Phloretin Attenuates Listeria monocytogenes Virulence Both In vitro and In vivo by Simultaneously Targeting Listeriolysin O and Sortase A

Abstract

The critical roles of sortase A (SrtA) and listeriolysin O (LLO) in Listeria monocytogenes pathogenicity render these two virulence factors as ideal targets for the development of anti-virulence agents against L. monocytogenes infection. Additionally, the structures of SrtA and LLO are highly conserved among the members of sortase enzyme family and cholesterol dependent toxin family. Here, phloretin, a natural polyphenolic compound derived from apples and pears that has little anti-L. monocytogenes activity, was identified to simultaneously inhibit LLO expression and neutralize SrtA catalytic activity. Phloretin neutralized SrtA activity by causing a conformational change in the protein's active pocket, which prevented engagement with its substrate. Treatment with phloretin simultaneously reduced L. monocytogenes invasion into host cells and blocked the escape of vacuole-entrapped L. monocytogenes into cytoplasm. Further, L. monocytogenes-infected mice that received phloretin showed lower mortality, decreased bacterial burden and reduced pathological injury. Our results demonstrate that phloretin is a promising anti-infective therapeutic for infections caused by L. monocytogenes due to its simultaneous targeting of SrtA and LLO, which may result in fewer side effects than those caused by other antibiotics.

Keywords: Listeria monocytogenes; anti-infective therapy; listeriolysin O; phloretin; sortase A.

Figure 1

Phloretin inhibits LLO expression in L. monocytogenes. (A) The chemical structure of phloretin. (B) The effect of phloretin on the hemolytic activities of bacterial culture supernatants. Inhibition of hemolytic activity in culture supernatants of L. monocytogenes after co-culture with phloretin. L. monocytogenes was co-cultured with various concentrations of phloretin until reaching a stationary phase, and the hemolytic activities of the culture supernatants from each sample were determined using hemolysis assays. However, no inhibition of hemolytic activity for bacterial culture supernatants was observed following a direct pre-incubation with various concentrations of phloretin. (C) Western blotting analysis to detect LLO levels in culture supernatants and cell pellets. L. monocytogenes was co-cultured with various concentrations of phloretin until reaching a stationary phase, and the LLO level in the culture supernatants and cell pellets were determined by immunoblotting. Lanes 1–5, samples treated with 0, 7.30, 14.59, 29.17, and 58.34 μM phloretin. (D,E) No inhibition of hly transcription was produced by phloretin. Bacteria containing phly-lacZ plasmids were incubated with various concentrations of phloretin in TSB agar (D) or TSB (E). (D) Phloretin was added at increasing concentrations in wells 1–6 (0–116.68 μM); well 8 contained a 40% benzalkonium bromide solution; and well 9 contained a 60% benzalkonium bromide solution. (E) β-galactosidase activity was evaluated using a β-galactosidase staining kit and the values were obtained by comparing with data of phloretin-free samples (taken as 100%). ^*P < 0.05; ^**P < 0.01.

Figure 2

Phloretin inhibits SrtA catalytic activity in L. monocytogenes. (A) Inhibition of SrtA catalytic activity by phloretin. Purified SrtA was pre-incubated with phloretin at 37°C for 30 min and then mixed with the fluorescent peptide substrate Dabcyl-QALPTTGEE-Edans. Following incubation at 37°C for 1 h, the fluorescence values of the reaction system were determined using emission and excitation wavelengths of 350 and 520 nm, respectively. (B) Growth curve of L. monocytogenes in the presence of various concentrations of phloretin. (C) The effects of phloretin on the catalytic activities of WT-SrtA and SrtA mutants.

Figure 3

The 3D structure of SrtA in complex with phloretin determined by molecular modeling. (A) The 3D structure of the SrtA-phloretin complex. (B) The RMSD values of the backbone atoms of SrtA during MD simulations of the binding of SrtA to phloretin. (C) Decomposition of binding energy on a per-residue basis for the residues in the binding site of SrtA as the SrtA-phloretin complex formed. (D,E) Distances between Ala53-Ile114 and His55-Arg125 as a function of time. The black line (Ala53-Ile114 in the protein complex), red line (His55-Arg125 in the protein complex), green line (Ala53-Ile114 in free protein) and blue line (His55-Arg125 in free protein) represent the distances between the same residues.

Figure 4

Phloretin reduces L. monocytogenes invasion into Caco-2 cells. (A) Caco-2 cells were infected with phloretin (58.34 μM) pre-treated L. monocytogenes or L. monocytogenes with or without phloretin treatment (58.34 μM) during infection for 1 h, and the total number of viable bacteria in cells was counted after lysing the cells. (B) L. monocytogenes pre-cultured with or without phloretin (58.34 μM) were co-incubated with host cells at an MOI of 100 in the presence or absence of phloretin (58.34 μM). At 1 h post-infection, extracellular and total bacteria were stained with Alexa594-conjugated antibodies (red) and Alexa488-conjugated antibodies (green), respectively. Consistent with the samples infected with the EGDeΔsrtA strain, only the cells infected with phloretin-pre-treated L. monocytogenes showed reduced bacterial entry. Scale bar, 10 μm. ^*P < 0.05; ^**P < 0.01.

Figure 5

Phloretin simultaneously inhibits L. monocytogenes invasion into host cells and escape from internalization vacuoles. L. monocytogenes pre-cultured with or without phloretin (58.34 μM) were co-incubated with host cells at an MOI of 100 in the presence or absence of phloretin (58.34 μM). At 1, 3, and 5 h post-infection, F-actin in the host cells and bacteria was stained with Alexa Fluor 488-conjugated phalloidin antibodies (green) and Alexa Fluor 594-conjugated antibodies (red), respectively; nuclei were stained blue with DAPI. Infection with EGDe cells showed invasion of bacteria into host cells and bacterial escape from vacuoles, as indicated by the numerous bacteria (red) observed in the cells at 1 h post-infection and the fact that most bacteria (red) were surrounded by actin (green) at 5 h post-infection. Significant defects in host cell invasion and bacterial escape from vacuoles were observed for the samples infected with EGDeΔsrtA or EGDeΔhly cells. Note that phloretin treatment remarkably inhibited the escape of EGDe cells from vacuoles into cytoplasm, indicated by the fact that the majority of bacteria were not surrounded by F-actin. Importantly, significantly reduced bacterial invasion was observed for the samples infected with EGDe cells pre-treated with phloretin, but not samples infected with EGDe cells without phloretin pre-treatment. Scale bar, 10 μm.

Figure 6

Inhibition of cytosolic L. monocytogenes multiplication and bacteria-induced cytotoxicity by phloretin. (A) Caco-2 cells were infected with L. monocytogenes in the presence or absence of phloretin (58.34 μM). After 1, 3, and 5 h of infection, cells were lysed with 0.2% Triton X-100, and CFUs were counted on coverslips to determine the intracellular growth rates of the bacteria. (B) Cells were infected with phloretin-pre-treated L. monocytogenes at the indicated concentrations or non-pre-treated L. monocytogenes for 5 h, and LDH release was detected using a Cytotoxicity Detection Kit to evaluate the cytotoxicity induced by L. monocytogenes. ^*P < 0.05; ^**P < 0.01.

Figure 7

Phloretin protects mice from L. monocytogenes infection. Mice were intraperitoneally infected with L. monocytogenes and treated with phloretin or DMSO as a control. The mice were sacrificed 48 h post-infection to determine the bacterial burden and analyze the pathological damage resulting from infection. (A) Survival analysis of infected or uninfected mice treated with phloretin over a 96-h period. (B) Bacterial burdens in the livers and spleens of the infected mice. Treatment with phloretin alleviated the gross pathological changes (C) and histopathological changes (D) observed in the livers and spleens of the infected mice. Scale bar, 50 μm. ^*P < 0.05; ^**P < 0.01.