Abstract
BackgroundBrucellosis, caused by several Brucella species, such as the bacterium Brucella melitensis, is considered one of the most severe zoonotic diseases worldwide. Not only does it affect ruminant animal populations, leading to a substantial financial burden for stockbreeders, but also poses severe public health issues. For almost four decades in southern Europe and elsewhere, eradication of the disease has been based on ambiguously effective programs, rendering massive sanitation of livestock urgent and indispensable. Gene therapy, which has been proved effective in the clinic, could possibly constitute an alternative option towards a permanent cure for brucellosis, by aiding in the deletion or inactivation of genes associated with the replication of Brucella within the host cells.ResultsWe infected ovine macrophages with B.melitensis, to simulate the host cell/microorganism interaction in vitro, and transduced the infected cells with CRISPR/Cas9 lentiviral vectors that target Brucella’s RNA polymerase subunit A (RpolA) or virulence-associated gene virB10 at a multiplicity of infection of 60. We demonstrate a significant decrease in the bacterial load per cell when infected cells are transduced with the RpolA vector and that the number of internalized brucellae per cell remains unaffected when macrophages are transduced with a conventional lentiviral vector expressing the green fluorescence protein, thus underlining the bactericidal effect of our CRISPR/Cas9 system.ConclusionsPending in vivo verification of our findings, overall, these results may prove critical not only for the treatment of human brucellosis, but for other infectious diseases in general.
Highlights
Brucellosis, caused by several Brucella species, such as the bacterium Brucella melitensis, is considered one of the most severe zoonotic diseases worldwide
We show that the number of internalized brucellae/cell is significantly decreased 1 and 4 days post transduction with the CRISPR/Cas9 vector against bacterial RNA polymerase subunit A (RpolA) at an multiplicity of infection (MOI) of 60
Pilot transduction of infected sheep macrophages with a conventional green fluorescence protein (GFP) lentiviral vector We proceeded to a pilot transduction of the Brucella-infected macrophages in order to test their performance in culture, as well as their potency towards gene transfer, under conditions of heavy bacterial loads produced by the MOI of 5000
Summary
Brucellosis, caused by several Brucella species, such as the bacterium Brucella melitensis, is considered one of the most severe zoonotic diseases worldwide. The REV-1 vaccine, despite its vast use for the prevention of brucellosis in sheep and goats [8], may cause abortions and infertility in pregnant and male animals respectively [9], leading to its extensive diffusion into the environment with vaginal discharges during post-partum period. Since it partially preserves its virulent properties, it is not entirely safe for clinical practitioners who administer the vaccine [9]. Even though vaccination with REV-1 is applied in certain cases [11], its suitability has not been clarified by the manufacturers for these animals
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