EH Lambertsen, N. Johnsen, T. Corydon, H. Prætorius¹

Aarhus University, Department of Biomedicine, 8000 Aarhus, Denmark

 

Background:

Uropathogenic E. coli (UPEC) is the leading cause of urinary tract infections (UTIs). UPEC causing severe disease express various virulence factors that allow the UPEC to colonise the bladder, ascend to the kidney and disseminate to urosepsis^1,2. Our preliminary data show that thrombocytes are important for acute clearing of UPEC from the blood in a murine model of urosepsis. UPEC are known to adhere to epithelia via type 1 fimbriae, recognising D-mannose-rich structures^3,4. Here, we investigate if thrombocyte/E. coli complexes are prevented by pre-incubating UPEC with DM or preincubating thrombocytes with concanavalin A, a soluble lectin that binds D-mannose.

 

Methods:

Here, we use an in vitro assay of GFP-expressing UPEC (165·10⁶ ml^-1) added to whole blood samples or isolated thrombocytes from humane healthy volunteers (Danish Research Ethics Committees: 1-10-72-202-17). Complex formation between UPEC and thrombocytes (using CD42b as a thrombocyte marker) was determined at various time points by flow cytometry (Accuri 6plus, BD Biosciences). Data are analysed using one-way ANOVA and given as mean±SEM.

 

Results:

In our assay, we could easily confirm the instant formation of UPEC-thrombocyte complexes similar to what we observe in a mouse model of urosepsis. The complexes also readily form after thrombocytes have been fixed with 0.4% paraformaldehyde. Although thrombocytes have previously been suggested to be able to adhere E. coli via surface lipopolysaccharide (LPS) binding to thrombocyte TLR4, we could not prevent complex formation by either preincubation with TLR4 antagonists, TLR4 antibodies or pre-incubating thrombocytes with LPS. Moreover, we have not been able to prevent complex formation with antibodies directed against FcγRII or CD41/CD61. Both D-mannose and concanavalin A cause a concentration-dependent reduction in UPEC/thrombocyte complexes. D-mannose reduced UPEC-thrombocyte complex formation from 6.71·10⁶±0.78·10⁶ counts/ml to 2.69·10⁶±0.56·10⁶ counts/ml equal to a ~60% decrease (n=8, p=0.023). Concanavalin A reduced complex formation from 3.21·10⁵±0.34·10⁵ counts/ml to 1.57·10⁵±0.91·10⁵ counts/ml equal to a ~51% decrease (n=7, p=0.004).

 

Conclusions:

We show that pre-treatment of UPEC with DM, and pre-treatment of thrombocytes with Concanavalin A reduced complex formation in vitro. These data suggest that type 1 fimbriae and not LPS are important for E. coli-thrombocyte interaction, and currently, we are gaining further evidence for this notion. Once confirmed, the relevant mannose-bearing interaction partners on thrombocytes will be determined by proteomics.