Insect digestive enzymes: properties, compartmentalization and function

A 120 kDa glycoprotein in the larval midgut membrane of the lepidopteran Manduca sexta, previously identified as a putative receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin, has been purified by a combination of protoxin affinity chromatography and anion exchange chromatography. In immunoblotting experiments, the purified glycoprotein has the characteristics predicted of the receptor: it binds CrylA(c) toxin in the presence of GlcNAc but not GalNAc; it binds the lectin SBA; but it does not bind CrylB toxin. N-terminal and internal amino acid sequences obtained from the protein show a high degree of similarity with the enzyme aminopeptidase N (EC 3.4.11.2). When assayed for aminopeptidase activity, purified receptor preparations were enriched 5.3-fold compared to M. sexta brush border membrane vesicles. We propose that the receptor for CrylA(c) toxin in the brush border membrane of the lepidopteran M. sexta is the metalloprotease aminopeptidase N.

/pdf/the-receptor-for-bacillus-thuringiensis-cryla-c-delta-vkd5m4fohf.pdf

The receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N

To investigate the biochemical basis of the differences in the insecticidal spectrum of Bacillus thuringiensis insecticidal crystal proteins (ICPs), we performed membrane binding and toxicity assays with three different ICPs and three lepidopteran species. The three ICPs have different toxicity patterns in the three selected target species. Binding studies with these 125I-labeled ICPs revealed high-affinity saturable binding to brush border membrane vesicles of the sensitive species. ICPs with no toxicity against a given species did not bind saturably to vesicles of that species. Together with previous data that showed a correlation between toxicity and ICP binding, our data support the statement that differences in midgut ICP receptors are a major determinant of differences in the insecticidal spectrum of the entire lepidopteran-specific ICP family. Receptor site heterogeneity in the insect midgut occurs frequently and results in sensitivity to more than one type of ICP.

Receptors on the brush border membrane of the insect midgut as determinants of the specificity of Bacillus thuringiensis delta-endotoxins.

Receptor binding studies show that resistance of a laboratory-selected Plodia interpunctella strain to a Bacillus thuringiensis insecticidal crystal protein (ICP) is correlated with a 50-fold reduction in affinity of the membrane receptor for this protein. The strain is sensitive to a second type of ICP that apparently recognizes a different receptor. Understanding the mechanism of resistance will provide strategies to prevent or delay resistance and hence prolong the usefulness of B. thuringiensis ICPs as environmentally safe insecticides.

https://science.sciencemag.org/content/sci/247/4938/72.full.pdf

Mechanism of insect resistance to the microbial insecticide Bacillus thuringiensis

Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are commercially successful in reducing pest damage, yet knowledge of resistance mechanisms that threaten their sustainability is incomplete. Insect resistance to the pore-forming Cry1Ac toxin is correlated with the loss of high-affinity, irreversible binding to the mid-gut membrane, but the genetic factors responsible for this change have been elusive. Mutations in a 12-cadherin-domain protein confer some Cry1Ac resistance but do not block this toxin binding in in vitro assays. We sought to identify mutations in other genes that might be responsible for the loss of binding. We employed a map-based cloning approach using a series of backcrosses with 1,060 progeny to identify a resistance gene in the cotton pest Heliothis virescens that segregated independently from the cadherin mutation. We found an inactivating mutation of the ABC transporter ABCC2 that is genetically linked to Cry1Ac resistance and is correlated with loss of Cry1Ac binding to membrane vesicles. ABC proteins are integral membrane proteins with many functions, including export of toxic molecules from the cell, but have not been implicated in the mode of action of Bt toxins before. The reduction in toxin binding due to the inactivating mutation suggests that ABCC2 is involved in membrane integration of the toxin pore. Our findings suggest that ABC proteins may play a key role in the mode of action of Bt toxins and that ABC protein mutations can confer high levels of resistance that could threaten the continued utilization of Bt–expressing crops. However, such mutations may impose a physiological cost on resistant insects, by reducing export of other toxins such as plant secondary compounds from the cell. This weakness could be exploited to manage this mechanism of Bt resistance in the field.

/pdf/an-abc-transporter-mutation-is-correlated-with-insect-28pr4f4du3.pdf

An ABC transporter mutation is correlated with insect resistance to Bacillus thuringiensis Cry1Ac toxin.

Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae)

Metagenomic next-generation sequencing of microbial cell-free DNA (mcfDNA) allows for non-invasive pathogen detection from plasma. However, there is little data describing the optimal role for this assay in real-world clinical decision making.We performed a single-center retrospective cohort study of adult patients for whom a mcfDNA (Karius©) test was sent between May 2019 and February 2021. Clinical impact was arbitrated after review and discussion of each case.A total of 80 patients were included. The most common reason for sending the assay was unknown microbiologic diagnosis (78%), followed by avoiding invasive procedures (14%). The test had a positive impact in 34 (43%), a negative impact in 2 (3%), and uncertain or no impact in 44 (55%). A positive impact was observed in solid organ transplant recipients (SOTR, 71.4%, p = 0.003), sepsis (71.4%, p = 0.003), and those receiving antimicrobial agents for less than 7 days prior to mcfDNA testing (i.e., 61.8%, p = 0.004). Positive impact was driven primarily by de-escalation of antimicrobial therapy.Clinical impact of mcfDNA testing was highest in SOTR, patients with sepsis and patients who had been on antimicrobial therapy for less than 7 days. Positive impact was driven by de-escalation of antimicrobial therapy which may highlight a potential role for mcfDNA in the realm of stewardship. 

/pdf/clinical-impact-of-a-metagenomic-microbial-plasma-cell-free-8uuxdm0y.pdf

Clinical impact of a metagenomic microbial plasma cell-free DNA next-generation sequencing assay on treatment decisions: a single-center retrospective study

Abstract Invasive fungal diseases cause significant morbidity and mortality, in particular affecting immunocompromised patients. Resistant organisms are of increasing importance, yet there are many notable differences in the ability to both perform and interpret antifungal susceptibility testing compared with bacteria. In this review, we will highlight the strengths and limitations of resistance data of pathogenic yeasts and moulds that may be used to guide treatment and predict clinical outcomes.

/pdf/clinical-utility-of-antifungal-susceptibility-testing-3t82xl42.pdf

Clinical utility of antifungal susceptibility testing


 Background

Metagenomic next-generation sequencing (mNGS) of microbial cell-free DNA (mcfDNA) allows for non-invasive pathogen detection from plasma. However, there is little data describing the optimal role for this assay in real-world clinical decision making.
Methods

We performed a single-center retrospective cohort study of adult patients for whom a mcfDNA test was sent between May 2019 and February 2021. Clinical impact was arbitrated after review and discussion of each case.
Results

A total of 80 patients were included. The most common reason for sending the assay was unknown microbiologic diagnosis (78%), followed by avoiding invasive procedures (14%). The test had a positive impact in 34 (43%), a negative impact in 2 (3%), and uncertain or no impact in 44 (55%). A positive impact was observed in solid organ transplant recipients (SOTR, 71.4%, p=0.003), sepsis (71.4%, p=0.003), and those receiving antimicrobial agents for less than 7 days prior to mcfDNA testing (i.e., 61.8%, p=0.004).
Conclusion

Clinical utility of mcfDNA testing was highest in SOTR and patients with sepsis. Prolonged antimicrobial use prior to mcfDNA testing limited the utility of the assay. Prospective studies evaluating the utility of mcfDNA assays should be performed to further clarify its role in clinical management.

/pdf/clinical-utility-and-impact-of-the-metagenomic-microbial-1ilk2byx.pdf

Clinical Utility and Impact of the Metagenomic Microbial Plasma Cell-Free DNA Next-Generation Sequencing Assay on Treatment Decision: A Single-Center Retrospective Study

P Luethy

Papers

Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae)

Clinical impact of a metagenomic microbial plasma cell-free DNA next-generation sequencing assay on treatment decisions: a single-center retrospective study

Clinical utility of antifungal susceptibility testing

Clinical Utility and Impact of the Metagenomic Microbial Plasma Cell-Free DNA Next-Generation Sequencing Assay on Treatment Decision: A Single-Center Retrospective Study

Point-of-Care Testing for the Diagnosis of Fungal Infections: Current Testing Applications and Potential for the Future.