Sequence edition of single domains modulates the final immune and antimicrobial potential of a new generation of multidomain recombinant proteins

AbstractCombining several innate immune peptides into a single recombinant antimicrobial and immunomodulatory polypeptide has been recently demonstrated. However, the versatility of the multidomain design, the role that each domain plays and how the sequence edition of the different domains affects their final protein activity is unknown. Parental multidomain antimicrobial and immunomodulatory protein JAMF1 and several protein variants (JAMF1.2, JAMF2 and AM2) have been designed and recombinantly produced to explore how the tuning of domain sequences affects their immunomodulatory potential in epithelial cells and their antimicrobial capacity against Gram-positive and Gram-negative bacteria. The replacement of the sequence of defensin HD5 and phospholipase sPLA2 by shorter active fragments of both peptides improves the final immunomodulatory (IL-8 secretion) and antimicrobial function of the multidomain protein against antimicrobial-resistant Klebsiella pneumoniae and Enterococcus spp. Further, the presence of Jun and Fos leucine zippers in multidomain proteins is crucial in preventing toxic effects on producer cells. The generation of antimicrobial proteins based on multidomain polypeptides allows specific immunomodulatory and antimicrobial functions, which can be easily edited by modifying of each domain sequence.

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Lipid droplets and the host–pathogen dynamic: FATal attraction?

The Journal of Cell Biology ◽

10.1083/jcb.202104005 ◽

2021 ◽

Vol 220 (8) ◽

Author(s):

Marta Bosch ◽

Matthew J. Sweet ◽

Robert G. Parton ◽

Albert Pol

Keyword(s):

Innate Immunity ◽

Lipid Droplets ◽

Innate Immune ◽

Metabolic Energy ◽

Eukaryotic Cells ◽

Bioactive Lipids ◽

Front Line ◽

Antimicrobial Proteins ◽

Current State ◽

Fatal Attraction

In the ongoing conflict between eukaryotic cells and pathogens, lipid droplets (LDs) emerge as a choke point in the battle for nutrients. While many pathogens seek the lipids stored in LDs to fuel an expensive lifestyle, innate immunity rewires lipid metabolism and weaponizes LDs to defend cells and animals. Viruses, bacteria, and parasites directly and remotely manipulate LDs to obtain substrates for metabolic energy, replication compartments, assembly platforms, membrane blocks, and tools for host colonization and/or evasion such as anti-inflammatory mediators, lipoviroparticles, and even exosomes. Host LDs counterattack such advances by synthesizing bioactive lipids and toxic nucleotides, organizing immune signaling platforms, and recruiting a plethora of antimicrobial proteins to provide a front-line defense against the invader. Here, we review the current state of this conflict. We will discuss why, when, and how LDs efficiently coordinate and precisely execute a plethora of immune defenses. In the age of antimicrobial resistance and viral pandemics, understanding innate immune strategies developed by eukaryotic cells to fight and defeat dangerous microorganisms may inform future anti-infective strategies.

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Antimicrobial proteins and peptides of blood: templates for novel antimicrobial agents

Blood ◽

10.1182/blood.v96.8.2664 ◽

2000 ◽

Vol 96 (8) ◽

pp. 2664-2672 ◽

Cited By ~ 118

Author(s):

Ofer Levy

Keyword(s):

Host Defense ◽

Blood Cells ◽

Antimicrobial Agents ◽

Severe Infection ◽

Innate Immune ◽

Antimicrobial Proteins ◽

Microbial Invasion ◽

Immune Mediators ◽

Rapid Responses ◽

Proteins And Peptides

Abstract The innate immune system provides rapid and effective host defense against microbial invasion in a manner that is independent of prior exposure to a given pathogen.1 It has long been appreciated that the blood contains important elements that mediate rapid responses to infection. Thus, anatomic compartments with ample blood supply are less frequently infected and recover more readily once infected, whereas regions with poor perfusion are prone to severe infection and may require surgical débridement. Blood-borne innate immune mediators are either carried in circulating blood cells (ie, leukocytes and platelets) or in plasma after release from blood cells or on secretion by the liver.

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Antimicrobial proteins and peptides of blood: templates for novel antimicrobial agents

Blood ◽

10.1182/blood.v96.8.2664.h8002664_2664_2672 ◽

2000 ◽

Vol 96 (8) ◽

pp. 2664-2672 ◽

Cited By ~ 1

Author(s):

Ofer Levy

Keyword(s):

Host Defense ◽

Blood Cells ◽

Antimicrobial Agents ◽

Severe Infection ◽

Innate Immune ◽

Antimicrobial Proteins ◽

Microbial Invasion ◽

Immune Mediators ◽

Rapid Responses ◽

Proteins And Peptides

The innate immune system provides rapid and effective host defense against microbial invasion in a manner that is independent of prior exposure to a given pathogen.1 It has long been appreciated that the blood contains important elements that mediate rapid responses to infection. Thus, anatomic compartments with ample blood supply are less frequently infected and recover more readily once infected, whereas regions with poor perfusion are prone to severe infection and may require surgical débridement. Blood-borne innate immune mediators are either carried in circulating blood cells (ie, leukocytes and platelets) or in plasma after release from blood cells or on secretion by the liver.

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VarQ: a tool for the structural analysis of Human Protein Variants

10.1101/277491 ◽

2018 ◽

Cited By ~ 1

Author(s):

Leandro Radusky ◽

Carlos Modenutti ◽

Javier Delgado ◽

Juan P. Bustamante ◽

Sebastian Vishnopolska ◽

...

Keyword(s):

Protein Interactions ◽

Protein Function ◽

Single Amino Acid ◽

Disease Development ◽

Functional Effect ◽

Single Nucleotide Variants ◽

Protein Activity ◽

Single Nucleotide ◽

Online Tool ◽

Protein Variants

AbstractUnderstanding the functional effect of Single Amino acid Substitutions (SAS), derived from the occurrence of single nucleotide variants (SNVs), and their relation to disease development is a major issue in clinical genomics. Even though there are several bioinformatic algorithms and servers that predict if a SAS can be pathogenic or not they give little or non-information on the actual effect on the protein function. Moreover, many of these algorithms are able to predict an effect that no necessarily translates directly into pathogenicity. VarQ Web Server is an online tool that given an UniProt id automatically analyzes known and user provided SAS for their effect on protein activity, folding, aggregation and protein interactions among others. VarQ assessment was performed over a set of previously manually curated variants, showing its ability to correctly predict the phenotypic outcome and its underlying cause. This resource is available online at http://varq.qb.fcen.uba.ar/.Contact: [email protected] Information & Tutorials may be found in the webpage of the tool.

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REP-X: An Evolution-guided Strategy for the Rational Design of Cysteine-less Protein Variants

10.1101/797969 ◽

2019 ◽

Author(s):

Kevin Dalton ◽

Tom Lopez ◽

Vijay Pande ◽

Judith Frydman

Keyword(s):

Rational Design ◽

Biochemical Characterization ◽

Cysteine Residue ◽

Rational Approach ◽

Protein Activity ◽

Methanococcus Maripaludis ◽

Site Specific ◽

Protein Variants ◽

Group Ii Chaperonin ◽

Reduced Activity

AbstractSite-specific labeling of proteins is often a prerequisite for biophysical and biochemical characterization. Chemical modification of a unique cysteine residue is among the most facile methods for site-specific labeling of proteins. However, many proteins have multiple reactive cysteines, which must be mutated to other residues to enable labeling of unique positions. This trial-and-error process often results in cysteine-free proteins with reduced activity or stability. Herein we describe a general methodology to rationally engineer cysteine-less proteins. Briefly, natural variation across orthologues is exploited to identify suitable cysteine replacements compatible with protein activity and stability. As a proof-of-concept, we recount the successful engineering of a cysteine-less mutant of the group II chaperonin from methanogenic archaeon Methanococcus maripaludis. A webapp, REP-X (Replacement at Endogenous Positions from eXtant sequences), which enables users to design their own cysteine-less protein variants, will make this rational approach widely available.

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Salivary antimicrobial proteins and stress biomarkers are elevated during a 6-month mission to the International Space Station

Journal of Applied Physiology ◽

10.1152/japplphysiol.00560.2019 ◽

2020 ◽

Vol 128 (2) ◽

pp. 264-275 ◽

Cited By ~ 2

Author(s):

Nadia H. Agha ◽

Forrest L. Baker ◽

Hawley E. Kunz ◽

Guillaume Spielmann ◽

Preteesh L. Mylabathula ◽

...

Keyword(s):

International Space Station ◽

Space Station ◽

Immune Defense ◽

Innate Immune ◽

Viral Reactivation ◽

Antimicrobial Proteins ◽

First Line ◽

International Space ◽

Long Duration ◽

Salivary Antimicrobial Proteins

As the international space community plans for manned missions to Mars, spaceflight-associated immune dysregulation has been identified as a potential risk to the health and safety of the flight crew. There is a need to determine whether salivary antimicrobial proteins, which act as a first line of innate immune defense against multiple pathogens, are altered in response to long-duration (>6 mo) missions. We collected 7 consecutive days of whole and sublingual saliva samples from eight International Space Station (ISS) crewmembers and seven ground-based control subjects at nine mission time points, ~180 and ~60 days before launch (L−180/L−60), on orbit at flight days ~10 and ~90 (FD10/FD90) and ~1 day before return (R−1), and at R+0, R+18, R+33, and R+66 days after returning to Earth. We found that salivary secretory (s)IgA, lysozyme, LL-37, and the cortisol-to-dehydroepiandrosterone ratio were elevated in the ISS crew before (L−180) and during (FD10/FD90) the mission. “Rookie” crewmembers embarking on their first spaceflight mission had lower levels of salivary sIgA but increased levels of α-amylase, lysozyme, and LL-37 during and after the mission compared with the “veteran” crew who had previously flown. Latent herpesvirus reactivation was distinct to the ~6-mo mission crewmembers who performed extravehicular activity (“spacewalks”). Crewmembers who shed at least one latent virus had higher cortisol levels than those who did not shed. We conclude that long-duration spaceflight alters the concentration and/or secretion of several antimicrobial proteins in saliva, some of which are related to crewmember flight experience, biomarkers of stress, and latent viral reactivation. NEW & NOTEWORTHY Spaceflight-associated immune dysregulation may jeopardize future exploration-class missions. Salivary antimicrobial proteins act as a first line of innate immune defense. We report here that several of these proteins are elevated in astronauts during an International Space Station mission, particularly in those embarking on their first space voyage. Astronauts who shed a latent herpesvirus also had higher concentrations of salivary cortisol compared with those who did not shed. Stress-relieving countermeasures are needed to preserve immunity and prevent viral reactivation during prolonged voyages into deep space.

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Differential Regional Expression Of Innate Immune Antimicrobial Proteins In Sinonasal Mucosa

Journal of Allergy and Clinical Immunology ◽

10.1016/j.jaci.2011.12.746 ◽

2012 ◽

Vol 129 (2) ◽

pp. AB68

Author(s):

S. Seshadri ◽

M. Rosati ◽

D.C. Lin ◽

R. Carter ◽

J. Norton ◽

...

Keyword(s):

Innate Immune ◽

Antimicrobial Proteins ◽

Sinonasal Mucosa

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Age-Related Loss of Innate Immune Antimicrobial Function of Dermal Fat Is Mediated by Transforming Growth Factor Beta

Immunity ◽

10.1016/j.immuni.2018.11.003 ◽

2019 ◽

Vol 50 (1) ◽

pp. 121-136.e5 ◽

Cited By ~ 24

Author(s):

Ling-juan Zhang ◽

Stella Xiang Chen ◽

Christian F. Guerrero-Juarez ◽

Fengwu Li ◽

Yun Tong ◽

...

Keyword(s):

Growth Factor ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Innate Immune ◽

Age Related ◽

Antimicrobial Function ◽

Growth Factor Beta

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Cell-Free DNA Promotes Thrombin Autolysis and Generation of Thrombin-Derived C-Terminal Fragments

Frontiers in Immunology ◽

10.3389/fimmu.2021.593020 ◽

2021 ◽

Vol 12 ◽

Author(s):

Rathi Saravanan ◽

Yeu Khai Choong ◽

Chun Hwee Lim ◽

Li Ming Lim ◽

Jitka Petrlova ◽

...

Keyword(s):

Critical Role ◽

Innate Immune ◽

Antimicrobial Proteins ◽

Cell Free Dna ◽

Thrombin Cleavage ◽

Free Dna ◽

Major Structural Component ◽

Protease Degradation ◽

Proteins And Peptides

Cell-free DNA (cfDNA) is the major structural component of neutrophil extracellular traps (NETs), an innate immune response to infection. Antimicrobial proteins and peptides bound to cfDNA play a critical role in the bactericidal property of NETs. Recent studies have shown that NETs have procoagulant activity, wherein cfDNA triggers thrombin generation through activation of the intrinsic pathway of coagulation. We have recently shown that thrombin binds to NETs in vitro and consequently can alter the proteome of NETs. However, the effect of NETs on thrombin is still unknown. In this study, we report that DNA binding leads to thrombin autolysis and generation of multiple thrombin-derived C-terminal peptides (TCPs) in vitro. Employing a 25-residue prototypic TCP, GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), we show that TCPs bind NETs, thus conferring mutual protection against nuclease and protease degradation. Together, our results demonstrate the complex interplay between coagulation, NET formation, and thrombin cleavage and identify a previously undisclosed mechanism for formation of TCPs.

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Small proline-rich proteins (SPRRs) are epidermally produced antimicrobial proteins that defend the cutaneous barrier by direct bacterial membrane disruption

10.1101/2021.09.01.458578 ◽

2021 ◽

Author(s):

Chenlu Zhang ◽

Zehan Hu ◽

Abdul G Lone ◽

Methinee Artami ◽

Marshall Edwards ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Bactericidal Activity ◽

Skin Infection ◽

Mouse Skin ◽

Systemic Infection ◽

Bacterial Membrane ◽

Antimicrobial Proteins ◽

Antimicrobial Function ◽

Skin Commensals ◽

Insight Into

Human skin functions as a physical barrier, preventing the entry of foreign pathogens while also accommodating a myriad of commensal microorganisms. A key contributor to the skin landscape is the sebaceous gland. Mice devoid of sebocytes are prone to skin infection, yet our understanding of how sebocytes function in host defense is incomplete. Here we show that the small proline-rich proteins, SPRR1 and SPRR2 are bactericidal in skin. SPRR1B and SPPR2A were induced in human sebocytes by exposure to the bacterial cell wall component lipopolysaccharide (LPS). Further, LPS injected into mouse skin triggered the expression of the mouse SPRR orthologous genes, Sprr1a and Sprr2a, through stimulation of MYD88. Both mouse and human SPRR proteins displayed potent bactericidal activity against MRSA (methicillin-resistant Staphylococcus aureus), Pseudomonas aeruginosa and skin commensals. Thus, Sprr1a-/-;Sprr2a-/- mice are more susceptible to MRSA and Pseudomonas aeruginosa skin infection. Lastly, mechanistic studies demonstrate that SPRR proteins exert their bactericidal activity through binding and disruption of the bacterial membrane. Taken together, these findings provide insight into the regulation and antimicrobial function of SPRR proteins in skin and how the skin defends the host against systemic infection.

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