scholarly journals TmSpz-like Plays a Fundamental Role in Response to E. coli but Not S. aureus or C. albican Infection in Tenebrio molitor via Regulation of Antimicrobial Peptide Production

2021 ◽  
Vol 22 (19) ◽  
pp. 10888
Author(s):  
Ho Am Jang ◽  
Bharat Bhusan Patnaik ◽  
Maryam Ali Mohammadie Kojour ◽  
Bo Bae Kim ◽  
Young Min Bae ◽  
...  
Keyword(s):  
Amino Acid ◽  
Antimicrobial Peptide ◽  
Intracellular Signaling ◽  
Critical Role ◽  
Malpighian Tubules ◽  
Whole Body ◽  
Amino Acid Sequence Similarity ◽  
E Coli ◽  
Cystine Knot ◽  
Encoding Genes

The cystine knot protein Spätzle is a Toll receptor ligand that modulates the intracellular signaling cascade involved in the nuclear factor kappa B (NF-κB)-mediated regulation of antimicrobial peptide (AMP)-encoding genes. Spätzle-mediated activation of the Toll pathway is critical for the innate immune responses of insects against Gram-positive bacteria and fungi. In this study, the open reading frame (ORF) sequence of Spätzle-like from T. molitor (TmSpz-like) identified from the RNA sequencing dataset was cloned and sequenced. The 885-bp TmSpz-like ORF encoded a polypeptide of 294 amino acid residues. TmSpz-like comprised a cystine knot domain with six conserved cysteine residues that formed three disulfide bonds. Additionally, TmSpz-like exhibited the highest amino acid sequence similarity with T. castaneum Spätzle (TcSpz). In the phylogenetic tree, TmSpz-like and TcSpz were located within a single cluster. The expression of TmSpz-like was upregulated in the Malpighian tubules and gut tissues of T. molitor. Additionally, the expression of TmSpz-like in the whole body and gut of the larvae was upregulated at 24 h post-E. coli infection. The results of RNA interference experiments revealed that TmSpz-like is critical for the viability of E. coli-infected T. molitor larvae. Eleven AMP-encoding genes were downregulated in the E. coli-infected TmSpz-like knockdown larvae, which suggested that TmSpz-like positively regulated these genes. Additionally, the NF-κB-encoding genes (TmDorX1, TmDorX2, and TmRelish) were downregulated in the E. coli-infected TmSpz-like knockdown larvae. Thus, TmSpz-like plays a critical role in the regulation of AMP production in T. molitor in response to E. coli infection.

Overproduction and domain structure of the glutamyl-tRNA synthetase of Escherichia coli

1989 ◽  
Vol 67 (8) ◽  
pp. 404-410 ◽  
Author(s):  
Anne Brisson ◽  
Yves V. Brun ◽  
Alexander W. Bell ◽  
Paul H. Roy ◽  
Jacques Lapointe
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Domain Structure ◽  
Sequence Similarity ◽  
Trna Synthetase ◽  
Amino Acid Sequence Similarity ◽  
Structural Domains ◽  
E Coli ◽  
Domain Structures

The charging of glutamate on tRNAGlu is catalyzed by glutamyl-tRNA synthetase, a monomer of 53.8 kilodaltons in Escherichia coli. To obtain the large amounts of enzyme necessary for the identification of structural domains, we have inserted the structural gene gltX in the conditional runaway-replication plasmid pOU61, which led to a 350-fold overproduction of glutamyl-tRNA synthetase. Partial proteolysis of this enzyme revealed the existence of preferential sites of attack that, according to their N-terminal sequences, delimit regions of 12.9, 2.3, 12.1, and 26.5 kilodaltons from the N- to C-terminal of the enzyme. Their sizes suggest that the 2.3-kilodalton fragment is a hinge structure, and that those of 12.9, 12.1, and 26.5 kilodaltons are domain structures. The 12.9-kilodalton domain of the glutamyl-tRNA synthetase of E. coli is the only long region of this enzyme displaying a good amino acid sequence similarity with the glutaminyl-tRNA synthetase of Escherichia coli.Key words: glutamyl-tRNA synthetase, structural domains, overproduction, runaway-replication plasmid, Escherichia coli.

scholarly journals Hypothalamic extended synaptotagmin-3 contributes to the development of dietary obesity and related metabolic disorders

2020 ◽  
Vol 117 (33) ◽  
pp. 20149-20158 ◽  
Author(s):  
Yi Zhang ◽  
Yunliang Guan ◽  
Susu Pan ◽  
Lihong Yan ◽  
Ping Wang ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Metabolic Disorders ◽  
Critical Role ◽  
Whole Body ◽  
Activator Protein 1 ◽  
Melanocyte Stimulating Hormone ◽  
Diet Induced Obesity ◽  
Hypothalamic Nuclei ◽  
Enhanced Expression ◽  
Dietary Obesity

The C2domain containing protein extended synaptotagmin (E-Syt) plays important roles in both lipid homeostasis and the intracellular signaling; however, its role in physiology remains largely unknown. Here, we show that hypothalamic E-Syt3 plays a critical role in diet-induced obesity (DIO). E-Syt3 is characteristically expressed in the hypothalamic nuclei. Whole-body or proopiomelanocortin (POMC) neuron-specific ablation ofE-Syt3ameliorated DIO and related comorbidities, including glucose intolerance and dyslipidemia. Conversely, overexpression of E-Syt3 in the arcuate nucleus moderately promoted food intake and impaired energy expenditure, leading to increased weight gain. Mechanistically,E-Syt3ablation led to increased processing of POMC to α-melanocyte-stimulating hormone (α-MSH), increased activities of protein kinase C and activator protein-1, and enhanced expression of prohormone convertases. These findings reveal a previously unappreciated role for hypothalamic E-Syt3 in DIO and related metabolic disorders.

scholarly journals Novel Cefotaximase (CTX-M-16) with Increased Catalytic Efficiency Due to Substitution Asp-240→Gly

2001 ◽  
Vol 45 (8) ◽  
pp. 2269-2275 ◽  
Author(s):  
R. Bonnet ◽  
C. Dutour ◽  
J. L. M. Sampaio ◽  
C. Chanal ◽  
D. Sirot ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Catalytic Efficiency ◽  
Enterobacter Cloacae ◽  
E Coli ◽  
Genes Encoding ◽  
Encoding Gene ◽  
Synergy Test ◽  
Encoding Genes

ABSTRACT Three clinical strains (Escherichia coli Rio-6,E. coli Rio-7, and Enterobacter cloacae Rio-9) collected in 1996 and 1999 from hospitals in Rio de Janeiro (Brazil) were resistant to broad-spectrum cephalosporins and gave a positive double-disk synergy test. Two bla CTX-M genes encoding β-lactamases of pl 7.9 and 8.2 were implicated in this resistance: the bla CTX-M-9 gene observed inE. coli Rio-7 and E. cloacae Rio-9 and a novel CTX-M-encoding gene, designated bla CTX-M-16, observed in E. coli strain Rio-6. The deduced amino acid sequence of CTX-M-16 differed from CTX-M-9 only by the substitution Asp-240→Gly. The CTX-M-16-producing E. coli transformant exhibited the same level of resistance to cefotaxime (MIC, 16 μg/ml) but had a higher MIC of ceftazidime (MIC, 8 versus 1 μg/ml) than the CTX-M-9-producing transformant. Enzymatic studies revealed that CTX-M-16 had a 13-fold higher affinity for aztreonam and a 7.5-fold higher kcat for ceftazidime than CTX-M-9, thereby showing that the residue in position 240 can modulate the enzymatic properties of CTX-M enzymes. The two bla CTX-M-9 genes and the bla CTX-M-16 gene were located on different plasmids, suggesting the presence of mobile elements associated with CTX-M-encoding genes. CTX-M-2 and CTX-M-8 enzymes were found in Brazil in 1996, and two other CTX-M β-lactamases, CTX-M-9 and CTX-M-16, were subsequently observed. These reports are evidence of the diversity of CTX-M-type extended-spectrum β-lactamases in Brazil.

scholarly journals DsdX Is the Second d-Serine Transporter in Uropathogenic Escherichia coli Clinical Isolate CFT073

2006 ◽  
Vol 188 (18) ◽  
pp. 6622-6628 ◽  
Author(s):  
Andrew T. Anfora ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Carbon Source ◽  
Clinical Isolate ◽  
Sole Carbon Source ◽  
Minimal Medium ◽  
Amino Acid Sequence Similarity ◽  
Predicted Amino Acid Sequence ◽  
E Coli ◽  
K 12

ABSTRACTd-Serine is an amino acid present in mammalian urine that is inhibitory toEscherichia colistrains lacking a functionaldsdAgene. Counterintuitively, adsdAstrain ofE. coliclinical isolate CFT073 hypercolonizes the bladder and kidneys of mice relative to wild type during a coinfection in the murine model of urinary tract infection. We are interested in the mechanisms for uptake ofd-serine in CFT073.d-Serine entersE. coliK-12 via CycA, thed-alanine transporter andd-cycloserine sensitivity locus. CFT073cycAcan grow on minimal medium withd-serine as a sole carbon source. ThedsdXgene of thedsdCXAlocus is a likely candidate for an additionald-serine transporter based on its predicted amino acid sequence similarity to gluconate transporters. In minimal medium, CFT073dsdXcan grow ond-serine as a sole carbon source; however, CFT073dsdX cycAcannot. Additionally, CFT073dsdXA cycAis not sensitive to inhibitory concentrations ofd-serine during growth on glycerol andd-serine minimal medium.d-[14C]serine uptake experiments with CFT073dsdX cycAharboringdsdXorcycArecombinant plasmids confirm thatd-serine is able to enterE. colicells via CycA or DsdX. In whole-celld-[14C]serine uptake experiments, DsdX has an apparentKmof 58.75 μM and aVmaxof 75.96 nmol/min/mg, and CycA has an apparentKmof 82.40 μM and aVmaxof 58.90 nmol/min/mg. Onlyd-threonine marginally inhibits DsdX-mediatedd-serine transport, whereasd-alanine, glycine, andd-cycloserine inhibit CycA-mediatedd-serine transport. DsdX or CycA is sufficient to transport physiological quantities ofd-serine, but DsdX is ad-serine-specific permease.

scholarly journals Reconstruction and optimized expression of a synthetic secretory leukocyte protease inhibitor (SLPI) gene in Escherichia coli BL21

2020 ◽  
Author(s):  
Mudyawati Kamaruddin
Keyword(s):  
Escherichia Coli ◽  
Wound Healing ◽  
Amino Acid ◽  
Fusion Protein ◽  
Amino Acid Sequences ◽  
E Coli ◽  
Usage Frequency ◽  
Sds Page ◽  
Double Digestion ◽  
Encoding Genes

An active substance that has the greatest effect on wound healing is Secretory Leukocyte ProteaseInhibitor (SLPI). It is known that the SLPI encoding genes can be isolated and expressed onamnion membrane. Previous studies, we isolated and optimized the SLPI gene throughEscherichia coli BL21 (DE3) mediated pET101/DTOPO, which expressed active recombinanthuman SLPI (rhSLPI ) stored in pET-ESLPI. However, the expression of the rhSLPI products hasnot yet been accomplished. In this study, we optimized SLPI expression by developing a syntheticSLPI gene based on amino acid sequences with codons and expressed in E. coli BL21 to give themaximum expression. We used pUC57 and pET-32a plasmids to promote the cloning of syntheticSLPI genes. A codon-optimized SLPI gene was successfully synthesized with codon adaptationindex value showing the distribution of codon usage frequency along the length of the genesequence. In addition, the pET-SLPIopt fusion protein was successfully optimized with band sizesof 5900bp (pET-32a) and 413bp (SLPI) by double-digestion of NcoI and EcoI restriction enzymes.After the pET-SLPIopt was induced with various IPTG concentrations (50, 100 and 500 uM) at30°C, both soluble and insoluble fractions were analyzed as a result of SDS-PAGE which showedthat the fusion protein, expressed predominantly in the supernatant, was 29.18 kDa. Our reportedfindings the recombinant protein of SLPI through pET-32a plasmid could be expressed indissolved form.

scholarly journals The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing β-Lactamases of Potential Clinical Relevance

2015 ◽  
Vol 60 (1) ◽  
pp. 151-160 ◽  
Author(s):  
Dereje Dadi Gudeta ◽  
Valeria Bortolaia ◽  
Greg Amos ◽  
Elizabeth M. H. Wellington ◽  
Kristian K. Brandt ◽  
...  
Keyword(s):  
Amino Acid ◽  
Clinical Relevance ◽  
Pathogenic Bacteria ◽  
Amino Acid Identity ◽  
Soil Samples ◽  
Clinical Samples ◽  
Soil Microbiota ◽  
Content Type ◽  
E Coli ◽  
Encoding Genes

ABSTRACTThe origin of carbapenem-hydrolyzing metallo-β-lactamases (MBLs) acquired by clinical bacteria is largely unknown. We investigated the frequency, host range, diversity, and functionality of MBLs in the soil microbiota. Twenty-five soil samples of different types and geographical origins were analyzed by antimicrobial selective culture, followed by phenotypic testing and expression of MBL-encoding genes inEscherichia coli, and whole-genome sequencing of MBL-producing strains was performed. Carbapenemase activity was detected in 29 bacterial isolates from 13 soil samples, leading to identification of seven new MBLs in presumptivePedobacter roseus(PEDO-1),Pedobacter borealis(PEDO-2),Pedobacter kyungheensis(PEDO-3),Chryseobacterium piscium(CPS-1),Epilithonimonas tenax(ESP-1),Massilia oculi(MSI-1), andSphingomonassp. (SPG-1). Carbapenemase production was likely an intrinsic feature inChryseobacteriumandEpilithonimonas, as it occurred in reference strains of different species within these genera. The amino acid identity to MBLs described in clinical bacteria ranged between 40 and 69%. Remarkable features of the new MBLs included prophage integration of the encoding gene (PEDO-1), an unusual amino acid residue at a key position for MBL structure and catalysis (CPS-1), and overlap with a putative OXA β-lactamase (MSI-1). Heterologous expression of PEDO-1, CPS-1, and ESP-1inE. colisignificantly increased the MICs of ampicillin, ceftazidime, cefpodoxime, cefoxitin, and meropenem. Our study shows that MBL producers are widespread in soil and include four genera that were previously not known to produce MBLs. The MBLs produced by these bacteria are distantly related to MBLs identified in clinical samples but constitute resistance determinants of clinical relevance if acquired by pathogenic bacteria.

scholarly journals TmDorX2 positively regulates antimicrobial peptides in Tenebrio molitor gut, fat body, and hemocytes in response to bacterial and fungal infection

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Maryam Keshavarz ◽  
Yong Hun Jo ◽  
Ki Beom Park ◽  
Hye Jin Ko ◽  
Tariku Tesfaye Edosa ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Antimicrobial Peptides ◽  
Post Infection ◽  
Fat Body ◽  
Tenebrio Molitor ◽  
Malpighian Tubules ◽  
Whole Body ◽  
Mrna Levels ◽  
E Coli ◽  
Pathogen Invasion

AbstractDorsal, a member of the nuclear factor-kappa B (NF-κB) family of transcription factors, is a critical downstream component of the Toll pathway that regulates the expression of antimicrobial peptides (AMPs) against pathogen invasion. In this study, the full-length ORF of Dorsal was identified from the RNA-seq database of the mealworm beetle Tenebrio molitor (TmDorX2). The ORF of TmDorX2 was 1,482 bp in length, encoding a polypeptide of 493 amino acid residues. TmDorX2 contains a conserved Rel homology domain (RHD) and an immunoglobulin-like, plexins, and transcription factors (IPT) domain. TmDorX2 mRNA was detected in all developmental stages, with the highest levels observed in 3-day-old adults. TmDorX2 transcripts were highly expressed in the adult Malpighian tubules (MT) and the larval fat body and MT tissues. After challenging the larvae with Staphylococcus aureus and Escherichia coli, the TmDorX2 mRNA levels were upregulated 6 and 9 h post infection in the whole body, fat body, and hemocytes. Upon Candida albicans challenge, the TmDorX2 mRNA expression were found highest at 9 h post-infection in the fat body. In addition, TmDorX2-knockdown larvae exposed to E. coli, S. aureus, or C. albicans challenge showed a significantly increased mortality rate. Furthermore, the expression of 11 AMP genes was downregulated in the gut and fat body of dsTmDorX2-injected larvae upon E. coli challenge. After C. albicans and S. aureus challenge of dsTmDorX2-injected larvae, the expression of 11 and 10 AMPs was downregulated in the gut and fat body, respectively. Intriguingly, the expression of antifungal transcripts TmTenecin-3 and TmThaumatin-like protein-1 and -2 was greatly decreased in TmDorX2-silenced larvae in response to C. albicans challenge, suggesting that TmDorX2 regulates antifungal AMPs in the gut in response to C. albicans infection. The AMP expression profiles in the fat body, hemocytes, gut, and MTs suggest that TmDorX2 might have an important role in promoting the survival of T. molitor larvae against all mentioned pathogens.

scholarly journals Critical Roles of Spätzle5 in Antimicrobial Peptide Production Against Escherichia coli in Tenebrio molitor Malpighian Tubules

2021 ◽  
Vol 12 ◽  
Author(s):  
Maryam Ali Mohammadie Kojour ◽  
Tariku Tesfaye Edosa ◽  
Ho Am Jang ◽  
Maryam Keshavarz ◽  
Yong Hun Jo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Peptide ◽  
Bacterial Infections ◽  
Tenebrio Molitor ◽  
Larval Survival ◽  
Malpighian Tubules ◽  
Control Group ◽  
Mrna Levels ◽  
E Coli ◽  
Pathway Activation

The dimeric cytokine ligand Spätzle (Spz) is responsible for Toll pathway activation and antimicrobial peptide (AMP) production upon pathogen challenge in Tenebrio molitor. Here, we indicated that TmSpz5 has a functional role in response to bacterial infections. We showed that the highest expression of TmSpz5 is induced by Candida albicans. However, TmSpz5 knockdown reduced larval survival against Escherichia coli and Staphylococcus aureus. To evaluate the molecular mechanism underlying the observed survival differences, the role of TmSpz5 in AMP production was examined by RNA interference and microbial injection. T. molitor AMPs that are active against Gram-negative and -positive bacteria, including Tmtenecins, Tmattacins, Tmcoleoptericins, Tmtaumatin-like-proteins, and Tmcecropin-2, were significantly downregulated by TmSpz-5 RNAi in the Malpighian tubules (MTs) following a challenge with E. coli and S. aureus. However, upon infection with C. albicans the mRNA levels of most AMPs in the dsTmSpz5-injected group were similar to those in the control groups. Likewise, the expression of the transcription factors NF-κB, TmDorX2, and TmRelish were noticeably suppressed in the MTs of TmSpz5-silenced larvae. Moreover, E. coli-infected TmSpz5 knockdown larvae showed decreased antimicrobial activity in the MTs and hindgut compared with the control group. These results demonstrate that TmSpz5 has a defined role in T. molitor innate immunity by regulating AMP expression in MTs in response to E. coli.

scholarly journals Tailoring a Global Iron Regulon to a Uropathogen

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Rajdeep Banerjee ◽  
Erin Weisenhorn ◽  
Kevin J. Schwartz ◽  
Kevin S. Myers ◽  
Jeremy D. Glasner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Urinary Tract ◽  
Amino Acid ◽  
Regulatory Network ◽  
Iron Homeostasis ◽  
Iron Limitation ◽  
Content Type ◽  
E Coli ◽  
General Stress ◽  
K 12

ABSTRACT Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O2-dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O2 to produce aerobactin. IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes.

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