Complete Core Structure Research Articles

Polymorphic Nature of Human T-Cell Leukemia Virus Type 1 Particle Cores as Revealed through Characterization of a Chronically Infected Cell Line

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 cell-to-cell transmission is dependent on the release of infectious virus particles into the virological synapse. The HTLV-1 particle structure is still poorly understood, and previous studies analyzed viruses produced by transformed lymphocytic cell lines chronically infected with HTLV-1, particularly the MT-2 cell line, which harbors truncated proviruses and expresses aberrant forms of the Gag protein. In this study, we demonstrate that the chronically infected SP cell line harbors a relatively low number of proviruses, making it a more promising experimental system for the study of the HTLV-1 particle structure. We first identified the genomic sites of integration and characterized the genetic structure of the gag region in each provirus. We also determined that despite encoding a truncated Gag protein, only the full-length Gag protein was incorporated into virus particles. Cryo-transmission electron microscopy analyses of the purified virus particles revealed three classes of particles based upon capsid core morphology: complete cores, incomplete cores, and particles without distinct electron densities that would correlate with the capsid region of a core structure. Observed cores were generally polygonal, and virus particles were on average 115 nm in diameter. These data corroborate particle morphologies previously observed for MT-2 cells and provide evidence that the known poor infectivity of HTLV-1 particles may correlate with HTLV-1 particle populations containing few virus particles possessing a complete capsid core structure.IMPORTANCE Studies of retroviral particle core morphology have demonstrated a correlation between capsid core stability and the relative infectivity of the virus. In this study, we used cryo-transmission electron microscopy to demonstrate that HTLV-1 particles produced from a distinct chronically infected cell line are polymorphic in nature, with many particles lacking organized electron densities that would correlate with a complete core structure. These findings have important implications for infectious HTLV-1 spread, particularly in the context of cell-to-cell transmission, a critical step in HTLV-1 transmission and pathogenesis.

Structural Studies of Lipopolysaccharide-defective Mutants from Brucella melitensis Identify a Core Oligosaccharide Critical in Virulence

The structures of the lipooligosaccharides from Brucella melitensis mutants affected in the WbkD and ManBcore proteins have been fully characterized using NMR spectroscopy. The results revealed that disruption of wbkD gives rise to a rough lipopolysaccharide (R-LPS) with a complete core structure (β-d-Glcp-(1→4)-α-Kdop-(2→4)[β-d-GlcpN-(1→6)-β-d-GlcpN-(1→4)[β-d-GlcpN-(1→6)]-β-d-GlcpN-(1→3)-α-d-Manp-(1→5)]-α-Kdop-(2→6)-β-d-GlcpN3N4P-(1→6)-α-d-GlcpN3N1P), in addition to components lacking one of the terminal β-d-GlcpN and/or the β-d-Glcp residues (48 and 17%, respectively). These structures were identical to those of the R-LPS from B. melitensis EP, a strain simultaneously expressing both smooth and R-LPS, also studied herein. In contrast, disruption of manBcore gives rise to a deep-rough pentasaccharide core (β-d-Glcp-(1→4)-α-Kdop-(2→4)-α-Kdop-(2→6)-β-d-GlcpN3N4P-(1→6)-α-d-GlcpN3N1P) as the major component (63%), as well as a minor tetrasaccharide component lacking the terminal β-d-Glcp residue (37%). These results are in agreement with the predicted functions of the WbkD (glycosyltransferase involved in the biosynthesis of the O-antigen) and ManBcore proteins (phosphomannomutase involved in the biosynthesis of a mannosyl precursor needed for the biosynthesis of the core and O-antigen). We also report that deletion of B. melitensis wadC removes the core oligosaccharide branch not linked to the O-antigen causing an increase in overall negative charge of the remaining LPS inner section. This is in agreement with the mannosyltransferase role predicted for WadC and the lack of GlcpN residues in the defective core oligosaccharide. Despite carrying the O-antigen essential in B. melitensis virulence, the core deficiency in the wadC mutant structure resulted in a more efficient detection by innate immunity and attenuation, proving the role of the β-d-GlcpN-(1→6)-β-d-GlcpN-(1→4)[β-d-GlcpN-(1→6)]-β-d-GlcpN-(1→3)-α-d-Manp-(1→5) structure in virulence.

Structural heterogeneity in the core oligosaccharide of the S-layer glycoprotein from Aneurinibacillus thermoaerophilus DSM 10155.

The surface layer glycoprotein of Aneurinibacillus thermoaerophilus DSM 10155 has a total carbohydrate content of 15% (by mass), consisting of O-linked oligosaccharide chains. After proteolytic digestion of the S-layer glycoprotein byPronase E and subsequent purification of the digestion products by gel permeation chromatography, chromatofocusing and high-performance liquid chromatography two glycopeptide pools A and B with identical glycans and the repeating unit structure -->4)-alpha-l-Rha p -(1-->3)-beta-d- glycero -d- manno -Hep p -(1--> (Kosma et al., 1995b, Glycobiology, 5, 791-796) were obtained. Combined evidence from modified Edman-degradation in combination with liquid chromatography electrospray mass-spectrometry and nuclear magnetic resonance spectroscopy revealed that both glycopeptides contain equal amounts of the complete core structure alpha-l-Rha p -(1-->3)-alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and the truncated forms alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and beta-d-Gal p NAc-(1-->O)-Thr/Ser. All glycopeptides possessed the novel linkage types beta-d-Gal p NAc-(1-->O)-Thr/Ser. The different cores were substituted with varying numbers of disaccharide repeating units. By 300 MHz proton nuclear magnetic resonance spectroscopy the complete carbohydrate core structure of the fluorescently labeled glyco-peptide B was determined after Smith-degradation of its glycan chain. The NMR data confirmed and complemented the results of the mass spectroscopy experiments. Based on the S-layer glycopeptide structure, a pathway for its biosynthesis is suggested.

Escherichia coli K12 regains its O antigen

Extant Escherichia coli K12 strains are phenotypically rough, their lipopolysaccharide having a complete core structure, but no O antigen. We used DNA hybridization and DNA sequencing to show that the rough phenotype of this strain is due to the presence of one of two independent mutations in the rfb gene cluster. The rfb-50 mutation, consisting of an IS5 insertion at the downstream end of rfb, is present in strain EMG2, which is representative of most K12 derivatives. The rfb-51 mutation is a deletion at the upstream end of rfb, and was found in strain WG1. A gene cloned from strain WG1 could complement the rfb-50 mutation in strain EMG2, and the complemented strain produced O antigen which was typed as O16 with cross reaction to O17.

The core region of Proteus mirabilis R110/1959 lipopolysaccharide.

The complete core structure present in the lipopolysaccharide of the R mutant R110/1959 from Proteus mirabilis (Proteus type II core) was investigated using methylation analysis and a number of degradation methods such as Smith degradation and beta-elimination. These studies combined with earlier work on a Rc-type mutant of P. mirabilis O28 (R4/O28) which shares the same inner core region, allowed formulation of the complete core structure of the Proteus type II core as shown in Scheme 1. (formula; see text) A characteristic feature of the Proteus core of type II is the presence of two units of D-galacturonic acid (DGalA); one in terminal, the other one in a chain-linked position. In addition, the presence of the two isomers of glycero-D-manno-heptose (LDHep and DDHep) and the lack of galactose are conspicuous. DDHep occupies a terminal position in the external core region, whereas the three units of LDHep in addition to dOclA form, as in other enterobacterial core types, the internal core region. The taxonomic significance of the presence of DGalA in the Proteus type II core, but also in all R cores of other Proteeae investigated so far, will be discussed.

Genetical studies on the lipopolysaccharide structure of Escherichia coli K12.

SUMMARY: Escherichia coli K12 strain 2578 was crossed with an E. coli O8 donor. Most of the recombinants selected for the donor his allele were agglutinable by anti-O8 serum. This indicated that the recombinants can synthesize O8-specific units and attach them to the K12 lipopolysaccharide (LPS) core in consequence of the transfer of the his-linked donor rfb cluster determining O8-chain synthesis. In serological tests and phage sensitivity tests most O8-reactive recombinants behaved like typical smooth E. coli O8 strains. This implies that E. coli K12 strain 2578 synthesizes a complete core structure, which is a prerequisite for the formation of smooth recombinants. Some O8-reactive recombinants displayed a partly rough phenotype. In addition, the typical smooth recombinants segregated partly rough forms. Thus the smooth recombinants appear to be partial diploids for some donor gene(s) involved in LPS synthesis. Serological tests and phage typing showed that the Escherichia coli K12 core is different from other known core types of Salmonella (Ra) and E. coli (R1–-R3). The genes (rfa) for the K12 core type were mapped by conjugation at approximately 72 min on the Escherichia coli linkage map.

Immunochemistry of R lipopolysaccharides of Escherichia coli. Studies on R mutants with an incomplete core, derived from E. coli O8:K27.

Acapsular (K−) forms were isolated from Escherichia coli strain E56b (serotype O8 :K27(A): H−). A number of different R strains were obtained from the K− forms by selection of spontaneous mutants and by phage selection. The lipopolysaccharides of these R strains (consisting of core and lipid A) were subjected to mild acid degradation and, after removal of insoluble lipid A, the mixtures were fractionated by gel chromatography. Results of chemical analyses showed that the core oligosaccharides of different R mutants differed with respect to their sugar constituents as well as to the molar ratios of these constituents. A group of R mutants did not contain phosphate in their core oligosaccharides (P− mutants). Enzyme determinations indicated that one R mutant had a block involving UDP glucose pyrophosphorylase, whereas the enzymes of activation and interconversion of component sugars were intact in the other R mutants. By allelic exchange (mating with S‐form Hfr strains) it could be demonstrated that with one exception the chromosomal site of the SR mutation of all mutants maps close to mtl (mannitol utilization). In Salmonella the rfa locus maps in the same region. All R mutants had an unimpaired (his‐linked) rfb locus. The groups of mutants which differed in the sugar composition of their core oligosaccharides could also be differentiated serologically and by their sensitivity to phages. There was serological cross reactivity between the lipopolysaccharides of some R mutants with R mutants of Salmonella minnesota which also have a very incomplete core. It is concluded that the lipopolysaccharides of the R mutants described in this paper have incomplete core structures which result from blocks at successive stages in the synthesis of the coli R1 core. In analogy to Salmonella these E. coli R mutants should, with one exception, be termed rfa type mutants.

Immunochemical studies on core lipopolysaccharides of Enterobacteriaceae of different genera.

1 R forms of Escherichia coli 0100:K(B) and 0111:K58 were obtained by genetic recombination transferring into the respective S forms a lesion in the his-linked som locus. Such mutants are not able to synthesize O-specific polysaccharide side chains but do have a complete core region. 2 R forms of Citrobacter 010,9b and Arizona 09a,9c were isolated at random as rough looking colonies and characterized as possessing complete core lipopolysaccharide. 3 These R forms and their respective lipopolysaccharides were compared in phage sensitivity, serological properties and in chemical composition with known prototypes of Salmonella and E. coli R mutants (coli R1 and R2) having a complete core structure. 4 According to the phage pattern, the R mutants could be subdivided into distinct groups. One group comprising the R forms of E. coli 0111 and Citrobacter showed a peculiar phage pattern hitherto not observed with other enterobacterial R mutants. The phage pattern of R mutants of Salmonella and Arizona proved to be identical. The R form of E. coli 0100 on the other hand gave the same phage pattern as described for E. coli R2 type mutants. 5 The results of phage typing were fully corroborated by the serological and chemical investigations upon the lipopolysaccharide. In serological studies lipopolysaccharides of all R types except coli R1 were found to cross react with each other, thus indicating structural similarities. In all cross reacting lipopolysaccharide preparations the same sugar constituents namely glucose, galactose, N-acetylglucosamine, l-glycero-d-manno-heptose and 3-deoxy-d-manno-octulosonic acid were present; but quantitative analysis revealed remarkable differences. Striking is the very low content of galactose in the lipopolysaccharide of the R mutants of E. coli 0111 and Citrobacter. 6 The results described show that the E. coli 0111 core represents a further core type which will be designated coli R3. 7 The serological and chemical investigation of the lipopolysaccharide of the Arizona R mutant confirms its identity with the Salmonella core type, thus indicating the close relationship of these bacterial groups.

Immunochemistry of R lipopolysaccharides of Escherichia coli. Different core regions in the lipopolysaccharides of O group 8.

1 Acapsular (K−) forms were isolated from E. coli strains A295b (serotype 08:K42(A):H−) and E56b (serotype O8:K27(A):H−). A number of R strains were obtained from them by selection of spontaneous mutants, phage selection and mating experiments. 2 The (his−) R mutants were crossed with a (his+) Hfr S strain (O8) and selected for his+. Over 95% of the his+ recombinants were S forms. This showed that the R forms have a genetic defect which is closely linked to his. They all have a complete core structure in their lipopolysaccharides. 3 When the R strains were tested as to their sensitivity towards a number of phages it appeared that they can be divided into two groups. The discrimination was based on the sensitivity of the R mutants towards phages Br2, FO and C21. 4 Serological investigations of the corresponding lipopolysaccharides (using passive haemagglutination and its inhibition) indicated that the lipopolysaccharides obtained from the R mutants of E. coli O8:K42− were distinct from those obtained from the R mutants of E. coli O8:K27−. Thus two groups of R structures are present in E. coli O8:coli R1 (derived from O8:K27−) and coli R2 (derived from O8:K42−). The lipopolysaccharides belonging to coli R2 cross react with that of S. minnesota R60 (RII type). 5 The lipopolysaccharides of types coli R1 and coli R2 were subjected to mild acid degradation and the polysaccharide moieties were fractionated by gel chromatography. The results of chemical analysis were the following: coli R1 and coli R2 represent chemically distinct group of E. coli R lipopolysaccharides. Coli R1 lipopolysaccharides contain galactose, glucose and heptose in the molar ratios of 2:3:3 and coli R2 lipopolysaccharides contain galactose, glucose, glucosamine and heptose in the molar ratios of 2:4:1:4. Both types of lipopolysaccharides contain 1–1.5 moles of2-keto-3-deoxyoctonate per mole of galactose. After acid degradation and column fractionation the bulk of the polysaccharide moiety was obtained as core fraction. The molar rations of the constituents in these core fractions, with the exception of 2-keto-3-deoxyoctonate, were the same as in the corresponding degraded polysaccharides. It appeared that during degradation 1–2 2-keto-3-deoxyoctonate units (on a molar basis) were liberated. 6 Degradation and fractionation of S lipopolysaccharides (O8) yielded the core fraction and a high molecular weight fraction (mol. wt. about 10000). The latter consisted almost exclusively of mannose and had serological O8 specificity. The core fractions had the same composition as those obtained from the corresponding R mutants. Thus the lipopolysaccarides of E. coli O8 seem to consist of an identical O specific polysaccharide linked to individual core structures. These results show that there is more than one core type in lipopolysaccarides from different E. coli strains. Until now only one core type has been found in Salmonella and a different one in Shigella. Thus the findings reported here are not only of biochemical but possibly also of taxonomic interest.