Biospies
Biospies
3
0019-9567/98/$04.00!0
Copyright © 1998, American Society for Microbiology
A growing body of evidence suggests that endogenous antibiotics contribute to the innate defense of
mammalian mucosal surfaces. In the cow, !-defensins constitute a large family of antibiotic peptides whose
members have been previously isolated from the respiratory and oral mucosa, as well as circulating phagocytic
cells. A novel bovine genomic clone with sequence related to those of these "-defensins was isolated and
characterized. The corresponding cDNA was isolated from a small intestinal library; its open reading frame
predicts a deduced sequence of a novel !-defensin, which we designate enteric !-defensin (EBD). Northern blot
analysis of a variety of bovine tissues revealed that EBD mRNA is highly expressed in the distal small intestine
and colon, anatomic locations distinct from those for previously characterized !-defensins. EBD mRNA was
further localized by in situ hybridization to epithelial cells of the colon and small intestinal crypts. Infection
of two calves with the intestinal parasite Cryptosporidium parvum induced 5- and 10-fold increases above control
levels of EBD mRNA in intestinal tissues. An anchored-PCR strategy was used to identify other !-defensin
mRNAs expressed in the intestine. In addition to that of EBD, several low-abundance cDNAs which corre-
sponded to other !-defensin mRNAs were cloned. Most of these clones encoded previously characterized
!-defensins or closely related isoforms, but two encoded a previously uncharacterized prepro-!-defensin.
Northern blot evidence supported that all of these other !-defensin genes are expressed at levels lower than
that of the EBD gene in enteric tissue. Furthermore, some of these !-defensin mRNAs were abundant in bone
marrow, suggesting that in enteric tissue their expression may be in cells of hematopoietic origin. Extracts of
small intestinal mucosa obtained from healthy cows have numerous active chromatographic fractions as
determined by an antibacterial assay, and one peptide was partially purified. The peptide corresponded to one
of the low-abundance cDNAs. This study provides evidence of !-defensin expression in enteric tissue and that
the mRNA encoding a major !-defensin of enteric tissue, EBD, is inducibly expressed in enteric epithelial cells.
These findings support the proposal that !-defensins may contribute to host defense of enteric mucosa.
A striking feature of the mammalian intestinal tract is the nisms protecting this and other mammalian mucosal surfaces
large surface area of the mucosal epithelium. This expansive (5, 30, 35, 48, 60, 66).
surface facilitates nutrient absorption but can, to the detriment Current understanding of mucosal defense suggests that the
of the host, also serve as a port of entry for invading microor- collective actions of multiple innate, nonclonal host defenses
ganisms. Colonizing microbes in the intestinal lumen continu- integrate with the specific clonal immune responses mediated
ously pose a potential threat of infection. The relatively low by lymphocytes (3, 9, 22, 23, 43, 60). In the gastrointestinal
occurrence of intestinally derived systemic infections, however, tract, examples of innate defenses include physical processes,
suggests the presence of effective host defense pathways. A such as peristalsis and shedding of epithelial cells, and chem-
more comprehensive understanding of these pathways may ical barriers, including gastric acidity, mucus, bile acids, and
define therapeutic targets for enhancing host defense. There- proteins (30). Among the proteins which contribute to local
fore, interest has focused on elucidating local defense mecha- defense against microbes are several antibiotic peptides re-
cently identified in extracts from gastrointestinal mucosa (1, 2,
17, 36, 42, 45, 50, 58). In situ hybridization analysis has dem-
onstrated that some of these peptides are synthesized by epi-
* Corresponding author. Mailing address: Department of Immunol- thelial cells (32, 33, 44, 51). Other antibiotic peptides appear to
ogy, NN 10, The Cleveland Clinic Foundation Research Institute, 9500 be products of leukocytes which have migrated to the bowel (1,
Euclid Ave., Cleveland, OH 44195. Phone: (216) 444-9107. Fax: (216) 2, 36).
444-9329. E-mail: bevinsc@cesmtp.ccf.org. The "-defensin class of antimicrobial peptides was unveiled
† Present address: Department of Pathology, The Johns Hopkins
University School of Medicine, Baltimore, MD 21287-6940. with the discovery of tracheal antimicrobial peptide (TAP), a
‡ Present address: Department of Anatomy, Cell Biology and Injury peptide expressed in bovine tracheal epithelial cells (13, 15).
Sciences, University of Medicine and Dentistry of New Jersey, Newark, Numerous genomic sequences related to the TAP gene were
NJ 07103. identified by Southern blot analysis (13), which suggested that
1045
1046 TARVER ET AL. INFECT. IMMUN.
EBD 242a........................................................5'-CCGCATCTCTTCCTTCTTTTACC-3'
EBD 271a........................................................5'-CGCAGTTTCTGTCTCTGCTTAGG-3'
EBD 285a........................................................5'-TTTCTGTCGAAGGCCGCAGTTTCTGTCTCTGCTTAGG-3'
EBD 9UTa ......................................................5'-AGAGGCTGCTCTTGCCTCTTTATAAAGGTCCCAGGTTCT-3'
TAP30s ............................................................5'-ATGAGGCTCCATCACCTGCTC-3'
TAP48a............................................................5'-CCAAGCAGACAGGACCAGGAAGAGGAGCGCGAGGAGCAGGTGATGGAGCCTCAT-3'
TAP286a..........................................................5'-GCTCTGTCAAAGGGCGCAGTTTCTGACTGGGCATTGA-3'
BNBD2/3-189a................................................5'-TCTACCACGACCTGCAGCATTTTATTCGGGGCCCGAA-3'
JR335.B1 .........................................................5'-CTTTTACCACTACCTGCAGCATTTTATTTGGGGCGCT-3'
JR300.C7 .........................................................5'-GGTCCAGGGCACCTGATCAGAATACAGATGCCTCCTT-3'
#Tub-632a .......................................................5'-GTGGTGTGGGTGGTGAGGATAGAGTTGTAGGGCTCAAC-3'
a large family of "-defensin genes exists in the cow. Additional Preliminary sequence analysis indicated that one of these clones, G11, encodes
"-defensin peptides have since been isolated from bovine neu- enteric "-defensin (EBD), and this clone was selected for further analysis.
was terminated by incubation at 65°C for 15 min. The solution was then diluted dried material was redissolved in 0.4 ml of 0.1% TFA and injected onto a C18
to 500 $l with 10 mM Tris-HCl–1 mM EDTA (pH 8.0). A 5-$l aliquot was reverse-phase HPLC column (220 by 4.6 mm; Vydac, Hesperia, Calif.) which had
amplified by PCR with 100 ng each of RACE dT17 adapter (24) and EBD 271a been equilibrated in 0.1% TFA in water (solvent A). The column was washed
in 2.5 mM MgCl2–50 mM KCl–200 $M dNTP–10 mM Tris-HCl (pH 8.3) in a with 5 ml of solvent A at 1 ml/min and then eluted with a 10-min linear gradient
total volume of 50 $l. The PCR products were amplified at denaturing and to 15% solvent B (0.08% TFA in acetonitrile), followed by a 49-min gradient to
extension temperatures of 94 and 72°C, respectively, by using a modification of 30% solvent B, an isocratic elution at 30% solvent B, and finally a linear gradient
a published ramping protocol (16). The annealing temperature was 42°C for five over 5 min to 80% solvent B. Aliquots (50 $l) of each fraction were dried,
cycles and then was increased to 60°C for two cycles. The annealing temperature resuspended in 5 $l of 0.01% acetic acid, and assayed for activity. Three peaks
was decreased 1°C every 2 cycles until it reached 52°C; here, 10 cycles of of activity were observed (eluting at 39 to 41, 52, and 59 min). The first (m/z )
amplification were executed and then the product ends were fully extended at 5,494) and third (m/z ) 6,249) active fractions had a blocked N terminus. The
72°C for 7 min. Each segment of the first five cycles lasted 1 min, and the second active fraction of this separation, eluting at 52 min, was analyzed as
segments of the remaining cycles lasted 30 s. A 0.5-$l aliquot of the resulting follows.
reaction product was amplified in a second PCR with 138 ng of RACE adapter Purified peptides were analyzed by a combination of automated Edman deg-
(24) and 100 ng of EBD 242a as primers. The PCR program was 25 cycles of 30 s radation andmatrix-assisted laser-desorption ionization time-of-flight (MALDI-
at 94°C, 30 s at 55°C, and 30 s at 72°C. This product was extracted after gel TOF) mass spectrometry (18, 19). Mass analysis (with 2% aliquots) was carried
electrophoresis with Mermaid (Bio 101, La Jolla, Calif.) and then phosphory- out with a model Voyager reverse-phase MALDI-TOF instrument (PerSeptive,
lated and filled in with T4 polynucleotide kinase and T4 DNA polymerase (40). Framingham, Mass.) in the linear mode and with #-cyano-4-hydroxycinnamic
The resulting products were subcloned into pBluescript II SK! at the SmaI site acid (Linear Sci., Reno, Nev.) as the matrix; a 30-kV ion acceleration voltage
and subjected to dideoxynucleotide termination sequence analysis. (grid voltage at 70%; guide wire voltage at 0.1%) and &2.0 kV multiplier voltage
For 3'-RACE, a protocol based on that described by Borson (7) was employed, were used. Chemical sequencing (on 95% of the sample) was done with a model
with reagents from Clontech Laboratories, Inc. Briefly, 1 $g of total RNA from 477A instrument from Applied Biosystems (AB) (Perkin-Elmer Corp., Norwalk,
bovine distal ileum was reverse transcribed by using the anchor primer 5'-CC Conn.). Stepwise liberated phenylthiohydantoin amino acids were identified by
FIG. 1. Structure and sequence of the EBD gene. (A) Restriction map of the bovine EBD gene including 0.9 kb of 5' flanking sequence. E, EcoRI; H, HindIII; P,
PstI; UTR, untranslated region; SIG, signal. Also shown is a diagram of the predicted precursor structure of EBD deduced from the gene and cDNA sequences. (B)
Nucleotide sequence of the EBD gene and alignment with the TAP gene sequence (13), with carets representing nucleotide identity. Exons (capital letters) were
determined by comparison with EBD cDNA sequences (Fig. 2). Consensus sequences for TATA boxes (underlined), NF–IL-6 sites (boldface underlined), and H-APF-1
(double underlined) are indicated (see text). The consensus splice junction residues are shown in boldface. The polyadenylation signal is boxed.
VOL. 66, 1998 ENTERIC "-DEFENSIN 1049
extra 5' sequence is contiguous with the adjacent transcribed EBD gene-specific probe, EBD 285a (Fig. 3). These results
genomic sequence, ruling out an alternative 5' exon. The 5' indicate that the EBD gene is a single-copy gene and address
nucleotide of this clone (&47) was 32 nucleotides downstream the specificity of hybridization conditions employed in this
of a second TATA sequence and thus appears to be a second study. The specificity of hybridization with this probe was fur-
site of transcription initiation. We conclude that EBD gene ther demonstrated by hybridizing dot blot panels of cloned
transcription is initiated as indicated in Fig. 2A and that EBD DNAs of various "-defensin genes. Only the EBD gene among
mRNA is derived from splicing of two exons as depicted in Fig. a collection of nine "-defensin-encoding gene sequences was
1 and 2B. recognized by EBD 285a under the same hybridization condi-
The sequences of the deduced prepropeptide and predicted tions as for the Southern and Northern blots (data not shown).
mature peptide of EBD are 72 and 67% identical to those of
TAP, respectively (Fig. 2B). A shared feature of the deduced
amino acid sequence is the cysteine array inherent to the fam-
ily of "-defensins (13, 28, 59). In addition, both sequences have
particularly high similarity of the 5' cDNA sequence and the
corresponding amino acids of the putative signal sequence
encoded by this region.
A search of the genomic sequence for motifs recognized by
transcription factors revealed several possible sites of EBD
gene regulation. For example, there is an NF–interleukin-6
(NF–IL-6) consensus binding site in the 5' flanking region of
EBD positioned where a putative NF-*B site is located in the
TAP gene (Fig. 1). Both the EBD and TAP genes have two
additional NF–IL-6 consensus binding sites similarly located 5'
relative to the respective NF–IL-6/NF-*B site (Fig. 1). The
presence of a consensus binding site for H-APF-1 (Fig. 1), a
factor known to cooperate with NF–IL-6 in gene activation
(39), further supports a possible functional significance of the
putative NF–IL-6 sites. A database search with the entire EBD
gene sequence revealed the presence of several highly repeti- FIG. 3. Southern blot hybridization analysis of the EBD gene. Bovine
genomic DNA (10 $g) was digested with restriction endonucleases, and the
tive elements of the bovine genome, including nucleotides products were size separated by agarose gel electrophoresis. The DNA was
&540 to &729, &370 to &540, and !1530 to !1580. The transferred to a nylon membrane and hybridized with 32P-end-labeled oligonu-
domains between these elements had similarity to the other cleotide EBD 285a (see Materials and Methods). Hybridization conditions were
bovine "-defensin genes previously characterized, but no other 5% SSC–1% SDS–5% Denhardt solution–40 $g of RNA per ml at 42°C in the
presence of 37.5% (vol/vol) formamide. The high-stringency wash of the filter
matches were notable. was with 2% SSC–0.1% SDS at 60°C for 60 min. The autoradiographic exposure
A Southern blot analysis of bovine genomic DNA revealed a was approximately 4 weeks. Numbers on the left indicate sizes (in basepairs) of
single band with each of five restriction enzymes by using an mobility standards.
1050 TARVER ET AL. INFECT. IMMUN.
FIG. 4. Northern blot analysis of EBD gene expression in bovine tissues. (A) Tissue distribution of "-defensins. Total RNA (20 $g) extracted from 14 different
tissues was resolved by denaturing gel electrophoresis, capillary blotted to a nylon filter, and probed with either EBD 285a as an EBD probe, TAP286a as a TAP-specific
probe, TAP48a as a common probe for "-defensins, or an #-tubulin probe. The hybridization and wash conditions were as described in Materials and Methods. Small
intestine samples designated #1 and #2 represent RNAs extracted from the tissues of two healthy cows. (B) Expression of EBD in colonic tissue and usage of the
putative upstream transcription start site. Total RNAs from the distal small (SM.) intestine, proximal colon (10 cm from the ileocecal junction), and distal colon (10
cm from the rectum) were analyzed as for panel A. The Northern blot was hybridized with EBD 285a to assess distribution of expression. The same blot was stripped
of probe and rehybridized with EBD 9UTa, a probe from the unique sequence found in the 5'-extended RACE clone (Fig. 2, clone APT131.5) (see text). (C)
Comparison of fetal and adult tissue expression of EBD. Total RNAs were isolated from the distal small intestine (S.I.) and colon of a bovine fetus at 4 months
gestational age and from corresponding tissues of an adult cow and then analyzed as for panel A. (D) Northern blot analysis of EBD mRNA in enteric tissue from a
calf infected with C. parvum. Total RNA was isolated from the distal 20 cm of small intestine from a C. parvum-infected calf and from a control uninfected calf. Analysis
was as for panel A.
Northern blot and in situ hybridization analysis of EBD colon and small intestine (Fig. 4A, EBD). The intensity of the
mRNA. Northern blot analysis of total RNAs extracted from signal detected in the distal small intestine was strong relative
several bovine tissues with EBD 285a as probe revealed a to the detectable mRNA in more-proximal segments of the
message of about 600 nucleotides abundantly expressed in the small intestine. Comparable levels of mRNA were detected in
VOL. 66, 1998 ENTERIC "-DEFENSIN 1051
the proximal and distal colon (Fig. 4B). Trace hybridization the small intestine and colon than in cells covering the small
was detected in the trachea, and EBD mRNA was not detected intestinal villi (absorptive cells) or on the surface of the colonic
in the bone marrow, adrenal gland, lung, spleen, kidney cortex, mucosa. For individual epithelial cells, the localization of EBD
or kidney medulla (Fig. 4A). Somewhat lower levels of EBD mRNA did not correlate with localization of C. parvum infec-
mRNA were detected in both the distal small intestine and tion, since the heavily infected villus tip cells produced little
colon of a bovine fetus at a gestational age of 4 months as EBD mRNA while the minimally infected crypt cells produced
compared to in an adult cow (Fig. 4C). Hybridization of the abundant EBD mRNA. Therefore, C. parvum does not appear
filter with an oligonucleotide sequence specific for mRNA to directly induce EBD mRNA production in individual in-
derived from the &47 transcription initiation site demon- fected cells but rather causes the hyperplasia of uninfected
strated an identical distribution of signal but uniformly at a crypt cells which produce EBD mRNA.
lower relative intensity (Fig. 4B, 9UTa, and data not shown). Identification of !-defensin isoforms expressed in intestinal
All samples had intact RNA as evidenced by hybridization to tissue. To investigate the possibility that additional "-defensin
an #-tubulin probe (Fig. 4A to C). Hybridization at low strin- mRNA isoforms are expressed in the bovine small intestine, an
gency with a probe from the 5' cDNA region with sequence anchored-PCR strategy was employed. Invariant nucleotide
nearly identical in all characterized members of the bovine sequences have been found in the 5' portions of all bovine
"-defensin gene family reveals the presence of abundant "-defensin cDNAs cloned to date (13, 53, 57, 65). A PCR
mRNA in several tissues (Fig. 4A, "-Defensin), consistent with primer, BTAP-27s, was selected from this region of high nu-
widespread expression of various "-defensins. The relative cleotide similarity. RNA from the distal ileum was reverse
FIG. 5. Detection of EBD mRNA by in situ hybridization. Paraffin-embedded sections of bovine ileum (A to D) and colon (E to H) were hybridized with EBD
riboprobes labeled with [35S]UTP, washed under high-stringency conditions, and processed as described in Materials and Methods. (A, C, E, and G) Normal, uninfected
intestinal tissue; (B, D, F, and H) intestinal tissue infected by C. parvum. The parasites are not visible at this magnification, but the blunting of the small intestinal villi
and the inflammation of the lamina propria induced by the infection are easily seen in panels B and D. In normal ileum and colon, EBD mRNA is localized within
epithelial cells at the base of the crypts (C and G). In C. parvum-infected ileum and colon, abundant EBD mRNA is present in epithelial cells throughout the elongated
crypt (D and H). Magnification, %80.
detected, and several were partially characterized. As de- composed of one component which had a molecular mass-to-
scribed in Materials and Methods, a combination of gel filtra- charge ratio (m/z) of 4,113 + 1.6 and minor species with m/z )
tion, ion-exchange chromatography, and reverse-phase chro- 7,220 and 14,440. The two minor species are almost certainly
matography was used to partially purify an active fraction related, with either the 7,220 species representing the doubly
relevant to this report, APT161G (Fig. 7A). This fraction was charged form of a 14,440-Da component or the 14,440 species
VOL. 66, 1998 ENTERIC "-DEFENSIN 1053
2) indicates a two-exon gene structure, similar to that of the "-defensin genes are highly similar to one another, the genes
TAP gene. Its nucleotide identity with the TAP gene is 84% have no detectable sequence similarity to those of mammalian
across the gene. This finding is most consistent with the two #-defensins (data not shown). Recent data indicate that #- and
genes arising from relatively recent gene duplication and/or "-defensin genes are located in the same chromosomal cluster
conversion events. Yount et al. have recently determined a (38), suggesting that they are part of a single gene family which
similar two-exon structure for the gene encoding the hemato- arose from a common ancestral gene. The lack of significant
poietic "-defensin BNBD-4 (65). The nucleotide identity be- sequence similarity suggests that these two subfamilies di-
tween the EBD and BNBD-4 genes is also quite high (88%) in verged long ago and/or that sequence divergence in this gene
the 969 nucleotides of available BNBD-4 gene sequence. All family has occurred at an accelerated pace. Nevertheless, the
three of these genes have been localized to bovine chromo- expression of "-defensin genes in tissues which come in fre-
some 27 (25), further supporting an evolutionary history of quent contact with bacteria and other microbes supports the
divergence from a common ancestral gene. A striking feature idea that this gene subfamily, like the #-defensin subfamily, is
of this comparison is the high similarity of nucleotide sequence part of a first-line host defense system.
throughout these three genes, given the dramatic difference in An anchored-reverse-transcription-PCR strategy has re-
tissue expression (Fig. 4) (65). Thus, an important question vealed that several additional "-defensins [TAP, TAP(S20N),
that remains to be addressed is the identity of sequences within LAP, BNBD-3, BNBD-4, BNBD-9, and BBD-C7] are also
these genes which mediate their distinct patterns of expression. expressed in the distal small intestine but at much lower rela-
A second feature is that although the sequences of these three tive abundances. The cellular sources of these low-abundance
VOL. 66, 1998 ENTERIC "-DEFENSIN 1055
23. Fiocchi, C. 1996. Cytokines and intestinal inflammation. Transplant Proc. 46. Patterson, S., J. Gross, and A. B. D. Webster. 1989. DNA probes bind
28:2442–2443. nonspecifically to eosinophils during in situ hybridization: carbol chromo-
24. Frohman, M. A., M. K. Dush, and G. R. Martin. 1988. Rapid production of trope blocks binding to eosinophils but does not inhibit hybridization to
full-length cDNAs from rare transcripts: amplification using a single gene- specific nucleotide sequences. J. Virol. Methods 23:105–109.
specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85:8998–9002. 47. Petersen, C. 1992. Cryptosporidiosis in patients infected with the humanim-
25. Gallagher, D. S., A. M. Ryan, G. Diamond, C. L. Bevins, and J. E. Womack. munodeficiency virus. Clin. Infect. Dis. 15:903–909.
1995. Somatic cell mapping of "-defensin genes to cattle syntenic group U25 48. Pleyer, U., and H. Baatz. 1997. Antibacterial protection of the ocular surface.
and fluorescent in situ hybridization localization to chromosome 27. Mamm. Ophthalmologica 1:2–8.
Genome 6:554–556. 49. Porter, E., L. Liu, A. Oren, P. Anton, and T. Ganz. 1997. Localization of
26. Gilman, M. 1987. In situ hybridization, sections 14.1–8 and 14.3–14. In R. M. human intestinal defensin 5 in Paneth cell granules. Infect. Immun. 65:2389–
Ausubel, R. Brent, R. E. Kinston, D. L. Moore, J. G. Seidman, J. Smith, and 2395.
K. Struhl (ed.), Current protocols in molecular biology. John Wiley and 50. Porter, E., E. van Dam, E. Valore, and T. Ganz. 1997. Broad-spectrum
Sons, New York, N.Y. antimicrobial activity of human intestinal defensin 5. Infect. Immun. 65:
27. Harder, J., J. Bartels, E. Christophers, and J. M. Schroder. 1997. A peptide 2396–2401.
antibiotic from human skin. Nature 387:861. (Letter.) 51. Reilly, D. S., N. Tomassini, C. L. Bevins, and M. Zasloff. 1994. A Paneth cell
28. Harwig, S. S. L., K. M. Swiderek, V. N. Kokryakov, L. Tan, T. D. Lee, E. A. analogue in Xenopus small intestine expresses antimicrobial peptide genes:
Panyutich, G. M. Aleshina, O. V. Shamova, and R. I. Lehrer. 1994. Galli- conservation of an intestinal host-defense system. J. Histochem. Cytochem.
nacins: cysteine-rich antimicrobial peptides of chicken leukocytes. FEBS 42:697–704.
Lett. 342:281–285. 52. Russell, J. P., and C. L. Bevins. Unpublished observations.
29. Huttner, K. M., C. A. Kozak, and C. L. Bevins. 1997. The mouse genome 53. Russell, J. P., G. Diamond, A. Tarver, and C. L. Bevins. 1996. Coordinate
encodes a single homolog of the antimicrobial peptide human b-defensin 1. induction of two antibiotic genes in tracheal epithelial cells exposed to the
FEBS Lett. 413:45–49. inflammatory mediators lipopolysaccharide and tumor necrosis factor alpha.
Editor: J. R. McGhee
ERRATA
Allelic Polymorphisms at the H-2A and HLA-DQ Loci Influence
the Response of Murine Lymphocytes to the Mycoplasma
arthritidis Superantigen MAM
BARRY C. COLE, ALLEN D. SAWITZKE, ELSAYED A. AHMED,
CURTIS L. ATKIN, AND CHELLA S. DAVID
Division of Rheumatology, University of Utah School of Medicine, Salt Lake City, Utah 84132,
and Department of Immunology, Mayo Clinic, Rochester, Minnesota 55095
Volume 65, no. 10, p. 4191, column 1, lines 31 and 32: “. . . genes in knockout mice lacking endogenous mouse class II molecules”
should read “. . . genes in transgenic mice with a B10.M background.”
Volume 66, no. 3, p. 1045, Abstract, line 4: “A novel bovine genomic clone with sequence related to those of these
"-defensins. . .” should read “A novel bovine genomic clone with !-defensin-related sequence. . .”
2399
2400 ERRATA INFECT. IMMUN.