Coevolution of ABC transporters and two-component regulatory systems as resistance modules against antimicrobial peptides in Firmicutes Bacteria

In Firmicutes bacteria, ATP-binding cassette (ABC) transporters have been recognized as important resistance determinants against antimicrobial peptides. Together with neighboring two-component systems (TCSs), which regulate their expression, they form specific detoxification modules. Both the transport permease and sensor kinase components show unusual domain architecture: the permeases contain a large extracellular domain, while the sensor kinases lack an obvious input domain. One of the best-characterized examples is the bacitracin resistance module BceRS-BceAB of Bacillus subtilis. Strikingly, in this system, the ABC transporter and TCS have an absolute mutual requirement for each other in both sensing of and resistance to bacitracin, suggesting a novel mode of signal transduction in which the transporter constitutes the actual sensor. We identified over 250 such BceAB-like ABC transporters in the current databases. They occurred almost exclusively in Firmicutes bacteria, and 80% of the transporters were associated with a BceRS-like TCS. Phylogenetic analyses of the permease and sensor kinase components revealed a tight evolutionary correlation. Our findings suggest a direct regulatory interaction between the ABC transporters and TCSs, mediating communication between both components. Based on their observed coclustering and conservation of response regulator binding sites, we could identify putative corresponding two-component systems for transporters lacking a regulatory system in their immediate neighborhood. Taken together, our results show that these types of ABC transporters and TCSs have coevolved to form self-sufficient detoxification modules against antimicrobial peptides, widely distributed among Firmicutes bacteria.     

Anatomy of the bacitracin resistance network in Bacillus subtilis

Protection against antimicrobial peptides (AMPs) often involves the parallel production of multiple, well‐characterized resistance determinants. So far, little is known about how these resistance modules interact and how they jointly protect the cell. Here, we studied the interdependence between different layers of the envelope stress response of Bacillus subtilis when challenged with the lipid II cycle‐inhibiting AMP bacitracin. The underlying regulatory network orchestrates the production of the ABC transporter BceAB, the UPP phosphatase BcrC and the phage‐shock proteins LiaIH. Our systems‐level analysis reveals a clear hierarchy, allowing us to discriminate between primary (BceAB) and secondary (BcrC and LiaIH) layers of bacitracin resistance. Deleting the primary layer provokes an enhanced induction of the secondary layer to partially compensate for this loss. This study reveals a direct role of LiaIH in bacitracin resistance, provides novel insights into the feedback regulation of the Lia system, and demonstrates a pivotal role of BcrC in maintaining cell wall homeostasis. The compensatory regulation within the bacitracin network can also explain how gene expression noise propagates between resistance layers. We suggest that this active redundancy in the bacitracin resistance network of B. subtilis is a general principle to be found in many bacterial antibiotic resistance networks.     

Identification of Regions Important for Resistance and Signalling within the Antimicrobial Peptide Transporter BceAB of Bacillus subtilis

In the low-G+C-content Gram-positive bacteria, resistance to antimicrobial peptides is often mediated by so-called resistance modules. These consist of a two-component system and an ATP-binding cassette transporter and are characterized by an unusual mode of signal transduction where the transporter acts as a sensor of antimicrobial peptides, because the histidine kinase alone cannot detect the substrates directly. Thus, the transporters fulfill a dual function as sensors and detoxification systems to confer resistance, but the mechanistic details of these processes are unknown. The paradigm and best-understood example for this is the BceRS-BceAB module of Bacillus subtilis, which mediates resistance to bacitracin, mersacidin, and actagardine. Using a random mutagenesis approach, we here show that mutations that affect specific functions of the transporter BceAB are primarily found in the C-terminal region of the permease, BceB, particularly in the eighth transmembrane helix. Further, we show that while signaling and resistance are functionally interconnected, several mutations could be identified that strongly affected one activity of the transporter but had only minor effects on the other. Thus, a partial genetic separation of the two properties could be achieved by single amino acid replacements, providing first insights into the signaling mechanism of these unusual modules.     

Condition-specific coregulation with cis-regulatory motifs and modules in the mouse genome

Deciphering genetic regulatory codes remains a challenge. Here, we present an effective approach to identifying in vivo condition-specific coregulation with cis-regulatory motifs and modules in the mouse genome. A resampling-based algorithm was adopted to cluster our microarray data of a stress response, which generated 35 tight clusters with unique expression patterns containing 811 genes of 5652 genes significantly altered. Database searches identified many known motifs within the 3-kb regulatory regions of 40 genes from 3 clusters and modules with six to nine motifs that were commonly shared by 60-100% of these genes. The upstream regulatory region contained the highest frequency of these common motifs. CisModule program predictions were comparable with the results from database searches and found four potentially novel motifs. This result indicates that these motifs and modules could be responsible for gene coregulation of the stress response in the lacrimal gland.     

转录因子及miRNA调控食管癌耐药机制研究

由于耐药性的存在,不同患者在使用相同药物时会导致治疗效果的差异.因此识别耐药性相关的关键生物学标记,有助于临床医生快速选择出适合的药物,延长患者的生存时间,对药物研发以及药物的作用机制的详细研究具有重要意义.首先在食管癌细胞系中筛选不同药物的耐药及敏感细胞系,从中找到不同药物耐药相关的基因,将这些计算得到的耐药相关基因...     

A Sensory Complex Consisting of an ATP-binding Cassette Transporter and a Two-component Regulatory System Controls Bacitracin Resistance in Bacillus subtilis

Resistance against antimicrobial peptides in many Firmicutes bacteria is mediated by detoxification systems that are composed of a two-component regulatory system (TCS) and an ATP-binding cassette (ABC) transporter. The histidine kinases of these systems depend entirely on the transporter for sensing of antimicrobial peptides, suggesting a novel mode of signal transduction where the transporter constitutes the actual sensor. The aim of this study was to investigate the molecular mechanisms of this unusual signaling pathway in more detail, using the bacitracin resistance system BceRS-BceAB of Bacillus subtilis as an example. To analyze the proposed communication between TCS and the ABC transporter, we characterized their interactions by bacterial two-hybrid analyses and could show that the permease BceB and the histidine kinase BceS interact directly. In vitro pulldown assays confirmed this interaction, which was found to be independent of bacitracin. Because it was unknown whether BceAB-type transporters could detect their substrate peptides directly or instead recognized the peptide-target complex in the cell envelope, we next analyzed substrate binding by the transport permease, BceB. Direct and specific binding of bacitracin by BceB was demonstrated by surface plasmon resonance spectroscopy. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the autophosphorylation activity of the histidine kinase. Taken together, our findings clearly show the existence of a sensory complex composed of TCS and ABC transporters and provide the first functional insights into the mechanisms of stimulus perception, signal transduction, and antimicrobial resistance employed by Bce-like detoxification systems.     

Properties of long myosin light chain kinase binding to F-actin in vitro and in vivo

Short and long myosin light chain kinases (MLCKs) are Ca(2+)/calmodulin-dependent enzymes that phosphorylate the regulatory light chain of myosin II in thick filaments but bind with high affinity to actin thin filaments. Three repeats of a motif made up of the sequence DFRXXL at the N terminus of short MLCK are necessary for actin binding (Smith, L., Su, X., Lin, P., Zhi, G., and Stull, J. T. (1999) J. Biol. Chem. 274, 29433-29438). The long MLCK has two additional DFRXXL motifs and six Ig-like modules in an N-terminal extension, which may confer unique binding properties for cellular localization. Two peptides containing either five or three DFRXXL motifs bound to F-actin and smooth muscle myofilaments with maximal binding stoichiometries consistent with each motif binding to an actin monomer in the filaments. Both peptides cross-linked F-actin and bound to stress fibers in cells. Long MLCK with an internal deletion of the five DFRXXL motifs and the unique NH(2)-terminal fragment containing six Ig-like motifs showed weak binding. Cell fractionation and extractions with MgCl(2) indicate that the long MLCK has a greater affinity for actin-containing filaments than short MLCK in vitro and in vivo. Whereas DFRXXL motifs are necessary and sufficient for short MLCK binding to actin-containing filaments, the DFRXXL motifs and the N-terminal extension of long MLCK confer high affinity binding to stress fibers in cells.     

Dipeptidyl-peptidase IV (CD26)--role in the inactivation of regulatory peptides

Dipeptidyl-peptidase IV (DPP IV/CD26) has a dual function as a regulatory protease and as a binding protein. Its role in the inactivation of bioactive peptides was recognized 20 years ago due to its unique ability to liberate Xaa-Pro or Xaa-Ala dipeptides from the N-terminus of regulatory peptides, but further examples are now emerging from in vitro and vivo experiments. Despite the minimal N-terminal truncation by DPP IV, many mammalian regulatory peptides are inactivated--either totally or only differentially--for certain receptor subtypes. Important DPP IV substrates include neuropeptides like neuropeptide Y or endomorphin, circulating peptide hormones like peptide YY, growth hormone-releasing hormone, glucagon-like peptides(GLP)-1 and -2, gastric inhibitory polypeptide as well as paracrine chemokines like RANTES (regulated on activation normal T cell expressed and secreted), stromal cell-derived factor, eotaxin and macrophage-derived chemokine. Based on these findings the potential clinical uses of selective DPP IV inhibitors or DPP IV-resistant analogues, especially for the insulinotropic hormone GLP-1, have been tested to enhance insulin secretion and to improve glucose tolerance in diabetic animals. Thus, DPP IV appears to be a major physiological regulator for some regulatory peptides, neuropeptides, circulating hormones and chemokines.     

Probing the sORF-Encoded Peptides of Deinococcus radiodurans in Response to Extreme Stress

Organisms have developed different mechanisms to respond to stresses. However, the roles of small ORF–encoded peptides (SEPs) in these regulatory systems remain elusive, which is partially because of the lack of comprehensive knowledge regarding these biomolecules. We chose the extremophile Deinococcus radiodurans R1 as a model species and conducted large-scale profiling of the SEPs related to the stress response. The integrated workflow consisting of multiple omics approaches for SEP identification was streamlined, and an SEPome of D. radiodurans containing 109 novel and high-confidence SEPs was drafted. Forty-four percent of these SEPs were predicted to function as antimicrobial peptides. Quantitative peptidomics analysis indicated that the expression of SEP068184 was upregulated upon oxidative treatment and gamma irradiation of the bacteria. SEP068184 was conserved in Deinococcus and exhibited negative regulation of oxidative stress resistance in a comparative phenotypic assay of its mutants. Further quantitative and interactive proteomics analyses suggested that SEP068184 might function through metabolic pathways and interact with cytoplasmic proteins. Collectively, our findings demonstrate that SEPs are involved in the regulation of oxidative resistance, and the SEPome dataset provides a rich resource for research on the molecular mechanisms of the response to extreme stress in organisms.     

Identification of functional toxin/immunity genes linked to contact-dependent growth inhibition (CDI) and rearrangement hotspot (Rhs) systems

Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiA/CdiB family of two-partner secretion proteins. Each CdiA protein exhibits a distinct growth inhibition activity, which resides in the polymorphic C-terminal region (CdiA-CT). CDI(+) cells also express unique CdiI immunity proteins that specifically block the activity of cognate CdiA-CT, thereby protecting the cell from autoinhibition. Here we show that many CDI systems contain multiple cdiA gene fragments that encode CdiA-CT sequences. These "orphan" cdiA-CT genes are almost always associated with downstream cdiI genes to form cdiA-CT/cdiI modules. Comparative genome analyses suggest that cdiA-CT/cdiI modules are mobile and exchanged between the CDI systems of different bacteria. In many instances, orphan cdiA-CT/cdiI modules are fused to full-length cdiA genes in other bacterial species. Examination of cdiA-CT/cdiI modules from Escherichia coli EC93, E. coli EC869, and Dickeya dadantii 3937 confirmed that these genes encode functional toxin/immunity pairs. Moreover, the orphan module from EC93 was functional in cell-mediated CDI when fused to the N-terminal portion of the EC93 CdiA protein. Bioinformatic analyses revealed that the genetic organization of CDI systems shares features with rhs (rearrangement hotspot) loci. Rhs proteins also contain polymorphic C-terminal regions (Rhs-CTs), some of which share significant sequence identity with CdiA-CTs. All rhs genes are followed by small ORFs representing possible rhsI immunity genes, and several Rhs systems encode orphan rhs-CT/rhsI modules. Analysis of rhs-CT/rhsI modules from D. dadantii 3937 demonstrated that Rhs-CTs have growth inhibitory activity, which is specifically blocked by cognate RhsI immunity proteins. Together, these results suggest that Rhs plays a role in intercellular competition and that orphan gene modules expand the diversity of toxic activities deployed by both CDI and Rhs systems.     

Synthetic biology for mammalian cell technology and materials sciences

The synthetic reconstruction of natural gene networks and the de novo design of artificial genetic circuits provide new insights into the cell's regulatory mechanisms and will open new opportunities for drug discovery and intelligent therapeutic schemes. We will present how modular synthetic biology tools like repressors, promoters and enzymes can be assembled into complex systems in order to discover small molecules to shut off antibiotic resistance in tubercle bacteria and to design self-sufficient therapeutic networks. The transfer of these synthetic biological modules to the materials science field enables the construction of novel drug-inducible biohybrid materials for biomedical applications.