Inhibitors of the Neisseria meningitidis PilF ATPase provoke type IV pilus disassembly.

Despite the availability of antibiotics and vaccines, Neisseria meningitidis remains a major cause of meningitis and sepsis in humans. Due to its extracellular lifestyle, bacterial adhesion to host cells constitutes an attractive therapeutic target. Here, we present a high-throughput microscopy-based approach that allowed the identification of compounds able to decrease type IV pilus-mediated interaction of bacteria with endothelial cells in the absence of bacterial or host cell toxicity. Compounds specifically inhibit the PilF ATPase enzymatic activity that powers type IV pilus extension but remain inefficient on the ATPase that promotes pilus retraction, thus leading to rapid pilus disappearance from the bacterial surface and loss of pili-mediated functions. Structure activity relationship of the most active compound identifies specific moieties required for the activity of this compound and highlights its specificity. This study therefore provides compounds targeting pilus biogenesis, thereby inhibiting bacterial adhesion, and paves the way for a novel therapeutic option for meningococcal infections.

Humanized mouse model to study bacterial infections targeting the microvasculature.

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Assessment of the interplay between blood and skin vascular abnormalities in adult purpura fulminans.

RATIONALE: Purpura fulminans in adults is a rare but devastating disease. Its pathophysiology is not well known. OBJECTIVES: To understand the pathophysiology of skin lesions in purpura fulminans, the interplay between circulating blood and vascular alterations was assessed. METHODS: Prospective multicenter study in four intensive care units. Patients with severe sepsis without skin lesions were recruited as control subjects. MEASUREMENTS AND MAIN RESULTS: Twenty patients with severe sepsis and purpura fulminans were recruited for blood sampling, and skin biopsy was performed in deceased patients. High severity of disease and mortality rates (80%) was observed. Skin biopsies in purpura fulminans lesions revealed thrombosis and extensive vascular damage: vascular congestion and dilation, endothelial necrosis, alteration of markers of endothelial integrity (CD31) and of the protein C pathway receptors (endothelial protein C receptor, thrombomodulin). Elevated plasminogen activating inhibitor-1 mRNA was also observed. Comparison with control patients showed that these lesions were specific to purpura fulminans. By contrast, no difference was observed for blood hemostasis parameters, including soluble thrombomodulin, activated protein C, and disseminated intravascular coagulation markers. Bacterial presence at the vascular wall was observed specifically in areas of vascular damage in eight of nine patients tested (including patients with Streptococcus pneumoniae, Neisseria meningitidis, Escherichia coli, and Pseudomonas aeruginosa infection). CONCLUSIONS: Thrombi and extensive vascular damage with multifaceted prothrombotic local imbalance are characteristics of purpura fulminans. A "vascular wall infection" hypothesis, responsible for endothelial damage and subsequent skin lesions, can be put forward.

Knockout of insulin-like growth factor-1 receptor impairs distal lung morphogenesis.

BACKGROUND: Insulin-like growth factors (IGF-I and -II) are pleiotropic regulators of somatic growth and development in vertebrate species. Endocrine and paracrine effects of both hormones are mediated by a common IGF type 1 receptor (IGF-1R). Lethal respiratory failure in neonatal IGF-1R knockout mice suggested a particular role for this receptor in pulmonary development, and we therefore investigated the consequences of IGF-1R inactivation in lung tissue. METHODS AND FINDINGS: We first generated compound heterozygous mutant mice harboring a hypomorphic (Igf1r(neo)) and a null (Igf1r(-)) allele. These IGF-1R(neo/-) mice express only 22% of normal IGF-1R levels and are viable. In adult IGF-1R(neo/-) mice, we assessed lung morphology and respiratory physiology and found normal histomorphometric characteristics and normal breathing response to hypercapnia. We then generated homozygous IGF-1R knockout mutants (IGF-1R(-/-)) and analyzed their lung development during late gestation using histomorphometric and immunohistochemical methods. IGF-1R(-/-) embryos displayed severe lung hypoplasia and markedly underdeveloped diaphragms, leading to lethal neonatal respiratory distress. Importantly, IGF-1R(-/-) lungs from late gestation embryos were four times smaller than control lungs and showed markedly thickened intersaccular mesenchyme, indicating strongly delayed lung maturation. Cell proliferation and apoptosis were significantly increased in IGF-1R(-/-) lung tissue as compared with IGF-1R(+/+) controls. Immunohistochemistry using pro-SP-C, NKX2-1, CD31 and vWF as markers revealed a delay in cell differentiation and arrest in the canalicular stage of prenatal respiratory organ development in IGF-1R(-/-) mutant mice. CONCLUSIONS/SIGNIFICANCE: We found that low levels of IGF-1R were sufficient to ensure normal lung development in mice. In contrast, complete absence of IGF-1R significantly delayed end-gestational lung maturation. Results indicate that IGF-1R plays essential roles in cell proliferation and timing of cell differentiation during fetal lung development.

Activation of peroxisome proliferator-activated receptor pathway stimulates the mitochondrial respiratory chain and can correct deficiencies in patients' cells lacking its components.

CONTEXT: The mitochondrial respiratory chain (RC) disorders are the largest group of inborn errors of metabolism and still remain without treatment in most cases. OBJECTIVE: We tested whether bezafibrate, a drug acting as a peroxisome proliferator-activated receptor (PPAR) agonist, could stimulate RC capacities. DESIGN: Fibroblasts or myoblasts from controls or patients deficient in complex I (CI), complex III (CIII), or complex IV (CIV) were cultured with or without bezafibrate. MAIN OUTCOME MEASURES: Enzyme activities, mRNA and protein expression, and respiration rates were measured. RESULTS: In control cells, bezafibrate increased the CI, CIII, and CIV enzyme activities (+42 to +52%), as well as RC mRNAs (+40 to +120%) and RC protein levels (+50 to +150%). Nine of 14 patient cell lines tested exhibited a significant increase in the activity of the deficient RC complex after bezafibrate treatment (+46 to +133%), and full pharmacological correction could be achieved in seven cell lines. Similar effects were obtained using a PPARdelta agonist. These changes were related to a drug-induced increase in the mutated mRNAs and RC protein levels. Finally, the molecular mechanisms by which the PPAR pathway could induce the expression of genes encoding structural subunits or ancillary proteins of the RC apparatus, leading to stimulate the activity and protein levels of RC complex, likely involved the PPARgamma coactivator-1alpha. CONCLUSIONS: This study suggests a rationale for a possible correction of moderate RC disorders due to mutations in nuclear genes, using existing drugs, and brings new insights into the role of PPAR in the regulation of the mitochondrial RC in human cells.

Peroxisome proliferator activated receptor delta (PPARdelta) agonist but not PPARalpha corrects carnitine palmitoyl transferase 2 deficiency in human muscle cells.

Type 2 carnitine palmitoyl transferase (CPT2) is involved in the transfer of long-chain fatty acid into the mitochondria. CPT2-deficient patients carry gene mutations associated with different clinical presentations, correlating with various levels of fatty acid oxidation (FAO) and residual CPT2 enzyme activity. We tested the hypothesis that pharmacological stimulation of peroxisome proliferator-activated receptors (PPAR) can stimulate FAO in CPT2-deficient muscle cells. Accordingly, we show that a 48-h treatment of CPT2-deficient myoblasts by bezafibrate restored FAO in patient cells. Specific agonists of PPARdelta (GWdelta 0742), and, to a lower extent, PPARalpha (GWalpha 7647) also stimulated FAO in control myoblasts. However, when tested in CPT2-deficient myoblasts, only the delta-agonist was able to restore FAO, whereas the alpha-agonist had no effect. GWdelta 0742 increased CPT2 mRNA levels, whereas no change in CPT2 transcripts was found in response to GWalpha 7647. Bezafibrate and GWdelta 0742 increased residual CPT2 activity and normalized long-chain acylcarnitine production by deficient cells. Finally, CPT1-B mRNA was also stimulated after PPAR agonist treatment, and this likely takes part in drug-induced increase of FAO in control muscle cells. In conclusion, this study clearly suggests that PPARs could be therapeutic targets for correction of inborn beta-oxidation defects in human muscle. Furthermore, these data also illustrate a selective control of beta-oxidation enzyme gene expression by PPARdelta, with no contribution of PPARalpha.