Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine.

UNLABELLED: Bacterial type IV secretion systems (T4SSs) are composed of two major subfamilies, conjugation machines dedicated to DNA transfer and effector translocators for protein transfer. We show here that the Escherichia coli pKM101-encoded conjugation system, coupled with chimeric substrate receptors, can be repurposed for transfer of heterologous effector proteins. The chimeric receptors were composed of the N-terminal transmembrane domain of pKM101-encoded TraJ fused to soluble domains of VirD4 homologs functioning in Agrobacterium tumefaciens, Anaplasma phagocytophilum, or Wolbachia pipientis A chimeric receptor assembled from A. tumefaciens VirD4 (VirD4At) mediated transfer of a MOBQ plasmid (pML122) and A. tumefaciens effector proteins (VirE2, VirE3, and VirF) through the pKM101 transfer channel. Equivalent chimeric receptors assembled from the rickettsial VirD4 homologs similarly supported the transfer of known or candidate effectors from rickettsial species. These findings establish a proof of principle for use of the dedicated pKM101 conjugation channel, coupled with chimeric substrate receptors, to screen for translocation competency of protein effectors from recalcitrant species. Many T4SS receptors carry sequence-variable C-terminal domains (CTDs) with unknown function. While VirD4At and the TraJ/VirD4At chimera with their CTDs deleted supported pML122 transfer at wild-type levels, ΔCTD variants supported transfer of protein substrates at strongly diminished or elevated levels. We were unable to detect binding of VirD4At's CTD to the VirE2 effector, although other VirD4At domains bound this substrate in vitro We propose that CTDs evolved to govern the dynamics of substrate presentation to the T4SS either through transient substrate contacts or by controlling substrate access to other receptor domains. IMPORTANCE: Bacterial type IV secretion systems (T4SSs) display striking versatility in their capacity to translocate DNA and protein substrates to prokaryotic and eukaryotic target cells. A hexameric ATPase, the type IV coupling protein (T4CP), functions as a substrate receptor for nearly all T4SSs. Here, we report that chimeric T4CPs mediate transfer of effector proteins through the Escherichia coli pKM101-encoded conjugation system. Studies with these repurposed conjugation systems established a role for acidic C-terminal domains of T4CPs in regulating substrate translocation. Our findings advance a mechanistic understanding of T4CP receptor activity and, further, support a model in which T4SS channels function as passive conduits for any DNA or protein substrates that successfully engage with and pass through the T4CP specificity checkpoint.

Caught in the act: the dialogue between bacteriophage R17 and the type IV secretion machine of plasmid R1.

Bacteria communicate with each other through contact-independent and -dependent signalling mechanisms. Sensory perception of both types of signals is needed for conjugative transfer of mobile DNA elements via type IV secretion systems (T4SSs) to bacterial or eukaryotic target cells. While the regulatory circuitries coupling extracellular quorum and environmental signals to transcription of T4SS genes are increasingly understood, it remains fundamentally unknown how a potential recipient cell stimulates donor conjugative DNA transfer upon contact. In this issue, Lang et al. (2011) report use of the male-specific bacteriophage R17, a phage that binds conjugative pili elaborated by IncF plasmid R1, to define requirements for phage-contact-mediated T4SS activation and phage penetration. They report that R17 penetrates only through T4SS channels engaged for delivery of their plasmid cargo to recipient cells. Engagement requires docking of catalytically active relaxase TraI bound at oriT with the TraD substrate receptor (also termed the T4CP). The data, together with recent ultrastructural and biochemical findings, support an intriguing new model that the T4CP cumulatively senses an intracellular signal (substrate docking) and an extracellular signal (pilus bound by phage or a recipient cell) to co-ordinate a late stage morphogenetic or gating reaction that enables bidirectional transmission of nucleoprotein substrates through the T4SS.

Do early skin care practices alter the risk of atopic dermatitis? A case-control study.

The rise in atopic dermatitis prevalence observed in industrialized countries is unexplained. We hypothesized that certain skin care practices early in life may increase the risk for developing atopic dermatitis. Our case-control study could not identify any one practice that increased the odds of developing atopic dermatitis, but it revealed that regular lotion use was very common in infants who later develop atopic dermatitis.

A pilot study of emollient therapy for the primary prevention of atopic dermatitis.

BACKGROUND: Prevention strategies in atopic dermatitis (AD) using allergen avoidance have not been consistently effective. New research reveals the importance of the skin barrier in the development of AD and possibly food allergy and asthma. Correcting skin barrier defects from birth may prevent AD onset or moderate disease severity. OBJECTIVE: We sought to determine the feasibility of skin barrier protection as a novel AD prevention strategy. METHODS: We enrolled 22 neonates at high risk for developing AD in a feasibility pilot study using emollient therapy from birth. RESULTS: No intervention-related adverse events occurred in our cohort followed up for a mean time of 547 days. Of the 20 subjects who remained in the study, 3 (15.0%) developed AD, suggesting a protective effect when compared with historical controls. Skin barrier measurements remained within ranges seen in normal-appearing skin. LIMITATIONS: No conclusions regarding efficacy can be made without a control group. CONCLUSIONS: Skin barrier repair from birth represents a novel and feasible approach to AD prevention. Further studies are warranted to determine the efficacy of this approach.

A pilot study of an oral phosphodiesterase inhibitor (apremilast) for atopic dermatitis in adults.

OBJECTIVE: To investigate the preliminary safety and efficacy of apremilast, an oral phosphodiesterase 4 inhibitor, for atopic dermatitis. DESIGN: This investigator-initiated, open-label pilot study evaluated 2 doses of apremilast in patients with atopic dermatitis. Differential gene analysis was performed from peripheral whole blood using data before and after treatment. SETTING: University-based dermatology clinical research unit. PATIENTS: Sixteen adult patients with atopic dermatitis. INTERVENTION: A specific phosphodiesterase 4 inhibitor, apremilast. MAIN OUTCOME MEASURES: The primary outcome was incidence of adverse events. Secondary outcomes included the differences in pruritus, Dermatology Life Quality Index (DLQI), and Eczema Area and Severity Index (EASI) scores between the baseline visit and end-ofstudy visit for each cohort. RESULTS: The group receiving apremilast, 20 mg twice daily, displayed a significant reduction from baseline of pruritus (P=.02) and the DLQI (P=.003) at 3 months. The group receiving apremilast, 30 mg twice daily, displayed a significant reduction of the EASI (P=.008) and the DLQI (P=.01) at 3 months. At 6 months, there was a significant reduction of the EASI (P=.002), the visual analog scale (P=.03), and the DLQI (P=.03). Gene ontologic analyses comparing baseline with samples during treatment revealed alterations in immune response pathways, especially those related to cyclic adenosine monophosphate­mediated signaling. CONCLUSIONS: These results support further development of apremilast for treatment of atopic dermatitis. Larger randomized controlled studies are needed to more adequately evaluate both safety and efficacy. Limitations include the small sample size and absence of a control. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01393158.

Is food allergy testing reliable in pediatric atopic dermatitis? A population-based study.

We sought to assess the value and reliability of serologic testing for predicting clinical food allergy in a population-based cohort of infants with atopic dermatitis (AD). Infants 3-18 months of age, recruited from the general population, were followed quarterly for 3 years and carefully evaluated for evidence of immediate reactions to foods. Specific serum IgE levels for six foods were assayed at 3-5 years. Parents were interviewed at each visit regarding past/current immediate food-specific reactions involving skin, gut or respiratory systems. Data were entered into Excel for calculations of performance characteristics. Nine of the 40 patients (23%) who completed 3 years of follow-up had reactions to one or more foods. Reactions occurred in 5, 11 and 18% to milk, peanut and egg ingestion, respectively. In contrast, 30% of food-specific serum IgE tests were above normal. Predictive reliability of tests was generally low unless values were in the high range for peanut and milk. Conversely, egg allergy could be seen across a nearly full-spectrum of IgE values, making prediction highly unreliable. We conclude that physician and patient misinterpretation of the relevance and reliability of allergy testing may misdirect proper prevention and therapy of AD.