Human Salivary Protein Histatin 5 Has Potent Bactericidal Activity against ESKAPE Pathogens

Abstract

ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa, and Enterobacter species) pathogens have characteristic multiple-drug resistance and cause an increasing number of nosocomial infections worldwide. Peptide-based therapeutics to treat ESKAPE infections might be an alternative to conventional antibiotics. Histatin 5 (Hst 5) is a salivary cationic histidine-rich peptide produced only in humans and higher primates. It has high antifungal activity against Candida albicans through an energy-dependent, non-lytic process; but its bactericidal effects are less known. We found Hst 5 has bactericidal activity against S. aureus (60-70% killing) and A. baumannii (85-90% killing) in 10 and 100 mM sodium phosphate buffer (NaPB), while killing of >99% of P. aeruginosa, 60-80% E. cloacae and 20-60% of E. faecium was found in 10 mM NaPB. Hst 5 killed 60% of biofilm cells of P. aeruginosa, but had reduced activity against biofilms of S. aureus and A. baumannii. Hst 5 killed 20% of K. pneumonia biofilm cells but not planktonic cells. Binding and uptake studies using FITC-labeled Hst 5 showed E. faecium and E. cloacae killing required Hst 5 internalization and was energy dependent, while bactericidal activity was rapid against P. aeruginosa and A. baumannii suggesting membrane disruption. Hst 5-mediated killing of S. aureus was both non-lytic and energy independent. Additionally, we found that spermidine conjugated Hst 5 (Hst5-Spd) had improved killing activity against E. faecium, E. cloacae, and A. baumannii. Hst 5 or its derivative has antibacterial activity against five out of six ESKAPE pathogens and may be an alternative treatment for these infections.

Keywords: Candida albicans; ESKAPE; Histatin 5; antimicrobial peptide; bactericidal activity.

Figure 1

Killing of ESKAPE cells by Hst 5. E. faecium (A), S. aureus (B), K. pneumoniae (C), A. baumannii (D), P. aeruginosa (E), and E. cloacae (F) cells in exponential growth were exposed to 30 μM Hst 5 in 10 mM NaPB or 100 mM NaPB for 1 min, 1, and 5 h. Aliquots taken at different time points were diluted and plated. CFU were determined after 24 h. Error bars represent the standard errors from at least three independent replicates of each strain.

Figure 2

Antibacterial activity of Hst 5 against E. faecium required internalization and is energy dependent. (A) E. faecium cells were exposed to F-Hst 5 (30 μM) and PI (2 μg/mL). F-Hst 5 (green) and PI (red) uptake were measured in parallel by time-lapse confocal microscopy. Images were recorded every 10 min and selected images of indicated time points were shown. (Scale bar: 5 μm) (B) Quantitative analysis of F-Hst 5 uptake (green line) and PI uptake. Error bars represent the standard errors from four different fields of image. (C) Cells pretreated with 10 mM NaN₃ at 37°C for 3 h showed decreased susceptibility to Hst 5. (^*P < 0.05, Student's t-test).

Figure 3

Antibacterial activity of Hst 5 against E. cloacae requires internalization and is energy dependent. (A) E. cloacae cells were exposed to F-Hst 5 (30 μM) and PI (2 μg/mL). The F-Hst 5 (green) and PI (red) uptake were measured in parallel by time-lapse confocal microscopy. Images were recorded every 10 min and selected images as indicated time points were shown. (Scale bar: 5 μm) (B) Quantitative analysis of F-Hst 5 uptake (green line) and PI uptake. Error bars represent the standard errors from four different fields of image. (C) Cells pretreated with 10 mM NaN₃ at 37°C for 3 h showed more resistance to Hst 5. (^***P < 0.001, Student's t-test).

Figure 4

Hst 5 bactericidal activity against P. aeruginosa cells is primarily mediated by membrane disruption. (A) P. aeruginosa cells were exposed to F-Hst 5 (30 μM) and PI (2 μg/mL). F-Hst 5 (green) and PI (red) uptake were measured in parallel by time-lapse confocal microscopy. Images were recorded every 10 min and selected images at indicated time points were shown (Scale bar: 5 μm) (B) Quantitative analysis of F-Hst 5 uptake (green line) and PI uptake. Error bars represent the standard errors from four different fields of image. (C) Cells pretreated with 10 mM NaN₃ at 37°C for 3 h did not show significant difference in susceptibility to Hst 5.

Figure 5

Activity of Hst 5 against A. baumannii is mediated in part by membrane disruption. (A) A. baumannii cells in exponential phase were exposed to F-Hst 5 (30 μM) and PI (2 μg/mL). The F-Hst 5 (green) and PI (red) uptake were measured in parallel by time-lapse confocal microscopy. Images were recorded every 2 min and selected images as indicated time points were shown. (Arrow, cells of F-Hst 5 uptake positive but without PI uptake; Scale bar: 5 μm). (B) Quantitative analysis of F-Hst 5 uptake (green line) and PI uptake. Error bars represent the standard errors from four different fields of image. (C) Cells pretreated with 10 mM NaN₃ at 37°C for 3 h did not show significant difference in susceptibility to Hst 5.

Figure 6

The activity of Hst 5 against S. aureus is mediated by energy-independent mechanisms. (A) S. aureus cells were exposed to F-Hst 5 (30 μM) and PI (2 μg/mL). F-Hst 5 (green) and PI (red) uptake were measured in parallel by time-lapse confocal microscopy. Images were recorded every 10 min and selected images of indicated time points were shown. (Scale bar: 5 μm). (B) Quantitative analysis of F-Hst 5 uptake (green line) and PI uptake. Error bars represent the standard errors from four different fields of image. (C) Cells pretreated with 10 mM NaN₃ at 37°C for 3 h did not show significant difference in susceptibility to Hst 5.

Figure 7

Hst 5 is minimally active against K. pneumoniae. (A) K. pneumoniae cells in exponential phase were exposed to F-Hst 5 (30 μM) and PI (2 μg/mL). F-Hst 5 (green) and PI (red) uptake were measured in parallel by time-lapse confocal microscopy. Images were recorded every 10 min and selected images of indicated time points were shown. (Scale bar: 5 μm). (B) Quantitative analysis of F-Hst 5 uptake (green line) and PI uptake. Error bars represent the standard errors from four different fields of image.

Figure 8

Killing of ESKAPE pathogens by Hst 5-Spd. E. faecium (A), S. aureus (B), K. pneumoniae (C), A. baumannii (D), P. aeruginosa (E), and E. cloacae (F) cells in exponential growth were exposed to 30 μM of Hst 5, F-Hst 5 and spermidine in 10 mM NaPB for 1 min, 1, and 5 h. Aliquots taken at different time points were diluted and plated. CFU were determined after 24 h. Error bars represent the standard errors from at least three independent replicates of each strain. Hst 5-Spd conjugate showed more killing efficiency against E. faecium (A), A. baumannii (D), and E. cloacae (F) (^**P < 0.01, ^***P < 0.001, Student's t-test).