Biology subjects

Stupak, J.

Publications and source records attributed to Stupak, J..

4 recordsLinked to original sources

bioRxiv2025

Biosynthesis of glycosylated 5-hydroxycytosine in the DNA of diverse viruses

Characterizing viral strategies for circumventing cellular defenses is critical to understanding the biology and therapeutic application of bacteriophages as antimicrobials. Many bacteriophages synthesize complex modifications (i.e., hypermodifications) to the nucleobases of their virion DNA in order to circumvent the endonuclease-based defenses of their hosts. To date, most hypermodified pyrimidines in viruses are synthesized via group transfer to a pre-existing hydroxyl moiety in DNA during the later stages of lytic development leading up to packaging of the viral chromosome into the capsid. Typically, these occur at hydroxymethylcytosine or hydroxymethyluracil. We find mono- and poly-arabinosylated cytidines completely replacing canonical cytidine in the native DNA of the Escherichia phage RB69 and Acinetobacter baumannii phage DLP3. In both cases, an arabinosyl moiety is connected directly to the pyrimidine through an ether linkage with the bridging oxygen originating from 5-hydroxycytosine (5hoC) in DNA. The 5hoC is synthesized as a mononucleotide by a virally encoded thymidylate synthase homolog. We find diverse virally encoded thymidylate synthase homologs, including the Rhizobium phage RL38J1, are capable of producing 5hoC suggesting this base modification is a starting point for glycodiverse cytosine derivatives. Characterizing these cytosine derivatives adds to our understanding of viral anti-defense mechanisms. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/659550v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@fcc165org.highwire.dtl.DTLVardef@1fced2aorg.highwire.dtl.DTLVardef@369ceaorg.highwire.dtl.DTLVardef@177df60_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

Safety and uptake of fully oxidized β-carotene

Spontaneous oxidation of {beta}-carotene yields a polymer-rich product (OxBC) also containing small amounts of many apocarotenoids. OxBC extends {beta}-carotenes benefits beyond vitamin A, finding utility in supporting health in livestock, pets, and humans. Although naturally occurring OxBC is consumed in foods and feeds, a direct demonstration here of safety of synthetic OxBC supports its increasing usage. A toxicological study in rats showed a maximum tolerated single oral dose, an LD50, and a NOAEL of 5,000, 30,079 and 1875 mg/kg body weight, respectively. The repeat-dose 90-day oral toxicity study showed no adverse physiological or pathological effects. A first study of OxBC uptake by mice over 2-5 days into a select set of tissues showed OxBC already was naturally present. The highest levels were in liver, lung, and hamstring. Despite dosing, no net increases occurred in liver, kidney, lung, and muscle. Net increases occurred in urine, intestinal content, plasma, feces, spleen, and cecum, consistent with processing of OxBC and preferential elimination of polymer. Compared to the 4:1 polymer: apocarotenoid ratio of OxBC, polymer was enriched in liver and spleen and depleted in lung, kidney, hamstring, and abdominal muscle. The apparent control of OxBC in major tissues further supports its safety.

pharmacology and toxicology↗

bioRxiv2018

A novel sialylation site on Neisseria gonorrhoeae lipooligosaccharide links heptose II lactose expression with pathogenicity

Sialylation of lacto-W-neotetraose (LNnT) extending from heptose I (HepI) of gonococcal lipooligosaccharide (LOS) contributes to pathogenesis. Previously, gonococcal LOS sialyltransterase (Lst) was shown to sialylate LOS in Triton X-100 extracts of strain 15253, which expresses lactose from both HepI and HepII, the minimal structure required for mAb 2C7 binding. Ongoing work has shown that growth of 15253 in cytidine monophospho-W-acetylneuraminic acid (CMP-Neu5Ac)-containing media enables binding to CD33/Siglec-3, a cell surface receptor that binds sialic acid, suggesting that lactose termini on LOS of intact gonococci can be sialylated. Neu5Ac was detected on LOSs of strains 15253 and a MS11 mutant with only lactose from HepI and HepII by mass spectrometry; deleting HepII lactose rendered Neu5Ac undetectable. Resistance of HepII lactose Neu5Ac to desialylation by 2-3-specific neuraminidase suggested an 2-6-linkage. Although not associated with increased factor H binding, HepII lactose sialylation inhibited complement C3 deposition on gonococci. 15253 mutants that lacked Lst or HepII lactose were significantly attenuated in mice, confirming the importance of HepII Neu5Ac in virulence. All 75 minimally passaged clinical isolates from Nanjing, China, expressed HepII lactose, evidenced by reactivity with mAb 2C7; mAb 2C7 was bactericidal against the first 62 (of 75) isolates that had been collected sequentially and were sialylated before testing. mAb 2C7 effectively attenuated 15253 vaginal colonization in mice. In conclusion, this novel sialylation site could explain the ubiquity of gonococcal HepII lactose in vivo. Our findings reiterate the candidacy of the 2C7 epitope as a vaccine antigen and mAb 2C7 as an immunotherapeutic antibody.

microbiology↗

bioRxiv2018

Genetics behind the biosynthesis of nonulosonic acid containing lipooligosaccharides in Campylobacter coli

Campylobacter jejuni and Campylobacter coli are the most common cause of bacterial gastroenteritis in the world. Ganglioside mimicry by C. jejuni lipooligosaccharide (LOS) is the triggering factor of Guillain-Barre syndrome (GBS), an acute polyneuropathy. Sialyltransferases from the glycosyltransferase (GT) family 42 are essential for the expression of ganglioside mimics in C. jejuni. Recently, two novel GT-42 genes, cstIV and cstV, have been identified in C. coli. Despite being present in [~]11% of currently available C. coli genomes, the biological role of cstIV and cstV is unknown. In the present study, mutation studies in two strains expressing either cstIV or cstV were performed and mass spectrometry was used to investigate differences in the chemical composition of LOS. Attempts were made to identify donor and acceptor molecules using in vitro activity tests with recombinant GT-42 enzymes. Here, we show that CstIV and CstV are involved in C. coli LOS biosynthesis. In particular, cstV is associated with LOS sialylation, while cstIV is linked to the addition of a diacetylated nonulosonic acid residue.\n\nIMPORTANCEDespite being a major foodborne pathogen, Campylobacter coli glycobiology has been largely neglected. The genetic makeup of the C. coli lipooligosaccharide biosynthesis locus was largely unknown until recently. C. coli harbour a large set of genes associated to lipooligosaccharide biosynthesis, including several putative glycosyltransferases involved in the synthesis of sialylated lipooligosaccharide in Campylobacter jejuni. In the present study, C. coli was found to express lipooligosaccharide structures containing sialic acid and other nonulosonate acids. These findings have a strong impact in understanding C. coli ecology, host-pathogen interaction, and pathogenesis.

microbiology↗