Search

During the different stages of the Plasmodium life cycle, surface-associated proteins establish key interactions with the host and play critical roles in parasite survival. The 6-cysteine (6-cys) protein family is one of the most abundant surface antigens and expressed throughout the Plasmodium falciparum life cycle. This protein family is conserved across Plasmodium species and plays critical roles in parasite transmission, evasion of the host immune response and host cell invasion. Several 6-cys proteins are present on the parasite surface as hetero-complexes but it is not known how two 6-cys proteins interact together. Here, we present a crystal structure of Pf12 bound to Pf41 at 2.85 Å resolution, two P. falciparum proteins usually found on the parasite surface of late schizonts and merozoites. Our structure revealed two critical interfaces required for complex formation with important implications on how different 6-cysteine proteins may interact with each other. Using structure-function analyses, we identified important residues for Pf12-Pf41 complex formation. In addition, we generated 16 nanobodies against Pf12 and Pf41 and showed that several Pf12-specific nanobodies inhibit Pf12-Pf41 complex formation. Using X-ray crystallography, we were able to describe the structural mechanism of an inhibitory nanobody in blocking Pf12-Pf41 complex formation. Future studies using these inhibitory nanobodies will be useful to determine the functional role of these two 6-cys proteins in malaria parasites. [ABSTRACT FROM AUTHOR]

Objectives: Escherichia coli ST131 is a globally disseminated MDR clone originally identified due to its association with the blaCTX-M-15 gene encoding an ESBL. It is thus assumed that blaCTX-M-15 is the major determinant for resistance to β-lactam antibiotics in this clone. The complete sequence of EC958, a reference strain for E. coli ST131, revealed that it contains a chromosomally located blaCMY-M-23 gene with an upstream ISEcp1 element as well as several additional plasmid-encoded β-lactamase genes. Here, we examined the genetic context of the blaCMY-M-23 element in EC958 and other E. coli ST131 strains and investigated the contribution of blaCMY-M-23 to EC958 resistance to a range of β-lactam antibiotics. Methods: The genetic context of blaCMY-M-23 and its associated mobile elements was determined by PCR and sequencing. Antibiotic susceptibility testing was performed using Etests. The activity of the blaCMY-M-23 promoter was assessed using lacZ reporter assays. Mutations were generated using λ-Red-recombination. Results: The genetic structure of the ISEcp1-IS5-blaCTX-M-23 mobile element was determined and localized within the betU gene on the chromosome of EC958 and five other E. coli ST131 strains. The transcription of blaCTX-M-23, driven by a previously defined promoter within ISEcp1, was significantly higher than other β-lactamase genes and could be induced by cefotaxime. Deletion of the blaCTX-M-23 gene resulted in enhanced susceptibility to cefoxitin, cefotaxime and ceftazidime. Conclusions: This is the first known report to demonstrate the chromosomal location of blaCTX-M-23 in E. coli ST131. In EC958, CMY-23 plays a major role in resistance to third-generation cephalosporins and cephamycins. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]