. In conclusion, eNOS gene transfer restores endothelium-dependent vasodilation and inhibits lesion formation in ApoE(-/-) mouse carotids. Further studies are needed to assess whether vasoprotection is maintained at later disease stages and to evaluate the long-term efficacy of eNOS gene therapy for primary arteriosclerosis.

Long-term efficacy of ciliary muscle gene transfer of three sFlt-1 variants in a rat model of laser-induced choroidal neovascularization.

Inhibition of vascular endothelial growth factor (VEGF) has become the standard of care for patients presenting with wet age-related macular degeneration. However, monthly intravitreal injections are required for optimal efficacy. We have previously shown that electroporation enabled ciliary muscle gene transfer results in sustained protein secretion into the vitreous for up to 9 months. Here, we evaluated the long-term efficacy of ciliary muscle gene transfer of three soluble VEGF receptor-1 (sFlt-1) variants in a rat model of laser-induced choroidal neovascularization (CNV). All three sFlt-1 variants significantly diminished vascular leakage and neovascularization as measured by fluorescein angiography (FA) and flatmount choroid at 3 weeks. FA and infracyanine angiography demonstrated that inhibition of CNV was maintained for up to 6 months after gene transfer of the two shortest sFlt-1 variants. Throughout, clinical efficacy was correlated with sustained VEGF neutralization in the ocular media. Interestingly, treatment with sFlt-1 induced a 50% downregulation of VEGF messenger RNA levels in the retinal pigment epithelium and the choroid. We demonstrate for the first time that non-viral gene transfer can achieve a long-term reduction of VEGF levels and efficacy in the treatment of CNV.Gene Therapy advance online publication, 27 June 2013; doi:10.1038/gt.2013.36.

Impact of an intervention to control risk associated with patient transfer.

QUESTION UNDER STUDY: Hospitals transferring patients retain responsibility until admission to the new health care facility. We define safe transfer conditions, based on appropriate risk assessment, and evaluate the impact of this strategy as implemented at our institution. METHODS: An algorithm defining transfer categories according to destination, equipment monitoring, and medication was developed and tested prospectively over 6 months. Conformity with algorithm criteria was assessed for every transfer and transfer category. After introduction of a transfer coordination centre with transfer nurses, the algorithm was implemented and the same survey was carried out over 1 year. RESULTS: Over the whole study period, the number of transfers increased by 40%, chiefly by ambulance from the emergency department to other hospitals and private clinics. Transfers to rehabilitation centres and nursing homes were reassigned to conventional vehicles. The percentage of patients requiring equipment during transfer, such as an intravenous line, decreased from 34% to 15%, while oxygen or i.v. drug requirement remained stable. The percentage of transfers considered below theoretical safety decreased from 6% to 4%, while 20% of transfers were considered safer than necessary. A substantial number of planned transfers could be "downgraded" by mutual agreement to a lower degree of supervision, and the system was stable on a short-term basis. CONCLUSION: A coordinated transfer system based on an algorithm determining transfer categories, developed on the basis of simple but valid medical and nursing criteria, reduced unnecessary ambulance transfers and treatment during transfer, and increased adequate supervision.

Transfer factor for carbon monoxide: a glance behind the scene.

The transfer factor for carbon monoxide (TLCO) is widely used in pulmonary function laboratories because it represents a unique non-invasive window on pulmonary microcirculation. The TLCO is the product of two primary measurements, the alveolar volume (VA) and the CO transfer coefficient (KCO). This test is most informative when VA and KCO are examined, together with their product TLCO. In a normal lung, a low VA due to incomplete expansion is associated with an elevated KCO, resulting in a mildly reduced TLCO. Thus, in case of low VA, a seemingly "normal KCO" must be interpreted as an abnormal gas transfer. The most common clinical conditions associated with an abnormal TLCO are characterised by a limited number of patterns for VA and KCO: incomplete lung expansion, discrete loss of alveolar units, diffuse loss of alveolar units, emphysema, pulmonary vascular disorders, high pulmonary blood volume, alveolar haemorrhage.

Relevé des consultations et des transferts de patients par enquête directe auprès des médecins: méthode d'une enquête, acceptabilité et limites d'interprétation. [Summary of consultations and transfer of patients by a direct survey of physicians: survey methods, acceptability and limits of interpretation].

A survey of medical ambulatory practice was carried out in February-March 1981 in the two Swiss cantons of Vaud and Fribourg (total population: 700,000), in which 205 physicians participated. The methodology used was inspired from the U.S. National Ambulatory Medical Care Survey, the data collection instrument of which was adapted to our conditions; in addition, data were gathered on all referrals prescribed by 154 physicians during two weeks. (The instruments used are presented.) The potential and limits of this type of survey are discussed, as well as the representativity of the participating physicians and of the recorded visits, which are a systematic sample of over 43,000 visits.

Life history analysis of integrative and conjugative element activation in growing microcolonies of Pseudomonas.

Integrative and conjugative elements (ICE) are in some ways parasitic mobile DNA that propagate vertically through replication with the bacterial host chromosome but at low frequencies can excise and invade new recipient cells through conjugation and reintegration (horizontal propagation). The factors that contribute to successful horizontal propagation are not very well understood. Here, we study the influence of host cell life history on the initiation of transfer of a model ICE named ICEclc in bacteria of the genus Pseudomonas. We use time-lapse microscopy of growing and stationary-phase microcolonies of ICEclc bearing cells in combination with physiological staining and gene reporter analysis in stationary-phase suspended cells. We provide evidence that cell age and cell lineage are unlikely to play a role in the decision to initiate the ICEclc transfer program. In contrast, cells activating ICEclc show more often increased levels of reactive oxygen species and membrane damage than nonactivating cells, suggesting that some form of biochemical damage may make cells more prone to ICEclc induction. Finally, we find that ICEclc active cells appear spatially at random in a microcolony, which may have been a selective advantage for maximizing ICEclc horizontal transmission to new recipient species.

Plutonium from above-ground nuclear tests in milk teeth: investigation of placental transfer in children born between 1951 and 1995 in Switzerland.

BACKGROUND: Occupational risks, the present nuclear threat, and the potential danger associated with nuclear power have raised concerns regarding the metabolism of plutonium in pregnant women. OBJECTIVE: We measured plutonium levels in the milk teeth of children born between 1951 and 1995 to assess the potential risk that plutonium incorporated by pregnant women might pose to the radiosensitive tissues of the fetus through placenta transfer. METHODS: We used milk teeth, whose enamel is formed during pregnancy, to investigate the transfer of plutonium from the mother's blood plasma to the fetus. We measured plutonium using sensitive sector field inductively coupled plasma mass spectrometry techniques. We compared our results with those of a previous study on strontium-90 ((90)Sr) released into the atmosphere after nuclear bomb tests. RESULTS: Results show that plutonium activity peaks in the milk teeth of children born about 10 years before the highest recorded levels of plutonium fallout. By contrast, (90)Sr, which is known to cross the placenta barrier, manifests differently in milk teeth, in accordance with (90)Sr fallout deposition as a function of time. CONCLUSIONS: These findings demonstrate that plutonium found in milk teeth is caused by fallout that was inhaled around the time the milk teeth were shed and not from any accumulation during pregnancy through placenta transfer. Thus, plutonium may not represent a radiologic risk for the radiosensitive tissues of the fetus.

Host and invader impact of transfer of the clc genomic island into Pseudomonas aeruginosa PAO1

Genomic islands, large potentially mobile regions of bacterial chromosomes, are a major contributor to bacteria evolution. Here, we investigated the fitness cost and phenotypic differences between the bacterium Pseudomonas aeruginosa PAO1 and a derivative carrying one integrated copy of the clc element, a 103-kb genomic island [and integrative and conjugative element (ICE)] originating in Pseudomonas sp. strain B13 and a close relative of genomic islands found in clinical and environmental isolates of P. aeruginosa. By using a combination of whole genome transcriptome profiling, phenotypic arrays, competition experiments, and biofilm formation studies, only few differences became apparent, such as reduced biofilm growth and fourfold stationary phase repression of genes involved in acetoin metabolism in PAO1 containing the clc element. In contrast, PAO1 carrying the clc element acquired the capacity to grow on 3-chlorobenzoate and 2-aminophenol as sole carbon and energy substrates. No fitness loss >1% was detectable in competition experiments between PAO1 and PAO1 carrying the clc element. The genes from the clc element were not silent in PAO1, and excision was observed, although transfer of clc from PAO1 to other recipient bacteria was reduced by two orders of magnitude. Our results indicate that newly acquired mobile DNA not necessarily invoke an important fitness cost on their host. Absence of immediate detriment to the host may have contributed to the wide distribution of genomic islands like clc in bacterial genomes

Helical tomotherapy (HT) for the treatment of anal canal cancer: preliminary clinical results, and dosimetric comparison between HT and intensity-modulated or 3D conformal radiotherapy

Background: To report a single-center experience in 19 patients (pts) with anal canal cancer treated with helical tomotherapy (HT) and concurrent chemotherapy, and compare the dosimetric results with fixed-field intensitymodulated radiotherapy (IMRT) and 3D conformal radiotherapy (3D RT). Materials and Methods: Between 2007 and 2008, 19 consecutive pts were treated with HT and concurrent CT for anal canal cancer. Median age was 59 years (range, 38−83), and female/male ratio was 14/5. The majority of the pts had T2 or T3 tumours (68.4%), and 52.6% had positive lymph nodes. In all 19 pts, pelvic and inguinal nodes, and tumour irradiation was given using HT upto a median dose of 36 Gy (1.8 Gy/fr) followed by a 1-week gap. A boost dose of 23.4 Gy (1.8 Gy/fr) was delivered to the tumour and involved nodes using 3DRT (n = 12), HT (n = 6), or IMRT (n = 1). Simultaneous integrated boost was used in none of the pts. All but one patient with a T1N0 tumour received concomitant mitomycin/5- fluorouracil (n = 12) or mitomycin/capecitabin (n = 7) CT. Toxicity was scored according to the Common Terminology Criteria for Adverse Events (NCICTCAE v3.0). HT plans and treatments were generated using Tomotherapy, Inc., software and hardware; and 3D or IMRT boost plans with the CMS treatment planning system (TPS), using 6−18 MV photons from a Siemens Primus accelerator. For dosimetric comparison, computed tomography data sets of 10 pts were imported into the TPS, and 3D and 5-field step-andshoot IMRT plans were generated for each case. Plans were optimized with the aim of assessing organs at risk (OAR) and healthy-tissue sparing while enforcing highly conformal target coverage, and evaluated by dose-volume histograms (DVH) of planning target volumes (PTV) and OAR. Results: With a median follow-up of 13 months (range, 3−18), all pts are alive and well; except one patient developing local recurrence at 12 months. No patient developed grade 3 or more acute toxicity. No unplanned treatment interruption was necessary because of toxicity. With 360-degree-of-freedom beam projection, HT showed an advantage over 3D or IMRT plans in terms of dose conformity around the PTV, and dose gradients were steeper outside the PTV, resulting in reduced doses to OARs. Using HT, acute toxicity was acceptable, and seemed to be better than historical standards. Conclusion: We conclude that HT combined with concurrent chemotherapy for anal canal cancer is effective and tolerable. Compared to 3DRT or 5-field IMRT, there is better conformity around the PTV, and OAR sparing.