Characterization of Early-Phase Neutrophil Extracellular Traps in Urinary Tract Infections

Neutrophils have an important role in the antimicrobial defense and resolution of urinary tract infections (UTIs). Our research suggests that a mechanism known as neutrophil extracellular trap (NET) formation is a defense strategy to combat pathogens that have invaded the urinary tract. A set of human urine specimens with very high neutrophil counts had microscopic evidence of cellular aggregation and lysis. Deoxyribonuclease I (DNase) treatment resulted in disaggregation of such structures, release of DNA fragments and a proteome enriched in histones and azurophilic granule effectors whose quantitative composition was similar to that of previously described in vitro-formed NETs. The effector proteins were further enriched in DNA-protein complexes isolated in native PAGE gels. Immunofluorescence microscopy revealed a flattened morphology of neutrophils associated with decondensed chromatin, remnants of granules in the cell periphery, and myeloperoxidase co-localized with extracellular DNA, features consistent with early-phase NETs. Nuclear staining revealed that a considerable fraction of bacterial cells in these structures were dead. The proteomes of two pathogens, Staphylococcus aureus and Escherichia coli, were indicative of adaptive responses to early-phase NETs, specifically the release of virulence factors and arrest of ribosomal protein synthesis. Finally, we discovered patterns of proteolysis consistent with widespread cleavage of proteins by neutrophil elastase, proteinase 3 and cathepsin G and evidence of citrullination in many nuclear proteins.

• Sample 33 was viscous and clumpy. In vitro culture and proteomic analysis revealed Enterococcus faecalis as the infectious agent. The extractions related to this experiment were performed after a freeze/thaw cycle. The sample 33 remained clumpy and viscous and was not homogenized upon incubation with PBS and PBS + 50 mM DTT (UP sol 1 and UP sol 2 fractions, respectively). The pellet derived from the UP sol 2 extraction step was incubated with deoxyribonuclease I (DNAse I)for 60 min, resulting in the fraction UP sol 3. • Incubation steps with 50 µg/ml mutanolysin and 100 µg/ml lysosome in PBS or TBS followed and 10 mM EDTA and 0.4% CHAPS were added. Samples were vortexed, left at 20°C for 10 min, and sonicated at the amplitude 6 in ten 30 sec on/off cycles using a Misonex 3000 sonicator while cooling on ice. Following centrifugation for 6 min at 8,000 x g, a supernatant termed UP sol 4 was isolated. The insoluble pellet was incubated with the SED solution (1% SDS, 0.3% Tween-20, 10 mM EDTA, 25 mM DTT) for 30 min followed by sonication at the amplitude 6 for 5 min (Misonex 3000 sonicator). The homogenate was vortexed, left at 20°C for 10 min, and heat-denatured at 95°C for 3 min. This was followed by repeated sonication step and centrifugation at 16,100 × g for 10 min to isolate the fraction UP sol 5.

Summary:
Step 1: extraction with PBS (UP sol 1); Step 2: two successive extraction steps with PBS + 50 mM DTT (UP sol 2a and UP sol 2b); Step 3: incubation/extraction with PBS and DNAse I (UP sol 3); Step 4: extraction/incubation with lysozyme and mutanolysin in the presence of the partially membrane-solubilizing reagents EDTA and CHAPS (UP sol 4); Step 5: incubation/heat extraction with denaturing solution (UP sol 5);  1: UP sol 1, ca. 10 ul extract 2: UP sol 2, ca. 10 ul extract 3: UP sol 3, ca. 10 ul extract 4: UP sol 5, ca. 10 ul extract 5: M r standard, 5 ul solution • Sample 9 was neither aggregated nor clumpy after resuspension in PBS. Proteomic analysis revealed presence of Gardnerella vaginalis with no evidence that the bacterium caused a urinary tract infection although some neutrophils were microscopically identified. The extraction experiments shown here were performed after a freeze/thaw cycle. The pellet of sample 9 was homogenized upon incubation with PBS and PBS + 50 mM DTT (UP sol 1 and UP sol 2 fractions, respectively). The pellet derived from the UP sol 2 extraction step was small and was incubated with DNAse I for 60 min, resulting in the fraction UP sol 3.
• In this experiment, the following extraction steps were identical to those described in Figure 1A, with the exception that the extraction step resulting in UP sol 4 was not performed.

Summary:
Step 1: extraction with PBS (UP sol 1); Step 2: extraction with PBS + 50 mM DTT (UP sol 2); Step 3: incubation/extraction with PBS and DNAse I (UP sol 3); Step 4: incubation/heat extraction with denaturing solution (UP sol 5);  • In this experiment, the following extraction steps were equivalent to those described in Figure 1A, with the exception that the extraction step resulting in UP sol 4 was not performed.

UMOD
Step 1: extraction with PBS (UP sol 1); Step 2: two successive extraction steps with PBS + 50 mM DTT (UP sol 2a and UP sol 2b); Step 3: incubation/extraction with PBS and DNAse I (UP sol 3); Step 4: incubation/heat extraction with denaturing solution (UP sol 5); UMOD: uromodulin DNAse I: deoxyribonuclease I (abbreviations also used in other images)   1 and UP sol 2 fractions, respectively). The pellet derived from the UP sol 2 extraction step was small and was incubated with DNAse I for 60 min, resulting in the fraction UP sol 3.

Figure 2c
• In this experiment, the following extraction steps were identical to those described in Figure 1A, with the exception that the extraction step resulting in UP sol 4 was not performed.

Summary:
Step 1: extraction with PBS (UP sol 1); Step 2: extraction with PBS + 50 mM DTT (UP sol 2); Step 3: incubation/extraction with PBS and DNAse I (UP sol 3); Step 4: incubation/heat extraction with denaturing solution (UP sol 5);   Proteomic analysis revealed presence of the uropathogen Klebsiella pneumoniae with no evidence that the bacterium elicited an immune response and urinary tract infection. Few neutrophils were microscopically identified. The extraction experiments shown here were performed after a freeze/thaw cycle. The pellet of sample 55 was homogenized upon incubation with PBS and PBS + 50 mM DTT (UP sol 1 and UP sol 2 fractions, respectively). The pellet derived from the UP sol 2 extraction step was small and was incubated with DNAse I for 60 min, resulting in the fraction UP sol 3.
• In this experiment, the following extraction steps were identical to those described in Figure 1A, with the exception that the extraction step resulting in UP sol 4 was not performed.

Summary:
Step 1: two extraction steps with PBS (UP sol 1a and UP sol 1b); Step 2: two extraction steps with PBS + 50 mM DTT (UP sol 2a and UP sol 2b); Step 3: incubation/extraction with PBS and DNAse I (UP sol 3); Step 4: incubation/heat extraction with denaturing solution (UP sol 5);  • Sample 64 was derived from a urinary sediment that was neither aggregated nor clumpy after resuspension in PBS or PBS-DTT. The human subject had a urethral catheter-associated infection (CAUTI), and this sample was obtained after the catheter-associated biofilm dispersed into the urine sediment. Proteomic analysis revealed Proteus mirabilis as the infectious agent. The extraction experiments shown here were performed after a first freeze/thaw cycle. The pellet of sample 64 was homogenized upon incubation with PBS and PBS + 50 mM DTT (UP sol 1 and UP sol 2 fractions, respectively). The pellet derived from the UP sol 2 extraction was relatively large. Incubation with DNAse I for 60 min did not change the pellet volume and resulted in fraction UP sol 3.
• In this experiment, the following extraction steps were identical to those described in Figure 1A, with the exception that the extraction step resulting in UP sol 4 was not performed.

Summary:
Step 1: extraction step with PBS (UP sol 1a); Step 2: two extraction steps with PBS + 50 mM DTT (UP sol 2a and UP sol 2b); Step 3: incubation/extraction with PBS and DNAse I (UP sol 3); Step 4: incubation/heat extraction with denaturing solution (UP sol 5);  The extraction experiments shown here were performed after a freeze/thaw cycle. The pellet of sample 88 was homogenized upon incubation with PBS and PBS + 50 mM DTT (UP sol 1 and UP sol 2 fractions, respectively). The pellet derived from the UP sol 2 extraction step was small and was incubated with DNAse I for 60 min, resulting in the fraction UP sol 3.
• In this experiment, the following extraction steps were identical to those described in Figure 1A, with the exception that the extraction step resulting in UP sol 4 was not performed.

Summary:
Step 1: extraction with PBS (UP sol 1); Step 2: extraction with PBS + 50 mM DTT (UP sol 2); Step 3: incubation/extraction with PBS and DNAse I (UP sol 3); Step 4: incubation/heat extraction with denaturing solution (UP sol 5);  • In this experiment, the following extraction steps were equivalent to those described in Figure 1A.

Figure 2g
Summary: Step 1: extraction with PBS (UP sol 1); Step 2: two successive extraction steps with PBS + 50 mM DTT (UP sol 2a and UP sol 2b); Step 3: incubation/extraction with PBS and DNAse I (UP sol 3); Step 4: extraction/incubation with lysozyme and mutanolysin in the presence of the partially membrane-solubilizing reagents EDTA and CHAPS (UP sol 4); Step 5: incubation/heat extraction with denaturing solution (UP sol 5);  In vitro culture and proteomic analysis revealed Klebsiella pneumoniae and Escherichia coli as the infectious agents. The extractions related to this experiment were performed after a freeze/thaw cycle. The pellet of sample 118 became less clumpy and viscous but was not completely homogenized upon incubation with PBS and PBS + 50 mM DTT (UP sol 1 and UP sol 2 fractions, respectively). Homogenization was achieved with the DNAse I incubation step.
• In this experiment, the following extraction steps were equivalent to those described in Figure 1A, with the exception that the extraction step resulting in UP sol 4 was not performed.

Summary:
Step 1: extraction with PBS (UP sol 1); Step 2: two extraction steps with PBS + 50 mM DTT (UP sol 2a and UP sol 2b); Step 3: incubation/extraction with PBS and DNAse I (UP sol 3); Step 4: incubation/heat extraction with denaturing solution (UP sol 5);  • In this experiment, the following extraction steps were equivalent to those described in Figure 1A, with the exception that the extraction step resulting in UP sol 4 was not performed.

Summary:
Step 1: extraction with PBS (UP sol 1) Step 2: extraction with PBS + 50 mM DTT (UP sol 2a); Step 3: re-extraction with PBS + 50 mM DTT (UP sol 2b); gel lane not shown Step 4: incubation/extraction with PBS and DNAse I (UP sol 3); Step 5: incubation/heat extraction with denaturing solution (UP sol 5);  • In this experiment, the following extraction steps were equivalent to those described in Figure 1A.

Figure 2k
Page • Sample 134 was viscous and clumpy. In vitro culture and proteomic analysis revealed Klebsiella pneumoniae as the infectious agent. The extractions related to this experiment were performed after two freeze/thaw cycles. The pellet of sample 134 became less clumpy and viscous but was not completely homogenized upon incubation with PBS and PBS + 50 mM DTT (UP sol 1 and UP sol 2 fractions, respectively). Homogenization was achieved with the DNAse I incubation step.
• In this experiment, the following extraction steps were equivalent to those described in Figure 1A, except that an additional wash step after DNAse I incubation was added.

Summary:
Step 1: extraction with PBS (UP sol 1); Step 2: two extraction steps with PBS + 50 mM DTT (UP sol 2a and UP sol 2b); Step 3: incubation/extraction with PBS and DNAse I (UP sol 3); Step 4: pellet wash step with PBS; Step 5: extraction/ incubation with lysozyme, EDTA and CHAPS (UP sol 4); Step 6: incubation/heat extraction with denaturing solution (UP sol 5); • Sample 20 was originally viscous and clumpy. In vitro culture and proteomic analysis revealed Proteus mirabilis as the infectious agent. The extractions related to this experiment were performed after two freeze/thaw cycles. The pellet of sample 20 was less clumpy and viscous upon thawing for the 2 nd time. It was homogenized upon incubation with PBS and PBS + 50 mM DTT (UP sol 1 and UP sol 2 fractions, respectively). DNA was released into the UP sol 1 and UP sol 2 fractions. No further homogenization was achieved with the DNAse I incubation step (UP sol 3 fraction). The DNA extraction was performed after a 3 rd freeze-thaw cycle.

Figure 2l
• In this experiment, the following extraction steps were equivalent to those described in Figure 1A, with that the extraction step resulting in UP sol 4 was not performed.

Summary:
Step 1: extraction with PBS (UP sol 1); Step 2: extraction with PBS + 50 mM DTT (UP sol 2); Step 3: incubation/extraction with PBS and DNAse I (UP sol 3); Step 4: incubation/heat extraction with denaturing solution (UP sol 5);  • In this experiment, the following extraction steps were equivalent to those described in Figure 1A, with that the extraction step resulting in UP sol 4 was not performed.

Summary:
Step 1: extraction with PBS (UP sol 1); Step 2: extraction with PBS + 50 mM DTT (UP sol 2); Step 3: incubation/extraction with PBS and DNAse I (UP sol 3); Step 4: incubation/heat extraction with denaturing solution (UP sol 5); • In this experiment, the following extraction steps were equivalent to those described in Figure 1A. Summary: Step 1: extraction with PBS (UP sol 1) Step 2: extraction with PBS + 50 mM DTT (UP sol 2a); Step 3: incubation/extraction with PBS and DNAse I (UP sol 3); Step 4: extraction/ incubation with lysozyme, EDTA and CHAPS (UP sol 4) Step 6: incubation/heat extraction with denaturing solution (UP sol 5);  1: UP sol 1, ca. 10 ul extract 2: UP sol 2, ca. 10 ul extract 3: UP sol 3, ca. 10 ul extract, 10 min DNAse incubation 4: UP sol 3, ca. 10 ul extract , 60 min DNAse incubation 5: UP sol 5, ca. 10 ul extract 6: M r standard, 10 ul solution UP sample 142 was not clumpy. In vitro culture and proteomic analysis revealed Serratia marcescens as the infectious agent. The extractions related to this experiment were performed from a UP sample obtained approximately 2-5 hours after urine specimen collection without cooling or freezing. The UP sample 142 became clumpy and aggregated after centrifugation and addition of PBS + 50 mM DTT. The aggregate was not homogenized upon incubation with PBS and PBS + 50 mM DTT (UP sol 1 and UP sol 2 fractions, respectively). The pellet derived from UP sol 2 extraction was incubated with DNAse I for 10 min, and then another 50 min, resulting in fraction UP sol 3. In this experiment, the following extraction steps were identical to those described in Figure 1A, with the exception that the extraction step resulting in UP sol 4 was not performed.

Summary:
Step 1: extraction with PBS (UP sol 1); Step 2: extraction with PBS + 50 mM DTT (UP sol 2); Step 3: incubation/extraction with PBS and DNAse I (UP sol 3); Step 4: incubation/heat extraction with denaturing solution (UP sol 5);  • Sample 151 was aggregated and clumpy. In vitro culture and proteomic analysis revealed Escherichia coli as the infectious agent. The extractions related to this experiment were performed from a UP sample obtained approximately 2-5 hours after urine specimen collection without cooling or freezing the neutrophils. The pellet of sample 151 remained clumpy and was not homogenized upon incubation with PBS and PBS + 50 mM DTT (UP sol 1 and UP sol 2 fractions, respectively). The pellet derived from UP sol 2 extraction was incubated with DNAse I for 60 min, resulting in fraction UP sol 3.
• In this experiment, the following extraction steps were identical to those described in Figure 1A, with the exception that the extraction step resulting in UP sol 4 was not performed.

Summary:
Step 1: extraction with PBS (UP sol 1); Step 2: extraction with PBS + 50 mM DTT (UP sol 2); Step 3: incubation/extraction with PBS and DNAse I (UP sol 3); Step 4: incubation/heat extraction with denaturing solution (UP sol 5);  The pellet of sample 157 remained clumpy and viscous and was not homogenized upon incubation with PBS and PBS + 50 mM DTT (UP sol 1 and UP sol 2 fractions, respectively). The pellet derived from UP sol 2 extraction was incubated with DNAse I for 60 min, resulting in fraction UP sol 3.

Figure 2q
• In this experiment, the following extraction steps were identical to those described in Figure 1A, with the exception that the extraction step resulting in UP sol 4 was not performed.

Figure 2r
M r 1 2 3 4 5 • In this experiment, the following extraction steps were identical to those described in Figure 1A, with the exception that the extraction step resulting in UP sol 4 was not performed.

Figure 2s
Date :  • Sample 146 was aggregated and clumpy. In vitro culture and proteomic analysis revealed Escherichia coli as the infectious agent. The extractions related to this experiment were performed from a UP sample obtained approximately 2-5 hours after urine specimen collection without cooling or freezing the neutrophils. The pellet of sample 146 remained clumpy and was not homogenized upon incubation with PBS and PBS + 50 mM DTT (UP sol 1 and UP sol 2 fractions, respectively). The pellet derived from UP sol 2 extraction was incubated with DNAse I for 60 min, resulting in fraction UP sol 3.
• In this experiment, the following extraction steps were identical to those described in Figure 1A, with the exception that the extraction step resulting in UP sol 4 was not performed.

Summary:
The reactivity of a major band at 50 kDa indicates that S. aureus protein A (Spa) from the recombinant protein (lane 3), two lysostaphin digests of S. aureus strains (lanes 4 and 5) and the UP sol 3 fraction of AUP sample #112 is recognized by the 2 nd antibody-HRP conjugate. In conclusion, Spa from the clinical sample (AUP sample #112) actively binds IgG which is a relevant activity in the defense against immunoglobulins and opsonization.