Bioengineered version of Lactobacillus can prevent listeria infections

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For pregnant women, the elderly and those with weakened immune systems, listeriosis is a serious foodborne illness often linked to deli meats, fresh produce and dairy products.

Even with antibiotic treatment, listeriosis is fatal for about 20 percent of patients, resulting in thousands of deaths annually.

Purdue University’s Arun Bhunia, a professor of food science, and postdoctoral researcher Rishi Drolia have developed a probiotic that could prevent infections in at-risk populations. A bioengineered version of Lactobacillus, a bacterium common in the human gut, can block the pathway the Listeria monocytogenes bacteria use to cross intestinal wall cells into the bloodstream, his team reports in the journal Nature Communications.

“The Lactobacillus bacteria we developed seeks out the same proteins as Listeria monocytogenes in the gut. When it attaches, it blocks the roadway for Listeria,” Bhunia said.

“This could be included in probiotic yogurts, capsules or gummies and used as a preventive measure to treat people who are at high risk of infection.”

In a previous study, Bhunia’s and Drolia’s work showed how Listeria crosses the epithelial barrier, a wall of cells in the gut that generally protects the bloodstream from harmful pathogens.

A protein in Listeria, called Listeria adhesion protein (LAP), interacts with heat shock protein in those epithelial cells and forces the cells apart. That gives Listeria access to the bloodstream.

Bhunia’s team has now isolated the Listeria adhesion protein from nonpathogenic strains of the listeria bacterium and added it to the Lactobacillus to make a probiotic. When introduced to human gut cells and in mice, the Lactobacillus probiotic stably colonized the intestine and attached to epithelial cells on the heat shock protein.

When pathogenic Listeria was introduced, it wasn’t able to attach to those gut cells and invade the bloodstream.

“This novel approach of engineering a probiotic strain with an adhesion protein from a nonpathogenic bacterium to exclude a pathogen significantly enhances the prophylactic use of such bioengineered probiotic bacteria without raising serious health or regulatory concerns and, thus, their potential as preventive agents against listeriosis,” the authors wrote.

“This study, thus, provides the first and direct evidence that rational engineering of probiotic strains allows them to outcompete and diminish the colonization of the pathogens by competing for the receptor binding adhesion sites.”

The Lactobacillus probiotic could also have potential for other gut illnesses such as celiac disease or inflammatory bowel disease.

“We’ve seen evidence that the same proteins Listeria adheres to are overactive in these other illnesses,” Drolia said.

“The probiotic has an anti-inflammatory effect and colonizes the gut for a week or more at a time. We’re interested in seeing how this could improve the lives of patients suffering from a variety of gut-related illnesses.”

Bhunia has applied for a patent for the bioengineered Lactobacillus probiotic and envisions licensing the technology.

“We’re also interested in using this model to look at other pathogenic bacteria such as E. coli or Salmonella,” Bhunia said. “If we can engineer bacteria properly, we may be able to use this technology to prevent a wider range of foodborne illnesses.”

Bioengineered probiotic could prevent Listeria infections
Bioengineered lactobacillus (red) interacts with surface heat shock protein 60 (white, yellow arrows), blocking Listeria monocytogenes (green, white arrows) from crossing into the bloodstream. Credit: Rishi Drolia and Arun Bhunia

Probiotic-Produced Bacteriocins for the Control of Microbial Infections

Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” (Hill et al., 2014). Most of the probiotics that have been reported and marketed to-date are LAB having the ability to inhibit certain pathogens at various body sites.

While some probiotics are targeted for animal use, the majority have principally been utilized to benefit human health via their effect on the composition of the gut microbiota (Hill et al., 2014). Applications include remediation of dysbiosis within the gut microbiota and the more specific treatment and prophylaxis of bacterial and fungal infections.

The mode of action of probiotics for the therapeutic treatment of bacterial and fungal infections is well documented (Stoyanova et al., 2012; Suvorov, 2013). There are, however, also reports indicating the successful application of probiotics in the treatment of various intestinal, respiratory, and urogenital diseases caused by viruses.

Such antiviral probiotics might be a preferable alternative to more conventional antiviral agents due to the escalating emergence of viruses that are resistant to commonly used antivirals (WHO, 2008). Suggested primary mechanisms of probiotic action against viruses include direct probiotic cell interaction with the targeted viruses, production of antiviral metabolites and modulation of the eukaryotic host’s immune system (Ichinohe et al., 2011; Wu et al., 2013; Al Kassaa et al., 2014; Drider et al., 2016).

Other possibilities range from modulation of the host microbiota to interaction with eukaryotic epithelial cells and effects on the electrolyte potential (Olaya Galán et al., 2016).

Virtually all bacteria, including the LAB probiotics, are known to produce ribosomally synthesized substances of proteinaceous nature that exhibit bactericidal activity. These substances are collectively called bacteriocins (Alvarez-Sieiro et al., 2016).

Many bacteriocins have been extensively studied, and some have been commercially developed due to their ability to preserve food and to exhibit therapeutic antimicrobial activity. Many bacteriocins are extremely thermostable and are active over a broad pH range. Furthermore, most bacteriocins are non-immunogenic, and are generally colorless, odorless, and tasteless.

These characteristics make bacteriocins particularly attractive for food preservation and health care applications (for reviews see: Alvarez-Sieiro et al., 2016). Due to the widespread emergence of resistance to most of the commonly used therapeutic antibiotics (Woolhouse et al., 2016), new classes of antimicrobial agents are desperately being sought. Tagg et al. (1976) have defined bacteriocins as peptides active against closely related bacteria. However, some of the more recently described bacteriocins are also active against relatively distantly related bacteria (Gupta et al., 2016; Belguesmia et al., 2020).

The sensitivity of a target bacterium to bacteriocins depends on the physico-chemical characteristics of the environment, of which pH, ionic strength, and the presence of neutralizing or membrane-disrupting molecules all play a major role (Belguesmia et al., 2010).

Probiotic bacteria often rely on bacteriocins to compete with other bacteria and to colonize a niche, such as in the gastrointestinal tract (Dobson et al., 2011a). While the intestinal microbiota comprises a dynamic community and plays an integral role in gut health, the bacteriocin thuricin CD inhibited the growth of Clostridium difficile in a colon model, without having a significant effect on the remainder of the microbiota (Rea et al., 2011).

Similarly, a bacteriocin produced by the probiotic strain Lactobacillus salivarius UCC118 protected mice against infection caused by Listeria monocytogenes (Corr et al., 2007). Other studies have shown that modulation of the gut microbiota by bacteriocins may lead to an increase in body mass (Murphy et al., 2013).

Metabolites such as lactic acid, hydrogen peroxide, and also bacteriocins produced by LAB, have been studied for their ability to decrease viral loads (Al Kassaa et al., 2014; Drider et al., 2016; Chikindas et al., 2018). Other studies have shown that bacteriocins may also play an important role in host defense and cell signaling (Cotter et al., 2005).

These antimicrobial proteins (AMPs) have a beneficial effect on the host microbiota and various organs of the body, and have shown promise in controlling potential pathogens (for reviews see: Drider et al., 2016; Chikindas et al., 2018). The antibacterial activity of bacteriocins is relatively well understood, while the basis for their antiviral activities has received far less attention and is only now being studied in depth.

Wachsman et al. (2000) reported that a peptide produced by Enterococcus faecium CRL35 inhibited the late stages of HSV-1 and HSV-2 replication (Wachsman et al., 2000). In addition, Serkedjieva et al. (2000) demonstrated that a 5.0 kDa-bacteriocin produced by Lactobacillus delbrueckii subsp. bulgaricus, highly specifically inhibited replication of influenza virus A/chicken/Germany, strain Weybridge (H7N7) and strain Rostock (H7N1).

Expression of the viral glycoproteins neuraminidase, hemagglutinin, and nucleoprotein on the surface of infected cells, virus-induced cytopathic effect, infectious virus yield, and hemagglutinin production were all reduced at concentrations of the peptide that were non-toxic for eukaryotic cells (Serkedjieva et al., 2000).

It would be interesting to determine if there is any relation between the antiviral activity of a bacteriocin and viral structure, e.g., naked versus enveloped. To the best of our knowledge, information on this relationship has not been published and any conclusions would be speculative. This knowledge gap merits further research.

Several other bacteriocins have been found to be active against viruses. Enterocin AAR-74 partially reduced viral replication, while enterocin AAR-71 and erwiniocin NA4 completely eliminated replication (Qureshi H. et al., 2006). Enterocins CRL35 and ST4V were active against strains of HSV-1 and HSV-2 in Vero and BHK-21 cells, inhibiting the late stages of viral replication (Wachsman et al., 1999; Todorov et al., 2005).

This antiviral activity of bacteriocins could be due to their blocking of receptor sites on host cells (Wachsman et al., 2003). Labyrinthopeptin A1 (LabyA1) is a carbacyclic lantibiotic which exhibited consistent and broad anti-HIV activity against cell-line adapted HIV-1 strains (Férir et al., 2013).

LabyA1 showed very consistent anti-HIV-1 activity against nine clinical isolates of HIV-1 and repressed intercellular transmission between HIV-infected T cells and uninfected CD4+ T cells (Férir et al., 2013). This inhibited the transmission of HIV from DC-SIGN+ cells to uninfected CD4+ T cells.

Synergistic anti-HIV-1 and anti-HSV-2 activity was confirmed using LabyA1 in dual combination with the antiretroviral agents raltegravir, acyclovir tenofovir, enfuvirtide, and saquinavir (Al Kassaa et al., 2014). Remarkably, labyrinthopeptins A1 and A2 were also able to inhibit the entry of the human respiratory syncytial virus (hRSV) into human carcinoma-derived lung cells in vitro (Haid et al., 2017).

Lange-Starke et al. (2014) demonstrated Inhibitory activity against murine norovirus (MNV) associated with a mixture of complex metabolites present in the cell-free supernatants (CFS) of L. curvatus 1. This bacterial strain-specific antiviral effect was subsequently confirmed in five trials (Botic et al., 2007).

Further studies were conducted to assess the antiviral properties of the CFS. Since no significant antiviral activity was present following heat treatment, it was suggested that the antiviral substance was of proteinaceous nature and possibly a bacteriocin. Potential interference by the active agent with virus replication or cell adsorption was suggested.

In another study, Serkedjieva et al. (2000) found that bacteriocin B1 from L. delbrueckii inhibits some intracellular virus replication steps. To date, no bacteriocin activity against SARS-CoV-2 has been reported, suggesting further exploration of this possibility is warranted.

Bacteriocin-Producing Probiotics for Combating/Prophylaxis of Viral Infections
Bacteriocins and Modulation of the Gut Microbiota

The assumed ability of bacteriocins to alter the gut microbiota by targeting detrimental components without having a negative influence on the beneficial microorganisms is an idealistic concept. The eukaryotic host is colonized by trillions of microbes in symbiotic association, and some of these microbes have the potential to become pathogenic during dysbiosis (Turnbaugh et al., 2007; Round and Mazmanian, 2009).

The disruption of the intestinal microbiota due to antibiotic treatment can trigger pathogenic behavior in some bacteria, which may result in their overgrowth within the host’s commensal microbiota. Several reports have described a high incidence of bacteriocin production among enterococci.

A large percentage of enterococci isolated from stool specimens do produce bacteriocins. Enterococci isolated from infections also frequently produce bacteriocins, such as bacteriocins 31 and 41 from Enterococcus faecalis (Tomita et al., 1996, 2008) and bacteriocins 43 (Todokoro et al., 2006), 32 (Nes et al., 1996), and 51 (Yamashita et al., 2011) from E. faecium.

Kommineni et al. (2015) suggested that bacteriocins produced by enterococci play a role in colonization of the GI tract. Bacteriocin production may enhance the stability of enterococcal communities, fostering their competition with closely related species and promoting the growth of the bacteriocin-producing strains (Corr et al., 2007; Hibbing et al., 2010).

It was found that E. faecalis producing bacteriocin 21 were able to better colonize the mouse GI tract than were bacteriocin non-producers. To test if bacteriocin-21, encoded by genes on plasmid pPD1, facilitates colonization by enterococci, the authors introduced an in-frame deletion of bacAB into pPD1.

The bacteriocin-depleted mutant lost its colonization advantage over wild-type E. faecalis. Colonization experiments with a bacteriocin 21 producer showed it to become more abundant in the feces and throughout the GI tract, suggesting more effective colonization (Kommineni et al., 2015).

The expression of bacteriocins by commensal bacteria may have a beneficial effect on their competition for niches within the GI tract. Bacteriocins produced by commensal strains in a specific niche may thereby also have a therapeutic role and may prevent the growth of multidrug-resistant bacteria without disrupting the indigenous microbiota.

The introduction of bacteriocin-producing commensal strains to the GI tract could be a method for the removal of antibiotic-resistant enterococci from the GI tract of patients and may also help in preventing the re-emergence of enteric infections (Kommineni et al., 2015).

The majority of studies to date have shown that bacteriocin-producing strains have the ability to inhibit the proliferation of well-established gut pathogens. Bacteriocins may thus be used to either prevent or treat infections (Hegarty et al., 2016).

Among other significant commensals, LAB play a vital role in keeping the human and animal gut microbiome in a balanced state (Gillor et al., 2008; Dicks and Botes, 2010; Mackowiak, 2013) and in maintaining the integrity of the gut wall (for reviews see Bajaj et al., 2015; Dicks et al., 2018). Other probiotic attributes include stimulation of the immune system, reduction of lactose, production of vitamins (B and C), prevention of colon cancer and Crohn’s disease, and reduction of cholesterol (reviewed by Dicks and Botes, 2010).

Probiotic LAB produce a wide variety of antimicrobial compounds ranging from the metabolites hydrogen peroxide, short-chain fatty acids, and lactic acid to bacteriocin-like inhibitory substances (BLIS) and bacteriocins (Dobson et al., 2011a). In this review, we are focused primarily on LAB probiotics and their bacteriocins, although it is commonly accepted that virtually all bacteria produce bacteriocins as defensive weaponry and as communication signals. Despite the many papers published on bacteriocin structures, functions, and food applications, unforgivably little research has been devoted to the medicinal properties of these peptides, including their activity against eukaryotic viruses, a feature most certainly of relevance for health promotion (Dicks et al., 2011; Chikindas et al., 2018).

The reason for this is largely due to the perception that bacteriocins have limited stability in the GIT, serum, liver, and kidneys (Joerger, 2003; McGregor, 2008). Although bacteriocins are typically degraded by proteolytic enzymes, some exceptions to the rule have been reported. When nisin F was injected into the peritoneal cavity of mice, it remained active against Staphylococcus aureus (in vivo) for 15 min (Brand et al., 2010). Nisin A (Bartoloni et al., 2004) and mutacin B-Ny266 (Mota-Meira et al., 2000) survived conditions in the peritoneal cavity, albeit for short periods.

Microbisporicin remained active for a few minutes when intravenously administered to mice (Castiglione et al., 2008). Lacticin 3147 prevented the systemic spread of S. aureus Xen 29 in mice and thuricin CD, a two-peptide bacteriocin produced by Bacillus thuringiensis 6431 (although not a LAB), inhibited the growth of Clostridium difficile in vivo (Rea et al., 2010).

When ingested, nisin F had a stabilizing effect on the bacterial population in the murine GIT (Van Staden et al., 2011). Enterococcus mundtii produced bacteriocin ST4SA when cells were exposed to conditions simulating the GIT (Granger et al., 2008). Plantaricin 423, produced by Lactobacillus plantarum, was expressed when cells were exposed to simulated gastric fluid (Ramiah et al., 2007). Nisin F, produced by Lactococcus lactis, prevented respiratory tract and subcutaneous skin infections instigated by S. aureus (De Kwaadsteniet et al., 2009, 2010).

While on the subject of bacteriocins, it should be made clear that these proteinaceous, ribosomally synthesized antimicrobials of bacterial origin are significantly different from conventional therapeutic antibiotics (Weeks and Chikindas, 2019). Unlike antibiotics, strains sensitive to bacteriocins seldom develop resistance, and the mechanisms of resistance are different from those reported for antibiotics (for review see de Freire Bastos et al., 2015).

Strains that do develop resistance undergo major structural changes in their cell walls and cell membranes. L. monocytogenes developed resistance to nisin after altering the fatty acid and phospholipid composition in its cell membrane (Abee et al., 1995). S. aureus and Bacillus subtilis increased the d-alanyl ester and galactose in their cell walls (Peschel et al., 1999; Vadyvaloo et al., 2002; Chatterjee et al., 2005).

Some strains of S. aureus prevented nisin from entering the cell by developing a mutation in the nsaS (nisin susceptibility-associated sensor) (Blake et al., 2011; Collins et al., 2012). Other Gram-positive strains developed thicker cell walls, which prevented nisin from docking with lipid II (Mantovani and Russell, 2001). L. monocytogenes gained resistance to mesentericin Y105 by inactivating the rpoN gene that encodes the σ54 subunit of bacterial RNA polymerase (Robichon et al., 1997).

Some Bacillus spp. produced non-proteolytic nisin-inactivating enzymes that reduced dehydroalanine (Jarvis, 1967; Jarvis and Farr, 1971). Other forms of resistance described have been less structure-based, such as developing a requirement for Mg2+, Ca2+, Mn2+, and Ba2+ (Ming and Daeschel, 1993; Mazzotta and Montville, 1997; Crandall and Montville, 1998). Resistance may also be pH related. In an acidic environment, L. lactis repelled nisin by binding high concentrations of the peptide to its cell surface (Hasper et al., 2006).

Bacteriocins and Immune Modulation Against Viral Infections

Modulation of the immune system is commonly considered a valid approach to the prophylaxis of viral infections, including COVID-19 (for review, see Jayawardena et al., 2020). The immunomodulatory properties of LAB are well documented. Intestinal strains have the ability to alter the roles of dendritic cells (DC), monocytes/macrophages, and T and B lymphocytes, thereby enhancing the phagocytosis of pathogenic bacteria (Grasemann et al., 2007).

In vitro studies have shown that LAB induce the release of the pro-inflammatory cytokines TNF-α and IL-6, thus stimulating non-specific immunity (Isolauri et al., 2001). Lactobacillus rhamnosus GG increased the number of rotavirus-specific IgM secreting cells in infants who had been administered an oral rotavirus vaccine (Cross, 2002). Feeding mice with Lactobacillus casei Shirota prior to an influenza virus challenge protected the upper respiratory tract significantly (Cross, 2002).

Yogurt supplemented with Lactobacillus acidophilus, Bifidobacterium infantis, and Bifidobacterium bifidum enhanced mucosal and systemic IgA responses to cholera toxin in mice (Kaur et al., 2002). As expected, most of reports have focused on species of LAB in the human GIT. It would be interesting to determine if transient strains of LAB have the same effect on the immune system.

Surprisingly, only a few reports have been published on the immunomodulatory properties of bacteriocins (Brand et al., 2013; Kindrachuk et al., 2013). This is an important area that requires further investigation, especially since one of the first studies (Kindrachuk et al., 2013) had shown that the immunomodulatory properties of nisin are superior to that reported for the human cationic peptide LL-37 (Scott et al., 2002).

Nisin, gallidermin, and Pep5 induced the discharge of multiple chemokines at concentrations similar to those recorded for LL-37 (Scott et al., 2002). Mice pre-treated with nisin, and then infected with Escherichia coli and Salmonella typhimurium, showed a significant reduction in bacterial cell numbers compared to the control group (Kindrachuk et al., 2013).

Since nisin is inactive against Gram-negative bacteria, protection against these pathogens was ascribed to increased immunity. Contradictory findings were reported by Brand et al. (2013). These authors did not observe a noticeable immune response with continuous in vivo injection of nisin F into the peritoneal cavity of mice. T

he activity of interleukin-6, interleukin-10, and tumor necrosis factor was stimulated, irrespective of treatment with active or inactive nisin F. However, the overall immune response was not high enough to trigger an abnormal increase in antigenic immune reactions (Brand et al., 2013).

Begde et al. (2011) reported the toxic side effects of a sample containing a combination of nisins A and Z against human lymphocytes and neutrophils. The administration of Nisaplin (a commercial form of nisin A) to mice for 30 and 75 days resulted in an increase in CD4 and CD8 T-lymphocytes, but a decrease in B-lymphocytes (De Pablo et al., 1999).

However, no side effects were recorded after 100 days of administration. Enhanced phagocytic activity of peritoneal cells was observed after long-term administration of Nisaplin. In another study, treatment with nisin resulted in immunostimulation of head kidney macrophages in fish (Villamil et al., 2003). Nisin, purified by RP-HPLC and vaginally administered to rats, was not toxic to host cells (Gupta et al., 2008). Nisin, pediocin, and peptide AS-48 have all shown immunogenic properties in antibody studies (Maqueda et al., 1993; Suárez et al., 1996; Martinez et al., 1997).

Ancovenin, a cinnamycin-like lantibiotic, inhibits angiotensin I converting enzyme (ACE), which plays an important role in regulating blood pressure through the conversion of angiotensin I to angiotensin II (Kido et al., 1983). Cinnamycin-like lantibiotics also inactivated phospholipase A2 by sequestering phosphatidylethanolamine (the substrate for phospholipase A2), thereby indirectly mediating inflammatory responses (Märki et al., 1991; Zhao, 2011).

Phospholipase A2 plays a role in the release of arachidonic acid. The latter is oxidized to eicosanoids, such as prostaglandins and leukotrienes, serving as strong mediators of the immune system. Most higher eukaryotic species, such as mammals, have CAMPs (cationic antimicrobial peptides) interacting with the innate immune system.

These peptides are usually positively charged, relatively small, and hydrophobic, which are also typical characteristics of lantibiotics (Sahl et al., 2005). It is thus not surprising that many researchers are advocating the use of CAMPs (including bacteriocins) as an alternative to antibiotics, especially for the killing of multidrug-resistant strains (Behrens et al., 2017). Bacteriocins modulate interleukin production (in vivo) and trigger the production of CD4(+) and CD8(+) T cells (Malaczewska et al., 2019).

Modulation of the immune system can protect eukaryotic cells against viral infections, as observed in studies conducted on poultry (Yitbarek et al., 2018). Bacteriocin producing probiotic strains could thus protect humans and other animals against viral infections, including Covid-19.

Dreyer et al. (2019) were the first to show in vitro that bacteriocins can cross the gut-blood barrier (GBB). The authors showed that 85% of plantaricin 423, 75% of nisin, and 82% of bacST4SA (a class IIa bacteriocin produced by E. mundtii) migrated across a Caco-2 cell monolayer within 3 h. In the case of HUVEC cells, 93% plantaricin 423, 88% nisin, and 91% bacST4SA migrated across the cell monolayers within 3 h (Dreyer et al., 2019).

Further research has to be done to determine the rate at which bacteriocins (either active or inactive) cross the GBB if they have the ability to re-enter tissue cells and if so, accumulate in organs. Dreyer et al. (2019) concluded that the rate at which bacteriocins cross cell membranes most likely depends on the physiological and biochemical state of the membrane.

The authors have also shown that class IIa bacteriocins retained a higher level of antibacterial activity compared to class I bacteriocins (lantibiotics). More bacteriocins will have to be studied to confirm these findings. It would also be interesting to determine the effects that molecular size, the number of sulfide bridges (folding of the peptide), hydrophobicity, and charge have on the migration of bacteriocins across epithelial cell membranes.

Antiviral Properties of Probiotic Bacteriocins

The first reports of LAB inactivating viruses were published around 30 years ago. At that time, antiviral activity was mostly ascribed to the protein denaturing reactions of hydrogen peroxide and lactic acid produced by Lactobacillus spp., leading to the inactivation of the human immunodeficiency virus type 1 (HIV-1) and human simplex virus type 2 (HSV-2) (Martin et al., 1985; Klebanoff and Coombs, 1991; Tuyama et al., 2006; Conti et al., 2009).

In other studies, a non-protein cell wall component of Lactobacillus brevis reduced the replication of HSV-2 (Mastromarino et al., 2011). Probiotic strains of Lactobacillus paracasei, Lactobacillus paracasei subsp. rhamnosus, L. plantarum, and Lactobacillus reuteri entrapped vesicular stomatitis viruses by adhering to the particles (Botic et al., 2007).

Similar modes of action were reported for the inhibition of influenza viruses by E. faecium NCIMB 10415 (Wang et al., 2013) and for the inhibition of HSV-2 by L. gasseri CMUL57 (Al Kassaa et al., 2014). Since then, a large number of reports have been published on the antiviral properties of LAB (reviewed by Al Kassaa et al., 2014).

Proposed modes of antiviral action include direct interaction between the LAB and viruses (Botic et al., 2007; Wang et al., 2013; Al Kassaa et al., 2014), production of antiviral substances (Martín et al., 2010; Maragkoudakis et al., 2010; Mastromarino et al., 2011), and stimulation of the host’s immune system (Szajewska and Mrukowicz, 2001; Yasui et al., 2004; De Vrese et al., 2006; Olivares et al., 2007; Boge et al., 2009; Rautava et al., 2009; Kawashima et al., 2011; Salva et al., 2011; Villena et al., 2011; Khania et al., 2012; Kiso et al., 2013).

Lactobacillus plantarum L-137 decreased the levels of influenza virus H1N1 in infected mice by eliciting a pro-inflammatory response (Maeda et al., 2009). Similar findings were reported for Lactobacillus fermentum CECT5716 and L. casei DN114-001. Both strains stimulated the formation of antibodies to H1N1 (Olivares et al., 2007; Boge et al., 2009).

A combination of L. gasseri PA 16/8, Bifidobacterium longum SP07/3, and B. bifidum MF 20/5 reduced symptoms of the common cold (De Vrese et al., 2006), L. rhamnosus GG reduced the incidence of respiratory virus infections (Rautava et al., 2009) and treatment with L. acidophilus strain NCFM reduced influenza-like symptoms (Leyer et al., 2009). These are all promising findings and warrant further research.

Most reports on the antiviral activities of LAB have focused on class IIa bacteriocins, including enterocin AAR-71 and enterocin AAR-74 from E. faecalis (Qureshi H. et al., 2006), enterocin ST5Ha from E. faecium (Todorov et al., 2010), enterocin ST4V and enterocin CRL35 from E. mundtii (Wachsman et al., 2003; Todorov et al., 2005), and a peptide designated by the authors as a “bacteriocin” from L. delbrueckii (Serkedjieva et al., 2000).

Enterocin AAR-74 reduced the proliferation of coliphage HSA 10-fold, whereas enterocin AAR-71 had no effect on phage HSA (Humaira et al., 2006). Enterocin ST4V inhibited herpes viruses HSV-1 and HSV-2 in a dose-dependent manner (Todorov et al., 2005). Although enterocin CRL35 inhibits late stages of HSV-1 and HSV-2 replication, the mode of action of enterocin ST5Ha is uncertain.

Interestingly, the non-LAB bacteriocin subtilosin A also inhibited HSV-1, its drug-resistant mutant, and HSV-2A, most likely acting at the late stage of virus replication. However, subtilosin A did not act on non-enveloped viruses (Torres et al., 2013; Quintana et al., 2014). Enterocins CRL35 and ST4V acted on the multiplication of virus particles (Wachsman et al., 1999, 2003; Todorov et al., 2005).

Small differences in amino acid sequence have seemingly had a huge effect on antiviral activity, as reported for enterocin CRL35. A derivative of enterocin CRL35, missing two cysteine residues, was inactive against herpes viruses (Salvucci et al., 2007). The same derivative was also inactive against bacteria. This, however, does not necessarily mean that the same peptide segment is responsible for both anti-bacterial and antiviral activities.

The carbacyclic lantibiotic labyrinthopeptin A1 (LabyA1) inactivated the HIV virus and prevented its transmission between CD4 cells (Férir et al., 2013). A bacteriocin produced by L. delbrueckii subsp. bulgaricus 1043 inhibited one of the influenza viruses (Serkedjieva et al., 2000). Thus far, most reports on bacteriocins having antiviral activity have been based on observations of inhibition of virus replication. Further research is now needed to elucidate the exact mode of antiviral activity.

Little is known about the pharmacodynamics of bacteriocins, including their interactions with eukaryotic cells and individual cellular components. We know probiotic LAB trigger anti-inflammatory responses in the innate immune system by signaling dendritic cells (DCs) to secrete anti-inflammatory cytokines such as interleukin 10 (IL-10) (de LeBlanc et al., 2011).

Probiotics can also down-regulate pro-inflammatory cytokines by interfering with inflammatory signaling pathways such as the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways (Yoon and Sun, 2011). Activation of these pathways increases the secretion of pro-inflammatory cytokines that may lead to damage of intestinal epithelial cells. It is not known if bacteriocins have the same effect on the immune system.

Probiotics and Prophylaxis of Viral Diseases
It is becoming increasingly evident that the variety, extent, and significance of viral zoonotic infections is far greater than has been previously recognized. The scientific community, healthcare, and animal welfare organizations must now prioritize the development of acceptable and effective new strategies to reduce the genesis and transmission of these infections.

This is where the use of probiotics could provide an effective prophylactic measure. Some bacteriocin-producing probiotics, including strains known to exhibit antiviral activity, have been used to affect modulation of the microbiota in livestock animals (Mingmongkolchai and Panbangred, 2018). The results of a pilot study by Meazzi et al. (2019) provide new insights into the feline gut microbiota.

It was discovered that the lactobacilli present in healthy cats, were not present in animals having infectious peritonitis, caused by feline coronavirus (Meazzi et al., 2019). This points to a possible antagonistic relationship between lactobacilli and these viruses. Aboubakr et al. (2014) have also reported upon the antiviral activity of probiotic LAB against feline calicivirus (Aboubakr et al., 2014) and Stoeker et al. (2013) observed improved intestinal homeostasis in cats infected with feline immunodeficiency virus following oral administration of L. acidophilus (Stoeker et al., 2013). These findings all support the antiviral activity of probiotics in cats.

In other studies, L. casei was used as an antigen carrier to prevent diarrhea in bovines (Wang Y. et al., 2019), and a combination of probiotics was used to study the humoral response in cattle vaccinated against rabies (Vizzotto-Martino et al., 2016). Other studies have reported beneficial outcomes from the prophylactic dosing of probiotics in pigs infected with vesicular stomatitis virus, influenza A virus, transmissible gastroenteritis virus, epidemic diarrhea virus, and rotaviruses (Wang et al., 2013, 2017; Sirichokchatchawan et al., 2018; Peng et al., 2019). Clearly, the close similarities in the anatomy and physiology of humans and pigs suggest an increased potential threat of the development of suid zoonoses (Wang et al., 2013).

Additionally, there are still no effective prophylactic and treatment schedules in place for a number of viral diseases of farm animals that currently appear unlikely to become a source of human infections. Rabbit hemorrhagic disease, along with myxomatosis, is amongst the most contagious and lethal of viral infections.

The gold standard for prevention of the above-mentioned diseases is vaccination; however, there are potential problems associated with its implementation, especially in relation to biosafety. In order to develop new protocols for the control of the rabbit hemorrhagic disease, Wang L. et al. (2019) suggested the use of an oral L. casei probiotic vaccine as an antigen delivery system to stimulate humoral immune responses against caliciviruses (Wang L. et al., 2019). However, there are currently no published studies on the use of probiotics to afford protection against myxomatosis. This indicates an opportunity for future research on this topic.

Probiotics are widely used in the poultry industry and have become a routine infection preventative in some large commercial populations. Details of the antiviral mechanisms of probiotics have, in many cases, yet to be elucidated (Dong et al., 2020). Recently, Shojadoost et al. (2019) discovered that probiotic lactobacilli stimulate cell-mediated immunity in chickens by augmenting antiviral macrophage responses. On the other hand, there also appears to be some probiotic-mediated antiviral humoral immune responses in chickens (Gonmei et al., 2019).

In aquaculture, probiotics are now also more frequently serving as preventive and management measures against a variety of microbial infections, including those that are viral. White spot syndrome virus and infectious hypodermal and hematopoietic necrosis virus have been responsible for extensive economic losses (Kuebutornye et al., 2020). A number of Bacillus species isolates have been used to enhance the antiviral immunity of crustaceans (Sánchez-Ortiz et al., 2016; Sekar et al., 2016; Pham et al., 2017; Kuebutornye et al., 2020).

In some cases, it has been shown that probiotic extracellular products can help protect animals against viral infections. The antiviral activities of probiotics include proteinaceous products such as bacteriocins as well as non-proteinaceous metabolites such as lactic acid and hydrogen peroxide (Abdelhamid et al., 2019). The principal antiviral mechanisms of probiotics described to date appear to include the inhibition of virus replication (Kanmani et al., 2018).

Action of this nature occurring in the initial stages of infection when virus numbers are relatively small is clearly likely to be more effective than in inflammatory stages of the infection. These observations support the contention that probiotics are, in general, more likely to be efficacious as a prophylactic measure rather than as a virus treatment. Figure 1 outlines basic strategies for the probiotic control of zoonotic viruses. Examples of viruses potentially transmissible to humans and their animal reservoirs are listed in Table 1.

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FIGURE 1
Scheme of possible mechanism of prevention of emerging viral outbreaks by probiotics: Progenitor virus (PV) developed in primary host is able to “jump” to human (JH) directly or through intermediate host (JIH – “jump” to intermediate host). Intensive human-to-human transmission (HHT) is favorable condition for PV genetic developing (GD) making it more relative to human. This in the end contributes to appearance of highly pathogenic novel human virus (NHV), causing epidemic or pandemic outbreaks. Because of antagonistic relationship, probiotic bacteria (PB) can possibly inhibit (I) virus replication in all hosts with reducing its development and transmissions, in perspective, preventing outbreaks. PV, progenitor virus; JDA, jumping to domestic animals; JH, jumping to humans; HHT, human-to-human transmission; GD, genetic development; NHV, novel human virus; P, probiotic; I, inhibition.

TABLE 1

Examples of emerging zoonotic viruses.

Animal reservoirs of virus progenitorVirusesTransmission routeReferences
BatsSARS-CoV-2Via respiratory droplets;Liu et al., 2019Guo et al., 2020Meo et al., 2020Wan et al., 2020
PangolinsVia contaminated surfaces
BatsMERS-CoVVia consumption of products from infected camel;Fehr et al., 2017Bradley and Bryan, 2019Widagdo et al., 2019Meo et al., 2020Yang et al., 2020
Dromedary CamelsVia contact with infected camel
BatsSARS-CoVVia consumption of products from infected civet;Wicker et al., 2017Meo et al., 2020Yang et al., 2020
CivetsVia contact with infected camel
PigsA(H1N1)pdm09Via respiratory droplets;Garten et al., 2009Watson et al., 2015Bradley and Bryan, 2019
BirdsVia contaminated surfaces
BatsEbolaVia body fluids;Caron et al., 2018Banerjee et al., 2020Markotter et al., 2020
Via respiratory droplets
BirdsWest Nile feverVia mosquito vectorJohnson et al., 2018Napp et al., 2018
PrimatesHIVVia sexual contacts;Pinto-Santini et al., 2017
Via contact with or transfer of blood, vaginal fluid, pre-ejaculate and semen

Can Health-Promoting Commensal and Probiotic Bacteria Defend Against Pathogenic Viruses?

The human body is extensively colonized by archaea, bacteria, fungi, and viruses, and indeed, each of us is comprised of more microbial than human cells (Sender et al., 2016a, b). Interestingly, the total number of viral particles actually exceeds the total number of microbes by around one hundred-fold.

It would be naïve to think that because we can not directly visualize our microbial inhabitants, they are unlikely to be interacting with one other. Viral-bacterial interactions have long been considered exclusively as an interaction between bacteria as prey and phage as the ultimate predator. However, even phage-bacterial relationships no longer appear to be so simplistic. More likely, phages and bacteria are involved in a complex and mutually beneficial relationship allowing them both to proliferate at the site of bacterial colonization.

It is also rather simplistic to view bacteria merely as objects of prey, hunted by the virus without any tools to retaliate. Among these tools are the bacteriocins, small proteinaceous anti-competitor molecules deployed by bacteria to combat excessive proliferation by their neighbors. Knowing that evolutional developments are generally economical, it is logical to suspect that bacteriocins are also antiviral weapons of bacteria.

In recent decades, the use of bacteriocins as alternatives or supplements to traditional antimicrobial, antifungal, and antiviral drugs has been increasingly investigated. Many positively charged cationic bacteriocins kill susceptible bacteria by membrane permeabilization of the target cells. Antimicrobial peptides of bacterial origin play a significant role in the maintenance of stable bacterial consortia by decreasing the total number of bacterial cells in the population and preventing incursions by heterogenous microorganisms into their pre-occupied ecological niche.

On the other hand, why shouldn’t virions also be a bacteriocin target? Indeed, evidence has been found for nisin activity against lactobacillus bacteriophage c2 via disruption of the viral capsid (Ly-Chatain et al., 2013). Also, coliphage can be inhibited by staphylococcin18, enterocins AAR-71, AAR-74, and erwiniocin NA4 (Qureshi T. A. et al., 2006). Not surprisingly, numerous examples of bacteriocin activity against eukaryotic viruses have also been reported (Table 2).

TABLE 2

Anti-viral activity of bacteriocins.

Bacteriocin nameProducing strainAntiviral activity testedMechanisms of actionReferences
Enterocin CRL35Enterococcus mundtii CRL35HSV-1 and HSV-2A late step of virus multiplication is hindered by the prevention of mainly late glycoprotein D (gamma protein) synthesis. Virus adsorption and penetration are not affected.Wachsman et al., 19992003
Enterocin ST4VE. mundtii ST4VHSV-1 and HSV-2, Poliovirus PV-3, Measles virus (strain MV/BRAZIL/001/91, an attenuated strain)The HSV-1 and HSV-2 replication is inhibited.Todorov et al., 2005
Mechanism also might involve aggregation of the virus particles or blocking of their receptor sites.
Staphylococcin 188Staphylococcus aureus AB188Newcastle disease virusUnknownSaeed et al., 2004
Enterocin BE. faecium L3HSV-1UnknownErmolenko et al., 2010
Enterocin ST5HE. faecium ST5HaHSV-1UnknownTodorov et al., 2010
Labyrinthopeptin A1Actinomadura namibiensis DSM 6313HIV-1 and HSV-1LabyA1 interacted with envelope proteins, but not with the cellular receptors and acts as an entry inhibitor.Férir et al., 2013
Subtilosin ABacillus subtilis KATMIRA 1933HSV-1 and HSV-2Acts on enveloped viruses, no activity on non-enveloped viruses. Most likely, inhibits late stages of protein synthesis. Also active against drug-resistant HSV-1.Torres et al., 2013Quintana et al., 2014
Enterocin BE. faecium L3À/Perth/16/2009(H3N2) and A/South Africa/3626/2013(H1N1) pdm influenza virusesUnknownErmolenko et al., 2018
Bacteriocin-containing cell free supernatantLactobacillus delbrueckiiInfluenza virus A/chicken/Germany, strain Weybridge (H7N7) and strain Rostock (H7N1) in cell cultures of chicken embryo fibroblasts (CEF)Reduces expression of viral glycoproteins hemagglutinin, neuraminidase, and nucleoprotein on the surface of infected cells, reduces virus-induced cytopathic effect, infectious virus yield, and hemagglutinin production. Crude bacteriocin-containing preparation did not protect cells from infection, did not affect adsorption, and slightly inhibited viral penetration into infected cells.Serkedjieva et al., 2000
Computer modeling Nisin- and subtilosin-derivativesIn silico designHepatitis E virus (HEV)Theoretical estimation: binding with the capsid protein.Quintero-Gil et al., 2017
Semi-purified bacteriocinsLactococcus lactis GLc03 and GLc05, E. durans GEn09, GEn12, GEn14 and GEn17Herpes simplex virus 1 (HVS-1) and Poliovirus (PV-1)Antiviral activity before virus adsorption was recorded against HSV-1 35 for GEn14 (58.7%) and GEn17 (39.2%). Antiviral activity after virus 36 adsorption was identified against PV-1 for GLc05 (32.7%), GEn09 (91.0%), GEn12 (93.7%) 37 and GEn17 (57.2%), and against HSV-1 for GEn17 (71.6%).Quintana et al., 2014
The inactivation of HVS-1 viral particles may have occurred due to its interaction with the phospholipids on the viral envelope, avoiding its binding to cell receptors.
The inhibition of PV-1 did not occur before its adsorption.
DuramycinStreptomyces cinnamoneusZika virus(An inhibitor of TIM1 receptor)Tabata et al., 2016
Duramycin, a peptide that binds phosphatidylethanolamine in enveloped virions and precludes TIM1 binding, reduced ZIKV infection in placental cells and explants.
West Nile, dengue and Ebola virusesInhibits the entry of West Nile, dengue, and Ebola viruses. The inhibitory effect of duramycin is specific manner: it inhibits TIM1-mediated, but not L-SIGN-mediated, virus infection, and it does so by blocking virus attachment to TIM1.Richard et al., 2015
Micrococcin P1Staphylococcus equorum WS2733Hepatitis CInhibited HCV entry in a pan-genotypic manner, and prevented cell-to-cell spread without affecting the secretion of infectious HCV particles. In addition, micrococcin P1 acted synergistically with selected HCV inhibitors, and could potentially be used as a cost-effective component in HCV combination therapies.Lee et al., 2016
NisinLactococcus lactis subsp. lactisBovine viral diarrhea virus (BVDV)Nisin decreased both the extracellular virus titre and theamount of intracellular viral RNA. The best effect wasMałaczewska et al., 2019
observed when nisin was present throughout the entire
duration of viral infection (adsorption + post-adsorption).
CytomegalovirusUnknownBeljaars et al., 2001
Bacteriophage c2 (DNA head and non-contractile tail) infecting Lactococcus strainsThe positively charged compounds can adsorb on viral capsid by also electrostatic interaction which inhibit viral adsorption on host cells.Ly-Chatain et al., 2013
Staphylococcin 18 enterocins AAR-71, AAR-74, and erwiniocin NA4Staphylococcus aureus AB188 E. faecalis/BLIS Erwinia carotovora NA4/BLISColiphage HSAUnknownQureshi H. et al., 2006

Although there is growing evidence documenting the action of bacteriocins against viruses, the details of the mechanism(s) of action remain largely ill-defined. Included amongst the possible suggested mechanisms are: (a) direct aggregation with viral particles thereby preventing viral entry in host cells; (b) inhibition of synthesis of viral structural proteins; (c) neutralization of virus entry due to the blocking of receptor sites on the host cell (Wachsman et al., 2003; Todorov et al., 2005; Torres et al., 2013), and (d), disruption of the capsid or supercapsid structure by the bacteriocins. Direct virucidal activity was shown for subtilosin, which inactivated HSV-1 viral particles at the non-cytotoxic concentration of 200 lg/mL.

Interestingly, at lower non-virucidal levels, subtilosin inhibited the HSV-1 multiplication cycle in a dose-dependent manner (Torres et al., 2013). The peptide duramycin has been shown to have a rather specific antiviral effect: it binds to phosphatidylethanolamine in enveloped virions, precluding virus attachment to TIM1 receptors on the host cells and reducing TIM1-mediated, but not L-SIGN-mediated, virus infection (Richard et al., 2015; Tabata et al., 2016). Labyrinthopeptin A1 (LabyA1) also acts as an entry inhibitor against HIV and HSV. It was revealed that LabyA1 interacted with the HIV envelope protein gp120, but not with HIV cellular receptors (Férir et al., 2013).

The hypothesis that bacteriocins as positively charged compounds can adsorb to viral capsids by electrostatic interaction, thereby inhibiting viral adsorption to host cells was tested in a bacteriophage-based test system (Ly-Chatain et al., 2013). Wachsman et al. (2003) showed that the inhibition of HSV replication by enterocin CRL35 was due to interference with intracellular viral multiplication via the prevention of glycoprotein D (gamma protein) synthesis. HSV adsorption and penetration were not affected. Bacteriocin ST4V, from E. mundtii, inhibited both HSV-1 and HSV-2 replication in a dose-dependent manner (Todorov et al., 2005).

This growing evidence of antiviral activity associated with some bacteriocins supports speculation that the beneficial biological attributes of probiotics are not limited to the previously well-documented evidence of their immune modulation and anti-bacterial activities.

We venture to speculate that at least in part, the anti-infection activities of some probiotic bacteria might include the capability of limiting or even eliminating the replication of pathogenic viruses. The evolutionary genesis of microbes capable of carrying out these actions appears quite logical as a protective response of a stabilized indigenous ecosystem to potential territorial invasion regardless of whether it is bacterial, fungal, or viral in origin.

As was rightfully pointed out in a recent review by Akour (2020), presently, there are no published reports on the implementation of probiotics for prophylaxis or combating COVID-19. However, there is a Phase II clinical trial to evaluate the previously explored anti-asthma “live cells” formulation MRx4DP0004 as an immunomodulatory drug for hospitalized COVID-19 patients6.

While the actual formulation is not reported, the patients received a daily dose of 4 × 109 to 4 × 1010 live cells in two doses, twice daily for 2 weeks. It is only logical to speculate that properly selected probiotics can serve as effective adjuvants for the prophylaxis and even treatments of COVID-19. Until the pathogenesis of novel coronavirus and its effect on gut microbiota is established, the use of probiotics may not be appropriate.

In addition, while foodborne transmission of COVID-19 is rather unlikely (Li et al., 2021), appropriate model studies should be conducted to exclude food as a possible vehicle for the virus transmission. Since immune-compromised humans are likely to be most vulnerable to COVIV-19, the use of probiotics as prophylactic agents or adjuvants in the treatment regiment should be carefully considered, bearing in mind that even the friendliest, most studied probiotics may cause septicemia in immune-compromised and otherwise challenged health status individuals (Kochan et al., 2011; Kulkarni and Khoury, 2014; Doron and Snydman, 2015; Koyama et al., 2019).

reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468459/


More information: Rishi Drolia et al. Receptor-targeted engineered probiotics mitigate lethal Listeria infection, Nature Communications (2020). DOI: 10.1038/s41467-020-20200-5

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