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Yeung M.,University of Toronto | Balma-Mena A.,University of Toronto | Shear N.,University of Toronto | Shear N.,Sunnybrook Health science Center | And 7 more authors.
Microbes and Infection | Year: 2011

Staphylococcus aureus promotes the onset and severity of atopic dermatitis (AD), which is exacerbated by superantigen toxins SEB and SEC. The genetic identity of these isolates, and their relationship to common hospital- or community-associated methicillin resistant S. aureus (HA-MRSA and CA-MRSA) has not been defined. We conducted spa typing, partial multi-locus sequence typing (MLST), and toxin profiling (seb, sec, lukS-PV) of S. aureus from 119 pediatric and 40 adult AD patients. MLST clonal complexes CC45, CC5, CC15, CC1, CC8 and CC30 accounted for 79% of isolates, representing the same major groups reported for nosocomial S. aureus in hospital intensive care units. The highest disease severity was associated with CC1, which was significantly greater relative to CC15 (p = 0.017) or CC30 (p = 0.040), but with no significant difference relative to CC45, CC5 or CC8. Although there were two few lukS-PV, seb or sec isolates to infer a role in disease severity, CC45 was identified as a source of SEC producing strains, and lukS-PVL was associated with a small number of CC5 pediatric isolates. CC1 harbored the only CA-MRSA that was identified, and was a source of isolates that expressed both seb and sec, and closely resembled the USA400 strain of CA-MRSA. © 2010 Institut Pasteur. Source

News Article
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What if a single vaccine could protect people from infection by many different viruses? That concept is a step closer to reality. Researchers at Washington University School of Medicine​ in St. Louis have identified “broadly neutralizing” antibodies that protect against infection by multiple, distantly related alphaviruses – including Chikungunya virus – that cause fever and debilitating joint pain. The discovery, in mice, lays the groundwork for a single vaccine or antibody-based treatment against many different alphaviruses. “There is a lot of emphasis on identifying and understanding broadly neutralizing antibodies for other viruses — HIV, hepatitis C virus, dengue virus, influenza virus — but most of those antibodies neutralize different strains of the same virus,” said senior author Michael Diamond, M.D., Ph.D., a professor of medicine and director of the Division of Infectious Diseases and Vaccine Development in the Center for Human Immunology and Immunotherapy Programs. “What we’ve identified here are antibodies that actually neutralize several different alphaviruses.” The research is available online in the journal Cell and will appear in the Nov. 19 print issue.​​​​​​​​​​​​​​​ The viruses Diamond and colleagues studied are arthritogenic alphaviruses, so-named for their characteristic symptoms of fever followed by arthritis-like joint pain. These mosquito-borne viruses typically cause only sporadic outbreaks, although Chikungunya has been identified in Africa, Asia, Europe, South America and even the southern United States. In recent years, the virus has caused millions of infections annually across the globe. So far in 2015, 650 cases have been seen in U.S. residents, largely among travelers returning from the Caribbean, where they were infected. There is no vaccine or treatment for Chikungunya or other alphaviruses. As part of the study, Diamond and his colleagues screened 60 neutralizing mouse and human antibodies against Chikungunya and determined that 10 react against three or more different alphaviruses that cause arthritis-like symptoms. They also identified a small piece of the alphavirus called an epitope that is identical across the arthritogenic alphavirus family. In follow-up work in cell cultures, they showed that antibodies that recognize this epitope also protect against infection by multiple alphaviruses. The antibodies blocked multiple steps in the viral life cycle, including the virus’s ability to enter or exit host cells. To confirm that the antibodies could protect animals from disease, the researchers infected mice with three different alphaviruses: Chikungunya, the closely related O’nyong’nyong virus, or the more distantly related Mayaro virus. The mice then were treated with the each of two of the most potent broadly neutralizing antibodies, and both antibodies markedly reduced joint disease caused by any of the viruses. The researchers then identified a section of an alphavirus protein as the key binding site for the cross-protective antibodies. When such an antibody binds to this site, it changes the three-dimensional structure of the proteins on the surface of the virus, thus providing an explanation for how these antibodies prevent viral infection. “If you can make an antibody response against this region, you may be able to protect against many viruses in the family,” said Diamond. “Our group is making proteins now that focus on this epitope, and we’re planning to start immunizing animals soon to see if we generate the right kinds of antibodies.” People who are infected with alphaviruses produce antibodies against many viral epitopes, some of which are not protective. This could lead to a weak immune response that gives the virus time to multiply and cause disease. People who are immunized with proteins expressing the key epitope identified by the researchers, however, should be able to quickly produce the “right” protective antibodies, thereby short-circuiting the disease process. “We have more work to do but are encouraged that targeting this epitope could be a viable strategy for developing vaccines or treatments against Chikungunya and other related viruses that cause significant disease worldwide,” Diamond said. The research is supported by the National Institutes of Health (NIH); the Dutch Organization for Scientific Research; the University Medical Center Groningen; and a NRSA-Infectious Diseases training grant.

News Article | April 6, 2016
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A research team at Washington University School of Medicine in St. Louis has established a mouse model for testing of vaccines and therapeutics to battle Zika virus. The mouse model mimics aspects of the infection in humans, with high levels of the virus seen in the mouse brain and spinal cord, consistent with evidence showing that Zika causes neurological defects in human fetuses. Interestingly, the researchers detected the highest levels of the virus in the testes of male mice, a finding that supports clinical data indicating the virus can be sexually transmitted. The new research is published April 5 in Cell Host & Microbe. “Now that we know the mice can be vulnerable to Zika infection, we can use the animals to test vaccines and therapeutics – and some of those studies are already underway – as well as to understand the pathogenesis of the virus,” said senior author Michael Diamond, M.D., Ph.D., a professor of medicine at Washington University. The new model of Zika virus infection, along with another recently identified by scientists at the University of Texas Medical Branch, are the first to be developed since 1976. The earlier models were not as clinically relevant because the infections were generated by injecting the virus directly into the brain. In the new models, infection occurs via the skin, much like the bite of the mosquito that spreads the virus. The ongoing Zika virus outbreak in Latin America and the Caribbean has created an urgent need for identifying small animal models as a first step toward developing vaccines and treatments to fight the infection. The infection has been linked to microcephaly, a condition in which infants are born with unusually small heads and brain damage. In adults, the virus is thought to be related to rare cases of Guillian-Barré syndrome, an illness that can cause temporary paralysis. For the new study, researchers in Diamond’s laboratory, led by first author Helen Lazear, Ph.D., now at the University of North Carolina at Chapel Hill, tested five strains of the Zika virus in the mice: the original strain acquired from Uganda in 1947; three strains that circulated in Senegal in the 1980s; and the French Polynesian strain, which caused infections in 2013 and is nearly identical to the strain causing the current outbreak. All yielded similar results in the animals, suggesting that there may not be much difference in the pathogenicity between individual strains, at least in this mouse model. Tests with the viral strains from the current Zika outbreak are ongoing. Because Zika typically has trouble establishing infections in mice, the researchers used animals that were genetically altered so that they could not produce interferon, a key immune system signaling molecule, thus dampening the animals’ immune response to the virus. “If you take away interferon, the Zika virus replicates quite well in the mouse and goes to the sites that we see it causing disease in humans,” said Diamond, an expert in viral immunology. He also is a professor of molecular microbiology and of pathology and immunology. The immune-deficient mice lost weight, became lethargic and died within 10 days of infection. In contrast, normal laboratory mice included in the study only developed severe symptoms of Zika infection if they were infected soon after birth, under one week of age, before their immune systems were developed. That finding parallels what is seen in humans. “It appears that pregnant women infected with Zika can pass the virus to babies in utero and that newborns also may be susceptible to infection,” said Diamond, also an associate director of the university’s Center for Human Immunology and Immunotherapy Programs. “Other than in infants, we don’t really see severe disease in most people with Zika, except for a small fraction who develop Guillian-Barré.” He was inspired to pursue Zika research after a meeting at the National Institutes of Health (NIH) in June 2015, where Brazilian scientists described accounts of a rise in birth defects related to a local Zika outbreak. He returned to St. Louis and shifted several members of his lab to studying Zika, including developing mouse models of the disease. As new clinical information becomes available about the virus in humans, Diamond has pivoted his research to investigate suspected links in mice. “We looked for evidence of Zika in the mouse testes mostly as an afterthought, due to mounting evidence of sexual transmission and were surprised that viral levels were the highest we saw in any tissue,” Diamond noted. “We are now doing subsequent tests to determine how  long those viral levels are sustained, which could help us estimate the length of time Zika can be transmitted sexually.”

Ruan X.,Center for Human Immunology | Loyola D.E.,University of Santiago de Chile | Marolda C.L.,Center for Human Immunology | Perez-Donoso J.M.,University of Santiago de Chile | And 2 more authors.
Glycobiology | Year: 2012

WaaL is a membrane enzyme that catalyzes a key step in lipopolysaccharide (LPS) synthesis: the glycosidic bonding of a sugar at the proximal end of the undecaprenyl-diphosphate (Und-PP) O-antigen with a terminal sugar of the lipid A-core oligosaccharide (OS). Utilizing an in vitro assay, we demonstrate here that ligation with purified Escherichia coli WaaL occurs without adenosine-5′-triphosphate (ATP) and magnesium ions. Furthermore, E. coli and Pseudomonas aeruginosa WaaL proteins cannot catalyze ATP hydrolysis in vitro. We also show that a lysine substitution of the arginine (Arg)-215 residue renders an active protein, whereas WaaL mutants with alanine replacements in the periplasmic-exposed residues Arg-215, Arg-288 and histidine (His)-338 and also the membrane-embedded aspartic acid-389 are nonfunctional. An in silico approach, combining predicted topological information with the analysis of sequence conservation, confirms the importance of a positive charge at the small periplasmic loop of WaaL, since an Arg corresponding to Arg-215 was found at a similar position in all the WaaL homologs. Also, a universally conserved H[NSQ]X 9GXX[GTY] motif spanning the C-terminal end of the predicted large periplasmic loop and the membrane boundary of the transmembrane helix was identified. The His residue in this motif corresponds to His-338. A survey of LPS structures in which the linkage between O-antigen and lipid A-core OS was elucidated reveals that it is always in the β-configuration, whereas the sugars bound to Und-PP are in the-configuration. Together, our biochemical and in silico data argue that WaaL proteins use a common reaction mechanism and share features of metal ion-independent inverting glycosyltransferases. © 2011 The Author. Source

Patel K.B.,Center for Human Immunology | Ciepichal E.,Polish Academy of Sciences | Swiezewska E.,Polish Academy of Sciences | Valvano M.A.,Center for Human Immunology | Valvano M.A.,University of Western Ontario
Glycobiology | Year: 2012

Two families of membrane enzymes catalyze the initiation of the synthesis of O-antigen lipopolysaccharide. The Salmonella enterica Typhimurium WbaP is a prototypic member of one of these families. We report here the purification and biochemical characterization of the WbaP C-terminal (WbaPCT) domain harboring one putative transmembrane helix and a large cytoplasmic tail. An N-terminal thioredoxin fusion greatly improved solubility and stability of WbaPCT allowing us to obtain highly purified protein. We demonstrate that WbaPCT is sufficient to catalyze the in vitro transfer of galactose (Gal)-1-phosphate from uridine monophosphate (UDP)-Gal to the lipid carrier undecaprenyl monophosphate (Und-P). We optimized the in vitro assay to determine steady-state kinetic parameters with the substrates UDP-Gal and Und-P. Using various purified polyisoprenyl phosphates of increasing length and variable saturation of the isoprene units, we also demonstrate that the purified enzyme functions highly efficiently with Und-P, suggesting that the WbaPCT domain contains all the essential motifs to catalyze the synthesis of the Und-P-P-Gal molecule that primes the biosynthesis of bacterial surface glycans. © 2011 The Author. Source

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