scholarly journals The cysteine-rich exosporium morphogenetic protein, CdeC, exhibits self-assembly properties that lead to organized inclusion bodies in Escherichia coli

2020 ◽  
Author(s):  
A. Romero-Rodríguez ◽  
S. Troncoso-Cotal ◽  
E. Guerrero-Araya ◽  
D. Paredes-Sabja
Keyword(s):  
Self Assembly ◽  
Inclusion Bodies ◽  
Self Organization ◽  
Obligate Anaerobe ◽  
E Coli ◽  
Clostridioides Difficile ◽  
Nosocomial Diarrhea ◽  
Morphogenetic Protein ◽  
Underlying Mechanisms

AbstractClostridioides difficile is an obligate anaerobe spore-forming, Gram-positive, pathogenic bacterium, considered the leading cause of nosocomial diarrhea worldwide. Recent studies have attempted to understand the biology of the outer-most layer of C. difficile spores, the exosporium, which is believed to contribute to early interactions with the host. The fundamental role of the cysteine-rich proteins CdeC and CdeM has been described. However, the molecular details behind the mechanism of exosporium assembly are missing. The underlying mechanisms that govern exosporium assembly in C. difficile remain poorly studied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. In this work, we observed that CdeC was able to form organized inclusion bodies in the E. coli BL21 (DE3) pRIL strain filled with lamellae-like structures separated by an interspace of 5-15 nm; however, this lamellae-like organization is lost upon overexpression in E. coli SHuffle T7 strain with an oxidative environment. Additionally, DTT treatment of CdeC inclusion bodies released monomeric soluble forms of CdeC. Three truncated versions of the CdeC protein were constructed. While all the variants were able to aggregate forming oligomers that are resistant to denaturation conditions, TEM micrographs suggest that the self-organization properties of CdeC may be attributed to the C-terminal domain. Overall, these observations have important implications in further studies implicated in elucidating the role of CdeC in the exosporium assembly of C. difficile spores.

scholarly journals The Clostridioides difficile Cysteine-Rich Exosporium Morphogenetic Protein, CdeC, Exhibits Self-Assembly Properties That Lead to Organized Inclusion Bodies in Escherichia coli

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
A. Romero-Rodríguez ◽  
S. Troncoso-Cotal ◽  
E. Guerrero-Araya ◽  
D. Paredes-Sabja
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Self Assembly ◽  
Inclusion Bodies ◽  
Content Type ◽  
Clostridioides Difficile ◽  
Nosocomial Diarrhea ◽  
Morphogenetic Protein ◽  
Underlying Mechanisms

ABSTRACT Clostridioides difficile is an obligately anaerobic, spore-forming, Gram-positive pathogenic bacterium that is considered the leading cause of nosocomial diarrhea worldwide. Recent studies have attempted to understand the biology of the outermost layer of C. difficile spores, the exosporium, which is believed to contribute to early interactions with the host. The fundamental role of the cysteine-rich proteins CdeC and CdeM has been described. However, the molecular details behind the mechanism of exosporium assembly are missing. The underlying mechanisms that govern exosporium assembly in C. difficile remain poorly studied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. In this work, we observed that CdeC was able to form organized inclusion bodies (IBs) in Escherichia coli filled with lamella-like structures separated by an interspace of 5 to 15 nm; however, CdeC expression in an E. coli strain with a more oxidative environment led to the loss of the lamella-like organization of CdeC IBs. Additionally, dithiothreitol (DTT) treatment of CdeC inclusion bodies released monomeric soluble forms of CdeC. Deletions in different portions of CdeC did not affect CdeC’s ability to aggregate and form oligomers stable under denaturation conditions but affected CdeC’s self-assembly properties. Overall, these observations have important implications in further studies elucidating the role of CdeC in the exosporium assembly of C. difficile spores. IMPORTANCE The endospore of Clostridioides difficile is the vehicle for transmission and persistence of the pathogen, and, specifically, the exosporium is the first contact between the host and the spore. The underlying mechanisms that govern exosporium assembly in C. difficile remain understudied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. Understanding the exosporium assembly’s molecular bases may be essential to developing new therapies against C. difficile infection.

Gonadal rejuvenation of mice by GDF11

2021 ◽  
Author(s):  
Yang Zhou ◽  
Shousheng Ni ◽  
Congjun Li ◽  
Lili Song ◽  
Shicui Zhang
Keyword(s):  
Antioxidant Enzymes ◽  
Oral Delivery ◽  
Marker Enzymes ◽  
Slowing Down ◽  
Testicular Cells ◽  
Morphogenetic Protein ◽  
Aging Properties ◽  
Potential Improvement ◽  
Underlying Mechanisms

Abstract Growth differentiation factor 11 (GDF11), also known as bone morphogenetic protein 11 (BMP11), has been shown to have rejuvenation and anti-aging properties, but little information is available regarding the role of GDF11 in reproductive system to date. In this study, we first confirmed the bioavailability of recombinant GDF11 (rGDF11) by oral delivery in mice. We also showed that dietary intake of rGDF11 had little influence on body and gonadal (ovary/testis) weights of recipient mice, indicating their general condition and physiology were not affected. Based on these findings, we started to test the function of rGDF11 in ovary and testis of mice and to explore the underlying mechanisms. It was found that to some extent, rGDF11 could attenuate the senescence of ovarian and testicular cells, and contribute to the recovery of ovarian and testicular endocrine functions. Moreover, rGDF11 could rescue the diminished ovarian reserve in female mice and enhance the activities of marker enzymes of testicular function (SDH and G6PD) in male mice, suggesting a potential improvement of fertility. Notably, rGDF11 markedly promoted the activities of antioxidant enzymes in the ovary and testis, and remarkably reduced the levels of lipid peroxidation, protein oxidation and ROS in the ovary and testis. Collectively, these results suggest that GDF11 can protect ovarian and testicular functions of aged mice via slowing down the generation of ROS through enhancing activities of antioxidant enzymes.

Graft-Versus-Host Disease Alters Intestinal Microbial Ecology by Inhibiting Production of Enteric Defensins

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 244-244
Author(s):  
Yoshihiro Eriguchi ◽  
Shuichiro Takashima ◽  
Noriko Miyake ◽  
Yoji Nagasaki ◽  
Nobuyuki Shimono ◽  
...  
Keyword(s):  
Paneth Cell ◽  
Intestinal Microbiome ◽  
Gram Negative Bacteria ◽  
Obligate Anaerobe ◽  
Gut Flora ◽  
Gram Negative ◽  
E Coli ◽  
Intestinal Gvhd ◽  
Intestinal Ecology

Abstract Abstract 244 Bacterial infection is a serious complication of bone marrow transplantation (BMT). Intestinal GVHD, in particular, significantly enhances the risk for Gram-negative septicemia. The majority of the intestinal microbiome of the hosts consists of non-cultivable obligate anaerobes, while the Gram-negative bacteria such as Escherichia coli (E. coli) make up a small proportion of the microflora. Thus, it remains unclear why Gram-negative septicemia is dominant in intestinal GVHD, while the role of systemic immunosuppression and use of antibiotics is well-appreciated. We evaluated gut flora changes in the course of GVHD in mouse models of BMT without giving antibiotic or immunosuppresive drugs. Lethally irradiated B6D2F1 (H-2b/d) or B6C3F1 (H-2b/k) mice were injected with 5 × 106 T-cell depleted BM alone (non-GVHD controls) or with 2 × 106 T cells (GVHD group) from MHC-mismatched B6 (H-2b) donors on day 0. Intestinal microflora was identified by using terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA gene libraries constructed from each sample of gut contents. It consisted of approximately 80% of obligate anaerobe, and 20% of anaerobe such as Lactobacilli and Clostridia with very few E. coli before BMT. After BMT, this diversity of gut flora was preserved in non-GVHD controls (Table). In contrast, GVHD mice showed a marked increase of E. coli with a significant decrease in the members of obligate anaerobe. This was associated with dissemination of E. coli to the mesenteric lymph nodes (mLNs) and liver, with elevated serum levels of lipopolysaccharide (LPS). Such a loss of diversity of gut flora with a flora shift towards E. coli was significantly associated with morbidity and mortality of GVHD. Numbers of T-RFLP peaks that indicate diversity of intestinal flora were inversely correlated with GVHD clinical scores (p<0.001). A degree of E. coli proportion has significant correlation with GVHD clinical scores (p<0.001) and GVHD mortality (80% in high E. coli group vs. 0% in low E. coli group). We then investigated the underlying mechanisms of the disruption of intestinal ecology in GVHD. Paneth cell derived α-defensins are essential regulators of intestinal microbial ecology. We therefore hypothesized that Paneth cell damage in GVHD inhibited production of enteric defensins and disrupted intestinal ecology. Immunohistochemistry for lysozyme that marks Paneth cells showed significant reduction of Paneth cells in GVHD and quantitative real-time PCR analysis showed dramatically reduced expression of enteric defensins including Defa21, Defa1, Defa4, Defa5, and Defcr-rs1 (Table). These results suggest that Paneth cell injury in GVHD could lead to lower expression of enteric defensins and a shift of gut flora from commensal microorganisms towards widespread prevelance of gram-negative bacteria in the intestinal microbiome, and the subsequent high risk for the development of life threatening Gram-negative septicemia. LPS derived from Gram-negative bacteria plays an important role in amplifying systemic GVHD. Thus, such an alteration of intestinal ecology may be related to exaggeration of systemic GVHD. These results thus uncover the previously unrecognized role of the intrinsic antimicrobial peptides and the crosstalk between hosts and the intestinal microbes in the pathogenesis of GVHD and infection after allogeneic BMT. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Expression of the Mitochondrial ADP/ATP Carrier inEscherichia coli

2001 ◽  
Vol 276 (15) ◽  
pp. 11499-11506 ◽  
Author(s):  
Simone Heimpel ◽  
Gabriele Basset ◽  
Sabine Odoy ◽  
Martin Klingenberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Inclusion Bodies ◽  
Positive Charge ◽  
Inescherichia Coli ◽  
Transport Activity ◽  
E Coli ◽  
The Matrix ◽  
A Charge

Previously, the role of residues in the ADP/ATP carrier (AAC) fromSaccharomyces cerevisiaehas been studied by mutagenesis, but the dependence of mitochondrial biogenesis on functional AAC impedes segregation of the mutational effects on transport and biogenesis. Unlike other mitochondrial carriers, expression of the AAC from yeast or mammalians inEscherichia coliencountered difficulties because of disparate codon usage. Here we introduce the AAC fromNeurospora crassainE. coli, where it is accumulated in inclusion bodies and establish the reconstitution conditions. AAC expressed with heat shock vector gave higher activity than with pET-3a. Transport activity was absolutely dependent on cardiolipin. The 10 single mutations of intrahelical positive residues and of the matrix repeat (+X+) motif resulted in lower activity, except of R245A. R143A had decreased sensitivity toward carboxyatractylate. The ATP-linked exchange is generally more affected than ADP exchange. This reflects a charge network that propagates positive charge defects to ATP4−more strongly than to ADP3−transport. Comparison to the homologous mutants of yeast AAC2 permits attribution of the roles of these residues more to ADP/ATP transport or to AAC import into mitochondria.

scholarly journals Invasiveness of Escherichia coli Is Associated with an IncFII Plasmid

Pathogens ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1645
Author(s):  
Lars Johannes Krall ◽  
Sabrina Klein ◽  
Sébastien Boutin ◽  
Chia Ching Wu ◽  
Aline Sähr ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bloodstream Infections ◽  
Intestinal Epithelial ◽  
Genetic Changes ◽  
E Coli ◽  
Underlying Mechanisms ◽  
Evolutionary Route ◽  
Invasion Assays

Escherichia coli is one of the most prevalent pathogens, causing a variety of infections including bloodstream infections. At the same time, it can be found as a commensal, being part of the intestinal microflora. While it is widely accepted that pathogenic strains can evolve from colonizing E. coli strains, the evolutionary route facilitating the commensal-to-pathogen transition is complex and remains not fully understood. Identification of the underlying mechanisms and genetic changes remains challenging. To investigate the factors involved in the transition from intestinal commensal to invasive E. coli causing bloodstream infections, we compared E. coli isolated from blood culture to isolates from the rectal flora of the same individuals by whole genome sequencing to identify clonally related strains and potentially relevant virulence factors. in vitro invasion assays using a Caco- 2 cell intestinal epithelial barrier model and a gut organoid model were performed to compare clonally related E. coli. The experiments revealed a correlation between the presence of an IncFII plasmid carrying hha and the degree of invasiveness. In summary, we provide evidence for the role of an IncFII plasmid in the transition of colonization to invasion in clinical E. coli isolates.

scholarly journals MB109 as bioactive human bone morphogenetic protein-9 refolded and purified from E. coli inclusion bodies

2014 ◽  
Vol 13 (1) ◽  
pp. 29 ◽  
Author(s):  
Mario Meng-Chiang Kuo ◽  
Phuong Nguyen ◽  
Yun-Hui Jeon ◽  
Subin Kim ◽  
So-Mi Yoon ◽  
...  
Keyword(s):  
Bone Morphogenetic Protein ◽  
Inclusion Bodies ◽  
Human Bone ◽  
E Coli ◽  
Morphogenetic Protein ◽  
Human Bone Morphogenetic Protein ◽  
Bone Morphogenetic Protein 9

Immunogold labeling of CMP-KDO synthetase produced by recombinant E. Coli cells

1987 ◽  
Vol 45 ◽  
pp. 924-925
Author(s):  
F. A. Durum ◽  
R. G. Goldman ◽  
T. J. Bolling ◽  
M. F. Miller
Keyword(s):  
Inclusion Bodies ◽  
Large Scale ◽  
Recombinant Dna ◽  
Test System ◽  
Cell Protein ◽  
Gram Negative Bacteria ◽  
Cytoplasmic Inclusions ◽  
Isolation And Purification ◽  
E Coli ◽  
Low Concentrations

CMP-KDO synthetase (CKS) is an enzyme which plays a key role in the synthesis of LPS, an outer membrane component unique to gram negative bacteria. CKS activates KDO to CMP-KDO for incorporation into LPS. The enzyme is normally present in low concentrations (0.02% of total cell protein) which makes it difficult to perform large scale isolation and purification. Recently, the gene for CKS from E. coli was cloned and various recombinant DNA constructs overproducing CKS several thousandfold (unpublished data) were derived. Interestingly, no cytoplasmic inclusions of overproduced CKS were observed by EM (Fig. 1) which is in contrast to other reports of large proteinaceous inclusion bodies in various overproducing recombinant strains. The present immunocytochemical study was undertaken to localize CKS in these cells.Immune labeling conditions were first optimized using a previously described cell-free test system. Briefly, this involves soaking small blocks of polymerized bovine serum albumin in purified CKS antigen and subjecting them to various fixation, embedding and immunochemical conditions.

Response Interference as a Mechanism Underlying Implicit Measures

2008 ◽  
Vol 24 (4) ◽  
pp. 218-225 ◽  
Author(s):  
Bertram Gawronski ◽  
Roland Deutsch ◽  
Etienne P. LeBel ◽  
Kurt R. Peters
Keyword(s):  
Task Performance ◽  
Psychological Research ◽  
Implicit Measures ◽  
Mediation Model ◽  
Response Interference ◽  
Relevant Evidence ◽  
Moderated Mediation Model ◽  
Stimulus Features ◽  
Underlying Mechanisms

Over the last decade, implicit measures of mental associations (e.g., Implicit Association Test, sequential priming) have become increasingly popular in many areas of psychological research. Even though successful applications provide preliminary support for the validity of these measures, their underlying mechanisms are still controversial. The present article addresses the role of a particular mechanism that is hypothesized to mediate the influence of activated associations on task performance in many implicit measures: response interference (RI). Based on a review of relevant evidence, we argue that RI effects in implicit measures depend on participants’ attention to association-relevant stimulus features, which in turn can influence the reliability and the construct validity of these measures. Drawing on a moderated-mediation model (MMM) of task performance in RI paradigms, we provide several suggestions on how to address these problems in research using implicit measures.

The More You Say, the Less They Hear

2015 ◽  
Vol 27 (4) ◽  
pp. 159-169 ◽  
Author(s):  
Elsbeth D. Asbeek Brusse ◽  
Marieke L. Fransen ◽  
Edith G. Smit
Keyword(s):  
Hearing Protection ◽  
Entertainment Education ◽  
Mediating Role ◽  
Attitude Measures ◽  
Underlying Mechanisms

Abstract. This study examined the effects of disclosure messages in entertainment-education (E-E) on attitudes toward hearing protection and attitude toward the source. In addition, the (mediating) role of the underlying mechanisms (i.e., transportation, identification, and counterarguing) was studied. In an experiment (N = 336), three different disclosure messages were compared with a no-disclosure condition. The results show that more explicit disclosure messages negatively affect transportation and identification and stimulate the generation of counterarguments. In addition, the more explicit disclosure messages affect both attitude measures via two of these processes (i.e., transportation and counterarguing). Less explicit disclosure messages do not have this effect. Implications of the findings are discussed.

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