Purified EDEM3 or EDEM1 alone produces determinant oligosaccharide structures from M8B in mammalian glycoprotein ERAD.

Sequential mannose trimming of N-glycan, from M9 to M8B and then to oligosaccharides exposing the α1,6-linked mannosyl residue (M7A, M6, and M5), facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). We previously showed that EDEM2 stably disulfide-bonded to the thioredoxin domain-containing protein TXNDC11 is responsible for the first step (George et al., 2020). Here, we show that EDEM3 and EDEM1 are responsible for the second step. Incubation of pyridylamine-labeled M8B with purified EDEM3 alone produced M7 (M7A and M7C), M6, and M5. EDEM1 showed a similar tendency, although much lower amounts of M6 and M5 were produced. Thus, EDEM3 is a major α1,2-mannosidase for the second step from M8B. Both EDEM3 and EDEM1 trimmed M8B from a glycoprotein efficiently. Our confirmation of the Golgi localization of MAN1B indicates that no other α1,2-mannosidase is required for gpERAD. Accordingly, we have established the entire route of oligosaccharide processing and the enzymes responsible

<i>Campylobacter jejuni pdxA</i> Affects Flagellum-Mediated Motility to Alter Host Colonization

<div><p>Vitamin B6 (pyridoxal-5'-phosphate, PLP) is linked to a variety of biological functions in prokaryotes. Here, we report that the <i>pdxA</i> (putative 4-hydroxy-L-threonine phosphate dehydrogenase) gene plays a pivotal role in the PLP-dependent regulation of flagellar motility, thereby altering host colonization in a leading foodborne pathogen, <i>Campylobacter jejuni</i>. A <i>C. jejuni pdxA</i> mutant failed to produce PLP and exhibited a coincident loss of flagellar motility. Mass spectrometric analyses showed a 3-fold reduction in the main flagellar glycan pseudaminic acid (Pse) associated with the disruption of <i>pdxA</i>. The <i>pdxA</i> mutant also exhibited reduced growth rates compared with the WT strain. Comparative metabolomic analyses revealed differences in respiratory/energy metabolism between WT <i>C. jejuni</i> and the <i>pdxA</i> mutant, providing a possible explanation for the differential growth fitness between the two strains. Consistent with the lack of flagellar motility, the <i>pdxA</i> mutant showed impaired motility-mediated responses (bacterial adhesion, ERK1/2 activation, and IL-8 production) in INT407 cells and reduced colonization of chickens compared with the WT strain. Overall, this study demonstrated that the <i>pdxA</i> gene affects the PLP-mediated flagellar motility function, mainly through alteration of Pse modification, and the disruption of this gene also alters the respiratory/energy metabolisms to potentially affect host colonization. Our data therefore present novel implications regarding the utility of PLP and its dependent enzymes as potent target(s) for the control of this pathogen in the poultry host.</p></div

Deletion of the <i>pdxA</i> gene impairs <i>in vitro</i> cellular responses and <i>in vivo</i> colonization.

<p>(A) INT407 cells were infected for 1 h with the <i>C. jejuni</i> WT, pdxA−, pdxA−/+, and flaA− strains. The number of cell-adherent bacteria was measured by counting the plates after washing three times with PBS. (B) ERK1/2 activation upon infection. Western blotting was performed to detect the levels of phosphorylated and total ERK1/2 in the lysates from infected cells. (C) IL-8 production in INT407 cells was measured at 4 h and 16 h <i>p.i. via</i> ELISA. The data are presented in sections A and C as the mean values ± standard deviations from samples run in duplicate in at least three experiments. (D) Disruption of the <i>pdxA</i> gene reduces the colonization of the chicken cecum by <i>C. jejuni</i>. Groups of 14-day-old chickens (n = 10 per group) were orally inoculated with approximately 3×10⁷ CFU of WT or <i>pdxA</i> mutant <i>C. jejuni</i>. At 1 week and 4 weeks <i>p.i.</i>, the ceca were aseptically removed from the infected animals (n = 5 for each time point) and homogenized. Serial dilutions of the suspensions were plated on mCCDA agar to count CFU numbers. The closed diamonds and open circles represent the numbers of WT and <i>pdxA</i> mutant CFUs recovered from the animals, respectively.</p

Representative metabolites that are altered between the <i>C. jejuni</i> WT and <i>pdxA</i> mutant strains.

<p>The detected metabolites exhibiting >2.0-fold differences between the WT and pdxA- strains are shown. Each mean represents average from two independent tests. Candidate compounds are identified based on the detection peak (<i>m</i>/<i>z</i>)^*1 and migration time (MT)^*2 through HTM database. ^*3 Relative mean of the pdxA-/WT ratio. Full lists are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070418#pone.0070418.s006" target="_blank">Table S2</a>.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Inactivation of the <i>pdxA</i> gene impairs the biosynthesis of vitamin B6 (PLP) in <i>C. jejuni</i>.

<p>(A) A scheme for the PLP production pathway (right box) in <i>C. jejuni</i> in relation to Pse biosynthesis (left box) is illustrated based on <i>in silico</i> pathway analysis performed using PATRIC (<a href="http://patricbrc.vbi.vt.edu/portal/portal/patric/Home" target="_blank">http://patricbrc.vbi.vt.edu/portal/portal/patric/Home</a>). (B) The <i>pdxA</i> mutant produced no PLP. The <i>C. jejuni</i> 81–176 WT, <i>pdxA</i> mutant, and the complemented strains were grown in 10ml of MH broth to an OD₆₀₀ of 0.60. The suspensions were then homogenized, serially diluted, and subjected to ELISA to quantify the amounts of PLP (μg 10 ml⁻¹). The data show the mean +/− standard deviations from three independent assays.</p

Reactivity of lectins against proteins in the mouse testicular TS fraction co-immunoprecipitated with Ts4.

<p>The immunoprecipitated proteins from testicular TS fraction with either Ts4 or normal control IgM (n.c.) were separated by SDS-PAGE under reducing conditions. Control experiments were conducted under the same conditions except for the absence of the TS fraction (buf). The testicular TS fraction was used as a positive control (IP (-)). Proteins were electroblotted onto PVDF membranes and then probed with E-PHA (A), PSA (B), WGA (C), DSA (D), L-PHA (E), DBA (F), or SJA (G). Arrowheads indicate the lectin-reactive bands corresponding to TEX101. Mr, molecular mass.</p