Activation of human TLR4/MD-2 by hypoacylated lipopolysaccharide from a clinical isolate of Burkholderia cenocepacia

Lung infection by Burkholderia species, in particular B. cenocepacia, accelerates tissue damage and increase post-lung transplant mortality in cystic fibrosis patients. Host- microbes interplay largely depends on interactions between pathogen specific molecules and innate immune receptors such as the Toll-like receptor 4 (TLR4), which recognizes the lipid A moiety of the bacterial lipopolysaccharide (LPS). The human TLR4/MD-2 LPS receptor complex is strongly activated by hexa-acylated lipid A and poorly activated by underacylated lipid A. Here, we report that B. cenocepacia LPS strongly activates human TLR4/MD-2 despite its lipid A having only five acyl chains. Further, we show that aminoarabinose residues in lipid A contribute to TLR4-lipid A interactions, and experiments in a mouse model of LPS-induced endotoxic shock confirmed the pro- inflammatory potential of B. cenocepacia penta-acylated lipid A. Molecular modeling, combined with mutagenesis of TLR4-MD2 interactive surfaces, suggests that longer acyl chains and the aminoarabinose residues in the B. cenocepacia lipid A allow exposure of the fifth acyl chain on the surface of MD-2 enabling interactions with TLR4 and its dimerization. Our results provide a molecular model for activation of the human TLR4/MD- 2 complex by penta-acylated lipid A, explaining the ability of hypoacylated B. cenocepacia LPS to promote pro- inflammatory responses associated to the severe pathogenicity of this opportunistic bacterium.

A Case of Canine Borreliosis in Iran Caused by Borrelia persica

Tick-borne relapsing fever is an endemic disease in Iran, with most cases attributed to infection by B. persica, which is transmitted by Ornithodoros tholozani soft ticks. Here, we report spirochetemia in blood of a puppy residing in Tehran, Iran. The causative species was identified by use of highly discriminative IGS sequencing; the 489 bp IGS sequence obtained in our study showed 99% identity (100% coverage) when compared with Borrelia persica sequences derived from clinical cases or from Ornithodoros tholozani ticks. Our IGS sequence also showed 99% similarity over 414 bp (85% coverage) with a strain from a domestic dog, and 96% over 328 bp (69% coverage) with a strain from a domestic cat. Pet-keeping in cosmopolitan cities like Tehran has become increasingly popular in recent years. Animals are often transported into the city in cages or cardboard boxes that might also harbour minute tick larvae and/or early stages of the nymphs bringing them into the urban environment. This may pose a threat to household members who buy and keep these puppies and as a result may come into close contact with infected ticks.

Malaria and tuberculosis co-infection:role for hemozoin immunosuppression?

Tese de doutoramento, Ciências Biomédicas (Microbiologia e Parasitologia), Universidade de Lisboa, Faculdade de Medicina, 2014

Human factors required for mycobacterium tuberculosis macrophage infection

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2014

Sonication of removed breast implants for improved detection of subclinical infection.

BACKGROUND: Capsular fibrosis is a severe complication after breast implantation with an uncertain etiology. Microbial colonization of the prosthesis is hypothesized as a possible reason for the low-grade infection and subsequent capsular fibrosis. Current diagnostic tests consist of intraoperative swabs and tissue biopsies. Sonication of removed implants may improve the diagnosis of implant infection by detachment of biofilms from the implant surface. METHODS: Breast implants removed from patients with Baker grades 3 and 4 capsular contracture were analyzed by sonication, and the resulting sonication fluid was quantitatively cultured. RESULTS: This study investigated 22 breast implants (6 implants with Baker 3 and 16 implants with Baker 4 capsular fibrosis) from 13 patients. The mean age of the patients was 49 years (range, 31-76 years). The mean implant indwelling time was 10.4 years (range, 3 months to 30 years). Of the 22 implants, 12 were used for breast reconstruction and 10 for aesthetic procedures. The implants were located subglandularly (n = 12), submuscularly (n = 6), and subcutaneously (n = 4). Coagulase-negative staphylococci, Propionibacterium acnes, or both were detected in the sonication fluid cultures of nine implants (41%), eight of which grew significant numbers of microorganisms (>100 colonies/ml of sonication fluid). CONCLUSIONS: Sonication detected bacteria in 41% of removed breast implants. The identified bacteria belonged to normal skin flora. Further investigation is needed to determine any causal relation between biofilms and capsular fibrosis.

Innate Immune Sensing of Fusarium culmorum by Mouse Dendritic Cells.

Chronic inhalation of grain dust is associated with asthma and chronic bronchitis in grain worker populations. Exposure to fungal particles was postulated to be an important etiologic agent of these pathologies. Fusarium species frequently colonize grain and straw and produce a wide array of mycotoxins that impact human health, necessitating an evaluation of risk exposure by inhalation of Fusarium and its consequences on immune responses. Data showed that Fusarium culmorum is a frequent constituent of aerosols sampled during wheat harvesting in the Vaud region of Switzerland. The aim of this study was to examine cytokine/chemokine responses and innate immune sensing of F. culmorum in bone-marrow-derived dendritic cells and macrophages. Overall, dendritic cells and macrophages responded to F. culmorum spores but not to its secreted components (i.e., mycotoxins) by releasing large amounts of macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, monocyte chemoattractant protein (MCP)-1, RANTES, and interleukin (IL)-12p40, intermediate amounts of tumor necrosis factor (TNF), IL-6, IL-12p70, IL-33, granulocyte colony-stimulating factor (G-CSF), and interferon gamma-induced protein (IP-10), but no detectable amounts of IL-4 and IL-10, a pattern of mediators compatible with generation of Th1 or Th17 antifungal protective immune responses rather than with Th2-dependent allergic responses. The sensing of F. culmorum spores by dendritic cells required dectin-1, the main pattern recognition receptor involved in β-glucans detection, but likely not MyD88 and TRIF-dependent Toll-like receptors. Taken together, our results indicate that F. culmorum stimulates potently innate immune cells in a dectin-1-dependent manner, suggesting that inhalation of F. culmorum from grain dust may promote immune-related airway diseases in exposed worker populations.

Host-parasite relations in a mycoparasitic system : alterations in membrane permeability and lipid composition in Choanephora cucurbitarum infected by Piptocephalis virginiana /

examined in Choanephora cucurbita rum during the early stages of infection by Piptocephalis virginiana » There was a small but consistent increase in the leakage of electrolytes, amino acids and sugars as a result of infection. These low levels of differential leakage in infected tissues are explained on the basis of the nature of this obligate, biotrophic, mycoparasitic system. Quantitative analysis of the twenty six amino acids and amino compounds detected in the leacheates — showed similar profiles in infected and control host and no new species of amino acids or amino compounds were detected in either infected or control host leacheates. Comparatively high amounts of aspartic acid, glutamic acid and alanine were found in the leacheates of host and infected host . Analyses of the sugars comprising the leacheates of infected and control host showed the presence of eight sugars, among which glucose was found in significant amounts (50-53%) ' The nutritional implication of this preferential leakage is discussed. No significant difference was observed in the leacheates of infected host sugar profiles compared with that of the control host. Profiles of the internal pool sugars of infected and control host did not reflect that obtained from the leacheate data, perhaps owing to leakage of sugars in a selective manner . Membrane lipid analyses yielded higher levels of lipid in infected host compared with the control, both at the 24 h and 36 h analyses. In addition, preliminary investigations of phosphorous-32 incorporation and turnover in phospholipids showed higher levels of 32p incorporation and turnover in infected host compared with the control. No apparent difference was noted in the profiles of the neutral lipid classes and the polar lipid classes of the membrane lipids as determined by one and two dimensional thin-layer chromatography respectively. However, a small but consistently higher degree of unsaturation was detected in the fatty acids of infected tissue compared with the control. Also, '^''-^^''^^'-'-^'^^c acid, a polyunsaturated fatty acid previously reported to show a direct correlation during the early stages of infection and the degree of parasitism of P. virginiana on C. cucurbitarum , was found in higher amounts in infected host membrane lipids compared with that of the control host. The implications of these membrane lipid alterations are discussed with particular reference to the small but consistently higher leakage of electrolytes, amino acids and sugars observed during infection in this study.

Permissivity of human HeLa cells to bovine adenovirus type 2 (BaV2 infection

Infection of hUlnan cells by bovine adenovirlls type 2 (BAV2) is abortive. To obtain a better understanding of this pllenomel1011, and in particular to identify Wllich steps in the viral replicative cycles are altered dllring this virlls-host cells interaction, we have llndertaken a detailed study of BAV2 infections of the nonpennissive hUlnan IIeLa cells. Using autoradiography and 3H-thymidine-labeled vvhole virus particles for infection of HeLa cells, vve determined that viral attachluent appears normal. Furthermore, Southern analysis revealed that internalization and transport to the nuclells occurs in BAV2 infected HeLa cells. To investigate viral DNi\ synthesis, infectivity assays involving hydroxyllrea, a viral DN-A synthesis inhibitor, were carried out. The results revealed that Bft:LV2 DNA synthesis does not occur in HeLa cells. Fllrtller investigations into viral early gene expression by northern blotting analyses indicated that HeLa cells fail to support expression of EIA. This suggested that abortive infection by BAV2 could be attributed to faiiure of EIA to express. To test the possibility that the failure to express ElA was due to the inability of the host cell to recognize the E lA prOlTIoter, ,ve carried out transient expression transfection experiments using plaslnids \vith the bacterial lacZ linder the control of either BAV2 or i\d5 EIA promoter. X-gal histochelIlical assays sho\ved expression of lacZ from the Ad5 ElA prOlnoter but no expression of lacZ [rOln the BAV2 EIA prOlTIoter. This further suggests that the abortive infection b:y BAV2 could be attributed to failure of EIA to express dlle to a nonfllnctional prOlTIoter in hlunan cells. Thus we speClllated that abortive infection of HeLa cells by adenoviruses may be averted by providing EtA functions in trans. To demonstrate this, we coinfected HeLa cells with Ad5 and BAV2, reasoning that Ad5 could cOlnpensate for EIA deficiency in BAV2. OUf results showed that BAV2 DNA synthesis was indeed Sllpported in HeLa cells coinfected with Ad5dlE3 as revealed by Southern analysis. In contrast, coinfection of HeLa cells \vith BAV2 and Ad5dlElE3 mutallt did not support BLt\V2 DNA synthesis. Interestingly, BAV2 failed to replicate in 293 cells which are constitlltively expressing the El genes. This could ilnply that El is necessary but not sufficient to avert the failllre ofBAV2 to undergo productive infection ofhulnan cells.

Étude de la régulation du facteur de transcription NF-kB dans l'infection par le RSV

L’infection par le Virus Respiratoire Syncytial cause des affections pulmonaires aiguës en pédiatrie caractérisée par une réponse inflammatoire excessive médiée par la production de cytokines par les cellules épithéliales des voies aériennes. Les gènes codant pour ces cytokines sont régulés par le facteur de transcription NF-κB (p50/p65) dont l’activation est classiquement induite par la phosphorylation de son inhibiteur IκBα, ce qui permet l’accumulation de l’hétérodimère au noyau. Par contre, nous avons récemment identifié la phosphorylation en sérine 536 de la sous-unité p65 comme une autre étape essentielle à son activation lors de l’infection des AEC par RSV. Le travail présenté dans ce mémoire a permis de démontrer que l’inhibition de l’expression de RIG-I, de Cardif ou de TRAF6, 3 protéines impliquées dans la reconnaissance cellulaire des virus, conduit à l’inhibition de cette phosphorylation en réponse à RSV. Nous avons également établi à l’aide d’inhibiteurs pharmacologiques et d’ARNi que, parmi les diverses kinases connues pour phosphoryler p65 en réponse à divers stimulus, IKKα/β sont essentielles à cette phosphorylation lors d’une stimulation par RSV. Puisque TRAF6 est bien connu dans la littérature pour activer le complexe IKK, nous proposons que TRAF6, après reconnaissance de l’ARN viral de RSV par RIG-I, active le complexe IKK qui induit la phosphorylation de la sousunité p65 de NF-κB, permettant l’expression de gènes cibles. D’autre part, nous avions précédemment démontré que Nox2, un isoforme de NADPH oxydase, contrôle l’activation de NF-κB en régulant les phosphorylations de IκBα et p65. Nous montrons ici que l’inhibition de Nox2 réduit fortement l’activité du complexe kinase IKK. De plus, la présence au niveau basal de Nox2 est critique pour le niveau d’ARN messager de Cardif. Nous proposons donc que la régulation de la phosphorylation de p65 en ser536 par Nox2 soit via son effet sur Cardif en permettant la fonctionnalité de la voie RIG-I.

Prévalence et facteurs de risque de l'infection par Coxiella Burnettii chez les ruminants d'élevage au Québec

Coxiella burnetii est une bactérie zoonotique affectant un grand nombre d’espèces animales. Chez les ruminants domestiques, l’infection est généralement asymptomatique, mais parfois associée à des problèmes reproducteurs. Néanmoins, le cycle de transmission de l’infection chez ceux-ci demeure peu connu. Dans ce contexte, nous avons réalisé une étude auprès de fermes bovines, ovines et caprines dans deux régions administratives du Québec afin d’estimer les prévalences de cette infection et d’identifier les facteurs de risque, aux niveaux individuel et troupeau, associés à la positivité. Nous avons estimé une prévalence de positivité au niveau troupeau de 44.6 % (IC95%=33.0-56.6) chez les bovins, de 70.8 % (IC95% =48.9-87.4) chez les ovins et de 66.7 % (IC95% =22.3-95.7) chez les caprins. Une association a été observée chez les troupeaux bovins entre leur positivité et la densité de petits ruminants par kilomètre carré dans un rayon de cinq kilomètres entourant la ferme. Chez les petits ruminants, une association avec la positivité des troupeaux a été observée avec la taille des troupeaux et la présence d’un chien sur la ferme. Au niveau individuel, le nombre de jours en lait ainsi que l’âge des petits ruminants étaient associés à la positivité, et ce dernier facteur était modulé par l’accès des animaux au pâturage. Aucun agrégat spatial de fermes positives n’a été détecté chez aucune des trois espèces. L’infection par Coxiella burnetii est donc fréquente dans les troupeaux de ruminants domestiques québécois et semble associée à certaines pratiques de régie et à la présence, ou proximité, d’autres animaux domestiques.

Control of Leveillula taurica in tomato by Acremonium alternatum is by induction of resistance, not Hyperparasitism

Spores of the hyperparasite Acremonium alternatum reduced powdery mildew infection by Leveillula taurica on greenhouse tomato. The effect was slightly increased when spores were applied killed, and therefore not due to direct parasitism. The effect was systemic, protecting untreated leaves above the treated ones. Spores killed by heat had more effect than when killed by UV, so the effect was presumably due to induction of host resistance by substances released when cells were heat killed. The size of the effect depended upon leaf age and level of infection. Effects on primary infection and expansion of successful infections appear to be under independent control.

Coxsackievirus B3-associated myocardial pathology and viral load reduced by recombinant soluble human decay-accelerating factor in mice

Coxsackievirus B3 (CVB3) infection can result in myocarditis, which in turn may lead to a protracted immune response and subsequent dilated cardiomyopathy. Human decay-accelerating factor (DAF), a binding receptor for CVB3, was synthesized as a soluble IgG1-Fc fusion protein (DAF-Fc). In vitro, DAF-Fc was able to inhibit complement activity and block infection by CVB3, although blockade of infection varied widely among strains of CVB3. To determine the effects of DAF-Fc in vivo, 40 adolescent A/J mice were infected with a myopathic strain of CVB3 and given DAF-Fc treatment 3 days before infection, during infection, or 3 days after infection; the mice were compared with virus alone and sham-infected animals. Sections of heart, spleen, kidney, pancreas, and liver were stained with hematoxylin and eosin and submitted to in situ hybridization for both positive-strand and negative-strand viral RNA to determine the extent of myocarditis and viral infection, respectively. Salient histopathologic features, including myocardial lesion area, cell death, calcification and inflammatory cell infiltration, pancreatitis, and hepatitis were scored without knowledge of the experimental groups. DAF-Fc treatment of mice either preceding or concurrent with CVB3 infection resulted in a significant decrease in myocardial lesion area and cell death and a reduction in the presence of viral RNA. All DAF-Fc treatment groups had reduced infectious CVB3 recoverable from the heart after infection. DAF-Fc may be a novel therapeutic agent for active myocarditis and acute dilated cardiomyopathy if given early in the infectious period, although more studies are needed to determine its mechanism and efficacy.

Differential induction of apoptosis in human colonic carcinoma cells (Caco-2) by Atopobium, and commensal, probiotic and enteropathogenic bacteria: mediation by the mitochondrial pathway

The induction of apoptosis in mammalian cells by bacteria is well reported. This process may assist infection by pathogens whereas for non-pathogens apoptosis induction within carcinoma cells protects against colon cancer. Here, apoptosis induction by a major new gut bacterium, Atopobium minutum, was compared with induction by commensal (Escherichia coli K-12 strains), probiotic (Lactobacillus rhamnosus, Bifidobacterium latis) and pathogenic (E. coli: EPEC and VTEC) gut bacteria within the colon cancer cell line, Caco-2. The results show a major apoptotic effect for the pathogens, mild effects for the probiotic strains and A. minutum, but no effect for commensal E. coli. The mild apoptotic effects observed are consistent with the beneficial roles of probotics in protection against colon cancer and suggest, for the first time, that A. minutum possesses similar advantageous, anti-cancerous activity. Although bacterial infection increased Caco-2 membrane FAS levels, caspase-8 was not activated indicating that apoptosis is FAS independent. Instead, in all cases, apoptosis was induced through the mitochondrial pathway as indicated by BAX translocation, cytorchrome c release, and caspase-9 and -3 cleavage. This suggests that an intracellular stimulus initiates the observed apoptosis responses.

Macrophage migration inhibitory factor is up-regulated in human first-trimester placenta stimulated by soluble antigen of Toxoplasma gondii, resulting in increased monocyte adhesion on villous explants

Considering the potential role of macrophage migration inhibitory factor (MIF) in the inflammation process in placenta when infected by pathogens, we investigated the production of this cytokine in chorionic villous explants obtained from human first-trimester placentas stimulated with soluble antigen from Toxoplasma gondii (STAg). Parallel cultures were performed with villous explants stimulated with STAB, interferon-gamma (IFN-gamma), or STAB plus IFN-gamma. To assess the role of placental MIF on monocyte adhesiveness to human trophoblast, explants were co-cultured with human myelomonocytic THP-1 cells in the presence or absence of supernatant from cultures treated with STAB (SPN), SPN plus anti-MIF antibodies, or recombinant MIF. A significantly higher concentration of MIF was produced and secreted by villous explants treated with STAB or STAB plus IFN-gamma after 24-hour culture. Addition of SPN or recombinant MIF was able to increase THP-1 adhesion, which was inhibited after treatment with anti-MIF antibodies. This phenomenon was associated with intercellular adhesion molecule expression by villous explants. Considering that the processes leading to vertical dissemination of T. gondii remain widely unknown, our results demonstrate that MIF production by human first-trimester placenta is up-regulated by parasite antigen and may play an essential role as an autocrine/paracrine mediator in placental infection by T. gondii.

Apoptosis of macrophages during pulmonary Mycobacterium bovis infection: correlation with intracellular bacillary load and cytokine levels

P>Apoptosis of macrophages infected with pathogenic mycobacteria is an alternative host defence capable of removing the environment supporting bacterial growth. In this work the influence of virulence and bacterial load on apoptosis of alveolar macrophages during the initial phase of infection by Mycobacterium bovis was investigated. BALB/c mice were infected intratracheally with high or low doses of the virulent (ATCC19274) or attenuated (bacillus Calmette-Guerin Moreau) strains of M. bovis. The frequency of macrophage apoptosis, the growth of mycobacteria in macrophages, and the in situ levels of the cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10) and IL-12 and of the anti-apoptotic protein Bcl-2 were measured at day 3 and day 7 post-infection. An increase of macrophage apoptosis was observed after infection with both strains but the virulent strain induced less apoptosis than the attenuated strain. On the 3rd day after infection with the virulent strain macrophage apoptosis was reduced in the high-dose group, while on the 7th day post-infection macrophage apoptosis was reduced in the low-dose group. Inhibition of apoptosis was correlated with increased production of IL-10, reduced production of TNF-alpha and increased production of Bcl-2. In addition, the production of IL-12 was reduced at points where the lowest levels of macrophage apoptosis were observed. Our results indicate that virulent mycobacteria are able to modulate macrophage apoptosis to an extent dependent on the intracellular bacterial burden, which benefits its intracellular growth and dissemination to adjacent cells.