Engineering Lactobacillus rhamnosus GG and GR-1 to express HIV-inhibiting griffithsin
Introduction
With human immunodeficiency virus (HIV) infections being one of the top infectious diseases worldwide, the interest in carbohydrate-binding agents (CBAs) as therapeutic agents that can block HIV is growing [1]. Two promising peptidic CBAs, with documented activity against HIV and thus far unknown side-effects for human host cells, are actinohivin (AH) and griffithsin (GRFT) [2], [3]. AH is a 12.5-kDa lectin isolated from the actinomycete Longisporum albida [4], [5]. AH consists of 114 amino acid residues, divided into three segments, forming three sugar-binding pockets to accommodate Man-α(1-2)-Man residues of N-linked glycans present on the surface of the HIV envelope glycoprotein gp120, therefore inhibiting HIV infection [3], [5], [6]. AH has been shown to prevent T-cell and macrophage (M)-tropic syncytium formation by HIV [4], has no mitogenic activity and does not induce secretion of inflammatory cytokines or chemokines in peripheral blood cells [7]. GRFT, isolated from the red alga Griffithsia sp., is a 12.7-kDa lectin consisting of 121 amino acids [2], [8]. The GRFT molecules form a domain-swapped dimer, providing six independent binding sites for monosaccharides [9]. GRFT binds strongly to the HIV-envelope glycoproteins gp120, gp160 and gp41, and at the same time shows an outstanding safety profile towards human epithelial and immune cells [10]. GRFT has been shown to inhibit HIV-induced cell killing in a concentration-dependent manner and to be active against T- and M-tropic strains of HIV-1 [2].
Conversely, lactobacilli that are present on (vaginal) mucosal surfaces are increasingly considered as natural factors that can promote the host defence against HIV [11]. Furthermore, they can also be applied as probiotics to improve human lives or to prevent HIV infections. Probiotics are known as “live microorganism that, when administrated in adequate amounts, confer health benefits in the host” [12]. For example, the well-known and widely used urogenital probiotic strain L. rhamnosus GR-1 can colonize the vaginal niche, as well as inhibit the growth and adhesion of a number of bacterial and viral pathogens [13], [14]. Furthermore, oral administration of L. rhamnosus GR-1 and Lactobacillus reuteri RC-14 to HIV/AIDS patients has been associated with the resolution of diarrhoea, nausea and flatulence [15], increased CD4+ T-cell counts and reported longer working days [16], [17]. Therefore, L. rhamnosus GR-1 can be considered as a good probiotic able to restore health conditions both in the gastrointestinal tract and in the vaginal niche. Since probiotic bacteria are able to exclude pathogens in the human host and to improve human health, they also have a great potential to serve as a delivery vehicle for microbicides, such as anti-HIV molecules. In this way, the probiotic strains can be used not only as a delivery system but also as adjuvants stimulating the immune function and as agents that reduce symptoms often associated with HIV infections (e.g., gastrointestinal problems).
In this study, the well-documented probiotic strains L. rhamnosus GG and L. rhamnosus GR-1 were genetically engineered for mucosal oral and/or vaginal delivery of AH and GRFT as anti-HIV CBAs.
Section snippets
Bacterial strains, plasmids and growth conditions
The bacterial strains and plasmids used in this study are listed in Table 1 and in more detail in Supplementary Tables S1 and S2. L. rhamnosus GG and GR-1 wild-type, as well as the corresponding recombinant strains, were routinely grown under anaerobic conditions in MRS medium (DifcoTM 288130, Belgium) [18] at 37 °C. Escherichia coli strains were grown in LB medium with aeration at 37 °C. If required, antibiotics were used at the following concentrations: 100 µg/mL ampicillin and 5 µg/mL (
AH cannot be expressed intracellularly and extracellularly by L. rhamnosus
Intracellular expression of AH monomer (AHm) and AH dimer (AHd) was investigated under the control of the msp1 promoter. The successfully constructed recombinant L. rhamnosus GG and GR-1 strains were checked for production of AH intracellularly by western blot with monoclonal anti-His6 antibodies and polyclonal anti-AH antibodies. Although at DNA level, the bacterial strains appeared to contain the constructs with the correct DNA sequence, no protein bands were detected at the expected size for
Discussion
In this study, we investigated the heterologous expression of two HIV-inhibiting lectins, namely AH and GRFT by two well-known Lactobacillus probiotic strains L. rhamnosus GG and GR-1 [25], [26]. These two lectins, AH and GRFT, have well-documented activity against HIV and unknown, if any, observed side-effects on the host cells [7], [27].
First, we optimized the genetic toolbox, including a selection of vectors with strong (endogenous) promoter for recombinant expression and constructs for
Acknowledgements
We thank Haruo Tanaka for kindly providing the AH genes and Kenneth E. Palmer for recombinant GRFT. We thank Sigrid De Keersmaecker for initiating part of this project and Sandra Claes and Evelyne Van Kerckhove for excellent technical assistance.
Declarations
None.
Funding
Mariya Petrova was supported by FWO Vlaanderen. Sarah Lebeer was supported by the FWO Vlaanderen and a research grant KaN 28960. Work at KU Leuven was supported by program financing from the KU Leuven (PF/10/018). The funding bodies had no role in study design, data collection and interpretation.
Competing Interests
The authors have no conflicts of interest to declare.
Ethical Approval
Not required.
References (34)
- et al.
Isolation and characterization of griffithsin, a novel HIV-inactivating protein, from the red alga Griffithsia sp
J Biol Chem
(2005) - et al.
Actinohivin, a novel anti-HIV protein from an actinomycete that inhibits syncytium formation: isolation, characterization, and biological activities
Biochem Biophys Res Commun
(2001) - et al.
Actinohivin, a novel anti-human immunodeficiency virus protein from an actinomycete, inhibits viral entry to cells by binding high-mannose type sugar chains of gp120
Biochem Biophys Res Commun
(2004) - et al.
Domain-swapped structure of the potent antiviral protein griffithsin and its mode of carbohydrate binding
Structure
(2006) - et al.
Disruption of urogenital biofilms by lactobacilli
Colloids Surf B Biointerfaces
(2011) - et al.
Potential of carbohydrate-binding agents as therapeutics against enveloped viruses
Med Res Rev
(2012) - et al.
Actinohivin: specific amino acid residues essential for anti-HIV activity
J Antibiot (Tokyo)
(2010) - et al.
Mechanism by which the lectin actinohivin blocks HIV infection of target cells
Proc Natl Acad Sci U S A
(2009) - et al.
Actinohivin, a broadly neutralizing prokaryotic lectin, inhibits HIV-1 infection by specifically targeting high-mannose-type glycans on the gp120 envelope
Antimicrob Agents Chemother
(2010) - et al.
Griffithsin, a potent HIV entry inhibitor, is an excellent candidate for anti-HIV microbicide
J Med Primatol
(2007)
Investigation of griffithsin's interactions with human cells confirms its outstanding safety and efficacy profile as a microbicide candidate
PLoS One
Vaginal microbiota and its role in HIV transmission and infection
FEMS Microbiol Rev
Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic
Nat Rev Gastroenterol Hepatol
Persistence of Lactobacillus fermentum RC-14 and Lactobacillus rhamnosus GR-1 but not L. rhamnosus GG in the human vagina as demonstrated by randomly amplified polymorphic DNA
Clin Diagn Lab Immunol
Yogurt containing probiotic Lactobacillus rhamnosus GR-1 and L. reuteri RC-14 helps resolve moderate diarrhea and increases CD4 count in HIV/AIDS patients
J Clin Gastroenterol
Effect of 25 weeks probiotic supplementation on immune function of HIV patients
Gut Microbes
Probiotic yogurt consumption is associated with an increase of CD4 count among people living with HIV/AIDS
J Clin Gastroenterol
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