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Unlabelled: 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.

To investigate the cell entry and intracellular trafficking of anthrax oedema factor (EF) and lethal factor (LF), they were C-terminally fused to the enhanced green fluorescent protein (EGFP) and monomeric Cherry (mCherry) fluorescent proteins. Both chimeras bound to the surface of BHK cells treated with protective antigen (PA) in a patchy mode. Binding was followed by rapid internalization, and the two anthrax factors were found to traffic along the same endocytic route and with identical kinetics, indicating that their intracellular path is essentially dictated by PA. Colocalization studies indicated that anthrax toxins enter caveolin-1 containing compartments and then endosomes marked by phoshatidylinositol 3-phoshate and Rab5, but not by early endosome antigen 1 and transferrin. After 40 min, both EF and LF chimeras were observed to localize within late compartments. Eventually, LF and EF appeared in the cytosol with a time-course consistent with translocation from late endosomes. Only the EGFP derivatives reached the cytosol because they are translocated by the PA channel, while the mCherry derivatives are not. This difference is attributed to a higher resistance of mCherry to unfolding. After translocation, LF disperses in the cytosol, while EF localizes on the cytosolic face of late endosomes., (© 2010 Blackwell Publishing Ltd.)

The adenylate cyclase toxin (CyaA) released by Bordetella pertussis is an essential virulence factor for colonization of the host. This toxin inhibits migration and activation of phagocytes, thereby preventing bacterial killing. In addition, CyaA interferes with the initiation of adaptive immunity by misdirecting dendritic cell differentiation to a suppressive rather than stimulatory phenotype. Here we show that CyaA directly affects adaptive responses by catalyzing cyclic AMP (cAMP) production in peripheral blood lymphocytes. Treatment with CyaA resulted in profound impairment of T-lymphocyte activation and chemotaxis. These effects resulted from inhibition of T-cell antigen receptor and chemokine receptor signaling via a cAMP/protein kinase A (PKA)-dependent pathway. A comparison of the activities of CyaA on T-cell and macrophage activation and migration revealed that the biological effects of the toxin were paralleled by inhibition of the activation of mitogen-activated protein (MAP) kinases, highlighting the conclusion that the ubiquitous and evolutionarily conserved MAP kinase modules are common targets of the PKA-mediated immunosuppressant activities of CyaA and underlining the potential of cAMP-elevating toxins as a means of evasion of immunity by bacterial pathogens.

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 cAMP-dependent 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 perinuclear localization of EdTx to its intoxication mechanism, indicating that this is a specific feature of its intoxication mechanism.

The entry and enzymatic activity of the anthrax edema factor (EF) in different cell types was studied by monitoring EF-induced changes in intracellular cAMP with biochemical and microscopic methods. cAMP was imaged in live cells, transfected with a fluorescence resonance energy transfer biosensor based on the protein kinase A regulatory and catalytic subunits fused to CFP and YFP, respectively. The cAMP biosensor was located either in the cytosol or was membrane-bound owing to the addition of a tag determining its myristoylation/palmitoylation. Real-time imaging of cells expressing the cAMP biosensors provided the time course of EF catalytic activity and an indication of its subcellular localization. Bafilomycin A1, an inhibitor of the vacuolar ATPase proton pump, completely prevented EF activity, even when added long after the toxin. The time course of appearance of the adenylate cyclase activity and of bafilomycin A1 action suggests that EF enters the cytosol from late endosomes. EF remains associated to these compartments and its activity shows a perinuclear localization generating intracellular cAMP concentration gradients from the cell centre to the periphery.

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.

The lethal and oedema toxins produced by Bacillus anthracis, the aetiological agent of anthrax, are made by association of protective antigen with lethal and oedema factors and play a major role in the pathogenesis of anthrax. In the present paper, we describe the production of peptide-based specific inhibitors in branched form which inhibit the interaction of protective antigen with lethal and oedema factors and neutralize anthrax toxins in vitro and in vivo. Anti-protective antigen peptides were selected from a phage library by competitive panning with lethal factor. Selected 12-mer peptides were synthesized in tetra-branched form and were systematically modified to obtain peptides with higher affinity and inhibitory efficiency.