Lysis of staphylococcal mastitis pathogens by bacteriophage
phi11endolysin
David M. Donovan, Michelle Lardeo & Juli Foster-Frey
Biotechnology and Germplasm Laboratory, ANRI, ARS, USDA, Beltsville, MD, USA
Correspondence: David M. Donovan,
Biotechnology and Germplasm Laboratory,
ANRI, ARS, USDA, Bldg. 230, Room 104,
BARC-East, 10300 Baltimore Ave, Beltsville,
MD 20705-2350, USA. Tel.: 11 301 504
9150; fax: 11 301 504 8571;
e-mail: ddonovan@anri.barc.usda.gov
Received 26 June 2006; accepted 25 August
2006.
First published online 19 October 2006.
DOI:10.1111/j.1574-6968.2006.00483.x
Editor: Mark Enright
Keywords
Staphylococcus aureus ; coagulase-negative
staphylococcus; phi11 endolysin; peptidoglycan
hydrolase; antimicrobial.
Abstract
The Staphylococcus aureus bacteriophage phi11 endolysin has two peptidoglycan
hydrolase domains (endopeptidase and amidase) and an SH3b cell wall-binding
domain. In turbidity reduction assays, the purified protein can lyse untreated
staphylococcal mastitis pathogens, Staphylococcus aureus and coagulase-negative
staphylococci (Staphylococcus chronogenes, Staphylococcus epidermidis, Staphylococcus
hyicus, Staphylococcus simulans, Staphylococcus warneri and Staphylococcus
xylosus), making it a strong candidate protein antimicrobial. This lytic activity is
maintained at the pH (6.7), and the `free' calcium concentration (3 mM) of milk.
Truncated endolysin-derived proteins containing only the endopeptidase domain
also lyse staphylococci in the absence of the SH3b-binding domain.
Introduction
Bovine mastitis, an infection of the mammary gland, exists
on every dairy farm, often with one-third of the animals
affected. This worldwide problem costs two billion dollars
annually to the US dairy industry (Sordillo & Streicher,
2002). Coagulase-negative staphylococcus (CoNS) and Staphylococcus
aureus are major US mastitis pathogens, accounting
for 22% and 18% of cases, respectively, in a study
of New York and Pennsylvania dairy herds (Wilson et al.,
1997). Antibiotics are the standard treatment but are often
less than 50% effective, at times resulting in premature
culling (Deluyker et al., 2005).
Antibiotic resistance in mastitis treatment is of concern
because mastitis is the single most common reason for
antimicrobial use in dairy herds (Erskine et al., 2002). In a
broad study of nine European countries, the USA and
Zimbabwe, 57% of 811 Staphylococcus aureus isolates from
bovine mastitis showed resistance to penicillin (De Oliveira
et al., 2000), and 44% of the mastitis strains identified in an
Ohio study demonstrated resistance to at least one antibiotic
(Rajala-Schultz et al., 2004). FDA, USDA and CDC promote
the development of antimicrobials that reduce risk
of resistance development (CDC Action Plan: http://
www.cdc.gov/drugresistance/actionplan/html/product.htm).
The use of pathogen-specific antimicrobials is expected to
reduce the incidence of resistance development (Walsh, 2003;
Nathan, 2004).
To reduce the use of broad-range antibiotics and thus
decrease the chance of antibiotic resistance development,
our goal is to develop pathogen-specific antimastitis agents
that can be expressed in mammary glands of transgenic
cattle (Kerr & Wellnitz, 2003; Donovan et al., 2005; Wall
et al., 2005). Bacteriophage endolysins specifically degrade
the peptidoglycan of their host cell wall, thus lysing the
bacteria, and allowing infective phage to escape. Bacteriophage
endolysins are of interest as antimicrobials against
Gram-positive organisms (Loessner, 2005) due to their high
host specificity and reports that Gram-positive bacteria are
highly unlikely to develop resistance to the peptidoglycan
hydrolyzing action of their bacteriophage endolysins (Loeffler
et al., 2001; Schuch et al., 2002). Peptidoglycan hydrolases
have been proposed for human antimicrobial
applications (Schuch et al., 2002; Fischetti, 2003, 2005) and
have demonstrated efficacy in mouse models of human
disease (Nelson et al., 2001; Jado et al., 2003; Cheng et al.,
2005) as well as transgenic murine (Kerr et al., 2001) and
bovine (Wall et al., 2005) mammary glands.
FEMS Microbiol Lett 265 (2006) 133­139 c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd.
No claim to original US government works
Peptidoglycan hydrolases can be divided into three general
types: glycosidases, amidases and endopeptidases
(Lopez & Garcia, 2004), and are often encoded by highly
conserved domains (Loessner, 2005). The conserved domains
are $200 amino acids in size (Bateman & Rawlings,
2003; Huard et al., 2003; Rigden et al., 2003), and are readily
identified using common domain databases (Uniprot:
http://www.pir.uniprot.org/index.shtml; Pfam: http://www.
sanger.ac.uk/cgi-bin/Pfam/getacc?PF04650; SMART: http://
smart.embl-heidelberg.de/, or NCBI conserved domain
database). The domains are referred to as modules because
they can often function independently of the remainder of
the native endolysin (Navarre et al., 1999; Morita et al., 2001;
Yokoi et al., 2005; Donovan et al., 2006), and maintain
activity when fused to create novel, recombinant, fusion
hydrolases (Diaz et al., 1990; Garcia et al., 1990; Donovan
et al., 2006).
The Staphylococcus aureus (NCTC 8325) phi11 prophage
endolysin gene was first isolated from the Staphylococcus
aureus genome as the LytA amidase (Wang et al., 1991) and
was later shown to be a phage lytic enzyme. It contains two
peptidoglycan hydrolase activities: a cysteine, histidinedependent
amidohydrolases/peptidase (CHAP) endopeptidase
(Bateman & Rawlings, 2003; Rigden et al., 2003)
that cleaves at D-alanyl-glycyl moieties, and an amidase
domain that cleaves at N-acetylmuramyl-L-alanyl bonds
(Navarre et al., 1999). The phi11 endolysin also contains a
C-terminal SH3b cell wall-binding domain (Baba & Schneewind,
1998). Many related endolysins with high amino acid
sequence identity to the phi11 endolysin sequence (Genbank
Accession # P24556) have been identified from staphylococcal
phage genomes [phi 80 alpha: 99% (AAB39699); bacteriophage
69: 91% (YP_239596); bacteriophage 29: 91%
(YP_240560); bacteriophage 92: 90% (YP_240773); bacteriophage
52A: 90% (YP_240634); bacteriophage 55: 90%
(YP_240484); bacteriophage mu 50: 89% (BAB57075);
bacteriophage 88: 89% (YP_240699); and bacteriophage 37:
76% (YP_240103)] (Kwan et al., 2005).
A weakly homologous (40% identical) Staphylococcus
warneri M phage phi WMYendolysin was recently described
and shown to share the same domain organization [Nterminal
CHAP endopeptidase ­ amidase ­ C-terminal
SH3b] as the phi11 endolysin (Yokoi et al., 2005). Via sitedirected
and deletion mutagenesis analysis, it was shown
that both the CHAP endopeptidase domain and the amidase
domains of the phi WMY endolysin contribute to degradation
of autoclaved staphylococci in sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) zymograms.
Despite knowledge of the peptidoglycan hydrolase
enzymatic activities of this and other Staphylococcus aureus
bacteriophage endolysins, only one published phage endolysin,
LysK, also sharing a similar modular organization as
previously described, has been shown to kill untreated
staphylococci. LysK also kills many CoNS and clinically
relevant, multi-drug-resistant strains of Staphylococcus aureus
(O'Flaherty et al., 2005).
We have examined the 481 amino acid phi11 endolysin for
its ability to lyse untreated Staphylococcus aureus and CoNS,
and for its pH and calcium concentration optima. We have
also truncated the phi11 endolysin to remove the SH3b
domain and thus test for its role in `lysis from without'.
Materials and methods
Bacterial strains and culture conditions
Cloning and vector constructs were maintained in Escherichia
coli DH5a cells (Invitrogen, Carlsbad, CA) or BL21
(DE3) (EMD Biosciences, San Diego, CA). Staphylococcus
aureus (ATCC 29740), and the mastitis isolates Streptococcus
agalactiae, Staphylococcus chromogenes, Staphylococcus epidermidis,
Staphylococcus hyicus, Staphylococcus simulans,
Staphylococcus warneri and Staphylococcus xylosus (USDA)
were grown at 37 1C in Brain Heart Infusion (DIFCO,
Franklin Lakes, NJ).
PCR cloning
The AmpR
plasmid pTZ18R contains the phi11 endolysin
on a 3 kb EcoRI fragment of the phi11 bacteriophage
genome (a gift from R. Jayaswal) (Jayaswal et al., 1990).
The endolysin gene was subcloned into pET21a (EMD
Biosciences, San Diego, CA) via PCR of the pTZ18R plasmid
template. High-fidelity PCR (Deep Vent Polymerase; New
England Biolabs, Ipswich, MA) with primers that introduce
unique restriction enzyme (RE) sites NdeI (CATATG, includes
an ATG translation initiation site) and XhoI (at the
translational stop sequence, amino acid 481) were used to
amplify the full-length phi11 endolysin-coding sequences
(phi11-481). Primers: F LytA NdeI, 50
-GTG GCG CAT ATG
CAA GCA AAA TTA AC-30
and R LytA XhoI 481 50
-T GAC
TAT GTC CTC GAG ACT GAT TTC-30
(the introduced RE
sites are underlined). These sites were designed to allow
subcloning into pET21a such that a six Histidine tag (in
pET21a) was added at the C-terminus of the phi11 protein.
Gradient thermocycling determined optimal primer annealing
temperature and PCR conditions. Gel-purified (Qiaex,
Qiagen, Valencia, CA) PCR products were digested, desalted
(Micro Bio-Spin 30 columns) (BioRad, Hercules, CA) or
gel-purified and ligated to a similarly digested, dephosphorylated
(Shrimp Alkaline Phosphatase, Roche, Basel,
Switzerland) and purified recipient vector (pET21a) using
standard techniques. In order to subclone truncated phi11
endolysin protein-coding sequences, two reverse primers
that introduce XhoI sites at amino acid 194 and 389 were
utilized [R LytA XhoI 194: 50
-ACT ACC ACG CTC GAG
TAG GTC -30
; R LytA XhoI 389: 50
-AGTACC ATATTT CTC
FEMS Microbiol Lett 265 (2006) 133­139c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd.
No claim to original US government works
134 D.M. Donovan et al.
GAG TTT CCA TGC-30
] with the NdeI forward primer F
LytA NdeI (above). These primers create a truncated protein
coding sequence that, when cloned into pET21a, fused the
truncated phi11 endolysin to the six x His tag codons of the
pET21a vector. Ligations were transformed into E. coli
DH5a (Invitrogen, Carlsbad, CA), DNA isolated (Qiagen,
Valencia, CA), RE site mapped and retransformed into
BL21(DE3) (EMD Biosciences, San Diego, CA) for expression
and purification of protein products. All constructs
tested positive for peptidoglycan hydrolase activity (in lieu
of DNA sequence analysis).
Extract preparation and protein purification
Escherichia coli BL21(DE3) cells harboring plasmid constructs
were grown in 500 mL Superbroth (Becton Dickenson,
Franklin Lakes, NJ) supplemented with 100 mg mL1
ampicillin at 37 1C with shaking. Mid log phase (OD600 nm
of 0.4­0.6) cultures were induced with 1 mM IPTG (isopropyl-beta-D-thiogalactopyranoside),
followed by 4 h shaking
at 37 1C. Cells were pelleted, washed with lysis buffer
(50 mM NaH2PO4, 300 mM NaCl, 10 mM Imidazole, pH 8)
and pellets frozen at  80 1C. Extracts were prepared
according to a modified procedure of Pritchard et al., 2004
(Neville et al., 1994). For nickel column-purified protein,
500 mL culture cell pellets were resuspended in 10 mL Lysis
buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM Imidazole,
pH 8), sonicated (15 Â 5 s pulses with 15 s rest between),
centrifuged at 6800 r.p.m. in a Sorvall HS4 rotor (8500 g),
supernatant decanted and added to 5 mL Ni-NTA (nickel
matrix) slurry according to the manufacturer's instructions
(Qiagen, Valencia, CA) with gentle rocking for 1 h at 4 1C.
The matrix was washed and protein batch eluted according
to the manufacturer's instructions. Protein eluates were
desalted in Micro Bio-Spin 30 columns (BioRad) prior to
protein determination with BCA protein assay (Pierce,
Rockford, Ill.). The various protein fractions and Kaleidoscope
protein standards (Invitrogen, Carlsbad, CA) were
analyzed with 15% SDS-PAGE run in Tris-Glycine buffer at
131 V for 1.5 h in the BioRad Mini-PROTEAN three gel
apparatus, according to the manufacturer's instructions.
Gels were stained in BioSafe Coomassie stain (BioRad,
Hercules, CA) for 1 h and then destained in 50% methanol/
10% Acetic Acid solution in distilled water overnight.
Turbidity assay
The turbidity assay measures a decline in optical density due
to lysis of the target bacteria with the phage endolysinderived
protein. Mid log phase (OD600 nm of 0.4­0.6) target
cells were grown in Brain Heart Infusion (Becton Dickenson,
Franklin Lakes, NJ) and concentrated in lysin buffer
A (LBA, 50 mM ammonium acetate, 10 mM CaCl2, 1 mM
DTT pH 6.2) to an OD600 nm $2.0. Turbidity assays were
performed with 25 mg of purified phi11 endolysin-derived
proteins in a cuvette and were initiated, by addition of target
cells to a final OD600 nm of $1.2 at room temperature.
Changes in OD were recorded for 1 h OD600 nm. Changes
in the cells alone sample were subtracted from samples
with lysin, before calculating the activity (change in
OD600 nm mg protein1
min1
). Optimum pH and Ca21
concentration were determined with modified LBA.
Changes in pH were performed with Glacial acetic acid or
ammonium hydroxide. CaCl2 was added at the appropriate
concentrations to LBA minus CaCl2. Results are often
reported as `representative' plots due to the instability
of the phi11-derived proteins, and the resulting high degree
of variability from assays performed on different days.
However, the trends identified were consistent among the
multiple preparations.
Results
C-terminal truncations of the phi11 endolysin
The full-length (481 codon) phi11 endolysin protein-coding
region was subcloned between the NdeI and XhoI sites of
pET21a. All constructs described in this work were derived
from this parental clone (Fig. 1). With the long-term goal to
express the phi11 endolysin in transgenic cattle mammary
glands, a series of C-terminal deletions were created in order
to 1) reduce the amount of protein engineering necessary to
express a bacterial protein in a eukaryotic system, and 2) to
determine whether the SH3b domain was essential for
pathogen lysis. The C-terminal truncation phi11-194 contains
the entire CHAP domain, bisects the amidase domain
and lacks the SH3b domain. The phi11-389 construct
contains both the CHAP domain and the amidase domain,
but lacks the SH3b domain. Partially purified nickel column
preparations of full length and truncated gene products are
CHAP Amidase SH3b
33518913115
1
194aa
389aa
481aa
6xHis
398 466
Fig. 1. Schematic of phi11 endolysin protein
structure and deletion constructs. phi11
194 and phi11 389 deletion constructs are
illustrated, along with the approximate
location of the CHAP, amidase and SH3b
cell wall-binding domains.
FEMS Microbiol Lett 265 (2006) 133­139 c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd.
No claim to original US government works
135Lysis of staphylococcal mastitis pathogens
visualized in SDS-PAGE (Fig. 2a). Both truncations and the
full-length protein lyse Staphylococcus aureus in turbidity
assays (Fig. 2b). None of the constructs were lytic towards
Streptococcus agalactiae (Fig. 2b). The CHAP domain's
ability to lyse Staphylococcus aureus in the absence of the
amidase domain is consistent with the peptidoglycan hydrolase
activity of a previous phi11 endolysin deletion, which
removed the amidase domain from the center of the protein,
fusing the phi11 CHAP domain to the SH3b cell wallbinding
domain (Navarre et al., 1999). The lytic activity of
the phi11-194 construct indicates that the CHAP domain
alone is sufficient for exolysis (`lysis from without') of the
target bacteria. The intact phi11-481 protein has significantly
greater activity than either the phi11-194 or phi11-
389, suggesting that the SH3b domain may not be essential,
but does improve the exolytic activity.
The phi11-389 construct has a slightly higher exolytic
activity compared with phi11-194, suggesting that the
amidase domain does contribute to exolysis of Staphylococcus
aureus, as opposed to the Acm `mid-protein' domain of
the streptococcal bacteriophage B30 endolysin, which is
essentially silent during exolysis of streptococci (Donovan
et al., 2006).
phi11 endolysin is active at the physiological pH
and Ca21
concentration of milk
The physiological pH and free calcium concentration
of bovine milk is 6.7 and 3 mM, respectively (http://
www.foodsci.uoguelph.ca/dairyedu/chem.html#overview)
(Pritchard et al., 2004). The phi11-481 endolysin has peak
activity in the turbidity assay at 2­3 mM CaCl2 (Fig. 3), and
is active at a broad range of physiologically relevant pHs
(Fig. 4).
Phi11 endolysin is lytic towards CoNS
The CoNS have been reported to contribute to 11% of
mastitis in one study from Pennsylvania and New York
(Wilson et al., 1997), and 12% in another study where
Staphylococcus simulans accounted for 53% of the 149 CoNS
isolates (Waage et al., 1999). The phi11-481 endolysin is lytic
for six representative strains (including Staphylococcus simulans)
that were isolated from mastitis infections (USDA) and
identity verified via the University of MD College Park
microbiology service (Fig. 5).
Discussion
Our goal is to avoid the use of broad-range antibiotics
through the identification of pathogen-specific protein
antimicrobials that can be expressed in transgenic cow
mammary glands. Staphylococcus aureus and CoNS are
major mastitis pathogens in the USA. The Staphylococcus
1.6
1.2
0.8
0.4
0
phi11
194
phi11
389
phi11
481
Specificactivity
M 1 2 3
kDa 100
75
50
37
25
20
15
(a) (b)
Fig. 2. SDS-PAGE and Turbidity assay results of full length and phi11 endolysin truncations. (a) 3 mg of nickel column-purified phi11 endolysin-derived
proteins. Lane M, Markers; 1, phi11-481; 2, phi11-389; 3, phi11-194. Carrots indicate phi11-derived proteins. (b) 25 mg of purified endolysin-derived
proteins are active against Staphyloccocus aureus (black bars). There is no activity against Streptococcus agalactiae (open bars). Specific Activity
(DOD600 nm mg1
min1
). Extracts from Escherichia coli harboring just pET21a vector do not have lytic activity in the turbidity assays against
Streptococcus agalactiae or S. aureus (data not shown).
0.4
0.3
0.2
0.1
0.0
0 5 10
[CaCI2]
15 20
Specificactivity
Fig. 3. Effect of CaCl2 concentration on lytic activity of phi11-481
endolysin. Specific Activity = OD600 nm mg1
min1
. (Graph is a representative
plot.) Twenty-five micrograms endolysin per sample.
FEMS Microbiol Lett 265 (2006) 133­139c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd.
No claim to original US government works
136 D.M. Donovan et al.
aureus phi11 endolysin has both an amidase and an endopeptidase
domain that are specific for and have been
shown to be enzymatically active on cell wall preparations of
Staphylococcus aureus (Navarre et al., 1999). We have extended
this work to demonstrate that the endolysin has
exolytic activity toward untreated Staphylococcus aureus and
mastitis-causing CoNS, and is active at the physiological pH
and calcium concentrations found in milk.
Previous work of Navarre et al. (1999) created a deletion
construct that removed the amidase mid-region of the
protein and fused the N-terminal CHAP endopeptidase to
the C-terminal SH3b-binding domain. This work demonstrated
that the CHAP domain did not require the amidase
domain for enzymatic activity but it was not shown whether
or not the CHAP domain requires the SH3b domain for
activity and specificity, nor did it demonstrate exolytic
activity on living cells. With the phi11-194 construct, we
demonstrate that the CHAP domain is sufficient to lyse
untreated Staphylococcus aureus cells without a need for
either the amidase domain or the SH3b domain. The
Staphylococcus warnei M phage phiWMY endolysin (lysWMY)
has a similar CHAP-amidase-SH3b protein organization
and is only 43% identical to the phi11 endolysin. The
lysWMY CHAP domain has lytic activity towards several
SDS-treated staphylococci in zymograms, including many
CoNS and Staphylococcus aureus (Yokoi et al., 2005), but was
not tested on live Staphylococcus aureus or CoNS. The
Staphylococcus aureus K phage endolysin, lysK, also has a
similar modular organization, and although only 41%
identical to the phi11 endolysin, is the only other staphylococcal
phage endolysin that has been reported to lyse
untreated Staphylococcus aureus and CoNS cells (in a plate
lysis assay) (O'Flaherty et al., 2005). For use as an antimastitis
agent, it is essential that the endolysin have exolytic
activity.
It is also important to our project that the antimicrobial
be specific to its pathogen group. The broad-range antibiotics
pirlimyacin and penicillin are widely used in the
treatment of mastitis (Cattell et al., 2001). Use of broadrange
antimicrobials, although successful, is discouraged
due to concerns that antibiotic resistance among mastitis
pathogens is on the rise (Werckenthin et al., 2001; RajalaSchultz
et al., 2004), and the fear that such strains might find
their way from the farm to the clinic (Ferber, 2002, 2003). To
reduce concerns about antimicrobial resistance among mastitis
pathogens, it is our goal to identify protein antimicrobials
that do not have activity against a broad range of
pathogens but rather are pathogen-specific (or lyse only
closely related species). We have shown that neither the full
length nor truncated phi11 endolysin constructs will lyse
Streptococcus agalactiae in the turbidity reduction assay.
Similarly, it is important that a mastitis antimicrobial be
active in a milk environment. We have characterized the fulllength
phi11-481 endolysin and demonstrate antimicrobial
activity at both the pH (6.7) and free-calcium concentration
(3 mM) consistent with milk (Pritchard et al., 2004). This
activity is comparable to that of lysostaphin (data not
shown) and this is encouraging because lysostaphin has a
proven track record of preventing Staphylococcus aureus
mammary infections in transgenic mammary glands in mice
(Kerr et al., 2001) and cattle (Wall et al., 2005).
Although the results of lysostaphin expression in transgenic
cattle and mice are encouraging, we believe that the
phi11 endolysin is potentially a better antimicrobial for
several reasons. First, it is known that lysostaphin has a
single endopeptidase function cleaving glycyl-glycyl bonds
2.0
1.5
1.0
0.5
0.0
5 6
pH
7 8
Specificactivity
Fig. 4. Effect of pH on lytic activity of phi11-481 endolysin targeting
Staphyloccocus aureus. [Graph is a representative plot.] Specific Activity
(OD600 nm mg1
min1
). Twenty-five micrograms endolysin per sample.
3.5
3
2.5
2
1.5
1
0.5
0
S.chrom
ogenesS.epiderm
idis
S.hyicusS.sim
ulans
S.w
arneri
S.xylosus
Specificactivity
Fig. 5. Turbidity assay results of phi11-481 endolysin lysis of CoNS.
Specific Activity (OD600 nm mg1
min1
). Twenty-five micrograms endolysin
per sample.
FEMS Microbiol Lett 265 (2006) 133­139 c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd.
No claim to original US government works
137Lysis of staphylococcal mastitis pathogens
of the peptidoglycan peptide cross bridge (Browder et al.,
1965) and that strains have developed resistance to this
activity by inserting serines into the cross bridge (Thumm &
G¨otz, 1997). Second, the phi11 endolysin potentially has two
putative antimicrobial activities: an endopeptidase and an
amidase, and it is known that for a pathogen to develop
resistance to two enzymatic activities would require two
simultaneous compensatory mutations in the same cell.
This is believed to be a rare event. Third, it is reported that
through the coevolution of bacteriophage and their hosts
the bacteriophage endolysins target essential bonds in the
peptidoglycan, such that despite significant effort to find
them, no resistant strains have been identified to those
phage endolysins that have been screened (Loessner, 2005).
Finally, the phi11 endolysin is potentially a better antimastitis
agent, because it lyses multiple mastitis pathogens (both
Staphylococcus aureus and CNS) while lysostaphin lyses
Staphylococcus aureus alone.
We are continuing to quantify the activity of the truncated
and full-length phi11 endolysins in milk and when
expressed in mammalian cells. The highly conserved, yet
apparently nonessential role of the SH3b-binding domain of
phage endolysins during cellular exolysis raises unanswered
questions about these complex modular proteins.
Acknowledgements
The authors would like to thank David Pritchard for helpful
review of the manuscript. Mention of trade names or
commercial products in this article is solely for the purpose
of providing specific information and does not imply
recommendation or endorsement by the U.S. Department
of Agriculture.
References
Baba T & Schneewind O (1998) Targeting of muralytic enzymes
to the cell division site of Gram-positive bacteria: repeat
domains direct autolysin to the equatorial surface ring of
Staphylococcus aureus. EMBO J 17: 4639­4646.
Bateman A & Rawlings ND (2003) The CHAP domain: a large
family of amidases including GSP amidase and peptidoglycan
hydrolases. Trends Biochem Sci 28: 234­237.
Browder HP, Zygmunt WA, Young JR & Travormina PA (1965)
Lysostaphin: ezymatic mode of action. Biochem Bio Phys
Res Commun 19: 383­389.
Cattell MB, Dinsmore RP, Belschner AP, Carmen J & Goodell G
(2001) Environmental gram-positive mastitis treatment: in
vitro sensitivity and bacteriologic cure. J Dairy Sci 84:
2036­2043.
Cheng Q, Nelson D, Zhu S & Fischetti VA (2005) Removal of
group B streptococci colonizing the vagina and oropharynx of
mice with a bacteriophage lytic enzyme. Antimicrob Agents
Chemother 49: 111­117.
Deluyker HA, Van Oye SN & Boucher JF (2005) Factors affecting
cure and somatic cell count after pirlimycin treatment of
subclinical mastitis in lactating cows. J Dairy Sci 88: 604­614.
De Oliveira AP, Watts JL, Salmon SA & Aarestrup FM (2000)
Antimicrobial susceptibility of Staphylococcus aureus isolated
from bovine mastitis in Europe and the United States. J Dairy
Sci 83: 855­862.
Diaz E, Lopez R & Garcia JL (1990) Chimeric phage-bacterial
enzymes: a clue to the modular evolution of genes. Proc Natl
Acad Sci USA 87: 8125­8129.
Donovan DM, Kerr DE & Wall RJ (2005) Engineering disease
resistant cattle. Transgenic Res 14: 563­567.
Donovan DM, Foster-Frey JA, Dong S, Rousseau G, Moineau S &
Pritchard DG (2006) The cell lysis activity of the Streptococcus
agalactiae bacteriophage B30 endolysin relies on the CHAP
endopeptidase domain. Appl Environ Microbiol 72: 5108­5112.
Donovan DM, Dong S, Garrett W, Rousseau G, Moineau S &
Pritchard DG (2006) Peptidoglycan hydrolase fusions
maintain their parental specificities. Appl Environ Microbiol
72: 2988­2996.
Erskine RJ, Walker RD, Bolin CA, Bartlett PC & White DG (2002)
Trends in antibacterial susceptibility of mastitis pathogens
during a seven-year period. J Dairy Sci 85: 1111­1118.
Ferber D (2002) Antibiotic resistance. Livestock feed ban
preserves drugs' power. Science 295: 27­28.
Ferber D (2003) Antibiotic resistance. WHO advises kicking the
livestock antibiotic habit. Science 301: 1027.
Fischetti VA (2003) Novel method to control pathogenic bacteria
on human mucous membranes. Ann N Y Acad Sci 987:
207­214.
Fischetti VA (2005) Bacteriophage lytic enzymes: novel antiinfectives.
Trends Microbiol 13: 491­496.
Garcia P, Garcia JL, Garcia E, Sanchez-Puelles JM & Lopez R
(1990) Modular organization of the lytic enzymes of
Streptococcus pneumoniae and its bacteriophages. Gene 86:
81­88.
Huard C, Miranda G, Wessner F, Bolotin A, Hansen J, Foster SJ &
Chapot-Chartier MP (2003) Characterization of AcmB, an
N-acetylglucosaminidase autolysin from Lactococcus lactis.
Microbiology 149: 695­705.
Jado I, Lopez R, Garcia E, Fenoll A, Casal J, Garcia P & Spanish
Pneumococcal Infection Study Network. (2003) Phage lytic
enzymes as therapy for antibiotic-resistant Streptococcus
pneumoniae infection in a murine sepsis model.
J Antimicrob Chemother 52: 967­973.
Jayaswal RK, Lee YI & Wilkinson BJ (1990) Cloning and
expression of a Staphylococcus aureus gene encoding a
peptidoglycan hydrolase activity. J Bacteriol 172: 5783­5788.
Kerr DE & Wellnitz O (2003) Mammary expression of new genes
to combat mastitis. J Anim Sci 81: (Suppl 3): 38­47.
Kerr DE, Plaut K, Bramley AJ, Williamson CM, Lax AJ, Moore K,
Wells KD & Wall RJ (2001) Lysostaphin expression in
FEMS Microbiol Lett 265 (2006) 133­139c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd.
No claim to original US government works
138 D.M. Donovan et al.
mammary glands confers protection against staphylococcal
infection in transgenic mice. Nat Biotechnol 19: 66­70.
Kwan T, Liu J, DuBow M, Gros P & Pelletier J (2005) The
complete genomes and proteomes of 27 Staphylococcus aureus
bacteriophages. Proc Natl Acad Sci USA 102: 5174­5179.
Loeffler JM, Nelson D & Fischetti VA (2001) Rapid killing of
Streptococcus pneumoniae with a bacteriophage cell wall
hydrolase. Science 294: 2170­2172.
Loessner MJ (2005) Bacteriophage endolysins­current state of
research and applications. Curr Opin Microbiol 8: 480­487.
Lopez R & Garcia E (2004) Recent trends on the molecular
biology of pneumococcal capsules, lytic enzymes, and
bacteriophage. FEMS Microbiol Rev 28: 553­580.
Morita M, Tanji Y, Orito Y, Mizoguchi K, Soejima A & Unno H
(2001) Functional analysis of antibacterial activity of Bacillus
amyloliquefaciens phage endolysin against Gram-negative
bacteria. FEBS Lett 500: 56­59.
Nathan C (2004) http://www.nature.com/news/2004/041018/pf/
431899a_pf.html
Navarre WW, Ton-That H, Faull KF & Schneewind O (1999)
Multiple enzymatic activities of the murein hydrolase from
staphylococcal phage phi11. Identification of a D-alanylglycine
endopeptidase activity. J Biol Chem 274: 15847­15856.
Nelson D, Loomis L & Fischetti VA (2001) Prevention and
elimination of upper respiratory colonization of mice by
group A streptococci by using a bacteriophage lytic enzyme.
Proc Natl Acad Sci USA 98: 4107­4712.
Neville MC, Keller RP, Casey C & Allen JC (1994) Calcium
partitioning in Human and bovine Milk. J Dairy Sci 77:
1964­1975.
O'Flaherty S, Coffey A, Meaney W, Fitzgerald GF & Ross RP
(2005) The recombinant phage lysin LysK has a broad
spectrum of lytic activity against clinically relevant
staphylococci, including methicillin-resistant Staphylococcus
aureus. J Bacteriol 187: 7161­7164.
Pritchard DG, Dong S, Baker JR & Engler JA (2004) The
bifunctional peptidoglycan lysin of Streptococcus agalactiae
bacteriophage B30. Microbiology 150: 2079­2087.
Rajala-Schultz PJ, Smith KL, Hogan JS & Love BC (2004)
Antimicrobial susceptibility of mastitis pathogens from first
lactation and older cows. Vet Microbiol 102: 33­42.
Rigden DJ, Jedrzejas MJ & Galperin MY (2003) Amidase domains
from bacterial and phage autolysins define a family of gammaD,L-glutamate-specific
amidohydrolases. Trends Biochem Sci
28: 230­234.
Schuch R, Nelson D & Fischetti VA (2002) A bacteriolytic
agent that detects and kills Bacillus anthracis. Nature 418:
884­889.
Sordillo LM & Streicher KL (2002) Mammary gland immunity
and mastitis susceptibility. J Mammary Gland Biol Neoplasia 7:
135­146.
Thumm G & G¨otz F (1997) Studies on prolysostaphin processing
and characterization of the lysostaphin immunity factor (Lif)
of Staphylococcus simulans biovar staphylolyticus. Mol
Microbiol 23: 1251­1265.
Waage S, Mork T, Roros A, Aasland D, Hunshamar A & Odegaard
SA (1999) Bacteria associated with clinical mastitis in dairy
heifers. J Dairy Sci 82: 712­719.
Wall RJ, Powell AM, Paape MJ, Kerr DE, Bannerman DD, Pursel
VG, Wells KD, Talbot NC & Hawk HW (2005) Genetically
enhanced cows resist intramammary Staphylococcus aureus
infection. Nat Biotechnol 23: 445­451.
Walsh C (2003) Where will new antibiotics come from? Nat Rev
Microbiol 1: 65­70.
Wang X, Wilkinson BJ & Jayaswal RK (1991) Sequence analysis of
a Staphylococcus aureus gene encoding a peptidoglycan
hydrolase activity. Gene 102: 105­109.
Werckenthin C, Cardoso M, Martel JL & Schwarz S (2001)
Antimicrobial resistance in staphylococci from animals with
particular reference to bovine Staphylococcus aureus, porcine
Staphylococcus hyicus, and canine Staphylococcus intermedius.
Vet Res 32: 341­362.
Wilson DJ, Gonzalez RN & Das HH (1997) Bovine mastitis
pathogens in New York and Pennsylvania: prevalence and
effects on somatic cell count and milk production. J Dairy Sci
80: 2592­2598.
Yokoi KJ, Kawahigashi N, Uchida M, Sugahara K, Shinohara M,
Kawasaki K, Nakamura S, Taketo A & Kodaira K (2005) The
two-component cell lysis genes holWMYand lysWMYof the
Staphylococcus warneri M phage var phiWMY: cloning,
sequencing, expression, and mutational analysis in Escherichia
coli. Gene 351: 97­108.
FEMS Microbiol Lett 265 (2006) 133­139 c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd.
No claim to original US government works
139Lysis of staphylococcal mastitis pathogens