Zika flavivirus infection during pregnancy appears to produce higher risk of microcephaly, and also causes multiple neurological problems such as Guillain–Barré syndrome. The Zika virus is now widespread in Central and South America, and is anticipated to become an increasing risk in the southern United States. With continuing global travel and the spread of the mosquito vector, the exposure is expected to accelerate, but there are no currently approved treatments against the Zika virus. The Zika NS2B/NS3 protease is an attractive drug target due to its essential role in viral replication. Our studies have identified several compounds with inhibitory activity (IC50) and binding affinity (KD) of ∼5-10 μM against the Zika NS2B-NS3 protease from testing 71 HCV NS3/NS4A inhibitors that were initially discovered by high-throughput screening of 40,967 compounds. Competition surface plasmon resonance studies and mechanism of inhibition analyses by enzyme kinetics subsequently determined the best compound to be a competitive inhibitor with a Ki value of 9.5 μM. We also determined the X-ray structure of the Zika NS2B-NS3 protease in a “pre-open conformation”, a conformation never observed before for any flavivirus proteases. This provides the foundation for new structure-based inhibitor design.

During pregnancy, the placenta protects the fetus against the maternal immune response, as well as bacterial and viral pathogens. Bacterial pathogens that have evolved specific mechanisms of breaching this barrier, such as Listeria monocytogenes, present a unique opportunity for learning how the placenta carries out its protective function. We previously identified the L. monocytogenes protein Internalin P (InlP) as a secreted virulence factor critical for placental infection. Here, we show that InlP, but not the highly similar L. monocytogenes internalin Lmo2027, binds to human afadin (encoded by AF-6), a protein associated with cell-cell junctions. A crystal structure of InlP reveals several unique features, including an extended leucine-rich repeat (LRR) domain with a distinctive Ca2+-binding site. Despite afadin’s involvement in the formation of cell-cell junctions, MDCK epithelial cells expressing InlP displayed a decrease in the magnitude of the traction stresses they could exert on deformable substrates, similar to the decrease in traction exhibited by AF-6 knock-out MDCK cells. L. monocytogenes ΔinlP mutants were deficient in their ability to form actin-rich protrusions from the basal face of polarized epithelial monolayers, a necessary step in the crossing of such monolayers (transcytosis). A similar phenotype was observed for bacteria expressing an internal in-frame deletion in inlP (inlP ΔLRR5) that specifically disrupts its interaction with afadin. However, afadin deletion in the host cells did not rescue the transcytosis defect. We conclude that secreted InlP targets cytosolic afadin to specifically promote L. monocytogenes transcytosis across the basal face of epithelial monolayers, which may contribute to the crossing of the basement membrane during placental infection.

Staphylococcus aureus, an invasive pathogen of humans and animals, requires a specialized ESS pathway to secrete proteins (EsxA, EsxB, EsxC, and EsxD) during infection. Expression of ess genes is required for S. aureus establishment of persistent abscess lesions following bloodstream infection; however, the mechanisms whereby effectors of the ESS pathway implement their virulence strategies were heretofore not known. Here, we show that EssE forms a complex with other members of the ESS secretion pathway and its substrates, promoting the secretion of EsxA, EsxB, EsxC, EsxD, and EssD. During bloodstream infection of mice, the S. aureus essE mutant displays defects in host cytokine responses, specifically in the production of interleukin-12 (IL-12) (p40/p70) and the suppression of RANTES (CCL5), activators of T H 1 T cell responses and immune cell chemotaxis, respectively. Thus, essE-mediated secretion of protein effectors via the ESS pathway may enable S. aureus to manipulate host immune responses by modifying the production of cytokines.IMPORTANCE Staphylococcus aureus and other firmicutes evolved a specialized ESS (EsxA/ESAT-6-like secretion system) pathway for the secretion of small subsets of proteins lacking canonical signal peptides. The molecular mechanisms for ESSdependent secretion and their functional purpose are still unknown. We demonstrate here that S. aureus EssE functions as a membrane assembly platform for elements of the secretion machinery and their substrates. Furthermore, S. aureus EssEmediated secretion contributes to the production or the suppression of specific cytokines during host infection, thereby modifying immune responses toward this pathogen.

Bacterial spores are the most resistant form of life known on Earth and represent a serious problem for (i) bioterrorism attack, (ii) horizontal transmission of microbial pathogens in the community, and (iii) persistence in patients and in a nosocomial environment. Stage II sporulation protein D (SpoIID) is a lytic transglycosylase (LT) essential for sporulation. The LT superfamily is a potential drug target because it is active in essential bacterial processes involving the peptidoglycan, which is unique to bacteria. However, the absence of structural information for the sporulation-specific LT enzymes has hindered mechanistic understanding of SpoIID. Here, we report the first crystal structures with and without ligands of the SpoIID family from two community relevant spore-forming pathogens, Bacillus anthracis and Clostridium difficile. The structures allow us to visualize the overall architecture, characterize the substrate recognition model, identify critical residues, and provide the structural basis for catalysis by this new family of enzymes.Sporulation is an evolutionarily conserved process that allows some Gram-positive bacteria to differentiate into a dormant cell type called a spore that allows them to resist chemical agents such as antibiotics and disinfectants and physical agents such as radiation, boiling and drying (1). Sporulation is characterized by an asymmetric cell division, followed by migration of the mother cells' membrane around the nascent spore in a phagocyte-like process (2, 3). Engulfment of what will become the spore requires PGN 2 degradation by the SpoIIM/SpoIIP/ SpoIID molecular machine (4, 5). In this complex, SpoIIM serves as a scaffold upon which the SpoIID and SpoIIP enzymes assemble (1, 4, 6). SpoIIP is single-pass transmembrane protein with a large extracellular portion that has both amidase and endopeptidase activities. This enzyme cleaves the cross-links connecting the stem peptides of PGN and removes the stem peptides from the glycan chains (Fig. 1a). SpoIID is a lytic transglycosylase (LT), an enzyme that cleaves the glycan strands of PGN (Fig. 1b). It also has a single-pass transmembrane domain and a large extracellular portion that can bind PGN but can only cleave glycan strands after the stem peptides of PGN have been removed by SpoIIP. The requirement for a peptide-free glycan strand defines an ordered and sequential set of events during the engulfment process. SpoIID also functions to stimulate SpoIIP activity (4).LTs represent a class of autolysin that cleaves the glycan chain of the PGN to facilitate biosynthesis, remodeling, degradation, and turnover of the bacterial cell wall (7). LTs differ from the other two classes of glycan strand-cleaving enzymes. Although muramidases hydrolyze the ␤-1,4 N-acetylmuramic acid (NAM)-N-acetylglucosamine (NAG) glycosidic bond and glucosaminidases hydrolyze the ␤-1,4 NAG-NAM glycosidic bond, LTs use the C-6 hydroxyl of NAM as a nucleophile in the cleavage reaction of the ␤-1,4 NAM-NAG glycosidic bond. Consequently, the product o...

Streptococcus pneumoniae pilus 1 is present in 30 to 50% of invasive disease-causing strains and is composed of three subunits: the adhesin RrgA, the major backbone subunit RrgB, and the minor ancillary protein RrgC. RrgB exists in three distinct genetic variants and, when used to immunize mice, induces an immune response specific for each variant. To generate an antigen able to protect against the infection caused by all pilus-positive S. pneumoniae strains, we engineered a fusion protein containing the three RrgB variants (RrgB321). RrgB321 elicited antibodies against proteins from organisms in the three clades and protected mice against challenge with piliated pneumococcal strains. RrgB321 antisera mediated complement-dependent opsonophagocytosis of piliated strains at levels comparable to those achieved with the PCV7 glycoconjugate vaccine. These results suggest that a vaccine composed of RrgB321 has the potential to cover 30% or more of all pneumococcal strains and support the inclusion of this fusion protein in a multicomponent vaccine against S. pneumoniae.