INFECTION AND IMMUNITY, Jan. 2007, p. 35–43 Vol. 75, No. 10019-9567/07/$08.00�0 doi:10.1128/IAI.00963-06Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Analysis of Bartonella Adhesin A Expression Reveals Differencesbetween Various B. henselae Strains�†

Tanja Riess,1 Gunter Raddatz,2 Dirk Linke,3 Andrea Schafer,1 and Volkhard A. J. Kempf1*Institut fur Medizinische Mikrobiologie und Hygiene, Eberhard-Karls-Universitat, Elfriede-Aulhorn-Str. 6, 72076 Tubingen,

Germany1; Max-Planck-Institut fur biologische Kybernetik, Spemannstr. 34, 72076 Tubingen, Germany2; andMax-Planck-Institut fur Entwicklungsbiologie, Abteilung Proteinevolution, Spemannstr. 35,

72076 Tubingen, Germany3

Received 16 June 2006/Returned for modification 16 August 2006/Accepted 6 October 2006

Bartonella henselae causes cat scratch disease and the vasculoproliferative disorders bacillary angiomatosisand peliosis hepatis in humans. One of the best known pathogenicity factors of B. henselae is Bartonella adhesinA (BadA), which is modularly constructed, consisting of head, neck/stalk, and membrane anchor domains.BadA is important for the adhesion of B. henselae to extracellular-matrix proteins and endothelial cells (ECs).In this study, we analyzed different B. henselae strains for BadA expression, autoagglutination, fibronectin (Fn)binding, and adhesion to ECs. We found that the B. henselae strains Marseille, ATCC 49882, Freiburg 96BK3(FR96BK3), FR96BK38, and G-5436 express BadA. Remarkably, BadA expression was lacking in a B. henselaeATCC 49882 variant, in strains ATCC 49793 and Berlin-1, and in the majority of bacteria of strain Berlin-2.Adherence of B. henselae to ECs and Fn reliably correlated with BadA expression. badA was present in all testedstrains, although the length of the gene varied significantly due to length variations of the stalk region.Sequencing of the promoter, head, and membrane anchor regions revealed only minor differences that did notcorrelate with BadA expression, apart from strain Berlin-1, in which a 1-bp deletion led to a frameshift in thehead region of BadA. Our data suggest that, apart from the identified genetic modifications (frameshiftdeletion and recombination), other so-far-unknown regulatory mechanisms influence BadA expression. Be-cause of variations between and within different B. henselae isolates, BadA expression should be analyzed beforeperforming infection experiments with B. henselae.

Bartonella henselae is a gram-negative, facultatively intracel-lular, fastidious, and slow-growing bacterium. Cats are thereservoir host of the bacteria, and transmission to humansoccurs through cat scratches or cat fleas (8). Usually, in immu-nocompetent patients, a B. henselae infection results in catscratch disease, an often self-limiting disease characterizedby lymphadenopathy. In immunocompromised patients, B.henselae infections can cause tumorous proliferations of endo-thelial cells (ECs) in the skin or inner organs, which are calledbacillary angiomatosis or peliosis hepatis, respectively (2). Theability to induce vasculoproliferations is a unique feature ofBartonella spp. Both in vitro and in vivo, B. henselae infectionslead to the activation of hypoxia-inducible factor 1 (HIF-1), thekey transcription factor involved in angiogenesis, and to thesecretion of vasculoproliferative cytokines, e.g., vascular endo-thelial growth factor (VEGF) (15, 17).

One important pathogenicity factor of B. henselae is Bar-tonella adhesin A (BadA) (29), originally described as a “typeIV-like pilus” (4). The 340-kDa BadA (monomer) consists ofan N-terminal head region; a long, highly repetitive stalk; anda C-terminal membrane anchor (29). Together with Yersiniaadhesin A (YadA) of Yersinia enterocolitica, Haemophilus in-

fluenzae adhesin (Hia) and Haemophilus surface fibrils (Hsf) ofH. influenzae, and ubiquitous surface protein A (UspA) ofMoraxella catarrhalis, BadA belongs to the class of trimericautotransporter adhesins (TAAs), which all share similar mod-ular architectures. The membrane anchor distinguishes TAAsfrom classical monomeric autotransporters; it is a highly con-served element, present throughout the protein family (14, 22),that has been shown to form trimers (19, 26, 34).

BadA is crucial for the adhesion of B. henselae to host cellsand extracellular-matrix proteins (fibronectin [Fn] and colla-gens). It is also important for the activation of HIF-1 and theinduction of VEGF secretion in infected host cells (17, 29).Variably expressed outer membrane proteins (Vomps) of Bar-tonella quintana (37) and Bartonella repeat protein A (BrpA)of Bartonella vinsonii (12), which are all highly homologous toBadA, are also members of the TAA family (22).

BadA expression is lost after multiple passaging of the bac-teria in vitro. This phenomenon has been described as “phasevariation” (4), but the underlying mechanism of gene regula-tion is not known. In addition, in many studies, the exactpassage number of the bacteria is unfortunately not stated, andmoreover, it is not clear whether these strains express BadA.This is somewhat problematic, as a lack of BadA expressionmight greatly influence the outcome of infection experimentswith B. henselae. The only BadA-negative B. henselae strainthat has been characterized functionally and genetically so faris a highly passaged variant of B. henselae Marseille (17), inwhich a deletion of the 5� end of badA and a large regionupstream of the gene occurred (29).

Here, we analyzed 10 B. henselae strains (human and cat

* Corresponding author. Mailing address: Institut fur MedizinischeMikrobiologie und Hygiene, Elfriede-Aulhorn-Str. 6, D-72076 Tubin-gen, Germany. Phone: 49-7071-2981526. Fax: 49-7071-295440. E-mail:volkhard.kempf@med.uni-tuebingen.de.

† Supplemental material for this article may be found at http://iai.asm.org/.

� Published ahead of print on 23 October 2006.

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isolates from different geographical regions) that are widelyused in infection experiments for (i) BadA expression, by im-munofluorescence and Western blotting; (ii) BadA-dependentautoagglutination; and (iii) the correlation between BadA ex-pression and the ability of the strains to infect ECs. We foundthat adherence to ECs and Fn reliably correlated with theexpression of BadA, confirming the importance of BadA inhost cell infection. Also, as the regulation of BadA expressionis not yet understood, the genes and the putative promoterregions were analyzed by PCRs and partial sequencing, show-ing that badA is present in all tested strains, although it is notexpressed in several isolates. In one strain (Berlin-1), a frame-shift deletion in the head region of badA correlated with a lackof BadA expression, whereas in three BadA-negative isolates(an ATCC 49882 variant, ATCC 49793, and Berlin-2), no ex-planation based on the analysis of the promoter region, thehead, and the membrane anchor domain can be given.

MATERIALS AND METHODS

Bacterial strains and growth conditions. The strains used in this study aresummarized in Table 1. All B. henselae strains were grown on Columbia bloodagar (Becton Dickinson, Heidelberg, Germany) in a humidified atmosphere at37°C and 5% CO2. Except for B. henselae Marseille wild type and B. henselaeMarseille variant strains, the passage numbers of the strains are not known.

Determination of autoagglutination. B. henselae strains were harvested fromagar plates after 5 days of growth and resuspended in phosphate-buffered saline(PBS). The optical density at 600 nm (OD600) was adjusted to 2.0 (�1 � 109

bacteria per ml), and bacteria were used at a concentration of �2 � 108 per ml.Three milliliters of each suspension was added to a plastic tube and incubated at37°C with 5% CO2 in a humidified atmosphere. After 60 min, 10 �l of thesuspension was taken from the bottom of each tube and transferred to a glassslide, air dried, and fixed with 3.75% PBS-buffered paraformaldehyde (PFA).After being washed with PBS, the bacteria were stained with 4�,6�-diamidino-2-phenylindole (DAPI) (1 �g/ml dissolved in PBS). Autoagglutination was evalu-ated by confocal laser scanning microscopy (CLSM) (see below). Images weredigitally processed with Photoshop 6.0 (Adobe Systems).

Culture and infection of endothelial cells. Human umbilical vein endothelialcells (HUVECs) were cultured in endothelial cell growth medium (PromoCell,Heidelberg, Germany), and infection experiments were performed in endothelialcell basal medium (PromoCell) as described previously (16). Briefly, 1 � 105 cellswere seeded onto coverslips the day before the experiment. For infections,bacteria were harvested from agar plates and the OD600 was adjusted to 2.0(�1 � 109 bacteria per ml). Bacteria were used at a multiplicity of infection of100 and sedimented onto cultured cells by centrifugation for 5 min at 300 � g atroom temperature. Infections were stopped after 30 min by adding 3.75% PBS-buffered PFA. Bacterial adhesion was analyzed by CLSM.

Immunostaining and CLSM. B. henselae was resuspended in PBS, air dried onglass slides, and fixed in 3.75% PBS-buffered PFA. Immunostaining was per-formed as described previously (29). Briefly, the bacteria were washed threetimes in PBS after fixation and each of the following incubation steps, andnonspecific binding was blocked by incubation with 0.2% bovine serum albuminfor 15 min. The bacteria were incubated with a BadA-specific rabbit immuno-globulin G antibody (Ab) for 1 h, followed by incubation with a fluoresceinisothiocyanate (FITC)-conjugated secondary anti-immunoglobulin G Ab (29).

For adhesion assays, HUVECs (1 � 105) were seeded onto coverslips andinfected with B. henselae, and infection was stopped after 30 min by adding3.75% PFA. Filamentous actin was stained with tetramethyl rhodamine isothio-cyanate (TRITC)-labeled phalloidin for 30 min, and bacterial and host cell DNAwas stained with DAPI for 10 min. FITC-conjugated secondary Ab and TRITC-labeled phalloidin were purchased from Dianova (Hamburg, Germany) andSigma (Deisenhofen, Germany). Cellular fluorescence was evaluated using aLeica DM IRE 2 confocal laser scanning microscope. Three different fluoro-chromes were detected, representing the green (FITC), red (TRITC), and blue(DAPI) channels. Images were digitally processed with Photoshop 6.0 (AdobeSystems).

Western blotting and detection of Fn binding. For Western blotting, B.henselae strains were harvested from agar plates after 5 days of cultivation, andthe OD600 was adjusted to 2.0 (�1.0 � 109 bacteria per ml) for normalization.Equal volumes of bacterial suspensions were centrifuged, and the resultingbacterial pellets (�1.0 � 107 bacteria) were lysed in sodium dodecyl sulfatesample buffer, heated at 98°C for 3 min and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis in 12% gels. For immunoblotting,proteins were transferred onto nitrocellulose membranes (Schleicher andSchuell, Dassel, Germany). Membranes were blocked for 1 h in 5% skim milkpowder in 25 mM Tris, pH 7.5, 0.15 M NaCl, and 0.05% Tween 20. For thedetection of BadA, membranes were incubated with a BadA-specific rabbit Ab(29) overnight. To analyze the Fn-binding capacity, the membranes were incu-bated with a monoclonal anti-Fn Ab (Becton Dickinson). The blots were devel-oped using horseradish peroxidase-conjugated secondary Abs, and signals werevisualized via chemiluminescence (ECL; Amersham).

PCRs. For PCR analysis of badA, bacterial genomic DNA was isolated usingeither the Genomic-tip 100/G kit (QIAGEN, Hilden, Germany) or the BacterialGenomic DNA kit (Metabion, Martinsried, Germany) according to the manu-facturer’s instructions.

Long-distance PCRs of full-length badA were performed with the ExpandLong Template PCR system (Roche, Mannheim, Germany) and the primersbadA-LRf1 (5�-TTACATACCGGATCCCACTCAATATAAAGAAACACTCG-3�) and badA-LRr2 (5�-ACTGCATAAGGATCCGACGTGTTTCACCAGCTGC-3�). Primer badA-LRf1 binds 484 bp upstream of badA, and primer badA-LRr2 binds 182 bp downstream of badA. The cycling conditions were as follows:denaturation at 92°C for 10 s, annealing at 58°C for 10 s, and extension at 68°Cfor 8 min for 10 cycles, followed by 15 cycles with denaturation at 92°C for 10 s,annealing at 58°C for 30 s, and extension at 68°C for 8 min, plus 20 s ofelongation for each successive cycle. Cycling was started with an initial 2-mindenaturation at 92°C and a final 7-min extension at 68°C. Long-distancePCRs of the badA stalk region were performed using the primers badAf6(5�-AAAGCATTAAGGGGAATGATATCAG-3�) and badAr7 (5�-CTCATA

TABLE 1. B. henselae strains used in this study

Designation Otherdesignation

Passageno. Characteristic(s) Reference or source

Marseille wild type URLLY-8 �10 Human isolate from a cat scratch disease patient; BadA� 11; D. Raoult, Marseille,France

Marseille variant URLLY-8 �50 BadA-negative variant of strain Marseille; extensivelypassaged; originally described as “pilus” negative

17

ATCC 49882 Houston-1 Unknown Human isolate from blood of an AIDS patient 1, 28ATCC 49882 variant Houston-1 Unknown Variant of ATCC 49882; laboratory strain A. Sander, Freiburg,

GermanyATCC 49793 87-66 Oklahoma Unknown Human isolate from blood 4, 33FR96BK3 Unknown Cat isolate, Freiburg, Germany 30FR96BK38 Unknown Cat isolate, Freiburg, Germany 30Berlin-1 Unknown Human isolate from a bacillary angiomatosis patient,

Berlin, Germany3

Berlin-2 Unknown Cat isolate, Berlin, Germany 3G-5436 Houston-1 Unknown Human isolate from blood 35, 36; CDC

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CTTAACAGCACTATCTGC-3�). The cycling conditions were as follows:denaturation at 92°C for 10 s, annealing at 55°C for 30 s, and extension at68°C for 6 min for 10 cycles, followed by 15 cycles with denaturation at 92°Cfor 10 s, annealing at 55°C for 30 s, and extension at 68°C for 6 min, plus 5 sof elongation for each successive cycle. Cycling was started with an initial2-min denaturation at 92°C and a final 7-min extension at 68°C. PCR productswere analyzed using 0.6% agarose gels.

All other PCRs were carried out according to standard protocols with Taqpolymerase purchased from Metabion. For analysis of the putative promoterregion of badA, primers badAPf1 (5�-TATTGGATCCTGAATTTACAGAGTGTAAGC-3�) and badAr9 (5�-TTACAGTTTCCAATGAGAGC-3�) were used.The primers for the amplification of the head region were badAf5 (5�-CCAATAATAAAACTGCATAATGATTCGACG-3�) and badAr8 (5�-TGATATCATGGATCCTTATGCTTTTAGCTGTGC-3�). PCRs of the membrane anchor re-gion were performed with primers badAf4 (5�-GATAGTACTGGCAAGAAAACG-3�) and badAr5 (5�-CCAATAATAAAACTGCATAATGATTCGACG-3�).The PCR products were sequenced, and sequence alignments were made inClustal W (6; http://www.ebi.ac.uk/clustalw/).

Nucleotide sequence accession numbers. The promoter, head, and membraneanchor sequences of the various B. henselae strains (Table 1) are given underGenBank accession numbers DQ779056 (ATCC 49882), DQ779057 (ATCC49882 variant), DQ779058 (ATCC 49793), DQ779059 (FR96BK3), DQ779060(FR96BK38), DQ779061 (Berlin-1), DQ779062 (Berlin-2), and DQ779063 (G-5436). The complete badA sequence and the putative promoter sequence of B.henselae strain Marseille are available under GenBank accession numbersDQ665674 and DQ779055, respectively (generation of the complete badA se-quence is not described in this report).

RESULTS

Analysis of BadA expression in various B. henselae strains.BadA has been characterized as an important pathogenicityfactor of B. henselae (29). Expression of BadA is lost afterbacteria are passaged in vitro (4, 17), but unfortunately, inmany studies the passage number of bacteria was not statedand expression of BadA was not tested. Therefore, we wantedto analyze different B. henselae strains for BadA expression.The strains we used were three cat isolates and five humanisolates from different geographical regions (Table 1). TheBadA-positive strain B. henselae Marseille (early passage) andthe highly passaged BadA-negative strain B. henselae Marseillevariant (originally described as B. henselae Pil�) (17, 29) wereused as positive and negative controls, respectively.

First, BadA expression was analyzed by immunofluores-cence using BadA-specific Abs (Fig. 1). The results showedthat B. henselae strains Marseille, ATCC 49882, Freiburg96BK3 (FR96BK3), FR96BK38, and G-5436 expressed BadA;in contrast, B. henselae strains Marseille variant, ATCC 49882variant, ATCC 49793, and Berlin-1 did not. In strain Berlin-2,the vast majority of bacteria were BadA negative and only

FIG. 1. BadA expression in different B. henselae strains. (A) BadA was detected on the surfaces of the strains Marseille, ATCC 49882,FR96BK3, FR96BK38, and G-5436 by immunofluorescence using a specific anti-BadA-Ab (var., variant strain). Note that in strain Berlin-2, onlya few bacteria are BadA positive. (B) For internal control, bacterial DNA was stained with DAPI. Scale bar, 8 �m.

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some expressed BadA. These data show that expression ofBadA differs between various B. henselae strains independentlyof their origin of isolation (human or cat) or geographic region(Europe or United States).

Correlation between BadA expression and biological func-tions of different B. henselae strains. It has been shown thatBadA of B. henselae and VompA to -C of B. quintana areimportant for autoagglutination (4, 37). Moreover, expressionof BadA is crucial for the adhesion of B. henselae to extracel-lular-matrix proteins (e.g., Fn) and to ECs (29). Therefore, wenext analyzed the different B. henselae strains for (i) Fn bind-ing, (ii) autoagglutination, and (iii) adhesion to ECs.

Bacterium-bound Fn was detected by Western blotting ofwhole bacterial lysates using anti-Fn Abs (Fig. 2). For controlreasons and to confirm the data obtained by immunofluores-cence (see above), expression of BadA was analyzed in parallelby Western blotting using anti-BadA Abs. The results clearlyshowed that only the BadA-positive strains (Marseille, ATCC49882, FR96BK3, FR96BK38, and G-5436) bound Fn, whereasBadA-negative strains (Marseille variant, ATCC 49882 variant,ATCC 49793, and Berlin-1) did not. In accordance with im-munofluorescence, strain Berlin-2 showed only very weak Fnbinding.

Autoagglutination of B. henselae was analyzed by CLSMafter incubation of bacteria in suspension. Only BadA-positivestrains (Marseille, ATCC 49882, FR96BK3, FR96BK38, andG-5436) autoagglutinated, whereas BadA-negative bacteria(Marseille variant, ATCC 49882 variant, ATCC 49793, andBerlin-1) were not self-adherent (Fig. 3).

Moreover, we investigated the adherence of the differentstrains to ECs. For this purpose, HUVECs were infected withB. henselae (multiplicity of infection, 100), and the number ofadherent bacteria was analyzed by CLSM 30 min after infec-tion (Fig. 4). The BadA-negative strains (Marseille variant,ATCC 49882 variant, ATCC 49793, and Berlin-1) adheredsignificantly less to HUVECs than BadA-positive strains (Mar-seille, ATCC 49882, FR96BK3, FR96BK38, and G-5436). B.

FIG. 2. Analysis of the Fn-binding capacity of B. henselae and de-tection of BadA expression by Western blotting. (A) Fn (240 kDa)bound to bacteria was detected in bacterial lysates using an anti-Fn Ab.Note that only BadA-positive strains bind Fn. Strain Berlin-2, in whichonly a few bacteria express BadA (see Fig. 1), shows very weak Fnbinding. (B) For internal control, BadA (�340 kDa) was detected inbacterial lysates by using a specific anti-BadA Ab. Multiple bands areinterpreted as degradation products of the high-molecular-weightBadA (29).

FIG. 3. BadA-dependent autoagglutination of different B. henselae strains. Bacteria were resuspended in PBS, incubated for 60 min, stainedwith DAPI, and analyzed by CLSM. Note that only BadA-positive strains show autoagglutination. Scale bar, 20 �m.

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henselae Berlin-2 appeared in these functional assays like theBadA-negative strains, most likely because most of the bacteriawere BadA negative (see above).

Taken together, BadA expression of different B. henselaestrains correlated perfectly with function (autoagglutination,Fn binding, and EC adherence). These results suggest that theproposed functions of BadA originally identified in the B.henselae Marseille strain (Fn binding and adherence to ECs)(29) or of the BadA-homologous VompA to -C in B. quintana(autoagglutination) (37) are valid for the whole genus Bar-tonella.

Genetic analysis of badA in different B. henselae strains. Lossof BadA expression after B. henselae was passaged in vitro onagar plates has been described as phase variation (4, 17). How-ever, the actual mechanism of gene regulation is not known. Sofar, the sequence of the genomic region harboring badA isavailable only for the special B. henselae ATCC 49882 sub-strain used in the genome-sequencing project (1) and for theMarseille (GenBank accession number DQ665674) and theBadA-negative Marseille variant strains (29).

The size of badA from the strain ATCC 49882, for whichthe genomic sequence has been published (1), is about 9.1 kb.It contains a 1-bp deletion at position 9054, leading to a pre-mature stop codon upstream of the membrane anchor se-quence (29). B. henselae Marseille contains a complete badAgene of 9.25 kb, while the corresponding BadA-negative vari-ant strain contains an 8.5-kb deletion spanning the 5� end andthe promoter region of badA (29). As the reason for divergentBadA expression of the strains used in this study is not known,we analyzed the respective badA genes and the putative pro-moter regions in greater detail.

First, the presence of badA was confirmed by long-distancePCR (see Materials and Methods). The PCR revealed ampli-

cons in all strains showing the presence of badA apart from theB. henselae Marseille variant (data not shown), in which thebinding site of the upstream primer is missing (29). Interest-ingly, badA genes differ in size, ranging from about 9 to 12 kb(Fig. 5A). Amplification and sequencing of the nonrepetitivehead and membrane anchor regions of badA revealed that theyare exactly the same size in every strain investigated here (Fig.5B). Therefore, we suggested that the differences in the size ofbadA are probably caused by variations in the lengths of thestalk sequences in various B. henselae strains. For this reason,we performed long-distance PCRs of the stalk region. In fact,we found the stalk region of badA to be highly variable inlength (�7 to 10 kb) (Fig. 5B). Notably, no deletions, such asare known from the B. henselae Marseille variant (deletionsize, 8.5 kb), leading to a loss of essential parts of the gene orthe promoter region (29) were detected, and moreover, itseems that there is no correlation between the size of the geneand BadA expression.

Analysis of the head domain. Next, the head-coding se-quences of badA were sequenced and analyzed for differencesthat could explain possible mechanisms of gene regulation.The data revealed 100% sequence homology of the differentbadA head regions (ending before the first neck sequence)either to that of B. henselae ATCC 49882 (the sequence ref-erence strain) (1) or to that of B. henselae Marseille (GenBankaccession number DQ665674). Although two clusters of badAhead domain sequences were detected (cluster A, B. henselaeMarseille, FR96BK3, and Berlin-2; cluster B, B. henselaeATCC 49882, ATCC 49882 variant, ATCC 49793, FR96BK38,and G-5436), these differences did not correlate with BadAexpression (Fig. 6A). Surprisingly, strain Berlin-1, belonging tocluster B, exhibited a single-base deletion at position 585. The

FIG. 4. BadA-dependent adhesion of different B. henselae strains to endothelial cells. HUVECs were infected with B. henselae, and adhesionof the bacteria was analyzed after 30 min by CLSM. Bacteria and host cell nuclei were stained with DAPI (blue signal); filamentous actin wasstained with TRITC-labeled phalloidin (red signal). Note that only BadA-positive strains adhere strongly to HUVECs (arrows). Scale bar, 20 �m.

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resulting frameshift introduced a stop codon within the headregion, explaining the BadA-negative phenotype of the strain.

Analysis of the membrane anchor domain. Analysis of themembrane anchor region (starting after the last neck se-quence) showed that none of the strains contains the same1-bp deletion as that known from the particular ATCC 49882type strain sequence (1, 29). Even the ATCC 49882 strain weused in this study contains the complete membrane anchorsequence without any frameshift mutations. Again, the strainscluster in two groups of identical sequences (cluster A,FR96BK3 and Berlin-2; cluster B, ATCC 49882, ATCC 49882variant, ATCC 49793, Berlin-1, FR96BK38, and G-5436), withthe B. henselae Marseille sequence as an intermediate betweenthe clusters (Fig. 6B), and sequence differences do not corre-late with BadA expression. As there are only minor amino acidexchanges between the clusters, we suggest that the membraneanchors of all strains are probably functional and should allowthe transport of the passenger domain of BadA.

Analysis of the putative promoter region of badA. For anal-ysis of the putative promoter region of badA, �650 bp up-stream of the start codon were sequenced (for an alignment of

the sequences, see Fig. S1 in the supplemental material). Se-quence comparison revealed only minor differences betweenthe strains. The sequences cluster in two groups of identicalsequences, as shown for the head and membrane anchor re-gions (cluster A, strains Marseille, FR96BK3, and Berlin-2;cluster B, ATCC 49882, ATCC 49882 variant, ATCC 49793,Berlin-1, and G-5436). The sequence of FR96BK38 is an in-termediate between the groups and contains some uniquebases. As both BadA-positive and BadA-negative strains arepresent in both clusters, it can be suggested that the differencesin the promoter region do not correlate with the expression ofBadA.

DISCUSSION

The TAA BadA (originally described as “pilus” [4]) is animportant pathogenicity factor of B. henselae (29). Loss ofBadA expression after passaging has been described as phasevariation (4, 17, 20), but the actual mechanism of gene regu-lation is not known. In the current study, we investigated whichstrains of B. henselae express BadA on their surfaces andwhether this expression correlates with function. Moreover, wetried to elucidate mechanisms involved in the regulation ofBadA expression. For this purpose, BadA expression by dif-ferent B. henselae strains isolated from humans and cats indifferent geographical regions was analyzed, revealing dra-matic differences between and within distinct B. henselaestrains.

Five of 10 B. henselae strains (Marseille, ATCC 49882,FR96BK3, FR96BK38, and G-5436) expressed BadA (Fig. 1and 2), whereas four strains (Marseille variant, ATCC 49882variant, ATCC 49793, and Berlin-1) did not. In one strain (B.henselae Berlin-2), only a few bacteria expressed BadA.Among the strains we investigated, no correlation betweenBadA expression and the source of the pathogen (human orcat isolates and different geographic regions of the first isola-tion) could be observed.

It was shown earlier that BadA is crucial for Fn binding andadherence to host cells (29). The VompA to -C proteins wereshown to mediate autoagglutination and collagen binding (37),and these Vomp proteins were recently described as represent-ing TAAs highly homologous to BadA (22). Therefore, weanalyzed whether the expression of BadA on the variousstrains correlates with Fn binding, adherence to ECs, andautoagglutination. In fact, all BadA-expressing strains showedthese biological characteristics, which were absent when BadAwas not expressed. These results confirm the crucial role ofBadA in the infection process that was described earlier for theparticular B. henselae Marseille strain (29).

Phase variation is defined as switching the expression of acertain phenotype on and off. Usually, this happens randomlyand at high frequency. One possible mechanism for phasevariation is slipped-strand mispairing, leading to variations inthe number of short sequence repeats located either in thepromoter or within a gene, influencing gene expression (13).Such a mechanism of gene regulation was shown, for example,for the BadA-homologous Neisseria adhesin A (NadA) of Neis-seria meningitidis: here, a TAAA repeat tract is located up-stream of the putative �35 element of the nadA promoter (25).Variations in the number of these repeats were associated with

FIG. 5. Presence and lengths of badA, badAhead, badAstalk, andbadAmembrane anchor of different B. henselae strains analyzed by PCR.(A) Long-distance PCRs were performed (for details, see Materialsand Methods), and the PCR products were analyzed in a 0.6% agarosegel. All B. henselae strains harbor badA, although there are differencesin the size of the gene (�9 to 12 kb). (B) Detection of the badAhead-(upper left), badAstalk- (lower middle) and badAmembrane anchor-codingsequences (upper right) in different B. henselae isolates. Note thatdifferences in size are detectable only in badAstalk. For reasons ofclarity, BadA is schematically depicted with the signal peptide (lightblue), the head sequence (gray and red), 24 neck/stalk repeats (brownand green, respectively) and the membrane anchor (orange).

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changes in the level of nadA expression by altering the bindingof the transcriptional regulator integration host factor to thenadA promoter (24). Similarly, expression of UspA1 of M.catarrhalis, another TAA highly homologous to BadA (22),exhibits phase variation, depending on the number of guanineresidues in a homopolymeric poly(G) tract upstream of uspA1(21). Based on these observations, we analyzed the putativepromoter regions of badA in all B. henselae strains tested herefor such sequence differences, which might explain a mecha-nism of phase variation. However, we found only minor single-nucleotide exchanges that did not correlate with BadA expres-sion. Therefore, it can be assumed that (in contrast to NadA ofN. meningitidis and UspA1 of M. catarrhalis) BadA expressionis not regulated by genetic changes in the promoter region.

Next, we analyzed the 5� end of badA (coding for the signalsequence and the head domain) for sequence repeats thatmight facilitate phase variation. We found that in B. henselaeBerlin-1, the deletion of one guanine in a stretch of six guanineresidues present in all other B. henselae strains (possibly causedby slipped-strand mispairing) leads to the introduction of astop codon within the head-coding sequence (Fig. 6). However,as this mutation occurs in only one BadA-negative strain, thisis probably not a common mechanism regulating BadA expres-sion. Remarkably, in the sequenced B. henselae ATCC 49882isolate, a similar phenomenon of a 1-bp deletion leads to aframeshift and to the introduction of a stop codon upstream ofthe membrane anchor-coding sequence (1, 29). Again, none ofthe strains we have tested contained this deletion. All otherdifferences we found in the head and membrane anchor se-quences resulted in only minor changes in the deduced aminoacid sequences that neither correlate with BadA expressionnor should affect the structure or function of BadA. Based onthese results, it might be speculated that single-base deletionsfrequently affect BadA expression. Because of the enormoussize of the badA gene (�9 to 12 kb) (Fig. 5A), mutations(including single-base deletions) might become more likely forstochastic reasons.

Additionally, it can be suggested that recombination eventslead to a loss of TAA expression. For instance, the deletion ofan 8.5-kb fragment spanning from the 5�-terminal end of theopen reading frame upstream of badA (BH01490) to the 5�-terminal end of badA causes the lack of BadA expression in theB. henselae Marseille variant (29). A similar observation wasmade for B. quintana, in which a deletion of two vomps (vompAand -B) within the vomp gene cluster (vompA to -D) wasdetected (37). It is worth mentioning that we generated asecond BadA-negative variant strain of B. henselae Marseilleafter 55 passages. In this strain, we could not detect a largedeletion, as is known for B. henselae Marseille variant (seeabove). Here, the insertion of one guanine residue at position4513 led to a stop codon within the stalk region, which is mostlikely responsible for the lack of BadA expression as shown byimmunofluorescence and immunoblotting (data not shown).From all of these data, it can be suggested that within onestrain at least two different events (e.g., recombination or sin-gle-base deletion or insertion) might cause the loss of BadAexpression.

The observed variations in the total size of badA are due tovariations in the length of the repetitive neck/stalk sequences;the sizes of the head and membrane anchor sequences turned

out to be equal in all B. henselae strains investigated here (Fig.5B). The highly repetitive nature of the neck/stalk fragmentsshould facilitate recombination events, resulting in an expan-sion or contraction of their copy numbers (22). This suggestionis supported by the analysis of the vomp genes of B. quintana,which are constructed very similarly to badA but differ mainlyin the number of their neck/stalk repeats (BadA, 24 neckrepeats; VompA to -C, 6 neck repeats; VompD, 7 neck re-peats) (32). It can be assumed that these recombinations leadto frameshifts in the neck/stalk repeats affecting BadA expres-sion (22) (Fig. 5). However, because of the enormous length(�7 to 10 kb) and the highly repetitive nature of the badAneck/stalk repeats, we were unfortunately not able to deter-mine the sequences of these parts of the gene in strains otherthan B. henselae Marseille and its variants. Why the number ofTAA neck/stalk repeats within the genus Bartonella differ by upto four times and whether this has any biological impact onthe infection process are unclear and need to be elucidatedfurther.

It might be speculated that expression of BadA is crucial forthe virulence of B. henselae when infecting humans or theanimal reservoir host. Because of its enormous size, expressionof BadA should be a highly energy-consuming process, limitingthe growth of freshly isolated and BadA-expressing wild-typestrains. Therefore, loss of BadA expression upon extensivepassaging on agar plates in vitro without any ecological pres-sure might improve the growth of BadA-negative variants. Thisis supported by the observation that BadA (“pilus”)-negativestrains grow much faster on agar plates than wild-type bacteria(4, 18, 28). It can be speculated that, once a mutation affectingBadA expression occurs, BadA-negative strains overgrow theslowly growing BadA-positive wild-type colonies. This obser-vation is supported by the presence of only a few BadA-posi-tive bacteria in the culture of B. henselae Berlin-2 (Fig. 1) andthe observation that the percentage of BadA-positive B.henselae Marseille decreases gradually with passage number(data not shown). According to this, further passaging of B.henselae Berlin-2 should result in a completely BadA-negativestrain.

It is important to note that work on the pathogenicity of B.henselae was often performed using bacteria with variable,unstated (7, 23, 27, 31), and sometimes high passage numbers(9, 10) that were not tested for BadA expression. Becauseseveral B. henselae substrains (e.g., the ATCC 49882 variant)do not express BadA, we strongly emphasize that BadA ex-pression should be evaluated first when performing infectionexperiments with B. henselae, as BadA determines host cellinteraction (15, 29). This observation is also important in termsof interpreting serological results obtained with B. henselaeantigen (5), as BadA has been shown to be an immunodomi-nant surface protein of B. henselae (29).

Taken together, our results represent the first systematicapproach analyzing BadA expression in different B. henselaestrains. Our data suggest that different events (single-base de-letions or insertions and recombinations) affect BadA expres-sion, resulting in the coexistence of BadA-positive and BadA-negative substrains within one B. henselae type strain (e.g., B.henselae ATCC 49882). Further analysis (e.g., functional pro-moter analysis) is needed to understand the mechanisms reg-

42 RIESS ET AL. INFECT. IMMUN.

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ulating the expression of this important pathogenicity factor ofB. henselae.

ACKNOWLEDGMENTS

We thank Ingo B. Autenrieth for continuous support, StephanSchuster and Christa Lanz for helping to sequence B. henselae Mar-seille badA, Andrei Lupas for critical discussion, and Diana Neumannfor excellent technical assistance.

This work was supported by grants from the Deutsche Forschungs-gemeinschaft to V.A.J.K. and from the University of Tubingen (Centerfor Interdisciplinary Clinical Research, junior group program).

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