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1.  Guideline contact dermatitis 
Allergo Journal International  2014;23(4):126-138.
doi:10.1007/s40629-014-0013-5
PMCID: PMC4484750  PMID: 26146602
contact dermatitis; guideline; epidemiology; symptoms; clinical picture; diagnosis; patch test; treatment
2.  Authorised allergen products for intracutaneous testing may no longer be available in Germany 
Allergo Journal International  2015;24(3):84-93.
Background:
Beside the skin prick test, the intracutaneous test represents the most important skin test method for detecting type-1 allergies. With the incorporation of European directives into national law, test allergens used for allergy diagnosis are deemed medicinal products within the meaning of the German Medicinal Products Act (Arzneimittelgesetz) and therefore require marketing authorisation for distribution in Germany. The high costs of acquiring and maintaining these authorisations have lead to no new finished intracutaneous test products being authorized in Germany for more than 20 years. Instead, most manufacturers have voluntarily withdrawn their existing marketing authorisations for intracutaneous test extracts. The last manufacturer to offer approved finished allergen products for intracutaneous tests recently announced that it would now cease production and distribution of these solutions.
Methods:
Research on the current European and German legislation; selective literature search in Medline, including national and international guidelines and Cochrane meta-analyses; licensing information on the Paul-Ehrlich-Institute homepage (www.pei.de) as well as in the Bundesanzeiger (Federal Gazette).
Results:
According to information on www.pei.de, marketing authorisations still existed as of 31.01.2015 for intracutaneous test solutions of six grass/cereal/herbal pollens, seven tree pollens, ten food allergens, twelve moulds and yeasts as well as two fungal mixtures, five house dust and storage mites and five animal epithelia/danders, all held by only one company in Germany. These marketing authorisations were granted between 16th March 1987 and 17th January 1992; more recent marketing authorisations do not exist.
Conclusions:
European legislation and the associated increase in production and licensing costs have already lead to numerous suppliers withdrawing their marketing authorisation for diagnostic test allergens — marketing authorisations for 443 diagnostic allergens were voluntarily withdrawn by manufacturers in 2013 alone. If the announced restrictions on the allergen portfolio go ahead, considerable problems in the management of allergy patients in Germany due to the discontinuation of the intracutaneous test are likely to be encountered. Moreover, the fact that a diagnostic procedure that has been established for decades seems set to disappear quite simply because all the requisite substances vanish from the market in one fell swoop may well be without parallel in modern medicine.
The situation for skin prick test allergens is less dramatic, although, here again, the available range is becoming increasingly limited.
doi:10.1007/s40629-015-0051-7
PMCID: PMC4479459  PMID: 26120551
intracutaneous testing; allergic rhinitis; diagnostic test allergens; allergen products; marketing authorisation
3.  Guideline on allergen-specific immunotherapy in IgE-mediated allergic diseases 
Allergo Journal International  2014;23(8):282-319.
Summary
The present guideline (S2k) on allergen-specific immunotherapy (AIT) was established by the German, Austrian and Swiss professional associations for allergy in consensus with the scientific specialist societies and professional associations in the fields of otolaryngology, dermatology and venereology, pediatric and adolescent medicine, pneumology as well as a German patient organization (German Allergy and Asthma Association; Deutscher Allergie- und Asthmabund, DAAB) according to the criteria of the Association of the Scientific Medical Societies in Germany (Arbeitsgemeinschaft der Wissenschaftlichen Medizinischen Fachgesellschaften, AWMF).
AIT is a therapy with disease-modifying effects. By administering allergen extracts, specific blocking antibodies, toler-ance-inducing cells and mediators are activated. These prevent further exacerbation of the allergen-triggered immune response, block the specific immune response and attenuate the inflammatory response in tissue.
Products for SCIT or SLIT cannot be compared at present due to their heterogeneous composition, nor can allergen concentrations given by different manufacturers be compared meaningfully due to the varying methods used to measure their active ingredients. Non-modified allergens are used for SCIT in the form of aqueous or physically adsorbed (depot) extracts, as well as chemically modified allergens (allergoids) as depot extracts. Allergen extracts for SLIT are used in the form of aqueous solutions or tablets.
The clinical efficacy of AIT is measured using various scores as primary and secondary study endpoints. The EMA stipulates combined symptom and medication scores as primary endpoint. A harmonization of clinical endpoints, e. g., by using the combined symptom and medication scores (CSMS) recommended by the EAACI, is desirable in the future in order to permit the comparison of results from different studies. The current CONSORT recommendations from the ARIA/GA2LEN group specify standards for the evaluation, presentation and publication of study results.
According to the Therapy allergen ordinance (TAV), preparations containing common allergen sources (pollen from grasses, birch, alder, hazel, house dust mites, as well as bee and wasp venom) need a marketing authorization in Germany. During the marketing authorization process, these preparations are examined regarding quality, safety and efficacy. In the opinion of the authors, authorized allergen preparations with documented efficacy and safety, or preparations tradeable under the TAV for which efficacy and safety have already been documented in clinical trials meeting WAO or EMA standards, should be preferentially used. Individual formulations (NPP) enable the prescription of rare allergen sources (e.g., pollen from ash, mugwort or ambrosia, mold Alternaria, animal allergens) for specific immunotherapy. Mixing these allergens with TAV allergens is not permitted.
Allergic rhinitis and its associated co-morbidities (e. g., bronchial asthma) generate substantial direct and indirect costs. Treatment options, in particular AIT, are therefore evaluated using cost-benefit and cost-effectiveness analyses. From a long-term perspective, AIT is considered to be significantly more cost effective in allergic rhinitis and allergic asthma than pharmacotherapy, but is heavily dependent on patient compliance.
Meta-analyses provide unequivocal evidence of the efficacy of SCIT and SLIT for certain allergen sources and age groups. Data from controlled studies differ in terms of scope, quality and dosing regimens and require product-specific evaluation. Therefore, evaluating individual preparations according to clearly defined criteria is recommended. A broad transfer of the efficacy of certain preparations to all preparations administered in the same way is not endorsed. The website of the German Society for Allergology and Clinical Immunology (www.dgaki.de/leitlinien/s2k-leitlinie-sit; DGAKI: Deutsche Gesellschaft für Allergologie und klinische Immunologie) provides tables with specific information on available products for AIT in Germany, Switzerland and Austria. The tables contain the number of clinical studies per product in adults and children, the year of market authorization, underlying scoring systems, number of randomized and analyzed subjects and the method of evaluation (ITT, FAS, PP), separately given for grass pollen, birch pollen and house dust mite allergens, and the status of approval for the conduct of clinical studies with these products.
Strong evidence of the efficacy of SCIT in pollen allergy-induced allergic rhinoconjunctivitis in adulthood is well-documented in numerous trials and, in childhood and adolescence, in a few trials. Efficacy in house dust mite allergy is documented by a number of controlled trials in adults and few controlled trials in children. Only a few controlled trials, independent of age, are available for mold allergy (in particular Alternaria). With regard to animal dander allergies (primarily to cat allergens), only small studies, some with methodological deficiencies are available. Only a moderate and inconsistent therapeutic effect in atopic dermatitis has been observed in the quite heterogeneous studies conducted to date. SCIT has been well investigated for individual preparations in controlled bronchial asthma as defined by the Global Initiative for Asthma (GINA) 2007 and intermittent and mild persistent asthma (GINA 2005) and it is recommended as a treatment option, in addition to allergen avoidance and pharmacotherapy, provided there is a clear causal link between respiratory symptoms and the relevant allergen.
The efficacy of SLIT in grass pollen-induced allergic rhinoconjunctivitis is extensively documented in adults and children, whilst its efficacy in tree pollen allergy has only been shown in adults. New controlled trials (some with high patient numbers) on house dust mite allergy provide evidence of efficacy of SLIT in adults.
Compared with allergic rhinoconjunctivitis, there are only few studies on the efficacy of SLIT in allergic asthma. In this context, newer studies show an efficacy for SLIT on asthma symptoms in the subgroup of grass pollen allergic children, adolescents and adults with asthma and efficacy in primary house dust mite allergy-induced asthma in adolescents aged from 14 years and in adults.
Aspects of secondary prevention, in particular the reduction of new sensitizations and reduced asthma risk, are important rationales for choosing to initiate treatment early in childhood and adolescence. In this context, those products for which the appropriate effects have been demonstrated should be considered.
SCIT or SLIT with pollen or mite allergens can be performed in patients with allergic rhinoconjunctivitis using allergen extracts that have been proven to be effective in at least one double-blind placebo-controlled (DBPC) study. At present, clinical trials are underway for the indication in asthma due to house dust mite allergy, some of the results of which have already been published, whilst others are still awaited (see the DGAKI table “Approved/potentially completed studies” via www.dgaki.de/Leitlinien/s2k-Leitlinie-sit (according to www.clinicaltrialsregister.eu)). When establishing the indication for AIT, factors that favour clinical efficacy should be taken into consideration. Differences between SCIT and SLIT are to be considered primarily in terms of contraindications. In individual cases, AIT may be justifiably indicated despite the presence of contraindications.
SCIT injections and the initiation of SLIT are performed by a physician experienced in this type of treatment and who is able to administer emergency treatment in the case of an allergic reaction. Patients must be fully informed about the procedure and risks of possible adverse events, and the details of this process must be documented (see “Treatment information sheet”; available as a handout via www.dgaki.de/Leitlinien/s2k-Leitlinie-sit). Treatment should be performed according to the manufacturer‘s product information leaflet. In cases where AIT is to be performed or continued by a different physician to the one who established the indication, close cooperation is required in order to ensure that treatment is implemented consistently and at low risk. In general, it is recommended that SCIT and SLIT should only be performed using preparations for which adequate proof of efficacy is available from clinical trials.
Treatment adherence among AIT patients is lower than assumed by physicians, irrespective of the form of administration. Clearly, adherence is of vital importance for treatment success. Improving AIT adherence is one of the most important future goals, in order to ensure efficacy of the therapy.
Severe, potentially life-threatening systemic reactions during SCIT are possible, but – providing all safety measures are adhered to – these events are very rare. Most adverse events are mild to moderate and can be treated well.
Dose-dependent adverse local reactions occur frequently in the mouth and throat in SLIT. Systemic reactions have been described in SLIT, but are seen far less often than with SCIT. In terms of anaphylaxis and other severe systemic reactions, SLIT has a better safety profile than SCIT.
The risk and effects of adverse systemic reactions in the setting of AIT can be effectively reduced by training of personnel, adhering to safety standards and prompt use of emergency measures, including early administration of i. m. epinephrine. Details on the acute management of anaphylactic reactions can be found in the current S2 guideline on anaphylaxis issued by the AWMF (S2-AWMF-LL Registry Number 061-025).
AIT is undergoing some innovative developments in many areas (e. g., allergen characterization, new administration routes, adjuvants, faster and safer dose escalation protocols), some of which are already being investigated in clinical trials.
Cite this as Pfaar O, Bachert C, Bufe A, Buhl R, Ebner C, Eng P, Friedrichs F, Fuchs T, Hamelmann E, Hartwig-Bade D, Hering T, Huttegger I, Jung K, Klimek L, Kopp MV, Merk H, Rabe U, Saloga J, Schmid-Grendelmeier P, Schuster A, Schwerk N, Sitter H, Umpfenbach U, Wedi B, Wöhrl S, Worm M, Kleine-Tebbe J. Guideline on allergen-specific immunotherapy in IgE-mediated allergic diseases – S2k Guideline of the German Society for Allergology and Clinical Immunology (DGAKI), the Society for Pediatric Allergy and Environmental Medicine (GPA), the Medical Association of German Allergologists (AeDA), the Austrian Society for Allergy and Immunology (ÖGAI), the Swiss Society for Allergy and Immunology (SGAI), the German Society of Dermatology (DDG), the German Society of Oto-Rhino-Laryngology, Head and Neck Surgery (DGHNO-KHC), the German Society of Pediatrics and Adolescent Medicine (DGKJ), the Society for Pediatric Pneumology (GPP), the German Respiratory Society (DGP), the German Association of ENT Surgeons (BV-HNO), the Professional Federation of Paediatricians and Youth Doctors (BVKJ), the Federal Association of Pulmonologists (BDP) and the German Dermatologists Association (BVDD). Allergo J Int 2014;23:282–319
doi:10.1007/s40629-014-0032-2
PMCID: PMC4479478  PMID: 26120539
allergen-specific immunotherapy; AIT; Hyposensitization; guideline; allergen; allergen extract; allergic disease; allergic rhinitis; allergic asthma
4.  Phage-mediated Dispersal of Biofilm and Distribution of Bacterial Virulence Genes Is Induced by Quorum Sensing 
PLoS Pathogens  2015;11(2):e1004653.
The microbiome and the phage meta-genome within the human gut are influenced by antibiotic treatments. Identifying a novel mechanism, here we demonstrate that bacteria use the universal communication molecule AI-2 to induce virulence genes and transfer them via phage release. High concentrations (i.e. 100 μM) of AI-2 promote dispersal of bacteria from already established biofilms, and is associated with release of phages capable of infecting other bacteria. Enterococcus faecalis V583ΔABC harbours 7 prophages in its genome, and a mutant deficient in one of these prophages (i.e. prophage 5) showed a greatly reduced dispersal of biofilm. Infection of a probiotic E. faecalis strain without lytic prophages with prophage 5 resulted in increased biofilm formation and also in biofilm dispersal upon induction with AI-2. Infection of the probiotic E. faecalis strain with phage-containing supernatants released through AI-2 from E. faecalis V583ΔABC resulted in a strong increase in pathogenicity of this strain. The polylysogenic probiotic strain was also more virulent in a mouse sepsis model and a rat endocarditis model. Both AI-2 and ciprofloxacin lead to phage release, indicating that conditions in the gastrointestinal tract of hospitalized patients treated with antibiotics might lead to distribution of virulence genes to apathogenic enterococci and possibly also to other commensals or even to beneficial probiotic strains.
Author Summary
All higher organisms live in intimate contact with bacteria and viruses in their direct environment. Some of these bacteria in our gut can switch between being harmless commensals and causing severe and sometimes lethal infections. This involves a tight regulation of the mechanisms needed to initially colonize and later to harm the host. Here we describe a novel mechanism by which phages (i.e. viruses that infect bacteria) contribute to virulence in commensal gut bacteria. Our results show that bacteria "sense" the number of bacteria present at any given moment through a process called quorum sensing and this provides them with the information needed to assess the specific step during the infectious process. At late stages of infection bacteria are usually present in high numbers, and at this point release viruses that can infect nearby bacteria and transfer genes that are needed to cause infection, thereby enabling previously harmless bacteria to become dangerous pathogens.
doi:10.1371/journal.ppat.1004653
PMCID: PMC4338201  PMID: 25706310
5.  Production of Siderophores Increases Resistance to Fusaric Acid in Pseudomonas protegens Pf-5 
PLoS ONE  2015;10(1):e0117040.
Fusaric acid is produced by pathogenic fungi of the genus Fusarium, and is toxic to plants and rhizobacteria. Many fluorescent pseudomonads can prevent wilt diseases caused by these fungi. This study was undertaken to evaluate the effect of fusaric acid on P. protegens Pf-5 and elucidate the mechanisms that enable the bacterium to survive in the presence of the mycotoxin. The results confirm that fusaric acid negatively affects growth and motility of P. protegens. Moreover, a notable increase in secretion of the siderophore pyoverdine was observed when P. protegens was grown in the presence of fusaric acid. Concomitantly, levels of enzymes involved in the biosynthesis of pyoverdine and enantio-pyochelin, the second siderophore encoded by P. protegens, increased markedly. Moreover, while similar levels of resistance to fusaric acid were observed for P. protegens mutants unable to synthesize either pyoverdine or enanto-pyochelin and the wild type strain, a double mutant unable to synthesize both kinds of siderophores showed a dramatically reduced resistance to this compound. This reduced resistance was not observed when this mutant was grown under conditions of iron excess. Spectrophotometric titrations revealed that fusaric acid binds not only Fe2+ and Fe3+, but also Zn2+, Mn2+ and Cu2+, with high affinity. Our results demonstrate that iron sequestration accounts at least in part for the deleterious effect of the mycotoxin on P. protegens.
doi:10.1371/journal.pone.0117040
PMCID: PMC4287623  PMID: 25569682
6.  A Tale of Two Machines: A review of the BLAST meeting, Tucson, AZ, January 20–24, 2013 
Molecular microbiology  2013;91(1):6-25.
Since its inception, Bacterial Locomotion and Signal Transduction (BLAST) meetings have been the place to exchange and share the latest developments in the field of bacterial signal transduction and motility. At the 12th BLAST meeting, held last January in Tucson AZ, researchers from all over the world met to report and discuss progress in diverse aspects of the field. The majority of these advances, however, came at the level of atomic level structures and their associated mechanisms. This was especially true of the biological machines that sense and respond to environmental changes.
doi:10.1111/mmi.12427
PMCID: PMC3964004  PMID: 24125587
7.  Identification of a Novel Nutrient-Sensing Histidine Kinase/Response Regulator Network in Escherichia coli 
Journal of Bacteriology  2014;196(11):2023-2029.
When carbon sources become limiting for growth, bacteria must choose which of the remaining nutrients should be used first. We have identified a nutrient-sensing signaling network in Escherichia coli that is activated at the transition to stationary phase. The network is composed of the two histidine kinase/response regulator systems YehU/YehT and YpdA/YpdB and their target proteins, YjiY and YhjX (both of which are membrane-integrated transporters). The peptide/amino acid-responsive YehU/YehT system was found to have a negative effect on expression of the target gene, yhjX, of the pyruvate-responsive YpdA/YpdB system, while the YpdA/YpdB system stimulated expression of yjiY, the target of the YehU/YehT system. These effects were confirmed in mutants lacking any of the genes for the three primary components of either system. Furthermore, an in vivo interaction assay based on bacterial adenylate cyclase detected heteromeric interactions between the membrane-bound components of the two systems, specifically, between the two histidine kinases and the two transporters, which is compatible with the formation of a larger signaling unit. Finally, the carbon storage regulator A (CsrA) was shown to be involved in posttranscriptional regulation of both yjiY and yhjX.
doi:10.1128/JB.01554-14
PMCID: PMC4010973  PMID: 24659770
8.  Translational stalling at polyproline stretches is modulated by the sequence context upstream of the stall site 
Nucleic Acids Research  2014;42(16):10711-10719.
The polymerization of amino acids into proteins occurs on ribosomes, with the rate influenced by the amino acids being polymerized. The imino acid proline is a poor donor and acceptor for peptide-bond formation, such that translational stalling occurs when three or more consecutive prolines (PPP) are encountered by the ribosome. In bacteria, stalling at PPP motifs is rescued by the elongation factor P (EF-P). Using SILAC mass spectrometry of Escherichia coli strains, we identified a subset of PPP-containing proteins for which the expression patterns remained unchanged or even appeared up-regulated in the absence of EF-P. Subsequent analysis using in vitro and in vivo reporter assays revealed that stalling at PPP motifs is influenced by the sequence context upstream of the stall site. Specifically, the presence of amino acids such as Cys and Thr preceding the stall site suppressed stalling at PPP motifs, whereas amino acids like Arg and His promoted stalling. In addition to providing fundamental insight into the mechanism of peptide-bond formation, our findings suggest how the sequence context of polyproline-containing proteins can be modulated to maximize the efficiency and yield of protein production.
doi:10.1093/nar/gku768
PMCID: PMC4176338  PMID: 25143529
9.  Dynamics of an Interactive Network Composed of a Bacterial Two-Component System, a Transporter and K+ as Mediator 
PLoS ONE  2014;9(2):e89671.
KdpD and KdpE form a histidine kinase/response regulator system that senses K+ limitation and induces the kdpFABC operon, which encodes a high-affinity K+ uptake complex. To define the primary stimulus perceived by KdpD we focused in this study on the dynamics of the Kdp response. Escherichia coli cells were subjected to severe K+ limitation, and all relevant parameters of the Kdp response, i.e., levels of kdpFABC transcripts and KdpFABC proteins, as well as extra- and intracellular K+ concentrations, were quantitatively analysed over time (0 to 180 min). Unexpectedly, induction of kdpFABC was found to follow a non-monotonic time-course. To interpret this unusual behaviour, a mathematical model that adequately captures the dynamics of the Kdp system was established and used for simulations. We found a strong correlation between KdpD/KdpE activation and the intracellular K+ concentration, which is influenced by the uptake of K+ via the KdpFABC complex. Based on these results a model is proposed in which KdpD/KdpE phosphorylation is inversely correlated with the intracellular K+ concentration. To corroborate this hypothesis an isogenic mutant that produces a defective KdpFABC complex, and the trans-complemented mutant that expresses the KtrAB high-affinity K+ uptake system of Vibrio alginolyticus were quantitatively analysed. Experimental data and simulations for the mutants consistently support the tight correlation between KdpD/KdpE activation and the intracellular K+ concentration. This study presents a striking example of the non-intuitive dynamics of a functional unit comprising signalling proteins and a transporter with K+ as mediator.
doi:10.1371/journal.pone.0089671
PMCID: PMC3938482  PMID: 24586952
10.  Single Cell Kinetics of Phenotypic Switching in the Arabinose Utilization System of E. coli 
PLoS ONE  2014;9(2):e89532.
Inducible switching between phenotypes is a common strategy of bacteria to adapt to fluctuating environments. Here, we analyze the switching kinetics of a paradigmatic inducible system, the arabinose utilization system in E. coli. Using time-lapse fluorescence microscopy of microcolonies in a microfluidic chamber, which permits sudden up- and down-shifts in the inducer arabinose, we characterize the single-cell gene expression dynamics of the araBAD operon responsible for arabinose degradation. While there is significant, inducer-dependent cell-to-cell variation in the timing of the on-switching, the off-switching triggered by sudden removal of arabinose is homogeneous and rapid. We find that rapid off-switching does not depend on internal arabinose degradation. Because the system is regulated via the internal arabinose level sensed by AraC, internal arabinose must be rapidly depleted by leakage or export from the cell, or by degradation via a non-canonical pathway. We explored whether the poorly characterized membrane protein AraJ, which is part of the arabinose regulon and has been annotated as a possible arabinose efflux protein, is responsible for rapid depletion. However, we find that AraJ is not essential for rapid switching to the off-state. We develop a mathematical model for the arabinose system, which quantitatively describes both the heterogeneous on-switching and the homogeneous off-switching. The model also predicts that mutations which disrupt the positive feedback of internal arabinose on the production of arabinose uptake proteins change the heterogeneous on-switching behavior into a homogeneous, graded response. We construct such a mutant and confirm the graded response experimentally. Taken together, our results indicate that the physiological switching behavior of this sugar utilization system is asymmetric, such that off-switching is always rapid and homogeneous, while on-switching is slow and heterogeneously timed at sub-saturating inducer levels.
doi:10.1371/journal.pone.0089532
PMCID: PMC3935871  PMID: 24586851
11.  Driving Factors for the Evolution of Species-Specific Echolocation Call Design in New World Free-Tailed Bats (Molossidae) 
PLoS ONE  2014;9(1):e85279.
Phylogeny, ecology, and sensorial constraints are thought to be the most important factors influencing echolocation call design in bats. The Molossidae is a diverse bat family with a majority of species restricted to tropical and subtropical regions. Most molossids are specialized to forage for insects in open space, and thus share similar navigational challenges. We use an unprecedented dataset on the echolocation calls of 8 genera and 18 species of New World molossids to explore how habitat, phylogenetic relatedness, body mass, and prey perception contribute to echolocation call design. Our results confirm that, with the exception of the genus Molossops, echolocation calls of these bats show a typical design for open space foraging. Two lines of evidence point to echolocation call structure of molossids reflecting phylogenetic relatedness. First, such structure is significantly more similar within than among genera. Second, except for allometric scaling, such structure is nearly the same in congeneric species. Despite contrasting body masses, 12 of 18 species call within a relatively narrow frequency range of 20 to 35 kHz, a finding that we explain by using a modeling approach whose results suggest this frequency range to be an adaptation optimizing prey perception in open space. To conclude, we argue that the high variability in echolocation call design of molossids is an advanced evolutionary trait allowing the flexible adjustment of echolocation systems to various sensorial challenges, while conserving sender identity for social communication. Unraveling evolutionary drivers for echolocation call design in bats has so far been hampered by the lack of adequate model organisms sharing a phylogenetic origin and facing similar sensorial challenges. We thus believe that knowledge of the echolocation call diversity of New World molossid bats may prove to be landmark to understand the evolution and functionality of species-specific signal design in bats.
doi:10.1371/journal.pone.0085279
PMCID: PMC3891751  PMID: 24454833
12.  Bat echolocation calls facilitate social communication 
Bat echolocation is primarily used for orientation and foraging but also holds great potential for social communication. The communicative function of echolocation calls is still largely unstudied, especially in the wild. Eavesdropping on vocal signatures encoding social information in echolocation calls has not, to our knowledge, been studied in free-living bats so far. We analysed echolocation calls of the polygynous bat Saccopteryx bilineata and found pronounced vocal signatures encoding sex and individual identity. We showed experimentally that free-living males discriminate approaching male and female conspecifics solely based on their echolocation calls. Males always produced aggressive vocalizations when hearing male echolocation calls and courtship vocalizations when hearing female echolocation calls; hence, they responded with complex social vocalizations in the appropriate social context. Our study demonstrates that social information encoded in bat echolocation calls plays a crucial and hitherto underestimated role for eavesdropping conspecifics and thus facilitates social communication in a highly mobile nocturnal mammal.
doi:10.1098/rspb.2012.1995
PMCID: PMC3497101  PMID: 23034703
eavesdropping; sex-specific signature; individual signature; Saccopteryx bilineata; vocal communication; Chiroptera
13.  Behavioral flexibility of the trawling long-legged bat, Macrophyllum macrophyllum (Phyllostomidae) 
We assessed the behavioral flexibility of the trawling long-legged bat, Macrophyllum macrophyllum (Phyllostomidae) in flight cage experiments by exposing it to prey suspended from nylon threads in the air and to food placed onto the water surface at varying distances to clutter-producing background (water plants). The bat revealed flexibility in foraging mode and caught prey in the air (aerial hawking) and from the water surface (trawling). M. macrophyllum was constrained in finding food very near to and within clutter. As echolocation was the prime sensory mode used by M. macrophyllum for detection and localization of food, the bat might have been unable to perceive sufficient information from prey near clutter as background echoes from the water plant increasingly overlapped with echoes from food. The importance of echolocation for foraging is reflected in a stereotypic call pattern of M. macrophyllum that resembles other aerial insectivorous and trawling bats with a pronounced terminal phase (buzz) prior to capture attempts. Our findings contrast studies of other phyllostomid bats that glean prey very near or from vegetation, often using additional sensory cues, such as prey-produced noise, to find food and that lack a terminal phase in echolocation behavior. In M. macrophyllum, acoustic characteristics of its foraging habitat have shaped its sonar system more than phylogeny.
doi:10.3389/fphys.2013.00342
PMCID: PMC3838978  PMID: 24324442
sensory ecology; aerial hawking; gleaning; bat echolocation; clutter; echo overlap
14.  A New Class of Quorum Quenching Molecules from Staphylococcus Species Affects Communication and Growth of Gram-Negative Bacteria 
PLoS Pathogens  2013;9(9):e1003654.
The knowledge that many pathogens rely on cell-to-cell communication mechanisms known as quorum sensing, opens a new disease control strategy: quorum quenching. Here we report on one of the rare examples where Gram-positive bacteria, the ‘Staphylococcus intermedius group’ of zoonotic pathogens, excrete two compounds in millimolar concentrations that suppress the quorum sensing signaling and inhibit the growth of a broad spectrum of Gram-negative beta- and gamma-proteobacteria. These compounds were isolated from Staphylococcus delphini. They represent a new class of quorum quenchers with the chemical formula N-[2-(1H-indol-3-yl)ethyl]-urea and N-(2-phenethyl)-urea, which we named yayurea A and B, respectively. In vitro studies with the N-acyl homoserine lactone (AHL) responding receptor LuxN of V. harveyi indicated that both compounds caused opposite effects on phosphorylation to those caused by AHL. This explains the quorum quenching activity. Staphylococcal strains producing yayurea A and B clearly benefit from an increased competitiveness in a mixed community.
Author Summary
While studying the potential interaction of staphylococci with Gram-negative bacteria, we came across another communication system in a Staphylococcus species group, which consists of closely related coagulase-positive bacterial species that play a role as zoonotic pathogens. We found that these species excrete two small compounds that inhibit both the expression of QS-controlled toxins and other QS-regulated compounds as well as growth in Gram-negative bacteria. The excreted compounds, which we named yayurea A and B, were isolated from S. delphini and structurally characterized. They represent new bacterial products, which quench the QS regulation in a wide spectrum of Gram-negative bacteria by stimulating the LuxN-mediated phosphorylation of LuxU. Furthermore, growth of yayurea A and B producing S. delphini is not suppressed by respiratory toxins when co-cultured with P. aeruginosa. This suggests that the quorum quenchers have a function in self-protection and competitiveness in natural environments shared with Gram-negatives. Here we show one of the rare cases of inter-phylum interference between firmicutes (Gram-positive) and beta-/gammaproteobacteria (Gram-negative).
doi:10.1371/journal.ppat.1003654
PMCID: PMC3784491  PMID: 24098134
15.  Identification of a Target Gene and Activating Stimulus for the YpdA/YpdB Histidine Kinase/Response Regulator System in Escherichia coli 
Journal of Bacteriology  2013;195(4):807-815.
Escherichia coli contains 30 two-component systems (TCSs), each consisting of a histidine kinase and a response regulator. Whereas most TCSs are well characterized in this model organism, little is known about the YpdA/YpdB system. To identify YpdB-regulated genes, we compared the transcriptomes of E. coli cells overproducing either YpdB or a control protein. Expression levels of 15 genes differed by more than 1.9-fold between the two strains. A comprehensive evaluation of these genes identified yhjX as the sole target of YpdB. Electrophoretic mobility shift assays with purified YpdB confirmed its interaction with the yhjX promoter. Specifically, YpdB binds to two direct repeats of the motif GGCATTTCAT separated by an 11-bp spacer in the yhjX promoter. yhjX encodes a cytoplasmic membrane protein of unknown function that belongs to the major facilitator superfamily of transporters. Finally, we characterized the pattern of yhjX expression and identified extracellular pyruvate as a stimulus for the YpdA/YpdB system. It is suggested that YpdA/YpdB contributes to nutrient scavenging before entry into stationary phase.
doi:10.1128/JB.02051-12
PMCID: PMC3562098  PMID: 23222720
16.  Perception of silent and motionless prey on vegetation by echolocation in the gleaning bat Micronycteris microtis 
Gleaning insectivorous bats that forage by using echolocation within dense forest vegetation face the sensorial challenge of acoustic masking effects. Active perception of silent and motionless prey in acoustically cluttered environments by echolocation alone has thus been regarded impossible. The gleaning insectivorous bat Micronycteris microtis however, forages in dense understory vegetation and preys on insects, including dragonflies, which rest silent and motionless on vegetation. From behavioural experiments, we show that M. microtis uses echolocation as the sole sensorial modality for successful prey perception within a complex acoustic environment. All individuals performed a stereotypical three-dimensional hovering flight in front of prey items, while continuously emitting short, multi-harmonic, broadband echolocation calls. We observed a high precision in target localization which suggests that M. microtis perceives a detailed acoustic image of the prey based on shape, surface structure and material. Our experiments provide, to our knowledge, the first evidence that a gleaning bat uses echolocation alone for successful detection, classification and precise localization of silent and motionless prey in acoustic clutter. Overall, we conclude that the three-dimensional hovering flight of M. microtis in combination with a frequent emission of short, high-frequency echolocation calls is the key for active prey perception in acoustically highly cluttered environments.
doi:10.1098/rspb.2012.2830
PMCID: PMC3574334  PMID: 23325775
prey perception; echolocation; acoustic clutter; narrow-space active gleaning insectivorous bat; Micronycteris microtis
17.  The Janthinobacterium sp. HH01 Genome Encodes a Homologue of the V. cholerae CqsA and L. pneumophila LqsA Autoinducer Synthases 
PLoS ONE  2013;8(2):e55045.
Janthinobacteria commonly form biofilms on eukaryotic hosts and are known to synthesize antibacterial and antifungal compounds. Janthinobacterium sp. HH01 was recently isolated from an aquatic environment and its genome sequence was established. The genome consists of a single chromosome and reveals a size of 7.10 Mb, being the largest janthinobacterial genome so far known. Approximately 80% of the 5,980 coding sequences (CDSs) present in the HH01 genome could be assigned putative functions. The genome encodes a wealth of secretory functions and several large clusters for polyketide biosynthesis. HH01 also encodes a remarkable number of proteins involved in resistance to drugs or heavy metals. Interestingly, the genome of HH01 apparently lacks the N-acylhomoserine lactone (AHL)-dependent signaling system and the AI-2-dependent quorum sensing regulatory circuit. Instead it encodes a homologue of the Legionella- and Vibrio-like autoinducer (lqsA/cqsA) synthase gene which we designated jqsA. The jqsA gene is linked to a cognate sensor kinase (jqsS) which is flanked by the response regulator jqsR. Here we show that a jqsA deletion has strong impact on the violacein biosynthesis in Janthinobacterium sp. HH01 and that a jqsA deletion mutant can be functionally complemented with the V. cholerae cqsA and the L. pneumophila lqsA genes.
doi:10.1371/journal.pone.0055045
PMCID: PMC3566124  PMID: 23405110
18.  First Insights into the Unexplored Two-Component System YehU/YehT in Escherichia coli 
Journal of Bacteriology  2012;194(16):4272-4284.
Two-component systems (TCSs) consisting of a membrane-anchored histidine kinase (HK) and a response regulator (RR) with DNA-binding activity. are major players in signal transduction in prokaryotes. Whereas most TCSs in Escherichia coli are well characterized, almost nothing is known about the LytS-like HK YehU and the corresponding LytTR-like RR YehT. To identify YehT-regulated genes, we compared the transcriptomes of E. coli cells overproducing either YehT or the RR KdpE (control). The expression levels of 32 genes differed more than 8-fold between the two strains. A comprehensive evaluation of these genes identified yjiY as a target of YehT. Electrophoretic mobility shift assays with purified YehT confirmed that YehT interacts directly with the yjiY promoter. Specifically, YehT binds to two direct repeats of the motif ACC(G/A)CT(C/T)A separated by a 13-bp spacer in the yjiY promoter. The target gene yjiY encodes an inner membrane protein belonging to the CstA superfamily of transporters. In E. coli cells growing in media containing peptides or amino acids as a carbon source, yjiY is strongly induced at the onset of the stationary-growth phase. Moreover, expression was found to be dependent on cyclic AMP (cAMP)/cAMP receptor protein (CRP). It is suggested that YehU/YehT participates in the stationary-phase control network.
doi:10.1128/JB.00409-12
PMCID: PMC3416263  PMID: 22685278
19.  Autoinducers Act as Biological Timers in Vibrio harveyi 
PLoS ONE  2012;7(10):e48310.
Quorum sensing regulates cell density-dependent phenotypes and involves the synthesis, excretion and detection of so-called autoinducers. Vibrio harveyi strain ATCC BAA-1116 (recently reclassified as Vibrio campbellii), one of the best-characterized model organisms for the study of quorum sensing, produces and responds to three autoinducers. HAI-1, AI-2 and CAI-1 are recognized by different receptors, but all information is channeled into the same signaling cascade, which controls a specific set of genes. Here we examine temporal variations of availability and concentration of the three autoinducers in V. harveyi, and monitor the phenotypes they regulate, from the early exponential to the stationary growth phase in liquid culture. Specifically, the exponential growth phase is characterized by an increase in AI-2 and the induction of bioluminescence, while HAI-1 and CAI-1 are undetectable prior to the late exponential growth phase. CAI-1 activity reaches its maximum upon entry into stationary phase, while molar concentrations of AI-2 and HAI-1 become approximately equal. Similarly, autoinducer-dependent exoproteolytic activity increases at the transition into stationary phase. These findings are reflected in temporal alterations in expression of the luxR gene that encodes the master regulator LuxR, and of four autoinducer-regulated genes during growth. Moreover, in vitro phosphorylation assays reveal a tight correlation between the HAI-1/AI-2 ratio as input and levels of receptor-mediated phosphorylation of LuxU as output. Our study supports a model in which the combinations of autoinducers available, rather than cell density per se, determine the timing of various processes in V. harveyi populations.
doi:10.1371/journal.pone.0048310
PMCID: PMC3482212  PMID: 23110227
20.  Single cell analysis of Vibrio harveyi uncovers functional heterogeneity in response to quorum sensing signals 
BMC Microbiology  2012;12:209.
Background
Vibrio harveyi and closely related species are important pathogens in aquaculture. A complex quorum sensing cascade involving three autoinducers controls bioluminescence and several genes encoding virulence factors. Single cell analysis of a V. harveyi population has already indicated intercellular heterogeneity in the production of bioluminescence. This study was undertaken to analyze the expression of various autoinducer-dependent genes in individual cells.
Results
Here we used reporter strains bearing promoter::gfp fusions to monitor the induction/repression of three autoinducer-regulated genes in wild type conjugates at the single cell level. Two genes involved in pathogenesis - vhp and vscP, which code for an exoprotease and a component of the type III secretion system, respectively, and luxC (the first gene in the lux operon) were chosen for analysis. The lux operon and the exoprotease gene are induced, while vscP is repressed at high cell density. As controls luxS and recA, whose expression is not dependent on autoinducers, were examined. The responses of the promoter::gfp fusions in individual cells from the same culture ranged from no to high induction. Importantly, simultaneous analysis of two autoinducer induced phenotypes, bioluminescence (light detection) and exoproteolytic activity (fluorescence of a promoter::gfp fusion), in single cells provided evidence for functional heterogeneity within a V. harveyi population.
Conclusions
Autoinducers are not only an indicator for cell density, but play a pivotal role in the coordination of physiological activities within the population.
doi:10.1186/1471-2180-12-209
PMCID: PMC3511230  PMID: 22985329
Bioluminescence; Exoprotease; Type III secretion; Autoinducer; Division of labor; Subpopulation
21.  Identification of ArgP and Lrp as Transcriptional Regulators of lysP, the Gene Encoding the Specific Lysine Permease of Escherichia coli▿† 
Journal of Bacteriology  2011;193(10):2536-2548.
Expression of lysP, which encodes the lysine-specific transporter LysP in Escherichia coli, is regulated by the concentration of exogenous available lysine. In this study, the LysR-type transcriptional regulator ArgP was identified as the activator of lysP expression. At lysine concentrations higher than 25 μM, lysP expression was shut off and phenocopied an argP deletion mutant. Purified ArgP-His6 bound to the lysP promoter/control region at a sequence containing a conserved T-N11-A motif. Its affinity increased in the presence of lysine but not in the presence of the other known coeffector, arginine. In vivo data suggest that lysine-loaded ArgP and arginine-loaded ArgP compete at the lysP promoter. We propose that lysine-loaded ArgP prevents lysP transcription at the promoter clearance step, as described for the lysine-dependent regulation of argO (R. S. Laishram and J. Gowrishankar, Genes Dev. 21:1258-1272, 2007). The global regulator Lrp also bound to the lysP promoter/control region. An lrp mutant exhibited reduced lysP expression in the absence of external lysine. These results indicate that ArgP is a major regulator of lysP expression but that Lrp modulates lysP transcription under lysine-limiting conditions.
doi:10.1128/JB.00815-10
PMCID: PMC3133163  PMID: 21441513
22.  Detection and function of an intramolecular disulfide bond in the pH-responsive CadC of Escherichia coli 
BMC Microbiology  2011;11:74.
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.
doi:10.1186/1471-2180-11-74
PMCID: PMC3096576  PMID: 21486484
23.  Helicobacter pylori Type IV Secretion Apparatus Exploits β1 Integrin in a Novel RGD-Independent Manner 
PLoS Pathogens  2009;5(12):e1000684.
Translocation of the Helicobacter pylori (Hp) cytotoxin-associated gene A (CagA) effector protein via the cag-Type IV Secretion System (T4SS) into host cells is a major risk factor for severe gastric diseases, including gastric cancer. However, the mechanism of translocation and the requirements from the host cell for that event are not well understood. The T4SS consists of inner- and outer membrane-spanning Cag protein complexes and a surface-located pilus. Previously an arginine-glycine-aspartate (RGD)-dependent typical integrin/ligand type interaction of CagL with α5β1 integrin was reported to be essential for CagA translocation. Here we report a specific binding of the T4SS-pilus-associated components CagY and the effector protein CagA to the host cell β1 Integrin receptor. Surface plasmon resonance measurements revealed that CagA binding to α5β1 integrin is rather strong (dissociation constant, KD of 0.15 nM), in comparison to the reported RGD-dependent integrin/fibronectin interaction (KD of 15 nM). For CagA translocation the extracellular part of the β1 integrin subunit is necessary, but not its cytoplasmic domain, nor downstream signalling via integrin-linked kinase. A set of β1 integrin-specific monoclonal antibodies directed against various defined β1 integrin epitopes, such as the PSI, the I-like, the EGF or the β-tail domain, were unable to interfere with CagA translocation. However, a specific antibody (9EG7), which stabilises the open active conformation of β1 integrin heterodimers, efficiently blocked CagA translocation. Our data support a novel model in which the cag-T4SS exploits the β1 integrin receptor by an RGD-independent interaction that involves a conformational switch from the open (extended) to the closed (bent) conformation, to initiate effector protein translocation.
Author Summary
Integrins are single transmembrane proteins present on almost all types of cells. They are composed of an α and a β subunit, which together form the ligand binding pocket, able to interact with extracellular matrix proteins. The best known binding domain on integrin ligands is the RGD domain. Many bacterial, but also viral pathogens exploit this ligand-binding domain to interact with integrins on the host cell. Helicobacter pylori, a common bacterial pathogen associated with gastric diseases, was recently added to this list. One of H. pylori's most important factors associated with gastric pathologies is the CagA protein. This protein is directly injected into host cells through the Cag Type IV Secretion System (cag-T4SS). Previous studies demonstrated that the cag-T4SS requires integrins for the injection (translocation) of CagA into cells. We provide evidence that three proteins, CagA, CagI and CagY, interact with integrins in an RGD-independent way. Additionally, our data point out that the Cag apparatus needs the physical capacity of a β1 integrin heterodimer to change from an active/extended conformation to a closed/bent conformation. This novel kind of integrin interaction opens a new way in which pathogens can use receptors on cells.
doi:10.1371/journal.ppat.1000684
PMCID: PMC2779590  PMID: 19997503
24.  Domain swapping reveals that the N-terminal domain of the sensor kinase KdpD in Escherichia coli is important for signaling 
BMC Microbiology  2009;9:133.
Background
The KdpD/KdpE two-component system of Escherichia coli regulates expression of the kdpFABC operon encoding the high affinity K+ transport system KdpFABC. The input domain of KdpD comprises a domain that belongs to the family of universal stress proteins (Usp). It has been previously demonstrated that UspC binds to this domain, resulting in KdpD/KdpE scaffolding under salt stress. However the mechanistic significance of this domain for signaling remains unclear. Here, we employed a "domain swapping" approach to replace the KdpD-Usp domain with four homologous domains or with the six soluble Usp proteins of E. coli.
Results
Full response to salt stress was only achieved with a chimera that contains UspC, probably due to unaffected scaffolding of the KdpD/KdpE signaling cascade by soluble UspC. Unexpectedly, chimeras containing either UspF or UspG not only prevented kdpFABC expression under salt stress but also under K+ limiting conditions, although these hybrid proteins exhibited kinase and phosphotransferase activities in vitro. These are the first KdpD derivatives that do not respond to K+ limitation due to alterations in the N-terminal domain. Analysis of the KdpD-Usp tertiary structure revealed that this domain has a net positively charged surface, while UspF and UspG are characterized by net negative surface charges.
Conclusion
The Usp domain within KdpD not only functions as a binding surface for the scaffold UspC, but it is also important for KdpD signaling. We propose that KdpD sensing/signaling involves alterations of electrostatic interactions between the large N- and C-terminal cytoplasmic domains.
doi:10.1186/1471-2180-9-133
PMCID: PMC2714519  PMID: 19589130
25.  Photorhabdus luminescens genes induced upon insect infection 
BMC Genomics  2008;9:229.
Background
Photorhabdus luminescens is a Gram-negative luminescent enterobacterium and a symbiote to soil nematodes belonging to the species Heterorhabditis bacteriophora. P.luminescens is simultaneously highly pathogenic to insects. This bacterium exhibits a complex life cycle, including one symbiotic stage characterized by colonization of the upper nematode gut, and a pathogenic stage, characterized by release from the nematode into the hemocoel of insect larvae, resulting in rapid insect death caused by bacterial toxins. P. luminescens appears to sense and adapt to the novel host environment upon changing hosts, which facilitates the production of factors involved in survival within the host, host-killing, and -exploitation.
Results
A differential fluorescence induction (DFI) approach was applied to identify genes that are up-regulated in the bacterium after infection of the insect host Galleria mellonella. For this purpose, a P. luminescens promoter-trap library utilizing the mCherry fluorophore as a reporter was constructed, and approximately 13,000 clones were screened for fluorescence induction in the presence of a G. mellonella larvae homogenate. Since P. luminescens has a variety of regulators that potentially sense chemical molecules, like hormones, the screen for up-regulated genes or operons was performed in vitro, excluding physicochemical signals like oxygen, temperature or osmolarity as variables. Clones (18) were obtained exhibiting at least 2.5-fold induced fluorescence and regarded as specific responders to insect homogenate. In combination with a bioinformatics approach, sequence motifs were identified in these DNA-fragments that are similar to 29 different promoters within the P. luminescens genome. By cloning each of the predicted promoters upstream of the reporter gene, induction was verified for 27 promoters in vitro, and for 24 promoters in viable G. mellonella larvae. Among the validated promoters are some known to regulate the expression of toxin genes, including tccC1 (encoding an insecticidal toxin complex), and others encoding putative toxins. A comparably high number of metabolic genes or operons were observed to be induced upon infection; among these were eutABC, hutUH, and agaZSVCD, which encode proteins involved in ethanolamine, histidine and tagatose degradation, respectively. The results reflect rearrangements in metabolism and the use of other metabolites available from the insect. Furthermore, enhanced activity of promoters controlling the expression of genes encoding enzymes linked to antibiotic production and/or resistance was observed. Antibiotic production and resistance may influence competition with other bacteria, and thus might be important for a successful infection. Lastly, several genes of unknown function were identified that may represent novel pathogenicity factors.
Conclusion
We show that a DFI screen is useful for identifying genes or operons induced by chemical stimuli, such as diluted insect homogenate. A bioinformatics comparison of motifs similar to known promoters is a powerful tool for identifying regulated genes or operons. We conclude that signals for the regulation of those genes or operons induced in P. luminescens upon insect infection may represent a wide variety of compounds that make up the insect host. Our results provide insight into the complex response to the host that occurs in a bacterial pathogen, particularly reflecting the potential for metabolic shifts and other specific changes associated with virulence.
doi:10.1186/1471-2164-9-229
PMCID: PMC2422844  PMID: 18489737

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