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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Bartonella henselae is a slow-growing microorganism and the causative pathogen of bacillary angiomatosis in man. Here, we analysed how interaction of B. henselae with endothelial cells might affect bacterial growth. For this purpose, bacterial rRNA production and ribosome content was determined by fluorescence in situ hybridization (FISH) using rRNA-targeted fluorescence-labelled oligonucleotide probes. B. henselae grown on agar plates showed no detectable rRNA content by means of FISH, whereas B. henselae co-cultured with endothelial cells showed a rapid increase of rRNA production within the first 18 h after inoculation. The increased rRNA synthesis was paralleled by a ∼1000-fold intracellular bacterial replication, whereas bacteria grown on agar base showed only a ∼10-fold replication within the first 48 h of culture. Pretreatment of host cells with paraformaldehyde prevented adhesion, invasion, intracellular replication and bacterial rRNA synthesis of B. henselae. In contrast, inhibition of host cell protein synthesis by cycloheximide did not affect bacterial adhesion and invasion, but prevented intracellular replication although bacterial rRNA content was increased. Inhibition of actin polymerization by cytochalasin D did not affect adhesion, invasion, increased rRNA content or intracellular replication of B. henselae. These results demonstrate that rRNA synthesis and replication of B. henselae is promoted by viable host cells with intact de novo protein synthesis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Bartonella henselae is a re-emerging human pathogen that has been associated with bacillary angiomatosis (BA), bacillary peliosis, cat scratch disease (CSD), bacteraemia and endocarditis ( Slater et al., 1990 , 1992; Schwartzman, 1992; Adal et al., 1994 ; Drancourt et al., 1996; Anderson and Neuman, 1997). Histological investigations of biopsy specimens from patients with BA revealed bacteria in close association with proliferating endothelial cells ( Schneider et al., 1993 ; Monteil et al., 1994 ). This angiogenic process is likely to be induced by B. henselae as bacterial eradication by antibiotic treatment results in a complete regression of angiomatous tumours ( Webster et al., 1992 ).

B. henselae attachment to, and entry into, human epithelial and endothelial cells has been reported previously ( Batterman et al., 1995; Dehio et al., 1997 ). Invasion of cultured epithelial cells by B. henselae may be preceded by cell adherence mediated by the expression of type 4-like pili ( Batterman et al., 1995 ). Outer membrane proteins of B. henselae appear to bind to endothelial cells ( Burgess and Anderson, 1998).

B. henselae is a slow-growing fastidious microorganism ( Slater et al., 1990; Anderson and Neuman, 1997). Visible colonies can be observed after incubation intervals of several days or weeks after subcultivation on, for example, sheep blood agar ( Welch et al., 1992 ; Anderson and Neuman, 1997). In contrast, co-culture with host cells is more efficacious for recovery of B. henselae from patients' materials ( LaScola and Raoult, 1999). The reason for the higher recovery of B. henselae upon co-culture with mammalian cells is unclear.

Because ribosomes provide the site of mRNA translation and protein synthesis, an increased growth rate of bacteria is accomplished by an increase in the number of ribosomes ( DeLong et al., 1989 ; Wallner et al., 1993 ; Condon et al., 1995 ). Therefore, RNA molecules which are abundant in dividing cells, but rare in resting cells, may be useful molecular indicators of bacterial growth ( Cangelosi and Brabant, 1997). In situ hybridization of bacteria by fluorescence-labelled rRNA-targeted oligonucleotide probes (fluorescence in situ hybridization; FISH) enables (i) bacteria by specific ribosomal sequences to be distinguished and (ii) the rRNA content of bacteria to be estimated ( Wallner et al., 1993 ; Amann et al., 1995 ). Quantification of probe-conferred fluorescence allows estimation of in situ growth rates of individual bacterial cells ( Amann et al., 1995 ).

In this study, we compared the synthesis of rRNA in B. henselae grown on agar base or in the presence of endothelial host cells by means of FISH. Moreover, the interaction of B. henselae with endothelial cells was modulated by altering host cell functions with paraformaldehyde (PFA), cycloheximide (CHX), cytochalasin D (Cy-D), nocodazole and taxol. Our results demonstrate that the presence of viable host cells promotes the efficacious increase of production of rRNA, ribosomes and, thus, replication of B. henselae.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Invasion of B. henselae into endothelial cells results in perinuclear localized intracellular bacteria and invasome formation

Infection of EA hy 926 cells ( Edgell et al., 1983 ) with B. henselae resulted in two different intracellular distribution patterns of B. henselae 18 h after infection, as shown by confocal laser scanning microscopy (CLSM; Fig. 1A and B): (i) an intracellular agglomerate of bacteria called an invasome ( Dehio et al., 1997 ) and (ii) perinuclearly localized single bacteria similar to a distribution pattern described earlier ( Zbinden et al., 1995 ). The majority of B. henselae-infected cells (∼90%) showed a distribution pattern of perinuclear localized single intracellular bacteria and the frequency of invasome formation was significantly lower (∼10% of infected cells; data not shown). Similar results were observed upon infection of human umbilical vein endothelial cells (HUVECS; data not shown). Electron microscopic examinations confirmed that B. henselae were internalized by endothelial cells as bacterial aggregates (invasomes) or as single bacteria by protrusions of the cells, suggesting a zipper-like uptake mechanism ( Fig. 2). Intracellular B. henselae were found exclusively within vacuoles but not in the cytosol ( Fig. 2). Moreover, dividing intracellular bacteria were frequently observed ( Fig. 2).

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Figure 1. Analysis of EA hy 926 cells infected with B. henselae by confocal laser scan microscopy.

A. Invasome formation (arrow).

B. Intracellular perinuclearly distributed bacteria (x, nucleus). Extracellular bacteria were labelled by FITC-conjugated antibodies (green signal), intracellular bacteria were labelled by Cy-5-conjugated antibodies (blue signal). Filamentous actin was stained with TRITC-labelled phalloidin (red signal). Specimens were analysed by xy and xz sectioning (magnification 126×). Scale bar = 10 µm.

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Figure 2. Transmission electron microscopic analysis of EA hy 926 cells 18 h after infection with B. henselae. Interaction of B. henselae with endothelial cells resulted in (A) large intracellular bacterial aggregates (invasomes, scale bar = 3 µm) or (B) uptake of single bacteria by cellular protrusions (scale bar = 0.5 µm), leading to intracellular B. henselae in vacuoles (C, scale bar = 1 µm). Dividing bacteria (D–G) were frequently observed intracellularly (scale bar = 0.5 µm).

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Ribosoma RNA synthesis of B. henselae is upregulated by interaction with host cells

The content of bacterial rRNA in B. henselae was determined by the FISH technique using an eubacterial ribosomal probe (EUB338) ( Kempf et al., 2000 ) . B. henselae from stock suspensions showed no detectable signal by FISH, suggesting a low rRNA content ( Fig. 3). Thirty minutes after infection of endothelial cells with B. henselae FISH revealed a positive signal that reached a maximum after 18 h, suggesting a strong increase of bacterial rRNA content ( Fig. 3). Bacterial aggregates associated with cells and single intra- and extracellularly located bacteria hybridized to the oligonucleotide probe and revealed a bright fluorescent signal ( Fig. 3) which decreased at later time intervals (48–72 h; data not shown).

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Figure 3. Infection of EA hy 926 cells with B. henselae and detection of B. henselae by fluorescence in situ hybridization (FISH) 5 min, 30 min, 2 h and 18 h after infection. Overlay of FISH (EUB338-Cy3, red signal) and phase contrast microscopy. Scale bar = 10 µm.

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Comparison of the rRNA content of B. henselae grown on agar plates with that of B. henselae co-cultured with endothelial cells revealed striking differences. Whereas only a small amount of bacterial rRNA was detectable by means of FISH for B. henselae grown on agar plates over 72 h (not all data shown), FISH revealed bright fluorescence signals, indicating a high rRNA content for B. henselae co-cultured with endothelial cells ( Fig. 4). This increase of bacterial rRNA content was also detectable in HUVECS, Vero or HeLa cells, indicating that increased bacterial rRNA content is not depending on a specific host cell (data not shown).

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Figure 4. Detection of B. henselae grown on Columbia agar base (upper row) or co-cultured with endothelial cells by FISH (EUB338-Cy3, red signal) and immunofluorescence using polyclonal anti-B. henselae antibodies (FITC, green signal) after various incubation intervals. For details, see Experimental procedures. Overlay of green and red fluorescence (yellow signal) indicates B. henselae cells with an increased rRNA content. Scale bar = 10 µm.

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Growth rates of B. henselae grown on agar base or in cell culture

Because B. henselae showed a strong increase of the rRNA content upon co-culture with host cells compared with exposure to solid agar bases, we compared growth rates of B. henselae subcultured on Columbia agar base with that of B. henselae co-cultured with endothelial cells. Whereas 18 h after inoculation of agar base B. henselae cell numbers did not increase, B. henselae co-cultured with endothelial cells replicated 50-fold, corresponding to a replication time of ∼3 h ( Fig. 5). Forty-eight hours after inoculation, B. henselae cultured on agar base replicated ∼10-fold, whereas B. henselae co-cultured with endothelial cells replicated 400-fold ( Fig. 5). The replication time according to these data was ∼15 h for B. henselae grown on agar base and ∼5 h for B. henselae co-cultured with endothelial cells. After 72 h, the number of viable B. henselae cultured on agar base increased ∼200-fold and the number of viable B. henselae co-cultured with endothelial cells increased ∼650-fold. Infection of EA hy 926 cells with non-invasive and invasive Escherichia coli revealed no intracellular replication ( Fig. 6).

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Figure 5. Growth rate of B. henselae grown on Columbia agar plates or co-cultured with endothelial cells. The values show the multiplicity of replication related to the number of bacteria determined 0.5 h after inoculation of bacteria. The values indicate means of triplicate samples ± SD. The asterisks indicate statistically significant differences (P < 0.05).

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Figure 6. Number of intracellular B. henselae 30 min, 2 h, 18 h, 48 h and 72 h after infection of EA hy 926 cells (for details, see Experimental procedures). As controls, non-invasive and invasive E. coli were used. The values indicate means of triplicate wells ± SD. The asterisks indicate statistically significant differences (P < 0.05). (◆) B. henselae, control; (▪) B. henselae, PFA; (▴) B. henselae, CHX; (▵) B. henselae, Cy-D; (*) E. coli inv–; (●) E. coli inv+.

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Modulation of host cell function alters adherence, invasion, rRNA content and growth rate of B. henselae

To investigate which cellular factors are required for growth promotion of B. henselae, host cell functions were modulated by PFA, CHX, Cy-D, nocodazole or taxol, and adhesion, invasion, growth rates and rRNA content of B. henselae were determined. PFA and glutaraldehyde were used to kill host cells without damaging the cell structure. CHX suppresses protein biosynthesis, Cy-D was used to depolymerize F-actin, nocodazole causes depolymerization of microtubuli and taxol stabilizes microtubuli ( Sampath and Pollard, 1991; Dehio et al., 1997 ).

In untreated control cells, B. henselae showed an adherence of 11.6 ± 2.6 bacteria per cell 30 min after infection at a multiplicity of infection (MOI) of 280, revealing 5% adherent bacteria. PFA-pretreated cells showed a significantly (P < 0.05) reduced adhesion of B. henselae (1.0 ± 0.3 bacteria per cell), suggesting that B. henselae does not adhere to non-viable host cells. CHX (8.3 ± 1.7 bacteria per cell) and Cy-D (9.1 ± 2.1 bacteria per cell) slightly, but not significantly, reduced adherence of B. henselae to host cells. From these data, we can conclude that adhesion of B. henselae to endothelial cells depends on cell viability but does not require de novo protein synthesis, actin polymerization or interaction of bacteria with microtubuli of the host cell.

Bacterial invasion into endothelial cells was determined using a gentamicin kill assay. Two hours after infection, ∼103 bacteria (less then 1% of the inoculated bacteria) invaded untreated control cells ( Fig. 6). This invasion was significantly reduced under the limit of detection in PFA (< log10 CFU: 1.4) and glutaraldehyde-pretreated cells (< log10 CFU: 1.4). CHX and Cy-D did not significantly reduce the invasion of B. henselae into host cells, suggesting that invasion of B. henselae into viable host cells does not require de novo protein synthesis or actin polymerization of the host cell. Nocodazole or taxol had no significant influence on the number of adherent bacteria (data not shown). From these data, we conclude that invasion of B. henselae into host cells depends on bacterial adherence to viable host cells which triggers bacterial invasion.

To determine whether B. henselae might replicate intracellularly after cell invasion, cells were pretreated with medium (control), PFA, CHX or Cy-D and were infected with B. henselae. Intracellular numbers of B. henselae were determined after 18, 48 and 72 h by gentamicin kill assay ( Fig. 6). The data show that the number of intracellular B. henselae in untreated control cells was ∼1000-fold increased after 48 h. In PFA- and glutaraldehyde-pretreated cells, the amount of B. henselae was below the limit of detection (< log10 CFU: 1.4). No significant intracellular bacterial growth was observed in the presence of CHX within 72 h. The results suggest that inhibition of protein biosynthesis of host cells by CHX prevents intracellular replication of B. henselae. In contrast, Cy-D did not significantly affect intracellular replication of B. henselae compared with control cells. Likewise, nocodazole or taxol had no significant influence on intracellular growth within the first 48 h after infection. From these data, we can conclude that intracellular replication of B. henselae does not require actin polymerization or organization of microtubuli.

Finally, we investigated whether the different growth rates of B. henselae in endothelial cells, modulated as described above, correlated with different bacterial rRNA contents. Therefore, 18 h after co-culture we analysed extra- and intracellular bacteria by immunofluorescence and bacterial rRNA content of B. henselae by FISH ( Fig. 7). B. henselae-infected untreated control cells showed the typical distribution pattern of B. henselae, and the content of rRNA of B. henselae was significantly increased compared with agar-grown bacteria as revealed by FISH ( Fig. 7B). Bacteria co-cultured with PFA or glutaraldehyde fixed cells did not show an increased rRNA content. ( Fig. 7).

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Figure 7. Interaction of B. henselae with EA hy 926 cells pretreated with medium (control), PFA, CHX or Cy-D 18 h after infection.

A. Specimens were stained by immunofluorescence with anti-B. henselae antibodies [extracellular bacteria, green signal; intracellular bacteria, blue signal (arrows); actin, red signal]. Specimens were analysed by confocal laser scan microscopy.

B. FISH (EUB338-Cy3, red signal) of B. henselae 18 h after infection. Immunofluorescence staining of B. henselae was performed using Cy-5-labelled anti-B. henselae polyclonal antibodies (blue signal). Actin was stained with FITC-labelled phalloidin (green signal). Scale bar = 10 µm.

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When cells were treated with CHX, fewer B. henselae were interacting with host cells compared with controls as shown by CLSM ( Fig. 7A). However, B. henselae showed an increased content of bacterial rRNA, as revealed by FISH ( Fig. 7B). In Cy-D-treated cell cultures, B. henselae invaded into host cells although without invasome formation ( Fig. 7A), as described previously ( Dehio et al., 1997 ). Moreover, B. henselae showed increased rRNA synthesis under these culture conditions. It should be noted that both intracellular and extracellular as well as unassociated B. henselae exhibited an increased rRNA content. However, fluorescence intensity was most intensive in B. henselae closely associated with host cells or located intracellularly, especially within invasomes ( Fig. 8).

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Figure 8. Detection of B. henselae rRNA by fluorescence in situ hybridization (FISH) and quantification of rRNA content by fluorescence intensity profiles. EA hy 926 cells were infected and FISH was performed using probe EUB338-Cy3. Photomicrographs showing true colour images (upper row) and fluorescence intensity profiles (lower row).

A and D. rRNA content 2 h after infection in singular cell-adherent (large arrow) and singular non-cell-adherent (small arrow) B. henselae.

B and E. rRNA content 18 h after infection in perinuclear localized B. henselae revealing increased (large arrow) and highly increased (small arrow) bacterial rRNA contents of singular intracellular bacteria.

C and F. rRNA content 18 h after infection of B. henselae forming an invasome (arrow). White equals maximal intensity, dark blue equals minimal fluorescence intensity [see calibration bar of fluorescence intensities, minimum (0) to maximum (256)]. Scale bar = 10 µm.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The major goal of this study was to determine the impact of endothelial cells on growth of B. henselae, the aetiological agent of BA in man ( Anderson and Neuman, 1997). Co-culture of B. henselae with mammalian cells enhances the recovery of B. henselae from patients' specimens compared with standard agar-based culture techniques ( Drancourt et al., 1996; LaScola and Raoult, 1999). By means of FISH using rRNA-targeted oligonucleotides, we have analysed the rRNA content and growth rate of B. henselae exposed to different environments. The most salient findings of our study were that (i) rRNA synthesis, and thus the growth rate, of B. henselae was rapidly increased upon co-culture with viable endothelial cells with intact protein synthesis and (ii) intracellular growth, but not increase of bacterial rRNA production, depends on de novo protein synthesis of the host cell.

A direct correlation between rRNA content and growth rate was first shown for Salmonella typhimurium ( Schaechter et al., 1958 ). Signals conferred by FISH correspond to the cellular rRNA content and growth rate ( DeLong et al., 1989 ; Wallner et al., 1993 ). Therefore, the amount of a fluorescent rRNA-targeted probe bound to bacteria reflects the growth rate and metabolic activity ( DeLong et al., 1989 ). Slowly growing bacteria, however, have a low cellular rRNA content ( Kemp et al., 1993 ; Kerkhof and Ward, 1993). B. henselae grows slowly on Columbia agar base. Accordingly, the rRNA content was marginal, as shown by FISH. However, a weak fluorescence intensity revealed by means of FISH could also result from a poor permeability of the fixed bacterial cells for the oligonucleotide probes ( DeLong et al., 1989 ). Because of bright fluorescence signals in internal controls including E. coli (data not shown) and B. henselae co-cultured with host cells over several time periods, we can conclude that the low fluorescence signal obtained by FISH of B. henselae grown on agar plates is in fact due to a low ribosomal content.

A strong increase of rRNA content was detectable starting 30 min after inoculation, especially in bacteria adherent to or that had invaded host cells, suggesting a significantly higher growth rate of adherent and intracellular B. henselae. Adherence to host cells appeared to be sufficient to induce an increased rRNA content which reached maximum levels after ∼18 h. This phenomenon was observed upon co-culture with different cell lines (data not shown) and different Bartonella strains (data not shown), indicating that these results are not a Bartonella strain- or cell line-specific phenomenon.

The brightest fluorescence signal by FISH was observed in bacteria located intracellularly as single perinuclear scattered bacteria or within invasomes, suggesting that B. henselae can replicate intracellularly ( Fig. 8). This observation was supported by transmission electron microscopy (TEM) studies that frequently revealed dividing bacterial cells. Moreover, determination of the intracellular growth rate by gentamicin kill assay demonstrated ∼1000-fold increased bacterial numbers within 48 h, suggesting a generation time of at least ∼5 h that was similar to other Proteobacteria, e.g. Rickettsia spp., Anaplasma marginale or Legionella pneumophila ranging from 4 to 17 h ( Hidalgo, 1975; Ristroph et al., 1981 ; Pang and Winkler, 1994). Therefore, B. henselae can be considered as a facultative intracellular pathogen. PFA- or glutaraldehyde-fixed endothelial cells did not promote an increased rRNA content in B. henselae, suggesting that interaction of B. henselae with viable cells is required for this phenomenon. Treatment of host cells with Cy-D, nocodazole or taxol did not affect either intracellular growth or fluorescence signals and thus rRNA content in B. henselae. Therefore, intracellular growth of B. henselae appears to be actin and microtubuli independent.

The increase of bacterial rRNA synthesis in B. henselae can be separated from bacterial replication as CHX prevented bacterial growth via suppression of eukaryotic de novo protein synthesis but not the increase of bacterial rRNA synthesis as revealed by FISH. Thus, protein starvation of the host cell prevents bacterial growth but not rRNA production. It has been shown that there is no clear relationship in starving bacterial cells between rRNA content and growth rate ( Koch, 1971; Flardh et al., 1992 ). For example, cells of marine Vibrio sp. strain S14 (CCUG 15956) have been deprived for exogenous carbon and were not growing, although they retained large numbers of ribosomes ( Flardh et al., 1992 ). It has been suggested that these non-growing carbon-starved Vibrio strains possess an excess protein synthesis capacity, which might be essential for their ability to immediately initiate an upshift programme when substrate is added ( Flardh et al., 1992 ). Similar results were observed for E. coli ( Koch, 1971). According to these observations, we speculate that B. henselae co-cultured with CHX-treated endothelial cells might be stimulated to sense for good growth conditions which would be accompanied by a bright FISH signal. However, starvation for, for example, host proteins would prevent bacterial replication.

The molecules required for initiating rRNA production and growth of B. henselae are not yet known. Moreover, increased rRNA content was observed not exclusively in bacteria associated with endothelial cells but also in cell-unassociated B. henselae. Whether products secreted or released by endothelial cells may be sufficient to increase the bacterial rRNA content rapidly appears to be unlikely as culture of B. henselae separated from host cells by a transwell system (pore size 0.4 µm) did not induce significant bacterial growth within the first 48 h (data not shown). Therefore, we cannot exclude that because of the fixation protocol single bacteria dissolved from the host cells may appear as cell-unassociated bacteria. Moreover, our observations may argue for a role of quorum sensing in regulation of rRNA synthesis in B. henselae, a phenomenon which is known in gene regulation from other Proteobacteria ( Schripsema et al., 1996; Zhu et al., 1998; Hastings and Greenberg, 1999).

Factors which regulate rRNA transcription, for example NTP concentration, provide a molecular explanation for the growth rate-dependent control and homeostatic regulation of ribosome synthesis ( Gaal et al., 1997 ). The rate of transcription initiation of the growth rate-dependent rrn promoters is physiologically connected to the metabolic state of the cell ( Gaal et al., 1997 ). RNA synthesis is efficiently regulated by prrnA and prrnB operons and several factors such as FIS, UP and antidetermination factors seem to be involved in RNA polymerase activity ( Zhang and Bremer, 1996; Gaal et al., 1997 ). However, none of these factors has yet been characterized in B. henselae.

B. henselae can interact with host cells in at least two different ways, leading (i) to perinuclear localized bacteria ( Batterman et al., 1995; Zbinden et al., 1995 ) or (ii) to bacterial aggregates, so called invasomes ( Dehio et al., 1997 ). In our investigations, invasome frequency was low (data not shown). Because of the higher amount of host cells infected with B. henselae distributed perinuclearly, we assume that this process might reflect the more common and relevant course of infection in host cells. TEM revealed single B. henselae adhering to host cells, leading to membrane protrusions of the cell surface as well as bacterial aggregates; intracellular bacteria were found in small vacuoles and in large invasomes. These observations suggest that at least two different ways of invasion into host cells may exist for B. henselae. As actin-dependent invasion processes are involved in invasome formation of B. henselae and Cy-D did not significantly reduce invasion of B. henselae, an actin-independent invasion process must be assumed. Such processes have been shown, for example, for Actinobacillus actinomycetemcomitans or Trypanosoma ( Rodriguez et al., 1996; Brissette and Fives, 1999).

Adherence of B. henselae to host cells may depend on the expression of pili ( Batterman et al., 1995 ) and a 43 kDa protein ( Burgess and Anderson, 1998). In fact, the B. henselae strain used in this study expressed pili, as revealed by TEM, and a 43 kDa protein (data not shown). Preincubation of host cells with PFA or glutaraldehyde abrogated adherence of B. henselae, suggesting that adherence of B. henselae depends on the host cell viability. The selective adherence to viable cells might be due to host cell functions such as membrane ruffling or clustering of cell-surface receptors.

In summary, our results show that viable host cells with intact protein biosynthesis promote rRNA synthesis and growth of B. henselae. Intracellular growth rates were related to increased rRNA content. Ongoing work in our laboratory is focusing on the cross-talk between B. henselae and host cells regulating bacterial rRNA synthesis and intracellular replication.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Bartonella henselae culture conditions

Bartonella henselae strain Marseille ( Drancourt et al., 1996 ) was grown on Columbia agar plates supplemented with 5% defibrinated sheep blood (Becton Dickinson) in a humidified atmosphere at 37°C and 5% CO2. For production of bacterial stock suspensions, bacteria were harvested from agar plates after 4 days of culture, resuspended in Luria–Bertani (LB) medium containing 40% glycerin, and stored at −80°C. For infection experiments, stocks were thawed, washed, suspended in cell culture medium (see below) and adjusted to the appropriate concentration. The number of viable bacteria in the frozen stocks was determined by plating serial dilutions of the suspension and calculating the number of bacteria.

Culture and infection of endothelial and epithelial cells

EA hy 926 cells were kindly provided by Dr Edgell (University of North Carolina, Chapell Hill, NC, USA) ( Edgell et al., 1983 ). These cells were cultured in Clicks/RPMI-1640 medium (Biochrom) with 10% heat-inactivated FCS (Sigma), 2 mM glutamine, 1 mM sodium pyruvate, non-essential amino acids, 10 µg ml−1 streptomycin and 100 U ml−1 penicillin (Biochrom). In some experiments, human umbilical vein endothelial cell (HUVEC) culture (passages 2 and 6) was performed as described previously ( Aepfelbacher et al., 1997 ) in endothelial growth medium (Promo Cell) containing EC growth supplement. Vero cells (ATCC catalogue number CRL-1586) were cultured in Clicks/RPMI-1640 with the ingredients described for EA hy 926 cells.

Infection experiments were performed as previously described ( Dehio et al., 1997 ) with some modifications. For infection, cells were seeded the day before the experiment in the appropriate antibiotic-free medium onto coverslips coated with collagen (Biochrom) and placed inside individual wells of a 24-well cell culture plate (Nunc). For infection, bacteria stock solutions were thawed, washed in PBS supplemented with 1 mM CaCl2 and 0.5 mM MgCl2 and diluted in antibiotic-free cell culture medium and added in a concentration of 1 × 107 bacteria per well (MOI 285). Bacteria were sedimented onto the cultured cells by centrifugation for 5 min at 1800 g. Infected cell cultures were maintained in a humidified atmosphere at 37°C and 5% CO2. The medium was replaced the following day to prevent bacterial overgrowth.

Non-invasive E. coli HB101 and the E. coli HB101 (pINV1914) strain expressing the Y. enterocolitica invasin ( Schulte and Autenrieth, 1998) were grown in LB broth. For infection experiments, overnight cultures were diluted to an optical density at 600 nm of 0.2 in LB broth and incubated for 3 h at 37°C. For infection, bacteria were collected by centrifugation and washed twice in sterile PBS (pH 7.4).

Antibodies

Rabbit polyclonal antibodies were raised against viable bacteria of B. henselae Marseille strain derived from culture on Columbia agar plates. Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibodies and Cy-5-conjugated goat anti-rabbit IgG Fab fragments were purchased from Dianova; FITC- and tetramethylrhodamine isothiocyanate (TRITC)-labelled phalloidin was purchased from Sigma .

Fluorescence in situ hybridization

In situ hybridization of bacteria on glass slides was performed as previously described ( Kempf et al., 1999 ) with the following modifications. The universal eubacterial oligonucleotide probe EUB-338 [GCT GCC TCC CGT AGG AGT, synthesized and 5′-labelled (Metabion) with the fluorochromes Cy3 (red signal) or FITC (green signal)] and the irrelevant control probe non-338 (CGA CGG AGG GCA TCC TCA) complementary to EUB-338 to exclude non-specific binding of the probes were used for this study ( Amann et al., 1990 , 1995). Coverslips with infected cells were incubated in ethanol. Thereafter, slides were washed in PBS, and 25 ng of each oligonucleotide was added in 50 µl of hybridization buffer containing 20% formamide for 90 min at 46°C before washing with the same stringency. For some investigations, an immunofluorescence staining was performed additionally. Citifluor (Citifluor) was used as a mounting medium on hybridized slides. Finally, the slides were analysed with a Leitz DM RBE microscope (see below).

Immunofluorescence labelling and confocal laser scanning microscopy

For all staining protocols, the PFA-fixed and/or alternatively hybridized cells were washed three times in PBS at the beginning and after each incubation step. For differential staining of extra- and intracellular B. henselae, fixed cells were sequentially incubated with 0.2% BSA (Biomol) in PBS for 15 min to block unspecific binding. Then, rabbit anti-B. henselae antiserum (diluted 1:2000 in PBS) was added for 1 h, followed by FITC-conjugated goat anti-rabbit IgG antibodies (diluted 1:100 in PBS) for 1 h. Then, cells were permeabilized by incubation with 0.1% Triton X-100 in PBS for 15 min. After a further blocking step with 0.2% BSA in PBS for 15 min, cells were incubated with rabbit anti-B. henselae antiserum (diluted 1:2000 in PBS) for 1 h followed by Cy5-conjugated goat anti-rabbit antibodies mixed with TRITC-labelled phalloidin (diluted 1:1000 or 1 µg ml−1 in PBS respectively) for 1 h. For immunostaining of hybridized probes, coverslips were sequentially incubated with 0.2% BSA in PBS for 15 min to block unspecific binding, with rabbit anti-B. henselae antiserum (diluted 1: 2000 in PBS) for 1 h and with FITC- or Cy5-conjugated goat anti-rabbit IgG antibodies mixed with FITC- or TRITC-labelled phalloidin (diluted 1:1000 or 1 µg ml−1 in PBS). Finally, the slides were analysed with a Leitz DM RBE microscope (Leica). Three different fluorochromes could be detected simultaneously with three different photomultipliers and represented the green (FITC), red (TRITC or Cy-3) and blue (Cy-5) channels. The corresponding images were digitally processed with Photoshop 7.0 (Adobe Systems).

Transmission electron microscopy

EA hy 926 cells were infected as previously described. After a short incubation with Trypsin/EDTA, cells were harvested, centrifuged and washed. After centrifugation at a speed of 150 g for 10 min, the resulting pellets were fixed for 2 h in 4% glutaraldehyde in a 0.05 M phosphate-buffered solution containing 0.15 M NaCl at pH 7.3 at room temperature. Post-fixation was based on 1% osmium tetroxide containing 1% potassium dichromate in 0.85% NaCl at pH 7.3 for 45 min. After embedding in glycide ether, the blocks containing cells were cut using an ultra microtome (Ultracut). Semi-thin sections (1 µm) were studied with a light microscope after staining with 1% toluidine blue and 1% pyronine G (Merck). The sections were viewed at a magnification of 400×. Ultrathin sections (80 nm) were stained with 0.5% uranyl acetate for 10 min at 30°C and 2.7% lead citrate for 5 min (Ultrastainer) at 20°C. Grids were examined using a Zeiss EM 902 transmission electron microscope (Zeiss) operating at 80 kV, at magnifications between 2000× and 50 0000×.

Recovery of bacteria from Columbia agar base

The growth rates of B. henselae on Columbia agar base were compared with growth rates of B. henselae co-cultured with endothelial cells. Bacteria (1 × 107B. henselae, 100 µl) were plated on the surface of Columbia agar plates. After the indicated time intervals, bacteria were harvested from the agar surface by extensive washing with PBS. Bacterial numbers were determined by plating serial dilutions on Columbia agar and counting colony-forming units after incubation of 1 week (37°C, 5% CO2, humidified atmosphere).

Adherence to and invasion of B. henselae into host cells

To determine the amount of adherent bacteria, reisolation of B. henselae from infected cell culture was performed after 30 min. Briefly, cell culture supernatants were gently removed, cells were extensively washed with Clicks/RPMI-1640 medium and osmotic lysis was performed as described above to calculate the total amount of bacteria.

A gentamicin kill assay was used to calculate the number of intracellular bacteria as described previously ( Mehock et al., 1998; Schulte and Autenrieth, 1998) with a few modifications. In brief, cell culture supernatants were gently removed, cells were extensively washed with Clicks/RPMI-1640 medium and gentamicin (100 µg ml−1) was added for 2 h to kill extracellular bacteria. Then cells were extensively washed with Clicks/RPMI-1640 medium to remove gentamicin. Cell culture supernatants were gently removed and osmotic lysis was performed by adding 900 µl sterile water. Additionally, cells were disrupted by using a 1 ml syringe and a 0.40 mm × 20 mm needle. Microscopic controls revealed that after 5 min all cells were lysed. Cell lysates including extra- and intracellular bacteria were resuspended with 10× PBS to overcome osmotic lysis. Bacterial numbers were determined by plating serial dilutions on Columbia agar. Reisolated B. henselae showed no gentamicin resistance by adding gentamicin for 3 h and subsequent subcultivation on agar plates excluded a change in gentamicin sensibility.

Modulation of B. henselae infection in endothelial cell culture

B. henselae infection of endothelial cells was modulated by preincubation of host cells with (i) PFA, (ii) CHX, (iii) Cy-D, (iv) nocodazole and (v) taxol. For all experiments, EA hy 926 cells were seeded the day before the experiment. The following day, medium was removed and medium containing various concentrations of CHX (2 µg ml−1, 20 µg ml−1, 200 µg ml−1) ( Deiwick and Hensel, 1999), of Cy-D (50 nM, 100 nM, 150 nM), nocodazole (1 µM, 2 µM) or of taxol (5 µM, 10 µM) ( Dehio et al., 1997 ) was added. Pretreatment of B. henselae with these substances revealed no bactericidal effects (data not shown). Treatment with taxol or nocodazole reduced the viability of EA hy 926 cells to less than 10%, as revealed by trypan exclusion assays when cultivated for longer than 48 h (data not shown).

To kill host cells, 1 ml of 3.75% PFA or 1 ml of 2% glutaraldehyde was added for 30 min and was removed afterwards by several wash steps. Infection of cell cultures was performed as described above in antibiotic-free cell culture medium containing the substances described herein.

Statistical analysis

All experiments were performed at least three times and revealed similar results. Differences between mean values of experimental and control groups were analysed by Student's t-test. A P-value of < 0.05 was considered statistically significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank Sook Trogisch for expert technical support, Elfriede Januschke for preparing transmission electron microscopy, Guntram Grassl for providing E. coli strains, Christoph Jacobi for confocal laser scanning assistance, Wolf-Dietrich Hardt for critical reading of the manuscript, Siv G. E. Andersson for stimulating discussions and Jürgen Heesemann for generous support. This work was supported by a grant from the Münchner Medizinische Wochenschrift.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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