CAMP factor homologues in Propionibacterium acnes: a new protein family differentially expressed by types I and II

Susanna Valanne1,{dagger}, Andrew McDowell1, Gordon Ramage1,{ddagger}, Michael M. Tunney1,§, Gisli G. Einarsson1, Seamus O'Hagan1,||, G. Brian Wisdom2, Derek Fairley3, Ajay Bhatia4, Jean-Francois Maisonneuve4, Michael Lodes4, David H. Persing4 and Sheila Patrick1

1 Department of Microbiology and Immunobiology, School of Medicine, Queen's University, Grosvenor Road, Belfast BT12 6BN, UK
2 School of Biology and Biochemistry, Medical Biology Centre, 97 Lisburn Road, Queen's University, Belfast BT9 7BL, UK
3 QUESTOR Centre, Queen's University, David Keir Building, Stranmillis Road, Belfast BT9 5AG, UK
4 Corixa Corporation, Infectious Disease Institute, Seattle, WA 98104, USA

Correspondence
Sheila Patrick
s.patrick{at}qub.ac.uk


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Analysis of the draft genome sequence of the opportunistic pathogen Propionibacterium acnes type strain NCTC 737 (=ATCC 6919) revealed five genes with sequence identity to the co-haemolytic Christie–Atkins–Munch-Peterson (CAMP) factor of Streptococcus agalactiae. The predicted molecular masses for the expressed proteins ranged from 28 to 30 kDa. The genes were present in each of the three recently identified recA-based phylogenetic groupings of P. acnes (IA, IB and II), as assessed by PCR amplification. Conserved differences in CAMP factor gene sequences between these three groups were also consistent with their previous phylogenetic designations. All type IA, IB and II isolates were positive for the co-haemolytic reaction on sheep blood agar. Immunoblotting and silver staining of SDS-PAGE gels, however, revealed differential protein expression of CAMP factors amongst the different groups. Type IB and II isolates produced an abundance of CAMP factor 1, detectable by specific antibody labelling and silver staining of SDS-PAGE gels. In contrast, abundant CAMP factor production was lacking in type IA isolates, although larger amounts of CAMP factor 2 were detectable by immunoblotting compared with type II isolates. While the potential role of the abundant CAMP factor 1 in host colonization or virulence remains to be determined, it should be noted that the type strain of P. acnes used in much of the published literature is a type IA isolate and is, therefore, lacking in this attribute.


Abbreviations: CAMP, Christie–Atkins–Munch-Peterson; IFM, immunofluorescence microscopy

The GenBank/EMBL/DDBJ accession numbers for the CAMP factor sequences of P. acnes strains NCTC 737 (IA), KPA171202 (IB), NCTC 10390 (II) and SG2 (II) are given in Table 3.

{dagger}Present address: Institute of Medical Technology, University of Tampere, Finland.

{ddagger}Present address: Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Glasgow, UK.

§Present address: School of Pharmacy, Queen's University, Belfast, UK.

||Present address: Withers Orthopaedic Unit, Musgrave Park Hospital, Belfast, UK.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
As a member of the resident human microbiota, the Gram-positive anaerobic coryneform bacterium Propionibacterium acnes is found predominantly in the sebaceous gland-rich areas of the skin in adults (Eady & Ingham, 1994). It can, however, also be isolated from the conjunctiva, the external ear canal, the mouth, the upper respiratory tract and, in some individuals, the intestine (Funke et al., 1997). It accounts for approximately half of the total skin microbiota (Tancrede, 1992), with an estimated density of 102 to 105–6 cm–2 (Leyden et al., 1998; McGinley et al., 1978). In most people it outnumbers coagulase-negative Staphylococcus spp. (CoNS) on the skin by 10- to 100-fold (Eady & Ingham, 1994). P. acnes is a well-recognized opportunistic pathogen, especially in relation to medical implants such as central nervous system shunts (Brook & Frazier, 1991), silicone implants (Ahn et al., 1996) and prosthetic hip joints, where it is recovered as frequently as CoNS (Tunney et al., 1998, 1999b). It is also responsible for ocular and periocular infections and endophthalmitis (Aldave et al., 1999; Clark et al., 1999) and has been implicated in periodontal and dental infections (Le Goff et al., 1997). Indeed, dental probing and treatment can lead to the dissemination of P. acnes in the bloodstream (Debelian et al., 1992), which is a recognized cause of endocarditis in relation to damaged or prosthetic heart valves. P. acnes is also considered to play a crucial role in inflammatory acne (Eady & Ingham, 1994), since antimicrobial therapy directed against P. acnes results in improvement, while the development of antibiotic resistance in P. acnes is associated with relapse (Leyden et al., 1998). The common form of acne, known as acne vulgaris, affects up to 80 % of the population at some time in their lives, making it the most common skin infection. There is also a strong association between severe forms of acne and joint pain, inflammation of the bone (osteitis) and arthritis. In patients suffering from this condition, known as SAPHO (synovitis, acne, pustulosis, hyperostosis and osteitis) syndrome, isolates of P. acnes have been recovered from bone biopsy samples, as well as synovial fluid and tissue (Schaeverbeke et al., 1998). Immunologists have long recognized and exploited the potent adjuvant activity of P. acnes and utilized it in models of inflammation, although many continue to refer to it as ‘Corynebacterium parvum’ (Tasaka et al., 1996).

Studies by Johnson & Cummins (1972) first revealed two distinct phenotypes of P. acnes, known as types I and II, based on serological agglutination tests and cell-wall sugar analysis. Recently, recA-based sequence analysis has revealed that P. acnes types I and II represent phylogenetically distinct groups (McDowell et al., 2005). Furthermore, a small subgroup of phylogenetically distinct type I strains with atypical mAb labelling characteristics, which we now designate type IB to distinguish them from other type I strains, designated type IA, were also described. The observation that the phenotypic differences between strains of the various P. acnes types reflect deeper differences in their phylogeny raises the possibility that they may also display variation in their expression of putative virulence factors.

P. acnes produces a co-haemolytic reaction with both sheep and human erythrocytes (Choudhury, 1978) similar to the Christie–Atkins–Munch-Petersen (CAMP) reaction first demonstrated in 1944 (Christie et al., 1944). The CAMP reaction describes the synergistic haemolysis of sheep erythrocytes by the CAMP factor from Streptococcus agalactiae and the {beta}-toxin (sphingomyelinase C) from Staphylococcus aureus, with the CAMP factor demonstrating non-enzymic affinity for ceramide (Bernheimer et al., 1979). Examination of sphingomyelinase-treated sheep erythrocytes has revealed the formation of discrete membrane pores by recombinant Streptococcus agalactiae CAMP factor (Lang & Palmer, 2003). In addition to the extensive study of the CAMP factor of Streptococcus agalactiae (Bernheimer et al., 1979; Brown et al., 1974; Jurgens et al., 1985, 1987; Ruhlmann et al., 1988; Skalka et al., 1980), a number of other Gram-positive and Gram-negative bacteria are known to produce a positive CAMP reaction, including Pasteurella haemolytica (Fraser, 1962), Aeromonas species (Figura & Guglielmetti, 1987), some Vibrio species (Kohler, 1988) and group G streptococci (Soedermanto & Lammler, 1996). Some of these species can also use phospholipase C ({alpha}-toxin) from Clostridium perfringens or phospholipase D from Corynebacterium pseudotuberculosis as a co-factor for haemolysis in addition to the Staphylococcus aureus {beta}-toxin (Frey et al., 1989). The CAMP factor genes of Actinobacillus pleuropneumoniae and Streptococcus uberis have also been identified, cloned and expressed in Escherichia coli (Frey et al., 1989; Jiang et al., 1996).

The precise role of the CAMP molecule in bacterial virulence remains unclear. It is likely that the co-haemolytic reaction represents a laboratory phenotype, or epiphenomenon, that is convenient for CAMP factor detection, but which may not be directly related to the role of the molecule in colonization and pathogenesis. The CAMP factor from Streptococcus agalactiae binds to the Fc region of IgG and IgM molecules, similar to the binding of IgG by Staphylococcus aureus protein A (Jurgens et al., 1987), and partial amino acid sequence similarity between the CAMP factor protein of Streptococcus agalactiae and Staphylococcus aureus protein A has been demonstrated (Ruhlmann et al., 1988). We now present evidence of differences amongst P. acnes types IA, IB and II in the expression of proteins with sequence similarity to the CAMP co-haemolysin.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial isolates, media and culture conditions.
The following reference strains were from the National Collection of Type Cultures (NCTC, Colindale, UK), the American Type Culture Collection (ATCC, Manassas, VA, USA) and the National Collections of Industrial, Marine and Food Bacteria (NCIMB, Aberdeen, UK): P. acnes NCTC 737 (=ATCC 6919) and NCTC 10390, Propionibacterium granulosum NCTC 10387, Staphylococcus aureus ATCC 25923, Actinomyces israelii NCTC 8047, Actinomyces naeslundii NCTC 10301, Micrococcus luteus NCIMB 13267 (formerly Fleming strain 2665) and Bacteroides fragilis NCTC 9343. A total of 112 isolates of P. acnes were examined. Sixty-seven isolates were recovered from failed prosthetic hip joints and associated bone, tissue and skin samples removed from patients attending Musgrave Park Orthopaedic Hospital, Belfast, as detailed previously (Tunney et al., 1998, 1999b). In addition, a further group of P. acnes isolates recovered from tissue samples removed during revision arthroplasty in Sweden (n=18), as well as acne (n=19) and dental (n=8) infections, were kind gifts. Isolates of Propionibacterium acidipropionici, Streptococcus agalactiae, Staphylococcus epidermidis, Streptococcus lactis, Streptococcus equi, Streptococcus sanguis, Streptococcus salivarius, E. coli DH5{alpha} and Propionibacterium avidum were obtained from the Department of Microbiology and Immunobiology culture collection, Queen's University, Belfast.

Bacterial culture.
All anaerobic strains were grown on anaerobic blood agar (ABA) (CM0972; Oxoid) or in brain heart infusion (BHI) (CM225; Oxoid) broth. Cultures were incubated at 37 °C in an anaerobic cabinet (MACS MG 1000; Don Whitley Scientific), in an atmosphere of 80 % N2, 10 % CO2 and 10 % H2. All Staphylococcus and Streptococcus strains were also grown at 37 °C on blood agar (BA). E. coli DH5{alpha} was grown aerobically on Luria–Bertani agar plates at 37 °C. Isolates of P. acnes were routinely identified using the API 20A multitest identification system (bioMérieux) in accordance with the manufacturer's instructions.

Co-haemolysis assay.
Co-haemolytic activity was monitored by a modification of the classical co-haemolysis reaction on sheep BA plates as originally described (Christie et al., 1944). Briefly, the Staphylococcus aureus strain ATCC 25923 was streaked vertically onto sheep BA and the test strain was then streaked horizontally outwards from either side, starting close to, but not touching, the Staphylococcus aureus streak. Plates were incubated anaerobically at 37 °C for 48 h. A butterfly-shaped zone of lysis at the junction of the streaks was caused by the co-effect of diffusing Staphylococcus aureus {beta}-toxin and co-haemolytic factor.

Production of mAb and rabbit polyclonal antisera.
The mAb QUBPa4 was generated using the protocol described previously (Harlow & Lane, 1988; Tunney et al., 1999a). Four BALB/c mice were immunized with killed whole cells (108 c.f.u. ml–1) of P. acnes. The hybridoma cell line producing QUBPa4 was then cloned by limiting dilution (Harlow & Lane, 1988).

Rabbit polyclonal antisera were prepared against the five CAMP proteins using recombinant products expressed in E. coli. CAMP genes were amplified from P. acnes NCTC 737 genomic DNA and subcloned into the plasmid vector pET17b (Stratagene). Ligation products were first transformed into E. coli XL-1 Blue competent cells (Stratagene) and the plasmid DNA isolated from XL-1 Blue transformants was subsequently transformed into E. coli BL21 (DE3) pLysE or pLysS host cells (Novagen). The recombinant proteins were expressed in E. coli with a poly-histidine tag at the N terminus and were purified from IPTG-induced batch cultures, in the presence of 8 M urea, by affinity chromatography using the one-step QIAexpress Ni-NTA agarose matrix (Qiagen). Purity of the recombinant proteins was assessed by SDS-PAGE, followed by Coomassie brilliant blue staining and N-terminal sequencing using Edman chemistry with a Procise 494 protein sequencer (Perkin-Elmer Applied Biosystems). All recombinant proteins were assayed for endotoxin contamination using the Limulus amoebocyte assay (BioWhittaker) and shown to contain less than 50 endotoxin units mg–1. Polyclonal rabbit antiserum was raised against all recombinant proteins by injecting New Zealand white rabbits (R&R rabbitry) with 200 µg purified recombinant protein in incomplete Freund's adjuvant (IFA) plus 100 µg muramyl dipeptide (Sigma). Serum was collected following two subsequent boosts separated by 3 weeks with 100 µg protein in IFA. As a result of problems with stability of the CAMP factor 1 and 5 recombinant proteins, only an N-terminal fragment of the CAMP 1 protein and N-terminal and C-terminal fragments of the CAMP 5 protein were used for immunization.

Patient serum.
Serum was prepared from venous blood (10 ml) taken pre-operatively from patients about to undergo either primary or revision total hip arthroplasty at Musgrave Park Hospital, Belfast, and from acne patients attending a Dermatology outpatients clinic in the Royal Victoria Hospital, Belfast. These procedures were approved by the local ethical committee and all patients gave full consent.

Immunofluorescence microscopy (IFM).
IFM was carried out as described previously (Patrick et al., 1995) with minor modification. Briefly, bacterial cultures were grown on ABA or BA and a suspension of 108 c.f.u. ml–1 was prepared in 0·01 M PBS (0·15 M NaCl, 0·0075 M Na2HPO4, 0·0025 M NaH2PO4.2H2O; pH 7·4). Samples (10 µl) were then applied to multiwell slides, air-dried and fixed in 100 % methanol for 10 min at –20 °C. Undiluted hybridoma cell culture supernatant containing mAb QUBPa4 (30 µl) was added to each well of the slides and incubated for 45 min at 37 °C. After washing in 0·01 M PBS for 20 min at room temperature, a 1 : 100 dilution of a fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody (Sigma) in PBS containing 0·1 % (w/v) Evans Blue (Merck Sharp & Dome) counterstain (30 µl) was applied to each well and incubated for 45 min at 37 °C. Control wells in which primary antibody was replaced with PBS were routinely included to monitor non-specific binding of the secondary antibody. Slides were then washed and mounted in glycerol-PBS, containing an anti-photobleaching agent (Citifluor; Agar Scientific), and examined using a Leitz Dialux 20 fluorescence microscope.

Preparation of bacterial extracts.
To obtain efficient extraction and reproducible recovery of loosely cell-associated and secreted CAMP factor 1, standardized whole cell bacterial preparations of 1x1010 c.f.u. ml–1 were obtained by suspending a culture grown for 6 days on ABA directly into PBS, such that a 1 : 100 dilution had an OD600 of 0·3. Cells were then disrupted by ultrasound (Soniprep 150; 26 µm amplitude) for 5 min at 4 °C. The sonicated suspension was brought to room temperature and Tween 20 (Bio-Rad) was added to a final concentration of 2 mM. The sample was then centrifuged and the resulting pellet discarded. Sodium azide was added to the supernatant (final concentration 0·02 % v/v) before storage at –20 °C.

To investigate the presence of secreted CAMP factor 1 protein in culture supernatant, bacteria were grown in BHI broth for 24 h and then centrifuged at 2370 g for 30 min (Mistral; MSE) at room temperature and the supernatant retained. An equal volume of ice-cold ethanol was then added to the supernatant followed by overnight incubation at 4 °C. The precipitated material was recovered by centrifugation at 12 000 g for 30 min (Sorvall) and the pellet was resuspended in 500 µl distilled water.

SDS-PAGE and immunoblotting.
Bacterial extracts, prepared as detailed above, were analysed using 9 % SDS-PAGE gels (Laemmli, 1970) and the resolved proteins were visualized using a silver staining kit (Amersham Pharmacia Biotech). To afford standardization and comparison of different P. acnes isolates for CAMP factor 1 expression, the colour development was carried out for between 110 and 120 s. Gels were washed three times for 5 min each in distilled water, placed in a drying solution [30 % (v/v) ethanol, 5·3 % (v/v) glycerol] for two periods of 30 min and preserved between cellophane sheets. The gels were then photographed using a Kodak DC290 digital camera fitted on a Kodak EDAS290 gel imaging hood and images were analysed using Kodak IK image analysis software version 3.5. Known positive and negative strains were included in each experiment as internal controls and indicated that the experimental system was reproducible.

Immunoblotting was carried out as described previously with a minor modification (Patrick & Lutton, 1990). In brief, nitrocellulose was blocked with 0·01 M PBS containing 0·05 % (v/v) Tween 20 (PBST) and 5 % (w/v) non-fat milk powder (Marvel; Premier brands). After washing with PBST, the nitrocellulose was incubated in undiluted mAb supernatant or an appropriate dilution of polyclonal antiserum in PBS. The nitrocellulose was then washed in PBST before incubation with alkaline phosphatase-conjugated goat anti-human IgG (H), anti-mouse IgG (H+L) or anti-rabbit IgG (H+L) (Sigma). Controls in which the primary antibody was replaced by PBS were routinely included to monitor non-specific binding of the secondary antibody. Bound antibodies were detected using an alkaline phosphatase conjugate substrate kit containing nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Bio-Rad).

Purification and analysis of CAMP factor 1 protein.
The mAb QUBPa4 was purified using a HiTrap protein G column (Amersham Biosciences), concentrated by ultrafiltration and immobilized on CNBr-activated agarose (Sigma). The antibody gel was packed into a column and equilibrated with PBS. Extracts of ABA-grown P. acnes (isolate SG2), prepared as detailed above, were diluted in PBS and recycled through the column overnight. The bound antigen was released with 100 mM glycine/HCl buffer, pH 2·7, and immediately neutralized with 1 M Tris before storage at –20 °C. For trypsin digestion, the eluted fractions from the affinity chromatography column, containing approximately 120 µg purified protein, were pooled and dialysed against distilled water at 4 °C overnight. The sample was then lyophilized using a Speed-Vac (Savant), the precipitate dissolved in 20 µl 8 M urea, 0·4 M NH4CO3 and the pH adjusted to between 7·5 and 8. DTT (0·25 µmol) was added and the suspension was incubated at 50 °C for 15 min. After cooling to room temperature, iodoacetic acid (0·5 µmol) was added and the suspension was incubated at room temperature for a further 15 min. The final concentration of urea was then reduced to 2 M (final volume of digest 80 µl) by the addition of 50 µl distilled water. Trypsin (2·5 mg ml–1; Roche) was added in a 1 : 25 ratio to the CAMP factor 1 protein (w/w) and the mixture was incubated at 37 °C for 24 h. Digestion was stopped by freezing the sample at –20 °C. Purified antigen and its fragments were subjected to SDS-PAGE and electroblotted onto PVDF membrane (Bio-Rad). The bands were stained with Coomassie brilliant blue, excised and sequenced by Edman chemistry at the Babraham Institute (Cambridge, UK).

PCR amplification and sequencing.
PCR was used to detect the five CAMP factor homologue genes in a selection of P. acnes strains. CAMP factor genes were amplified using primers directed to downstream and upstream flanking sequences of each ORF (based on the P. acnes NCTC 737 genome sequence), thus facilitating accurate sequence determination of the 5' and 3' ends of each ORF. Preparation of bacterial genomic DNA and PCR amplifications were carried out essentially as described previously (McDowell et al., 2005). PCR samples contained 1x PCR buffer, 200 µM of each dNTP (Amersham Pharmacia Biotech), 200 µM of each appropriate CAMP factor oligonucleotide primer (Table 1), 1·5 mM MgCl2, 1·25 U Platinum Taq DNA polymerase (Invitrogen Life Technologies) and 2·5 µl bacterial lysate, in a total volume of 25 µl. Samples were initially heated at 95 °C for 3 min, followed by 35 cycles of 1 min at 95 °C, 30 s at the appropriate annealing temperature (Table 1) and 1 min at 72 °C. The PCR was completed with a final extension step at 72 °C for 10 min. A negative water control was included in all experiments. All PCR products were analysed as described before (McDowell et al., 2005). Sequencing reactions were performed using ABI PRISM Ready Reaction Terminator cycle sequencing kits (Perkin Elmer Applied Biosystems) according to the manufacturer's instructions. Samples were analysed on an ABI PRISM 3100 DNA sequencer (Perkin Elmer Applied Biosystems).


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Table 1. PCR primers used in this study for amplification of P. acnes CAMP factor genes

 
Sequence and phylogenetic analyses.
A draft sequence of the P. acnes NCTC 737 genome was commissioned from Genset (Evry, France) by Corixa Corporation (Seattle, WA, USA). A total of 26 million bases of DNA were sequenced, representing approximately 10 genome equivalents. An annotated database of ORFs was created using seven Genemark predictive models, as well as BLASTP, Psort, SignalP, Pfamm, InterProScan and other bioinformatic applications. Comparison with the P. acnes KPA171202 genome sequence (Bruggemann et al., 2004) was carried out using the Artemis Comparison Tool (ACT; http://www.sanger.ac.uk/Software/ACT/). Consensus trees showing protein and nucleotide sequence relationships were generated using the Data Analysis in Molecular Biology and Evolution software (DAMBE; http://aix1.uottawa.ca/~xxia/software/software.htm). Multiple sequence alignments were performed using the CLUSTAL W algorithm (Thompson et al., 1994) and exported into the DAMBE program. For nucleotide analysis, consensus trees were constructed using the maximum-parsimony method and the neighbour-joining method using the Jukes–Cantor-based algorithm. Sequence input order was randomized and bootstrapping resampling statistics were performed using 100 datasets for each analysis.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identification of multiple genes with CAMP factor sequence identity
Analysis of the draft genome sequence of the P. acnes type IA strain NCTC 737 (=ATCC 6919) (property of Corixa Corporation) identified five related genes, sited at different locations within the genome, with similarity to the Streptococcus agalactiae co-haemolysin or CAMP factor protein (GenBank accession no. X72754.1) originally described by Christie, Atkins and Munch-Petersen in 1944 (Christie et al., 1944). The resulting CAMP factor protein sequences had predicted molecular masses that ranged from approximately 28 to 30 kDa. All five genes have a putative N-terminal signal peptide cleavage site (Table 2).


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Table 2. Characteristics of the predicted P. acnes CAMP factor proteins of NCTC 737, KPA 171202 and NCTC 10390

Strains belong to types IA (NCTC 737), IB (KPA171202) and II (NCTC 10390).

 
Construction of a bootstrapping consensus tree (using 100 bootstrapping replications) based on the different P. acnes CAMP factor protein sequences of NCTC 737, along with the CAMP factor sequences from Streptococcus agalactiae (GenBank accession no. NP_736433.1), Streptococcus uberis (accession no. AAA789101) and Streptococcus pyogenes (accession no. NP_802366.1), revealed that CAMP factor 3 was more distantly related to the other P. acnes CAMP factor proteins (Fig. 1). The P. acnes CAMP factors 1 and 5 were found to be more closely related to one another, as were CAMP factors 2 and 4.



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Fig. 1. Consensus tree illustrating the relationship between CAMP factor protein sequences from P. acnes NCTC 737 (type IA), Streptococcus agalactiae, Streptococcus uberis and Streptococcus pyogenes. Multiple sequence alignments were performed and the resulting phylogenetic tree was rooted with the Streptococcus pyogenes sequence. Bootstrapping resampling statistics were applied to the trees (100 datasets) and bootstrap values are shown at each node of the tree.

 
Nucleotide sequence analysis of CAMP factor genes in P. acnes types I and II
The presence of all five CAMP factor genes in other strains of P. acnes type IA, as well as strains of type IB and type II, was revealed by PCR with primers designed to downstream and upstream flanking sequences of each CAMP factor ORF. PCR analysis of 12 isolates representative of P. acnes type IA (NCTC 737, RM1, PV37), type IB (CK17, LED2, RM9, W1392, W1998) and type II (NCTC 10390, SG2, RM4 and P24) resulted in PCR products of the correct size for all five CAMP factor genes, in all the isolates examined (data not shown). Nucleotide sequence analysis of all five CAMP factor genes from strains selected to represent type IA (NCTC 737) and type II (NCTC 10390 and SG2) was carried out (Table 3). These sequences were then compared with the CAMP factor gene sequences from the recently published genome sequence of P. acnes KPA171202 (Bruggemann et al., 2004; accession no. AE017283), which we identified as a type IB strain based on recA and tly phylogenetic analysis. Consensus trees of the individual CAMP factor gene sequences from these strains were constructed using the maximum-parsimony and neighbour-joining methods. After 100 bootstrapping replications, the consensus trees derived using the two methods gave identical topologies. For CAMP factors 1, 2, 4 and 5, the phylogenetic relationships between the IA, IB and II strains were identical to that determined previously by recA and tly gene analysis (illustrated in Fig. 2a for CAMP factor 1). For CAMP 3, however, the consensus tree differed, as the type IA sequence was found to have a closer relationship to the type II sequence, with the type IB sequence being more distinct (Fig. 2b).


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Table 3. GenBank accession numbers for CAMP factor sequences of P. acnes NCTC 737 (IA), KPA171202 (IB), NCTC 10390 (II) and SG2 (II)

 


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Fig. 2. Consensus trees illustrating the relationship between P. acnes types IA, IB and II CAMP factor genes. Multiple alignments of CAMP factor 1 (a) and CAMP factor 3 (b) gene sequences from strains NCTC 737 (type IA), KPA171202 (type IB) and SG2 (type II) were performed, along with the published CAMP sequence of Streptococcus pyogenes, which was used as an outgroup for the phylogenetic analysis. Consensus trees were constructed using the neighbour-joining method (with Jukes–Cantor-based algorithm). Bootstrapping resampling statistics were applied to the trees (100 datasets) and bootstrap values are shown at each node on the trees.

 
Comparison was also made between strains NCTC 737 (IA) and KPA171202 (IB) for nucleotide and amino acid sequence differences in proteins putatively involved in the degradation of host molecules, as well as those thought to be cell-surface-associated. Analysis revealed variable numbers of nucleotide and amino acid sequence differences between the two strains (Table 4).


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Table 4. Percentage identity and number of nucleotide/amino acid differences between KPA171202 (type IB) and NCTC 737 (type IA) CAMP factors, selected putative host cell molecule-degrading factors and antigens

 
CAMP factor homologues of P. acnes and IgG-binding domains of protein A
As IgG binding by the Streptococcus agalactiae CAMP factor (Jurgens et al., 1987), as well as partial amino acid sequence similarity of the molecule to protein A of Staphylococcus aureus (Ruhlmann et al., 1988), has been demonstrated, sequence comparisons were made between the P. acnes CAMP factors and staphylococcal protein A. CLUSTAL W analyses identified a protein domain between positions 108 and 157 in CAMP factor 1 with 25 % or more identity to the IgG-binding domains E, B and C of Staphylococcus aureus protein A (Table 5). In addition, CAMP factor 3 showed approximately 25 % identity to domain A between positions 104 and 142. The CAMP factor of Streptococcus agalactiae was found to have considerably lower percentage identity.


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Table 5. Comparison of predicted CAMP factor amino acid sequences of P. acnes type IA (NCTC 737) and type II (NCTC 10390) with the IgG-binding domains of Staphylococcus aureus protein A

Values are percentages of identity. Where type IA and II differ, the values are listed before (NCTC 737) and after (NCTC 10390) the solidus (/).

 
Co-haemolytic (CAMP) reactivity of P. acnes types I and II
Fifty isolates of P. acnes (types IA, IB and II) recovered from failed prosthetic hip joints and associated bone and tissue samples, as well as skin and dental sources and strains of Staphylococcus epidermidis, A. israelii, A. naeslundii and Streptococcus agalactiae, were positive for the co-haemolytic CAMP reaction on sheep BA using a positive {beta}-toxin-producing strain of Staphylococcus aureus (ATCC 25923). All P. granulosum strains were negative for the CAMP reaction, as were strains of P. avidum, P. acidipropionici, M. luteus, Streptococcus equi, Streptococcus sanguis, Streptococcus salivarius, Streptococcus lactis, E. coli DH5{alpha} and B. fragilis.

Differences in CAMP factor protein expression between P. acnes types I and II
Expression of the five CAMP factor proteins by type I and II isolates was investigated by immunoblotting with rabbit polyclonal antisera raised against recombinant forms of each NCTC 737 CAMP factor protein, as well as a mouse mAb specific for CAMP factor 1. Representative immunoblots of NCTC 737 (type IA) and the prosthetic hip joint isolate SG2 (type II) are presented in Fig. 3. Labelled bands were observed in the correct region for the predicted molecular mass of the proteins, minus the signal sequence. Bands of lower molecular mass than that of the predicted secreted protein were sometimes observed and are believed to relate to degradation of a predicted labile N-terminal region. Isolates of P. acnes type II were found to produce large amounts of CAMP factor 1 compared with type IA strains. A corresponding abundant protein band was also observed on silver-stained SDS-PAGE gels of P. acnes type II, but was absent in type IA isolates (Fig. 4) and NCTC 10390 (type II; data not shown). Larger quantities of CAMP 2, however, were detectable in type IA isolates compared with type II by immunoblotting, although levels of the protein were still considerably less than CAMP factor 1 production (Fig. 3). Analysis of strains of P. acnes type IB by immunoblotting and silver staining of SDS-PAGE gels revealed a pattern of CAMP factor expression similar to that of type II organisms, with the production of large amounts of CAMP factor 1 and reduced expression of CAMP factor 2 compared with type IA strains (data not shown). Nucleotide sequences immediately upstream of the CAMP genes were compared to determine whether sequence differences in these regions could be influencing expression. Analysis revealed conservation of putative core Shine–Dalgarno sequences amongst the different P. acnes types for CAMP factors 1, 2, 3 and 5, but one base difference in the CAMP factor 1 Shine–Dalgarno sequence of NCTC 10390 was observed. The upstream sequences of CAMP factor 4 were more variable. The CAMP factor 4 gene for all isolates examined had a GTG start codon, whereas the start codon for all other CAMP factors was ATG (Fig. 5).



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Fig. 3. Immunoblots probed with rabbit anti-CAMP factor protein polyclonal antisera and mouse anti-CAMP factor 1 mAb (QUBPa4). (a) P. acnes NCTC 737 (type IA); (b) P. acnes SG2 (type II). C, Control with no primary antibody; U, undiluted.

 


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Fig. 4. Analysis of P. acnes extracts by SDS-PAGE followed by silver staining (top) or immunoblotting with a mouse anti-CAMP factor 1 mAb (QUBPa4) (bottom). Lanes: 1–3 and 5, type II P. acnes isolates JJK1 (lane 1), HMJ3 (2), SG2 (3) and PC3 (5); 4 and 6, type IA P. acnes isolates SB1 (4) and AT3 (6). Lanes M, molecular mass markers (Bio-Rad, low range).

 


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Fig. 5. Comparison of upstream regions of P. acnes CAMP factor genes. Consensus sequence for P. acnes NCTC 737 (type IA), KPA171202 (type IB) and SG2 (type II). Base matches representing equivalent DNA sequence of putative core Shine–Dalgarno sequences are illustrated in bold. *, Mismatch in NCTC 10390.

 
Comparison of CAMP factor 1 expression by P. acnes type I and II isolates
Comparison of CAMP factor 1 expression between types IA, IB and II was performed by silver staining of bacterial extracts resolved on SDS-PAGE gels. Confirmation that the abundant protein band observed was CAMP factor 1 was obtained by immunoblotting with our mAb specific for the CAMP factor 1 protein (QUBPa4; Fig. 4). Specificity of the mAb was confirmed by N-terminal sequencing of affinity-purified complete and trypsin-digested protein (data not shown). The staining intensity of the 28 kDa CAMP factor 1 band in extracts from NCTC 737 and NCTC 10390 was compared, as well as 76 type IA, 5 type IB and 31 type II isolates recovered from a variety of sources. The type IA strain NCTC 737 was used as an internal standard on all gels (arbitrary value of 1·0). All 31 type II P. acnes isolates, from failed prosthetic hip joints and associated bone and tissue samples, as well as one acne isolate, had a band intensity for CAMP factor 1 that was at least sixfold greater than that for NCTC 737 (mean=14). The type II strain NCTC 10390 was an exception and lacked an intense band in this region of the gel, consistent with its absence after immunoblotting (not illustrated). The five type IB isolates recovered from dental sources (n=2), a failed prosthetic hip joint (n=1), a prosthetic hip joint-associated tissue sample (n=1) and a skin sample from the hip of a patient undergoing revision arthroplasty (n=1), also had at least sixfold greater band intensity for CAMP factor 1 (mean=18). Type IA isolates recovered from dental sources (n=6), failed prosthetic hip joints and associated bone and tissue samples (n=52) and patients with acne (n=18) had a band intensity in the range of 1–9 (mean=3).

Immunofluorescence labelling of CAMP factor 1 on whole cells of P. acnes
IFM with mAb QUBPa4 indicated that the CAMP factor 1 protein was both cell-associated and secreted (Fig. 6a). Mouse mAbs of the same isotype as QUBPa4, specific for cell-surface components of P. acnes I and II, respectively, did not label extracellular material by IFM (Fig. 6b, c). A centrifugation wash in PBS of bacteria harvested from agar plates was sufficient to remove most of the CAMP factor 1 protein (Fig. 6d, e). In addition, we also found that the protein could be obtained from broth culture after 50-fold concentration by ethanol precipitation. P. acnes type II strain NCTC 10390, which did not react with QUBPa4 by immunoblotting, was also non-reactive by IFM. IFM revealed no reactivity of QUBPa4 with P. granulosum, P. acidipropionici, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus agalactiae (data not shown). Although A. israelii and A. naeslundii showed no reaction with QUBPa4 by IFM, reactivity was observed after SDS-PAGE and immunoblotting (data not shown).



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Fig. 6. Immunofluorescence micrographs of P. acnes clinical isolates labelled with mouse isotype IgG2b mAbs. (a)–(c) Type II isolate labelled with QUBPa4, specific for CAMP 1 (a), type II isolate labelled with QUBPa2, specific for P. acnes type II-associated carbohydrate antigen (b) and P. acnes type IA clinical isolate labelled with mouse isotype IgG2b mAb QUBPa5, specific for a protein antigen associated with type IA (c). (d)–(e) Unwashed and washed P. acnes type IA clinical isolate labelled with CAMP factor 1-specific QUBPa4: bacterial suspension dried and fixed directly onto slide (d) and bacterial suspension first centrifuged and then resuspended in PBS (e). Images were viewed with fluorescein-detecting filter (a, b) or with combined fluorescein- and Evans Blue-detecting filter (c–e). Microscope magnification, x1000.

 
Reactivity with patient antisera
The sera from acne patients (n=9) and patients undergoing primary total hip arthroplasty (n=10) and revision arthroplasty (n=11) were examined by immunoblotting at a single dilution of 1 : 500 for their reactivity against immunoaffinity-purified CAMP factor 1 protein from a type II isolate (SG2). In total, sera from three patients with acne, five patients undergoing primary arthroplasty and four patients undergoing revision arthroplasty were reactive.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A total of five individual genes with sequence identity to the co-haemolytic CAMP factor, originally described in Streptococcus agalactiae (Christie et al., 1944), were identified in the draft genome sequence of the P. acnes type IA strain NCTC 737. Recently, these genes have been independently identified in the genome sequence of the P. acnes strain KPA171202 (Bruggemann et al., 2004), which we identified as type IB based on comparison of recA and tly sequences (McDowell et al., 2005). PCR analysis of a selection of type IA, IB and type II isolates indicated that all five genes are present in each of the different P. acnes types. To our knowledge, multiple CAMP homologues have not been described in any other organisms. The conserved nucleotide and amino acid sequence differences observed with the CAMP factors from all three P. acnes types provided additional evidence for the phylogenetically distinct nature of the groups and confirmed previous results obtained with recA and tly genes (McDowell et al., 2005). In contrast, CAMP factor sequences from two serotypes of Streptococcus agalactiae (serotype III strain NEM316 and serotype V strain 2603V/R) have been shown to be identical (Glaser et al., 2002). In addition, varying numbers of nucleotide and amino acid differences were observed between the sequences of NCTC 737 (1A) and KPA171202 (1B) for a selection of putative secreted and cell-surface-associated proteins, providing further evidence for their designation as distinct phylogenetic groups within type I (McDowell et al., 2005).

Analysis of CAMP factor protein expression by immunoblotting and silver staining of SDS-PAGE gels revealed an abundance of CAMP factor 1 production by type II and type IB isolates, but not IA organisms. The type II strain NCTC 10390, however, was an exception, as abundant CAMP factor 1 protein was not detected. As the genes for all five CAMP factors are present in all three P. acnes groups, observed differences reflected different levels of expression rather than missing genes. One factor amongst many that could influence expression levels is the interaction between a Shine–Dalgarno ribosome-binding site and 16S rRNA. Strong Shine–Dalgarno sequences (e.g. GGAG, GAGG or AGGA) are associated with genes that are predicted to be highly expressed (Karlin & Mrazek, 2000). Interestingly, we identified a base difference in the putative Shine–Dalgarno sequence associated with the CAMP factor 1 gene of NCTC 10390, a type II strain that lacks abundant CAMP factor 1 production and is not reactive with our specific mAb by IFM. Whether or not this reduces the efficiency of interaction with the 16S ribosomal subunit, and therefore the level of expression of the protein, remains to be determined. The observation that NCTC 10390 differs from other type II organisms with respect to CAMP factor 1 production indicates that it is not the most appropriate representative strain of P. acnes type II.

Immunoblotting also revealed that type IA isolates express greater quantities of CAMP factor 2 compared with type II and type IB isolates; however, neither this nor the other CAMP factors were produced in quantity by IA strains, as the abundant protein band was not detectable by SDS-PAGE and silver staining. The putative Shine–Dalgarno sequences of CAMP factors 1 and 2 are conserved for all three phylogenetic groups; therefore, this cannot explain the differences in expression. No striking differences were observed with respect to the expression of CAMP factor proteins 3, 4 and 5, although CAMP factor 4 reacted only weakly in immunoblotting experiments with type IA and II isolates. The putative Shine–Dalgarno sequences upstream of the CAMP factor 4 genes were more varied amongst the three phylogenetic groupings, but comparison with the Shine–Dalgarno sequences of the other CAMP genes did not reveal any clear relationship that could explain the different expression levels. CAMP factor 4 does, however, have a GTG start codon, which is reported to be a weaker translational initiator than ATG (Ringquist et al., 1992).

All of the CAMP factor sequences contain a putative signal sequence cleavage site (Table 2) and molecular mass comparison of the proteins by SDS-PAGE and immunoblotting was in keeping with the loss of this signal sequence. IFM analysis and detection in ethanol-precipitated supernatant from broth culture confirmed that the CAMP factor 1 protein was secreted, although it was also detected on the surface of the P. acnes cells. Studies with Streptococcus agalactiae similarly detected CAMP factor protein in the external milieu, as well as on the cell surface (Jurgens et al., 1987). None of the CAMP factor homologues from P. acnes or Streptococcus agalactiae contain a C-terminal Leu-Pro-X-Thr-Gly (LPXTG) motif, although 25 genes encoding other proteins with an LPXTG motif have been described in the genome of P. acnes strain KPA171202 (Bruggemann et al., 2004).

Despite our observations of differential expression of the five CAMP factor homologues amongst P. acnes types IA, IB and II, all isolates from both groups were positive for the co-haemolytic phenotype. The P. acnes co-haemolytic reaction is therefore likely to be mediated by more than one CAMP factor protein. We are currently addressing the relationship between individual CAMP factors and the co-haemolytic phenotype. In our studies, some sequence identity between the P. acnes CAMP factor 1 and 3 proteins and Staphylococcus aureus protein A was demonstrated within the Fc-binding region, suggesting they may have immunoglobulin-binding activity. Interestingly, the percentage identities were greater than those of the Streptococcus agalactiae CAMP factor, which has been shown to bind IgG (Jurgens et al., 1987). It may be that the multiple P. acnes CAMP factors have arisen from divergence of a replicated common ancestral gene and now have divergent functions.

In addition to the P. acnes isolates studied, A. israelii and A. naeslundii produced a co-haemolytic reaction whereas P. granulosum did not. It would be interesting to determine whether this difference reflects the relative pathogenic potential of these organisms. The positive reaction between purified CAMP factor 1 protein and human sera obtained from patients with acne, as well as those undergoing primary or revision hip arthroplasty, indicates that the protein is expressed by P. acnes during human colonization. Whether its production relates to virulence remains to be determined, as the serum samples used were from a single time point and therefore give no indication of rising titre.

In conclusion, we have identified five genes with sequence identity to the co-haemolytic CAMP factor of Streptococcus agalactiae in strains of P. acnes types IA, IB and II. Differential protein expression of the CAMP factors amongst the various P. acnes phylogenetic groupings was observed; in particular, the extracellular and cell-associated CAMP factor 1 protein was produced in striking abundance by type IB and type II isolates. The observation of differential expression of putative virulence determinants amongst the various P. acnes types will have important consequences. In particular, it will impact on our interpretation of previously published virulence data that have been based on the study of only one isolate type, such as NCTC 737, which has often been used as a model organism for studies of P. acnes virulence (Roszkowski et al., 1980; Webster et al., 1985). More generally, such data, in combination with the recent demonstration that P. acnes is genetically heterogeneous, will serve to challenge our current understanding of the virulence and pathogenic potential of clinical isolates of P. acnes.


   ACKNOWLEDGEMENTS
 
We would like to thank A. Eady, C. Kamme and W. Wade for supplying a selection of Propionibacterium species. A. McD. and G. G. E. were funded by a Programme Grant from the Northern Ireland Health and Personal Social Services Research and Development Office (NIHPSS R & D Office). S. V. was funded by an NIHPSS R & D Office studentship and a grant from the British Orthopaedic Wishbone Trust. G. R. was funded by a Department of Employment and Learning Northern Ireland Studentship, M. M. T. was funded by the Arthritis Research Campaign, UK (project grant no. P0554), and S. O'H. was funded by Action Research, UK.


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Received 25 November 2004; revised 11 February 2005; accepted 16 February 2005.



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