Michael Hayward

Enteric defensins are essential regulators of intestinal microbial ecology.

Antimicrobial peptides are important effectors of innate immunity throughout the plant and animal kingdoms. In the mammalian small intestine, Paneth cell α-defensins are antimicrobial peptides that contribute to host defense against enteric pathogens. To determine if α-defensins also govern intestinal microbial ecology, we analyzed the intestinal microbiota of mice expressing a human α-defensin gene (DEFA5) and in mice lacking an enzyme required for the processing of mouse α-defensins. In these complementary models, we detected significant α-defensin-dependent changes in microbiota composition, but not in total bacterial numbers. Furthermore, DEFA5-expressing mice had striking losses of segmented filamentous bacteria and fewer interleukin 17 (IL-17)-producing lamina propria T cells. Our data ascribe a new homeostatic role to α-defensins in regulating the makeup of the commensal microbiota.

Technetium-99m sestamibi uptake in human breast carcinoma cell lines displaying glutathione-associated drug-resistance

An in vitro study was designed to evaluate the uptake of sestamibi (MIBI) in P-glycoprotein (Pgp) and glutathione-associated (GSH) multidrug-resistant (MDR) cell lines. MIBI uptake was studied in various human breast carcinoma cell lines, i.e. in wild-type (MCF7/wt) cells, in adriamycin-resistant (MCF7/adr) cells which express Pgp and in melphalan-resistant (MCF7/mph) cells with increased levels of GSH. The effects of buthiomine sulphoximine (BSO) and verapamil on MIBI uptake were also studied in the MCF7/mph and MCF7/adr cells respectively. The cells were incubated for 1 h with a dose of 0.1 MBq thallium-201 and technetium-99m MIBI. Both MIBI and201Tl uptakes were higher for MCF7/mph cells than for the other cells studied. The mean MIBI uptake in MCF7/adr cells was significantly lower than that in MCF7/wt cells (1.9%±0.5% vs 3.1%.0.6%;P <0.01). Verapamil treatment increased the MIBI uptake in MCF7/adr cells (to 2.6%.0.3%;P <0.05). Treatment of MCF7/mph cells with BSO resulted in a significant reduction in GSH content (from 243.2±81.1 nmoUmg protein to 17.6±4.4 nmol/mg protein;P <0.001). However, MIBI uptake in BSO-treated and untreated MCF7/mph cells was similar (4.43%±0.5% and 5.93%±1.7%, respectively;P >0.1). This study suggests that the uptake of MIBI is not diminished by glutathione-associated drug resistance and that MIBI uptake in a tumour sample does not necessarily indicate that a cancer is sensitive to drugs.

Sulfonic acids: l-Homocysteinesulfonic acid

Publisher Summary Sulfonic acids (RSO 3 H) are strongly acidic compounds containing organic sulfur in its most oxidized state. Sulfonic acids are among the most stable of organic sulfur derivatives. The chemical stability of cysteic acid (cysteinesulfonic acid) and the ease with which less oxidized forms of cyst(e)ine can be converted to that compound account for the frequent determination of protein cyst(e)ine as cysteic acid during ion-exchange amino acid analysis. Cysteinesulfonate has also received attention in biochemical studies as an analog of aspartate and cysteinesulfinate. Homocysteinesulfonate is of interest as a glutamate, homocysteinesulfinate, or aspartate analog in enzymological , neurophysiological, and amino acid transport studies. Sulfonic acids are easily prepared by oxidation of thiols, disulfides, sulfenic acids and sulfinic acids; the choice o f starting material is governed by availability. A variety of oxidizing agents have been used: bromine, hydrogen peroxide, performic acid, p - nitroperbenzoic acid, and dimethyl sulfoxide. The procedure given in the chapter for the synthesis of L-homocysteinesulfonic acid is based on studies by Friedmann and Greenstein and Winitz. In the synthesis of L-homocysteinesulfonic acid, Comparable yields are obtained from homocysteine and homocysteine thiolactone. Although the D- and L-enantiomers of homocyst(e)ine are available commercially, for larger scale preparations it may be preferable to prepare them by reduction of the much less expensive D- and L-methionine using sodium in liquid ammonia. With larger scale preparations, the temperature may increase markedly during the addition of bromine, for example, to 60°.

Intestinal Microbiota Disruption Reduces Regulatory T Cells and Increases Respiratory Viral Infection Mortality Through Increased IFNγ Production

Alterations in gastrointestinal microbiota indirectly modulate the risk of atopic disease, but effects on respiratory viral infections are less clear. Using the murine paramyxoviral virus type 1, Sendai virus (SeV), we examined the effect of altering gastrointestinal microbiota on the pulmonary antiviral immune response. C57BL6 mice were treated with streptomycin before or during infection with SeV and resulting immune response studied. Ingestion of the non-absorbable antibiotic streptomycin led to a marked reduction in intestinal microbial diversity without a significant effect on lung microbiota. Reduction in diversity in the gastrointestinal tract was followed by greatly increased mortality to respiratory viral infection (p < 0.0001). This increase in mortality was associated with a dysregulated immune response characterized by decreased lung (p = 0.01) and intestinal (p = 0.03) regulatory T cells (Tregs), and increased lung IFNγ (p = 0.049), IL-6 (p = 0.015), and CCL2 (p = 0.037). Adoptive transfer of Treg cells or neutralization of IFNγ prevented increased mortality. Furthermore, Lin−CD4+ cells appeared to be a potential source of the increased IFNγ. Together, these results demonstrate gastrointestinal microbiota modulate immune responses at distant mucosal sites and have the ability to significantly impact mortality in response to a respiratory viral infection.

Engineered E. coli Nissle 1917 for the reduction of vancomycin-resistant Enterococcus in the intestinal tract

Abstract Vancomycin‐resistant Enterococcus (VRE) poses a serious threat in hospitals where they densely colonize the intestinal tracts of patients. In vulnerable hosts, these pathogens may translocate to the bloodstream and become lethal. The ability to selectively reduce VRE in the intestinal tracts of patients could potentially prevent many of these translocation events and reduce the spread of the pathogen. Herein, we have engineered Escherichia. coli Nissle 1917 to produce and secrete three antimicrobial peptides, Enterocin A, Enterocin B, and Hiracin JM79, to specifically target and kill Enterococcus. These peptides exhibited potent activity against both Enterococcus faecium and Enterococcus faecalis, the two most prominent species responsible for VRE infections. We first discuss the optimization of the system used to express and secrete the peptides. We then show that by simultaneously expressing these peptides, both E. faecium and E. faecalis were drastically inhibited. We then demonstrate a suppression of the development of resistance when supernatant from the E. coli producer strains was used to treat E. faecium. Finally, we tested the efficacy of the probiotic in a VRE colonization model in mice. These studies showed that administration of the engineered probiotic significantly reduced the levels of both E. faecium and E. faecalis in the feces of male Balb/cJ mice.

Modulators of Enterococcus faecalis cell envelope integrity and antimicrobial resistance influence stable colonization of the mammalian gastrointestinal tract

ABSTRACT The Gram-positive bacterium Enterococcus faecalis is both a colonizer of the gastrointestinal tract (GIT) and an agent of serious nosocomial infections. Although it is typically required for pathogenesis, GIT colonization by E. faecalis is poorly understood. E. faecalis tolerates high concentrations of GIT antimicrobials, like cholate and lysozyme, leading us to hypothesize that resistance to intestinal antimicrobials is essential for long-term GIT colonization. Analyses of E. faecalis mutants exhibiting defects in antimicrobial resistance revealed that IreK, a determinant of envelope integrity and antimicrobial resistance, is required for long-term GIT colonization. IreK is a member of the PASTA kinase protein family, bacterial transmembrane signaling proteins implicated in the regulation of cell wall homeostasis. Among several determinants of cholate and lysozyme resistance in E. faecalis, IreK was the only one found to be required for intestinal colonization, emphasizing the importance of this protein to enterococcal adaptation to the GIT. By studying ΔireK suppressor mutants that recovered the ability to colonize the GIT, we identified two conserved enterococcal proteins (OG1RF_11271 and OG1RF_11272) that function antagonistically to IreK and interfere with cell envelope integrity, antimicrobial resistance, and GIT colonization. Our data suggest that IreK, through its kinase activity, inhibits the actions of these proteins. IreK, OG1RF_11271, and OG1RF_11272 are found in all enterococci, suggesting that their effect on GIT colonization is universal across enterococci. Thus, we have defined conserved genes in the enterococcal core genome that influence GIT colonization through their effect on enterococcal envelope integrity and antimicrobial resistance.