Where is fimbriae found

However, the response was divergent in different bacterial strains. The physiological heterogeneity in static cultures might be the cause of the strain-dependent variation detected. Our experiments using shaken cultures growing under uniformly aerated conditions gave identical results with all the strains used. Moreover, an increased fimA transcriptional expression in a CRP-cAMP deficient strain could be extracted from a microarray dataset on the effect of the crp mutation on the global pattern of expression in E.

When using early log phase cultures of the phase variation proficient strain CBP and its cya counterpart Fig. The differences in the kinetic of the response suggest that the dual role of CRP-cAMP on fimA expression might be achieved by distinct mechanisms: a direct stimulation of the fimA promoter activity and an indirect role in the overall negative effect of CRP-cAMP on type 1 fimbriae expression.

So far, we have described that CRP-cAMP deficiency causes: i a higher degree of type 1 fimbriation, ii an increase in the expression of type 1 fimbriae in phase variation proficient strains, and iii a reduction in fimA promoter activity in phase variation deficient strains.

These results suggest that the percentage of fimbriated cells ON-cells in both crp and cya mutant strains is elevated when compared to wt, causing the overall increase in fimA expression. To test this prediction, the percentage of ON-cells in the population was monitored by plating cultures of the phase variable fimA-lacZ reporter strains on indicator plates containing X-gal. As predicted, a significant increase in the percentage of ON-cells in both the crp and cya mutant strains was observed Fig.

S1 and used to detect and quantify the subpopulations having the invertible element either in the ON or in the OFF orientation [40]. Moreover, the cya deficiency was chemically complemented by addition of exogenous cAMP in the culture medium.

Similar results were obtained when using the reporterless and type 1 fimbriation-proficient strain MG and its cya mutant derivative Fig. Moreover, when derivatives of the uropathogenic isolate J96 were used, a significant increase in the percentage of ON-cells was detected in crp derivatives.

While most of the cells in the wt population were in the OFF orientation under the culture conditions used, a subpopulation of cells with the invertible element in the ON orientation was clearly detected in the mutant derivative Fig. To confirm these results, the level of fimA transcript in cultures of J96 and its derivative J96 crp were quantified by Northern blot analysis Fig. Mean values and standard deviations from three independent experiments are shown. Mean values and standard deviations of three independent experiments are shown.

C ON-OFF diagnostic of mid-log phase cultures of the J96 strain and its crp derivative; the arrowhead highlights the fragment corresponding to ON-cells detected in the J96 crp samples.

A control showing the band pattern of an OFF population was included for comparison. The pictures in panels C and E are electronically inverted images of ethidium bromide stained acrylamide gels. As depicted in Fig. Taken together, our results suggest that CRP-cAMP acts on the phase variation process by causing a decrease in the percentage of fimbriated cells in the population. Type 1 fimbriation is growth phase dependent [31] , [32]. As previously described, fimA expression in wt cultures was low in the early growth stages, increased in the middle of the logarithmic phase, and stayed constantly high throughout stationary phase Fig.

In contrast, fimA expression in the crp mutant peaked during early logarithmic phase and then dropped down to almost wt levels during late-logarithmic phase. The analysis of fimA expression through the growth curve suggests that CRP-cAMP represses type 1 fimbriation in actively growing cells, while during growth arrest, the repression is released and other global regulators such as RpoS and ppGpp assume the control [31] , [32].

As assessed by Northern blot analysis, crp transcriptional expression is high in early exponential phase and significantly reduced in stationary phase [44]. B Quantification of the percentage of fimA -expressing cells in the population of strain CBP wt on indicator plates.

The effect of glucose on the expression of type 1 fimbriae was monitored. A modest but significant increase in the percentage of fimA -expressing cells could be observed when CBP wt cultures were grown in M9-glucose medium compared with cultures grown in M9-glycerol Fig. The stimulatory effect of the presence of glucose on transcriptional expression of type 1 fimbriae was also observed by microarray analysis on the effect of glucose in the general expression pattern in E.

However, growth in media that significantly increases the growth rate of the crp strain, i. LB medium containing glucose 32 and 48 minutes generation time for CBP and CBP, respectively , did not alter the difference in the expression of type 1 fimbriae between the wt and the crp strains data not shown , suggesting that the CRP specific effect on type 1 fimbriae expression is not coupled to the growth rate. Also supporting our results, it was reported that the FimB-mediated switching frequency from OFF to ON is 3-fold higher in the presence of glucose i.

The orientation of the plasmid-encoded fim invertible element was determined after 3 h incubation by using the PCR-based assay see Materials and Methods. Results are provided in the bar diagram as percentage of invertible elements in the ON orientation.

The picture in the right part of the Figure illustrates an ethidium bromide stained gel from one of the experiments used to obtain the data shown.

A similar assay as presented in B was performed. Extracts were mixed with the template plasmid pMM36 fim invertible element in the ON orientation and analyzed as in B. In both B and C, mean values and standard deviations from four independent experiments are shown.

The percentage of ON-cells was quantified from cultures of two independent clones. In A and D, the image corresponds to the upper half of an ethidium bromide stained gel. Although higher expression of fimB was observed in crp derivatives as compared to wt counterparts in both J96 and MG strains Fig. To further test this hypothesis, in vivo experiments were performed under conditions of constitutive fimB expression using plasmid pPKL9, which contains the fimB gene under the control of the tet promoter Fig.

Control experiments by Northern blot analyses verified that the fimB expression levels from plasmid pPKL9 were essentially identical in CRP-cAMP proficient and deficient genetic backgrounds data not shown.

Comparable results were obtained when using MG derivative strains data not shown. Altogether, our results both in vivo and in vitro indicate that the CRP-cAMP complex has a negative effect on the switching process independently of the intracellular concentration of the FimB recombinase. No obvious alteration in the FimB-mediated switch was detected, strongly suggesting that CRP-cAMP does not directly interact with the nucleoprotein complex that is the substrate for the FimB recombinase.

Accordingly, no effect was observed in the outcome of in vitro recombination assays when purified CRP was added to extracts obtained from a crp strain data not shown. Extracts were mixed with the template plasmid pJL-2 in absence or presence of increasing amounts of cAMP 1 to 50 mM final concentration. B Effect of increasing amounts of the gyrase inhibitor novobiocin on fimA expression.

Mean values and standard deviations from two independent experiments are shown. C Effect of DNA gyrase inhibition on the orientation of the fim invertible element in vivo.

Both panels depict electronically inverted images of the upper half of acrylamide gels after ethidium bromide staining. Mean values and standard deviations in brackets of the estimated percentage of invertible elements in the ON orientation from four independent experiments are given as numbers below each lane.

The images correspond to ethidium bromide stained gels from a representative experiment used to obtain the data shown. At most, a low affinity binding was detected in case of the fragment containing the fimA promoter PCR7; Fig. However, when DNase I footprinting analysis of this putative CRP binding site was performed, no binding was observed data not shown.

Although it is possible that such low affinity CRP binding site s may exist in the fimA promoter region, our experimental evidence Fig.

A possible involvement of the putative CRP binding site s in the positive control of the fimA promoter activity Fig. DNA gyrase is involved in the regulation of DNA topology, but also in other processes such as replication or illegitimate recombination [50] , [51]. In crp deficient strains, low levels of gyrA expression and DNA gyrase activity, monitored as alterations in the topology of plasmid DNA, were detected [52].

Addition of increasing amounts of novobiocin DNA gyrase inhibitor in wt cultures caused a concomitant increase in fimA expression, consistent with previously reported data [29] , [48]. Remarkably, the fimA expression level was essentially unaltered by addition of novobiocin in cultures of the cya mutant strain. In agreement with the hypothesis proposed, the novobiocin mediated inhibition of the DNA gyrase caused an increase in the percentage of ON-cells in the wt strain, but not in the cya derivative Fig.

Results that further corroborated our hypothesis were obtained by inducing overexpression of DNA gyrase from cloned gyrAB genes in the cya strain CMM Both repression of fimA expression and reduction in the percentage of ON-cells were detected Fig. Moreover, when fimE mutant derivatives were used, thus only reflecting FimB-mediated inversion, an identical response to novobiocin was observed, indicating that the recombination process that was responsive to gyrase inhibition in vivo is FimB-specific Fig.

To rule out the possibility that the lacZYA sequences present in the fimA - lacZYA fusion might cause alterations in the regional DNA supercoiling and consequently affect the phase variation, similar experiments were performed using reporterless derivatives of strains MG and J Similar results were obtained: i an increase in the percentage of ON-cells in the wt strains was observed after addition of increasing novobiocin concentration 5-fold and 2-fold increase with the highest concentration of novobiocin tested in MG and J96 strains, respectively , ii the level of ON-cells was not altered by novobiocin treatment in the CRP-cAMP deficient derivatives, and iii the percentage of ON-cells in the wt achieved by novobiocin treatment was similar to the level detected in the CRP-cAMP deficient derivatives data no shown.

It is noteworthy that in all approaches Fig. To corroborate the in vivo results obtained, in vitro analyses were performed where increasing amounts of novobiocin were added to the wt strain extract. Moreover, the unaltered fimA expression in the cya mutant strain by addition of novobiocin, together with the in vitro switching data, indicates that the fimA promoter is indifferent to changes in the DNA gyrase activity, in agreement with previous data [48].

The fact that inhibition of the DNA gyrase activity in vitro stimulated the FimB-mediated recombination suggests an active role of the DNA gyrase during the recombination process itself. Altogether, our data provide evidence that the induction of type 1 fimbriation detected in the CRP-cAMP deficient strains is a process mediated by the alteration in DNA gyrase activity and therefore can be mimicked by the specific inhibition of this enzymatic activity by novobiocin. Recombination at the fim invertible element requires Lrp, a DNA bending protein that directly binds to specific sites within the invertible element and stimulates DNA inversion [26].

Kelly et al. These results suggest a possible link between the CRP and Lrp regulons. Interestingly, a direct demonstration of CRP dependent regulation of Lrp expression has not been done, although two putative CRP sites have been predicted in the promoter region of the lrp gene [53] , suggesting a possible direct regulation by CRP-cAMP.

An increase in the level of the lrp transcript in the crp derivatives was detected as compared with wt 2. A Lrp levels in different genetic backgrounds. Numbers below each lane represent the average of the signal intensity from three independent experiments relative to the corresponding wt value set as one.

C Effect of overexpression of lrp on the percentage of ON-cells. Cultures of strain CBP transformed with a plasmid that carries the lrp gene under the inducible P ara -promoter pAAG6 were grown to mid-log phase in presence of the indicated concentrations of arabinose. The induced levels of lrp were assessed by immunoblotting using Lrp-specific antiserum.

Simultaneously, quantification of the percentage of ON-cells in the populations was performed by using the PCR-based method.

The lower image corresponds to the upper half of a representative gel used for ON-OFF diagnostic; the results are given as numbers below each lane. Interestingly, when the levels of intracellular Lrp were monitored in the same cultures as in Fig. In a wt strain, the Lrp levels were strongly elevated at the highest concentration of novobiocin, where the amount of Lrp was apparently identical to the amount detected in the cya strain in absence of novobiocin, which again might be explained by the low DNA gyrase activity detected in the CRP-cAMP deficient background [52].

We tested whether increased levels of Lrp by itself would cause an alteration in the FimB-mediated switching process. Our results clearly indicate that Lrp overexpression per se did not result in any significant changes in the percentage of ON-cells when no alteration in DNA gyrase activity was induced Fig. The expression of type 1 fimbriae implies the allocation of an important part of the asset of the bacterial cell for the production of those proteinaceous appendages, as considerable amounts of energy and amino acids are needed for their synthesis.

A tightly regulated expression of such organelles is therefore expected. Considering the important metabolic effort performed by the bacterial cells committed to be fimbriated, regulation by phase variation can be seen as a selective advantage for the bacterial population, in addition of providing phenotypical heterogeneity in an otherwise genetically homogeneous population.

In previous works, we have shown that the expression of type 1 fimbriae is stimulated when intracellular levels of the stringent response alarmone ppGpp are raised [32] , [56].

The level of ppGpp in the cell increases under amino acid starvation and energy stress [57]. In this report, the role of CRP-cAMP, a regulatory complex that is associated with the energy state of the cell, has been included in the extensive list of regulatory networks controlling type 1 fimbriation.

Interestingly, many of the global regulators that affect type 1 fimbriae expression, such as H-NS, RpoS, Lrp, and now CRP-cAMP, have been shown to interplay among each other, thereby orchestrating gene regulation cascades in response to the growth conditions [58]. We dissected the initial observation that the crp derivatives of J96 showed an increased ability to agglutinate yeast cells and conclude that CRP-cAMP represses type 1 fimbriation, as schematically shown in Fig.

Interestingly, CRP-cAMP has a dual effect on type 1 fimbriation by repressing phase variation and promoting promoter activity.

Further studies will be required for fully understanding the underlying mechanisms by which CRP-cAMP affects both levels of regulation of type 1 fimbriation. Green arrows indicate stimulatory effects, whereas red lines indicate repressing effects. In Salmonella , crp cya mutants are avirulent in a mouse model [59] and it has been reported that the crp and the cya genes are strongly repressed during infection of macrophages [60]. Thank you for visiting nature. You are using a browser version with limited support for CSS.

To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. To survive antibiotics, bacteria use two different strategies: counteracting antibiotic effects by expression of resistance genes or evading their effects e.

Since bacterial adhesins provide access to the shielded, intracellular niche and the adhesin type 1 fimbriae increases bacterial survival chances inside macrophages, we asked if fimbriae also influenced survival by antibiotic evasion. Combined gentamicin survival assays, flow cytometry, single cell microscopy and kinetic modeling of dose response curves showed that type 1 fimbriae increased the adhesion and internalization by macrophages. This was caused by strongly decreased off-rates and affected the number of intracellular bacteria but not the macrophage viability and morphology.

Fimbriae thus promote antibiotic evasion which is particularly relevant in the context of chronic infections. While internalization of microbes by immune cells often leads to their intracellular degradation, survival within host cells is a potent mechanism of bacterial virulence and antibiotic evasion 1 , 2 , 3 , 4.

The internalization of bacteria into eukaryotic host cells can be triggered either by the host cells, e. Although immune cells have optimized their ability to recognize and kill bacteria, some pathogenic bacteria specifically target cells of the immune system and achieve intracellular survival.

The resulting failure of infected immune cells to clear bacteria, in combination with their highly migratory and tissue-invasive properties, can lead to a spread of the surviving bacteria and result in systemic and chronic infections 2 , 7 , 8 , 9.

Intracellular survival strategies of pathogens include escape from the phagosome into the cytosol and adaptation to their hostile surroundings by changing the biochemistry of their compartment as well as their own physiology 1 , 4 , Many pathogens have developed strategies to actively enter host cells and bacterial adhesion plays a critical role in optimizing survival chances 4 , 5 , Bacterial adhesion to host cells precedes their internalization and is a critical step in the progression of bacterial infection caused by intracellular pathogens 5 , 7 , Among the large variety of bacterial adhesins that promote internalization into host cells, type 1 fimbriae are remarkably versatile virulence factors 3 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , They mediate force activated catch bonds to mannosylated surfaces and cell receptors 12 , 14 , thus stabilizing the adhesion to host urinary epithelium under shear stress.

Consequently, fimbriae were found to be essential for the virulence of many uropathogenic E. They were also found to correlate with increased survival inside their predators by affecting intracellular trafficking of E.

In accordance with this, the expression of type 1 fimbriae is often associated with virulence of E. However, commensal E. Therefore, an understanding of the direct dependencies between bacterial adhesion and intracellular survival in phagocytes is of great medical relevance. Phagocytes such as macrophages and neutrophils are immune cells specialized in the recognition and removal of foreign particles, which involves uptake and intracellular degradation by aggressive chemicals and enzymes.

Many bacteria have thus evolved strategies to prevent recognition and phagocytosis by immune cells 5 , It has been hypothesized that bacterial adhesins like fimbriae promote internalization into epithelial cells in which they may survive, but avoid adhesion to phagocytes, which are their predators 5 , However, some phagocytes have fimbriae-specific receptors that trigger fimbriae-specific internalization 17 , Surprisingly, fimbriae-dependent phagocytosis of bacteria was found to result in higher intracellular survival chances than other types of phagocytosis 17 , This suggests that fimbriae-mediated adhesion to macrophages helps E.

Several studies compared survival of non-pathogenic and pathogenic bacteria, the latter of which often express additional virulence factors and may vary in the expression levels of these 15 , Different pathogens may therefore vary in their adhesion efficiency to macrophages and a quantification of the effects of adhesion efficiency on intracellular survival in macrophages is still lacking In particular, the question remains how the expression of fimbriae influences E.

Asking the question how fimbriation affects bacterial infection of macrophages and survival inside has additional clinical significance, since macrophages have been reported to release living pathogens which then continued to progress through their normal lifecycle Our main goal was to quantify how adhesion by fimbriated E.

Here, intracellular survival referred to bacteria that were internalized by macrophages and remained able to multiply after being extracted from the macrophage again. Internalization was assumed to occur via phagocytosis by the predatory immune cells and was expected to decrease the survival chances of E. We first assessed bacterial survival chances for three different non-pathogenic strains of E.

The pSH2 plasmid contains the whole fimbriae gene cluster and is a well-established expression system for investigations on overexpression of fimbriae 27 , We then asked if intracellular survival correlated with the efficiency of bacteria to adhere to macrophages and the number of bacteria per macrophage, i. To address this question, we established a dose-response curve for bacterial adhesion to macrophages.

We inhibited the functionality of fimbriae and the bacterial burden with two approaches. We used fimbriae-specific inhibitors to inhibit adhesion and cytoskeletal inhibitors to inhibit phagocytosis. The bacteria to macrophage ratios were systematically varied in a range from 0.

These ratios have often been referred to as multiplicities of infection MOI 17 , 19 , 30 which implicates, sometimes incorrectly, a linear relationship between bacterial concentration and host cell infection.

However, such linear relationship might not exist. We furthermore asked if we could identify a mechanistic model underlying bacterial adhesion to macrophages that could quantify the adhesion parameters in dependence of fimbriae expression.

Finally, we asked if expression of fimbriae and bacterial burden influenced the macrophage morphology in response to bacteria.

Such morphological changes could give indications to a fimbriae-specific activation or suppression of a characteristic macrophage response. The macrophage morphology was thus quantified regarding macrophage spreading, viability, proliferation and surface adhesion.

Our first goal was to investigate the specific impact of fimbriation on the survival chances of E. The exposure to the antibiotic gentamicin ensured that all bacteria that had not been internalized by the phagocytes were rendered incapable of replication. Bacterial survivors were quantified from single colonies based on their ability to replicate on agar plates after macrophage lysis Fig.

For a fimbriae-mediated survival advantage, we expected to find different behavior of bacterial clearance by macrophages, i. This would not require differences in the initial survivor numbers. However, we found large differences in the absolute numbers of surviving bacteria from the first time point onwards. Box plot whiskers indicate the S. Upper-case letters mark significant differences based on post hoc Tukey and Bonferroni tests. Error bars are S. The variance of population means was analyzed in the same way as in c.

Normalization to the absolute numbers of colony forming units to the 0. The finding on normalized survival is in good agreement with previous studies 17 , 23 , while the absolute numbers indicated that more bacteria were internalized from the beginning on when they overexpressed fimbriae.

This had to the best of our knowledge not been reported before. While this assay showed that more bacteria survived when they overexpressed fimbriae, it was not possible to differentiate whether the presence of fimbriae yielded overall higher numbers of infected macrophages with low bacterial burden or just increased the bacterial burden and fraction of survivors in a small macrophage subpopulation Fig. To test if differences in lysosomal acidification could explain the observed differences in survivors, we performed a series of lysosomal assessments using the fluorescent dye LysoID 31 Fig.

Since a parallel pathway for bacterial intracellular trafficking by autophagy was reported 19 , we blocked autophagy with the inhibitor 3-methyladenine 3-MA to test if this would increase the flux through the lysosomal pathway. This indicated that blocking autophagy at 0. We next quantified infection efficiency and bacterial burden, i. Towards this end, we investigated how fimbriae affected bacterial adhesion to macrophages using GFP-expressing E. Macrophages that bind or internalize GFP-expressing bacteria were detected by their fluorescence signal using flow cytometry Fig.

To quantify the bacterial burden on macrophages, the ratio of GFP-expressing bacteria to macrophages was systematically varied from 0. Serum-free media was used for 0. A potential effect of serum on adhesion efficiency was tested by using serum-containing media also during the adhesion assay Supplementary Fig. S1a and yielded the same trend of adhesion behavior. Fimbriae overexpression yielded higher adhesion efficiency, bacterial burden and intracellular survival of E.

Macrophages were incubated with E. Images of 25 randomly chosen macrophages were analyzed for total counts of adherent bacteria. GFP-expressing bacteria are colored in green, actin-binding phalloidin is colored in grey. Upper-case letters mark significant differences based on a post hoc Tukey and Bonferroni test. Notice that we carefully ensured that this comparison was made for the same bacteria to macrophage ratio of Fig. Since the efficiency of macrophage infection showed a clear dependency on fimbriae expression and adhesion to macrophages Fig.

The molecular basis of fimbriae mediated adhesion is their binding to mannose sugars on host cell receptors by the mannose-specific lectin FimH. We also used Latrunculin B LatB , a potent inhibitor of actin polymerization to inhibit actin-dependent internalization of bacteria. Incubation with LatB prevented internalization of bacteria while not inhibiting the adhesion to macrophages per se. LatB treated macrophages were unable to retain bacteria with the help of actin driven protrusions, since LatB blocks the actin polymerization required for this process and a smaller GFP-positive macrophage population was observed in all LatB treated samples Fig.

These results showed that fimbriae increased the efficiency by which bacteria bind to and are taken up by macrophages, thereby also suggesting a larger bacterial burden. It should be noted that all data from the flow cytometry assay, excluding those with LatB treatment, will inherently show a mix of surface-attached and internalized bacteria. We did not see any differences in fluorescence intensity between surface-attached and internalized bacteria Supplementary Fig.

S1b , which is in agreement with the constitutive expression of GFP from the E. To test if bacterial burden itself could contribute to intracellular survival, we quantified the bacterial burden by counting bacteria on macrophages using confocal fluorescence microscopy 0.

This intensity shift indicated several stably adherent bacteria per macrophage Supplementary Fig. S1c and was also confirmed by the number of bacteria per macrophage Fig. We thus showed that fimbriation resulted in an increased bacterial burden per macrophage. Comparison between experiments with the differently fimbriated bacteria showed the same, i. To clarify whether intracellular survival and adhesion efficiencies were functionally linked, we next asked if inhibiting bacterial adhesion via fimbriae would have consequences on intracellular survival.

The gentamicin protection assay was repeated under the influence of inhibitors Fig. S1d to test if the inhibitors could modulate intracellular survival chances in a similar manner since they changed adhesion behavior. Incubation with LatB however, did not yield any colony forming units Supplementary Fig. This finding is consistent with the implicit assumption of the assay that only intracellular bacteria can survive antibiotic treatment.

Bacterial adhesion to the outer plasma membrane of macrophages alone is thus not sufficient to protect bacteria from the effect of extracellular antibacterial drugs. The results derived from quantifying the modulation of bacterial burden therefore suggest that functional fimbriae have a larger impact on intracellular survival than the sole effect of bacterial burden and finding that had not been shown before. To further increase our mechanistic understanding of the E.

The data points from the flow cytometric determination of adhesion efficiencies Supplementary Fig. S1b, S1c were fit to two mathematical models. The best fit was obtained using a model inspired by Michaelis Menten kinetics Fig.

The model describes an adhesion process with a first step under an equilibrium assumption of adhesion and unbinding and a second irreversible and thus rate-limiting step Fig. By adapting the Michaelis Menten kinetics to bacteria-macrophage adhesion Fig. In principle, this complex is transient and can dissociate again. The second step is an irreversible and thus rate limiting step leading to stable adhesion of bacteria and their internalization.

The overall uptake rate R x of bacteria by macrophages is described as function of the percentage of macrophage population that can bind bacteria M max Fig. A quantitative model adapting Michaelis Menten kinetics to bacteria-macrophage interactions allowed to estimate infection doses and rate constants.

Inset graph shows the full range to enable a comparison with the graph in b. The value of M max is proportional to the rate constant of the second, rate-limiting step leading to internalization and thus determines the infection efficiency by the maximum possible number of infected macrophages. A detailed description of two alternate models can be found in the Supplementary information , including tests for the quality of a second model for an alternative adhesion mechanism assuming one irreversible adhesion step see Supplementary equations 9 — 12 , Supplementary Fig.

S2a, S2b. In summary, the Michaelis Menten-derived two-step model approximates the uptake kinetics and thus provides insights how the same numbers of bacteria that surround macrophages yield different infection efficiencies Fig. According to this kinetic model, type 1 fimbriae lead to a strongly decreased dissociation rate due to a decreased off-rate of initial binding.

This shifts the equilibrium towards the bound state Fig. Thus, the expression of fimbriae shifted the initial equilibrium of adhesion strongly to the side of stable adhesion. While the experiments here were conducted without flow, the presence of flow is expected to additionally decrease the off-rate since the FimH-mannose complex can form force-activated catch bonds Finally, we asked whether the increased bacterial survival was a consequence of differences in macrophage viability.

The viability of macrophages was assessed by staining with the calcein dye, which stains only cells with intact membranes Fig. Only the dead cells stain with propidium iodide, since it can enter only those cells with lysed or permeable membranes.

Second, we also observed that the number of macrophages per field of view was larger for cells in non-infected control, while the cell spreading area was smaller. This analysis showed that proliferation was indeed inhibited when macrophages were incubated with bacteria Fig. The results suggest that macrophages increased their interaction with the surface by increased spreading after encountering bacteria.

Substantial changes in the macrophage morphology occur upon exposure to E. Cells in the control were not exposed to E. Upper-case letters mark significant differences based on a post hoc Tukey test. Two randomly chosen images from vinculin immunostainings are shown on the left.

Data points belonging to the pictures on the left have no fill. To investigate if the adhesion strength to the surface was also increased and if fimbriae played a role in this process, we investigated the morphology of surface-adherent macrophages by immunostaining the protein vinculin. Changes in the morphology and spreading of macrophages have been described previously 33 , 34 , 35 and are instrumental for their role in anti-inflammatory behavior as well as infection-associated inflammation.

The biosynthesis of these fimbriae requires two specific and unique proteins, a periplasmic chaperone and an outer membrane located molecular usher 'doorkeeper'. Molecular and structural aspects of the secretion of fimbrial subunits across the cytoplasmic membrane, the interaction of these subunits with periplasmic molecular chaperone, their translocation to the inner site of the outer membrane and their interaction with the usher protein, as well as the ordered translocation of the subunits across the outer membrane and their assembly into a growing fimbrial structure will be described.

A model for K88 fimbriae is presented.