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Elsa N. Bou Ghanem Grant S. Jones Tanya Myers-Morales Pooja D. Patil Achmad N. Hidayatullah Sarah E. F. D'Orazio 《PLoS pathogens》2012,8(11)
Intestinal Listeria monocytogenes infection is not efficient in mice and this has been attributed to a low affinity interaction between the bacterial surface protein InlA and E-cadherin on murine intestinal epithelial cells. Previous studies using either transgenic mice expressing human E-cadherin or mouse-adapted L. monocytogenes expressing a modified InlA protein (InlAm) with high affinity for murine E-cadherin showed increased efficiency of intragastric infection. However, the large inocula used in these studies disseminated to the spleen and liver rapidly, resulting in a lethal systemic infection that made it difficult to define the natural course of intestinal infection. We describe here a novel mouse model of oral listeriosis that closely mimics all phases of human disease: (1) ingestion of contaminated food, (2) a distinct period of time during which L. monocytogenes colonize only the intestines, (3) varying degrees of systemic spread in susceptible vs. resistant mice, and (4) late stage spread to the brain. Using this natural feeding model, we showed that the type of food, the time of day when feeding occurred, and mouse gender each affected susceptibility to L. monocytogenes infection. Co-infection studies using L. monocytogenes strains that expressed either a high affinity ligand for E-cadherin (InlAm), a low affinity ligand (wild type InlA from Lm EGDe), or no InlA (ΔinlA) showed that InlA was not required to establish intestinal infection in mice. However, expression of InlAm significantly increased bacterial persistence in the underlying lamina propria and greatly enhanced dissemination to the mesenteric lymph nodes. Thus, these studies revealed a previously uncharacterized role for InlA in facilitating systemic spread via the lymphatic system after invasion of the gut mucosa. 相似文献
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It has been found that with mutation of two surface residues (Lys22 → Glu and His104 → Arg) in human purine nucleoside phosphorylase (hPNP), there is an enhancement of catalytic activity in the chemical step. This is true although the mutations are quite remote from the active site, and there are no significant changes in crystallographic structure between the wild-type and mutant active sites. We propose that dynamic coupling from the remote residues to the catalytic site may play a role in catalysis, and it is this alteration in dynamics that causes an increase in the chemical step rate. Computational results indicate that the mutant exhibits stronger coupling between promotion of vibrations and the reaction coordinate than that found in native hPNP. Power spectra comparing native and mutant proteins show a correlation between the vibrations of Immucillin-G (ImmG):O5′…ImmG:N4′ and H257:Nδ…ImmG:O5′ consistent with a coupling of these motions. These modes are linked to the protein promoting vibrations. Stronger coupling of motions to the reaction coordinate increases the probability of reaching the transition state and thus lowers the activation free energy. This motion has been shown to contribute to catalysis. Coincident with the approach to the transition state, the sum of the distances of ImmG:O4′…ImmG:O5′…H257:Nδ became smaller, stabilizing the oxacarbenium ion formed at the transition state. Combined results from crystallography, mutational analysis, chemical kinetics, and computational analysis are consistent with dynamic compression playing a significant role in forming the transition state. Stronger coupling of these pairs is observed in the catalytically enhanced mutant enzyme. That motion and catalysis are enhanced by mutations remote from the catalytic site implicates dynamic coupling through the protein architecture as a component of catalysis in hPNP. 相似文献
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Transition-state structures of human and bovine of purine nucleoside phosphorylases differ, despite 87% homologous amino acid sequences. Human PNP (HsPNP) has a fully dissociated transition state, while that for bovine PNP (BtPNP) has early SN1 character. Crystal structures and sequence alignment indicate that the active sites of these enzymes are the same within crystallographic analysis, but residues in the second-sphere from the active sites differ significantly. Residues in BtPNP have been mutated toward HsPNP, resulting in double (Asn123Lys; Arg210Gln) and triple mutant PNPs (Val39Thr; Asn123Lys; Arg210Gln). Steady-state kinetic studies indicated unchanged catalytic activity, while pre-steady-state studies indicate that the chemical step is slower in the triple mutant. The mutant enzymes have higher affinity for inhibitors that are mimics of a late dissociative transition state. Kinetic isotope effects (KIEs) and computational chemistry were used to identify the transition-state structure of the triple mutant. Intrinsic KIEs from [1'-3H], [1'-14C], [2'-3H], [5'-3H], and [9-15N] inosines were 1.221, 1.035, 1.073, 1.062 and 1.025, respectively. The primary intrinsic [1'-14C] and [9-15N] KIEs indicate a highly dissociative SN1 transition state with low bond order to the leaving group, a transition state different from the native enzyme. The [1'-14C] KIE suggests significant nucleophilic participation at the transition state. The transition-state structure of triple mutant PNP is altered as a consequence of the amino acids in the second sphere from the catalytic site. These residues are implicated in linking the dynamic motion of the protein to formation of the transition state. 相似文献
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Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a nonspecific diesterase that enables Escherichia coli to utilize alkyl phosphodiesters, such as diethyl phosphate, as the sole phosphorus source. The catalytic properties of GpdQ were determined, and the best substrate found was bis(p-nitrophenyl) phosphate with a kcat/Km value of 6.7 x 10(3) M-1 s-1. In addition, the E. aerogenes diesterase was tested as a catalyst for the hydrolysis of a series of phosphonate monoesters which are the hydrolysis products of the highly toxic organophosphonate nerve agents sarin, soman, GF, VX, and rVX. Among the phosphonate monoesters tested, the hydrolysis product of rVX, isobutyl methyl phosphonate, was the best substrate with a kcat/Km value of 33 M-1 s-1. The ability of GpdQ to hydrolyze the phosphonate monoesters provides an alternative selection strategy in the search of enhanced variants of the bacterial phosphotriesterase (PTE) for the hydrolysis of organophosphonate nerve agents. This investigation demonstrated that the previously reported activity of GpdQ toward the hydrolysis of methyl demeton-S is due to the presence of a diester contaminant in the commercial material. Furthermore, it was shown that GpdQ is capable of hydrolyzing a close analogue of EA 2192, the most toxic and persistent degradation product of the nerve agent VX. 相似文献
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The effects of the trichothecene mycotoxins (acetyl T-2 toxin, T-2 toxin, HT-2 toxin, palmityl T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), and T-2 tetraol) on bovine platelet function were examined in homologous plasma stimulated with platelet activating factor (PAF). The mycotoxins inhibited platelet function with the following order of potency: acetyl T-2 toxin > palmityl T-2 toxin = DAS > HT-2 toxin = T-2 toxin. While T-2 tetraol was completely ineffective as an inhibitor, DON exhibited minimal inhibitory activity at concentrations above 10×10?4M. The stability of the platelet aggregates formed was significantly reduced in all mycotoxin treated platelets compared to that of the untreated PAF controls. It is suggested that the increased sensitivity of PAF stimulated bovine platelets to the more lipophilic mycotoxins may be related to their more efficient partitioning into the platelet membrane compared to the more hydrophilic compounds. 相似文献