Extragenital cutaneous warts - clinical presentation, diagnosis and treatment.

Extragenital cutaneous warts are benign epidermal tumors caused by human papillomaviruses (HPVs) and a frequent reason for patients to consult a dermatologist. Depending on wart type and site involved, the clinical presentation is highly varied. Given that warts represent a self-limiting condition, a wait-and-see approach may be justified. However, treatment is always indicated if the lesions become painful or give rise to psychological discomfort. Factors to be considered in this context include subjective disease burden, patient age, site affected, as well as the number and duration of lesions. Destructive treatment methods involve chemical or physical removal of diseased tissue. Nondestructive methods consist of antimitotic and antiviral agents aimed at inhibiting viral proliferation in keratinocytes. Some of the various immunotherapies available not only have localized but also systemic effects and are thus able to induce remission of warts located at any distance from the injection site. Especially patients with warts at multiple sites benefit from this form of treatment. Intralesional immunotherapy using the mumps-measles-rubella (MMR) vaccine is a particularly promising option for the treatment of recalcitrant warts in adult patients. For children, on the other hand, HPV vaccination is a novel and promising approach, even though it has not been approved for the treatment of cutaneous warts. At present, there is no universally effective treatment available. Moreover, many frequently employed therapies are currently not supported by conclusive clinical trials.

The adaptive bacterial immune system CRISPR-Cas and its therapeutic potential. / Eine neue Ära in der Molekularbiologie.

The bacterial CRISPR-Cas-system is an adaptive and inheritable immune system for the defense against invasive genetic elements such as viral DNA or plasmids. CRISPR-Cas immunity acts by integrating short sequences of non-self DNA in the cell's CRISPR locus which allows the cell to recognize, to remember and to destroy the invasive element. In the last years the type-II-system CRISPR-Cas9 was developed as a powerful and universal tool for the sequence-specific modification of the genome also known as genome editing. Type-II-systems rely solely on one single protein, Cas9, and a non-coding, trans-activating RNA that leads the Cas9 protein to its target DNA. The RNA-guided molecular scissor Cas9 is predestinated to correct mutated genes in line with a gene therapy and to heal chronic diseases like HIV infections or virus-induced forms of cancer via deleting viral DNA that is integrated as a provirus in the host's genome. In addition, catalytically inactive Cas9 variants (dCas9) fused to effector domains can be used to specifically tag genome sections, regulate gene expression and modify epigenetic markers.

New insights into the interplay between the lysine transporter LysP and the pH sensor CadC in Escherichia coli.

The coordination of signal transduction and substrate transport represents a sophisticated way to integrate information on metabolite fluxes into transcriptional regulation. This widely distributed process involves protein-protein interactions between two integral membrane proteins. Here we report new insights into the molecular mechanism of the regulatory interplay between the lysine-specific permease LysP and the membrane-integrated pH sensor CadC, which together induce lysine-dependent adaptation of E. coli under acidic stress. In vivo analyses revealed that, in the absence of either stimulus, the two proteins form a stable association, which is modulated by lysine and low pH. In addition to its transmembrane helix, the periplasmic domain of CadC also participated in the interaction. Site-directed mutagenesis pinpointed Arg265 and Arg268 in CadC as well as Asp275 and Asp278 in LysP as potential periplasmic interaction sites. Moreover, a systematic analysis of 100 LysP variants with single-site replacements indicated that the lysine signal is transduced from co-sensor to sensor via lysine-dependent conformational changes (upon substrate binding and/or transport) of LysP. Our results suggest a scenario in which CadC is inhibited by LysP via intramembrane and periplasmic contacts under non-inducing conditions. Upon induction, lysine-dependent conformational changes in LysP transduce the lysine signal via a direct conformational coupling to CadC without resolving the interaction completely. Moreover, concomitant pH-dependent protonation of periplasmic amino acids in both proteins dissolves their electrostatic connections resulting in further destabilization of the CadC/LysP interaction.

Deactivation of the E. coli pH stress sensor CadC by cadaverine.

At acidic pH and in the presence of lysine, the pH sensor CadC activates transcription of the cadBA operon encoding the lysine/cadaverine antiporter CadB and the lysine decarboxylase CadA. In effect, these proteins contribute to acid stress adaptation in Escherichia coli. cadBA expression is feedback inhibited by cadaverine, and a cadaverine binding site is predicted within the central cavity of the periplasmic domain of CadC on the basis of its crystallographic analysis. Our present study demonstrates that this site only partially accounts for the cadaverine response in vivo. Instead, evidence for a second, pivotal binding site was collected, which overlaps with the pH-responsive patch of amino acids located at the dimer interface of the periplasmic domain. The temporal response of the E. coli Cad module upon acid shock was measured and modeled for two CadC variants with mutated cadaverine binding sites. These studies supported a cascade-like binding and deactivation model for the CadC dimer: binding of cadaverine within the pair of central cavities triggers a conformational transition that exposes two further binding sites at the dimer interface, and the occupation of those stabilizes the inactive conformation. Altogether, these data represent a striking example for the deactivation of a pH sensor.

Detection and function of an intramolecular disulfide bond in the pH-responsive CadC of Escherichia coli.

BACKGROUND: In an acidic and lysine-rich environment Escherichia coli induces expression of the cadBA operon which encodes CadA, the lysine decarboxylase, and CadB, the lysine/cadaverine antiporter. cadBA expression is dependent on CadC, a membrane-integrated transcriptional activator which belongs to the ToxR-like protein family. Activation of CadC requires two stimuli, lysine and low pH. Whereas lysine is detected by an interplay between CadC and the lysine-specific transporter LysP, pH alterations are sensed by CadC directly. Crystal structural analyses revealed a close proximity between two periplasmic cysteines, Cys208 and Cys272. RESULTS: Substitution of Cys208 and/or Cys272 by alanine resulted in CadC derivatives that were active in response to only one stimulus, either lysine or pH 5.8. Differential in vivo thiol trapping revealed a disulfide bond between these two residues at pH 7.6, but not at pH 5.8. When Cys208 and Cys272 were replaced by aspartate and lysine, respectively, virtually wild-type behavior was restored indicating that the disulfide bond could be mimicked by a salt bridge. CONCLUSION: A disulfide bond was found in the periplasmic domain of CadC that supports an inactive state of CadC at pH 7.6. At pH 5.8 disulfide bond formation is prevented which transforms CadC into a semi-active state. These results provide new insights into the function of a pH sensor.

The regulatory interplay between membrane-integrated sensors and transport proteins in bacteria.

Bacteria sense environmental stimuli and transduce this information to cytoplasmic components of the signal transduction machinery to cope with and to adapt to ever changing conditions. Hence, bacteria are equipped with numerous membrane-integrated proteins responsible for sensing such as histidine kinases, chemoreceptors and ToxR-like proteins. There is increasing evidence that sensors employ transport proteins as co-sensors. Transport proteins are well-suited information carriers as they bind low-molecular-weight molecules in the external medium and transport them into the cytoplasm, allowing them to provide dynamic information on the metabolic flux. This review explores the sensing capabilities of secondary permeases, primary ABC-transporters, and soluble substrate-binding proteins. Employing transporters as co-sensors seems to be a sophisticated and probably widely distributed mechanism.

Induction kinetics of a conditional pH stress response system in Escherichia coli.

The analysis of stress response systems in microorganisms can reveal molecular strategies for regulatory control and adaptation. In this study, we focused on the Cad module, a subsystem of Escherichia coli's response to acidic stress that is conditionally activated at low pH only when lysine is available. When expressed, the Cad system counteracts the elevated H(+) concentration by converting lysine to cadaverine under the consumption of H(+) and exporting cadaverine in exchange for external lysine. Surprisingly, the cad operon displays a transient response, even when the conditions for its induction persist. To quantitatively characterize the regulation of the Cad module, we experimentally recorded and theoretically modeled the dynamics of important system variables. We established a quantitative model that adequately describes and predicts the transient expression behavior for various initial conditions. Our quantitative analysis of the Cad system supports negative feedback by external cadaverine as the origin of the transient response. Furthermore, the analysis puts causal constraints on the precise mechanism of signal transduction via the regulatory protein CadC.

How are signals transduced across the cytoplasmic membrane? Transport proteins as transmitter of information.

In order to adapt to ever changing environmental conditions, bacteria sense environmental stimuli, and convert them into signals that are transduced intracellularly. Several mechanisms have evolved by which receptors transmit signals across the cytoplasmic membrane. Stimulus perception may trigger receptor dimerization and/or conformational changes. Another mechanism involves the proteolytic procession of a receptor whereby a diffusible cytoplasmic protein is generated. Finally, there is increasing evidence that transport proteins play an important role in transducing signals across the membrane. Transport proteins either directly translocate signaling molecules into the cytoplasm, or transmit information via conformational changes to their interacting partners such as membrane-integrated or soluble components of signal transduction cascades. Employing transport proteins as sensors and regulators of signal transduction represents a sophisticated way of interconnecting metabolic flux and transcriptional regulation in cells.