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Enterohemorrhagic (EHEC) is a major cause of foodborne gastrointestinal illness. EHEC uses a specialized type III secretion system (T3SS) to form attaching and effacing lesions in the colonic epithelium and outcompete commensal gut microbiota to cause disease. A recent report in (E. A. Cameron, M. M. Curtis, A. Kumar, G. M. Dunny, et al., mBio 9:e02204-18, 2018, https://doi.org/10.1128/mBio.02204-18) describes a new role for gut commensals in potentiating disease caused by EHEC. Proteases produced by EHEC and the prevalent human commensal cleave proteins in the EHEC T3SS translocon that modulate T3SS function. protease activity promotes translocation of bacterial effectors required for lesion formation. These results describe a new role for the microbiota in gastrointestinal disease that could uncover future treatments to prevent the spread of gastroenteritis.
Copyright © 2019 Palmer and Skaar.
Calprotectin is a potent antimicrobial that inhibits the growth of pathogens by tightly binding transition metals such as Mn and Zn, thereby preventing their uptake and utilization by invading microbes. At sites of infection, calprotectin is abundantly released from neutrophils, but calprotectin is also present in non-neutrophil cell types that may be relevant to infections. We show here that in patients infected with the Lyme disease pathogen Borreliella (Borrelia) burgdorferi, calprotectin is produced in neutrophil-free regions of the skin, in both epidermal keratinocytes and in immune cells infiltrating the dermis, including CD68 positive macrophages. In culture, B. burgdorferi's growth is inhibited by calprotectin, but surprisingly, the mechanism does not involve the classical withholding of metal nutrients. B. burgdorferi cells exposed to calprotectin cease growth with no reduction in intracellular Mn and no loss in activity of Mn enzymes including the SodA superoxide dismutase. Additionally, there is no obvious loss in intracellular Zn. Rather than metal depletion, we find that calprotectin inhibits B. burgdorferi growth through a mechanism that requires physical association of calprotectin with the bacteria. By comparison, calprotectin inhibited E. coli growth without physically interacting with the microbe, and calprotectin effectively depleted E. coli of intracellular Mn and Zn. Our studies with B. burgdorferi demonstrate that the antimicrobial capacity of calprotectin is complex and extends well beyond simple withholding of metal micronutrients.
State-of-the-art strategies for proteomics are not able to rapidly interrogate complex peptide mixtures in an untargeted manner with sensitive peptide and protein identification rates. We describe a data-independent acquisition (DIA) approach, microDIA (μDIA), that applies a novel tandem mass spectrometry (MS/MS) mass spectral deconvolution method to increase the specificity of tandem mass spectra acquired during proteomics experiments. Using the μDIA approach with a 10 min liquid chromatography gradient allowed detection of 3.1-fold more HeLa proteins than the results obtained from data-dependent acquisition (DDA) of the same samples. Additionally, we found the μDIA MS/MS deconvolution procedure is critical for resolving modified peptides with relatively small precursor mass shifts that cause the same peptide sequence in modified and unmodified forms to theoretically cofragment in the same raw MS/MS spectra. The μDIA workflow is implemented in the PROTALIZER software tool which fully automates tandem mass spectral deconvolution, queries every peptide with a library-free search algorithm against a user-defined protein database, and confidently identifies multiple peptides in a single tandem mass spectrum. We also benchmarked μDIA against DDA using a 90 min gradient analysis of HeLa and Escherichia coli peptides that were mixed in predefined quantitative ratios, and our results showed μDIA provided 24% more true positives at the same false positive rate.
Blood vessel epicardial substance (BVES), or POPDC1, is a tight junction-associated transmembrane protein that modulates epithelial-to-mesenchymal transition (EMT) via junctional signaling pathways. There have been no in vivo studies investigating the role of BVES in colitis. We hypothesized that BVES is critical for maintaining colonic epithelial integrity. At baseline, Bves mouse colons demonstrate increased crypt height, elevated proliferation, decreased apoptosis, altered intestinal lineage allocation, and dysregulation of tight junctions with functional deficits in permeability and altered intestinal immunity. Bves mice inoculated with Citrobacter rodentium had greater colonic injury, increased colonic and mesenteric lymph node bacterial colonization, and altered immune responses after infection. We propose that increased bacterial colonization and translocation result in amplified immune responses and worsened injury. Similarly, dextran sodium sulfate (DSS) treatment resulted in greater histologic injury in Bves mice. Two different human cell lines (Caco2 and HEK293Ts) co-cultured with enteropathogenic E. coli showed increased attaching/effacing lesions in the absence of BVES. Finally, BVES mRNA levels were reduced in human ulcerative colitis (UC) biopsy specimens. Collectively, these studies suggest that BVES plays a protective role both in ulcerative and infectious colitis and identify BVES as a critical protector of colonic mucosal integrity.
Complex II (SdhABCD) is a membrane-bound component of mitochondrial and bacterial electron transport chains, as well as of the TCA cycle. In this capacity, it catalyzes the reversible oxidation of succinate. SdhABCD contains the SDHA protein harboring a covalently bound FAD redox center and the iron-sulfur protein SDHB, containing three distinct iron-sulfur centers. When assembly of this complex is compromised, the flavoprotein SDHA may accumulate in the mitochondrial matrix or bacterial cytoplasm. Whether the unassembled SDHA has any catalytic activity, for example in succinate oxidation, fumarate reduction, reactive oxygen species (ROS) generation, or other off-pathway reactions, is not known. Therefore, here we investigated whether unassembled SdhA flavoprotein, its homolog fumarate reductase (FrdA), and the human SDHA protein have succinate oxidase or fumarate reductase activity and can produce ROS. Using recombinant expression in , we found that the free flavoproteins from these divergent biological sources have inherently low catalytic activity and generate little ROS. These results suggest that the iron-sulfur protein SDHB in complex II is necessary for robust catalytic activity. Our findings are consistent with those reported for single-subunit flavoprotein homologs that are not associated with iron-sulfur or heme partner proteins.
Individuals with sporadic colorectal cancer (CRC) frequently harbor abnormalities in the composition of the gut microbiome; however, the microbiota associated with precancerous lesions in hereditary CRC remains largely unknown. We studied colonic mucosa of patients with familial adenomatous polyposis (FAP), who develop benign precursor lesions (polyps) early in life. We identified patchy bacterial biofilms composed predominately of and Genes for colibactin () and toxin (), encoding secreted oncotoxins, were highly enriched in FAP patients' colonic mucosa compared to healthy individuals. Tumor-prone mice cocolonized with (expressing colibactin), and enterotoxigenic showed increased interleukin-17 in the colon and DNA damage in colonic epithelium with faster tumor onset and greater mortality, compared to mice with either bacterial strain alone. These data suggest an unexpected link between early neoplasia of the colon and tumorigenic bacteria.
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
Flavin is covalently attached to the protein scaffold in ~10% of flavoenzymes. However, the mechanism of covalent modification is unclear, due in part to challenges in stabilizing assembly intermediates. Here, we capture the structure of an assembly intermediate of the Escherichia coli Complex II (quinol:fumarate reductase (FrdABCD)). The structure contains the E. coli FrdA subunit bound to covalent FAD and crosslinked with its assembly factor, SdhE. The structure contains two global conformational changes as compared to prior structures of the mature protein: the rotation of a domain within the FrdA subunit, and the destabilization of two large loops of the FrdA subunit, which may create a tunnel to the active site. We infer a mechanism for covalent flavinylation. As supported by spectroscopic and kinetic analyses, we suggest that SdhE shifts the conformational equilibrium of the FrdA active site to disfavor succinate/fumarate interconversion and enhance covalent flavinylation.
There remains a need for new non-ionic detergents that are suitable for use in biochemical and biophysical studies of membrane proteins. Here we explore the properties of n-dodecyl-β-melibioside (β-DDMB) micelles as a medium for membrane proteins. Melibiose is d-galactose-α(1→6)-d-glucose. Light scattering showed the β-DDMB micelle to be roughly 30 kDa smaller than micelles formed by the commonly used n-dodecyl-β-maltoside (β-DDM). β-DDMB stabilized diacylglycerol kinase (DAGK) against thermal inactivation. Moreover, activity assays conducted using aliquots of DAGK purified into β-DDMB yielded activities that were 40% higher than those of DAGK purified into β-DDM. β-DDMB yielded similar or better TROSY-HSQC NMR spectra for two single-pass membrane proteins and the tetraspan membrane protein peripheral myelin protein 22. β-DDMB appears be a useful addition to the toolbox of non-ionic detergents available for membrane protein research.
The Complex II homolog quinol:fumarate reductase (QFR, FrdABCD) catalyzes the interconversion of fumarate and succinate at a covalently attached FAD within the FrdA subunit. The SdhE assembly factor enhances covalent flavinylation of Complex II homologs, but the mechanisms underlying the covalent attachment of FAD remain to be fully elucidated. Here, we explored the mechanisms of covalent flavinylation of the QFR FrdA subunit. Using a Δ strain, we show that the requirement for the assembly factor depends on the cellular redox environment. We next identified residues important for the covalent attachment and selected the FrdA residue, which contributes to proton shuttling during fumarate reduction, for detailed biophysical and structural characterization. We found that QFR complexes containing FrdA have a structure similar to that of the WT flavoprotein, but lack detectable substrate binding and turnover. In the context of the isolated FrdA subunit, the anticipated assembly intermediate during covalent flavinylation, FrdA variants had stability similar to that of WT FrdA, contained noncovalent FAD, and displayed a reduced capacity to interact with SdhE. However, small-angle X-ray scattering (SAXS) analysis of WT FrdA cross-linked to SdhE suggested that the FrdA residue is unlikely to contribute directly to the FrdA-SdhE protein-protein interface. We also found that no auxiliary factor is absolutely required for flavinylation, indicating that the covalent flavinylation is autocatalytic. We propose that multiple factors, including the SdhE assembly factor and bound dicarboxylates, stimulate covalent flavinylation by preorganizing the active site to stabilize the quinone-methide intermediate.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
The ability to de novo synthesize purines has been associated with the intracellular survival of multiple bacterial pathogens. Uropathogenic Escherichia coli (UPEC), the predominant cause of urinary tract infections, undergoes a transient intracellular lifestyle during which bacteria clonally expand into multicellular bacterial communities within the cytoplasm of bladder epithelial cells. Here, we characterized the contribution of the conserved de novo purine biosynthesis-associated locus cvpA-purF to UPEC pathogenesis. Deletion of cvpA-purF, or of purF alone, abolished de novo purine biosynthesis but did not impact bacterial adherence properties in vitro or in the bladder lumen. However, upon internalization by bladder epithelial cells, UPEC deficient in de novo purine biosynthesis was unable to expand into intracytoplasmic bacterial communities over time, unless it was extrachromosomally complemented. These findings indicate that UPEC is deprived of purine nucleotides within the intracellular niche and relies on de novo purine synthesis to meet this metabolic requirement.
Copyright © 2016 American Society for Microbiology.