Summary

Natural products are often large, synthetically intractable molecules, yet frequently offer surprising inroads into previously unexplored chemical space for enzyme inhibitors. Argifin is a cyclic pentapeptide that was originally isolated as a fungal natural product. It competitively inhibits family 18 chitinases by mimicking the chitooligosaccharide substrate of these enzymes. Interestingly, argifin is a nanomolar inhibitor of the bacterial-type subfamily of fungal chitinases that possess an extensive chitin-binding groove, but does not inhibit the much smaller, plant-type enzymes from the same family that are involved in fungal cell division and are thought to be potential drug targets. Here we show that a small, highly efficient, argifin-derived nine-atom fragment is a micromolar inhibitor of the plant-type chitinase ChiA1 from the opportunistic pathogen Aspergillus fumigatus. Evaluation of the binding mode with the first crystal structure of an A. fumigatus plant-type chitinase reveals that the compound binds the catalytic machinery in the same manner as observed for argifin with the bacterial-type chitinases. The structure of the complex was used to guide synthesis of derivatives to explore a pocket near the catalytic machinery. This work provides synthetically tractable plant-type family 18 chitinase inhibitors from the repurposing of a natural product.

Legionnaires’ disease is caused by a lethal colonization of alveolar macrophages with the Gram-negative bacterium Legionella pneumophila. LpGT (L. pneumophila glucosyltransferase; also known as Lgt1) has recently been identified as a virulence factor, shutting down protein synthesis in the human cell by specific glucosylation of EF1A (elongation factor 1A), using an unknown mode of substrate recognition and a retaining mechanism for glycosyl transfer. We have determined the crystal structure of LpGT in complex with substrates, revealing a GT-A fold with two unusual protruding domains. Through structure-guided mutagenesis of LpGT, several residues essential for binding of the UDP-glucose-donor and EF1A-acceptor substrates were identified, which also affected L. pneumophila virulence as demonstrated by microinjection studies. Together, these results suggested that a positively charged EF1A loop binds to a negatively charged conserved groove on the LpGT structure, and that two asparagine residues are essential for catalysis. Furthermore, we showed that two further L. pneumophila glycosyltransferases possessed the conserved UDP-glucose-binding sites and EF1A-binding grooves, and are, like LpGT, translocated into the macrophage through the Icm/Dot (intracellular multiplication/defect in organelle trafficking) system.

Background

Although the risk of hepatitis B virus (HBV) was reported to be higher in military personnel than the general population in Saudi Arabia (SA), there is lack of studies assessing HBV awareness among them. The objective was to evaluate the knowledge, attitude and practice (KAP) of HBV infection among military personnel.

Methods

An intervention design with pre- and post-education KAP questionnaire was completed among National Guard soldiers working in Jeddah during January 2009. Educational intervention was provided through educational leaflets, group and individual discussions, visual show, and a lecture. A score was created from the correct answers to 58 questions.

Results

A total of 400 male soldiers with mean age 30.7 ± 6.1 years completed both questionnaires. The majority had school education (96.8%) and in the lower military ranks (66.0%). Only 19.5% of soldiers reported HBV vaccine intake. The low median and inter-quartile range of the pre-intervention score (16, 6–26) markedly increased after education (to 53, 50–55, p<0.001). The overall improvement of mean KAP score (204%) was also observed in all its component scores; disease nature (272%), methods of transmission (206%), prevention and control (109%), attitude (155%), and practice (192%). The improvement was evident irrespective of socio-demographic characteristics and history of HBV vaccine. KAP scores were significantly associated with higher educational levels, higher monthly income, administrative jobs, and higher job ranks.

Conclusion

We are reporting a low level of HBV awareness among Saudi military population. The study confirms the need and effectiveness of focused multifaceted educational campaigns among the military population.

O-GlcNAcylation of TAB1 modulates TAK1-mediated cytokine release

The protein kinase TAK1 plays an important role in pro-inflammatory cytokine signalling. Interleukin-1- and osmotic stress-induced O-GlcNAcylation of its regulatory subunit TAB1 is required for full TAK1 activation to induce downstream cytokine production, linking this protein modification to innate immunity signalling.

Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is a key serine/threonine protein kinase that mediates signals transduced by pro-inflammatory cytokines such as transforming growth factor-β, tumour necrosis factor (TNF), interleukin-1 (IL-1) and wnt family ligands. TAK1 is found in complex with binding partners TAB1–3, phosphorylation and ubiquitination of which has been found to regulate TAK1 activity. In this study, we show that TAB1 is modified with N-acetylglucosamine (O-GlcNAc) on a single site, Ser395. With the help of a novel O-GlcNAc site-specific antibody, we demonstrate that O-GlcNAcylation of TAB1 is induced by IL-1 and osmotic stress, known inducers of the TAK1 signalling cascade. By reintroducing wild-type or an O-GlcNAc-deficient mutant TAB1 (S395A) into Tab1−/− mouse embryonic fibroblasts, we determined that O-GlcNAcylation of TAB1 is required for full TAK1 activation upon stimulation with IL-1/osmotic stress, for downstream activation of nuclear factor κB and finally production of IL-6 and TNFα. This is one of the first examples of a single O-GlcNAc site on a signalling protein modulating a key innate immunity signalling pathway.

Vibrio cholerae is a bacterial pathogen that colonizes the chitinous exoskeleton of zooplankton as well as the human gastrointestinal tract. Colonization of these different niches involves an N-acetylglucosamine binding protein (GbpA) that has been reported to mediate bacterial attachment to both marine chitin and mammalian intestinal mucin through an unknown molecular mechanism. We report structural studies that reveal that GbpA possesses an unusual, elongated, four-domain structure, with domains 1 and 4 showing structural homology to chitin binding domains. A glycan screen revealed that GbpA binds to GlcNAc oligosaccharides. Structure-guided GbpA truncation mutants show that domains 1 and 4 of GbpA interact with chitin in vitro, whereas in vivo complementation studies reveal that domain 1 is also crucial for mucin binding and intestinal colonization. Bacterial binding studies show that domains 2 and 3 bind to the V. cholerae surface. Finally, mouse virulence assays show that only the first three domains of GbpA are required for colonization. These results explain how GbpA provides structural/functional modular interactions between V. cholerae, intestinal epithelium and chitinous exoskeletons.

Author Summary

Vibrio cholerae is the bacterium that causes cholera, a disease endemic in developing countries with poor sanitation. The bacterium colonizes aquatic organisms that serve as a reservoir of transmission to humans. Our work has focused on GbpA, a protein that is secreted by V. cholerae and appears to facilitate growth of the bacteria both in the human intestine and on the exoskeletons of marine organisms. We show that the protein possesses an unusual three-dimensional structure consisting of four separate domains. Two of the domains are similar to proteins that are known to bind chitin, an exoskeleton biopolymer, and our data show that these domains indeed harbour the chitin binding properties of GbpA. One of these domains is also capable of binding intestinal mucus. The two remaining domains are required for interacting with the bacterium itself, creating a stable interface between the bacterium and the human/marine host, facilitating colonization. Finally, work with a cholera mouse model shows that only the first three domains of GbpA are required for colonization. These results show how GbpA provides structural/functional modular interactions between V. cholerae, the intestinal epithelium and chitinous exoskeletons.

BACKGROUND AND OBJECTIVES:

Few studies have been conducted in Saudi Arabia to estimate the prevalence of visual impairment and its causes. The objective of this study was to estimate the prevalence of visual impairment, and identify its causes and associated factors among the adult population attending primary health care (PHC) centers in Aljouf province, in northern Saudi Arabia.

DESIGN AND SETTING:

A cross-sectional study during the year 2005 in PHC centers in Aljouf province in northern Saudi Arabia

PATIENTS AND METHODS:

A sample of 620 Saudi adults, of age 18 years and older, from the catchment area of the Aljouf PHC centers, were randomly selected through a multistage random sampling technique. Data were collected using a questionnaire about socioeconomic and related information and a visual acuity test was performed using the Snellen chart (E). Diagnosis was established according to World Health Organization (WHO) criteria. Visual impairment was categorized into blindness for a visual acuity of less than 3/60 (20/400, 0.05) in the better eye with the best correction and low vision for a best corrected visual acuity of less than 6/18 (20/60, 0.3) but not less than 3/60 (20/400, 0.05) in the better eye. Regression analysis was used to identify the predictors of visual impairment.

RESULTS:

Of 617 adult Saudis interviewed and examined, 269 (43.6%) were females. The mean (SD) age was 38.6 (16.2) years. The overall prevalence of visual impairment was 13.9% (95% CI: 11.4%-16.9%). The main medical causes of visual impairments were refractive errors (36.0%) followed by cataract (29.1%) and diabetic retinopathy (20.9%), and the least leading cause was glaucoma (5.8%). The most prominent determinants of visual impairment were age (P<.05), sex (P<.001), and a history of previous eye injury (P<.05).

CONCLUSION:

Prevalence of visual impairment in the study population from the Aljouf area is high. It is recommended that regular checks of visual acuity be conducted for all Saudis of age 50+ years, who attend the PHC centers.

Post-translational modification of protein serines/threonines with N-acetylglucosamine (O-GlcNAc) is dynamic, inducible and abundant, regulating many cellular processes by interfering with protein phosphorylation. O-GlcNAcylation is regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase, both encoded by single, essential, genes in metazoan genomes. It is not understood how OGT recognises its sugar nucleotide donor and performs O-GlcNAc transfer onto proteins/peptides, and how the enzyme recognises specific cellular protein substrates. Here, we show, by X-ray crystallography and mutagenesis, that OGT adopts the (metal-independent) GT-B fold and binds a UDP-GlcNAc analogue at the bottom of a highly conserved putative peptide-binding groove, covered by a mobile loop. Strikingly, the tetratricopeptide repeats (TPRs) tightly interact with the active site to form a continuous 120 Å putative interaction surface, whereas the previously predicted phosphatidylinositide-binding site locates to the opposite end of the catalytic domain. On the basis of the structure, we identify truncation/point mutants of the TPRs that have differential effects on activity towards proteins/peptides, giving first insights into how OGT may recognise its substrates.