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Expression of two distinct types of pili by a hospital-acquired Enterococcus faecium isolate

¹ Department of Medical Microbiology, University Medical Center Utrecht, Heidelberglaan 100, Rm G04.614, 3584 CX, Utrecht, The Netherlands
² Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Heidelberglaan 100, Rm G04.614, 3584 CX, Utrecht, The Netherlands

Correspondence
Antoni P. A. Hendrickx
a.hendrickx{at}umcutrecht.nl

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Surface filamentous structures designated pili, and implicated in virulence, have been found on the surfaces of several Gram-positive pathogens. This work describes the conditional expression of two phenotypically distinct pilus-like structures, designated PilA and PilB, on the surface of a hospital-adapted Enterococcus faecium bloodstream isolate. E. faecium is an emerging Gram-positive opportunistic pathogen that can cause severe disease, particularly in immunocompromised patients. Expression of PilA- and PilB-type pili was analysed during different phases of growth in broth culture. During growth, PilA and PilB pilin subunits were expressed around the cross-wall in early-exponential-phase cells. Polymerization and migration of short PilB-type pili towards the poles occurred in cells from the exponential phase and long polymerized pili were expressed at the poles of cells grown to stationary phase. In contrast, PilA-type pili were not expressed in broth culture, but only when cells were grown on solid media. Furthermore, surface expression of the PilA- and PilB-type pili was regulated in a temperature-dependent manner, as polymerization of two distinct types of pili at the surface only occurred when cells were grown at 37 °C; no pili were observed on cells grown at 21 °C. Hospital-aquired E. faecium isolates were specifically enriched in pilin gene clusters, suggesting that conditional expression of pili may contribute to E. faecium pathogenesis.

The GenBank/EMBL/DDBJ accession numbers for the DNA sequences of PGC-1 and PGC-3 of E. faecium strain E1165 reported in this article are EU909697 and EU909696.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Enterococci have rapidly emerged as one of the most important nosocomial pathogens causing infections in debilitated patients (Murray, 2000). Although most hospital-acquired infections are still caused by Enterococcus faecalis, the ratio of E. faecalis to Enterococcus faecium infections is rapidly changing in favour of E. faecium in hospitals all across the world (Iwen et al., 1997; Murdoch et al., 2002; Top et al., 2007; Treitman et al., 2005). Hospital-acquired E. faecium isolates recovered from clinical sites of infection and responsible for nosocomial outbreaks (Leavis et al., 2007; Willems et al., 2005) show high-level resistance to a variety of antibiotics, including ampicillin and ciprofloxacin (Leavis et al., 2006b), and are enriched in a putative pathogenicity island (Leavis et al., 2004), genes encoding LPXTG-like cell-wall-anchored proteins (Hendrickx et al., 2007), insertion sequence elements and genes encoding novel metabolic pathways (Leavis et al., 2007). The cumulative acquisition of resistance determinants and putative virulence genes may have contributed to adaptation and selective advantage of specific E. faecium clones in the hospital environment (Leavis et al., 2006a).

Recently, pili (also called fimbriae) have been identified in Gram-positive bacteria (Lauer et al., 2005; Yeung & Ragsdale, 1997; Yeung et al.,1998). Pili are proteinaceous, non-flagellar, covalently joined multimers protruding from the bacterial cell wall, and function as a surface organelle (Marraffini & Schneewind, 2006; Ton-That & Schneewind, 2003). Pili of Gram-positive bacteria have been implicated in biofilm formation and endocarditis (E. faecalis) (Nallapareddy et al., 2006), macrophage resistance (Maisey et al., 2008), invasion of and adherence to brain microvascular endothelial cells (Streptococcus agalactiae) (Maisey et al., 2007), and adhesion to human tonsil epithelium and skin (Streptococcus pyogenes) (Abbot et al., 2007) and to human pharyngeal epithelia (Corynebacterium diphtheriae) (Mandlik et al., 2007). Typical heterotrimeric pili are composed of a major subunit which forms the pilus backbone, a minor subunit and a tip, each protein having characteristics of Gram-positive cell-wall-anchored proteins. The major pilin subunit contains an N-terminal signal sequence for initiation of Sec-mediated translocation across the plasma membrane and a C-terminal cell wall sorting signal (CWS), which comprises an LPXTG-like sortase substrate motif, a hydrophobic domain and positively charged amino acids (Schneewind et al., 1992, 1993; Ton-That & Schneewind, 2003). A pilus-dedicated sortase catalyses the cleavage of the LPXTG-like motif and mediates pilus polymerization, which involves specific motifs such as the YPK pilin motif with a lysine (K) residue required for pilin polymerization and a conserved E box sequence (YxLxETxAPxGY) with a glutamate (E) residue to incorporate minor pilin subunits (Gaspar & Ton-That, 2006; Swaminathan et al., 2007; Swierczynski & Ton-That, 2006; Ton-That et al., 2004). After polymerization, a housekeeping sortase anchors the pilus via the threonine residue of the last pilin to the amino group of the lipid II precursor within the peptidoglycan (Dramsi et al., 2006; Ton-That & Schneewind, 2003).

The presence of pili has not been documented previously in E. faecium. Here, we report the identification of two genetic clusters encoding two distinct types of pili (designated PilA and PilB, previously referred to as Orf1904 and Orf2569, respectively) at the surface of a clinical E. faecium isolate, which are both expressed in a growth condition- and temperature-dependent manner.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Bacterial strains and growth conditions.
E. faecium strain E1165 is a hospital-acquired wound isolate and strain E1002 is a faecal isolate from a human healthy volunteer. E. faecium strains were grown aerobically at 21 °C or 37 °C on Trypticase Soy Agar II supplemented with 5 % sheep blood (TSA; Becton Dickinson) or on Brain Heart Infusion (BHI) agar or broth. Growth curves were prepared from cultures in BHI broth inoculated with plate-grown E. faecium cells, standardized to OD₆₆₀ 0.1 and allowed to grow at 21 °C or 37 °C in an environmental air shaker at 225 r.p.m. A total of 131 E. faecium isolates collected from 20 countries worldwide were used in this study (Hendrickx et al., 2007). These isolates include 49 hospital-acquired E. faecium strains, representing clinical (n=31) and hospital outbreak (n=18) isolates from hospitalized patients, and 82 non-hospital-acquired isolates including human surveillance isolates (n=60, from individuals without known history of E. faecium infections) and isolates from various animals (n=12), from food products (2) and from the environment (8).

In silico search for genes encoding putative major pilin subunits.
The incomplete E. faecium TX0016 (TEX16, DO) genome deposited at DDBJ/EMBL/GenBank under the project accession number AAAK00000000 was searched for proteins annotated as cell wall surface anchor family protein, von Willebrand factor type A, CnaB-type or surface protein from Gram-positive cocci (anchor region). Furthermore, putative major pilin subunits were identified by searching for the presence of an N-terminal signal peptide sequence, a consensus WxxxVxVYPKN pilin motif, a conserved ETxAPxGY E box motif and an LPXTG-like sortase substrate motif (where X denotes any amino acid), a hydrophobic domain and at least one positively charged amino acid within the last eight residues of the C terminus. The LPXTG-like motif was compared to the consensus [FILMPSVY][AP]X[ATS][GAKNS] and NPX[ST][DGNS] patterns representing distinct sortase substrate motifs (Boekhorst et al., 2005). The presence of an N-terminal signal sequence was analysed using the SignalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP) (SignalP score >0.6) (Bendtsen et al., 2004). The presence of the conserved pilin, E box and LPXTG-like motifs, hydrophobic amino acids and one or more positively charged amino acids within the last 45 residues of the C terminus was examined visually. To identify similarities, BLAST analyses on protein sequences were performed at the NCBI site (http://www.ncbi.nlm.nih.gov/BLAST/). Alignments were made using the Weblogo 2.8.2 site (http://weblogo.berkeley.edu/).

Identification of pilin gene clusters (PGCs) in E. faecium E1165.
PGC-1 and PGC-3 were amplified from strain E1165 using primers 1900_fw and Int1_rv for PGC-1 and primers 2567_fw and Int2_rv (Table 1) for PGC-3 to yield 6980 bp and 8202 bp fragments, respectively. PCR products were sequenced by using the BigDye Terminator 3.1 reaction kit and an ABI PRISM 3100 capillary DNA sequencer (both from Applied Biosystems) to determine sequence similarity to the corresponding clusters of E. faecium TX0016.

Anti-peptide polyclonal rabbit antisera.
Highly specific polyclonal rabbit antisera were prepared by Eurogentec (Seraing, Belgium) according to their classic 87 day anti-peptide polyclonal antibody protocol, by immunizing two specific-pathogen-free rabbits with unique 15 amino acid residue keyhole limpet haemocyanin-conjugated peptides directed against the N-terminal region of PilA (H₂N-EDVTQKTPPEKVNITC-CONH₂) and PilB (H₂N-CKMTDLPDPLIQNSGK-CONH₂) of the E. faecium TX0016 genome sequence. Rabbits were exsanguinated at day 87; the serum was collected and stored at –20 °C.

Western immunoblot analysis.
To detect PilA and PilB pilin protein expression, E1165 bacteria were resuspended in PBS and harvested by centrifugation (1560 g, 5 min). To extract cell-surface proteins, cell pellets were suspended in 70 % formic acid and incubated at 65 °C for 30 min as described previously (Ton-That & Schneewind, 2003; Gaspar & Ton-That, 2006), followed by trichloroacetic acid precipitation and acetone washing. Protein pellets were resuspended in 50 µl PBS+50 µl sample buffer (100 mM Tris/HCl, 5 % dithiothreitol, 2 % SDS, 0.004 % bromophenol blue and 20 % glycerol) and boiled for 5 min. Equal amounts of protein sample of E. faecium E1165 were analysed by Western blotting as described previously (Hendrickx et al., 2007).

Dot-blot analysis.
Dot-blot analysis was performed on chromosomal DNA isolated from E. faecium E1002 to determine the presence or absence of LPXTG-like surface-protein genes from PGC-1 and PGC-3. Chromosomal DNA was isolated as described previously (Hendrickx et al., 2007). The chromosomal DNA was denatured with 0.5 M NaOH in a 96-well microtitre plate for 15 min, transferred to a Bio-Rad Bio dot apparatus, vacuum blotted onto a Hybond-N⁺ nylon membrane and fixed onto the membrane by incubation for 2 min in 0.4 M NaOH followed by neutralization in 10x SSC for 1 min. Membranes were hybridized overnight at 42 °C with 100 ng probe. Gene-specific probes were amplified by PCR using primer pairs specific for orf1901, pilA, pilB, orf2570 and orf2571 as described previously (Hendrickx et al., 2007). Amplified DNA probes were purified with a PCR purification kit (Qiagen) and labelled according to the ECL nucleic acid labelling kit (GE Healthcare). The membranes were exposed to Hyperfilm ECL (GE Healthcare).

mRNA expression analysis by reverse transcription (RT-)PCR.
For mRNA expression of two putative major pilin subunit genes (pilA and pilB) cells were resuspended into PBS to an OD₆₆₀ of 1.0 (1x10⁹ c.f.u. ml^–1) and pelleted by centrifugation (6500 g for 1 min). Total RNA was isolated according to Cheung et al. (1994). RNA purification and cDNA generation was done as described previously (Hendrickx et al., 2007; Nallapareddy & Murray, 2006). cDNA was used as template for PCR using primer pairs PilA_fw and PilA_rv and PilB_fw and PilB_rv (Table 1). As an internal control, the housekeeping gene ddl (encoding D-alanine–D-alanine ligase) was amplified using primers ddl_fw and ddl_rv (Table 1). RNA samples not treated with reverse transcriptase were used as control to detect DNA contamination in the total RNA preparations.

Electron microscopy and immunogold labelling.
Transmission immunoelectron microscopy was performed as described previously with some modifications (Hendrickx et al., 2007; Van Wamel et al., 2007). In brief, copper grids (mesh Formvar-carbon coated) were incubated for 30 min with the carbon side on a drop containing E. faecium E1165 cells (1x10⁹ c.f.u. ml^–1) to detect surface-exposed PilA and PilB expression. Grids were washed three times for 5 min on drops of 0.02 M glycine in PBS and subsequently blocked for 30 min on drops of 1 % BSA in PBS (PBSb). The PilA- and PilB-type pili were labelled for 1 h on drops of undiluted specific peptide rabbit immune serum or pre-immune serum in PBSb. Grids were washed four times for 2 min on drops of 0.1 % BSA in PBS. Pilus–antibody complexes were labelled by incubation for 20 min on drops with 1 : 55 diluted protein A-gold label (10 nm) in PBSb. Grids were washed four times for 2 min on drops of PBS, fixed by incubation on drops of 1 % glutaraldehyde in PBS and washed again eight times for 2 min on drops of H₂O. For double labelling experiments, the labelling was repeated similarly with a second immune serum, and a 1 : 70 diluted protein A-gold label (5 nm). Bacteria were stained by incubation of the grids for 5 min on drops containing 1.8 % methylcellulose (25 centipoise; Sigma-Aldrich) and 0.4 % uranyl acetate (pH 4) and subsequently air-dried for 10 min. Grids were examined using a JEOL 1010 transmission electron microscope (JEOL Europe).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Expression of putative pilus-like structures at the surface of E. faecium
To examine E. faecium for the presence of putative pilus-like appendages at the surface of the cell, transmission electron microscopy (TEM) was performed on E. faecium strain E1165, a clinical hospital-acquired isolate. This strain exhibited good binding to the carbon-coated copper grids, in contrast to many other strains. TEM identified putative pilus-like structures, typically 50 nm to 1 µm long, expressed at the poles of the cell (Fig. 1A).

View larger version (45K): [in this window] [in a new window]   Fig. 1. Putative pili, pilin gene clusters and major pilin subunit proteins in E. faecium. (A) TEM micrograph of a negatively stained E. faecium E1165 cell. Bar, 100 nm. (B) Schematic representation of the genomic organization of four putative pilin gene clusters identified from the E. faecium TX0016 genome sequence as published at DDBJ/EMBL/GenBank. The position and presumed direction of transcription of ORFs is indicated by arrows and the ORF number is depicted above the arrows. The black arrows indicate putative sortase genes; grey arrows indicate putative major pilin genes; white arrows indicate putative minor or tip pilin genes or genes with unknown function. Thin lines connect genes encoding putative major pilin subunits. The numbers at the beginning and the end of the pilin gene clusters are positions relative to the start of the contig. (C) Structural organization of the putative major pilin proteins. Signal sequences (SS) are depicted in grey, conserved pilin motifs in dotted grey, E box motifs in black, and the CWSs are striped. (D) Alignment of regions of the conserved pilin motifs and E box motifs found in the four putative major pilin proteins. Amino acid sequences in large letters are highly conserved.

PGC-2 contains four ORFs, orf2007, orf2008, orf2009 and orf2010 (Fig. 1B). The orf2007 gene product resembles a putative pilus-dedicated sortase, showing similarity (44 %) to the EF1094 pilus-associated sortase of E. faecalis V583. The orf2008 gene is predicted to encode a major pilin subunit protein with an N-terminal signal sequence, a putative pilin motif, an E box motif, and a canonical LPATG sortase substrate recognition sequence (Fig. 1C). The orf2008 gene product has 32 % similarity to the Bee protein of E. faecalis, which has been implicated in biofilm formation (Tendolkar et al., 2006). Interestingly, orf2009 also contains, in addition to all features of a Gram-positive LPXTG-like surface protein, a putative pilin motif, and may represent a minor pilin subunit. orf2010 is predicted to encode the largest LPXTG-like protein in this PGC.

PGC-3 contains four ORFs, orf2568 to orf2571 (Fig. 1B). This cluster closely resembles the organization of and has high similarity to the ebpABC–srtC (previously designated EF1091–EF1094) PGC of E. faecalis V583 (Nallapareddy et al., 2006). A putative sortase (orf2568) is located upstream of a putative major pilin subunit, pilB, previously designated orf2569. PilB contains a pilin and E box motif and exhibits 74 % similarity to the putative major EbpC (EF1093) pilin protein of E. faecalis V583 (Fig. 1C). orf2570 and orf2571 (the latter contains an E box motif) share similarities of 48 % with ebpB and 57 % with ebpA of E. faecalis and putatively encode a minor subunit and a pilus tip, respectively.

We recently identified a cluster of three LPXTG-like surface protein genes and a sortase gene (orf903 to orf908) located on a putative pathogenicity island, which was specifically enriched in hospital-acquired E. faecium (Hendrickx et al., 2007). This cluster possibly constitutes a fourth E. faecium PGC (Fig. 1B). In the majority of the hospital-acquired E. faecium isolates, the presence of multiple premature stop codons suggests that this is a non-functional locus. However, in 9.2 % of the isolates we analysed, this cluster comprises ORFs encoding three surface-exposed proteins, orf903, orf904.5 and orf906.7, of which the orf904.5 product contains a pilin motif and is a putative major pilin subunit (Fig. 1C). orf908 tentatively encodes a pilus-dedicated sortase, since it shares 52 % similarity to the EF1094 pilus-associated sortase protein of E. faecalis V583.

Alignment of the identified putative major pilin subunits of E. faecium did not reveal a significant degree of amino acid identity, except for the pilin and E box motifs, which were highly conserved among the E. faecium pilin proteins analysed (Fig. 1D). The consensus pilin motif of putative major pilin proteins of E. faecium is LxxIHLYPKNxx, with a conserved HLYPK motif, which is different from the consensus Gram-positive pilin motif, WxxxVxVYPKN (Mandlik et al., 2008). Similarly, the consensus E box motif of E. faecium, YxxxETxAPExY, was also different from the consensus E box motif (YxxxETxAPxGY) of other Gram-positive major pilin subunits.

PGCs-1 and -3 of E. faecium TX0016 and E1165 are conserved
In this paper, the focus is on the pilA and pilB genes located in PGC-1 and PGC-3, respectively. To identify homologues of pilA and pilB of E. faecium TX0016 in strain E1165, PGC-1 and PGC-3 were amplified by PCR from genomic E. faecium E1165 DNA and sequenced. PGC-1 and PGC-3 of E. faecium E1165 exhibit very high overall similarity (99 and 100 %, respectively) to PGC-1 and PGC-3 of E. faecium TX0016. The organization and transcriptional direction of the ORFs in PGC-1 and PGC-3 are similar, demonstrating that both PGCs are conserved in E. faecium E1165.

Putative pilin subunit genes are enriched among hospital-acquired E. faecium isolates
The presence of the putative pilin subunit genes from the four PGCs was analysed among 131 E. faecium isolates, of which 49 were hospital-acquired and 82 non-hospital-acquired. Based on PCR and Southern hybridization, both orf1901 and pilA (PGC-1) were present in the genomes of 71 % (35/49) of the hospital-acquired isolates and in 48 % (39/82) of the non-hospital E. faecium isolates (P=0.004). The three putative pilin subunit genes from PGC-2 (orf2008–orf2010) were detected in 80 % (39/49) of the hospital-acquired isolates and in only 43 % (35/82) of the non-hospital strains (P<0.001). The ORFs pilB to orf2571 were present in 94 % (46/49) of the hospital-acquired strains and in 74 % (61/82) of the non-hospital strains (P=0.002). These data, in combination with the previously reported distribution of PGC-4 (Hendrickx et al., 2007), show that hospital-acquired E. faecium isolates are significantly enriched for putative pilin genes.

Expression of PilA and PilB multimers at 37 °C
Expression of the PilA and PilB pilin proteins (of PGC-1 and -3) in E. faecium strain E1165 grown at 37 °C on TSA plates was studied at both the mRNA and protein level. mRNA transcripts of pilA and pilB were detected in strain E1165 (data not shown). Using specific rabbit anti-peptide immune sera directed against the two putative major pilin subunits, monomeric forms of PilA and PilB pilin proteins were detected at the predicted molecular masses of 64 and 61 kDa respectively (Fig. 2A). In addition, as observed for other Gram-positive pili, bands of high molecular mass were detected corresponding to PilA and PilB multimers, and PilA and PilB monomers coupled to sortase enzymes or associated with peptidoglycan. Rabbit pre-immune sera did not react with these protein monomers or multimeric pilin species (data not shown). The patterns of PilA and PilB multimeric pilin species were different, suggesting that pilA and pilB encode two different types of pili. To exclude potential cross-reactivity of the rabbit anti-PilA peptide immune serum, the E1002 PilB pili overexpression strain was included in this study. E. faecium strain E1002 is deficient for orf1901 and pilA from PGC-1, and harbours the pilB-orf2570-orf2571 genes from PGC-3 (Fig. 2B) as determined by PCR and DNA–DNA hybridizations using gene specific probes. The anti-PilA peptide immune serum did not react with PilB monomers or high-molecular-mass pilin species (Fig. 2A, lane 3), whereas the anti-PilB peptide immune serum did react with the PilB momoner and pilin species (Fig. 2A, lane 4).

View larger version (65K): [in this window] [in a new window]   Fig. 2. (A) Western blots of surface protein extracts of E. faecium E1165 and E1002 grown on TSA plates at 37 °C. Blots were probed with rabbit anti-PilA (lanes 1 and 3) and rabbit anti-PilB (lanes 2 and 4) immune sera followed by goat anti-rabbit IgG-HRP conjugate. Predicted monomeric PilA (64 kDa) and PilB (61 kDa) pilin proteins are indicated by arrows, high-molecular-mass pilin species are indicated by the bracket. (B) Presence and absence of PGC-1 and PGC-3 in strains E1165 and E1002 as determined by PCR and dot-blot hybridizations. + indicates presence and – indicates absence of the LPXTG-like surface protein genes in the PGC.

View larger version (100K): [in this window] [in a new window]   Fig. 3. TEM micrographs of PilA-type and PilB-type pili of E. faecium E1165. E1165 was grown on TSA plates at 37 °C and subsequently labelled with anti-PilA or anti-PilB peptide immune serum followed by protein A-gold (10 nm particles) and negative staining. (A) E. faecium cells incubated with anti-PilA rabbit immune serum, showing details of a PilA pilus, decorated with 10 nm gold particles. (B) Highly expressed PilA-type pili at the poles appearing as rigid and thin (2 nm) appendages, relatively short (to 300 nm) in length. (C) Similar to (B), but occasionally pili extended up to 1 µm long. (D) E. faecium cells incubated with PilA rabbit pre-immune serum not displaying gold labelling. (E) E. faecium cells incubated with anti-PilB rabbit immune serum, showing details of a PilB pilus, decorated with 10 nm gold particles. (F) PilB-type pili appear flexible, thick (4 nm) and less abundantly expressed at the poles of the cell compared to PilA-type pili. (G) Peritrichous PilB-type pili expression occurs in single cells. (H) E. faecium cells incubated with PilB rabbit pre-immune serum not displaying gold labelling. Bars, 200 nm.

View larger version (103K): [in this window] [in a new window]   Fig. 4. TEM micrograph of PilA- and PilB-type pili expressed on a single cell. E. faecium E1165 was labelled with rabbit anti-PilB immune serum followed by protein A-gold (5 nm particles), fixed, and consecutively labelled with anti-PilA immune serum followed by protein A-gold (10 nm particles). The PilA-type pili are indicated by black arrows, PilB-type pili by white arrows. Bar, 100 nm.

View larger version (57K): [in this window] [in a new window]   Fig. 5. Temperature-dependent downregulation of PilA- and PilB-type pili expression at 21 °C. E. faecium E1165 was grown on TSA plates at 21 °C. (A) Surface protein extracts analysed by Western blots probed with rabbit anti-PilA (left) and rabbit anti-PilB (right) immune sera. Only PilA and PilB monomeric pilin subunits are expressed, as indicated by the arrow, and no multimers were observed (compare Fig. 2A). (B) TEM micrographs of cells grown at 21 °C and labelled with anti-PilA (upper part) or anti-PilB (lower part) immune serum followed by protein A-gold (10 nm) and negative staining. Major pilin subunits are associated with the cell wall and no surface-exposed pili are expressed. Bars, 200 nm.

View larger version (54K): [in this window] [in a new window]   Fig. 6. Expression of PilA- and PilB-type pili in different phases of growth. (A) Growth curve of E. faecium E1165 in BHI at 37 °C. Cells were harvested in the early-exponential (EE), exponential (E) and stationary phase (S) as indicated by the arrows. (B) mRNA expression of pilA, pilB and ddl in cells isolated in EE, E and S phases of growth. In the upper part, mRNA of pilB is depicted, which decreased from EE- to S-phase cells. The pilA gene is constitutively expressed in all growth phases. Control ddl PCRs on total mRNA preparations in which the RT reaction was omitted were all negative, demonstrating absence of DNA contamination. Control ddl RT-PCRs (internal housekeeping control) with E. faecium specific ddl primers were all positive. The results are presented as amplified PCR products electrophoresed on the same ethidium-bromide-stained 1.5 % agarose gel. (C) Surface protein extracts of EE-, E- and S-phase cells analysed by Western blots incubated with rabbit anti-PilB (left) and rabbit anti-PilA (right) immune sera. The PilB pilin protein levels increase from EE- to S-phase cells. The monomeric PilA pilin protein is constitutively expressed (arrow) while pilin multimers are absent.

View larger version (136K): [in this window] [in a new window]   Fig. 7. TEM micrographs of PilB-type pili expression in different phases of growth. E. faecium cells were harvested in early-exponential (EE), exponential (E) and stationary (S) phase, and labelled with anti-PilB immune serum followed by protein A-gold (10 nm) and negative staining. (A) Cells harvested from an EE culture display PilB pilin protein expression around the cross-wall (black arrows). (B) Similar to (A); detailed micrograph of low-level of PilB-type pili polymerization at the cross-wall (black arrows). (C) Cells in E phase display PilB pilin protein expression around the cross-wall, to a lesser extent compared to EE cells, and pili are observed at the poles. (D) S-phase cells do not show pilin protein expression around the cross-wall; instead PilB pilin subunits and long (1 µm) PilB-type pili are observed. Bars, 200 nm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
We describe conditional expression of pili on the surface of hospital-acquired E. faecium. Pili are ‘hair-like’ surface-exposed structures involved in adhesion to multiple types of human cells and biofilm formation, two processes critical in the pathogenesis of bacterial disease. In contrast to pili of Gram-negative bacteria, little is known concerning conditional pilus expression in Gram-positives. We demonstrate that E. faecium can express two distinct types of pili at the surface of a single cell, and that these pili are encoded by two different and physically separated PGCs. Both pilA and pilB encode two different major pilin subunits that polymerize into distinct multimeric pilus structures. Five lines of evidence support this interpretation. First, in silico analysis revealed that the PilA and PilB proteins contain an N-terminal signal sequence to facilitate Sec-mediated translocation, a pilin motif required for pilus polymerization, an E box motif possibly involved in incorporation of minor pilin subunits and a CWS containing an LPXTG-like sortase recognition sequence. Second, expression analysis by RT-PCR detected two specific mRNA transcripts and Western blotting revealed two distinct PilA and PilB multimeric pilin species. Third, the rabbit anti-PilA immune serum did not cross-react with the PilB monomeric subunit or the PilB high-molecular-mass pilin species. Fourth, in TEM experiments, specific anti-PilA and anti-PilB peptide sera reacted with two phenotypically distinct pilus structures at the surface of E. faecium cells grown at 37 °C. The PilA-type pili were thin, rigid, relatively short and occasionally expressed as long polymers at the poles of dividing cells. The PilB-type pili were thicker than PilA-type pili, more flexible and expressed as long polymers at the poles of dividing cells. The observation that PilB-type pili are thick suggests incorporation of additional pilin proteins along the pilus shaft or a bundled multi-fibre structure (Hilleringmann et al., 2008). Finally, double-labelling TEM demonstrated expression of two distinct types of pili simultaneously on a single E. faecium cell. The fact that PilA- and PilB-type pili were differentially expressed to form a heterogeneous population of E. faecium cells expressing PilA- or PilB-type, both types or no pili at all indicates that cells grown in colonies on solid media are in different phases of growth and may favour phase variation, which plays a role in immune evasion by limiting exposure of pilin antigens to the immune system, or adaptation and survival in changing environments (Holden & Gally, 2004; Kearns et al., 2004; van der Woude, 2006).

Expression of PilA- and PilB-type pili at the surface of E. faecium was regulated in a temperature-dependent manner. In contrast to high-level expression of both types of pili on surfaces of E. faecium cells grown at 37 °C, no pili were observed when the bacteria were grown at 21 °C. Instead, cell-wall-associated major pilin subunits were detected at the surface. This suggests that during prolonged survival of E. faecium in the hospital environment, both types of pili are not expressed. Contamination of skin and indwelling medical devices by E. faecium, accompanied by a temperature shift to 37 °C, results in upregulation of pili biogenesis and may initiate intimate attachment, followed by colonization and finally infection.

The PilA and PilB major pilin subunits are expressed around the cross-wall in cells grown to early-exponential phase. The cross-wall is a thick layer of newly synthesized peptidoglycan at which the cell separates into two newly formed cells (Giesbrecht et al., 1998). A similar finding was recently reported for the LPXTG-like cell-wall-anchored protein A of Staphlococcus aureus (DeDent et al., 2007). This suggests that PilB pilin proteins are possibly translocated around the cross-wall and deposited into newly synthesized murein sacculi during separation of the cross-wall at its midline at cell division. Murein sacculi are rigid exoskeletal organelles in which surface proteins are immobilized (Salton, 1952; Cole & Hahn, 1962; Ghuysen, 1968; Navarre & Schneewind, 1999). Possibly, the pilus-dedicated sortase is also located at these cell division sites, as a low level of polymerization of PilB-type pili was observed, but this remains to be investigated. During further growth, novel murein sacculi arise and migration of old murein sacculi towards the poles occurs. Indeed, labelled PilB pilin proteins migrated towards the poles and the pili were longer than those of early-exponential-phase cells. These pili were typically 300 nm long and not abundantly expressed at the surface. In stationary phase, cells display high-level pili expression and pilin subunits were detected predominantly at the old poles. The finding of differential PilB expression and biogenesis during growth suggests that pili of this type are expressed in a growth-phase-dependent manner. However, environmentally relevant cues, such as fluctuations in pH, oxygen and carbon dioxide concentrations, increasing cell density, and cell-to-cell signalling in different phases of growth in liquid media may also influence pili expression. Which of these factors affect PilB expression remains to be elucidated.

The PilA-type pili were expressed when the bacteria were grown on solid media, but not in liquid media, and expression was not growth phase-dependent. These pili are possibly expressed upon cell–cell or cell–substratum contact. Alternatively, shearing, enzymic cleavage or degradation of PilA pili may occur in broth cultures, and could account for the lack of polymerized PilA structures at the surface of cells grown in liquid cultures. The localization of pilA between a putative housekeeping and a putative pilus-dedicated sortase gene in PGC-1 suggests that polymerization by the pilus-dedicated sortase may be followed instantly by cell wall anchoring of the PilA pilus by the housekeeping sortase. Possibly, the PilA-type pili may be involved in initial adherence of E. faecium.

The finding that PGCs were enriched in hospital-acquired E. faecium isolates implies that conditional pili expression may confer a selective advantage in the hospital environment and may promote E. faecium pathogenesis. Recently, active and passive immunization with recombinant pilus subunits of Streptococcus pneumoniae protected mice against a lethal challenge, suggesting that Gram-positive pili are promising novel targets for immune therapy to treat infections (Gianfaldoni et al., 2007). Hospital-acquired E. faecium are multi-resistant, which limits the choice of antibiotics and complicates effective therapy to treat infected patients. A novel strategy to combat these multi-antibiotic-resistant bacteria could be the development of vaccines that target pili.

	ACKNOWLEDGEMENTS

 
We thank Willem van Schaik for critical reading of the manuscript, Marc van Peski for help with the TEM micrographs and Willem van Wamel, George Posthuma and members of our laboratory for discussions. This work was supported by ZonMW research grant 6100.008 from The Netherlands Organization for Heath, Research and Development and by a grant from the European Union Sixth Framework Programme under contract LSHE-CT-2007-037410.

Edited by: K. E. Weaver

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Received 28 May 2008; revised 30 July 2008; accepted 31 July 2008.

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