Natural History of Innate Host Defense Peptides | SpringerLink

Probiotics & Antimicro. Prot. (2009) 1:97–112 DOI 10.1007/s12602-009-9031-x

Natural History of Innate Host Defense Peptides A. Linde • B. Wachter • O. P. Ho¨ner • L. Dib • C. Ross • A. R. Tamayo • F. Blecha • T. Melgarejo

Published online: 5 December 2009 Ó Springer Science + Business Media, LLC 2009

Abstract Host defense peptides act on the forefront of innate immunity, thus playing a central role in the survival of animals and plants. Despite vast morphological changes in species through evolutionary history, all animals examined to date share common features in their innate immune defense strategies, hereunder expression of host defense peptides (HDPs). Most studies on HDPs have focused on humans, domestic and laboratory animals. More than a thousand different sequences have been identified, yet data on HDPs in wild-living animals are sparse. The biological functions of HDPs include broad-spectrum

A. Linde F. Blecha Department of Anatomy and Physiology, Kansas State University, Manhattan, KS, USA B. Wachter O. P. Ho¨ner Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany L. Dib T. Melgarejo (&) Department of Human Nutrition, College of Human Ecology, Kansas State University, 143A Justin Hall, Manhattan, KS 66506-1407, USA e-mail: [email protected] T. Melgarejo Ancillary Faculty in Anatomy & Physiology, College of Veterinary Medicine, Kansas State University, 143A Justin Hall, Manhattan, KS 66506-1407, USA C. Ross School of Veterinary Medicine, Oklahoma State University, Stillwater, OK, USA A. R. Tamayo Research Institute of Veterinary Science, Autonomous University of Baja California, Mexicali, Mexico

antimicrobial activity and immunomodulation. Natural selection and coevolutionary host-pathogen arms race theory suggest that the extent and specificity of the microbial load influences the spectrum and potency of HDPs in different species. Individuals of extant species— that have lived for an extended period in evolutionary history amid populations with intact processes of natural selection—likely possess the most powerful and welladapted ‘‘natural antibiotics’’. Research on the evolutionary history of the innate defense system and the host in context of the consequences of challenges as well as the efficacy of the innate immune system under natural conditions is therefore of immediate interest. This review focuses on evolutionary aspects of immunophysiology, with emphasis on innate effector molecules. Studies on host defense in wild-living animals may significantly enhance our understanding of inborn immune mechanisms, and help identify molecules that may assist us to cope better with the increasing microbial challenges that likely follow from the continuous amplification of biodiversity levels on Earth. Keywords Innate immunity Defensins Cathelicidins Comparative genomics Vertebrate evolution Molecular timescale

The Origin of Inborn Host Defense Evolution of host defense falls into two main branches: (1) An ancient germ-line encoded, non-specific immune response—innate (natural) immunity and (2) A more ‘‘recently’’ evolved, specific system with molecular memory—adaptive (acquired) immunity [49]. Adaptive immunity is unique to vertebrates, whereas innate immune mechanisms are a shared feature between widely different

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species across the animal and plant kingdoms [49]. Immunophylogenic studies have shown a great degree of commonality in innate immunity between species—making non-vertebrate models, such as the fruit fly (Drosophilia melanogaster) or the soil nematode (Caenorhabditis elegans), useful for comparative immunological studies [43, 44]. Paleozoology dates the age of multi-cellular life to approximately one billion years in the context of Earth’s 4.6 billion-years-history [8, 128]. The origin of these early and most primitive multi-cellular organisms established a natural need for an immediate host defense system to ensure survival, thus promoting development of ‘‘innate immunity’’ (Fig. 1). In comparison to ‘‘adaptive immunity’’, which evolved with the derivation of the first vertebrate species around 500 million-years ago [4, 73], innate immunity is therefore the most ancient of the two defense systems [49]. Within the framework of innate immunity, different components evolved pending specific needs of individual animals—subsequently resulting in some degree of ‘‘species-specific’’ HDP composition [72, 148]. Still, rather striking commonalities are observed in innate host defense between otherwise quite dissimilar species [68]. Host defense peptides, such as defensins, are examples of central innate immune elements, which are widespread natural defense molecules free of monopoly by any one species or population [119]. Intriguingly, the cellular behavior of macrophages exhibits similarities to amoebas in that both engulf foreign matters [49]. The origin of innate immune elements may therefore be traced even further back in evolution to primordial unicellular amoebae, and thus closer toward the origin of life approximately 3.5–4.0 billion-years ago (Fig. 1) [65, 128]. Van Valen’s ‘‘The Red Queen Hypothesis’’ [131] (inspired by Lewis Carroll’s novel ‘‘Through the Looking Glass’’) states that species must evolve (‘‘run’’) to remain extant (‘‘stay in the same place’’). Therefore, in coexisting species, evolutionary changes in one species (such as a host or prey) lead to adaptive changes in competing species (i.e., microorganism or predator, respectively)—which relatively speaking appears like a standstill, because each

Fig. 1 Evolution of immune defense systems. Innate immunity is generally considered the evolutionary most ancient of the two main branches of immunological defense systems dating back to the first multi-cellular organisms or earlier (note change in time scale—Byr billion years, Myr million years)

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Origin of Life

Origin of Multi-Cellular Life

Early Hominids

1st Vertebrates

Age of Earth 4.5 Byr

side of the arms race reacts to change. This biological chess game theoretically leaves microbes and host-organisms in a continual evolutionary battle—although systems will typically evolve to the limit of their adaptive competence (also dubbed the ‘‘Peter’s Principle’’). This host-pathogen co-evolution has resulted in a layered structure of the immune system over time, as newly evolved mechanisms of defense typically complement previous ones [19]. New species emerge when the fitness of others proves inadequate for survival in a given ecological niche, and enhanced biodiversity—accompanied by a broader HDP repertoire—over time is a logical sequel. Throughout human history, the most dreaded causes of death were poison and plague, with epidemics unsettling cities or even countries with regular intervals leaving trails of social and economical affliction [113]. Historically, the main focus of immunological research has been directed toward mechanisms underlying immune memory [6]. In the old Roman Empire, the king of Pontus, Mithridates VI, made a habit of exposing himself to increasing amounts of poison in an attempt to provide resistance against attacks on his life [113]. Ancient Greek physicians and historians recognized the body’s capability of ‘‘memorizing’’ prior infections and a specific disease. However, the scientific field of immunology is only around 135 years old, counting from the first actual immunological experiments conducted by Louis Pasteur in the 1870s [6]. The following decades presented studies in immunology that were dominated by two schools directed by Ilya Ilyich Mechnikov’s (Elie Metchnikoff) research on cellular immunity (mainly phagocytic cells), and Paul Ehrlich’s work on humoral immunity (antibodies) [6, 113]. Investigations on the innate immune system were, however, scarce up until the past two decades. The 1980s saw an increased focused on innate immune receptors, the Toll-like receptors (TLRs), and the first reports on antimicrobial peptides—also known as ‘‘host defense peptides’’ (HDPs)—emerged in the eighties and early 1990s [29]. Despite an evolutionary ancient history, the receptors and effector molecules of innate immunity have thus remained ‘‘unsung heroes’’ until fairly recently [92].

3.5

1.0

500 Myr

Present

Homo Sapiens 7

100,000 yr

Innate Immune Defense Strategies

Acquired Immunity

Time


99

On the Frontiers of Innate Immunity

The myeloid C-type lectin receptors (CLRs) include PRRs such as dectin-1, which recognizes fungal beta-glucans and act in synergy with e.g. TLR signaling to induce inflammatory cytokines. Certain CLRs can furthermore identify glycosylation pattern changes on cancerous cells [100]. Another transmembrane receptor family of immunological relevance is the ‘‘triggering receptors expressed on myeloid cells’’, or TREM proteins, which can also act in synergism with other PRRs, including TLRs and NLRs [55]. Table 1 highlights some of the currently known PRRs with examples of their physiological role, while Fig. 2 outlines the cellular location of each receptor-group within context of the ‘‘danger model’’. The NF-jB transcription factor and down-stream signaling components comprise one of the main pathways responsible for HDP production, while signaling involving MAPK (mitogen-activated protein kinases) and JAK/STAT (Janus kinase/signal transducer and activator of transcription signaling pathway) have been listed as alternate routes [59]. The production of HDPs can be either following a pattern of continuity or be evoked by a number of different stressors, such as infection and inflammation [22, 32, 71]. Defensins and cathelicidins comprise two of the major groups of HDPs known to date [29]. Peptides belonging to the cathelicidin family have traditionally been derived from circulatory cells in a variety of species [147]. Within the defensin family, a total of five subgroups have been classified, and three of these peptide types are found in vertebrates [29, 32]. Alpha-defensins are typically derived from bone marrow cells and neutrophils, while betadefensins are classical epithelial host defense molecules expressed across several species [29]. Theta-defensins, on the other hand, have only been identified in the leukocytes of select primates [123]. The current review is focused primarily on the defensin and cathelicidin peptides. However, it should be noted that the literature lists a large number of other host defense substances of equal importance to an apt host defense response—including factors such as lysozyme, RNAses and lactoferrin. Broadly defined, ‘‘antimicrobial peptides’’ cover all oligo- or polypeptides with inhibitory and/or microbicidal activity, while ‘‘antimicrobial substances’’ encompass an even wider spectrum ranging from small inorganic molecules (e.g. hydrogen peroxide) to larger protein complexes (e.g. complement proteins) [30, 42]. The scope of this paper, however, is not to provide an exhaustive list of known HDPs because individual peptides can be localized through already existing HDP databases (see also Table 2). Host defense peptides are not only ubiquitous in their expression pattern, but they also cover a wide range of biological functions—e.g. members of the RNase families exhibit antibacterial and antiviral activities, but are also of

The survival of individuals (or organisms) has depended mainly upon the immediate responsiveness of innate immunity throughout eons. The innate immune system fights off the vast majority of impending threats, thus keeping living organisms at health, long before adaptive immune mechanisms are evoked [49]. Modern perception in immunology describes ‘‘danger-signals’’ as the true activators of an innate immune response [79], where ‘‘danger’’ encompasses infectious as well as non-infectious molecular patterns. The three major conserved components of an instantaneous host response include the following: (1) Pattern recognition receptors (PRRs), (2) Down-stream signaling via transcriptional factors (classically of the NFjB-Rel family) and (3) Production of effector molecules (mainly HDPs); which have all been identified in diverse species across different taxa (Fig. 2) [29, 49, 72]. Living organisms perceive ‘‘dangers’’ instantaneously through a rather ubiquitous expression of PRRs, which detect pathogen associated molecular patterns (PAMPs), such as LPS (lipopolysaccharide from the cell wall of Gram negative bacteria), or endogenous alarm signals (e.g. uric acid) released secondary to cellular damage (Fig. 2) [72, 99, 112]. More recently, the term ‘‘alarmin’’ was introduced to the literature, and here used to describe ‘‘an endogenous mediator capable of enhancing innate and adaptive immune responses through induction of concomitant recruitment and activation of antigen-presenting cells’’ [145]. The term is thus aimed at perhaps better describing the nature of these effector molecules that serve to signal the presence of ‘‘danger’’—whether it being due to microbial invasion or non-infectious tissue injury [145]. It is here also important to highlight the interrelationship and functional overlap between HDPs, such as defensins, and certain chemokines [57, 143, 144]. While some defensins exhibit chemokine-like activity, certain chemokines have antimicrobial activities [57, 143, 144]. The functional importance of such as scheme is significant to species survival. If evolution had not ensured a geneencoded response system aimed at detecting and addressing not only pathogenic intruders but also endogenous (non-pathogenic) damage in living systems, extant species would in all likelihood instead have faced extinction [5]. The TLRs are classical surface PRRs present also in the endosomal or lysosomal compartments of the cell, with over a dozen different members that recognize different types of PAMPs, while the NLRs (nodlike receptors) are PRRs operating from within the cytoplasm [27, 49]. Although the TLR family unquestionably plays a key role in innate immunity, all elements of a host response cannot be explained by the sole actions of TLRs [27, 91].

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PAMPs - exogenous ligands from bacteria, virus, fungi, parasites DAMPs/Alarmins - endogenous “alarm signals” from cell damage

Examples of PAMPs/DAMPS: dsRNA

LPS

PGN

“Dual-Recognition”

Uric acid

“Receptor Collaboration”

TLRs

CLRs

SRs

Extracellular PRR Sensors

TREMs

Intracellular PRR Sensors RLHs

NLRs

TLRs Recognition of “Danger” via PRRs Activation of Adaptor & Signaling Proteins (e.g. MyD88) “Cross-talk & Synergy”

Activation of Transcription Factors (e.g.NFkB Pathway)

Activation of Target Genes

Triggering of Host Defense Response

HDPs - Inflammatory Cytokines – Chemokines – Adhesion Molecules Enzymes – Secondary Inflammatory Mediators

Fig. 2 Simplified ‘‘danger model’’ and pattern recognition. The figure depicts a schematic of the ‘‘danger-model’’ concept. Geneencoded pattern recognition receptors (PRRs), such as toll-like receptors (TLRs) or nod-like receptors (NLRs), recognize different pathogen/danger-associated molecular patterns (PAMPs/DAMPs) or endogenous alarm signals (‘‘alarmins’’), leading to activation of downstream transcriptional factors (classically nuclear factor kappaB, NFjB) and production of immune effector components—including host defense peptides (e.g. defensins and cathelicidins). The Toll-like receptor family is the most studied of the PRR families. Other types of PRRs have, however, been described also as exemplified by the figure (see also Table 1). The schematic provides a simplified overview of the sequence of events from the innate detecting of

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‘‘danger’’ at the cell surface or in the cytosolic compartment to the activation of relevant genes leading to the production of various innate immune components (such as pro-inflammatory cytokines, chemokines and host defense peptides). It is important to also note that distinct recognition mechanisms involved in the detection of a given pathogen can cooperate synergistically, and that cross-talk exist among different receptors as well as downstream adaptor and signaling proteins—adding to the overall complexity. Synergistic effects have been described between NLRs and TLRs, CLRs and TLRs, as well as between TREM and TLRs or NLRs [18, 129]. The figure is as such intended as an overview only. A comprehensive database of different PRRs is also available at: http://www.imtech. res.in/raghava/prrdb/ [66]


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Table 1 Overview of innate pattern recognition receptors and their physiological role Family

Subclasses & examples

Localization Cell types (examples) Physiological relevance (examples)

References

TLRs

Toll-like receptors (incl. 13 TLRs)

Membrane Cytosolic

Epithelium, endothelium, neutrophils, macrophages, dendritic cells

Recognition of PAMPs (bacterial, viral, fungal, protozoal) chemokinetic— activate cells of the immune system

[37, 52, 53, 60, 61]

CLRs/ C-type lectin & lectin-like receptors LLRs (e.g. mannose receptor, dectin-1, DC-SIGN, selectins)

Membrane

Neutrophils, macrophages, dendritic cells, endothelium

Recognition of PAMPs, phagocytosis of pathogens, recognition of endogenous ligands (DAMPs), cell adhesion, leukocyte homing & extravasation

[10, 28, 37, 48]

TREMs Triggering receptor expressed on myeloid cells (incl. 4 TREMs)

Membrane

Neutrophils, monocytes, macrophages, dendritic cells

Inflammation, calcium mobilization, autoimmunity

[25, 54, 55]

SRs

Scavenger receptors (incl. subclasses A-H)

Membrane

Macrophages, platelets, endothelium

Lipoprotein homeostasis, atherogenesis, phagocytosis, apoptotic cell clearance, antimicrobial

[37, 70, 114]

NLRs

Nucleotide binding oligomerization domain-like receptors (incl. 5 NODs, 14 NALPs, plus [ 34 NLR genes in the mouse genome)

Cytosolic

Leukocytes and various tissues, including epithelium and endothelium

Inflammation, antibacterial response, detection of DAMPs, cell death— intracellular microbial sensors

[50, 53, 77, 81]

RLRs/ Retinoic acid-inducible gene-like RLHs helicases (incl. RIG-I, MDA5, LGP2)

Cytosolic

Ubiquitously expressed

Detection of viral PAMPs (ssRNA), [52, 53, antiviral response—intracellular sensors 58, 81, 83, 156]

Table 2 Overview of antimicrobial & host defense peptide databases Database AMSDb

Website—academic institution—publication year

Content

Entries

http://www.bbcm.units.it/*tossi/amsdb.html

Eukaryotic AMPs

880

Natural AMPs

1,788 [possibly inactive]

AMPs

1,518

University of Trieste, Italy (2002) ANTIMIC

http://research.i2r.a-star.edu.sg/Templar/DB/ANTIMIC/ Institute of Infocomm Research, Singapore (2004)

APD2

http://aps.unmc.edu/AP/main.php University of Nebraska Medical Center, USA (2004)

APPDb

http://ercbinfo1.ucd.ie/APPDb/

AMPs & Proteins

St. Vincent’s University Hospital, Ireland (2002) BAPDb

http://www.bbcm.units.it/*tossi/amsdb.html

— [Currently inactive]

Prokaryotic AMPs

University of Trieste, Italy

— [Under construction]

Defensin

http://defensins.bii.a-star.edu.sg/ Bioinformatics Institute, Singapore (2006)

Defensins

424

MiniCOPE

http://www.copewithcytokines.org/

Innate HDPs

1,760

Synthetic AMPs

260

http://penbase.immunaqua.com/

Penaeidins

29

University of Montpellier, France (2006)

[shrimp AMPs]

http://www.cryst.bbk.ac.uk/peptaibol

Peptaibols

University of London, UK (2004)

[fungal AMPs]

Author: Horst Ibelgaufts (2008, Ver.5.24) SAPD

http://oma.terkko.helsinki.fi:8080/*SAPD/ University of Helsinki, Finland (2002)

PenBase Peptaibol

[300

Adapted from: Tossi A. [127], University of Trieste, Italy & Wang G. [133], University of Nebraska Medical Center, USA

significance to nutrition, and scavenging of intestinal phosphate and nitrogen [41]. Similarities have been identified between RNase proteins in animals and plants, and it

is plausible that host defense represents a primordial function of this gene lineage [7, 41, 98, 101]. The evolution of these proteins may in part have been driven by selection

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102


Direct Action Host Defense Peptides

a)

b)

c)

a) HDPs attach to microbial membrane, resulting in b) pore-formation and c) cell death

Indirect Action neutrophils monocytes lymphocytes

Control of inflammation and microbial load Adaptive response

HDPs modulate recruitment & activation of immune cells

Fig. 3 Biological function of host defense peptides. Direct and indirect biological actions of host defense peptides (HDPs). Top panel schematic conceptualization of the Shai-Matsuzaki-Huang model (small circular structures represent HDPs, while larger (purple)

structure represents a generic microorganism). Bottom panel HDPs furthermore act as immunomodulators through recruitment of immune cells (larger (blue) structure represents a generic immune cell). Figure modified after Hancock [40]. (Color figure online)

for individual functional characteristics as well as energyefficacy in relation to enzyme size. Classical HDPs, such as defensins, are small cationic amphipathic peptides characterized by six disulfide-paired cysteines [34, 90]. The role of HDPs entails a versatility of functions (Fig. 3), including broad-spectrum antimicrobial activity toward a range of different microorganisms, including Gram-positive and Gram-negative bacteria, enveloped viruses, yeast and even certain parasites [31]. A theoretical concept (the Shai-Matsuzaki-Huang Model) is generally accepted as explanatory of how most HDPs conduct their antimicrobial activity through pore-formation in the microbial surface membrane leading to the pathogen’s demise [78, 109, 146]. Adding to this already impressive functional capacity are reports on chemotactic activity toward inflammatory cells, and cytotoxicity against carcinogenous cells [33, 90, 143]—functions which have sparked an interest in investigating the capacity of naturally occurring HDPs as potential model molecules for development of new antibiotics and immunomodulatory compounds [104].

logically created a foundation for the newer subspecialties evolutionary immunology and eco-immunology. Here, we discuss the two in combination, even though patterns in the variation of immunity on an individual level (ecological) may differ from evolutionary, species-specific immunological variation [67]. Contrary to the high number of immunologic studies in the veterinary and biomedical literature, reports on vertebrate ecological immunology and non-model species are scarce [67]. At the center of ecological immunology lies the assumption that trade-offs occur between immune defense mechanisms and other attributes, which contribute to an individual’s overall fitness (such as reproduction, development, and growth), if these share common and limited resources [67, 110]. The shape of an individual’s—and subsequently the species’— immune defense is a function of multifaceted interactions involving life history traits, pathogen exposure, body size and phylogeny (Fig. 4) [67]. Despite a significant amount of different variables influencing the immune system, sufficient generality exists in the cost/benefit association between various immunological defense systems across disparate taxa to allow formulation of a common heuristic model aimed at outlining a broader picture [67]. The model put forth by Lee makes a distinction between ‘‘slow-living’’ (e.g. African elephant (Loxodonta Africana)) and ‘‘fast-living’’ species (e.g. wild-living mice (Mus musculus), as well as most insects), and proposes a relationship between these generic types of species’ life history traits and the metabolic costs of their immune defense preferences. The model suggests that species with fast reproductive/short developmental/low survival rates (i.e., ‘‘fast-living’’) rely more on non-specific innate immunity and a constitutive defense system, while species with slow reproductive/long developmental/high

Evolutionary & Eco-Immunology The components of an immune system are a reflection of an individual’s genetic composition and the environmental factors to which it is exposed. The main powers that drive immunological variation over time are ecological (i.e., forces affecting the distribution and abundance of living organisms and their interactions with the environment) and evolutionary (i.e., forces affecting the change in a population’s inherited characteristics, or traits, from generation to generation) [3, 67, 110]. These considerations have

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103

Pathogen Exposure Body Size “Dangers”

HDPs Immune Cells

Host surface

Immune barrier

Phylogeny

Life History Traits

Fig. 4 Development of immune defense mechanisms. The cartoon outlines the factors that are believed to influence immunological variation in the individual and species over time

survival rates (i.e., ‘‘slow-living’’) tend to depend more on adaptive immunity and a less constitutive response [67]. Adding to the model are considerations regarding individual variation within species and across different seasons, expected to result in a lateral shift toward a less constitutive immune response in times of higher intensity efforts, such as during the breeding season [67]. Intraspecific (individual) immunological variation can therefore result in quite different expectations for a predicted type of immune response, pending on the time of year, as well as body-condition, gender-specific traits and age [67]. It should here be noted that many experimental studies related to immunity are conducted in ‘‘fast living’’ animals (rodents) and data subsequently extrapolated to humans (‘‘long living’’). However, it is important to keep in mind the basic difference between wild-living mice and the inbred mice strains encountered in most laboratories, given

that biomedical research relies heavily on rodent strains that have been genetically manipulated and artificially designed to specifically mimic human disease. Operation of an immune system is associated with certain metabolic costs, and a correlation with body-size is anticipated [67, 139]. Smaller species often rely more on a constitutive defense, because this tend to be more ‘‘costeffective’’ compared to an induced [67]. Pathogen exposure is a third element of immediate importance for the composition of immune defense strategies. The selective microbial pressure and frequency of pathogen challenges are components that affect both the selective advantage of a particular defense strategy in an individual, species or population and may supersede pressures imposed by the more general costs/ benefits of different immune mechanisms [67]. Specifications on the evolutionary implications of immune mechanisms in different taxa or genera remain a

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104


work in progress [132]. The majority of evolutionists would probably concur that all living organisms originated from a common ancestral life form on Earth [132]. Close to 95% of all enzyme activities are shared between pro- and eukaryotes, and basic metabolic pathways, such as glycolysis and the citric acid cycle, are essentially similar across all life forms [132]. Quantitatively higher evolutionary levels emerged in periods of so-called ‘‘evolutional revolutions’’, such as the appearance of the first eukaryotes in early Precambrium (Fig. 5), which created a natural need of recognition mechanisms capable of distinguishing self from non-self [132]. The evolution of multi-cellular life-forms (metazoans) in the ‘‘Cambrian Explosion’’ and an increasing organizational hierarchy led to fundamental improvements, including development of basic homeostatic

Myr 0

250

Tertiary Cretaceous Jurassic Triassic Permian Carboniferous Devonian Silurian Ordovician Cambrian

Time Periods

65

Phanerozoic Eon

Geologic Time Scale

Quaternary

3,,500 3,500

Proterozoic

Archean

Hadean 4 4,500 ,50 50 00 0

Eons

2,500

““Precambrian” “P Pr

550 55 50

Fig. 5 Geologic time scale. The time-line (note varying scale) shows the eons and major periods of Earth’s history. ‘‘Precambrian’’ covers the Hadean, Archean and Proterozoic eons (i.e., largest geologic measures of time)—which account for about 90% of Earth’s history— while the Phanerozoic eon (incl. associated periods) covers the remaining 10% of the history until present time. Major ‘‘evolutionary revolutions’’ are indicated by the explosions (orange) on the left of the time scale. ‘‘Oldest fossils’’ refer in this context to the earliest known (biotic?) stromatolites, which conceivably may have been formed by cyanobacteria (Based on information from University of California Museum of Paleontology (Berkeley), American Geological Institute, and Paleontology & Geobiology at Harvard [56]. (Color figure online)

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body schemes, and hereunder early defense mechanisms [132]. The requirements for increasingly more sophisticated defense systems would therefore increase as cell populations to a larger extent began specializing into tissues and organs. The basic body plan of an animal phylum reflects the most suitable morphofunctional outline according to genome and environment [132]. Specific animal groups (birds, mammals etc.) share a common ancestry and body plan, resulting in a common immune strategy in taxonomically related animals [132]. The evolutionary transition from a marine to terrestrial environment is another factor that in phylogenetic terms caused a big leap in immune defense strategies. The distribution of pathogens in an aquatic environment tends to be relatively more homogeneous and dilute, and microorganisms are frequently entirely capable of living without a host organism in such surroundings [65]. This may also help explain why a lobster lives up to four decades without the protection of a complicated immune system, whereas a mouse depends on a more elaborate system of defense to survive through an average life span of 3 years [65]. One can speculate further on the logic behind a seemingly more complex immune system in a relatively short-lived terrestrial species (mouse) when compared to a longer-lived aquatic species, such as the lobster—and whether this apparent difference has more to do with each species’ body complexity, general lifestyle and/or environments. Importantly, terrestrial species diversity is reportedly greater than aquatic—even though more than 70% of the Earth’s surface is covered by oceans [20] (thus making the marine environment the largest contiguous habitat on Earth) [51], and with aquatic life being significantly older than terrestrial life. Moreover, an interrelationship presumably exists between the diversity seen in microorganisms versus their host-species [122]. Combined, this might help further explain the basic need of a more ‘‘evolved’’ immune system in the mouse (terrestrial) versus the lobster (aquatic)— since the former (on a theoretical level) is facing the most ‘‘dangers’’ in form of a broader and more concentrated variety of pathogens. Further insight into immune mechanisms of ‘‘evolutionarily successful’’ animals—emphasizing present day animals living wild in their natural habitat—and the phylogenic aspects of immunity may lead to identification of novel effector molecules of potential future application in development of new pharmaceutical compounds to address infectious and inflammatory diseases.

Innate Host Defense Molecules Shaped by Time The overview aims to sharpen the focus on the immune system as a set of intrinsic host defense mechanisms, which


are capable of equivalently protecting living organisms ranging anywhere from a moth to an elephant. It is consequently clear that the immune system did evolve, and in the following section, we will look at how some of the most prevalent host defense peptides, the defensins and cathelicidins, developed over time—although it should be stressed that the world of HDPs/AMPs involved in innate host defense goes beyond the peptides mentioned here [2, 21, 88, 104, 118, 120]. The defensin family comprises members across the plant and animal kingdoms—and beyond [84, 154]. The five groups of defensins contained within the family share common structural features, including the characteristic cysteine-rich motif, suggesting a phylogenetic connection. The evolutionary relationships between the mammalian defensins (alpha, beta and theta), and mammalian, invertebrate and plant defensins have, however, been a controversial topic [142]. Some support that obvious similarities in biological function and spatial organization between mammalian defensins would favor the idea of a common origin [142]. Others note that a closer relationship seems to exist between b-defensins and insect defensins, compared to that between a- and b-defensins from vertebrate species [46]. With a focus on recently duplicated mammalian defensin genes, one study found a distinct pattern of nucleotide substitutions in the gene-region encoding the mature peptide, thereby suggesting that natural selection has had an impact on the diversification of defensins at the amino-acid level [46]. Interestingly, eukaryotic defensins seem to have originated from one ancestral myxobacterial DLP (defensin-like peptide) [154, 155], and sequence similarities between fungal (eukaryotic) DLPs and bacterial DLPs [84, 154] could furthermore suggest that these yeast peptides represent early precursors of the defensins found in higher eukaryotes. Of the vertebrate defensins, beta-defensins are thought to be the evolutionary most ancient of the three sub-groups, from which alpha-defensins and subsequently thetadefensins have derived [142]. Beta-defensins have been identified in most vertebrate species, whereas alphadefensins are specific to mammals—more specifically glires and primates [93]. Theta-defensins have thus far been reported only in primates [86, 123]. Different computational analysis studies [47, 93, 94, 108, 142] have focused on cross-species investigation of the beta- and alpha-defensin gene families in an attempt to clarify the evolution of innate host defense, thereby possibly leading to an improved understanding of intrinsic mechanisms of immunity. Screening of the chicken genome has revealed a single b-defensin cluster with a total of 13 different genes, notably with an absence of other defensins in the genome of this species [142]. Based on comparative analysis of

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chicken, mouse and human defensin clusters, it is likely that vertebrate defensins originated with a common ancestral b-defensin-like gene [155]. During evolution, this gene has presumably been subject to fast duplication, positive diversification and chromosomal translocation within different lineages of vertebrate species resulting in several gene clusters with diverse chromosomal locations [142]. Clustering of some chicken b-defensins with their mammalian homologues would furthermore suggest that large groups of b-defensins evolved approximately 165– 175 million years ago around the time of the first (prehistoric) mammals—compared to others that likely originated much earlier with the first birds around 310 million years ago [62, 142]. The mammalian b-defensin gene family has been studied through cross-species analysis revealing a total of 39, 37, 43, 43 and 52 b-defensin genes/pseudogenes in the human, chimpanzee, dog, rat and mouse genome, respectively [94]. This study also found that the b-defensin genes tend to cluster in four to five chromosomal regions across species, and phylogenetic analyses furthermore indicate that the majority of b-defensins appears to have originated before the last common mammalian ancestor—while some subgroups are species-specific, suggesting that these evolved after the inter-species divergence of these mammals [94]. The same study furthermore noted certain b-defensins’ involvement in fertility, adding to their already established role in host defense [94]. Collectively, mammalian b-defensins appear to have evolved through a very dynamic and active process, resulting in a significant number of different b-defensins, which have likely allowed individual animal species to better cope with dissimilar environmental microorganisms [94]. While b-defensins have been identified in most vertebrates, a-defensins are specific to glires (i.e., rabbit, guinea pig, rat and mouse) and primates (including human, chimpanzee, olive baboon and rhesus macaque) [93]. Complete repertoires of a-defensins in the human, chimpanzee, rat and mouse genome have been reported [93]. Interestingly, the a-defensin genes in rodents and primates are located within a b-defensin cluster, and because of this physical proximity (as well as similarities in the spatial structure and biological activities of the peptides) it is believed that mammalian a-defensins have evolved from two independent ancestral genes originated from the b-defensin family [93, 94]. Contrary to b-defensins, which are mostly conserved across mammalian species, the a-defensins appear to form species-specific clusters— especially in non-primates—which would suggest that several of these peptides diverged more recently in evolution around the time of the mouse/rat split approximately 40 million years ago [62, 93]. Theta-defensins (found in

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primate leukocytes only) are thought to have evolved subsequently from the a-defensins [123] and thus originated even more recently on an evolutionary time scale [93]. Members of the cathelicidin family have been reported in different vertebrate species and constitute a second group of HDPs of central importance to inborn immunity [126]. A remarkable degree of molecular diversity has been noted within this gene family; however, a well-conserved feature across evolutionary distant species is an N-terminal cathelin-domain [126]. The antimicrobial activity of cathelicidins tends to be more robust compared to other HDPs [126]. Structural diversity between cathelicidins has likely allowed for distinct biological functions within species, including varying antimicrobial potency and activity, while still maintaining the typical cationic and amphipathic characteristic of HDPs [126]. Differential selective pressure has seemingly resulted in a rather extreme range of expression in various animals, given that some species have a single cathelicidin gene, while up to a dozen has been identified in others [126]. Although only one cathelicidin gene has been identified in the dog, human and mouse, at least 3, 8 and 11 cathelicidin sequences are contained in the horse, cow/sheep and pig genome [126]. Computational analysis places cathelicidins from the equine, bovine and porcine species in one cluster with two clades separated from all other mammalian species [126]. The most ancient cathelicidins identified to date are intestinal HDPs from the Atlantic hagfish, suggesting that the cathelicidin gene can be traced as far back as 300– 500 million years in evolution [126, 130]. Divergence of genes coding for immune effector molecules can result in either functional redundancy or acquisition of novel properties, either of which would help strengthen the host’s overall defense capacity [93]. Since different animals are subjected to varying ecological conditions and pathogen exposure it makes sense that a certain extent of specificity in the HDP repertoire allows for an apt immunological response directed specifically at a relevant set of microbial challenges under the given ecological conditions [93].

Host Defense in Wild-Living Animals The number of reports on HDPs in domestic animals and humans is growing steadily from when the first antimicrobial peptides (the so-called cecropins) were first isolated from silk moth (Hyalophora cecropia) by Boman’s group in 1981 [117]. Few years later, Zasloff’s group isolated amphibian-derived AMPs (the magainins) from skin of the African clawed frog (Xenopus laevis) [149], which lead to a huge expansion of the field—and frog skin is now known

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Probiotics & Antimicro. Prot. (2009) 1:97–112 Fig. 6 Host defense across kingdoms & in select extant species. Host c defense peptides (HDPs) are ancient and omnipresent elements of the innate immune response. The illustration includes examples of different antimicrobial/host defense peptides sampled from a pool of over one thousand different HDPs reported in various species from across the biological kingdoms (see also Table 2). The content as such serves primarily to provide an impression of the ubiquitous expression pattern of HDPs, and is not meant to be exhaustive. A combined ‘‘three-domain-system’’ [140] and ‘‘five kingdom structure’’ [135, 136] is depicted here as a compromise on a longdivided discussion. * Obsolete taxonomic term, however, used here for the sake of simplicity in the overview

as one of the richest natural sources of HDPs, which is also reflected by the rather large number of studies on this topic [74]. The data on innate immune components of nondomesticated vertebrate species are, however, quite limited. A wide range of HDPs has been reported in different invertebrate species, and Drosophila has been the focus of much research interest. Figure 6 provides an overview of the ubiquitous distribution of HDPs throughout the different biological kingdoms, and includes examples of host defense substances isolated from various life forms. This section, however, is intended as an overview of what is currently known about HDPs isolated from wild-living (non-model) vertebrate species. Mammalia The platypus (Ornithorhynchus anatinus) is perhaps one of the most fascinating mammals with a unique anatomy that blends features from different classes of animals. The platypus anatomy entails the so-called crural system, which includes hollow venomous spurs on each rear leg that are used by males for secretion of venom during the annual breeding season [137]. The platypus-venom is produced from three Orinthorhynchus venom defensin-like peptide genes (OvDLP) [137], which further highlights the broad range of biological activities that is covered by these peptides. Aves Two papers have focused on avian b-defensins (spheniscin1 and -2) isolated from stomach content of the male King Penguin (Aptenodytes patagonicus) [64, 125]. These compounds are 38-residue HDPs with a salt-independent, broad spectrum of antimicrobial activity and thought to primarily act in preservation of food for the newly hatched chick while the female forage at sea after egg laying [125]. Reptilia Studies on reptile HDPs include one report on a b-defensin-like peptide (turtle egg white protein, TEWP) from the

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ARCHAEA BACTERIA

Five Kingdom Structure

EUCARYA

Three Domain System


MONERA

PROTISTA

FUNGI

PLANTAE

ANIMALIA

Production of a Broad Variety of Host Defense Peptides

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Loggerhead Sea Turtle (Caretta caretta) [12]. This protein replaces lysozyme in the egg white of this species and exhibits significant antibacterial and antiviral activity [12]. A defensin-like peptide (pelovaterin) with strong antimicrobial activity against Gram-negative bacteria has also been isolated from eggshell of the Chinese Soft-shelled turtle (Pelodiscus sinensis), and likely serves to protect the egg content against bacterial invasion [63]. Most recently, a novel beta-defensin peptide was isolated from leukocytes of the European pond turtle (Emys orbicularis) and appropriately named TBD-1 (turtle beta-defensin-1). This peptide exhibits potent antibacterial and antifungal activity in vitro [116]. Other reptilian HDPs reported in the literature include cathelicidins from snake species such as Chinese Cobra (Naja atra), Banded Krait (Bungarus fasciatus) and King Cobra (Ophiophagus Hannah) [153], in addition to different snake venom proteins with antibacterial activity [85]. One study moreover suggests that leukocytes from the American alligator (Alligator mississippiensis) contain cationic peptides with broad antimicrobial activity [80]. Amphibia Several reports exist on amphibian HDPs, more specifically ‘‘frog skin antimicrobial peptides’’ (HDPs). Around 5,000 different frog species are known, with an estimated 10–20 different HDPs each [87]. Different families of frog HDPs have been identified, including magainins, brevinins, esculetins, ranatuerins, ranalexin, temporins and others [36, 75, 82, 103, 149]. A complex array of different skin HDPs has been isolated from North American frogs with antimicrobial activity against Gram-positive and Gram-negative bacteria and yeast [36]. Other frog HDPs (temporin A and B) have furthermore shown pronounced antiparasitic activity toward Leishmania in a concentration dependent fashion [76]. Other amphibian HDPs have been isolated from the skin of the terrestial Salamander (Plethodon cinereus), which is a lung-less species relying on cutaneous respiration [26]. The granular glands of the salamander skin produce HDPs with select activity against Grampositive bacteria, which are most commonly found on cutaneous surfaces [26]. Pisces A total of six cathelicidin gene sequences have been identified in fish species, including the Rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar) and the Atlantic hagfish (Myxine glutinosa) [11, 126, 130]. The hagfish is unique in that it is the oldest living jawless fish, relying solely on innate defense mechanisms [130]. Hagfish intestinal antimicrobial peptides have proven potent

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broad-spectrum antibiotics, and it is possible that this primordial gut-associated innate immune capacity created the architectural framework, which evolved into gut-associated lymphatic tissue as well as the spleen in higher vertebrate species [130]. Furthermore, a general trend seems to be that fish cathelicidins have a shorter signal peptide and a longer cathelin-like domain compared to their mammalian counterparts [11]. Significant variations in ecological conditions have likely dictated the evolution of HDPs in individual species, and it is clear that HDPs reach further than simply acting as ‘‘natural antibiotics’’.

Antimicrobial Resistance in the 21st Century A significant and worldwide problem today is the growing microbial resistance toward commercially available antibiotics. Communicable diseases are responsible for around 20% of the total number of deaths per year globally, or an annual death toll of approximately 10 million lives according to The World Health Report [138]. The past four decades of research efforts have, however, failed in producing an actual novel class of antibiotics. New, more potent antibiotics are therefore urgently needed. Studies on innate effector molecules have received an increased amount of research attention since the 1980s, and more focus is now directed at naturally derived HDPs as potential model molecules for development of novel pharmaceutical compounds [40]. The HDPs are attractive in that they generally display broad and potent antimicrobial activity [152]. Moreover, studies have noted these peptides’ immunomodulatory capacity and thus their potential application in the treatment of non-infectious types of pathologies. Resistance to natural HDPs is rare and does not involve cross-resistance to conventional antimicrobial [9]. Still, it should be mentioned that some bacterial pathogens have developed strategies to try and limit the overall efficacy of HDPs [96, 97] (e.g. expulsion of HDPs through energy-dependent channels, protease cleaving, modification of anionic molecules to reduce negative surface charge and alteration of membrane fluidity) [9, 97, 102]. Over a dozen HDPs and peptide analogues are currently in different phases of pharmaceutical development [40], and it is thus likely that intensified research efforts in this field will present the market with one or more new commercially available antibiotic(s) in a not too distant future.

Summary Immunological research endeavors have traditionally focused mainly on elements of the adaptive immune


response. The past few decades have, however, seen a redirection of focus to the branch of inborn immunity, which in actuality keeps the majority of all pathogens at bay before disease is established. Innate host defense has been studied in different species of interest to human and veterinary medicine, yet only few studies have looked at innate immunity of animals living in their natural environment where processes of natural selection are still intact. It is possible that studies on innate immune mechanisms of animal species living wild under extreme environmental conditions and microbial loads will provide valuable insight, which can help in identifying novel natural antibiotics with application to modern medicine. Evolutionary considerations and a ‘‘travel through time’’ may consequently lead to the discovery of new host defense peptides with unprecedented antimicrobial potency toward emerging as well as known pathogens currently jeopardizing world health. Acknowledgments We wish to acknowledge the reviewers for their constructive input on an earlier version of the manuscript. We also wish to thank the K-State COBRE (NIH grant P20-RR017686) and KState’s MCAT research program for their support.

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