Extraintestinal pathogenic Escherichia coli survives within neutrophils

Abstract

Extracellular pathogenic Escherichia coli (ExPEC) strains are common causes of a variety of clinical syndromes, including urinary tract infections, abdominal infections, nosocomial pneumonia, neonatal meningitis, and sepsis. ExPEC strains are extracellular bacterial pathogens; therefore, the innate immune response (e.g., professional phagocytes) plays a crucial role in the host defense against them. Studies using the model ExPEC strain CP9 demonstrated that it is relatively resistant to neutrophil-mediated bactericidal activity. Although this could be due to resistance to phagocytosis, the ability of CP9 to survive the intracellular killing mechanisms of neutrophils is another possibility. Using a variation of the intracellular invasion assay, we studied the survival of CP9 within peripheral blood-derived human neutrophils. Our results indicated that CP9 did survive within human neutrophils, but we were unable to demonstrate that intracellular replication occurred. This finding was not unique to CP9, since when a conservative assessment of survival was used, four of six additional ExPEC strains, but not an E. coli laboratory strain, were also capable of survival within neutrophils. Initial studies in which we began to decipher the mechanisms by which CP9 is able to successfully survive intracellular neutrophil-mediated bactericidal activity demonstrated that CP9 was at least partially susceptible to the neutrophil oxidative burst. Therefore, absolute resistance to the oxidative burst is not a mechanism by which ExPEC survives within neutrophils. In addition, electron microscopy studies showed that CP9 appeared to be present in phagosomes within neutrophils. Therefore, avoidance of phagosomal uptake or subsequent escape from the phagosome does not appear to be a mechanism that contributes to CP9's survival. These findings suggest that survival of ExPEC within neutrophils may be an important virulence mechanism.

FIG. 1.

Flow cytometric analysis to determine the efficacy of cytochalasin D inhibition of phagocytosis. Purified neutrophils that were or were not pretreated with 20 μM cytochalasin D for 10 min at 37°C were or were not mixed with fluorescein isothiocyanate-labeled zymosan particles for 60 min at 37°C and then quenched for 1 min with 0.4% trypan blue, and samples were subjected to flow cytometric analysis. (A) Quenched neutrophils not pretreated with cytochalasin D and not exposed to zymosan particles. (B) Quenched neutrophils not pretreated with cytochalasin D and exposed to zymosan particles. (C) Quenched neutrophils pretreated with cytochalasin D and not exposed to zymosan particles. (D) Quenched neutrophils pretreated with cytochalasin D and exposed to zymosan particles. cyto. D, cytochalasin D.

FIG. 2.

Confirmation of the efficacy of DPI for inhibiting the neutrophil oxidative burst. A total of 1 × 10⁶ purified human neutrophils that were either not treated or pretreated with DPI were stimulated with various reagents. The generation of reactive oxygen species was measured by flow cytometric analysis using a Phagoburst assay kit. (Top left panel) Neutrophils only, autofluorescence. (Top right panel) Neutrophils exposed to only DMSO (DPI diluent). (Middle left panel) Neutrophils exposed to phorbol 12-myristate 13-acetate (PMA). (Middle right panel) Neutrophils pretreated with DPI and exposed to phorbol 12-myristate 13-acetate. Inhibition is expressed as the percent decrease compared with the untreated sample (middle left panel). (Bottom left panel) Neutrophils exposed to CP9. (Bottom right panel) Neutrophils pretreated with DPI and exposed to CP9. Inhibition is expressed as the percent decrease compared with the untreated sample (bottom left panel).

FIG. 3.

Neutrophil-mediated bactericidal activity against ExPEC strain CP9. Approximately 1 × 10³, 1 × 10⁴, and 1 × 10⁵ CFU of CP9 in 1 ml of 1× PBS containing 10% heat-inactivated autologous plasma, 1% anti-CP9 rabbit polyclonal antibody, and 1% active autologous plasma were incubated with and without 5 × 10⁵ human neutrophils isolated from peripheral blood. Samples were mixed with a Nutator at 37°C, and at 0, 30, 60, and 90 min aliquots were removed and bacterial titers were determined. CP9 survival over time in the presence and absence of neutrophils is shown for each titer.

FIG. 4.

Survival of ExPEC strain CP9 within human neutrophils. The standard assay for measuring intracellular survival was used (see Materials and Methods). The level of intracellular survival was calculated by determining the difference between the bacterial titer obtained for untreated neutrophils and the bacterial titer obtained for cytochalasin D-pretreated neutrophils. (A) Bacterial titers obtained for neutrophils that were pretreated (cytochalasin D-pretreated neutrophils) and were not pretreated (untreated neutrophils) with cytochalasin D. (B) Calculated levels of survival for various titers of CP9.

FIG. 5.

Survival of ExPEC strain CP9 within human neutrophils over time. The standard assay for measuring survival of E. coli within neutrophils over time was used (see Materials and Methods). (A) Calculated levels of survival of CP9, obtained by determining the difference between the bacterial titer obtained for untreated neutrophils (PMNs plus CP9, not pretreated) and the bacterial titer obtained for cytochalasin D-pretreated neutrophils (PMNs plus CP9, pretreated). (B) Multisizer Coulter Counter readings for neutrophils over time, obtained concomitant with the intracellular survival assay. PMNs-no bacteria represents neutrophils that were incubated at 37°C but not exposed to CP9 or cytochalasin D.

FIG. 6.

Intracellular survival of additional E. coli strains. The standard intracellular survival assay was performed with ExPEC strains CP9 (O4:K54), 743 (O6:K2), 470 (O25:K5), K1/Y (O7:K1), 104 (unknown serotype), IA2 (O4:K12), and CFT073 (O6:K2) and laboratory strain HB101, as described in Materials and Methods. The levels of intracellular survival were calculated by determining the difference between the bacterial titer obtained for untreated neutrophils and the bacterial titer obtained for cytochalasin D-pretreated neutrophils.

FIG. 7.

Survival of ExPEC strain CP9 within neutrophils in which the oxidative burst was inhibited. The standard assay for measuring survival of E. coli within neutrophils in which the oxidative burst has been inhibited was used (see Materials and Methods). Neutrophils were (i) not treated, (ii) pretreated with the NADPH oxidase inhibitor DPI, (iii) pretreated with cytochalasin D, and (iv) pretreated with cytochalasin D and DPI. The levels of intracellular survival were calculated by determining the difference between the bacterial titer obtained for untreated neutrophils and the bacterial titer obtained for cytochalasin D-pretreated neutrophils and the difference between the bacterial titer obtained for DPI-pretreated neutrophils and the bacterial titer obtained for DPI- and cytochalasin D-pretreated neutrophils. The asterisk indicates that the P value was <0.05, as determined by an unpaired t test.

FIG. 8.

Visualization of ExPEC strain CP9 within neutrophils by TEM. TEM was performed with neutrophils from a standard assay to measure survival of E. coli as described in Materials and Methods. Both images were obtained from the same sample. (A) CP9 in a “spacious” phagosome. (B) CP9 in a “tight” phagosome.

FIG. 9.

Effects of macropinocytosis inhibitors on the survival of ExPEC strain CP9 within neutrophils. Purified human neutrophils were not treated or were pretreated with either cytochalasin D (20 μM), amiloride (3 mM), dimethyl amiloride (500 μM), rottlerin (2 μM), a combination of cytochalasin D and amiloride, or a combination of cytochalasin D and dimethyl amiloride (DMA) for 15 min before the intracellular survival assay was performed. The intracellular survival assay was performed as described in the text.