scholarly journals 599. LiaF is an Activator of the LiaR-Mediated Response Against Daptomycin and Antimicrobial Peptides in Multidrug-Resistant Enterococcus faecalis (Efs)

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S281-S281
Author(s):  
Laura C Ortiz-Velez ◽  
Sandra L Rincon ◽  
Jesse Degani ◽  
Yousif Shamoo ◽  
Truc T Tran ◽  
...  
Keyword(s):  
Stress Response ◽  
Stop Codon ◽  
Response Regulator ◽  
Transmembrane Protein ◽  
Premature Stop Codon ◽  
Regulatory System ◽  
Laboratory Strain ◽  
Positive Interaction ◽  
Anionic Phospholipid ◽  
Membrane Architecture

Abstract Background Daptomycin (DAP) is a key first-line agent for the treatment of vancomycin-resistant enterococcal infections. Resistance to DAP in enterococci is regulated by the liaFSR three-component regulatory system that consists of a histidine kinase sensor (LiaS), a response regulator (LiaR) and a transmembrane protein of unknown function (LiaF). Previous studies indicate that deletion of isoleucine in position 177 of LiaF results in DAP tolerance and is sufficient to change membrane architecture. Here, we dissect the role of LiaF in DAP resistance Methods We generated three liaF mutants in OG1RF, a DAP-susceptible laboratory strain of Efs (DAP MIC = 2 µg/mL): (i) a non-polar, C-terminal truncation of liaF (OG1RFliaF∆152), (ii) a null liaF mutant with a premature stop-codon (OG1RFliaF*11), and (iii) an isoleucine deletion at position 177 (OG1RFliaF177). We determined DAP MIC by Etest and characterized the localization of anionic phospholipids microdomains using 10-nonyl-acridine-orange (NAO). The expression of the liaXYZ (the main target of LiaR) and liaFSR clusters were evaluated by qRT-PCR and relative expression ratios (Log2 fold change) were calculated by normalizing to gyrB expression. We assessed activation of LiaFSR by evaluating surface exposure of LiaX by ELISA. We used the bacterial adenylate cyclase two-hybrid system (BACTH) to evaluate the protein-protein interaction between LiaF and LiaS. Results Full deletion of liaF or the C-terminal truncation of LiaF did not have any effect on DAP MICs, membrane architecture or a significant increase in LiaX surface exposure compared with parental strain OG1RF. In contrast, deletion of the codon encoding isoleucine in position 177 of LiaF caused a major increase (8-fold) in LiaX exposure and redistribution of anionic phospholipid microdomains away from the septum without changes in the actual DAP MIC. Transcriptional analyses indicated upregulation (>2 log2-fold) in the liaXYZ gene cluster indicating activation of the stress response. We also observed a positive interaction between LiaF and LiaS. Conclusion LiaF is likely a key activator of the LiaFSR stress response and the critical regulatory domain appears to be located in a stretch of four isoleucines toward the C-terminal of the protein. Disclosures All authors: No reported disclosures.

scholarly journals The Cpx Envelope Stress Response Affects Expression of the Type IV Bundle-Forming Pili of Enteropathogenic Escherichiacoli

2005 ◽  
Vol 187 (2) ◽  
pp. 672-686 ◽  
Author(s):  
Anna Z. Nevesinjac ◽  
Tracy L. Raivio
Keyword(s):  
Stress Response ◽  
Protein Misfolding ◽  
Response Regulator ◽  
Regulatory System ◽  
Laboratory Strain ◽  
General Role ◽  
E Coli ◽  
Type Iv ◽  
Envelope Stress ◽  
Assembly Pathways

ABSTRACT The Cpx envelope stress response mediates adaptation to potentially lethal envelope stresses in Escherichia coli. The two-component regulatory system consisting of the sensor kinase CpxA and the response regulator CpxR senses and mediates adaptation to envelope insults believed to result in protein misfolding in this compartment. Recently, a role was demonstrated for the Cpx response in the biogenesis of P pili, attachment organelles expressed by uropathogenic E. coli. CpxA senses misfolded P pilus assembly intermediates and initiates increased expression of both assembly and regulatory factors required for P pilus elaboration. In this report, we demonstrate that the Cpx response is also involved in the expression of the type IV bundle-forming pili of enteropathogenic E. coli (EPEC). Bundle-forming pili were not elaborated from an exogenous promoter in E. coli laboratory strain MC4100 unless the Cpx pathway was constitutively activated. Further, an EPEC cpxR mutant synthesized diminished levels of bundle-forming pili and was significantly affected in adherence to epithelial cells. Since type IV bundle-forming pili are very different from chaperone-usher-type P pili in both form and biogenesis, our results suggest that the Cpx envelope stress response plays a general role in the expression of envelope-localized organelles with diverse structures and assembly pathways.

scholarly journals Progress Overview of Bacterial Two-Component Regulatory Systems as Potential Targets for Antimicrobial Chemotherapy

Antibiotics ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 635
Author(s):  
Hidetada Hirakawa ◽  
Jun Kurushima ◽  
Yusuke Hashimoto ◽  
Haruyoshi Tomita
Keyword(s):  
Drug Resistance ◽  
Antimicrobial Chemotherapy ◽  
Response Regulator ◽  
Regulatory System ◽  
Laboratory Strain ◽  
Regulatory Systems ◽  
Two Component ◽  
Bacterial Genes ◽  
Conventional Antibiotics ◽  
External Stimuli

Bacteria adapt to changes in their environment using a mechanism known as the two-component regulatory system (TCS) (also called “two-component signal transduction system” or “two-component system”). It comprises a pair of at least two proteins, namely the sensor kinase and the response regulator. The former senses external stimuli while the latter alters the expression profile of bacterial genes for survival and adaptation. Although the first TCS was discovered and characterized in a non-pathogenic laboratory strain of Escherichia coli, it has been recognized that all bacteria, including pathogens, use this mechanism. Some TCSs are essential for cell growth and fitness, while others are associated with the induction of virulence and drug resistance/tolerance. Therefore, the TCS is proposed as a potential target for antimicrobial chemotherapy. This concept is based on the inhibition of bacterial growth with the substances acting like conventional antibiotics in some cases. Alternatively, TCS targeting may reduce the burden of bacterial virulence and drug resistance/tolerance, without causing cell death. Therefore, this approach may aid in the development of antimicrobial therapeutic strategies for refractory infections caused by multi-drug resistant (MDR) pathogens. Herein, we review the progress of TCS inhibitors based on natural and synthetic compounds.

scholarly journals Disruption of a Cadherin Gene Associated with Resistance to Cry1Ac δ-Endotoxin of Bacillus thuringiensis in Helicoverpa armigera

2005 ◽  
Vol 71 (2) ◽  
pp. 948-954 ◽  
Author(s):  
Xinjun Xu ◽  
Liangying Yu ◽  
Yidong Wu
Keyword(s):  
Bacillus Thuringiensis ◽  
Helicoverpa Armigera ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Recessive Gene ◽  
Laboratory Strain ◽  
Cross Resistance ◽  
Resistance Pattern ◽  
Bt Cotton ◽  
Helicoverpa Armígera

ABSTRACT A laboratory strain (GY) of Helicoverpa armigera (Hübner) was established from surviving larvae collected from transgenic cotton expressing a Bacillus thuringiensis var. kurstaki insecticidal protein (Bt cotton) in Gaoyang County, Hebei Province, People's Republic of China, in 2001. The GYBT strain was derived from the GY strain through 28 generations of selection with activated Cry1Ac delivered by diet surface contamination. When resistance to Cry1Ac in the GYBT strain increased to 564-fold after selection, we detected high levels of cross-resistance to Cry1Aa (103-fold) and Cry1Ab (>46-fold) in the GYBT strain with reference to those in the GY strain. The GYBT strain had a low level of cross-resistance to B. thuringiensis var. kurstaki formulation (Btk) (5-fold) and no cross-resistance to Cry2Aa (1.4-fold). Genetic analysis showed that Cry1Ac resistance in the GYBT strain was controlled by one autosomal and incompletely recessive gene. The cross-resistance pattern and inheritance mode suggest that the Cry1Ac resistance in the GYBT strain of H. armigera belongs to “mode 1,” the most common type of lepidopteran resistance to B. thuringiensis toxins. A cadherin gene was cloned and sequenced from both the GY and GYBT strains. Disruption of the cadherin gene by a premature stop codon was associated with a high level of Cry1Ac resistance in H. armigera. Tight linkage between Cry1Ac resistance and the cadherin locus was observed in a backcross analysis. Together with previous evidence found with Heliothis virescens and Pectinophora gossypiella, our results confirmed that the cadherin gene is a preferred target for developing DNA-based monitoring of B. thuringiensis resistance in field populations of lepidopteran pests.

scholarly journals The Cpx Envelope Stress Response Is Controlled by Amplification and Feedback Inhibition

1999 ◽  
Vol 181 (17) ◽  
pp. 5263-5272 ◽  
Author(s):  
Tracy L. Raivio ◽  
Daniel L. Popkin ◽  
Thomas J. Silhavy
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Virulence Factors ◽  
Histidine Kinase ◽  
Feedback Inhibition ◽  
Response Regulator ◽  
Regulatory System ◽  
Concomitant Increase ◽  
Envelope Stress ◽  
Two Component

ABSTRACT In Escherichia coli, the Cpx two-component regulatory system activates expression of protein folding and degrading factors in response to misfolded proteins in the bacterial envelope (inner membrane, periplasm, and outer membrane). It is comprised of the histidine kinase CpxA and the response regulator CpxR. This response plays a role in protection from stresses, such as elevated pH, as well as in the biogenesis of virulence factors. Here, we show that the Cpx periplasmic stress response is subject to amplification and repression through positive and negative autofeedback mechanisms. Western blot and operon fusion analyses demonstrated that the cpxRA operon is autoactivated. Conditions that lead to elevated levels of phosphorylated CpxR cause a concomitant increase in transcription ofcpxRA. Conversely, overproduction of CpxP, a small, Cpx-regulated protein of previously unknown function, represses the regulon and can block activation of the pathway. This repression is dependent on an intact CpxA sensing domain. The ability to autoactivate and then subsequently repress allows for a temporary amplification of the Cpx response that may be important in rescuing cells from transitory stresses and cueing the appropriately timed elaboration of virulence factors.

scholarly journals Antimicrobial sensing coupled with cell membrane remodeling mediates antibiotic resistance and virulence inEnterococcus faecalis

2019 ◽  
Vol 116 (52) ◽  
pp. 26925-26932 ◽  
Author(s):  
Ayesha Khan ◽  
Milya Davlieva ◽  
Diana Panesso ◽  
Sandra Rincon ◽  
William R. Miller ◽  
...  
Keyword(s):  
Stress Response ◽  
Response Regulator ◽  
Cell Envelope ◽  
Multidrug Resistant ◽  
Innate Immune ◽  
Evolutionary Strategies ◽  
Phospholipid Content ◽  
Anionic Phospholipid ◽  
Extracellular Milieu ◽  
Terminal Domain

Bacteria have developed several evolutionary strategies to protect their cell membranes (CMs) from the attack of antibiotics and antimicrobial peptides (AMPs) produced by the innate immune system, including remodeling of phospholipid content and localization. Multidrug-resistantEnterococcus faecalis,an opportunistic human pathogen, evolves resistance to the lipopeptide daptomycin and AMPs by diverting the antibiotic away from critical septal targets using CM anionic phospholipid redistribution. The LiaFSR stress response system regulates this CM remodeling via the LiaR response regulator by a previously unknown mechanism. Here, we characterize a LiaR-regulated protein, LiaX, that senses daptomycin or AMPs and triggers protective CM remodeling. LiaX is surface exposed, and in daptomycin-resistant clinical strains, both LiaX and the N-terminal domain alone are released into the extracellular milieu. The N-terminal domain of LiaX binds daptomycin and AMPs (such as human LL-37) and functions as an extracellular sentinel that activates the cell envelope stress response. The C-terminal domain of LiaX plays a role in inhibiting the LiaFSR system, and when this domain is absent, it leads to activation of anionic phospholipid redistribution. Strains that exhibit LiaX-mediated CM remodeling and AMP resistance show enhanced virulence in theCaenorhabditis elegansmodel, an effect that is abolished in animals lacking an innate immune pathway crucial for producing AMPs. In conclusion, we report a mechanism of antibiotic and AMP resistance that couples bacterial stress sensing to major changes in CM architecture, ultimately also affecting host–pathogen interactions.

scholarly journals Identification and Application of BhAPRR2 Controlling Peel Colour in Wax Gourd (Benincasa hispida)

2021 ◽  
Vol 12 ◽  
Author(s):  
Lianlian Ma ◽  
Zhengguo Liu ◽  
Zhikui Cheng ◽  
Jiquan Gou ◽  
Jieying Chen ◽  
...  
Keyword(s):  
Stop Codon ◽  
Response Regulator ◽  
Bulked Segregant Analysis ◽  
Premature Stop Codon ◽  
Fruit Peel ◽  
Functional Region ◽  
White Skin ◽  
Peel Color ◽  
Benincasa Hispida ◽  
Wax Gourd

Peel color is an important factor affecting commodity quality in vegetables; however, the genes controlling this trait remain unclear in wax gourd. Here, we used two F2 genetic segregation populations to explore the inheritance patterns and to clone the genes associated with green and white skin in wax gourd. The F2 and BC1 trait segregation ratios were 3:1 and 1:1, respectively, and the trait was controlled by nuclear genes. Bulked segregant analysis of both F2 plants revealed peaks on Chr5 exceeding the confidence interval. Additionally, 6,244 F2 plants were used to compress the candidate interval into a region of 179 Kb; one candidate gene, Bch05G003950 (BhAPRR2), encoding two-component response regulator-like protein Arabidopsis pseudo-response regulator2 (APRR2), which is involved in the regulation of peel color, was present in this interval. Two bases (GA) present in the coding sequence of BhAPRR2 in green-skinned wax gourd were absent from white-skinned wax gourd. The latter contained a frameshift mutation, a premature stop codon, and lacked 335 residues required for the protein functional region. The chlorophyll content and BhAPRR2 expression were significantly higher in green-skinned than in white-skinned wax gourd. Thus, BhAPRR2 may regulate the peel color of wax gourd. This study provides a theoretical foundation for further studies of the mechanism of gene regulation for the fruit peel color of wax gourd.

scholarly journals Characterization of the CpxRA Regulon in Haemophilus ducreyi

2010 ◽  
Vol 78 (11) ◽  
pp. 4779-4791 ◽  
Author(s):  
Maria Labandeira-Rey ◽  
Chad A. Brautigam ◽  
Eric J. Hansen
Keyword(s):  
Stress Response ◽  
Response Regulator ◽  
Cell Envelope ◽  
Bacterial Species ◽  
Regulatory System ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Envelope Stress ◽  
Two Component ◽  
Genes Encoding

ABSTRACT The H aemophilus ducreyi 35000HP genome encodes a homolog of the CpxRA two-component cell envelope stress response system originally characterized in E scherichia coli. CpxR, the cytoplasmic response regulator, was shown previously to be involved in repression of the expression of the lspB-lspA2 operon (M. Labandeira-Rey, J. R. Mock, and E. J. Hansen, Infect. Immun. 77:3402-3411, 2009). In the present study, the H. ducreyi CpxR and CpxA proteins were shown to closely resemble those of other well-studied bacterial species. A cpxA deletion mutant and a CpxR-overexpressing strain were used to explore the extent of the CpxRA regulon. DNA microarray and real-time reverse transcriptase (RT) PCR analyses indicated several potential regulatory targets for the H. ducreyi CpxRA two-component regulatory system. Electrophoretic mobility shift assays (EMSAs) were used to prove that H. ducreyi CpxR interacted with the promoter regions of genes encoding both known and putative virulence factors of H. ducreyi, including the lspB-lspA2 operon, the flp operon, and dsrA. Interestingly, the use of EMSAs also indicated that H. ducreyi CpxR did not bind to the promoter regions of several genes predicted to encode factors involved in the cell envelope stress response. Taken together, these data suggest that the CpxRA system in H. ducreyi, in contrast to that in E. coli, may be involved primarily in controlling expression of genes not involved in the cell envelope stress response.

scholarly journals 602. Mechanism of LiaY-Mediated Daptomycin Resistance in Enterococcus faecalis

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S282-S282
Author(s):  
April Nguyen ◽  
Truc T Tran ◽  
Diana Panesso ◽  
Ayesha Khan ◽  
Eugenia Mileykovskaya ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Transmembrane Protein ◽  
Logarithmic Phase ◽  
Positive Interaction ◽  
Anionic Phospholipid ◽  
Biochemical Basis ◽  
Double Deletion ◽  
Genes Encoding ◽  
Lipopeptide Antibiotic ◽  
Two Hybrid

Abstract Background Daptomycin (DAP) is a lipopeptide antibiotic that targets the cell membrane (CM) at the division septum. DAP resistance (DAP-R) in E. faecalis (Efs) has been linked to mutations in genes encoding the LiaFSR stress response system and lipid biosynthetic enzymes, including cardiolipin synthase (Cls). The signature phenotype of DAP-R is redistribution of CM anionic phospholipid (APL) microdomains. Using a genetic approach, we have identified a transmembrane protein (LiaY) as a major mediator of cell membrane APL redistribution associated with DAP-R. Here, we explore the mechanism of LiaY-mediated changes in the CM under the hypothesis that CM remodeling occurs through interactions with Cls. Methods Efs encodes two cls genes (cls1 and cls2). Deletion mutants of both cls genes were generated using the Crispr/cas9 system in the daptomycin-sensitive strain Efs OG117 and Efs OG117∆liaX (a DAP-R derivative of OG117). DAP minimum inhibitory concentration (MIC) was determined using E-test on Mueller–Hinton II agar. Visualization of APL microdomains was performed by staining mid-logarithmic phase cells with 1 µM of 10-N-nonyl-acridine orange (NAO) and fluorescence microscopy. Bacterial two-hybrid system was used to study interactions between LiaY with Cls1 or Cls2. Results Single or double deletion of cls1 or cls2 in Efs OG117 did not affect DAP MIC, and no changes in CM architecture were seen by NAO staining. In contrast,deletion of cls1 (alone or in conjunction with a deletion of cls2) in a DAP-R derivative of OG117 OG117∆liaX, resulted in a marked decrease in DAP MIC, and NAO staining of Efs OG117∆liaX∆cls1∆cls2 shows a restoration of septal APL microdomain localization.In the same DAP-R background, deletion of cls2 alone did not have any effect on DAP MIC or APL microdomain distribution. Additionally, bacterial two-hybrid assays showed a positive interaction of LiaY with Cls1 but not with Cls2. Conclusion We have identified the biochemical basis for DAP-R associated CM remodeling. In a proposed model, the LiaR-mediated activation of the LiaY triggers specific interactions with Cls1 displacing the protein away from the septum, resulting in local generation of APL microdomains that prevents DAP-mediated damage to the CM. Disclosures All authors: No reported disclosures.

scholarly journals Highly multiplexed AmpliSeq technology identifies novel variation of flowering time-related genes in soybean (Glycine max)

DNA Research ◽  
2019 ◽  
Vol 26 (3) ◽  
pp. 243-260 ◽  
Author(s):  
Eri Ogiso-Tanaka ◽  
Takehiko Shimizu ◽  
Makita Hajika ◽  
Akito Kaga ◽  
Masao Ishimoto
Keyword(s):  
Flowering Time ◽  
Core Collection ◽  
Target Genes ◽  
Sequence Data ◽  
Stop Codon ◽  
Response Regulator ◽  
Premature Stop Codon ◽  
Mini Core Collection ◽  
Novel Variants ◽  
Two Component

Abstract Whole-genome re-sequencing is a powerful approach to detect gene variants, but it is expensive to analyse only the target genes. To circumvent this problem, we attempted to detect novel variants of flowering time-related genes and their homologues in soybean mini-core collection by target re-sequencing using AmpliSeq technology. The average depth of 382 amplicons targeting 29 genes was 1,237 with 99.85% of the sequence data mapped to the reference genome. Totally, 461 variants were detected, of which 150 sites were novel and not registered in dbSNP. Known and novel variants were detected in the classical maturity loci—E1, E2, E3, and E4. Additionally, large indel alleles, E1-nl and E3-tr, were successfully identified. Novel loss-of-function and missense variants were found in FT2a, MADS-box, WDR61, phytochromes, and two-component response regulators. The multiple regression analysis showed that four genes—E2, E3, Dt1, and two-component response regulator—can explain 51.1–52.3% of the variation in flowering time of the mini-core collection. Among them, the two-component response regulator with a premature stop codon is a novel gene that has not been reported as a soybean flowering time-related gene. These data suggest that the AmpliSeq technology is a powerful tool to identify novel alleles.

Acinetobacter baumannii regulates its stress responses via the BfmRS two-component regulatory system

2021 ◽  
Author(s):  
Samantha Palethorpe ◽  
John M. Farrow ◽  
Greg Wells ◽  
Morgan E. Milton ◽  
Luis A. Actis ◽  
...  
Keyword(s):  
Stress Response ◽  
Acinetobacter Baumannii ◽  
Stress Responses ◽  
Capsular Polysaccharide ◽  
Response Regulator ◽  
Regulatory System ◽  
Hospital Environment ◽  
Nosocomial Pathogen ◽  
Polysaccharide Biosynthesis ◽  
Two Component

Acinetobacter baumannii is a common nosocomial pathogen that utilizes numerous mechanisms to aid its survival in both the environment and in the host. Coordination of such mechanisms requires an intricate regulatory network. We report here that A. baumannii can directly regulate several stress-related pathways via the two-component regulatory system, BfmRS. Similar to previous studies, results from transcriptomic analysis showed that mutation of the BfmR response regulator causes dysregulation of genes required for the oxidative stress response, the osmotic stress response, the misfolded protein/heat shock response, Csu pili/fimbriae production, and capsular polysaccharide biosynthesis. We also found that the BfmRS system is involved in controlling siderophore biosynthesis and transport, and type IV pili production. We provide evidence that BfmR binds to various stress-related promoter regions and show that BfmR alone can directly activate transcription of some stress-related genes. Additionally, we show that the BfmS sensor kinase acts as a BfmR phosphatase to negatively regulate BfmR activity. This work highlights the importance of the BfmRS system in promoting survival of A. baumannii . Importance Acinetobacter baumannii is a nosocomial pathogen that has extremely high rates of multidrug resistance. This organism’s ability to endure stressful conditions is a key part of its ability to spread in the hospital environment and cause infections. Unlike other members of the γ-proteobacteria, A. baumannii does not encode a homolog of the RpoS sigma factor to coordinate its stress response. Here, we demonstrate that the BfmRS two-component system directly controls the expression of multiple stress resistance genes. Our findings suggest that BfmRS is central to a unique scheme of general stress response regulation by A. baumannii .

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