Anthrax edema factor, voltage-dependent binding to the protective antigen ion channel and comparison to LF binding.

Anthrax toxin complex consists of three different molecules, the binding component protective antigen (PA, 83 kDa), and the enzymatic components lethal factor (LF, 90 kDa) and edema factor (EF, 89 kDa). The 63-kDa N-terminal part of PA, PA(63), forms a heptameric channel that inserts at low pH in endosomal membranes and that is necessary to translocate EF and LF in the cytosol of the target cells. EF is an intracellular active enzyme, which is a calmodulin-dependent adenylate cyclase (89 kDa) that causes a dramatic increase of intracellular cAMP level. Here, the binding of full-length EF on heptameric PA(63) channels was studied in experiments with artificial lipid bilayer membranes. Full-length EF blocks the PA(63) channels in a dose, temperature, voltage, and ionic strength-dependent way with half-saturation constants in the nanomolar concentration range. EF only blocked the PA(63) channels when PA(63) and EF were added to the same side of the membrane, the cis side. Decreasing ionic strength and increasing transmembrane voltage at the cis side of the membranes resulted in a strong decrease of the half-saturation constant for EF binding. This result suggests that ion-ion interactions are involved in EF binding to the PA heptamer. Increasing temperature resulted in increasing half-saturation constants for EF binding to the PA(63) channels. The binding characteristics of EF to the PA(63) channels are compared with those of LF binding. The comparison exhibits similarities but also remarkable differences between the bindings of both toxins to the PA(63) channel

Binding of N-terminal fragments of anthrax edema factor (EFN) and lethal factor (LFN) to the protective antigen pore

AbstractAnthrax toxin consists of three different molecules: the binding component protective antigen (PA, 83 kDa), and the enzymatic components lethal factor (LF, 90 kDa) and edema factor (EF, 89 kDa). The 63 kDa C-terminal part of PA, PA63, forms heptameric channels that insert in endosomal membranes at low pH, necessary to translocate EF and LF into the cytosol of target cells. In many studies, about 30 kDa N-terminal fragments of the enzymatic components EF (254 amino acids) and LF (268 amino acids) were used to study their interaction with PA63-channels. Here, in experiments with artificial lipid bilayer membranes, EFN and LFN show block of PA63-channels in a dose, voltage and ionic strength dependent way with high affinity. However, when compared to their full-length counterparts EF and LF, they exhibit considerably lower binding affinity. Decreasing ionic strength and, in the case of EFN, increasing transmembrane voltage at the cis side of the membranes, resulted in a strong decrease of half saturation constants. Our results demonstrate similarities but also remarkable differences between the binding kinetics of both truncated and full-length effectors to the PA63-channel

Diabetic foot complicated by vertebral osteomyelitis and epidural abscess

Vertebral osteomyelitis (or spondylodiscitis) is steadily increasing in Western countries and often results from hematogenous seeding, direct inoculation during spinal surgery, or contiguous spread from an infection in the adjacent soft tissue. We present the case of a 67-year-old white patient with type 2 diabetes who went to Hospital for high fever, back pain, and worsening of known infected ulcers in the left foot. Despite intravenous antibiotic treatment and surgical debridement of the foot infection, high fever and lower back pain continued. Bone biopsy and two consecutive blood cultures were positive for Staphylococcus aureus. A spinal magnetic resonance imaging (MRI) was performed, revealing serious osteomyelitis in L4 and L5 complicated by an epidural abscess. Contiguous or other distant focuses of infection were not identified. In this case, diabetic foot could be considered as a primary distant focus for vertebral osteomyelitis. Clinicians should consider vertebral osteomyelitis as a 'possible' diagnosis in patients with type 2 diabetes complicated by foot infection that is associated with fever and lower back pain. LEARNING POINTS: Vertebral osteomyelitis is increasing in Western countries, especially in patients with type 2 diabetes.The primary focus of infection is the genitourinary tract followed by skin, soft tissue, endocarditis, bursitis, septic arthritis, and intravascular access.Diabetic foot could be a rare primary focus of infection for vertebral osteomyelitis, and, however, vertebral osteomyelitis could be a serious, albeit rare, complication of diabetic foot.Clinicians should keep in mind the many potential complications of diabetic foot ulcerations and consider vertebral osteomyelitis as a "possible" diagnosis in patients with type 2 diabetes and foot ulcers associated with nonspecific symptoms such as lower back pain.Early diagnosis and correct management of vertebral osteomyelitis are crucial to improve clinical outcomes

Anthrax Edema Toxin Modulates PKA- and CREB-Dependent Signaling in Two Phases

Background: Anthrax edema toxin (EdTx) is an adenylate cyclase which operates in the perinuclear region of host cells. However, the action of EdTx is poorly understood, especially at molecular level. The ability of EdTx to modulate cAMPdependent signaling was studied in Jurkat T cells and was compared with that of other cAMP-rising agents: Bordetella pertussis adenylate cyclase toxin, cholera toxin and forskolin. Methodology/Principal Findings: EdTx caused a prolonged increase of the intracellular cAMP concentration. This led to nuclear translocation of the cAMP-dependent protein kinase (PKA) catalytic subunit, phosphorylation of cAMP response element binding protein (CREB) and expression of a reporter gene under control of the cAMP response element. Neither p90 ribosomal S6 kinase nor mitogen- and stress-activated kinase, which mediate CREB phosphorylation during T cell activation, were involved. The duration of phospho-CREB binding to chromatin correlated with the spatio-temporal rise of cAMP levels. Strikingly, EdTx pre-treated T cells were unresponsive to other stimuli involving CREB phosphorylation such as addition of forskolin or T cell receptor cross-linking. Conclusions/Significance: We concluded that, in a first intoxication phase, EdTx induces PKA-dependent signaling, which culminates in CREB phosphorylation and activation of gene transcription. Subsequently CREB phosphorylation is impaired and therefore T cells are not able to respond to cues involving CREB. The present data functionally link the perinuclea

Early downregulation of hsa-miR-144-3p in serum from drug-naïve Parkinson's disease patients.

Advanced age represents one of the major risk factors for Parkinson's Disease. Recent biomedical studies posit a role for microRNAs, also known to be remodelled during ageing. However, the relationship between microRNA remodelling and ageing in Parkinson's Disease, has not been fully elucidated. Therefore, the aim of the present study is to unravel the relevance of microRNAs as biomarkers of Parkinson's Disease within the ageing framework. We employed Next Generation Sequencing to profile serum microRNAs from samples informative for Parkinson's Disease (recently diagnosed, drug-naïve) and healthy ageing (centenarians) plus healthy controls, age-matched with Parkinson's Disease patients. Potential microRNA candidates markers, emerging from the combination of differential expression and network analyses, were further validated in an independent cohort including both drug-naïve and advanced Parkinson's Disease patients, and healthy siblings of Parkinson's Disease patients at higher genetic risk for developing the disease. While we did not find evidences of microRNAs co-regulated in Parkinson's Disease and ageing, we report that hsa-miR-144-3p is consistently down-regulated in early Parkinson's Disease patients. Moreover, interestingly, functional analysis revealed that hsa-miR-144-3p is involved in the regulation of coagulation, a process known to be altered in Parkinson's Disease. Our results consistently show the down-regulation of hsa-mir144-3p in early Parkinson's Disease, robustly confirmed across a variety of analytical and experimental analyses. These promising results ask for further research to unveil the functional details of the involvement of hsa-mir144-3p in Parkinson's Disease

Heterogeneity of prodromal Parkinson symptoms in siblings of Parkinson disease patients

Abstract: A prodromal phase of Parkinson’s disease (PD) may precede motor manifestations by decades. PD patients’ siblings are at higher risk for PD, but the prevalence and distribution of prodromal symptoms are unknown. The study objectives were (1) to assess motor and non-motor features estimating prodromal PD probability in PD siblings recruited within the European PROPAG-AGEING project; (2) to compare motor and non-motor symptoms to the well-established DeNoPa cohort. 340 PD siblings from three sites (Bologna, Seville, Kassel/Goettingen) underwent clinical and neurological evaluations of PD markers. The German part of the cohort was compared with German de novo PD patients (dnPDs) and healthy controls (CTRs) from DeNoPa. Fifteen (4.4%) siblings presented with subtle signs of motor impairment, with MDS-UPDRS-III scores not clinically different from CTRs. Symptoms of orthostatic hypotension were present in 47 siblings (13.8%), no different to CTRs (p = 0.072). No differences were found for olfaction and overall cognition; German-siblings performed worse than CTRs in visuospatial-executive and language tasks. 3/147 siblings had video-polysomnography-confirmed REM sleep behavior disorder (RBD), none was positive on the RBD Screening Questionnaire. 173/300 siblings had <1% probability of having prodromal PD; 100 between 1 and 10%, 26 siblings between 10 and 80%, one fulfilled the criteria for prodromal PD. According to the current analysis, we cannot confirm the increased risk of PD siblings for prodromal PD. Siblings showed a heterogeneous distribution of prodromal PD markers and probability. Additional parameters, including strong disease markers, should be investigated to verify if these results depend on validity and sensitivity of prodromal PD criteria, or if siblings’ risk is not elevated

Studi sul meccanismo di azione cellulare delle tossine prodotte da Bacillus anthracis

The major determinants of anthrax pathogenesis are anthrax toxins. They are composed of three proteins, PA, LF and EF. PA binds to specific cellular receptors and the complex PA+LF+EF is endocytosed. Once inside the cell, the toxins follow the endocytic route until pH-dependent translocation of LF and EF occurs. LF is a metal-dependent protease that cleaves MEKs, whereas EF is a calmodulin-dependent adenylate cyclase. Informations about the site of EF release into the citosol are lacking. Biochemical analysis of intracellular cAMP increase in cultured cells reveals that EF activity begins after 30 minutes from intoxication and cAMP levels are still high after 4 hours. These results can provide two kinds of informations. The first one suggests that a long travel through the endocytic pathway may occur before EF can reach the cytosol, in accordance with translocation from late endosomes. The second one suggests a severe impairment of cellular signalling, since for several hours the cell is unable to overcome the massive production of cAMP. To investigate the site of EF translocation we performed FRET imaging of cAMP dynamics induced by the toxin. Mapping of narrow regions inside intoxicated cells and FRET efficiency measurements revealed a preferential site of cAMP increase, corresponding to perinuclear regions. This result is in agreement with the hypothesis of translocation from late endosomes. Following another approach, we fused LF and EF to green and red fluorescent proteins. Those chimerae provide a tool for tracking the route of the toxins inside the cell. Simultaneous visualization of ETx and LTx could clarify the mechanisms of their synergistic activity. Moreover, colocalization with cellular markers could define the pattern of toxicity and the advantages of translocating closely to the nucleus. Finally, we performed RT-PCR to test the presence of both anthrax receptors, TEM8 and CMG2, on the same cells that were used in our experiments. CMG2 has the highest affinity for PA and requires a low pH to allow dissociation and PA pore formation. Thus, the presence of CMG2 validates the hypothesis of EF translocation from late endosomes. The clarification of the precise intracellular localization of anthrax toxins is compulsory to understand their final effect, since microdomains with a broad variety and concentration of signal molecules are present in a cell.Le tossine di antrace hanno un ruolo centrale nella patogenesi dell’infezione. Sono composte da tre proteine, PA, LF e EF. PA si lega a specifici recettori cellulari e il complesso PA+LF+EF è endocitato. Le tossine dentro la cellula iniziano a percorrere la via endocitica fino a che LF e EF traslocano nel citosol con un meccanismo pH-dipendente. LF è una metalloproteasi che taglia le MEKs e EF è un’adenilato ciclasi calmodulina-dipendente. Ci sono poche informazioni riguardo il sito di rilascio di EF nel citoplasma. La misura dei livelli di ciclico AMP intracellulare rivela che l’attività di EF inizia dopo 30 minuti dall’intossicazione e dopo 4 ore i livelli sono ancora alti. Queste osservazioni portano due diverse considerazioni. La prima è che la tossina debba compiere un lungo precorso lungo la via di endocitosi prima di raggiungere il citosol. Infatti, i tempi di inizio dell’attività sono compatibili con una traslocazione dai late endosomes. La seconda considerazione è che per diverse ore dall’intossicazione la cellula non riesce a fronteggiare il massiccio aumento di cAMP indotto da ETx e resta a lungo sotto l’effetto di una condizione di disregolazione. Per indagare il percorso intracellulare e il sito di traslocazione della tossina è stata usata la tecnica della FRET, che permette di analizzare le dinamiche dell’aumento di cAMP indotto da EF. La mappatura di piccole zone all’interno della cellula ha rivelato che il cAMP aumenta prima nelle regioni perinucleari. Questo è in accordo con l’ipotesi della traslocazione dai late endosomes. Un altro approccio è stato quello di fondere LF e EF a green e red fluorescent proteins. Queste chimere sono uno strumento per tracciare il cammino delle tossine dentro la cellula. Inoltre, la visualizzazione simultanea di ETx e LTx può chiarire le basi della loro azione sinergica. In futuro si potranno fare delle colocalizzazioni con markers cellulari per chiarire i siti di interazione con i componenti dell’ospite

PAPILLOMAVIRUS IN HEALTHY SKIN AND MUCOSA OF WILD RUMINANTS IN THE ITALIAN ALPS

We investigated healthy skin and mucosal specimens of wild ruminants in the Italian Alps. We identified bovine papillomavirus (BPV)-2 DNA in the healthy skin of wild ruminants and documented coinfection of BPV-1 and Cervus elaphus papillomavirus (CePV)-1 in a healthy red deer (Cervus elaphus). We also demonstrated cross-infections of BPVs of the genus Xipapillomavirus, both as single virus infection and also in association with Deltapapillomavirus types 1 and 2, confirming that host tropism of papillomaviruses is not as species-speci\ufb01c as previously thought. Our results suggest that subclinical infections could be linked to the presence of domestic ruminants sharing the same habitat with wild species and that the wildlife may act as a reservoir for papillomaviruses affecting domestic species