Peptidoglycan-Associated Cyclic Lipopeptide Disrupts Viral Infectivity - PubMed (original) (raw)

. 2019 Oct 29;93(22):e01282-19.

doi: 10.1128/JVI.01282-19. Print 2019 Nov 15.

Adam Hage 1, Birte Kalveram 2, Megan Mears 2, Jessica A Plante 3 1, Sergio E Rodriguez 1, Zhixia Ding 2 4, Xuemei Luo 5 6, Dennis Bente 1, Shelton S Bradrick 5, Alexander N Freiberg 2, Vsevolod Popov 2 4, Ricardo Rajsbaum 1 7, Shannan Rossi 1 2, William K Russell 5 6, Vineet D Menachery 8 7

Affiliations

Peptidoglycan-Associated Cyclic Lipopeptide Disrupts Viral Infectivity

Bryan A Johnson et al. J Virol. 2019.

Abstract

Enteric viruses exploit bacterial components, including lipopolysaccharides (LPS) and peptidoglycan (PG), to facilitate infection in humans. Because of their origin in the bat enteric system, we wondered if severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome CoV (MERS-CoV) also use bacterial components to modulate infectivity. To test this question, we incubated CoVs with LPS and PG and evaluated infectivity, finding no change following LPS treatment. However, PG from Bacillus subtilis reduced infection >10,000-fold, while PG from other bacterial species failed to recapitulate this. Treatment with an alcohol solvent transferred inhibitory activity to the wash, and mass spectrometry revealed surfactin, a cyclic lipopeptide antibiotic, as the inhibitory compound. This antibiotic had robust dose- and temperature-dependent inhibition of CoV infectivity. Mechanistic studies indicated that surfactin disrupts CoV virion integrity, and surfactin treatment of the virus inoculum ablated infection in vivo Finally, similar cyclic lipopeptides had no effect on CoV infectivity, and the inhibitory effect of surfactin extended broadly to enveloped viruses, including influenza, Ebola, Zika, Nipah, chikungunya, Una, Mayaro, Dugbe, and Crimean-Congo hemorrhagic fever viruses. Overall, our results indicate that peptidoglycan-associated surfactin has broad viricidal activity and suggest that bacteria by-products may negatively modulate virus infection.IMPORTANCE In this article, we consider a role for bacteria in shaping coronavirus infection. Taking cues from studies of enteric viruses, we initially investigated how bacterial surface components might improve CoV infection. Instead, we found that peptidoglycan-associated surfactin is a potent viricidal compound that disrupts virion integrity with broad activity against enveloped viruses. Our results indicate that interactions with commensal bacterial may improve or disrupt viral infections, highlighting the importance of understanding these microbial interactions and their implications for viral pathogenesis and treatment.

Keywords: MERS-CoV; SARS-CoV; coronavirus; cyclic lipopeptide; microbiome; surfactin.

Copyright © 2019 American Society for Microbiology.

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Figures

FIG 1

FIG 1

Peptidoglycan from Bacillus subtilis reduces coronavirus infectivity. (A) Bacterial envelope components such as LPS are bound to CoVs, increasing their thermostability (right) relative to that of untreated samples (left). (B) Relative infectivity of HCoV-229E (n = 4) and MERS-CoV (n = 5) after treatment with PBS alone (black), 1 mg/ml LPS from E. coli (gray), or 1 mg/ml PG from B. subtilis (green) following 2 h of incubation at 37°C. (C) HCoV-299E (circles) and MERS-CoV (triangles) infectivity after treatment for 2 h at 37°C with peptidoglycan from the indicated bacterial species (n = 3). (D) HCoV-229E and (E) MERS-CoV after treatment with 1 mg/ml PG from B. subtilis at room temperature (RT), 32°C, and 37°C (n = 3). For all dot plots, the centered bar represents the group mean, while the error bars represent standard deviation (SD). P values are based on the two-tailed Student’s t test, indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 2

FIG 2

Identification of surfactin from B. subtilis peptidoglycan. (A and B) PG from B. subtilis in a PBS solution was clarified, washed with the indicated solvents, and clarified again. Supernatants were decanted and retained, while the insoluble fractions were resuspended in PBS. The (A) insoluble fraction and (B) supernatants were then used to treat HCoV-229E, and relative infectivity was determined (n = 3). (C, D, and E) Mass spectrometry was performed on PG (C) and ethanol wash (D). The peak corresponding to the molecular mass of 1,058 kDa in the ethanol wash was then further fragmented (E) to determine the identity of the molecule. Representative spectra are shown (n = 3). For all dot plots, the centered bar represents the group mean and error bars the SD.

FIG 3

FIG 3

Characterization of CoV inhibition by surfactin. (A) HCoV-229E, (B) MERS-CoV, and (C) SARS-CoV (MA15) were treated with PBS alone (black) or 100 μg/ml surfactin (red) and at room temperature (RT), 32°C, and 37°C, and infectivity was determined (n = 3). HCoV-229E (D) and MERS-CoV (E) were treated for the indicated time at 4°C (blue), RT (red), 32°C (green), or 37°C (purple), and infectivity was determined (n = 3). (F) HCoV-229E (blue), MERS-CoV (orange), and SARS-CoV MA15 (green) were diluted in concentrations ranging from 0.5 to 200 μg/ml, and viral infectivity was determined (n = 3). For dot plots, each point represents the titer from an independent experiment, while the group mean is indicated by a line. Each point on the line graph represents the group mean. All error bars represent SD. The two-tailed Student’s t test was used to determine P values, indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4

FIG 4

Surfactin disrupts CoV structural integrity. (A) HUH7 cells were first treated with either PBS or 100 μg/ml surfactin solution. Cells were then incubated with recombinant MERS-CoV S1-Fc protein and then stained with an anti-human IgG-FITC secondary in order to examine DPP4 levels. Unstained (red), PBS treated (blue), and surfactin treated (gold). Shown is a representative figure from 3 independent experiments. (B) MERS-CoV were treated with 100 μg/ml surfactin or PBS, as indicated, and then immunoblotted for MERS-CoV spike. Shown is a representative figure from 3 independent experiments. (C) HCoV-229E and (D) MERS-CoV were treated with 0 (ctrl) 10, 20, 30, 100, and 200 μg/ml of surfactin, as indicated. Viral infectivity was then determined (red) or samples were then treated with RNase I, RNA was extracted, and viral genome copy number determined by RT-qPCR (black). Bar graph bars represent the group mean for each experiment, and error bars represent SD (n = 3). (E to G) PBS- or 100 μg/ml surfactin-treated HCoV-229E samples were negatively stained and examined by TEM, and intact and disrupted virions were counted. A representative micrograph shown in panel E, with intact (i) and disrupted (ii) virions (left). Additional examples of intact and disrupted virions are also shown (right). Total counts of intact (F) and disrupted (G) virions are shown at various concentrations, with no less than 3 independent experiments having been performed for each concentration of surfactin. Horizontal lines represent group mean, while error bars represent SD. A two-tailed Student’s t test determined significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 5

FIG 5

In vivo characterization of surfactin treatment on SARS-CoV infection. (A and B) BALB/c mice were then intranasally infected with 104 PFU of PBS- (black) or 100 μg/ml surfactin (red)-treated SARS-CoV MA15 and (A) monitored for weight loss over 4 days. Dotted lines and triangles represent mock-infected animals receiving PBS alone (black) or surfactin alone (red). (B) Lung tissue was harvested and viral titer determined at days 2 and 4. n = 4 for all infected groups, n = 2 for mock groups. (C and D) BALB/c mice were pretreated intranasally with 50 μl of either PBS (black) or surfactin in PBS (red). Eighteen h later, BALB/c mice were infected with 104 PFU of SARS-CoV (MA15) and (C) monitored for weight loss over 4 days. (D) Lung titer determined 2 (n = 5) and 4 days postinfection (n = 10). Dots on line graphs and bars on bar graphs represent the group mean. ND, no titers were detected. All error bars represent SD. P values were calculated using the two-tailed Student’s t test, with *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

FIG 6

FIG 6

Surfactin, but not other cyclic lipopeptides, broadly reduces the infectivity of enveloped viruses. (A) Biochemical models of each of the seven cyclic lipopeptides tested. The number of amino acids present in the cyclic ring is shown in parentheses. (B) HCoV-229E (blue) and MERS-CoV (gray) were treated with PBS or 100 μg/ml of the indicated cyclic lipopeptides in PBS and incubated for 2 h at 37°C. Viral infectivity was then determined (n = 3). (C) The indicated viruses were diluted in PBS (black) or 100 μg/ml of surfactin (red), incubated for 2 h at 37°C, and viral infectivity was determined (n = 3). Viruses are as follows: coxsackievirus (CVB3), Dugbe (DUGV), Crimean-Congo hemorrhagic fever virus (CCHFV), Zika virus (ZIKV), Nipah virus (NiV), chikungunya virus (CHIKV), Una virus, Mayaro virus, influenza A strains H1N1 and H3N2, and Ebola virus (EBOV). Bar graph bars represent the group means (n = 3). Error bars represent SD. ND, no titers were detected. The Student’s t test was used to calculate P values, with *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

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