Membrane Association and Destabilization by Aggregatibacter Actinomycetemcomitans Leukotoxin Requires Changes in Secondary Structures

Summary: Aggregatibacter actinomycetemcomitans is a common inhabitant of the upper aerodigestive tract of humans and non-human primates and is associated with disseminated infections, including lung and brain abscesses, pediatric infective endocarditis, and localized aggressive periodontitis. Aggregatibacter actinomycetemcomitans secretes a repeats-in-toxin protein, leukotoxin, which exclusively kills lymphocyte function-associated antigen-1-bearing cells. The toxin\u27s pathological mechanism is not fully understood; however, experimental evidence indicates that it involves the association with and subsequent destabilization of the target cell\u27s plasma membrane. We have long hypothesized that leukotoxin secondary structure is strongly correlated with membrane association and destabilization. In this study, we tested this hypothesis by analysing lipid-induced changes in leukotoxin conformation. Upon incubation of leukotoxin with lipids that favor leukotoxin-membrane association, we observed an increase in leukotoxin α-helical content that was not observed with lipids that favor membrane destabilization. The change in leukotoxin conformation after incubation with these lipids suggests that membrane binding and membrane destabilization have distinct secondary structural requirements, suggesting that they are independent events. These studies provide insight into the mechanism of cell damage that leads to disease progression by A. actinomycetemcomitans. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Isolation of Salmonella spp. and bacteriophage active against Salmonella spp. from commercial swine

Bacteriophage are viruses that prey on bacteria and may be a potential strategy to reduce foodborne pathogemc bactena in the gastromtestlnal tract of food animals Phages are fairly common in the gastrointestinal microbial ecosystem of mammals, but the incidence is unknown. If phage are to be an intervention strategy, we must understand their role in the microbial ecology of the gut. From a regulatory perspective, knowing incidence of phage is crucial. Therefore the current study was designed to determine the incidence of phage active against Salmonella spp in the feces of commercial finishing swine in the United States. Fecal samples (n=60) were collected from each of six commercial swine finishing operations. Samples were collected from 10 randomly selected pens throughout each operation. Total number of fecal samples collected in this study was n=360 Salmonella spp were found in 66% of the fecal samples Salmonella spp. were isolated from only 2 farms and the serotypes represented were Schwarzengrund, Anatum, Ohio and Heidelberg Bacteriophages were isolated from fecal sample through 2 parallel methods, 1) initlal enrichment in Salmonella Typhimunum, or 2) initial ennchment in E. colt B (a strain very sensitive to phages), followed by direct spot-testing against Salmonella Typhimurium Bacteriophages active against Salmonella Typhimunum were isolated from 1.1% 4/360) of the individual fecal samples when initially enriched in Salmonella Typhimurium, but E coli S-killing phages were 1solated from 43.8% (158/360) of the fecal samples but only 2 of these Isolates were capable of k1ll1ng Salmonella Typhimunum. Our results mdicate that bactenophage capable of killing Salmonella Typh1murium are fairly w1despread across commercial swine production facilities but may be present at relatively low populat1ons These results md1cate that phage (predator) populations may vary along w1th Salmonella (prey) populations and that phage could potentially be used as a food safety pathogen reduction strategy

Reduction of Campylobacter and Salmonella in pigs treated with A select nitrocompound

The aim of this study was to test the effectiveness of administering a select nitrocompound (S-NO) on reducing naturally colonized Campylobacter and experimentally infected Salmonella in the weaned pig gut. Pigs were divided into four groups; control (0 g S-NO/pig), 1X (0.2 g S-NO/pig), 5X (1 g S-NO/pig), and 10X (2 g S-NO/pig). Treatments were administered via oral gavage 24 h before sacrifice. Mean ± SD populations (log10 cfu/g) of Campylobacter in the cecum were reduced (P \u3c 0.05) in pigs receiving the 10X dose when compared with untreated controls (1.64 ± 1.30 vs 5.31 ± 0.58, respectively). Campylobacter concentrations in rectal contents from pigs administered the 5X dose were reduced (P \u3c 0.05) compared to control (2.65 ± 2.86 vs 5.90 ± 0.94, respectively). Rectal Salmonella concentrations were reduced (P \u3c 0.05) in all of the S-NO-treated groups. Adverse effects of S-NO on pig health were not observed. These results demonstrate that S-NO may have potential as an intervention to reduce pig colonization by Campylobacter and Salmonella

Effect of thymol or diphenyliodonium chloride on feed intake, average daily gain and gut Campylobacter concentrations in growing swine

Food producing animals can be reservoirs of Campylobacter, a leading bacterial cause of human foodborne illness. Campylobacter differ from most other gut bacteria in that they do not ferment carbohydrates but can utilize amino acids as major energy substrates, a process that can be inhibited by thymol and diphenyliodonium chloride (DIC). To evaluate palatability issues pertaining to feeding thymol or DIC, growing pigs were provided ad libitum access to standard growing diets supplemented with or without 0.0067 or 0.0201% thymol or 0.0001 4 or 0.00042% DIC in a replicated study design (n=4 pens per treatment per replicate, 2 pigs/pen). The diets were offered 2X/day for 7 days during which time average daily feed intake (2.39 ± 0.06 kg d-1; mean ± SEM) and average daily gain (0.62 ± 0.04 kg d-1) were not affected (P \u3e 0.05) by treatment

Weak Segregation Theory and Non-Conventional Morphologies in the Ternary ABC Triblock Copolymers

The Leibler weak segregation theory in molten diblock copolymers is generalized with due regard for the 2nd shell harmonics contributions defined in the paper and the phase diagrams are built for the linear and miktoarm ternary ABC triblock copolymers. The symmetric linear copolymers with the middle block non-selective with respect to the side ones are shown to undergo the continuous ODT not only into the lamellar phase but also into various non-conventional cubic phases (depending on the middle block composition it could be the simple cubic, face-centered cubic or non-centrosymmetric phase revealing the symmetry of space group No.214 first predicted to appear in molten block copolymers). For asymmetric linear ABC copolymers a region of compositions is found where the weakly segregated gyroid (double gyroid) phase exists between the planar hexagonal and lamellar or one of the non-conventional cubic phases up to the very critical point. In contrast, the miktoarm ABC block copolymers with one of its arm non-selective with respect to the two others are shown to reveal a pronounced tendency towards strong segregation, which is preceded by increase of stability of the conventional BCC phase and a peculiar weakly segregated BCC phase (BCC3), where the dominant harmonics belong to the 3rd co-ordination sphere of the reciprocal lattice. The validity region of the developed theory is discussed and outlined in the composition triangles both for linear and miktoarm copolymers.Comment: 61 pages, 12 figure

SNAREs Interact with Retinal Degeneration Slow and Rod Outer Segment Membrane Protein-1 during Conventional and Unconventional Outer Segment Targeting

The authors would like to thank Mr. Marc Banworth, Mr. Justin Burnett, and Ms. Jamie Watson for their technical assistance, Drs. Muayyad Al-Ubaidi and David Sherry for their comments on the manuscript, and Drs. Roger Janz, Roderick McInnes, Neeraj Agarwal, Vadim Arshavsky, Robert Molday and Anand Swaroop for the provision of reagents as indicated in the text.Mutations in the photoreceptor protein peripherin-2 (also known as RDS) cause severe retinal degeneration. RDS and its homolog ROM-1 (rod outer segment protein 1) are synthesized in the inner segment and then trafficked into the outer segment where they function in tetramers and covalently linked larger complexes. Our goal is to identify binding partners of RDS and ROM-1 that may be involved in their biosynthetic pathway or in their function in the photoreceptor outer segment (OS). Here we utilize several methods including mass spectrometry after affinity purification, in vitro co-expression followed by pull-down, in vivo pull-down from mouse retinas, and proximity ligation assay to identify and confirm the SNARE proteins Syntaxin 3B and SNAP-25 as novel binding partners of RDS and ROM-1. We show that both covalently linked and non-covalently linked RDS complexes interact with Syntaxin 3B. RDS in the mouse is trafficked from the inner segment to the outer segment by both conventional (i.e., Golgi dependent) and unconventional secretory pathways, and RDS from both pathways interacts with Syntaxin3B. Syntaxin 3B and SNAP-25 are enriched in the inner segment (compared to the outer segment) suggesting that the interaction with RDS/ROM-1 occurs in the inner segment. Syntaxin 3B and SNAP-25 are involved in mediating fusion of vesicles carrying other outer segment proteins during outer segment targeting, so could be involved in the trafficking of RDS/ROM-1.Yeshttp://www.plosone.org/static/editorial#pee