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. 2021 Dec 8;13(12):2121.
doi: 10.3390/pharmaceutics13122121.

Antiviral Activity of the Rhamnolipids Mixture from the Antarctic Bacterium Pseudomonas gessardii M15 against Herpes Simplex Viruses and Coronaviruses

Rosa Giugliano  1 , Carmine Buonocore  2   3 , Carla Zannella  1 , Annalisa Chianese  1 , Fortunato Palma Esposito  2 , Pietro Tedesco  2 , Anna De Filippis  1 , Massimiliano Galdiero  1 , Gianluigi Franci  4 , Donatella de Pascale  2   3
Affiliations

Antiviral Activity of the Rhamnolipids Mixture from the Antarctic Bacterium Pseudomonas gessardii M15 against Herpes Simplex Viruses and Coronaviruses

Rosa Giugliano et al. Pharmaceutics. .

Abstract

Emerging and re-emerging viruses represent a serious threat to human health at a global level. In particular, enveloped viruses are one of the main causes of viral outbreaks, as recently demonstrated by SARS-CoV-2. An effective strategy to counteract these viruses could be to target the envelope by using surface-active compounds. Rhamnolipids (RLs) are microbial biosurfactants displaying a wide range of bioactivities, such as antibacterial, antifungal and antibiofilm, among others. Being of microbial origin, they are environmentally-friendly, biodegradable, and less toxic than synthetic surfactants. In this work, we explored the antiviral activity of the rhamnolipids mixture (M15RL) produced by the Antarctic bacteria Pseudomonas gessardii M15 against viruses belonging to Coronaviridae and Herpesviridae families. In addition, we investigated the rhamnolipids' mode of action and the possibility of inactivating viruses on treated surfaces. Our results show complete inactivation of HSV-1 and HSV-2 by M15RLs at 6 µg/mL, and of HCoV-229E and SARS-CoV-2 at 25 and 50 µg/mL, respectively. Concerning activity against HCoV-OC43, 80% inhibition of cytopathic effect was recorded, while no activity against naked Poliovirus Type 1 (PV-1) was detectable, suggesting that the antiviral action is mainly directed towards the envelope. In conclusion, we report a significant activity of M15RL against enveloped viruses and demonstrated for the first time the antiviral effect of rhamnolipids against SARS-CoV-2.

Keywords: Antarctic bacteria; SARS-CoV-2; TEM; antiviral; coronavirus; enveloped virus; herpes; microbial biosurfactants; rhamnolipids.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Antiviral activity of M15RL, presented as percentage of inhibition, against (a) HSV-1 and (b) HCoV-229E in the co-treatment assay (cells treated with virus and M15RL at the same time) and pre-treatment assay (virus treated with M15RL for 1 h and then titrated on cells). The inhibition percentage was calculated by comparing the number of plaques found in the presence of M15RL with respect to the untreated virus (CTRL–) (Formula (2)). The Greco extract [42] at 50 µg/mL was used as a positive control (CTRL+). Data are means of three independent experiments. Statistical analyses were determined by two-way ANOVA with Sidak’s test for multiple comparisons. Significances are referred to the negative control (CTRL–). ** p < 0.0021; **** p < 0.0001, ns (not significant).
Figure 2
Figure 2
Antiviral activity of M15RL against (a) HSV-2, (b) HCoV-OC43, (c) SARS-CoV-2, and (d) poliovirus PV-1 in the virus pre-treatment assay (virus treated with M15RL for 1h and then added on cells). The Greco extract [42] at 50 µg/mL was used as a positive control (CTRL+). Data are means of three independent experiments. Statistical analyses were determined by ANOVA with Dunnett’s test for multiple comparisons. Significances are referred to the negative control (CTRL–). *** p < 0.0002, **** p < 0.0001, ns (not significant).
Figure 3
Figure 3
Antiviral activity of M15RL against GFP-HSV-1. Plaques can be visualized in fluorescent (a) and RGB microscopy (d) in cells treated with 3 µg/mL. No plaques are present in (b) and (e) where cells have been treated with 6 µg/mL of M15RL, while (c) and (f) show untreated infected cells in fluorescence and RGB microscopy, respectively. (g) Quantification of mRNA levels of the spike proteins expressed in Vero cells infected by SARS-CoV-2. Data are presented as ratio between the reference (GAPDH) and target (S) genes. Statistical analyses were determined by ANOVA with Dunnett’s test for multiple comparisons.
Figure 4
Figure 4
The M15RL’s mode of action on HSV-1 was investigated through (a) attachment assay and (b) expression levels of UL52, an early gene coding for DNA primase, and UL27, a late gene coding for glycoprotein B, respectively. Data are means of three independent experiments. Statistical analyses were determined by ANOVA with Dunnett’s test for multiple comparisons. Significances are referred to the negative control (CTRL–). **** p < 0.0001, ns (not significant).
Figure 5
Figure 5
Transmission electron microscopy images of HSV-1 (a) and (b), and SARS-CoV-2 (c) and (d) virions. In (a), red arrows show untreated HSV-1 viral particles surrounded by the envelope (white ring) and tegument. The tegument represents the amorphous space between the nucleocapsid (black) and the envelope. In (c), spike proteins of untreated SARS-CoV-2 virions are indicated by yellow arrows. In (b) and (d), white arrows indicate naked viral particles of HSV-1 and SARS-CoV-2, respectively, treated with 50 µg/mL of M15RL. Green arrows indicate SARS-CoV-2 particles without S proteins. The black squares in (c) and (d) represent a magnification of a virion in the respective conditions.
Figure 6
Figure 6
Virus inactivation on surfaces treated with M15RL. Wells bottom surfaces of a 12-well plate were coated with different quantities of M15RL. Afterward, 104 PFU of HSV-1 (a) and HCoV-229E (b) were put in touch with the treated surfaces for 5 min, then the contact was interrupted, and the viruses were titrated on cells to test the virulence through plaque reduction assay. Data are means of three independent experiments. Statistical analyses were determined by ANOVA with Dunnett’s test for multiple comparisons. Significances are referred to the negative control (Untreated). ** p < 0.0002, **** p < 0.0001, ns (not significant).
Figure 7
Figure 7
Cell viability evaluation by MTT assay on (a) Vero and (b) HaCaT cells after treatment with rhamnolipids for 24 h. 100 µL of DMSO was used as negative control (CTRL–). Data are means of three independent experiments. Statistical analyses were determined by one-way ANOVA with Dunnett’s test for multiple comparisons. Significances are referred to the untreated cells. **** p < 0.0001, ns (not significant).
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