scholarly journals In vivocross-linking and transmembrane helix dynamics support a bidirectional non-piston model of signaling withinE. coliEnvZ

2017 ◽  
Author(s):  
Rahmi Yusuf ◽  
Tuyết Linh Nguyễn ◽  
Annika Heininger ◽  
Robert J. Lawrence ◽  
Benjamin A. Hall ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Histidine Kinase ◽  
In Silico ◽  
Transmembrane Domain ◽  
Coarse Grained ◽  
Sensor Histidine Kinase ◽  
Rotational Components ◽  
Stimulus Perception ◽  
Standing Question

AbstractIn Gram-negative bacteria, porins span the outer membrane and control the influx of several prominent groups of antibiotics. Thus, it should not be surprising that expression of these porins is often altered in clinical isolates exhibiting multidrug resistance (MDR). The major regulator of porin expression inEscherichia coliis EnvZ, a canonical sensor histidine kinase (SHK). It allosterically processes periplasmic interactions with MzrA and cytoplasmic osmosensing into a single unified change in the ratio of its kinase and phosphatase activities. Unfortunately, the role of the EnvZ transmembrane domain (TMD) in bidirectional communication of these signals remains not well understood. Here, we employedin vivosulfhydryl-reactivity to probe the dynamics of the TM2 helices and demonstrate that upon stimulus perception, only the region proximal to the periplasm undergoes conformational rearrangement. Furthermore,in silicocoarse-grained molecular dynamics (CG-MD) simulations with aromatically tuned variants of EnvZ TM2 demonstrate the existence of both tilting and azimuthal rotational components to transmembrane communication while ruling out piston-type repositioning of TM2. Finally, in contrast to a similar analysis of TM1, we identified position-specific mutants possessing a “flipped” phenotype by dual-color fluorescent reporter analysis suggesting that both the periplasmic and cytoplasmic ends of TM2 are critical for maintenance of EnvZ signal output. Taken together, these data strongly support that EnvZ employs a non-piston-type mechanism during transmembrane communication. We conclude by discussing these results within the context of allosteric processing by EnvZ and propose that these results can be used to predict and classify transmembrane communication by various SHKs.ImportanceThe EnvZ sensor histidine kinase serves as the major regulator of porin expression withinEscherichia coli. A long-standing question is how stimulus perception by a bacterial receptor on one side of a biological membrane is transmitted to the opposite side of the membrane. To address this question, we monitored the dynamics of the transmembrane domain of EnvZin vivoand coupled these results within silicosimulations of membrane-embedded EnvZ transmembrane domains. Taken together, these results demonstrate that detection of osmotic stress by the cytoplasmic domain of EnvZ results in non-piston communication across the inner membrane ofE. coli.Thus, in addition to understanding how EnvZ regulates porin balance and antibiotic influx, these results contribute to answering the long-standing question of how transmembrane communication is performed by bacterial receptors. Our work concludes with a framework that correlates receptor domain composition and signal transduction mechanisms that could be employed by other research groups on their particular receptors of interest.

scholarly journals C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 200010
Author(s):  
Navaneethan Palanisamy ◽  
Mehmet Ali Öztürk ◽  
Emir Bora Akmeriç ◽  
Barbara Di Ventura
Keyword(s):  
Escherichia Coli ◽  
In Silico ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Time Lapse ◽  
Enhanced Yellow Fluorescent Protein ◽  
Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

Identification of Escherichia coli enterotoxin inhibitors from traditional medicinal herbs by in silico, in vitro, and in vivo analyses

2009 ◽  
Vol 121 (3) ◽  
pp. 372-378 ◽  
Author(s):  
Jaw-Chyun Chen ◽  
Tin-Yun Ho ◽  
Yuan-Shiun Chang ◽  
Shih-Lu Wu ◽  
Chia-Cheng Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
In Silico ◽  
Medicinal Herbs

scholarly journals In silico analysis and in vivo assessment of a novel epitope-based vaccine candidate against uropathogenic Escherichia coli

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sara Hasanzadeh ◽  
Mehri Habibi ◽  
Mohammad Ali Shokrgozar ◽  
Reza Ahangari Cohan ◽  
Khadijeh Ahmadi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
In Silico ◽  
Vaccine Candidate ◽  
In Silico Analysis ◽  
T Cell Epitopes ◽  
Toll Like Receptor 4 ◽  
Virulence Proteins ◽  
Tract Infections

Abstract Uropathogenic Escherichia coli (UPEC) are common pathogens in urinary tract infections (UTIs), which show resistance to antibiotics. Therefore, there is a need for a vaccine to reduce susceptibility to the infection. In the present study, bioinformatics approaches were employed to predict the best B and T-cell epitopes of UPEC virulence proteins to develop a multiepitope vaccine candidate against UPEC. Then, the efficacy of the candidate was studied with and without Freund adjuvant. Using bioinformatics methods, 3 epitope-rich domains of IutA and FimH antigens were selected to construct the fusion. Molecular docking and Molecular dynamics (MD) simulation were employed to investigate in silico interaction between designed vaccine and Toll-like receptor 4 (TLR4). Our results showed that the levels of IgG and IgA antibodies were improved in the serum and mucosal samples of the vaccinated mice, and the IgG responses were maintained for at least 6 months. The fusion protein was also able to enhance the level of cytokines IFN.γ (Th1), IL.4 (Th2), and IL.17. In challenge experiments, all vaccine combinations showed high potency in the protection of the urinary tract even after 6 months post first injection. The present study indicates that the designed candidate is able to evoke strong protective responses which warrant further studies.

scholarly journals Effects of Escherichia coli Physiology on Growth of Phage T7 In Vivo and In Silico

2002 ◽  
Vol 184 (7) ◽  
pp. 1888-1894 ◽  
Author(s):  
Lingchong You ◽  
Patrick F. Suthers ◽  
John Yin
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
In Silico ◽  
Physiological State ◽  
Rise Rate ◽  
Host Growth ◽  
Phage T7 ◽  
Phage Growth ◽  
Pool Sizes

ABSTRACT Phage development depends not only upon phage functions but also on the physiological state of the host, characterized by levels and activities of host cellular functions. We established Escherichia coli at different physiological states by continuous culture under different dilution rates and then measured its production of phage T7 during a single cycle of infection. We found that the intracellular eclipse time decreased and the rise rate increased as the growth rate of the host increased. To develop mechanistic insight, we extended a computer simulation for the growth of phage T7 to account for the physiology of its host. Literature data were used to establish mathematical correlations between host resources and the host growth rate; host resources included the amount of genomic DNA, pool sizes and elongation rates of RNA polymerases and ribosomes, pool sizes of amino acids and nucleoside triphosphates, and the cell volume. The in silico (simulated) dependence of the phage intracellular rise rate on the host growth rate gave quantitatively good agreement with our in vivo results, increasing fivefold for a 2.4-fold increase in host doublings per hour, and the simulated dependence of eclipse time on growth rate agreed qualitatively, deviating by a fixed delay. When the simulation was used to numerically uncouple host resources from the host growth rate, phage growth was found to be most sensitive to the host translation machinery, specifically, the level and elongation rate of the ribosomes. Finally, the simulation was used to follow how bottlenecks to phage growth shift in response to variations in host or phage functions.

scholarly journals The Sensor Histidine Kinase RgfC Affects Group B Streptococcal Virulence Factor Expression Independent of Its Response Regulator RgfA

2015 ◽  
Vol 83 (3) ◽  
pp. 1078-1088 ◽  
Author(s):  
Claire Gendrin ◽  
Annalisa Lembo ◽  
Christopher Whidbey ◽  
Kellie Burnside ◽  
Jessica Berry ◽  
...  
Keyword(s):  
Histidine Kinase ◽  
Response Regulator ◽  
Group B Streptococci ◽  
Altered Expression ◽  
Content Type ◽  
Sensor Histidine Kinase ◽  
Opportunistic Bacteria ◽  
Extracellular Matrix Components ◽  
Group B

Group B streptococci (GBS;Streptococcus agalactiae) are beta-hemolytic, Gram-positive bacteria that are common asymptomatic colonizers of healthy adults. However, these opportunistic bacteria also cause invasive infections in human newborns and in certain adult populations. To adapt to the various environments encountered during its disease cycle, GBS encodes a number of two-component signaling systems. Previous studies have indicated that the TCS comprising the sensor histidine kinase RgfC and the response regulator RgfA mediate GBS binding to extracellular matrix components, such as fibrinogen. However, in certain GBS clinical isolates, a point mutation inrgfAresults in premature truncation of the response regulator. The truncated RgfA protein lacks the C-terminal DNA binding domain necessary for promoter binding and gene regulation. Here, we show that deletion ofrgfCin GBS strains lacking a functional RgfA increased systemic infection. Furthermore, infection with thergfCmutant increased induction of proinflammatory signaling pathwaysin vivo. Phosphoproteomic analysis revealed that 19 phosphopeptides corresponding to 12 proteins were differentially phosphorylated at aspartate, cysteine, serine, threonine, or tyrosine residues in thergfCmutant. This included aspartate phosphorylation of a tyrosine kinase, CpsD, and a transcriptional regulator. Consistent with this observation, microarray analysis of thergfCmutant indicated that >200 genes showed altered expression compared to the isogenic wild-type strain and included transcriptional regulators, transporters, and genes previously associated with GBS pathogenesis. Our observations suggest that in the absence of RgfA, nonspecific RgfC signaling affects the expression of virulence factors and GBS pathogenesis.

scholarly journals Altering the bilayer motif in ERBB2 HER2 TMD and in ErbB HER TMD dimer causing in vitro and in vivo tumor suppression

2020 ◽  
Author(s):  
Laszlo David Menyhert ◽  
Miguel Tejeda
Keyword(s):  
Tumor Suppression ◽  
In Silico ◽  
Transmembrane Domain ◽  
Strong Relationship ◽  
Ligand Docking ◽  
Tyrosine Kinase Receptor ◽  
Tumor Models ◽  
Mutant Complex

AbstractHuman ERBB2 is a transmembrane signaling tyrosine kinase receptor, which seems an ideal target of human WNT16B, the secreted growth factor possibly causes transmembrane domain (TMD) mutations. There is a strong relationship between the chemical nature of the TMD mutations and the potency with which they activate HER2. In silico, we modeled the possible docking conformation of human WNT16B and human ERBB2 TMD homodimer, resulted a mutant complex. The ribbon structure, the C-terminal and N-terminal and GG4-like motif structures are similar in HER2 TMD and HER TMD, we modeled WNTl6B’s possible docking conformation to the HER1 TMD (ErbB), also resulted a mutant complex. If there is a strong relationship between TMD mutations improving the active dimer interface or stabilizing an activated conformation and the potency with which they activate HER2 (and possibly also HER), than the TMD dimerization part seems ideal reagent-target. The agent we tested – the 4-(Furan-2-yl)hepta-1,6-dien-4-ol (AKOS004122375) – has very good connectivity attributes by its several rotatable bonds, and according to the in silico inspection of close residues intermolecular bonds, and the ligand docking, it can straight connect to human ERBB2 TMD (HER2), and to the ErbB TMD (HER1) dimer bilayer motif as well. In silico, we also tested the agent ligand’s docking into the residues of human WNT16B and human ERBB2 TMD (HER2) mutant complex, and human WNT16B and human ErbB TMD (HER1) mutant complex. We tested the agent ligand in vitro and in vivo in several tumor models, highlighting that targeting the EGFR’s TMD with an agent not only reduces treatment-induced metastasis, but radically decreases the tumor growth as well. Because of the analogous structure of HER2 TMD and HER TMD, this dimerization motif-targeting can also be successful in HER and HER2 EGFR signaling. In vitro, we reached 80-94% proliferation percentage in different tumor models, in vivo we reached 35-61% tumor suppression in different tumor models, the metastasis inhibition effect of the compound was 82-87% in different tumor models.

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