Cytolytic leukotoxins of the repeat in toxin (RTX) family are large proteins excreted by gram-negative bacterial pathogens through the type 1 secretion system (T1SS). Due to low yields and poor stability in cultures of the original pathogens, it is useful to purify recombinant fatty-acylated RTX cytolysins from inclusion bodies produced in E. coli. Such preparations are, however, typically contaminated by high amounts of E. coli lipopolysaccharide (LPS or endotoxin). We report a simple procedure for purification of large amounts of biologically active and endotoxin-free RTX toxins. It is based on the common feature of RTX cytolysins that are T1SS-excreted as unfolded polypeptides and fold into a biologically active toxin only upon binding of calcium ions outside of the bacterial cell. Mimicking this process, the RTX proteins are solubilized from inclusion bodies with buffered 8 M urea, bound onto a suitable chromatographic medium under denaturing conditions and the contaminating LPS is removed through extensive on-column washes with buffers containing 6 to 8 M urea and 1% Triton X-100 or Triton X-114. Extensive on-column rinsing with 8 M urea buffer removes residual detergent and the eluted highly active RTX protein preparations then contain only trace amounts of LPS. The procedure is exemplified using four prototypic RTX cytolysins, the Bordetella pertussis CyaA and the hemolysins of Escherichia coli (HlyA), Kingella kingae (RtxA), and Actinobacillus pleuropneumoniae (ApxIA).
- MeSH
- bakteriální proteiny izolace a purifikace toxicita MeSH
- cytotoxiny izolace a purifikace toxicita MeSH
- detergenty chemie MeSH
- erytrocyty účinky léků MeSH
- Escherichia coli metabolismus MeSH
- hemolýza MeSH
- hemolyziny izolace a purifikace toxicita MeSH
- lidé MeSH
- lipopolysacharidy analýza MeSH
- močovina chemie MeSH
- nádorové buněčné linie MeSH
- oktoxynol chemie MeSH
- ovce MeSH
- THP-1 buňky MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Four local Bacillus thuringiensis (Bt) isolates that had been serologically identified as Bt var. kurstaki (Btk2, Btk3, and Btk66) and Bt var. mexicanensis (Btm27), in addition to two reference strains (4D20 and 4AC1), were laboratory assayed as microbial control agents against the Egyptian cotton leafworm Spodoptera littoralis (Boisd.). Polymerase chain reaction (PCR) amplification analysis revealed that each of the six experimental strains carries, at least, a cry1 type gene which expresses a protein toxin active against lepidopterous insects. Additionally, PCR amplification results demonstrated that 4D20 and Btk66 contain the Lepidoptera- and Diptera-active cry2 type gene and that Btk66 contains Coleoptera-active cry7 and cry8 genes. Among the six strains, Btk66 and Btm27 were the most promising microbial control agents against S. littoralis. The present findings were the first to report that Btm27 (classified as B. thuringiensis var. mexicanensis) is a very potent microbial control agent against S. littoralis-tested larvae. For more characterization of these two isolates, the sspO gene was investigated as a molecular chronometer. The DNA sequencing results proved that Btk66 and Btm27 carry sspO open reading frames with identical nucleotide sequences, suggesting a strong phylogenetic relationship between the two strains.
- MeSH
- analýza přežití MeSH
- Bacillus thuringiensis klasifikace genetika izolace a purifikace patogenita MeSH
- bakteriální proteiny biosyntéza genetika toxicita MeSH
- biotest MeSH
- DNA bakterií chemie genetika MeSH
- endotoxiny biosyntéza genetika toxicita MeSH
- Gossypium parazitologie MeSH
- hemolyziny biosyntéza genetika toxicita MeSH
- larva účinky léků MeSH
- molekulární sekvence - údaje MeSH
- polymerázová řetězová reakce MeSH
- sekvenční analýza DNA MeSH
- Spodoptera mikrobiologie fyziologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Egypt MeSH
The delta-endotoxins (delta-ETX) of four native strains (RT7, RT19, RT25, and RT25), and one reference strain (4L1) of Bacillus thuringiensis were biochemically and molecularly characterized to determine their potential toxic activity against lepidopteran larvae. Crystals of delta-ETX were purified through a two-phase system to determine their morphology, molar mass, solubility, and resistance to proteinases. Toxic activity and cry gene content were also determined. Crystals from native strains exhibited polyhedral, irregular and cuboidal shapes, while those from 4L1 were bipyramidal. Seven proteins with estimated molar mass approximately 30-134 kDa were detected as the main components of the native delta-ETX. Only crystals from 4L1, RT24, and RT25 underwent complete solubilization at pH >12.0. Crystals from all strains produced trypsin-resistant peptides. None of the cry genes associated with toxicity in lepidopterans (cry1, cry2, cry9) was found in the native strains; however, 4L1 strain harbors cry1 and cry2 genes. Strains RT19 and RT25 caused significant mortality against Trichoplusia ni larvae with partial solubilization at pH 10, strain 4L1 caused 100 % mortality. Toxicity of native strains may come from a novel cry gene.
- MeSH
- analýza přežití MeSH
- Bacillus thuringiensis fyziologie MeSH
- bakteriální proteiny chemie izolace a purifikace toxicita MeSH
- endotoxiny chemie izolace a purifikace toxicita MeSH
- financování organizované MeSH
- hemolyziny chemie izolace a purifikace toxicita MeSH
- larva účinky léků MeSH
- Lepidoptera účinky léků MeSH
- molekulová hmotnost MeSH
- proteasy metabolismus MeSH
- rozpustnost MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- MeSH
- Bacteria MeSH
- bakteriální toxiny * izolace a purifikace toxicita MeSH
- bakteriologie trendy MeSH
- hemolyziny izolace a purifikace toxicita MeSH
- leukocidiny izolace a purifikace toxicita MeSH
- lidé MeSH
- Staphylococcaceae enzymologie izolace a purifikace patogenita MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- MeSH
- agar MeSH
- antibakteriální látky farmakologie MeSH
- antibiotická rezistence MeSH
- celofán MeSH
- druhová specificita MeSH
- elektroforéza MeSH
- erytrocyty imunologie MeSH
- hemolýza MeSH
- hemolyziny biosyntéza toxicita MeSH
- injekce intradermální MeSH
- injekce intraperitoneální MeSH
- králíci MeSH
- kultivační média MeSH
- metody MeSH
- mikrobiální testy citlivosti MeSH
- morčata MeSH
- myši MeSH
- ovce MeSH
- Pseudomonas aeruginosa metabolismus růst a vývoj účinky léků MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- morčata MeSH
- myši MeSH
- zvířata MeSH
- MeSH
- agar MeSH
- antibakteriální látky farmakologie MeSH
- antibiotická rezistence MeSH
- celofán MeSH
- druhová specificita MeSH
- elektroforéza MeSH
- erytrocyty imunologie MeSH
- hemolýza MeSH
- hemolyziny biosyntéza toxicita MeSH
- injekce intradermální MeSH
- injekce intraperitoneální MeSH
- králíci MeSH
- kultivační média MeSH
- metody MeSH
- mikrobiální testy citlivosti MeSH
- morčata MeSH
- myši MeSH
- ovce MeSH
- Pseudomonas aeruginosa metabolismus růst a vývoj účinky léků MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- morčata MeSH
- myši MeSH
- zvířata MeSH
- Klíčová slova
- atypické korynebaktérie,
- MeSH
- bakteriologické techniky metody využití MeSH
- biomedicínský výzkum * MeSH
- Corynebacterium pyogenes izolace a purifikace klasifikace MeSH
- Corynebacterium * izolace a purifikace klasifikace patogenita MeSH
- hemolyziny izolace a purifikace toxicita MeSH
- lidé MeSH
- morčata MeSH
- zoonózy MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- morčata MeSH
- zvířata MeSH
- Klíčová slova
- corynebacterium pyogenes bovis,
- MeSH
- antibakteriální látky diagnostické užití terapeutické užití MeSH
- bakteriologické techniky * metody využití MeSH
- biomedicínský výzkum * MeSH
- experimenty na zvířatech MeSH
- hemolyziny izolace a purifikace toxicita MeSH
- játra účinky léků MeSH
- lidé MeSH
- srdce účinky léků MeSH
- zoonózy MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
