Fig 1.
Urinary pellet lysates and solubilization of UP aggregates incubating with deoxyribonuclease I (DNase I).
(A) SDS-PAGE gels with 4–12% acrylamide gradients were stained with Coomassie Brilliant Blue G-250. The left lane contains Mr standards. The protein extracts displayed here are #36, #88, and #64 (DUP samples), #20 and #118 (AUP samples with a phenotype showing complete or moderate loss of aggregation after 2 freeze-thaw cycles), and six AUP samples where DNase I treatment was required to homogenize the pellets under moderate agitation. The proteins UMOD and MPO marked in the gel image were identified by LC-MS/MS. The bars at the bottom show proteomic identifications (PID) of microbial species and, if available, leukocyte counts/ml in the original urine sediments. Acronyms denote the following: NC, neutrophil counts in a high power field per ml urine; tntc, leukocytes too numerous to count; Gv, Gardnerella vaginalis; Pm, Proteus mirabilis; Ec, Escherichia coli; Sa, Staphylococcus aureus; Ef, Enterococcus faecalis; Kp, Klebsiella pneumoniae; n.d., not determined. (B) Photos of UP samples prior to and after incubation with DNase I in PBS at 37°C for 15 to 60 min.
Fig 2.
Evidence of neutrophil death, disintegration and multi-cellular aggregation in AUP samples.
Urine sediment samples were centrifuged at 1,300 x g for 5 min and gently resuspended in PBS. Aliquots were stained with Trypan Blue (1:10 dilution) and subjected to phase-contrast microscopy (40-fold magnification). White arrows denote bright viable cells; yellow arrows denote dead cells, some with a flattened morphology and larger and some with visible nuclei stained blue; red arrows denote ruptured cells with membrane borders no longer clearly visible, some with dark patches of apparently released chromatin (especially in AUP samples #151 and #157).
Fig 3.
Sequential extraction of DNA and proteins from AUP samples.
(A) Samples #122 and #134 were analyzed in 0.5% agarose gels and stained with ethidium bromide. DNA standards (St) are denoted in kilobase pairs (kbp). Lane numbers 1–5 match fraction numbers UPsol1, UPsol2, etc. (1 μl extract each). In the gel image for #134, lane 2’ pertains to a repeated incubation step with PBS and DTT, and lane 3’ pertains to a shorter 10 min incubation step with DNase I. UPsol3 (lane 3) represents incubation for 75 min. AUP sample #122 resisted disintegration of the pellet prior to the addition of DNase I; large size DNA was even retained even in fraction UPsol5. The aggregate in sample #134 released DNA in a decreasing size range with each incubation step. DNase I can cleave nucleic acids to nucleosome monomers (~ 0.2 kbp). (B) Protein extracts of samples #112 and #122 visualized in SDS-PAGE gels. Lane numbers 1 to 5 match fraction numbers UPsol1, UPsol2, etc. Ten μl extract were used in each lane. Low Mr proteins were abundant suggesting protein degradation in AUP samples. The positions of LTF, α-defensin 1, and MPO (all identified by LC-MS/MS) are marked in the gel image.
Fig 4.
Protein profiles of UPsol fractions show molecular evidence of NET formation in UTI cases.
Proteomic quantification for UPsol3 fractions shows consistent similarities with profiles observed for in vitro generated NETs. Twenty-one highly abundant proteins were included in the graphic. Each colored segment in a stacked bar represents the relative amount of a protein in the total proteome of a fraction (including the UPsol1 and combined UPsol4/5 fractions; terms UP-s1, UP-s3, and UP-s4/5 are used on the x-axis). Each protein segment has a color corresponding to the color code displayed on the very right in the order of occurrence in the stacked bars. Protein names are UniProt short names. The bar displayed on the right (term ‘exp NETs’) represents the relative quantity of 13 proteins from a publication on in vitro generated NETs [14]. Black arrows on top of stacked bars denote samples in which the retention of insoluble DNA in UP fractions prior to the enzymatic digestion was high. Red arrows denote samples with partial release of DNA fragments prior to DNase incubation, which was in agreement with the gradual release of histones in the UPsol1 fraction. Green arrows denote samples with high cytokeratin (KRT1) and/or UMOD contents. The y-axis value of 1 represents 100% of the proteome using the proteomic quantification tool MaxQuant (all identified protein quantities are listed in S3 Data).
Fig 5.
Analysis of DNA-protein complexes in solubilized extracts of four AUP samples using native PAGE.
Non-denaturing 3% acrylamide gels were stained with CBB to visualize proteins (A) and ethidium bromide to visualize DNA (B) in the same gel. Stained areas with high Mr values (protein stain) and large sizes (DNA stain) match and are thus indicative of DNA-protein complexes. These areas were excised, digested with trypsin, and analyzed by LC-MS/MS. Proteins identified are denoted in the gel image with UniProt short names that were also used in Fig 4. BIK is the urinary protein bikunin. Lane and UPsol fraction numbers listed below the sample ID (1 and 3) match. Sample #20 showed DNA release without adding DNase I into the fraction UPsol1. In contrast, the enzyme was responsible for the release of DNA fragments into the fraction UPsol3 for the samples #94, #151, and #157. The lack of DNA solubilization in prior extraction steps is shown for #94 (fraction UPsol1), and proteins in this fraction have generally lower Mr values forming sharper bands, consistent with the absence of association with DNA.
Fig 6.
Citrullinated proteins in NET structures.
Mass spectral data for the histone H1 peptide E55rNGLSLAALK65 (left) and the histone H3 peptide V24ArKSAPATGGVK36 (right), r = citrulline; m/z values for the y- and b-ion series confirmed R56 and R26 deamidations, respectively. Both spectra, derived from sample #112, were acquired using high-energy collisional dissociation MS/MS in the Orbitrap mass analyzer with a resolution of 17,500 and a mass accuracy of ~1 ppm. The human protein database was searched in the Proteome Discoverer software (v1.4) using a FDR ≤ 1%. The XCorr scores were 2.80 and 2.47 for the H1 and H3 peptide spectra, respectively. Analysis with the MaxQuant software tool confirmed the peptide citrullination sites.
Fig 7.
Proteolytic degradation in extracts of AUP samples.
(A) Western Blots were performed with polyclonal antibodies specific for NE, MPO, LTF, and histone H4A. The lane numbers match fraction numbers (UPsol1, UPsol3, and UPsol5) derived from samples #33, #94, and #112. The Mr standard consists of ten proteins denoted with kDa values. Green arrowheads point to Mr values observed for full-length proteins, including the heavy and light chains of MPO. Full-length NE (29 kDa) and a H4A fragment (8–10 kDa) were detected only in fractions of sample #94. LTF and MPO were represented by full length protein bands in UPsol3 fractions. The S. aureus protein A (SpA) was detected in a Mr range corresponding to its post-translationally modified, cell wall-immobilized forms for sample #112 (red arrowheads). (B) Cleavage sites identified in the peptide sequences of MPO and H4A that were in agreement with the preferred P1 site specificities of the proteases NE and PRTN3. This data is deduced from peptide termini identified via LC-MS/MS from five AUP samples. The N- and C-termini of peptide segments shaded in green include experimentally generated trypsin-specific cleavage sites and PRTN3/NE-specific sites apparently formed as a consequence of the in vivo inflammatory process. Red arrowheads denote sequence positions resulting from protein maturation of precursors. Other arrowheads denote PRTN3 and NE cleavage sites (C-terminal to A, V, L, I, S, T, C, M); if colored black, the site was identified in three or more of the five examined AUP datasets.
Fig 8.
Proteins in AUP samples are degraded to the level of peptides consistent with activities of cathepsin G, proteinase 3 and, elastase.
(A) Peptide maps for protein S100-A9, prolifin-1, and histone H2B. This data is derived from peptidome analyses combining the fractions UPsol1, UPsol2, and UPsol3 from both sample #94 and sample #134. No enzymatic cleavage sites were pre-selected in the database searches. The NE, PRTN3, and CTSG specific cleavage sites determined from peptide termini are mapped along each the respective protein sequence. Peptide termini not consistent with preferred cleavage sites of the three proteases were rare. The peptides, which are highlighted in the form of red and blue bars along the amino acid sequence to mark where they end, revealed peptide clusters around common cores. (B) Relative abundances of peptides associated with protein localization or functional groups comparing peptidome (PEP) and equivalent shotgun proteome (PROT) datasets. Quantification of peptides is based here on peptide-spectral counts. Protein groups denoted on the right of the graphic have color codes pertaining to the following names, functions, and localizations: NG, neutrophil granules; HIST, histones; CYT, cytosol; ACT1, actin; CSK, cytoskeleton (except actin) and keratins; RBC/COAG, red blood cells and coagulation.
Fig 9.
Detection of early-phase NETs by IF microscopy.
AUP aliquots were paraformaldehyde-fixed on glass slides on the day of specimen collection and stored at 4°C until further use. IF staining was performed with an MPO-specific polyclonal antibody followed by an anti-rabbit IgG conjugate to the dye CFl-555, and counterstaining with DAPI. Oil immersion microscopy (not confocal) was used for imaging with phase contrast and in blue and red channels. (a-d) sample #142; (e-h) sample #146; (i-l) sample #151; (m-p) sample #157. Sample #142 shows evidence of lobulated nuclei and no evidence of extracellular chromatin release (a), intact granular structures and well-defined cell perimeters (b), MPO staining in accordance with intact granules (c), and no co-localized MPO/chromatin staining (d). Sample #146 has intact neutrophils, but also some cells where nuclei fill the entire cell space (e) and granules are diminished in the perimeter of cells according to staining for MPO (g). Co-localization of nuclei and MPO is visible in the cell perimeter suggesting the emerging loss of nuclear membranes (h). Sample #151 shows less regularly shaped nuclei with fainter staining in their perimeters suggesting nuclear membrane disintegration (i), and patchy granular staining as described above (k); MPO and nuclear staining with DAPI overlap (l). Sample #157 shows areas of flattened and disintegrating cells (m, n) and streaks of extracellular DNA (m) that co-localizes with MPO staining (o, p). The closed white arrows point to cells with intact nuclei and well-distributed cytoplasmic granules. Open white arrows point to cells filled with chromatin and a patchy staining pattern for granules (MPO). Open yellow arrows point to disintegrated cells releasing chromatin from nuclei that co-localizes with MPO.
Fig 10.
Microbial cell viability staining for infectious agents in AUP samples.
Images are from oil immersion microscopy with the green (fluorescein) channel for sample #122, and green and red (rhodamine) channels for samples #146, #151 and #157. Image #122: AUP sample aliquot was incubated with SYTOX-green (5 μM in TBS) in the dark for 15 min, fixed on a glass slide at low heat (40°C), washed with water, and air-dried. C. albicans yeast forms are clearly visible. Red arrows point to dead cells (SYTOX-green stained), white arrows point to intact cells (unstained). Images #146 to #157: Sample aliquots were incubated with the live/dead differential staining kit (5 μM SYTO9 and 55 μM propidium iodide in TBS) in the dark for 15 min followed by centrifugation at 800 x g for 3 min, re-suspension in TBS, a 2nd centrifugation step, and fixation with 4% paraformaldehyde for 15 min. #146: rod-shaped E. coli cells propidium iodide-stained (red arrow) are dead. #151: filamentous K. pneumoniae rods stained with SYTO9 are living cells (white arrow). Neutrophils (bright yellow stain) are surrounded by bacterial cells suggesting a failure of phagocytosis. #157: S. aureus cocci are trapped in NET-like structures with red and green dots indicating death and survival (red and white arrows, respectively). The #157 insert shows a cluster of dead bacterial cells.
Fig 11.
S. aureus proteome comparing in vitro cultures of the isolate from AUP sample #112 to in vivo data derived from sequential extraction of the clinical specimen.
The UPsol fractions were defined in the main text. The isolate was grown in LB media to mid-exponential (EXP) and stationary (STAT) phases. Protein quantification was based on MaxQuant analysis, and proteins were assigned to three groups based on their roles in ribosomal protein synthesis, energy metabolism and cell envelope localization as annotated in the database UniProt. Black-rimmed boxes are the combined quantities of the cell wall enzymes Atl and IsaA. Detailed bacterial protein profiles are provided in S7 Data.