Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Gram-negative bacteria naturally shed particles that consist of outer membrane lipids, outer membrane proteins, and soluble periplasmic components. These particles have been proposed for use as vaccines but the yield has been problematic. We developed a high yielding production process of genetically derived outer membrane particles from the human pathogen Shigella sonnei. Yields of approximately 100 milligrams of membrane-associated proteins per liter of fermentation were obtained from cultures of S. sonnei ΔtolR ΔgalU at optical densities of 30–45 in a 5 L fermenter. Proteomic analysis of the purified particles showed the preparation to primarily contain predicted outer membrane and periplasmic proteins. These were highly immunogenic in mice. The production of these outer membrane particles from high density cultivation of bacteria supports the feasibility of scaling up this approach as an affordable manufacturing process. Furthermore, we demonstrate the feasibility of using this process with other genetic manipulations e.g. abolition of O antigen synthesis and modification of the lipopolysaccharide structure in order to modify the immunogenicity or reactogenicity of the particles. This work provides the basis for a large scale manufacturing process of Generalized Modules of Membrane Antigens (GMMA) for production of vaccines from Gram-negative bacteria.

Background: GMMA from Gram-negative bacteria are an attractive vaccine technology, but lipopolysaccharide (LPS) reactogenicity limits use.Results: Genetic LPS modification resulting in penta-acylation reduced Shigella GMMA reactogenicity to a TLR2-mediated limit. Modifications resulting in palmitoleoylated hexa-acylated LPS triggered higher TLR4-mediated reactogenicity.Conclusion: Use of GMMA as vaccines will likely require LPS penta-acylation.Significance: Understanding the relative contribution of TLR activation guides GMMA vaccine development.

Invasive nontyphoidal Salmonella (iNTS) disease is a neglected disease with high mortality in children and HIV-positive individuals in sub-Saharan Africa, caused primarily by Africa-specific strains of Salmonella enterica serovars Typhimurium and Enteritidis. A vaccine using GMMA (generalized modules for membrane antigens) from S. Typhimurium and S. Enteritidis containing lipid A modifications to reduce potential in vivo reactogenicity is under development. GMMA with penta-acylated lipid A showed the greatest reduction in the level of cytokine release from human peripheral blood monocytes from that for GMMA with wild-type lipid A. Deletion of the lipid A modification genes msbB and pagP was required to achieve pure penta-acylation. Interestingly, ΔmsbB ΔpagP GMMA from S. Enteritidis had a slightly higher stimulatory potential than those from S. Typhimurium, a finding consistent with the higher lipopolysaccharide (LPS) content and Toll-like receptor 2 (TLR2) stimulatory potential of the former. Also, TLR5 ligand flagellin was found in Salmonella GMMA. No relevant contribution to the stimulatory potential of GMMA was detected even when the flagellin protein FliC from S. Typhimurium was added at a concentration as high as 10% of total protein, suggesting that flagellin impurities are not a major factor for GMMA-mediated immune stimulation. Overall, the stimulatory potential of S. Typhimurium and S. Enteritidis ΔmsbB ΔpagP GMMA was close to that of Shigella sonnei GMMA, which are currently in phase I clinical trials.

No abstract

Shigella is the leading cause for dysentery worldwide. Together with several virulence factors employed for invasion, the presence and length of the O antigen (OAg) of the lipopolysaccharide (LPS) plays a key role in pathogenesis. S. flexneri 2a has a bimodal OAg chain length distribution regulated in a growth-dependent manner, whereas S. sonnei LPS comprises a monomodal OAg. Here we reveal that S. sonnei, but not S. flexneri 2a, possesses a high molecular weight, immunogenic group 4 capsule, characterized by structural similarity to LPS OAg. We found that a galU mutant of S. sonnei, that is unable to produce a complete LPS with OAg attached, can still assemble OAg material on the cell surface, but a galU mutant of S. flexneri 2a cannot. High molecular weight material not linked to the LPS was purified from S. sonnei and confirmed by NMR to contain the specific sugars of the S. sonnei OAg. Deletion of genes homologous to the group 4 capsule synthesis cluster, previously described in Escherichia coli, abolished the generation of the high molecular weight OAg material. This OAg capsule strongly affects the virulence of S. sonnei. Uncapsulated knockout bacteria were highly invasive in vitro and strongly inflammatory in the rabbit intestine. But, the lack of capsule reduced the ability of S. sonnei to resist complement-mediated killing and to spread from the gut to peripheral organs. In contrast, overexpression of the capsule decreased invasiveness in vitro and inflammation in vivo compared to the wild type. In conclusion, the data indicate that in S. sonnei expression of the capsule modulates bacterial pathogenesis resulting in balanced capabilities to invade and persist in the host environment.

Cholesterol homeostasis is critical for cellular proliferation. Liver X receptor (LXR) ␣ and ␤ are the nuclear receptors responsible for regulation of cholesterol metabolism. In physiological conditions, high intracellular cholesterol levels cause increased synthesis of oxysterols, which activate LXR, thus triggering a transcriptional response for cholesterol secretion and catabolism. Here we employed a mouse model of partial hepatectomy (PH) to dissect the molecular pathways connecting cholesterol homeostasis, cellular proliferation, and LXR. First, we show that hepatic cholesterol content increases after PH, whereas the entire LXR transcriptome is down-regulated. Although LXR messenger RNA (mRNA) levels are unmodified, LXR target genes are significantly down-regulated on day 1 after PH and restored to control levels on day 7, when the liver reaches normal size. The inactivation of LXR following PH is related to the reduced oxysterol availability by way of decreased synthesis, and increased sulfation and secretion. On the contrary, cholesterol synthesis is up-regulated, and extracellular matrix remodeling is enhanced. Second, we show that reactivation of LXR by way of a synthetic ligand determines a negative modulation of hepatocyte proliferation. This effect is sustained by the reactivation of hepatic cholesterol catabolic and secretory pathways, coupled with a significant reduction of cholesterol biosynthesis. Our data unveil a previously unrecognized and apparently paradoxical scenario of LXR modulation. During liver regeneration LXR activity is abated in spite of increasing intracellular cholesterol levels. Turning off LXR-transcriptional pathways is crucial to guaranteeing the requisite intracellular cholesterol levels of regenerating hepatocytes. In line with this hypothesis, pharmacological LXR reactivation during PH significantly reduces liver regeneration capacity. (HEPATOLOGY 2010;51:1334-1344 C holesterol is not only the precursor of steroid hormones but also a regulator of embryonic development and cell proliferation, being a key component of the cell membrane. 1,2 Thus, there is a need of coordinate tuning of cholesterol metabolism and lipogenic gene expression during membrane synthesis, cellular differentiation, and proliferation. Proliferating cells satisfy their cholesterol demand by increasing their uptake of exogenous cholesterol 3 through an increase in low-density lipoprotein receptor. 4 When cholesterol synthesis is inhibited by HMG-CoA reductase inhibitor, cell growth is reduced, 5 whereas the subsequent addition of choles-

The opportunistic pathogen Escherichia coli, a common member of the human gut microbiota belonging to the Enterobacteriaceae family, is the causative agent of the majority of urinary tract infections (UTIs). The gut microbiota serves as a reservoir for uropathogenic E. coli where they are shed in feces, colonize the periurethral area, and infect the urinary tract. Currently, front line treatment for UTIs consists of oral antibiotics, but the rise of antibiotic resistance is leading to higher rates of recurrence, and antibiotics cause collateral damage to other members of the gut microbiota. It is commonly believed that incorporation of the American cranberry, Vaccinium macrocarpon, into the diet is useful for reducing recurrence of UTIs. We hypothesized such a benefit might be explained by a prebiotic or antimicrobial effect on the gut microbiota. As such, we tested cranberry extracts and whole cranberry powder on a human gut microbiome-derived community in a gut simulator and found that cranberry components broadly modulate the microbiota by reducing the abundance of Enterobacteriaceae and increasing the abundance of Bacteroidaceae. To identify the specific compounds responsible for this, we tested a panel of compounds isolated from cranberries for activity against E. coli, and found that salicylate exhibited antimicrobial activity against both laboratory E. coli and human UTI E. coli isolates. In a gut simulator, salicylate reduced levels of Enterobacteriaceae and elevated Bacteroidaceae in a dose dependent manner.