Persistent Bacteriuria and Antibodies Recognizing Curli/eDNA Complexes From Escherichia coli Are Linked to Flares in Systemic Lupus Erythematosus

Abstract

Objective: Infections contribute to morbidity and mortality in systemic lupus erythematosus (SLE). Uropathogenic Escherichia coli (UPEC) are known to trigger urinary tract infections (UTIs) and form biofilms, which are multicellular communities of bacteria that are strengthened by amyloids such as curli. We previously reported that curli naturally form complexes with bacterial extracellular DNA (eDNA), and these curli/eDNA complexes induce hallmark features of lupus in mouse models. The present study was undertaken to investigate whether anti-curli/eDNA complex antibodies play a role in the pathogenesis of SLE or development of flares in SLE.

Methods: In total, 96 SLE patients who met at least 4 Systemic Lupus International Collaborating Clinics disease criteria were investigated. Anti-curli/eDNA complex antibodies in the plasma were tested for both IgG and IgA subclasses. Results were compared to that in 54 age-, sex-, and race/ethnicity-matched healthy controls. Correlations of the levels of anti-curli/eDNA antibodies with clinical parameters, lupus disease status, and frequency of bacteriuria were assessed.

Results: Anti-curli/eDNA antibodies were detected in the plasma of SLE patients and healthy controls, and their levels correlated with the presence of asymptomatic persistent bacteriuria and occurrence of disease flares in lupus patients. Persistent bacteriuria contained curli-producing UPEC, and this was associated with an inflammatory phenotype. Finally, curli/eDNA complexes cross-reacted with lupus autoantigens, such as double-stranded DNA, in binding autoantibodies.

Conclusion: These results suggest that UTIs and persistent bacteriuria are environmental triggers of lupus and its flares. Antibodies against curli/eDNA could serve as a sign of systemic exposure to bacterial products in SLE.

Figure 1.. Plasma anti-curli/eDNA Abs correlate with flares in SLE patients.

A) IgG Abs against curli/eDNA purified from E. coli biofilm were measured by ELISA in plasma of SLE patients, median = 1.47 O.D. and age- and sex-matched healthy controls median = 1.65 O.D.. B) Levels of IgG anti-curli/eDNA Abs in SLE patients in flare, median = 2.15, and in remission, median = 1.34 O.D.. Statistical significance was calculated with unpaired t-test with Welch correction.

Figure 2.. Curli/eDNA complexes cross-react with lupus autoantigen dsDNA.

ELISA for A) anti-dsDNA IgG Abs and (B) anti-curli/eDNA IgG Abs was performed on a representative SLE patient after co-incubation with calf thymus dsDNA or curli/eDNA complexes in suspension. C) ELISA used dsDNA as antigen and a mouse monoclonal Ab against dsDNA. D) ELISA used curli/eDNA as antigen and a rabbit polyclonal Ab against curli/eDNA complexes. In A-D, results are representative of 3 experiments. ELISA for anti-K12 E. coli DNA IgG (E), anti-calf thymus dsDNA IgG (F) and anti-curli/eDNA IgG Abs (G) in SLE patients anti-dsDNA Ab positive or negative by hospital records vs Healthy Controls (HC). Competitive ELISA with SLE plasma for anti-curli/eDNA IgG Abs (H) and anti-dsDNA Abs (I) after co-incubation with CsgA, curli/eDNA, and K12 DNA. Statistical significance was calculated with ANOVA and post-hoc t-test (E-I).

Figure 3.. IgA anti-curli/eDNA antibodies are higher in Lupus patients than healthy controls without correlation with flares.

A) Anti-curli/eDNA IgA Abs were measured by specific ELISA in plasma of SLE patients and age- and sex-matched healthy controls. B) Levels of anti-curli/eDNA IgA for SLE patients in flare and in remission. C) Correlation between levels of anti-curli/eDNA IgG and IgA Abs in SLE patients. *p <0.05 **p <0.01. Statistical significance was calculated with unpaired t-test with Welch correction (A-B) and Bivariate Normal Ellipse (C).

Figure 4.. Association between Bacteriuria, IgG anti-curli/eDNA antibodies and disease activity.

A) Frequency of bacteriuria in SLE patients as measured by routine UA ranged from never, to intermittent (<100%), to persistent at each visit (100%). B) Levels of anti-curli/eDNA Abs (IgG) and C) anti-dsDNA IgG Abs were compared between patients with increasing frequency of bacteriuria. Wilcoxon test. D) Levels of anti-curli/eDNA against Salmonella (shown) are compared between patients with bacteriuria, as detected by routine urine analyses with microscopy and calculated using a +, ++, +++ grading system. Unpaired two tailed t-test. E-F) Anti-curli/eDNA IgG and IgA Abs levels were compared between sexes in patients who had at least three urinalyses. Welch’s t-test. G) UPEC in lupus patients produce curli containing biofilms. Colony morphologies of E. coli wild type, curli and cellulose mutants as well as three UTI isolates from SLE patients with bacteriuria were grown using YESCA plates supplemented with Congo Red and Brilliant Blue. Curli production is shown by the wrinkled rdar morphology. H). Crystal violet assay was performed to measure biomass of the biofilm generated by three E. coli isolates from urine of SLE patients in comparison to the reference E. coli WT (UTI89) and its isogenic CsgA deficient mutant (curli-).