Candidacidal activity of human salivary histatin recombinant variants produced by site-directed mutagenesis

GENE AN (NTERNATIONAL ,JOURNAL GENES AND GENOMES

ELSEVIER

ON

Gene 177 (1996)29 34

Candidacidal activity of human salivary histatin recombinant variants produced by site-directed mutagenesis James Driscoll a, Chunni D u a n a, Yi Zuo a, Tao Xu a, Robert Troxler a,b, Frank G. Oppenheim a,b,. a Department of Periodontology and Oral Biology, School of Graduate Dentistry, School of Medicine, Boston University Medical Center, Boston, MA 02118, USA b Department of Biochemistry, School of Medicine, Boston University Medical Center, Boston, MA 02118, USA Received 12 December 1995; revised 19 February 1996; accepted 21 February 1996

Abstract

Histatin 5 (Hst5) is a 24-amino acid (aa) member of the Hst family that is found in human salivary secretions and exhibits candidacidal activity. Hst5 contains a 13-aa region that alone is capable of killing fungal pathogens and is referred to as the functional domain. To investigate the role of specific aa located within the functional domain, the pRSET bacterial expression system was used to produce recombinant Hst5 (re-Hst5) and several re-variants that were generated by site-directed mutagenesis. The vector pRSETC expresses genes of interest as fusion proteins attached to the carboxy end of an N-terminal His 6 tag that binds to nickel (Ni2+). The re-variants were generated using the polymerase chain reaction (PCR) and had Gly substituted for either the His, Glu or Lys/Arg within the functional domain. PCR products that encoded either the wild-type or variant forms of re-Hst5 were inserted into pRSETC and produced as fusion proteins which were affinity purified from cell lysates by Ni2+-Sepharose chromatography. Fusion proteins were digested with CNBr and re-Hsts were purified by reversed-phase high performance liquid chromatography (RP-HPLC). Re-Hsts were tested in bioassays to measure the ability to kill both Candida albicans (C. albicans) blastoconidia and spheroplasts which were generated by removal of the cell wall. In both assays, re-Hst5 displayed dose-dependent candidacidal activity that was nearly identical to that of native Hst5 purified from human salivary secretions. Re-Hst5 variants with either Glu or Lys/Arg substitutions demonstrated significantly lower candidacidal activity in both assays, while the variant with His mutated showed essentially no activity at physiological concentrations. These results indicate that acidic and basic aa within the functional domain contribute to candidacidal activity and that the His are essential for candidacidal activity. Additionally, since C. albicans spheroplasts were also susceptible to Hsts, the cell wall is not an essential component in the Hst mechanism of candidacidal action. Keywords: Salivary antimicrobial; pRSET expression system; Niz+-Sepharose chromatography; Blastoconidium; Spheroplast

1. Introduction

Histatins (Hsts) comprise a family of histidine (His)-rich proteins secreted by human parotid and sub-

* Corresponding author. Tel. + 1 617 6384942; Fax + 1 617 6384924; e-mail: [email protected] Abbreviations: A, absorbance (1 cm); aa, amino acid(s); Ap, ampicillin; Arg, arginine; C., Candida; Da, dalton; Glu, glutamic acid; Gly, glycine; HPS, human parotid saliva, Hst, histatin(s); His, histidine; Hst, gene (DNA) encoding Hst; IPTG, isopropy113-D-thiogalactopyranoside; LB, Luria-Bertani(medium); LDso , lethal dose required for 50% cell killing; oligo, oligodeoxyribonucleotide; Lys, lysine; PAGE, polyacrylamidegel electrophoresis; PCR, polymerase chain reaction; re-, recombinant; re-Hst3, recombinant Hst3; re-Hst5, recombinant Hst5; RP-HPLC, reversed-phase high performance liquid chromatography; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid. 0378-1119/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PH S 0 3 7 8 - 1 1 1 9 ( 9 6 ) 0 0 2 6 5 - X

mandibular glands (Oppenheim et al., 1988; Troxler et al., 1980). Hsts have been established as major hostdefense molecules that exert antimicrobial activity within the oral cavity (MacKay et al., 1984; Oppenheim et al., 1988; Xu et al., 1991) and recent evidence indicates that Hsts bear therapeutic promise for gene transfer to salivary glands in the treatment or prevention of orallyrelated candidiasis (Baum and O'Connell, 1995). The major members of the Hst family are Hst 1, 3 and 5 which consist of 38, 32 and 24 aa, respectively, and demonstrate highly homologous primary strucutures (Oppenheim et al., 1986,Oppenheim et al., 1988). To identify a functional domain within Hsts responsible for candidacidal activity, various peptides were synthesized based upon the primary structure of Hst3 and Hst5 and tested for candidacidal activity against the opportunistic oral pathogen Candida albicans (Xu and Oppenheim,

30

J. Driscoll et al./Gene 177 (1996) 29 34

a PCR-Amplified Hst Fragments

Vector pRSETC

5". BamHI Hst 5 Insert

I PT7 RBS ATG BamHI EcoRl Term ]

EcoRl 5"

~0

2 . 0 kb

[~

"lfBamHIIEcoRI T4 DNA ligase

I PT7- ATG- HIS-

BamHI- Hst 5 cDNA- EcoRI-

Term.]

CONSTRUCTS pHst 5 pAGlu pALys/Arg pAHis

b Expression vectors and primary structure of resulting re-proteins generated by site-directed mutagenesis

Expression vector

Re-protein

Mass, daltons

Primary structure 1

10

20

24

pHsl5

re-Hst5

3037

D-S-H-A-K-R-H-H-G Y-K-R K-F-H-E-K-H-H-S-H-R-G-Y

pHst5AHis

re-HsLSAHis

2717

D S H A-K-R-H-H-G-Y-K-R-K-F-G-E-K-G-G-S-G-R-G-Y

pHst5AGlu

re-Hst5AGlu

2965

D S H A-K-R-H-H-G-Y K R-K F-H-G-K-H-H-S-H-R-G-Y

pHst5ALys/Arg

re-Hst5ALys/Asg

2697

D-S-H A K R-H-H-G-Y-K-G-G-F-H-E-G-H-H-S-H-G-G-Y

1

1

1

10

10

10

20

20

20

24

24

24

Fig. 1. Schematic representation of pHst5 expression vectors and the resulting re-proteins. (a) Construction of pHst5 expression vectors. PCRgenerated fragments were digested with BamHI + EcoRI and inserted into pRSETC at the indicated restriction sites. (b) Expression vectors and primary structure of resulting re-proteins generated by site-directed mutagenesis. Amino acids (aa) are given in the single-letter code. The functional domain is underlined and aa substituted by site-directed mutagenesis are indicated in bold. Methods: The sense oligo (5'-GCGCGC G G A T C C C A G C T G C T G C C A T G G A T T C A ) and antisense oligo ( 5 ' - G C G C G A A T T C C T C A T C A A T A G C C T C G A T G T G A ) were used to amplify an insert that encoded Hst5 by using pHst3 as a template for PCR. The sense oligo added sequence that encoded a BamHI site and a Met directly adjacent to the codon that encoded aa 1 (Asp) of re-Hst5. The antisense oligo added sequence that encoded a stop codon and an EcoRI site. The fragment was inserted into p R S E T C as indicated by incubation with T4 ligase overnight at 16°C and the resulting plasmid was called pHst5. The sense oligo used above was also used to construct the three additional plasmids that encoded the re-Hst5 variants. First, the sense oligo and the antisense oligo ( 5 ' - G C G C G A A T T C C T C A T C A A T A G C C T C G A C C T G A A C C A C C C T T T T C A C C G A A ) were used to PCR-amplify a Hst5 insert that encoded a variant in which His 15, His TM, His 19 and His zl were converted to Gly. The PCR-amplified insert was digested with BamHl + EcoRI and inserted into pRSETC as above and the resulting plasmid was called pHst5AHis. The same sense oligo and the antisense oligo ( 5 ' - G C G C G A A T T C C T C A T C A A T A G C C T C G A C C T G A A C C A C C C T T T T C A C C G A A ) were used to amplify a Hst5 insert that encoded a variant in which the Glu 16 was converted to Gly. The insert was digested with BamHI+EcoRI, inserted into pRSETC and the resulting plasmid called pHst5AGlu. The sense oligo was used along with the antisense oligo ( 5 ' - G C G A A T T C T C A T C A A T A G C C T C C A T G T G A A A T G A T G C T T T T C A T G G A A T C C T C C T T ) were used to generate a PCR-amplified Hst5 insert that encoded a variant in which Arg 12, Lys 13, Lys 17 and Arg z2 were converted to Gly. The fragment was inserted into pRSETC and the resulting plasmid called pHst5ALys/Arg. Correct insertion of the Hst5 c D N A was confirmed by D N A sequencing using the dideoxynucleotide termination method.

J. Driscoll et al./Gene 177 (1996) 29 34 1993; R a j et al., 1993). T a k e n

31

bility in the design of Hst variants with altered primary

together, these studies for

s t r u c t u r e s . T h e specific a i m o f t h e p r e s e n t s t u d y w a s t o

candidacidal activity. To investigate the structure-function relationship of H s t s a n d t h e r o l e o f specific a a l o c a t e d w i t h i n t h e

generate re-Hst5 and several re-variants by site-directed

indicated

that

aa

12-24 of Hst5

was responsible

m u t a g e n e s i s o f H s t 5 c D N A ( L a n d t et al., 1990). W i l d type re-Hst5 and variants that contained site-directed aa substitutions within the functional domain were pro-

f u n c t i o n a l d o m a i n , w e d e v e l o p e d efficient s y s t e m s f o r the bacterial production and affinity purification of r e - H s t s a n d r e - v a r i a n t s ( Z u o et al., 1995; D r i s c o l l et al.,

d u c e d a n d t e s t e d f o r c a n d i d a c i d a l a c t i v i t y a g a i n s t C. albicans blastoconidia and spheroplasts generated by r e m o v a l o f t h e cell wall. T h e s e s t u d i e s h a v e i d e n t i f i e d a a

1996). T h e m o l e c u l a r b i o l o g i c a l a p p r o a c h g a v e h i g h y i e l d s o f f u n c t i o n a l l y a c t i v e r e - H s t s a n d p r o v i d e d flexi-

that are either involved in or are essential for Hst activity.

a

b kDa

1

2

3

4

5

6

7

8

HPS

43

2s

1

2

3

4

m

14

Hst3>Hs~5~Fig. 2. Gel electrophoresis of purified re-Hsts. (a) SDS-PAGE. Lanes: 1, molecular weight markers; 2, native Hstl (2 gg); 3, native Hst3 (2 gg); 4, native Hst5 (2 gg); 5, re-Hst5 (2 lag); 6, reHst5AGlu (2 lag); 7, re-Hs5ALys/Arg (2 lag); 8, re-Hst5AHis (2 I.tg). (b) Cationic Gel electrophoresis. Lanes: 1, HPS (160 lag); 2, re-Hst5 (2 lag); 3, reHst5AGlu (2 gg); 4, re-Hst5ALys/Arg (2 lag); 5, re-Hst5AHis (2 lag). Methods: Plasmids were transformed into cells from bacterial strain BL21 (DE3) and inoculated into Luria-Bertani (LB) media containing 100 lag/mL ampicillin (Ap). Large-scale cultures were grown to an A6oo,m of 0.8 and IPTG (0.4 mM) was added. Cultures were incubated for an additional 3 h before harvesting of cells by centrifugation. Pellets were resuspended, sonicated and lysed in 20 raM. phosphate pH 7.8/0.5 M NaC1/6 M guanidine • HC1. Lysates were centrifuged and the supernatant was passed through a 0.2 mm filter (Acrodisc, Gelman). NiZ+-Sepharose chromatography: The filtrate was applied to a 1.5 x 11 cm Ni 2+-Sepharose (ProBond ~ Resin, Invitrogen) column equilibrated in denaturing buffer (20 mM Na •phosphate pH 7.8/0.5 M NaC1/8 M urea). Following application of the sample, most bacterial protein was found in the unadsorbed fraction. Some nonspecifically bound protein was eluted at pH 6 and the fusion protein was eluted at pH 4. The yield of fusion protein was about 8 mg/L culture. Protein elution was monitored by Az8o ,m and fractions containing fusion protein were pooled, dialyzed against water, and lyophilized. Since a Met was introduced by PCR at the junction between the fusion carrier and the first aa (Asp) of the re-Hsts, fusions were digested with CNBr and then purified by RP-HPLC. Fusion protein cleavage with CNBr: Fusions were dissolved as a 1% protein solution in 70% formic acid. CNBr was added at a 100-fold molar excess relative to the Met content and cleavage proceeded for 24 h. The mixture was then diluted 15-fold with water and lyophilized. Purification of re-Hst5 and re-variants by RP-HPLC: CNBr digests were resuspended in 2% acetic acid and subjected to RP-HPLC on a TSK-ODS (120T) C18 column. Solvent A was 0.1% triftuoroacetic acid in water, and solvent B was 0.1% trifluoroacetic acid in 80% acetonitrile, 20% water. Eluted proteins were evaporated to dryness in a flash evaporator, redissolved in 2% acetic acid, and re-chromatographed at a concentration of solvent B 8% lower than that which released the protein from the column initially. The aa composition was determined using a Waters PicoTAG station (Xu et al., 1980). The aa composition of re-Hst5 was identical to that of native Hst5 within experimental error and the aa composition of re-Hst5 variants was identical to that deduced from the cDNA sequence. The yield of purified re-Hst5 and variants was approximately 4 mg/L of bacterial culture. Samples were electrophoresed on 15% SDS-polyacrylamide slab gels (16 x 20 cm) as described (Laemmli, 1970). Cationic PAGE was performed using 15% polyacrylamide gels (16 x 20 cm) essentially as described (Oppenheim et al., 1986). Gels were fixed in 20% TCA and stained in 0.1% Coomassie Brilliant blue and destained in 5% methanol/7% acetic acid.

J. Driscoll et al./Gene 177 (1996) 29-34

32

a

loo r o~

/

~

60

.__ N

40

_~ m

2o

c o o

'

Histatin5 Physiological c°ncentrati°nrange~ ~ - - - - - ~

~ ~ ' ~

0 0.2

0.5

0.8

1.1

1.4

1.7

Hst concentration, log nmol/mL

b 100 Histatin 5 Physiologic= concentration range

o~

Native

80

Hs~

re-Hst5 ~

60

re-Hst5AGlu

c °

-

LLys/Arg

-

Q.

4o

re-Hst5•His

e-e~

~

20

0

I

I

I

I

I

0.2

0.5

0.8

1.1

1.4

.7

Hst concentration, log nmol/mL Fig. 3. Candidacidal activity of native Hst5, re-Hst5 and re-variants. (a) Blastoconidia killing assay. Hsts were added to wells of a microtiter plate containing 1 x 1 0 4 C. albicans cells and incubated at 37°C for 1 h. Cells were washed, overlayed with molten Sabouraud's dextrose broth, incubated for 6 h at 30°C and candidacidal activity was determined as described (Zuo et al., 1995). Values shown represent the mean of triplicate measurements at each Hst concentration from a single experiment. Error bars represent the standard deviation calculated from the triplicate measurements and the mean value. Comparable results were obtained in a second experiment. (b) Spheroplast killing assay. Spheroplasts were prepared by treatment of mid-log phase blastoconidia with lyticase as described (Torres-Bauza and Riggsby, 1980). Values shown represent the mean of duplicate measurements in a single experiment. Methods: C. albicans (strain 44505) was grown on Sabouraud's dextrose agar plates at 30°C for 18 h, harvested, and suspended in 10 mM K. phosphate, pH 7.4 (suspension buffer) at 1 × 105 cells/mE Cells (100 I~L) were added to wells of a 96-well microtiter plate and combined with 100 ~tL of Hsts at final concentrations that ranged from 50 nmol/mL to 1.6 nmol/mL. After preincubation for 1 h at 37°C, wells were washed three times with 150 ~L of suspension buffer, 100 ~tL of molten Sabouraud's dextrose broth containing 2% agarose at 45°C was added to each well and the plate was incubated at 30°C for 6 h. Under these conditions a living cell will divide and form a colony while a dead cell will remain a single cell. To determine the percentage of blastoconidia killed, a total of 100 cells or colonies was counted under a Nikon inverted microscope. Candidicidal activity was calculated according to the formula:

J. Driscoll et al./Gene 177 (1996) 29-34

2. Experimental and discussion 2.1. Construction of expression vectors encoding re-Hst5 and variants To produce re-Hsts, we used the pRSET bacterial expression system that is designed specifically for the integrated high-level expression and affinity purification of re-proteins. The pRSET vectors express foreign sequences as fusion proteins with a 4.4-kDa N-terminal 'carrier' that contains a His 6 tag that binds to nickel (Ni2+). Previously, we constructed a pRSET vector, called pHst3, that produced high yields of biologically active the re-Hst3, 32-aa member of the Hst family (Zuo et al., 1995). Hst5 is derived by the post-translational cleavage of Hst3 and, consequently, the primary structure of Hst5 is identical to the first 24 aa of Hst3 (Troxler et al., 1980). Therefore, we used Hst3 cDNA as a PCR template to generate a Hst5 fragment that was inserted into pRSETC and the resulting plasmid was called pHst5 (Fig. la). Since native Hst5 does not contain any Met, the PCR sense oligo contained an ATG codon directly 5' to the codon encoding the first aa (Asp) of re-Hst5. The Met addition provided a CNBr cleavage site between the carrier and the first aa of the re-Hst5 moiety. Site-directed mutagenesis by PCR was then used to generate variants of re-Hst5 in which either the His, acidic or basic aa within the functional domain were converted to Gly. Since Gly has a small, non-polar and non-ionizable R-group, substitution for aa in the functional domain that have imidazole (His), negative (acidic) or positive (basic) R-groups could be readily detected in the resulting re-proteins. PCR-generated fragments were digested with BamHI + EcoRI, inserted into the IPTGinducible vector pRSETC and the resulting plasmids were called pHst5AHis, pHst5AGlu and pHst5ALys/Arg, respectively. The primary structure and mass of each resulting re-Hst variant is shown (Fig. lb). 2.2. Expression, purification and analysis of re-Hst5 and variants To express the Hst fragments that encoded re-Hst5 and the re-variants, the plasmids constructed above were

33

individually transformed into cells from bacterial strain BL21(DE3). Large-scale (8L) cultures were grown, induced with IPTG and cellular lysates prepared. Fusion proteins were purified from bacterial lysates in one step by Ni2+-Sepharose chromatography and affinity-purified fusions were digested with CNBr and purified by RP-HPLC similar to that described previously (Zuo et al., 1995). Upon SDS-PAGE, re-Hst5 and each of the re-variants exhibited the expected electrophoretic mobilities (Fig. 2a). The absence of other Coomassie blue staining proteins indicated that the re-Hsts were highly purified. Purified proteins were also analyzed by cationic PAGE which provides better resolution of Hsts than does SDS-PAGE since the Hsts migrate at characteristic positions based upon net charge (Oppenheim et al., 1986). Re-Hst5 migrated at a position similar to that of native Hst5 present in HPS. Cationic PAGE indicated that, relative to native Hst5 and re-Hst5, re-Hst5AGlu was more basic, re-Hst5ALys/Arg was slightly more acidic and that re-Hst5AHis was notably more acidic (Fig. 2b).

2.3. Candidacidal activity of re-Hsts The candidacidal activity of re-Hst5 was compared to that of native Hst5 in the blastoconidium killing assay (Fig. 3a). At all test protein concentrations, the activity of re-Hst5 was nearly identical to that of native Hst5 and exhibited a comparable LDso value (2.5 nmol/mL for re-Hst5 and 2.0 nmol/mL for native Hst5). Notably, the re-Hst5 variants all displayed lower activity (Fig. 3a). Re-Hst5AGlu and re-Hst5ALys/Arg displayed lower activity at all doses tested, while re-Hst5AHis was essentially inactive except at high doses. The candidacidal activity of native Hst5, re-Hst5 and the re-Hst5 variants were also tested against spheroplasts which were generated by lyticase treatment to remove the cell wall from blastoconidia. Importantly, both native Hst5 and re-Hst5 were effective in killing spheroplasts at doses comparable to those that killed blastoconidia (Fig. 3b). Re-Hst5AGlu was somewhat less effective than re-Hst5 against spheroplasts whereas re-Hst5ALys/Arg

1 - - (coloniest~¢.t~ d sample)

% blastoconidia killed =

× 100To generate spheroplasts, C. albicans blastoconidia grown to mid-log phase (A6o onm = 0 . 6 ) , (coloniesoo,trol) were suspended in 4 m L of spheroplasting buffer (1 M sorbitol, 50 m M K . p h o s p h a t e , (pH 7.5) and 15 m M 13-mercaptoethanol), EDTA was added to 1 m M and lyticase (100 units) was added. Cells were incubated at 30°C for 30 rain with gentle shaking. Following lyticase treatment, cells were pelleted, washed twice with spheroplasting buffer, resuspended in suspension buffer and added to microtiter plate wells at a concentration of 1 × 105 spheroplasts/well. Re-Hsts were then added at concentrations that ranged from 50 to 1.6 n m o l / m L and were incubated with spheroplasts at 37°C for 30 rain. Following incubation with re-Hsts, 5 gL aliquots of spheroplasts were removed and added to Sabouraud's dextrose agar plates. Plates were then placed at 30°C and after overnight incubation colonies that formed were counted. In control experiments, spheroplasts treated with suspension buffer alone generated 250 colonies on a plate/5 gL aliquot. To determine the percentage of spheroplasts killed, the total number of colonies formed on a plate was counted and activity was calculated according to the formula: % spheroplasts killed =

1 - (COIonieStreatea sample) (colonies ...... l)

× 100

34

J. Driscoll et al./Gene 177 (1996) 29 34

a n d r e - H s t 5 A H i s were m u c h less effective in killing s p h e r o p l a s t s (Fig. 3b).

Acknowledgement We t h a n k M a r i s a L a b a n c a a n d J o n a t h a n Brewer for their assistance d u r i n g the course of these studies which were s u p p o r t e d by N I H / N I D R g r a n t s D E O 7 6 5 2 a n d DEO5672.

3. Conclusions References R e - H s t 5 a n d v a r i a n t s were p r o d u c e d , purified a n d used to explore the s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p of c a n d i d a c i d a l activity. R e - H s t 5 d i s p l a y e d c a n d i d a c i d a l activity c o m p a r a b l e to that of native Hst5 o b t a i n e d from H P S . T h e a v a i l a b i l i t y of an expression system for re-Hst5 will facilitate investigation of the H s t m e c h a n i s m of a c t i o n since r e - p r o t e i n s can be r a d i o l a b e l e d m e t a b o l i cally, purified from b a c t e r i a a n d used in b i n d i n g studies to identify the site(s) of H s t interaction. We have similarly p r o d u c e d a n d c h a r a c t e r i z e d several re-Hst5 v a r i a n t s to define a a within the functional d o m a i n t h a t c o n t r i b u t e to c a n d i d a c i d a l activity. The results indicate that the G l u a n d L y s / A r g c o n t r i b u t e to activity, t h a t the His are essential for activity a n d extend the p r e v i o u s c h a r a c t e r i z a t i o n of the Hst functional d o m a i n i n i t i a t e d using synthetic p e p t i d e s (Xu a n d O p p e n h e i m , 1993). It is n o t e w o r t h y that r e - H s t 5 A G l u c o n t a i n e d only one m o d i f i e d residue while the o t h e r two v a r i a n t s c o n t a i n e d four m o d i f i e d residues each. F u t u r e studies will a d d r e s s the role of i n d i v i d u a l a a within the functional d o m a i n with respect to H s t c a n d i d a c i d a l activity. I m p o r t a n t l y , the sequence R - K - F - H - E - K - H - H S - H - R is c o n s e r v e d identically in all m a j o r h u m a n Hst a n d in the Hst i s o l a t e d from the s u b h u m a n p r i m a t e , M a c a c a f a s c i c u l a r i s (Xu et al., 1980) a n d this sequence is c o n t a i n e d within the H s t functional d o m a i n . This sequence has n o t been detected in a n y o t h e r a n t i m i c r o b i als c h a r a c t e r i z e d in h u m a n s a n d highlights the n o v e l t y of H s t as host-defense molecules. T h e cell wall is n o t r e q u i r e d for the Hsts to elicit c a n d i d a c i d a l activity since s p h e r o p l a s t s were also susceptible to their effects. H o w e v e r , the results also show t h a t the cell wall m a y p r o v i d e p r o t e c t i o n a g a i n s t H s t - m e d i a t e d killing. These finding m a y facilitate identification of H s t b i n d i n g site(s) a n d further the u n d e r s t a n d i n g of the H s t m e c h a n i s m of a n t i m i c r o b i a l action.

Baum, B.J. and O'Connell, B.C. (1995) The impact of gene therapy on dentistry. JADA 126, 179 188. Driscoll, J., Zuo, Y., Xu, T., Choi, J.R., Troxler, R.F. and Oppenheim, F.G. (1996) Functional comparison of native and recombinant forms of human salivary histatin 1. J. Dent. Res. (in press). Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 685. Landt, O., Grunert, H. and Hahn, U. (1990) A general method for rapid site-directed mutagenesis using the polymerase chain reaction. Gene 96, 125-128. MacKay, B.J., Denepitiya, L., Iacono, V.J., Krost, S.B. and Pollock, J.J. (1984) Growth-inhibitory and bactericidal effects of human parotid salivary histidine-rich polypeptides on Streptococcus mutans. Infect. Immun. 44, 695-701. Oppenheim, F.G., Yang, Y.C., Diamond, R.D., Hyslop, D., Offner, G.D. and Troxler, R.F. (1986) The primary structure and functional characterization of the neutral histidine-rich polypeptide from human parotid saliva. J. Biol. Chem. 261, 1177 1182. Oppenheim, F.G., Xu, T., McMillian, F.M., Levitz, S.M., Diamond, R.D., Offner, G.D. and Troxler, R.F. (1988) Histatins, a novel family of histidine-rich proteins in human parotid secretion, isolation, characterization, primary structure and fungistatic effects on Candida albicans. J. Biol. Chem. 263, 7472 7477. Raj, P.A., Soni, S.-D. and Levine, M.J. (1993) Membrane-induced helical conformation of an active candidacidal fragment of salivary histatins. J. Biol. Chem. 269, 9610-9619. Torres-Bauza, L.J. and Riggsby, W.S. (1980) Protoplasts from yeast and mycelial forms of Candida albicans. J. Gen. Microb. 119, 341-349. Troxler, R.F., Offner, G.D., Xu, T., Vanderspek, J.C. and Oppenheim, F.G. (1980) Structural relationship between human salivary histatins. J. Dent. Res. 69, 2-6. Xu, T., Tesler, E., Troxler, R.F. and Oppenheim, F.G. (1980) Primary structure and anticandidal activity of the major histatins from parotid secretion of the subhuman primate Macacafascicularis. J. Dent. Res. 69, 1717 1723. Xu, T., Levitz, S.M., Diamond, R.D. and Oppenheim, F.G. (1991) Anticandidal activity of major human salivary histatins. Infect. Immun. 59, 2549 2554. Xu, T. and Oppenheim, F.G.(1993) Salivary antimicrobials: Where are we? In: Bowen, W.H. and Tabak, L.A. (Eds.), Cariology for the Nineties. University of Rochester Press, Rochester, NY, pp. 117 131. Zuo, Y., Xu, T., Li, J., Troxler, R.F., Driscoll, J. and Oppenheim, F.G. (1995) Histatins: functional domain duplication enhances candidacidal activity. Gene 161.87-91.