An In Vivo High-Throughput Screening Approach Targeting the Type IV Secretion System Component VirB8 Identified Inhibitors of Brucella abortus 2308 Proliferation

Abstract

As bacterial pathogens develop resistance against most currently used antibiotics, novel alternatives for treatment of microbial infectious diseases are urgently needed. Targeting bacterial virulence functions in order to disarm pathogens represents a promising alternative to classical antibiotic therapy. Type IV secretion systems, which are multiprotein complexes in the cell envelope that translocate effectors into host cells, are critical bacterial virulence factors in many pathogens and excellent targets for such "antivirulence" drugs. The VirB8 protein from the mammalian pathogen Brucella was chosen as a specific target, since it is an essential type IV secretion system component, it participates in multiple protein-protein interactions, and it is essential for the assembly of this translocation machinery. The bacterial two-hybrid system was adapted to assay VirB8 interactions, and a high-throughput screen identified specific small-molecule inhibitors. VirB8 interaction inhibitors also reduced the levels of VirB8 and of other VirB proteins, and many of them inhibited virB gene transcription in Brucella abortus 2308, suggesting that targeting of the secretion system has complex regulatory effects in vivo. One compound strongly inhibited the intracellular proliferation of B. abortus 2308 in a J774 macrophage infection model. The results presented here show that in vivo screens with the bacterial two-hybrid assay are suited to the identification of inhibitors of Brucella type IV secretion system function.The increasing resistance to classical antibiotics necessitates the development of alternative therapeutic strategies against microbial infectious diseases (36, 47). Genomics-based approaches, which are aimed at identifying novel targets (29), have potential to yield new therapeutic approaches; it is nevertheless foreseeable that resistance will eventually develop against drugs that target vital cell functions. Alternative strategies comprise phage therapy, the stimulation of the host immune system, and the development of "antivirulence" drugs that specifically target bacterial virulence functions but not vital cell functions (4,7,16,30). The rationale underlying the latter approach is that these molecules will disarm pathogens, permitting their elimination from the body by the immune system, and that the selection pressure for the development of resistance mutations will be reduced, as they do not target vital cellular functions. Recent years have seen significant advances in this area, especially in type III secretion (T3S) systems, where promising molecules were discovered (22, 34). Interestingly, many of the active molecules belong to the class of salicylidene acylhydrazides and have broad-spectrum activity against Yersinia, Chlamydia, Salmonella, and Shigella species (33,37,39,46). These molecules were isolated using cell-based high-throughput screening (HTS) measuring T3S system functions in living cells, and their targets have not been unequivocally identified. In contrast, we have pursued a different ap...

Pathogenesis and Immunobiology of Brucellosis

“…This endeavor identified several potent and specific inhibitors of T4SS function. X-ray crystallography and docking studies were used to demonstrate that VirB8 inhibitors bind to a surface groove opposite to the dimerization interface, 88,90 thereby providing insight into the mechanism of action of this potential therapeutic.…”

A Lysozyme-Like Protein in Brucella abortus Is Involved in the Early Stages of Intracellular Replication

“…Samples were submitted to SDS-PAGE (12.5%) and transferred to nitrocellulose membranes. The presence of VirB5 and VirB8 proteins was carried out by immunoblot analysis using rabbit polyclonal antibodies specific for VirB5 and VirB8 (dilution, 1:2,000) (20) and IRDye secondary anti-rabbit antibody (LI-COR, Inc.). Detection was performed using the Odyssey Imaging System (LI-COR, Inc.).…”

Serial Kinetics of the Antibody Response against the Complete Brucella melitensis ORFeome in Focal Vertebral Brucellosis

“…The BMEII0032 (VirB8) protein has been noted to be an important virulence factor in Brucella spp. (14), most recently demonstrated with a lethal murine model of Brucella microti infection as well as for other newly identified Brucella species (7).…”