Chicken cathelicidin-B1, an antimicrobial guardian
at the mucosal M cell gateway

Ryo Goitsuka*†, Chen-lo H. Chen‡§, Lesley Benyon‡, Yusuke Asano*, Daisuke Kitamura*, and Max D. Cooper‡§¶
**††
*Research Institute for Biological Sciences, Tokyo University of Science, Noda, Chiba 278-0022, Japan; ‡Division of Developmental and Clinical Immunology,
University of Alabama at Birmingham, Birmingham, AL 35294-2812 ; and Departments of ¶Medicine,
Pediatrics, §Microbiology, and **Pathology, University
of Alabama at Birmingham, Birmingham, AL 35294-3300
Contributed by Max D. Cooper, July 26, 2007 (sent for review May 25, 2007)

Mucosal epithelial M cells provide an efficient portal of entry for
microorganisms. Initially defined by their irregular microvilli and
abundant transcytotic channels in the avian bursa of Fabricius,Mcells
also are found in the lymphoid follicle-associated epithelium of the
mammalian appendix, Peyer’s patches, and other mucosal surfacelymphoid
interfaces. We describe here a previously unrecognized
cathelicidin gene in chickens, chCATH-B1, that is expressed exclusively
in the epithelium of the bursa of Fabricius. Like the mature peptides
of previously identified cathelicidins, the carboxyl-terminal peptide of
chCATH-B1 has broad antimicrobial activity against Gram-positive
and Gram-negative bacteria. chCATH-B1 expression is restricted to
the secretory epithelial cell neighbors of the M cells, whereas its
mature peptide is transported to become concentrated on the fibrillar
network surrounding basolateral surfaces of the M cells that overlie
the bursal lymphoid follicles. We conclude that chCATH-B1 is well
placed to serve a protective antimicrobial role at the M cell gateway.
antimicrobial peptides  follicle-associated epithelium  innate immunity 
bursa of Fabricius
The survival of all multicellular organisms depends on an
effective immune response to microbial pathogens. The first
hurdle to microbial entry is provided by our epithelial surfaces.
Microbial pathogens that mount this barrier encounter innate
immunity elements as a first line of defense. Innate immune
responses also facilitate the ensuing adaptive immune responses
that vertebrates use to clear infectious agents. The mucosal M
cells are an important microbial portal of entry because of their
highly efficient pinocytotic channels. Initially identified as specialized
epithelial cells overlying the lymphoid follicles of the
avian bursa of Fabricius that have irregular microvilli and
efficient transcytotic capability (1),Mcells were also found to be
conserved in the lymphoid follicle-associated epithelium of
mammalian appendix and intestinal Peyer’s patches (1, 2). The
Mcells have since been found in other mucosal lymphoid tissues,
including those of the upper and lower airways, oropharynx,
salivary glands, stomach, colon, and eye (3, 4), where they
provide an efficient conduit for transporting microorganisms
and other antigenic substances into the underlying lymphoid
structures to initiate immune responses (5, 6). Despite the
physiological importance of this entry portal, there is limited
information about the differentiation of the M cells, their
transport mechanism(s), and how the microbes that constantly
enter the body via the M cells are rendered noninvasive.
Antimicrobial peptides are well known as front-line participants
in microbial defense (7–10). Two evolutionary groups of
antimicrobial peptides, the cathelicidins and the defensins, provide
endogenous peptide-based defense against microbial invasion
(11–13). Cathelicidins and defensins are produced by many
cell types and have broad spectrum antimicrobial activity against
bacteria, fungi, and viruses. As one example, the human cathelicidin
LL-37 (also called hCAP-18, FALL-39, and CAMP) is
produced by neutrophils, B cells,
 T cells, natural killer cells,
monocytes, and macrophages (14–16); it is also found in the
squamous epithelium of the mouth, tongue, and esophagus, as
well as in the colonic and bronchial mucosal epithelium (17).
LL-37 expression is negligible in normal skin, but epidermal cells
are induced to express high levels of LL-37 in inflammatory
conditions, such as psoriasis and contact dermatitis (18). Conversely,
deficiencies in the LL-37 cathelicidin and the HBD-2
defensin may underlie the Staphylococcus aureus skin infections
that plague patients with atopic dermatitis (19). Recent studies
have also indicated the importance of epithelial cathelicidin in
the maintenance of the sterility of the human urinary tract (20).
Remarkably, neither cathelicidins nor defensins have been identified
at the M cell interface, where one might anticipate their
need. We report here an avian cathelicidin that appears to fulfill
this expectation. This peptide was identified during a search for
genes expressed preferentially in the bursa of Fabricius.
Results
Identification of a Bursa-Specific Cathelicidin, chCATH-B1. Our search
for bursa-specific genes began with the cloning of bursal cDNA
subtracted by splenic cDNA and yielded cDNA clones, some of
which have been reported (21). Among these, BFG7 is expressed
exclusively in the bursa of Fabricius as shown by Northern blot
analysis (Fig. 1). Sequence analysis of a full-length BFG7 cDNA did
not yield a match with then-reported genes. However, a BLAST
search of the National Center for Biotechnology Information
protein database revealed aBFG7cathelin domain sequence, which
is a conserved hallmark of the cathelicidin gene family (Fig. 2).
Although conserved cathelin regions of mammalian cathelicidins
share 50% or greater amino acid identity (12), the BFG7 cathelin
region has only 20–30% homology with mammalian cathelicidins.
This is in the ‘‘twilight’’ zone of sequence similarity but is within the
range of identity shared by many avian and mammalian orthologs
(22). Provisionally, we have named this cathelicidin relative
‘‘chicken cathelicidin-B1,’’ or chCATH-B1, in view of its selective
expression in the bursa of Fabricius.
Proteolytic cleavage of mammalian cathelicidin proproteins
yields mature C-terminal peptides with antimicrobial activity. In
this context, a comparative alignment of chCATH-B1 with
mammalian cathelin region sequences predicted a cationic peptide
of 40 C-terminal amino acid residues with a high pI value
(pI
12.2) (Fig. 2).

This peptide sequence appears to belong to
Author contributions: R.G., C.-l.H.C., and M.D.C. designed research; R.G., C.-l.H.C., L.B., and
Y.A. performed research; C.-l.H.C. contributed new reagents/analytic tools; R.G., C.-l.H.C.,
D.K., and M.D.C. analyzed data; and R.G., C.-l.H.C., and M.D.C. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The sequences reported in this paper have been deposited in the DNA
Data Bank of Japan database, www.ddbj.nig.ac.jp (accession nos. AB307733 and AB308318
for chCATH-B1).
†To whom correspondence may be addressed at: Division of Development and Aging, 2669
Yamazaki, Noda, Chiba 278-0022, Japan. E-mail: ryogoi@rs.noda.tus.ac.jp.
††To whom correspondence may be addressed at: University of Alabama at Birmingham,
401 Shelby Research Building, 1825 University Boulevard, Birmingham, AL 35294-2812.
E-mail: max.cooper@ccc.uab.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0707037104/DC1.
© 2007 by The National Academy of Sciences of the USA