Phage-Related Ribosomal Protease (Prp) of Staphylococcus aureus: In Vitro Michaelis-Menten Kinetics, Screening for Inhibitors, and Crystal Structure of a Covalent Inhibition Product Complex.

Phage-related ribosomal proteases (Prps) are essential for the assembly and maturation of the ribosome in Firmicutes, including the human pathogens Staphylococcus aureus, Streptococcus pneumoniae, and Clostridium difficile. These bacterial proteases cleave off an N-terminal extension of a precursor of ribosomal protein L27, a processing step that is essential for the formation of functional ribosomes. This essential role of Prp in these pathogens has identified this protease as a potential antibiotic target. In this work, we determine the X-ray crystal structure of a covalent inhibition complex at 2.35 Å resolution, giving the first complete picture of the active site of a functional Prp. We also characterize the kinetic activity and screen for potential inhibitors of Prp. This work gives the most complete characterization of the structure and specificity of this novel class of proteases to date.

Molecular Targets and Strategies for Inhibition of the Bacterial Type III Secretion System (T3SS); Inhibitors Directly Binding to T3SS Components.

The type III secretion system (T3SS) is a virulence apparatus used by many Gram-negative pathogenic bacteria to cause infections. Pathogens utilizing a T3SS are responsible for millions of infections yearly. Since many T3SS knockout strains are incapable of causing systemic infection, the T3SS has emerged as an attractive anti-virulence target for therapeutic design. The T3SS is a multiprotein molecular syringe that enables pathogens to inject effector proteins into host cells. These effectors modify host cell mechanisms in a variety of ways beneficial to the pathogen. Due to the T3SS's complex nature, there are numerous ways in which it can be targeted. This review will be focused on the direct targeting of components of the T3SS, including the needle, translocon, basal body, sorting platform, and effector proteins. Inhibitors will be considered a direct inhibitor if they have a binding partner that is a T3SS component, regardless of the inhibitory effect being structural or functional.

Fluorescence Detection of Type III Secretion Using a Glu-CyFur Reporter System in Citrobacter rodentium.

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea worldwide. EPEC and the closely related murine model of EPEC infection, Citrobacter rodentium, utilize a type III secretion system (T3SS) to propagate the infection. Since the T3SS is not essential for the bacteria to survive or propagate, inhibiting the virulence factor with a therapeutic would treat the infection without causing harm to commensal bacteria. Studying inhibitors of the T3SS usually requires a BSL-2 laboratory designation and eukaryotic host cells while not indicating the mechanism of inhibition. We have designed a BSL-1 assay using the murine model C. rodentium that does not require mammalian cell culture. This CPG2-reporter assay allows for more rapid analysis of secretion efficiency than Western blotting and is sensitive enough to differentiate between partial and total inhibition of the T3SS. Here we present our method and the results of a small collection of compounds we have screened, including known T3SS inhibitors EGCG, regacin, and aurodox and related quorum sensing inhibitors tannic acid and ellagic acid. We have further characterized EGCG as a T3SS inhibitor and established its IC50 of 1.8 ± 0.4 µM. We also establish tannic acid as a potent inhibitor of the T3SS with an IC50 of 0.65 ± 0.09 µM.

Delivery of Heterologous Proteins, Enzymes, and Antigens via the Bacterial Type III Secretion System.

The Type III Secretion System (T3SS) is a multimeric protein complex composed of over 20 different proteins, utilized by Gram-negative bacteria to infect eukaryotic host cells. The T3SS has been implicated as a virulence factor by which pathogens cause infection and has recently been characterized as a communication tool between bacteria and plant cells in the rhizosphere. The T3SS has been repurposed to be used as a tool for the delivery of non-native or heterologous proteins to eukaryotic cells or the extracellular space for a variety of purposes, including drug discovery and drug delivery. This review covers the methodology of heterologous protein secretion as well as multiple cases of utilizing the T3SS to deliver heterologous proteins or artificial materials. The research covered in this review will serve to outline the scope and limitations of utilizing the T3SS as a tool for protein delivery.

Natural Product Type III Secretion System Inhibitors.

Many known inhibitors of the bacterial type III secretion system (T3SS), a virulence factor used by pathogenic bacteria to infect host cells, are natural products. These compounds, produced by bacteria, fungi, and plants, may have developed as prophylactic treatments for potential attack by bacterial pathogens or as an attempt by symbiotic organisms to protect their hosts. Regardless, better understanding of the structures and mechanisms of action of these compounds may open opportunities for drug development against diseases caused by pathogens utilizing the T3SS. This review will cover selected known natural products of the T3SS and detail what is known of their origin and mechanism of action. These inhibitors highlight nature's ability to modulate interactions between organisms at a cellular level.