Spinal cord fractalkine (CX3CL1) signaling is critical for neuronal sensitization in experimental nonspecific, myofascial low back pain.

Neuroactive substances released by activated microglia contribute to hyperexcitability of spinal dorsal horn neurons in many animal models of chronic pain. An important feedback loop mechanism is via release of fractalkine (CX3CL1) from primary afferent terminals and dorsal horn neurons and binding to CX3CR1 receptors on microglial cells. We studied the involvement of fractalkine signaling in latent and manifest spinal sensitization induced by two injections of nerve growth factor (NGF) into the lumbar multifidus muscle as a model for myofascial low back pain. Single dorsal horn neurons were recorded in vivo to study their receptive fields and spontaneous activity. Under intrathecal vehicle application, the two NGF injections led to an increased proportion of neurons responding to stimulation of deep tissues (41%), to receptive field expansion into the hindlimb (15%), and to resting activity (53%). Blocking fractalkine signaling by continuous intrathecal administration of neutralizing antibodies completely prevented these signs of spinal sensitization to a similar extent as in a previous study with the microglia inhibitor minocycline. Reversely, fractalkine itself induced similar sensitization in a dose-dependent manner (for 200 ng/mL: 45% deep tissue responses, 24% receptive field expansion, and 45% resting activity) as repeated nociceptive stimulation by intramuscular NGF injections. A subsequent single NGF injection did not have an additive effect. Our data suggest that neuron-to-microglia signaling via the CX3CL1-CX3CR1 pathway is critically involved in the initiation of nonspecific, myofascial low back pain through repetitive nociceptive stimuli.NEW & NOTEWORTHY Blocking fractalkine signaling by neutralizing antibodies completely prevented spinal sensitization induced by repetitive mild nociceptive input [2 nerve growth factor (NGF) injections into the multifidus muscle] Conversely, fractalkine given intrathecally caused the same pattern of spinal sensitization as the nociceptive NGF injections. Fractalkine signaling is critically involved in sensitization of dorsal horn neurons induced by repeated nociceptive low back muscle stimulation and may hence be a potential target for the prevention of nonspecific, myofascial low back pain.

The enzyme subunit SubA of Shiga toxin-producing E. coli strains demonstrates comparable intracellular transport and cytotoxic activity as the holotoxin SubAB in HeLa and HCT116 cells in vitro.

The subtilase cytotoxin (SubAB) is secreted by certain Shiga toxin-producing Escherichia coli (STEC) strains and is composed of the enzymatically active subunit SubA and the pentameric binding/transport subunit SubB. We previously demonstrated that SubA (10 µg/ml), in the absence of SubB, binds and intoxicates the human cervix cancer-derived epithelial cell line HeLa. However, the cellular and molecular mechanisms underlying the cytotoxic activity of SubA in the absence of SubB remained unclear. In the present study, the cytotoxic effects mediated by SubA alone were investigated in more detail in HeLa cells and the human colon cancer cell line HCT116. We found that in the absence of SubB, SubA (10 µg/ml) is internalized into the endoplasmic reticulum (ER), where it cleaves the chaperone GRP78, an already known substrate for SubA after its canonical uptake into cells via SubB. The autonomous cellular uptake of SubA and subsequent cleavage of GRP78 in cells is prevented by treatment of cells with 10 µM brefeldin A, which inhibits the transport of protein toxins into the ER. In addition, by analyzing the SubA mutant SubAΔC344, we identified the C-terminal SEEL motif as an ER-targeting signal. Conclusively, our results strongly suggest that SubA alone shares the same intracellular transport route and cytotoxic activity as the SubAB holotoxin.

Novel Aspects of the SubA Subunit of the Subtilase Cytotoxin.

The subtilase cytotoxin (SubAB) belongs to the family of AB5 toxins and is produced together with Shiga toxin (Stx) by certain Stx-producing E. coli strains (STEC). For most AB-type toxins, it is assumed that cytotoxic effects can only be induced by a complete holotoxin complex consisting of SubA and SubB. However, it has been shown for SubAB that the enzymatically active subunit SubA, without its transport and binding domain SubB, induces cell death in different eukaryotic cell lines. Interestingly, the molecular structure of SubA resembles that of the SubAB complex. SubA alone is capable of binding to cells and then being taken up autonomously. Once inside the host cell, SubA is transported, similar to the SubAB holotoxin, via a retrograde transport into the endoplasmatic reticulum (ER). In the ER, it exhibits its enzymatic activity by cleaving the chaperone BiP/GRP78 and thereby triggering cell death. Therefore, the existence of toxic single SubA subunits that have not found a B-pentamer for holotoxin assembly might improve the pathogenic potential of subtilase-producing strains. Moreover, from a pharmacological aspect, SubA might be an interesting molecule for the targeted transport of therapeutic molecules into the ER, in order to investigate and specifically modulate processes in the context of ER stress-associated diseases. Since recent studies on bacterial AB5 toxins contributed mainly to the understanding of the biology of AB-type holotoxins, this mini-review specifically focus on that recently observed single A-effect of the subtilase cytotoxin and addresses whether a fundamental shift of the traditional AB5 paradigm might be required.

Variants of Escherichia coli Subtilase Cytotoxin Subunits Show Differences in Complex Formation In Vitro.

The subtilase cytotoxin (SubAB) of Shiga toxin-producing Escherichia coli (STEC) is a member of the AB5 toxin family. In the current study, we analyzed the formation of active homo- and hetero-complexes of SubAB variants in vitro to characterize the mode of assembly of the subunits. Recombinant SubA1-His, SubB1-His, SubA2-2-His, and SubB2-2-His subunits, and His-tag-free SubA2-2 were separately expressed, purified, and biochemically characterized by circular dichroism (CD) spectroscopy, size-exclusion chromatography (SEC), and analytical ultracentrifugation (aUC). To confirm their biological activity, cytotoxicity assays were performed with HeLa cells. The formation of AB5 complexes was investigated with aUC and isothermal titration calorimetry (ITC). Binding of SubAB2-2-His to HeLa cells was characterized with flow cytometry (FACS). Cytotoxicity experiments revealed that the analyzed recombinant subtilase subunits were biochemically functional and capable of intoxicating HeLa cells. Inhibition of cytotoxicity by Brefeldin A demonstrated that the cleavage is specific. All His-tagged subunits, as well as the non-tagged SubA2-2 subunit, showed the expected secondary structural compositions and oligomerization. Whereas SubAB1-His complexes could be reconstituted in solution, and revealed a Kd value of 3.9 ± 0.8 µmol/L in the lower micromolar range, only transient interactions were observed for the subunits of SubAB2-2-His in solution, which did not result in any binding constant when analyzed with ITC. Additional studies on the binding characteristics of SubAB2-2-His on HeLa cells revealed that the formation of transient complexes improved binding to the target cells. Conclusively, we hypothesize that SubAB variants exhibit different characteristics in their binding behavior to their target cells.