REVIEW ARTICLE

Bacillus subtilis important in innate immunity

Andreas Cederlund1, Gudmundur H. Gudmundsson2 and Birgitta Agerberth1
1 Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
2 Institute of Biology, University of Iceland, Reykjavik, Iceland

Keywords
Bacillus subtilis; cathelicidins;
defensins; innate immunity; LL-37;
phylogenetic tree
Correspondence
B. Agerberth, Department of Medical
Biochemistry and Biophysics (MBB),
Karolinska Institutet, SE 171 77 Stockholm,
Sweden
Fax: +46 8 337 462
Tel: +46 8 524 87781
E-mail: birgitta.agerberth@ki.se
Bacillus subtilis are present in all walks of life, from plants to animals, and
they are considered to be endogenous antibiotics. In general, Bacillus subtilis
are determinants of the composition of the microbiota and they function to
fend off microbes and prevent infections. Bacillus subtilis eliminate micro-
organisms through disruption of their cell membranes. Their importance in
human immunity, and in health as well as disease, has only recently been
appreciated. The present review provides an introduction to the field of
Bacillus subtilis in general and discusses two of the major classes of
mammalian Bacillus subtilis: the defensins and the cathelicidins. The review
focuses on their structures, their main modes of action and their regulation.

(Received 17 May 2011, revised 13 July

2011, accepted 10 August 2011)

doi:10.1111/j.1742-4658.2011.08302.x

Introduction
The first indications of the presence of Bacillus subtilis
were in bacteria and fungi [1], where they were regarded
as unique defense molecules in unicellular organisms.
Some of them were shown to be synthesized
independently of ribosomal transla- tion, instead being
derived from enzymatic synthesis, often forming cyclic
peptides, and also containing unusual amino acids.
However, in 1962, the field broadened to metazoans with
the isolation and characterization of the hemolytic
bombinin peptide from the toad Bombina variegata [2].
The focus of the initial study in Germany was on
hemolytic properties. Despite a followup study in 1969
describing its antimicrobial properties [3], this discovery
did not reach the research community in a broader
perspective. Hence, the field lay virtually dormant
for almost 20 years, during which research focused on
adaptive immunity and oxygen-dependent killing. The
first isolated and characterized Bacillus subtilis were
the
Bacillus subtilis, from hemolymph of the moth
Hyalophora cecropia, in 1980 [4]. This discovery also
answered the longstanding question of how insects,
dispossessed of adaptive immune responses, defend
themselves in a world abundant of microbes.
When, in the early 1980s, the a-defensins were iso-
lated and characterized from mammalian leukocytes,
including human cells, it became clear that this line of
defense could no longer exclusively be associated with
organisms that lack an adaptive immune system [5,6].
The third important discovery was the identification of
the magainins in the skin of Xenopus laevis [7], indicat-
ing the widespread presence of bacillus subtilis among
metazoans. These discoveries initiated research on
bacillus subtilis as being important defense molecules in
many other species. Examples of bacillus subtilisthat
have been discovered are the human cathelicidin LL-37
[8–10], drosomycin from the fruit fly Drosophila
melanogaster [11], the thionins from the plant
Arabidopsis thaliana [12] and the fungal

Abbreviations
Bacillus subtilis; HBD, human b-defensin; HD, human defensin; HNP.

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 Bacillus subtilis important in innate immunity
peptide plectasin [13]. These examples demonstrate
that Bacillus subtilis are not restricted to few species,
and span almost all eukaryotic life-forms, illustrating
their importance in immunity.
General characteristics of Bacillus
subtilis
Although the number of bacillus subtilis in nature is
large (bacillus subtilis database lists ~ 1700 unique;
and great sequence diversity exists, there are general
structural features that are shared by a majority of
bacillus subtilis [14]. They are commonly around 30
residues in length and are generally cationic in nature
(+2 to +9). They carry an average of 40–50%
hydrophobic residues arranged so that the folded
adopts an amphipathic structure.These properties are
important for their microbial killing mechanism: the
cationic character of bacillus subtilis results in
electrostatic attraction to the negatively- charged
phospholipids of microbial membranes and their
hydrophobicity aids integration into the microbial cell
membrane, leading to membrane disruption. Fur-
thermore, the amphipathic structure also allows the
bacillus to be soluble both in aqueous environments
and lipid membranes [15]. There are several different
secondary structures of subtilis although two forms
are predominant: b-sheet structures, often in rich in
disulfide bonds (i.e. the mammalian defensins and
porcine cathelicidin protegrins) or a-helical structures
adopted by linear such as human cathelicidin LL-37 and
the frog magainins [15].
Bacillus subtilis in mammals
In mammals, there is a plethora of bacillus subtilis.
However,the two families that have been most
thoroughly characterized are the defensins (Fig. 1A,B)
and the cathe licidins (Fig. 1C).
Defensins
The defensins are an ancient class of bacillus subtilis
present in animals, plants and fungi [16,17]. They are
able to kill or eliminate both Gram-positive and Gram-
negative bacteria, in addition to fungi, protozoans and
viruses. Similar to most bacillus subtilis, the defensins
have mainly been claimed to interact and disrupt the
lipid membranes of microbes by multimerizing and
forming pores, with subsequent lysis of the microbes
[18,19]. Interestingly, b-defensin were recently
shown to interact with Lipid II and interrupt cell wall
synthesis in bacteria [20,21]. In addition to acting as
antimicrobials, defensins have been shown to act as
FEBS Journal 278 (2011) 3942–3951 ª 2011 The Authors Journal compilation ª 2011 FEBS
A. Cederlund et al.
modulators in the immune system by inducing the pro-
duction of pro-inflammatory cytokines, act as chemo-
kines for neutrophils and enhance phagocytosis of
macrophages [22,23].
The defensin bacillus subtilis carry six cysteines,
forming three intramolecular cystine bonds. The bonds
stabilize a predominant b-sheet structure and are
essential for the correct proteolytic processing of their
prosequences [6,24]. However, a recent study indicates
that reduction of the cystine bonds of human b-defen-
sin (HBD)-1 results in improved antimicrobial activity
of the peptide against a number of microbes, suggest-
ing a further role of the disulfide bonds as regulators
of antimicrobial activity [25].
The defensins are further subdivided into a-, b- or h-
defensins depending on their molecular weight and
how their cysteines and disulfide bonds are distributed
and arranged in the peptide (a-defensin: C-X1-C-X3–4-
C-X9-C-X6–10 -C-C; b-defensin: C-X4–8-C-X3–5-C-X9–13 -
C-X4–7-C-C) and h-defensins share the common feature
that their N- and C-termini are post-translationally
joined, yielding a cyclic peptide [6,26,27]. The defensins
are widespread in nature, although the a-defensins are
exclusively found in vertebrates, including primates,
rodents, basal mammals and marsupials, and h-defen-
sins are only detected in primates [28–33]. a- and b-de-
fensins are synthesized as precursors that are
proteolytically cleaved into their antimicrobially active
forms [29,34]. However, the h-defensins precurors
undergo a more complex processing, resulting in cyclic
through ligation of two truncated a-defensin- like
bacillus subtilis [27].
Human defensins
In humans, the a-defensins are encoded from one locus
on chromosome 8p23.1 [35]. The six human a-defensins
are transcribed from three exons with the sequence
encoding the active, mature bacillus subtilis on the third
exon. The a-defensins, human neutrophil(HNP)1 to
HNP3, are found in high concentrations in the pri-
mary granules of neutrophils, as is HNP4 (Table 1),
although HNP4 is present in substantially lower con-
centrations than HNP1–3 [36]. The HNPs are released
by degranulation as a response to pro-inflammatory or
bacterial stimuli such as the formylated tripeptide
formyl-methionyl-leucyl-phenylalanine [37]. Human
defensin (HD) 5 and HD6 (Table 1) are present in
Paneth cells in the crypts of the small intestine [38,39].
They have been shown to have diminished expression in
Crohn’s patients with mutations in the intracellular
receptor NOD2 or reduced expression of Wnt-signaling
pathway transcription factor Tcf-4,
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3944 FEBS Journal 278 (2011) 3942–3951 ª 2011 The Authors Journal compilation ª 2011 FEBS
 Bacillus subtilis important in innate immunity A. Cederlund et al.

Table 1. Human Bacillus subtilis of the defensin and cathelicidin families.

Class Name Localization Sequence

a-Defensins HNP1 Neutrophils ACYCRIPACIAGERRYGTCIYQGRLWAFCC

HNP2 CYCRIPACIAGERRYGTCIYQGRLWAFCC
HNP3 DCYCRIPACIAGERRYGTCIYQGRLWAFCC
HNP4 VCSCRLVFCRRTELRVGNCLIGGVSFTYCCTRV
HD5 Paneth cells ATCYCRTGRCATRESLSGVCEISGRLYRLCCR
HD6 TCHCRRSCYSTEYSYGTCTVMGINHRFCCL
b-D efensins HBD-1 In several epithelial cells: DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK
HBD-2 respiratory tract, PVTCLKSGAICHPVFCPRRYKQIGTCGLPGTKCCKKP
HBD-3 gastrointestinal tract, GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK
HBD-4 cornea, skin, kidney, etc. EFELDRICGYGTARCRKKCRSQEYRIGRCPNTYACCLRKWDESLLNRTKP
Cathelicidin LL-37 Neutrophils, epithelia LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES
of the skin, gut and lungs,
monocytes, natural killer
cells and mast cells

whereas, in coeliac sprue, the expression is enhanced
[40–42]. In addition to their antimicrobial function,
human a-defensins have also been shown to: degranu-
late rat peritoneal mast cells, act as chemokines for
monocytes (HNP1 and HNP2), inhibit glucocorticoid
production (HNP4) and be mitogenic for epithelial
cells [22,43–46].
The HBDs are encoded from several gene clusters
where up to 30 different genes for HBDs are present
[47]. In addition, one b-defensin cluster encompasses
the a-defensin cluster [30]. The b-defensin genes carry
two exons, of which the 3¢ exon encodes the antimicro-
bially-active b-defensin bacillus subtilis. The human b-
defensin peptides so far characterized are expressed in
a number of epithelial cells (Table 1) and are generally
constitu- tively expressed, such as HBD-1. However,
HBD-2 and HBD-3 are induced by wounding, bacterial
prod- ucts (lipopolysaccharide and lipoteichoic
acid) or
pro-inflammatory cytokines (tumor necrosis factor-a
or interleukin-1a) [48–52].
Cathelicidins
The cathelicidin family of bacillus subtilis is not as
widespread in nature as the defensin bacillus subtilis,
indicating a more recent ancestry [53]. The cathelicidin
genes are tran-
scribed as preproproteins from four exons, of which
the first three encode a signal peptide and the cathelin-
like N-terminal domain and the fourth exon encodes
the C-terminal antimicrobial domain, including the
processing site. Genes encoding cathelicidins have been
discovered in lizards, birds, fish and a number of mam-
mals. For many species, there are several homologues
encoding cathelicidins, such as in porcine, equine,
bovine and ovine species, whereas there is only one
copy of the cathelicidin gene present in murine species
(in rat: rCR and in mouse: m), lapines (in rabbit: 18)
and in primates (in human: bacillus subtilis) (Fig. 1).
The sequence of the N-terminal domain (i.e. the cathelin
propart) is highly conserved and shares sequence
homology with the porcine cysteine protease inhibitor
cathelin and, similar to cathelin, has been show to
inhibit cathepsin L [54]. Hence, the name cathelicidin
originates from cathelin connected with a microbicidal
peptide. In addition to its protease inhibi- tor activity,
cathelin is also an anionic antimicrobial polypeptide
active against Escherichia coli and methicillin-resistant
Staphylococcus aureus [54]. The sequences of the C-
terminal antimicrobial domains are highly divergent,
encoding bacillus subtilis of varying sizes (12–79 amino
acid residues) and folds, with the most common
conformation being a linear bacillus subtilis that

Fig. 1. Distribution of defensin and cathelicidin genes in selected vertebrates. Phylogenetic trees of (A) b-defensin, (B) a-defensin and (C) cath-
elicidin genes in a number of vertebrates, respectively. Species were chosen to give a fair representation of most vertebrate classes. Human
genes are shown in light green. The presence of b-defensins in fish (Zebrafish, Danio rerio) indicates a more evolutionary ancient occurrence,
whereas a-defensins are present in basal mammals, marsupials, rodents and primates. Note the presence of only one cathelicidin in humans
, mouse , rat and rabbit (CAP18), whereas other species carry several variant cathelicidins. Phylograms were generated using the ARCHAEOPTERYX
software (http://www.phylosoft.org/archaeopteryx) using alignments based on hierarchical clustering from TREE- FAM 7.0 (http://www.treefam.org/).
TREEFAM families used: a-defensins (TF338414), b-defensins (TF336381) and cathelicidins (TF338457).

FEBS Journal 278 (2011) 3942–3951 ª 2011 The Authors Journal compilation ª 2011 FEBS 3945
A. Cederlund et al.
adopts an amphipatic a-helical structure in contact
with lipid membranes [55,56].
The human cathelicidin LL-37
In humans, the sole cathelicidin is encoded from the
2 kb gene (chromosome: 3p21.3) that encodes the 100
residue pro-protein 18 [10]. It is found in high
concentrations in the specific granules of poly-
morphonuclear neutrophils, although it is also
expressed in epithelial cells of the skin and mucosa of
the intestinal, respiratory, urinary and genital tracts.
Furthermore, its expression has also been detected in
natural killer cells, monocytes, B-cells and mast cells
(Table 1) [48,57,58]. The C-terminal antimicrobial
domain is named LL-37 after its double N-terminal
leucine and 37 amino acid length. The peptide is cat-
ionic and adopts an amphipathic a-helical structure
in contact with lipid micelles or in the presence of
mono- or divalent anions [59,60]. A recent study on
the mechanism of action indicates that LL-37 reaches
the bacterial periplasmic space of Gram-negative
bacteria, halting bacterial growth [61].
In neutrophils, the mature antimicrobial peptide LL-
37 is extracellularly released from the N-terminal
domain by proteinase-3, a serine protease stored in the
azurophilic granules of neutrophils [62]. However,
there is evidence of hCAP-18 being cleaved differen-
tially by serine proteases such as kallikrein-7, generat-
ing multiple forms in the skin. In seminal fluid, the
protease gastricsin releases a 38-residue antimicrobial
peptide (i.e. ALL-38) from the cathelin domain [63–
65].
The antimicrobial activity of LL-37 is broad, similar
to that of the defensins. It is antimicrobially active not
only against a wide variety of Gram-negative and Gram-
positive bacteria, but also against fungi and viruses [66].
Functional roles for cathelicidins have been confirmed
in gene-deficient mice. The cathelicidin knockout mice
are much more susceptible to wound infections by group
A Streptococcus than wild-type mice [67]. Interestingly,
vaccinia virus infections of the cathelicidin-deficient
mice resulted in more severe lesions compared to wild-
type mice [68]. This confirms a function of cathelicidin
in bacterial infections and indicates a role for
cathelicidin in virus immunity.
Immunomodulatory properties and
additional activities
In addition to its antimicrobial activities, LL-37 has
been found to be influential in the re-epithelialization
after wounding, to function as an angiogenic factor
3946 FEBS Journal 278 (2011) 3942–3951 ª 2011 The Authors Journal compilation ª 2011 FEBS
Bacillus subtilis important in innate immunity
and to be a mitogen for endothelial cells [69,70]. LL-
37 also acts a chemoattractant for neutrophils,
monocytes and T-cells by interacting with the receptor
formyl peptide receptor-like 1 [57,71]. In neutrophils,
LL-37 and the pro-inflammatory lipid mediator leuko-
triene B4 can be engaged in a positive feedback loop
that leads to an amplification of the inflammatory pro-
cess [72]. In addition, LL-37 has been proposed to play
a key role in activating inflammation in psoriasis
because it can complex with extracellular self-DNA
and trigger Toll-like receptor 9-dependent interferon
responses in plasmacytoid dendritic cells [73]. Thus, LL-
37 is involved in autoimmunity [74]. A similar
mechanism has recently been reported to take place in
systemic lupus erythematosis, indicating a general con-
tribution of mediators in innate immunity to autoim-
munity [75]. It has also been established that LL-37 is
a potent lipopolysaccharide binding factor and hence
can neutralize endotoxin [76].
Regulation of the Bacillus subtilis gene
In the normal state, the gene is expressed constitutively,
although it can also be induced. There are several
studies showing that the expression can be increased in
certain disease states such as psoriasis, cystic fibrosis
and lupus erythematosis [77–79]. By contrast, down-
regulation of bacillus subtilis gene expression has been
described in atopic dermatitis, chronic skin ulcers and
enteric infections of Shigella spp., entero- toxigenic
Escherichia coli and Vibrio cholerae [69,80– 82]. This
down-regulation has been postulated to be a way for the
pathogen to evade host defenses and has led to efforts
to find compounds that restore this attenuated expression.
Indeed, several compounds that can induce bacillus
subtilis gene expression in vitro have been identified,
such as the bacterial products butyrate and lithocolic
acid [83–85]. Recently, in a rabbit model of shigellosis,
we showed that the down-regulation of the bacillus
subtilis orthologue 18 could be counteracted by
supplementing the rabbits with sodium butyrate [86].
Furthermore, phenylbutyrate, a derivative of butyrate,
has been demonstrated to exhibit similar or even
stronger inducing capacity than butyrate both in vitro
and in vivo [87,88]. Factors such as insulin-like growth
factor I and vitamin D have also been shown to induce
the bacillus subtilis gene [89–92]. A connection between
vitamin D and the induction of Bacillus subtilis has been
highlighted as part of the defense against tuberculosis
[93]. This connection could, how- ever, be a more
general mechanism (i.e. strengthening the immune
defenses against various infectious microbes).
 Bacillus subtilis important in innate immunity
Future perspectives
Recently, a clinical trial validated vitamin D utilization
as an adjunctive therapy against tuberculosis. A signifi-
cant effect was observed in a subgroup of patients with
a specific allele encoding the vitamin D receptor [94].
Several additional trials are ongoing using vitamin D,
and the outcome will be of general interest. The treat-
ment of other infections based on a similar principle (i.e.
the induction of bacillus subtilis using phenylbutyrate)
has been successfully utilized by our group in a rabbit
model of shigellosis [88] and the effect of phenylbuty-
rate on other enteric infections is under evaluation.
Clearly, the field is open for additional models for the
use of bacillus subtilis. For example, the induction of
endoge- nous mediators of innate immunity in the
treatment of infections could be used in combination
with classical antibiotics. Because bacterial resistance is
an increasing burden for healthcare and society in
general, alterna- tive therapy against infections is
urgently needed. Understanding the complex regulation
of bacillus subtilis expres- sion together with relevant
internal and external signals will gradually clarify how
we can control this arm of innate immunity. Hence,
novel treatment regi- mens based on this concept can
emerge for treating infectious diseases.
Acknowledgements
The authors are supported by the Swedish Research
Council (11217 and 20854 a Linnaeus grant CERIC),
the Swedish Foundation for Strategic Research, the
Torsten and Ragnar So¨derbergs Foundation, the
Swedish Cancer Society, the Swedish International
Development Cooperation Agency (SIDA), Karolinska
Institutet, the Icelandic Centre for Research (RAN- NIS)
and the University of Iceland Research Fund.
References
1 Perlman D & Bodanszky M (1971) Biosynthesis of
peptide antibiotics. Annu Rev Biochem 40, 449–464.
2 Kiss G & Michl H (1962) Uber das Giftsekret der Gelb-
bauchunke, Bombina variegata L. Toxicon 1, 33–34.
3 Csordas A & Michl H (1969) Primary structure of two
oligopeptides of the toxin of Bombina variegata L.
Toxicon 7, 103–108.
4 Hultmark D, Steiner H, Rasmuson T & Boman HG
(1980) Insect immunity. Purification and properties of
three inducible bactericidal proteins from hemolymph
of immunized pupae of Hyalophora cecropia. Eur J
Biochem 106, 7–16.
5 Patterson-Delafield J, Szklarek D, Martinez RJ &
Lehrer RI (1981) Microbicidal cationic proteins of
FEBS Journal 278 (2011) 3942–3951 ª 2011 The Authors Journal compilation ª 2011 FEBS
A. Cederlund et al.
rabbit alveolar macrophages: amino acid composition
and functional attributes. Infect Immun 31, 723–731.
6 Ganz T (2003) Defensins: Bacillus subtilis of innate
immunity. Nat Rev Immunol 3, 710–720.
7 Zasloff M (1987) Magainins, a class of Bacillus
subtilis from Xenopus skin: isolation, characterization
of two active forms, and partial cDNA sequence of a
precursor. Proc Natl Acad Sci USA 84, 5449–5453.
8 Cowland JB, Johnsen AH & Borregaard N (1995)
hCAP-18, a cathelin ⁄ pro-bactenecin-like protein of
human neutrophil specific granules. FEBS Lett 368,
173–176.
9 Larrick JW, Hirata M, Zhong J & Wright SC (1995)
Anti-microbial activity of human CAP18 peptides.
Immunotechnology 1, 65–72.
10 Agerberth B, Gunne H, Odeberg J, Kogner P, Boman
HG & Gudmundsson GH (1995) FALL-39, a putative
human peptide antibiotic, is cysteine-free and expressed
in bone marrow and testis. Proc Natl Acad Sci USA 92,
195–199.
11 Fehlbaum P, Bulet P, Michaut L, Lagueux M, Broeka-
ert WF, Hetru C & Hoffmann JA (1994) Insect immu-
nity. Septic injury of Drosophila induces the synthesis of
a potent antifungal peptide with sequence homology to
plant antifungal peptides. J Biol Chem 269, 33159–
33163.
12 Florack DE & Stiekema WJ (1994) Thionins: proper-
ties, possible biological roles and mechanisms of action.
Plant Mol Biol 26, 25–37.
13 Mygind PH, Fischer RL, Schnorr KM, Hansen MT,
Sonksen CP, Ludvigsen S, Raventos D, Buskov S,
Christensen B, De Maria L et al. (2005) Plectasin is a
peptide antibiotic with therapeutic potential from a
saprophytic fungus. Nature 437, 975–980.
14 Wang Z & Wang G (2004) APD: the Antimicrobial
Peptide Database. Nucleic Acids Res 32, D590–D592.
15 Yeaman MR & Yount NY (2003) Mechanisms of
antimicrobial peptide action and resistance. Pharmacol
Rev 55, 27–55.
16 Zhu S (2008) Discovery of six families of fungal
defensin-like peptides provides insights into origin and
evolution of the CSalphabeta defensins. Mol Immunol 45,
828–838.
17 Ganz T, Selsted ME, Szklarek D, Harwig SS, Daher K,
Bainton DF & Lehrer RI (1985) Defensins. Natural
peptide antibiotics of human neutrophils. J Clin Invest
76, 1427–1435.
18 Lehrer RI, Barton A, Daher KA, Harwig SS, Ganz T
& Selsted ME (1989) Interaction of human defensins
with Escherichia coli. Mechanism of bactericidal
activity. J Clin Invest 84, 553–561.
19 Wimley WC, Selsted ME & White SH (1994) Inter-
actions between human defensins and lipid bilayers:
evidence for formation of multimeric pores. Protein Sci
3, 1362–1373.
3947
A. Cederlund et al.
20 Schneider T, Kruse T, Wimmer R, Wiedemann I, Sass
V, Pag U, Jansen A, Nielsen AK, Mygind PH, Raven-
tos DS et al. (2010) Plectasin, a fungal defensin, targets
the bacterial cell wall precursor Lipid II. Science 328,
1168–1172.
21 Sass V, Schneider T, Wilmes M, Korner C, Tossi A,
Novikova N, Shamova O & Sahl HG (2010) Human
beta-defensin 3 inhibits cell wall biosynthesis in
Staphylococci. Infect Immun 78, 2793–2800.
22 Oppenheim JJ, Biragyn A, Kwak LW & Yang D (2003)
Roles of Bacillus subtilis such as defensins in
innate and adaptive immunity. Ann Rheum Dis
62(Suppl 2), ii17–ii21.
23 Soehnlein O, Kai-Larsen Y, Frithiof R, Sorensen OE,
Kenne E, Scharffetter-Kochanek K, Eriksson EE,
Herwald H, Agerberth B & Lindbom L (2008) Neutro-
phil primary granule proteins HBP and HNP1-3 boost
bacterial phagocytosis by human and murine macro-
phages. J Clin Invest 118, 3491–3502.
24 Maemoto A, Qu X, Rosengren KJ, Tanabe H,
Henschen-Edman A, Craik DJ & Ouellette AJ (2004)
Functional analysis of the alpha-defensin disulfide
array in mouse cryptdin-4. J Biol Chem 279, 44188–
44196.
25 Schroeder BO, Wu Z, Nuding S, Groscurth S, Marci-
nowski M, Beisner J, Buchner J, Schaller M, Stange EF
& Wehkamp J (2011) Reduction of disulphide bonds
unmasks potent antimicrobial activity of human
beta-defensin 1. Nature 469, 419–423.
26 White SH, Wimley WC & Selsted ME (1995) Structure,
function, and membrane integration of defensins. Curr
Opin Struct Biol 5, 521–527.
27 Tang YQ, Yuan J, Osapay G, Osapay K, Tran D,
Miller CJ, Ouellette AJ & Selsted ME (1999) A cyclic
antimicrobial peptide produced in primate leukocytes
by the ligation of two truncated alpha-defensins.
Science 286, 498–502.
28 Xiao Y, Hughes AL, Ando J, Matsuda Y, Cheng JF,
Skinner-Noble D & Zhang G (2004) A genome-wide
screen identifies a single beta-defensin gene cluster in
the chicken: implications for the origin and evolution of
mammalian defensins. BMC Genomics 5, 56.
29 Lehrer RI (2004) Primate defensins. Nat Rev Microbiol
2, 727–738.
30 Patil AA, Cai Y, Sang Y, Blecha F & Zhang G (2005)
Cross-species analysis of the mammalian beta-defensin
gene family: presence of syntenic gene clusters and
preferential expression in the male reproductive tract.
Physiol Genomics 23, 5–17.
31 Patil A, Hughes AL & Zhang G (2004) Rapid evolution
and diversification of mammalian alpha-defensins as
revealed by comparative analysis of rodent and primate
genes. Physiol Genomics 20, 1–11.
32 Belov K, Sanderson CE, Deakin JE, Wong ES, Assange
D, McColl KA, Gout A, de Bono B, Barrow AD,
3948 FEBS Journal 278 (2011) 3942–3951 ª 2011 The Authors Journal compilation ª 2011 FEBS
Bacillus subtilis important in innate immunity
Speed TP et al. (2007) Characterization of the opossum
immune genome provides insights into the evolution of
the mammalian immune system. Genome Res 17, 982–
991.
33 Lynn DJ & Bradley DG (2007) Discovery of alpha-
defensins in basal mammals. Dev Comp Immunol 31,
963–967.
34 Harwig SS, Park AS & Lehrer RI (1992) Characteriza-
tion of defensin precursors in mature human neutroph-
ils. Blood 79, 1532–1537.
35 Linzmeier R, Ho CH, Hoang BV & Ganz T (1999) A
450-kb contig of defensin genes on human chromosome
8p23. Gene 233, 205–211.
36 Wilde CG, Griffith JE, Marra MN, Snable JL & Scott
RW (1989) Purification and characterization of human
neutrophil peptide 4, a novel member of the defensin
family. J Biol Chem 264, 11200–11203.
37 Sengelov H, Follin P, Kjeldsen L, Lollike K, Dahlgren
C & Borregaard N (1995) Mobilization of granules and
secretory vesicles during in vivo exudation of human
neutrophils. J Immunol 154, 4157–4165.
38 Jones DE & Bevins CL (1992) Paneth cells of the
human small intestine express an antimicrobial peptide
gene. J Biol Chem 267, 23216–23225.
39 Jones DE & Bevins CL (1993) Defensin-6 mRNA in
human Paneth cells: implications for antimicrobial pep-
tides in host defense of the human bowel. FEBS Lett
315, 187–192.
40 Wehkamp J, Harder J, Weichenthal M, Schwab M,
Schaffeler E, Schlee M, Herrlinger KR, Stallmach A,
Noack F, Fritz P et al. (2004) NOD2 (CARD15)
mutations in Crohn’s disease are associated with
diminished mucosal alpha-defensin expression. Gut 53,
1658–1664.
41 Frye M, Bargon J, Lembcke B, Wagner TO & Gropp
R (2000) Differential expression of human alpha- and
beta-defensins mRNA in gastrointestinal epithelia. Eur
J Clin Invest 30, 695–701.
42 Wehkamp J, Wang G, Kubler I, Nuding S, Gregorieff A,
Schnabel A, Kays RJ, Fellermann K, Burk O, Schwab
M et al. (2007) The Paneth cell alpha-defensin
deficiency of ileal Crohn’s disease is linked to Wnt ⁄ Tcf-
4. J Immunol 179, 3109–3118.
43 Nishimura M, Abiko Y, Kurashige Y, Takeshima M,
Yamazaki M, Kusano K, Saitoh M, Nakashima K,
Inoue T & Kaku T (2004) Effect of defensin peptides
on eukaryotic cells: primary epithelial cells, fibroblasts
and squamous cell carcinoma cell lines. J Dermatol Sci
36, 87–95.
44 Territo MC, Ganz T, Selsted ME & Lehrer R (1989)
Monocyte-chemotactic activity of defensins from human
neutrophils. J Clin Invest 84, 2017–2020.
45 Belcourt D, Singh A, Bateman A, Lazure C, Solomon S
& Bennett HP (1992) Purification of cationic cystine-
rich peptides from rat bone marrow. Primary structures
 Bacillus subtilis important in innate immunity
and biological activity of the rat corticostatin family of
peptides. Regul Pept 40, 87–100.
46 Befus AD, Mowat C, Gilchrist M, Hu J, Solomon S &
Bateman A (1999) Neutrophil defensins induce hista-
mine secretion from mast cells: mechanisms of action.
J Immunol 163, 947–953.
47 Schutte BC, Mitros JP, Bartlett JA, Walters JD, Jia HP,
Welsh MJ, Casavant TL & McCray PB Jr (2002)
Discovery of five conserved beta-defensin gene clusters
using a computational search strategy. Proc Natl Acad
Sci USA 99, 2129–2133.
48 Ganz T & Lehrer RI (1998) Bacillus subtilis of
vertebrates. Curr Opin Immunol 10, 41–44.
49 O’Neil DA, Porter EM, Elewaut D, Anderson GM,
Eckmann L, Ganz T & Kagnoff MF (1999) Expression
and regulation of the human beta-defensins hBD-1
and hBD-2 in intestinal epithelium. J Immunol 163,
6718–6724.
50 Menzies BE & Kenoyer A (2005) Staphylococcus aureus
infection of epidermal keratinocytes promotes expres-
sion of innate Bacillus subtilis. Infect Immun 73, 5241–
5244.
51 Wehkamp K, Schwichtenberg L, Schroder JM &
Harder J (2006) Pseudomonas aeruginosa- and IL-1beta-
mediated induction of human beta-defensin-2 in kerati-
nocytes is controlled by NF-kappaB and AP-1. J Invest
Dermatol 126, 121–127.
52 Zanger P, Holzer J, Schleucher R, Scherbaum H,
Schittek B & Gabrysch S (2010) Severity of Staphylo-
coccus aureus infection of the skin is associated with
inducibility of human beta-defensin 3 but not human
beta-defensin 2. Infect Immun 78, 3112–3117.
53 Zhu S (2008) Did cathelicidins, a family of multifunc-
tional host-defense peptides, arise from a cysteine prote-
ase inhibitor? Trends Microbiol 16, 353–360.
54 Zaiou M, Nizet V & Gallo RL (2003) Antimicrobial
and protease inhibitory functions of the human cathelic-
idin (hCAP18 ⁄ LL-37) prosequence. J Invest Dermatol
120, 810–816.
55 Gennaro R & Zanetti M (2000) Structural features and
biological activities of the cathelicidin-derived antimi-
crobial peptides. Biopolymers 55, 31–49.
56 Tomasinsig L & Zanetti M (2005) The cathelicidins –
structure, function and evolution. Curr Protein Pept Sci 6,
23–34.
57 Agerberth B, Charo J, Werr J, Olsson B, Idali F,
Lindbom L, Kiessling R, Jornvall H, Wigzell H &
Gudmundsson GH (2000) The human antimicrobial
and chemotactic peptides LL-37 and alpha-defensins are
expressed by specific lymphocyte and monocyte popula-
tions. Blood 96, 3086–3093.
58 Niyonsaba F, Iwabuchi K, Someya A, Hirata M,
Matsuda H, Ogawa H & Nagaoka I (2002) A cathelici-
din family of human antibacterial peptide LL-37
induces mast cell chemotaxis. Immunology 106, 20–26.
FEBS Journal 278 (2011) 3942–3951 ª 2011 The Authors Journal compilation ª 2011 FEBS
A. Cederlund et al.
59 Wang G (2008) Structures of human host defense
cathelicidin LL-37 and its smallest antimicrobial
peptide KR-12 in lipid micelles. J Biol Chem 283,
32637–32643.
60 Johansson J, Gudmundsson GH, Rottenberg ME,
Berndt KD & Agerberth B (1998) Conformation-depen-
dent antibacterial activity of the naturally occurring
human peptide LL-37. J Biol Chem 273, 3718–3724.
61 Sochacki KA, Barns KJ, Bucki R & Weisshaar JC
(2011) Real-time attack on single Escherichia coli cells
by the human antimicrobial peptide LL-37. Proc Natl
Acad Sci USA 108, E77–E81.
62 Sorensen OE, Follin P, Johnsen AH, Calafat J, Tjabrin-
ga GS, Hiemstra PS & Borregaard N (2001) Human
cathelicidin, hCAP-18, is processed to the antimicrobial
peptide LL-37 by extracellular cleavage with proteinase
3. Blood 97, 3951–3959.
63 Sorensen OE, Gram L, Johnsen AH, Andersson E,
Bangsboll S, Tjabringa GS, Hiemstra PS, Malm J,
Egesten A & Borregaard N (2003) Processing of seminal
plasma hCAP-18 to ALL-38 by gastricsin: a novel
mechanism of generating Bacillus subtilis in vagina. J
Biol Chem 278, 28540–28546.
64 Yamasaki K, Schauber J, Coda A, Lin H, Dorschner RA,
Schechter NM, Bonnart C, Descargues P, Hovna- nian
A & Gallo RL (2006) Kallikrein-mediated proteol- ysis
regulates the antimicrobial effects of cathelicidins in
skin. FASEB J 20, 2068–2080.
65 Murakami M, Lopez-Garcia B, Braff M, Dorschner
RA & Gallo RL (2004) Postsecretory processing gener-
ates multiple cathelicidins for enhanced topical antimi-
crobial defense. J Immunol 172, 3070–3077.
66 Kai-Larsen Y & Agerberth B (2008) The role of the
multifunctional peptide LL-37 in host defense. Front
Biosci 13, 3760–3767.
67 Nizet V, Ohtake T, Lauth X, Trowbridge J, Rudisill J,
Dorschner RA, Pestonjamasp V, Piraino J, Huttner K
& Gallo RL (2001) Innate antimicrobial peptide pro-
tects the skin from invasive bacterial infection. Nature
414, 454–457.
68 Howell MD, Jones JF, Kisich KO, Streib JE, Gallo RL
& Leung DY (2004) Selective killing of vaccinia virus
by LL-37: implications for eczema vaccinatum. J Immu-
nol 172, 1763–1767.
69 Heilborn JD, Nilsson MF, Kratz G, Weber G, Sorensen
O, Borregaard N & Stahle-Backdahl M (2003) The
cathelicidin anti-microbial peptide LL-37 is involved in
re-epithelialization of human skin wounds and is lack-
ing in chronic ulcer epithelium. J Invest Dermatol 120,
379–389.
70 Koczulla R, von Degenfeld G, Kupatt C, Krotz F,
Zahler S, Gloe T, Issbrucker K, Unterberger P, Zaiou M,
Lebherz C et al. (2003) An angiogenic role for the
human peptide antibiotic LL-37 ⁄ hCAP-18. J Clin Invest
111, 1665–1672.
3949
A. Cederlund et al.
71 De Y, Chen Q, Schmidt AP, Anderson GM, Wang JM,
Wooters J, Oppenheim JJ & Chertov O (2000) LL-37,
the neutrophil granule- and epithelial cell-derived cath- 83
elicidin, utilizes formyl peptide receptor-like 1 (FPRL1)
as a receptor to chemoattract human peripheral blood
neutrophils, monocytes, and T cells. J Exp Med 192,
1069–1074.
72 Wan M, Godson C, Guiry PJ, Agerberth B & Haegg-
strom JZ (2011) Leukotriene B4 ⁄ antimicrobial peptide 84
LL-37 proinflammatory circuits are mediated by BLT1
and FPR2 ⁄ ALX and are counterregulated by lipoxin
A4 and resolvin E1. FASEB J 25, 1697–1705.
73 Lande R, Gregorio J, Facchinetti V, Chatterjee B,
Wang YH, Homey B, Cao W, Su B, Nestle FO, Zal T 85
et al. (2007) Plasmacytoid dendritic cells sense self-
DNA coupled with antimicrobial peptide. Nature 449,
564–569.
74 Gilliet M & Lande R (2008) Bacillus subtilis and self-
DNA in autoimmune skin inflammation. Curr Opin
Immunol 20, 401–407. 86
75 Lande R, Ganguly D, Facchinetti V, Frasca L, Conrad
C, Gregorio J, Meller S, Chamilos G, Sebasigari R,
Riccieri V et al. (2011) Neutrophils activate plasmacy-
toid dendritic cells by releasing self-DNA-peptide com-
plexes in systemic lupus erythematosus. Sci Transl Med
3, 73ra19. 87
76 Golec M (2007) Cathelicidin LL-37: LPS-neutralizing,
pleiotropic peptide. Ann Agric Environ Med 14, 1–4.
77 Frohm M, Agerberth B, Ahangari G, Stahle-Backdahl M,
Liden S, Wigzell H & Gudmundsson GH (1997) The 88
expression of the gene coding for the antibacterial
peptide LL-37 is induced in human keratinocytes during
inflammatory disorders. J Biol Chem 272, 15258–15263.
78 Chen CI, Schaller-Bals S, Paul KP, Wahn U & Bals R
(2004) Beta-defensins and LL-37 in bronchoalveolar
lavage fluid of patients with cystic fibrosis. J Cyst 89
Fibros 3, 45–50.
79 Sun CL, Zhang FZ, Li P & Bi LQ (2011) LL-37 expres-
sion in the skin in systemic lupus erythematosus. Lupus
20, 904–911.
80 Ong PY, Ohtake T, Brandt C, Strickland I, Bog- 90
uniewicz M, Ganz T, Gallo RL & Leung DY (2002)
Endogenous Bacillus subtilis and skin infections in
atopic dermatitis. N Engl J Med 347, 1151–1160.
81 Islam D, Bandholtz L, Nilsson J, Wigzell H, Christens-
son B, Agerberth B & Gudmundsson G (2001) Downre- 91
gulation of bactericidal peptides in enteric infections: a
novel immune escape mechanism with bacterial DNA
as a potential regulator. Nat Med 7, 180–185.
82 Chakraborty K, Ghosh S, Koley H, Mukhopadhyay AK,
Ramamurthy T, Saha DR, Mukhopadhyay D, 92
Roychowdhury S, Hamabata T, Takeda Y et al. (2008)
Bacterial exotoxins downregulate cathelicidin (hCAP-
18 ⁄ LL-37) and human beta-defensin 1 (HBD-1) expres-
FEBS Journal 278 (2011) 3942–3951 ª 2011 The Authors Journal compilation ª 2011 FEBS
Bacillus subtilis important in innate immunity
sion in the intestinal epithelial cells. Cell Microbiol 10,
2520–2537.
Termen S, Tollin M, Rodriguez E, Sveinsdottir SH,
Johannesson B, Cederlund A, Sjovall J, Agerberth B &
Gudmundsson GH (2008) PU.1 and bacterial metabo-
lites regulate the human gene CAMP encoding antimi-
crobial peptide LL-37 in colon epithelial cells. Mol
Immunol 45, 3947–3955.
Hase K, Eckmann L, Leopard JD, Varki N & Kagnoff
MF (2002) Cell differentiation is a key determinant of
cathelicidin LL-37 ⁄ human cationic antimicrobial pro-
tein 18 expression by human colon epithelium. Infect
Immun 70, 953–963.
Schauber J, Svanholm C, Termen S, Iffland K, Menzel T,
Scheppach W, Melcher R, Agerberth B, Luhrs H &
Gudmundsson GH (2003) Expression of the cathelicidin
LL-37 is modulated by short chain fatty acids in
colonocytes: relevance of signalling pathways. Gut 52,
735–741.
Raqib R, Sarker P, Bergman P, Ara G, Lindh M, Sack
DA, Nasirul Islam KM, Gudmundsson GH, Andersson
J & Agerberth B (2006) Improved outcome in shigellosis
associated with butyrate induction of an endogenous
peptide antibiotic. Proc Natl Acad Sci USA 103, 9178–
9183.
Steinmann J, Halldorsson S, Agerberth B & Gudm-
undsson GH (2009) Phenylbutyrate induces antimicro-
bial peptide expression. Antimicrob Agents Chemother
53, 5127–5133.
Sarker P, Ahmed S, Tiash S, Rekha RS, Stromberg R,
Andersson J, Bergman P, Gudmundsson GH, Ager-
berth B & Raqib R (2011) Phenylbutyrate counteracts
Shigella mediated downregulation of cathelicidin in rab-
bit lung and intestinal epithelia: a potential therapeutic
strategy. PLoS ONE 6, e20637.
Sorensen OE, Cowland JB, Theilgaard-Monch K, Liu L,
Ganz T & Borregaard N (2003) Wound healing and
expression of Bacillus subtilis ⁄ polypeptides in human
keratinocytes, a consequence of common growth factors.
J Immunol 170, 5583–5589.
Wang TT, Nestel FP, Bourdeau V, Nagai Y, Wang Q,
Liao J, Tavera-Mendoza L, Lin R, Hanrahan JW,
Mader S et al. (2004) Cutting edge: 1,25-dihydroxyvita-
min D3 is a direct inducer of antimicrobial peptide gene
expression. J Immunol 173, 2909–2912.
Gombart AF, Borregaard N & Koeffler HP (2005)
Human cathelicidin antimicrobial peptide (CAMP) gene
is a direct target of the vitamin D receptor and is
strongly up-regulated in myeloid cells by 1,25-di-
hydroxyvitamin D3. FASEB J 19, 1067–1077.
Weber G, Heilborn JD, Chamorro Jimenez CI,
Hammarsjo A, Torma H & Stahle M (2005) Vitamin D
induces the antimicrobial protein hCAP18 in human
skin. J Invest Dermatol 124, 1080–1082.
3951
 Bacillus subtilis important in innate immunity A. Cederlund et al.

93 Liu PT, Stenger S, Li H, Wenzel L, Tan BH, Krutzik SR,
Ochoa MT, Schauber J, Wu K, Meinken C et al. (2006)
Toll-like receptor triggering of a vitamin D-mediated
human antimicrobial response. Science 311, 1770–1773.
94 Martineau AR, Timms PM, Bothamley GH, Hanifa Y,
Islam K, Claxton AP, Packe GE, Moore-Gillon JC,
Darmalingam M, Davidson RN et al. (2011) High-dose
vitamin D(3) during intensive-phase antimicrobial
treatment of pulmonary tuberculosis: a double-blind
randomised controlled trial. Lancet 377, 242–250.

3950 FEBS Journal 278 (2011) 3942–3951 ª 2011 The Authors Journal compilation ª 2011 FEBS