Centre for Infectious Diseases and Microbiology, Level 3 ICPMR Building, University of Sydney, Westmead Hospital, Institute Road, Westmead, NSW 2145, Australia
Correspondence
Jonathan Iredell
joni{at}icpmr.wsahs.nsw.gov.au
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ABSTRACT |
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INTRODUCTION |
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Phase variation generally refers to reversible change in a defined phenotype, often due to variable expression of important surface structures such as lipopolysaccharide (LPS), flagellae or adhesins (Henderson et al., 1999). Phase variations are usually random events, occurring at a relatively high frequency in vitro (>10-5 per generation), which may be influenced or modulated by environmental conditions (Gally et al., 1993
). A slow-growing, agar-pitting, dry colony phenotype is reported to be typical of primary clinical isolates of B. henselae cultivated on chocolate agar plates (Arvand et al., 1998
; Slater et al., 1990
). Twitching motility is a characteristic feature of these isolates (Welch et al., 1992
), as it is of other organisms expressing type IV pili (Tennent & Mattick, 1994
). However, serial passage of dry agar-pitting isolates on solid media in vitro is associated with switching to a faster-growing, non-adherent and more-mucoid colony phenotype (Anderson & Neuman, 1997
). This is associated with loss of pilin expression and the ability to invade human epithelial cells in tissue culture (Batterman et al., 1995
), and to stimulate angiogenesis in vitro (Kempf et al., 2001
). Most available data on the pathogenicity of B. henselae have been derived using the type strain (Houston-1), which is of variable and usually unstated passage number in different studies. Reversal of the reported attenuation with serial passage has not been formally described, and questions therefore remain about the interpretation of pathogenicity studies using stored or highly passaged isolates.
DNA fingerprinting methods have been used to distinguish subgroups within human B. henselae isolates (Arvand et al., 2001; Dillon et al., 2002
; Sander et al., 1998
), and isolates with a 16S rDNA sequence matching that of the type strain (Houston-1) may be more common as a human pathogen (Dillon et al., 2002
; Sander et al., 1999
). SmaI PFGE demonstrated at least seven different clonal types among 19 feline isolates of B. henselae from Berlin, most of which were 16S type II (Arvand et al., 2001
), and the question of inter-strain variability in virulence remains open (Dillon et al., 2002
; O'Reilly et al., 1999
; Relman, 1998
; Chang et al., 2002
). It may be that recombinatorial events, evident as phenotypic phase variation, comprise a basic virulence mechanism in B. henselae, as described for other small vector-borne bacterial pathogens (Brayton et al., 2001
). A bacteriophage has also been demonstrated in B. henselae (Anderson et al., 1994
). We have previously shown that all of 59 undifferentiated B. henselae isolates were positive by PCR for the phage-associated papA gene (Dillon et al., 2002
), but this is of uncertain significance and it is unclear whether this gene is present in all primary isolates and whether it may be lost with serial passage in vitro.
Two distinct serological types of B. henselae have been reported (Drancourt et al., 1996), but inherent and phase-variable differences in LPS and outer-membrane proteins (OMPs) have not been systematically evaluated. There is considerable genetic heterogeneity within the species, but the extent to which genotypic and phenotypic variation confounds or informs the study of virulence remains unclear. A range of feline and human B. henselae isolates from Australia and New Zealand were therefore evaluated in order to determine the extent to which phase variation influences in vitro virulence-associated phenotypes of B. henselae, including growth characteristics, biofilm formation, and the expression of major OMPs, pili and LPS.
This study was presented in part at the American Society for Rickettsiology/Bartonella Joint Conference 2001 (Big Sky, MT, USA; 1722 August, 2001).
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METHODS |
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Growth curves of B. henselae.
Growth and viability curves in isogenic sets were compared in broth media. Each strain was harvested from CBA and suspended in PBS to an OD550 value of 0·25; duplicate broths were then inoculated 1 : 20 with the cell suspension. Aliquots were removed post-inoculation and then again every 24 h over the 10 day growth period. Bacteria were washed three times with PBS, then heated to 95 °C for 10 min and subjected to a freezethaw step using liquid nitrogen and a 65 °C water bath. Protein concentrations were determined using the Coomassie Plus protein assay.
Enrichment of B. henselae OMPs.
Preparation of membrane fractions was performed on 10 to 20 lawns of B. henselae (per strain) which were washed three times with PBS (pH 7·4) before the cell pellet was resuspended in 0·05 M HEPES buffer (pH 7·4) and lysed by sonication for 35 min on ice (Branson Sonic Power Sonifier 450; 60 % cycle, 20 % output). The sonicated suspension was freezethawed twice then centrifuged for 15 min at 3000 g (4 °C). The supernatant was centrifuged for 45 min at 229 600 g (4 °C) and the resulting pellet was resolubilized in 4·3 ml HEPES buffer. Forty-eight microlitres of 1 mM MgCl2 and 0·48 ml of a 20 % (v/v) Triton X-100 solution were added to the mixture, which was then incubated at room temperature for 20 min. The sample was centrifuged for 45 min at 303 800 g (4 °C). The insoluble pellet (outer-membrane fraction) was resuspended in HEPES (pH 7·4), and the protein concentration was determined using the Coomassie Plus protein assay. Bacterial membrane proteins were electrophoresed on SDS 420 % (w/v) polyacrylamide gels and stained with Coomassie brilliant blue, as described previously (Laemmli, 1970; Lugtenberg et al., 1975
).
LPS preparation and LPS- (SDS) PAGE.
Small-scale preparations of LPS were prepared by proteinase K digestion of whole-cell lysates, as described previously (Hitchcock & Brown, 1983; Morona et al., 1995
) but with minor modifications. Five bacterial lawns per B. henselae strain were washed three times with PBS (pH 7·4) and pelleted by centrifugation for 2 min at 7000 g before resuspension in 50 µl lysing buffer [2 % (w/v) SDS, 4 % (v/v) 2-mercaptoethanol, 10 % (v/v) glycerol, 1 M Tris (pH 6·8) and 0·1 % (w/v) bromophenol blue]. The suspensions were boiled for 10 min, and protein was digested by the addition of 10 µl lysing buffer containing proteinase K (2·5 mg ml-1; Astral Scientific); the suspensions were then incubated at 60 °C for 5 h. Lysing buffer without proteinase K was added to paired controls before incubation. Bacterial LPS controls were also prepared as above (Salmonella ser. Typhimurium, full LPS; E. coli DH5
, rough LPS; H. influenzae, oligo LPS). Stored LPS preparations (4 °C) were heated to 100 °C for 5 min before SDS 20 % (w/v) PAGE with a constant current of 1214 mA for 1824 h; the tracking dye was allowed to run off for 60120 min. LPS was detected with silver staining as described by Tsai & Frasch (1982)
.
Biofilm-formation assays.
Experiments were performed in triplicate and repeated twice in a minor modification of an established method (Watnick et al., 1999). A clinical isolate of V. cholerae (El Tor) grown in plain nutrient broth (Voss & Attridge, 1993
) and E. coli ATCC 25922 were utilized as controls (Pratt & Kolter, 1999
). Ehrlenmeyer flasks were utilized as surfaces for bacterial attachment. Duplicate broth cultures were incubated until an OD55O value of between 0·6 and 0·9 was reached. Each flask was rinsed vigorously with distilled water to remove any non-adherent cells. A small loopful of selected biofilms was removed off the glass surface and analysed by transmission electron microscopy. Each flask was filled with a 0·1 % (w/v) crystal violet solution, allowed to incubate for 30 min and then rinsed vigorously with distilled water. Biofilm formation was quantified by measuring the OD570 value of a solution produced by extracting cell-associated dye with DMSO (Sigma).
Transmission electron microscopy (TEM).
Preparation of nickel-coated 400 mesh grids for TEM was performed as described by Voss & Attridge (1993); the grids were counterstained with 1 % (v/v) uranyl acetate for 30 s before rinsing in distilled water.
PCR and sequencing: papA, gltA, 16S23S rRNA intergenic region (ITS) and 16S rDNA.
pap PCR was carried out according to previously published methods using Pap-1 and Pap-2 primers (Anderson et al., 1997). BH1 and BH2 primers were used in conjunction with the broad-host-range primer 16SF according to the method of Bergmans et al. (1996)
for PCR and sequencing. 16S23S ITS PCR was performed as described by Matar et al. (1993)
, with minor modifications. External primers RPC5 (5'-AAGTCGTAACAAGGTA-3') and R23S2693 (5'-TACTGGTTCACTATCGGTCA-3') (200 nmol each) were used in a 100 µl PCR mixture (Matar et al., 1993
). Internal primers BHITSF (5'-AAGAGGATGCCCGGGAAGGT-3') and BHITSR (5'-GCGTTCTCTGCCTTGTGCAA-3') (Dillon et al., 2002
) (200 nmol each) were used to amplify a partial ITS region for sequence analysis as follows: 5 min denaturation at 95 °C; 35 cycles at 95 °C for 30 s, 50 °C for 60 s and 72 °C for 90 s; final extension at 72 °C for 10 min. Previously described primers (BhCS.1137, 5'-AATGCAAAAAGAACAGTAAACA-3'; CS140f, 5'-TTACTTATGATCCKGGYTTTA-3'; 200 nmol each) were used to generate and sequence gltA amplicons (Birtles & Raoult, 1996
).
Infrequent restriction site (IRS)-PCR with EagI/HhaI.
This method employs the use of an infrequent (EagI) and a frequent (HhaI) restriction enzyme to digest genomic DNA, which is then amplified with primers and adapters that target the extremities of the restricted fragments (Riffard et al., 1998). It offers a robust PCR-based tool for sampling restriction sites of choice across the genome. By amplifying only those fragments that have both restriction sites, it generates less bands and thus easier analyses than a PFGE-based approach using the same enzymes. PCR reagent concentrations and amplification conditions have been described by Handley & Regnery (2000)
. DNA amplification was performed in a PC-960C thermal cycler (Corbett Research). PCR products were separated on a 6·5 % (w/v) polyacrylamide gel in 1x TBE buffer for 3 h at 150 V, and visualized using ethidium bromide staining and an ultraviolet transilluminator using 667 Polaroid film.
Statistical analysis.
Data were analysed by the Student's t test using the SPSS package (version 11.0.1) for Windows.
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RESULTS |
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DAP revertants can be derived from phase-variant colonies
Occasional sectored colonies and frequent dry, granular outgrowths from aged SNP colonies were observed in many subcultures, especially those that had recently undergone transition. HC60' (the prime symbol denoting the transition to SNP), NU4714', ATCC 49882 (Houston-1) and ATCC 49793 were therefore aged on chocolate blood agar for 1845 days. Sectoring and outgrowths were not observed in our culture of Houston-1 (SNP type) nor in our culture of ATCC 49793 (SNP type), with these two strains being of relatively high passage number. While aged (>30-day-old) colonies of the SNP phenotype HC60' (S12) reliably yielded a DAP form (which we named PK60) on subculture, DAP forms could not be similarly obtained from aged SNP colonies of NU4714' on several attempts. PK60 behaved essentially in the same way as HC60 (its DAP phenotype switching after six further subcultures), and the SNP phenotype of PK60' remained stable with further rapid passage in vitro. An agar-pitting (DAP) form of B. henselae Houston-1 was recovered after passage of 29-day-old colonies onto fresh chocolate blood agar. The small dry colonies formed deep pits in the solid agar, but subculture of a sweep of these colonies yielded only SNP forms again on solid agar. A stable DAP variant was not obtained on three attempts. We were unable to select agar-pitting isolates from aged SNP forms of NU4714' (S12) and ATCC 49793, although agar-pitting was observed under 45-day-old colonies of ATCC 49793 which failed to grow on subculture.
The non-agar-pitting phenotype is associated with increased growth rates
We confirmed previous observations (Batterman et al., 1995) of significantly faster growth rates in non-agar-pitting isolates (Fig. 1
). SNP forms consistently grew faster than isogenic DAP parent cultures, with similar results obtained for all SNP and DAP isolates. Initial subcultures of HC35 (e.g. S4) grew slower than those of higher passage number (e.g. S20), despite persistence of the DAP phenotype, suggesting that the changes in growth rate may be independent of the change in colony phenotype. Furthermore, the most striking difference was seen in the NU4714/NU4714' pairing, in which reversibility of the phenotype could not be demonstrated.
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Biofilm formation by B. henselae in vitro
Biofilm formation has not been previously characterized in B. henselae, although early observers noted adherence of the organism to flasks (Regnery et al., 1992). Formation of a wash-resistant biofilm on the inert (pyrex) surface of Ehrlenmeyer flasks was greatest in strain HC35, while NU4714' (S12) formed the smallest biofilm out of the strains tested (Fig. 2
).
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DISCUSSION |
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We compared three sets of organisms, including one that was reversibly phase-variant, and another in which the original DAP phenotype persisted upon multiple passages. We also found that the two ATCC strains we obtained were unable to stably maintain a DAP phenotype. One of these (ATCC 49793) is the quality assurance strain widely used in Australian diagnostic laboratories and we know this strain to have been extensively passaged in vitro. Outer-membrane profiles were relatively similar between all strains tested. However, SNP isolates HC60', NU4714' and Houston-1 all have a prominent band at approximately 23 kDa. This band does not appear in HC35 (S20), nor is it present in the heavily passaged strain ATCC 49793. Strain ATCC 49793 is clearly quite attenuated, with loss of pilin expression and associated phenotypes, and therefore may not reliably reflect early changes associated with phase variation (as seen in HC60' and NU4714'). Whether the 23 kDa OMP seen on SDS-PAGE analysis reflects a compensatory or regulatory event remains to be determined, but there is evidence of genetic change or rearrangement occurring in the strains presenting this protein. IRS-PCR with EagI/HhaI has been used to separate the DNA fingerprints' of Houston-1 and Marseille (Handley & Regnery, 2000). We find, however, that the distinguishing (ca. 200 bp) band for Houston-1 is in fact absent from our copy of Houston-1. It is present in ATCC 49793, which has Houston-type 16S rDNA and gltA sequences, but is also absent from NU4714'. Like Houston-1, NU4714' never yielded stable DAP revertants. Another, more subtle, band (ca. 350 bp) appears to come and go in the HC60-derived isolates, suggesting a relevant genetic change, but one which does not affect the presence or amplicon size obtained from the papA gene. The exact significance of such changes and the extent to which they are useful guides to phase variation is uncertain, but emphasizes that morphological change is associated with one or more genetic events. Comparison with NU4714 demonstrates that these events are not identical and that they may not be causal.
Our copy of Houston-1 is clearly a non-agar-pitting form of B. henselae, but it is well piliated, strongly auto-agglutinates and forms a biofilm. This contrasts with the poorly piliated strain ATCC 49793, which does not auto-agglutinate or form a biofilm, consistent with a pilin-dependent agar-pitting or twitching phenotype but illustrating that adhesins sufficient to promote strong intercellular and inert surface attachment may be present in its absence. Biofilm formation is likely to be a basic characteristic of B. henselae, as suggested in initial descriptions of the organism (Regnery et al., 1992). Pilin expression appears to be necessary for biofilm formation, and the associated in vitro phenotype may alter with serial passage in a way that is influenced by auto-agglutination, with coarse granules precipitating out of solution. The agar-pitting phenotype is most likely to be a reflection of solid-phase (twitching) motility, and pili are necessary but insufficient for this phenotype. The ability to migrate to fresh nutrient sources may thus explain the observed selection of DAP forms in those (piliated) SNP forms that retain the potential for reversion, and this may occur as a reversible genetic event, accompanied by direct or compensatory alterations in the expression of specific OMPs. The predictable development of SNP forms from DAP forms may be explained by competitive success of the faster-growing (SNP) forms in nutritious media, but (presumably without any solid-phase motility) exhausting nutrient supply and dying in aged colonies while slower-growing pitting/spreading DAP forms persist. Ultimately, an event resulting in loss of pilin expression or other irreversible events may make the return to the DAP form impossible. This is consistent with observations that primary isolates which may be detected in tissue culture are poorly recovered on solid media (La Scola & Raoult, 1999
) and with pili of a type which, in other organisms, confer solid-phase (twitching) motility (Batterman et al., 1995
). Thus, the passaged, non-reverting strains may include phase-variant isolates with acquired secondary and/or compensatory mutations, some of which may make it impossible to revert to the DAP phenotype.
In conclusion, and in contrast to published data, it is now clear that the growth characteristics associated with phase variation in B. henselae are not necessarily associated with loss of piliation, may be associated with variation in OMP expression and apparent genotype, and are variably reversible. Biofilm formation and expression of putative type IV pili appear to be basic characteristics of this organism. Genetic markers previously thought to distinguish between serotypes Houston and Marseille are shown to be misleading and probably reflect unrelated genetic events which may yet give us some insights into phase variation in B. henselae. Future work should be directed at understanding the genetic basis of phase variation in B. henselae, and detailed studies of proven primary isolates are needed. Workers in the field are cautioned to consider the genetic and phenotypic stability of isolates from which data regarding virulence and pathogenicity are generated.
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ACKNOWLEDGEMENTS |
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Received 23 September 2002;
revised 25 October 2002;
accepted 2 December 2002.