The molecular mechanism of Shiga toxin Stx2e neutralization by a single-domain antibody targeting the cell receptor-binding domain.

Shiga toxin Stx2e is the major known agent that causes edema disease in newly weaned pigs. This severe disease is characterized by neurological disorders, hemorrhagic lesions, and frequent fatal outcomes. Stx2e consists of an enzymatically active A subunit and five B subunits that bind to a specific glycolipid receptor on host cells. It is evident that antibodies binding to the A subunit or the B subunits of Shiga toxin variants may have the capability to inhibit their cytotoxicity. Here, we report the discovery and characterization of a VHH single domain antibody (nanobody) isolated from a llama phage display library that confers potent neutralizing capacity against Stx2e toxin. We further present the crystal structure of the complex formed between the nanobody (NbStx2e1) and the Stx2e toxoid, determined at 2.8 Å resolution. Structural analysis revealed that for each B subunit of Stx2e, one NbStx2e1 is interacting in a head-to-head orientation and directly competing with the glycolipid receptor binding site on the surface of the B subunit. The neutralizing NbStx2e1 can in the future be used to prevent or treat edema disease.

Suppression of type 1 pilus assembly in uropathogenic Escherichia coli by chemical inhibition of subunit polymerization.

OBJECTIVES: To identify and to characterize small-molecule inhibitors that target the subunit polymerization of the type 1 pilus assembly in uropathogenic Escherichia coli (UPEC). METHODS: Using an SDS-PAGE-based assay, in silico pre-filtered small-molecule compounds were screened for specific inhibitory activity against the critical subunit polymerization step of the chaperone-usher pathway during pilus biogenesis. The biological activity of one of the compounds was validated in assays monitoring UPEC type 1 pilus biogenesis, type 1 pilus-dependent biofilm formation and adherence to human bladder epithelial cells. The time dependence of the in vivo inhibitory activity and the overall effect of the compound on UPEC growth were determined. RESULTS: N-(4-chloro-phenyl)-2-{5-[4-(pyrrolidine-1-sulfonyl)-phenyl]-[1,3,4]oxadiazol-2-yl sulfanyl}-acetamide (AL1) inhibited in vitro pilus subunit polymerization. In bacterial cultures, AL1 disrupted UPEC type 1 pilus biogenesis and pilus-dependent biofilm formation, and resulted in the reduction of bacterial adherence to human bladder epithelial cells, without affecting bacterial cell growth. Bacterial exposure to the inhibitor led to an almost instantaneous loss of type 1 pili. CONCLUSIONS: We have identified and characterized a small molecule that interferes with the assembly of type 1 pili. The molecule targets the polymerization step during the subunit incorporation cycle of the chaperone-usher pathway. Our discovery provides new insight into the design and development of novel anti-virulence therapies targeting key virulence factors of bacterial pathogens.

Response of Porphyromonas gingivalis to heme limitation in continuous culture.

Porphyromonas gingivalis is an anaerobic, asaccharolytic, gram-negative bacterium that has essential requirements for both iron and protoporphyrin IX, which it preferentially obtains as heme. A combination of large-scale quantitative proteomic analysis using stable isotope labeling strategies and mass spectrometry, together with transcriptomic analysis using custom-made DNA microarrays, was used to identify changes in P. gingivalis W50 protein and transcript abundances on changing from heme-excess to heme-limited continuous culture. This approach identified 160 genes and 70 proteins that were differentially regulated by heme availability, with broad agreement between the transcriptomic and proteomic data. A change in abundance of the enzymes of the aspartate and glutamate catabolic pathways was observed with heme limitation, which was reflected in organic acid end product levels of the culture fluid. These results demonstrate a shift from an energy-efficient anaerobic respiration to a less efficient process upon heme limitation. Heme limitation also resulted in an increase in abundance of a protein, PG1374, which we have demonstrated, by insertional inactivation, to have a role in epithelial cell invasion. The greater abundance of a number of transcripts/proteins linked to invasion of host cells, the oxidative stress response, iron/heme transport, and virulence of the bacterium indicates that there is a broad response of P. gingivalis to heme availability.

Chemical attenuation of pilus function and assembly in Gram-negative bacteria.

Bacteria express a multitude of hair-like adhesive appendages on their cell surfaces, together referred to as pili or fimbriae. In Gram-negative bacteria, these proteinaceous structures are assembled through a number of dedicated secretion pathways including the chaperone-usher pathway, the nucleation/precipitation pathway and the type IV pilus pathway. Pili are prevalent in pathogenic strains and play important roles in the establishment and persistence of bacterial infections by mediating host cell adhesion, cell invasion or biofilm formation. Their indispensible roles in pathogenesis render them attractive targets for directed therapeutic intervention. Here, we describe the recent advances in the chemical attenuation of pilus-associated virulence in Gram-negative bacteria.