A novel initiation mechanism of death in Streptococcus pneumoniae induced by the human milk protein-lipid complex HAMLET and activated during physiological death

Abstract

To cause colonization or infection, most bacteria grow in biofilms where differentiation and death of subpopulations is critical for optimal survival of the whole population. However, little is known about initiation of bacterial death under physiological conditions. Membrane depolarization has been suggested, but never shown to be involved, due to the difficulty of performing such studies in bacteria and the paucity of information that exists regarding ion transport mechanisms in prokaryotes. In this study, we performed the first extensive investigation of ion transport and membrane depolarization in a bacterial system. We found that HAMLET, a human milk protein-lipid complex, kills Streptococcus pneumoniae (the pneumococcus) in a manner that shares features with activation of physiological death from starvation. Addition of HAMLET to pneumococci dissipated membrane polarity, but depolarization per se was not enough to trigger death. Rather, both HAMLET- and starvation-induced death of pneumococci specifically required a sodium-dependent calcium influx, as shown using calcium and sodium transport inhibitors. This mechanism was verified under low sodium conditions, and in the presence of ionomycin or monensin, which enhanced pneumococcal sensitivity to HAMLET- and starvation-induced death. Pneumococcal death was also inhibited by kinase inhibitors, and indicated the involvement of Ser/Thr kinases in these processes. The importance of this activation mechanism was made evident, as dysregulation and manipulation of physiological death was detrimental to biofilm formation, a hallmark of bacterial colonization. Overall, our findings provide novel information on the role of ion transport during bacterial death, with the potential to uncover future antimicrobial targets.

FIGURE 1.

HAMLET displays dose-dependent bactericidal activity against S. pneumoniae. S. pneumoniae D39ΔlytA cells were incubated in the presence of various concentrations of HAMLET. Bacterial viability was assessed and presented as log₁₀ CFU per ml of bacterial suspension, and represents the mean of three separate experiments, with error bars representing the S.D.

FIGURE 2.

Disruption of pneumococcal membrane polarity and integrity. Mid-log phase D39ΔlytA pneumococci were incubated with the fluorescent indicator dyes DiBAC₄(3) and propidium iodide (PI) concurrently to detect (A) membrane depolarization and (B) membrane rupture, respectively, by measuring fluorescence over time. HAMLET was added at time 0 (arrow). The detergent sodium deoxycholate (0.05%) was included as a positive control for both events. The results presented are from a representative experiment.

FIGURE 3.

HAMLET-induced depolarization is not sufficient to induce death. A, E. coli JM109 (ECOLI); B, S. aureus I7 (STAAU); and C, S. pneumoniae D39ΔlytA (STRPN) were incubated with the potential-sensitive indicator dye DiBAC₄(3) to monitor membrane depolarization by measuring fluorescence over time. HAMLET was added at time 0 (arrow), 250 μg/ml to the E. coli and S. aureus cultures, and 31 μg/ml to the pneumococci. After 1 h, the untreated (Untr) and HAMLET-treated (HL) bacteria were serially diluted and viability was determined (D). Viability data represent the mean of three individual experiments, with S.D. bars, and significance calculated using the paired t test with a 95% confidence interval (* = p < 0.05).

FIGURE 4.

HAMLET induces K⁺ efflux that is not central to the bactericidal activity. A, the fluorescence of PBFI-loaded (K⁺-indicator) S. pneumoniae strain R36A was recorded to monitor concentrations of intracellular K⁺. After baseline fluorescence was recorded for 1 min, either 10 μm valinomycin (positive control) or HAMLET was added (arrow) to the bacteria and the resulting fluorescence was recorded every second for the next 4 min. Tracings from a representative experiment are shown. B, D39ΔlytA pneumococci were incubated with HAMLET for 1 h in the presence of K⁺-related transport inhibitors, while monitoring membrane depolarization (gray bars) and death (black bars), using DiBAC fluorescence and viable counts, respectively. Membrane depolarization and death in the inhibitor-treated samples are expressed as a percentage of the HAMLET-alone treated samples (HAMLET-alone value was set to 100%, represented by dotted line). C, PBFI fluorescence was monitored in R36A to measure the HAMLET-induced decrease in intracellular K⁺ in the presence of ion transport inhibitors. K⁺ efflux after 4 min is expressed as a percentage of that observed with HAMLET alone (dotted line, 100% K⁺ efflux). The data in panels B and C represent the mean ± S.D. of three individual experiments with error bars, and significance was calculated using the paired t test with a 95% confidence interval (* = p < 0.05; ** = p < 0.01). Inhibitors: high-K⁺ PBS buffer (HKPBS; 60 mm extracellular K⁺), gadolinium (GAD), and RuR.

FIGURE 5.

HAMLET triggers influx of Ca²⁺ that is critical to its bactericidal mechanism. A, R36A pneumococci were loaded with the Ca²⁺-sensitive dye Fura-2, and B, D39ΔlytA pneumococci were incubated with the radioisotope ⁴⁵Ca²⁺ (2.5 μCi/ml). After recording baseline readings, PBS (untreated), the Ca²⁺ ionophore ionomycin (1 μm in panel A and 10 μm in panel B), or HAMLET were added (arrow) to the bacteria and fluorescence or radioactivity was measured over time. Results from a representative experiment are shown for each. B, inset, HAMLET-induced ⁴⁵Ca²⁺ uptake was recorded over 10 min, demonstrating “overshoot.” C, D39ΔlytA pneumococci were incubated with HAMLET for 1 h in the presence of RuR, nifedipine (NIF), verapamil (VPL), or sodium orthovanadate (SOV), while monitoring membrane depolarization (gray bars) and death (black bars), and were expressed as a percentage of the HAMLET-alone treated samples (HAMLET-alone value was set to 100%, represented by dotted line). The effect of ion transport inhibitors on HAMLET-induced Ca²⁺ uptake was assessed by monitoring (D) fluorescence (light blue bars) or ⁴⁵Ca²⁺ uptake (dark blue bars) after 5 min of treatment. Results are expressed as the percentage of HAMLET-alone treated samples (dotted line, 100% Ca²⁺ influx). E, D39ΔlytA pneumococci were incubated with HAMLET for 1 h in the absence or presence of 2.5 μm ionomycin and viability was determined. F, D39ΔlytA pneumococci were incubated with HAMLET for 1 h in the presence of amiloride (Amil) or DCB, while monitoring depolarization (gray bars) and death (black bars). Results are presented as in panel C. Panels C–F, data represent the mean of three individual experiments, with S.D. error bars, and significance was calculated using the paired t test with a 95% confidence interval (* = p < 0.05; ** = p < 0.01).

FIGURE 6.

HAMLET-induced ⁴⁵Ca²⁺ uptake and death are sodium-dependent. A, graphical representation of the pneumococcal NCX, which transports Na⁺ out of the pneumococcus against its electrochemical potential gradient while bringing Ca²⁺ into the cell in the direction of its substantial gradient. Using the radioisotope ⁴⁵Ca²⁺, HAMLET-induced Ca²⁺ uptake in D39ΔlytA pneumococci was recorded in the presence of (B) the Na⁺ ionophore monensin and (C) conditions of low extracellular Na⁺ (PBS with 25% of standard Na⁺ levels; PBS(25%)). cpm from representative experiments are shown for each panel. D, after 1 h of HAMLET treatment in both of these conditions, the bacteria were serially diluted for viable counts, and the log₁₀ of HAMLET-induced death were calculated. The viability data represent three individual experiments, with S.D. indicated with error bars, and significance calculated using the paired t test with a 95% confidence interval (* = p < 0.05; ** = p < 0.01).

FIGURE 7.

Inhibition of kinase activity protects pneumococci from HAMLET-induced death. S. pneumoniae D39 were incubated with HAMLET in the absence or presence of the protein kinase inhibitor staurosporine (20 μm). After 1 h, cells were serially diluted and plated for viable counts, which are presented as log₁₀ of HAMLET-induced death. The data represent the mean ± S.D. of three individual experiments with error bars, and significance was calculated using the paired t test with a 95% confidence interval (** = p < 0.01).

FIGURE 8.

Augmenting sensitivity to the natural pneumococcal death pathway autolysis. A, pneumococci were grown to stationary phase, at which point 1× PBS (Untreated), 10 μm ionomycin, or 100 μm monensin was added to samples of the culture, with the A_{600 nm} subsequently plotted over time. B, pneumococci were grown from lag phase in the presence of broth alone (Untreated), 30 μm RuR or 15 μm DCB, with the A_{600 nm} after late stationary phase displayed in the graph. C, pneumococci were grown from lag phase in the absence (Untreated) or presence of 20 μm staurosporine, with the A_{600 nm} after late stationary phase displayed in the graph.

FIGURE 9.

Inhibitors of autolysis limit pneumococcal biofilm formation. D39 was seeded into biofilms over a prefixed substratum of NCI-H292 cells in chemically defined medium. At each media change, PBS (negative control), 30 μm RuR, or 100 μm DCB was added so that it was present throughout the 48-h growth period. The data represent the total biomass of three individual biofilms per sample after 48 h of incubation, with S.D. indicated with error bars and significance calculated using the paired t test with a 95% confidence interval (* = p < 0.05; ** = p < 0.01).

FIGURE 10.

Summary model of membrane changes, ion transport events, and kinase activation during HAMLET-induced pneumococcal death. HAMLET encounters S. pneumoniae and triggers membrane perturbations including depolarization (detected by monitoring DiBAC fluorescence) and loss of integrity (propidium iodide fluorescence), with depolarization not sufficient for inducing death. Specific ion fluxes that are induced include an efflux of K⁺ (PBFI fluorescence) and an influx of Ca²⁺ (Fura-2 and Fluo-4 fluorescence; ⁴⁵Ca²⁺ uptake). Although the efflux of K⁺ does not appear to be required for the bactericidal activity of HAMLET, the influx of Ca²⁺ does, and is tightly linked to sodium gradients, suggesting a sodium-calcium exchange mechanism. These HAMLET-induced transport events and death can be modulated by introducing related channel inhibitors or altering ion gradients. Additionally, staurosporine, the calcium-related kinase inhibitor, blocks bactericidal activity of HAMLET.