A comparison of viral immune escape strategies targeting the MHC class I assembly pathway.

Peptide fragments from proteins of intracellular pathogens such as viruses are displayed at the cell surface by MHC class I molecules thus enabling surveillance by cytotoxic T cells. Peptides are produced in the cytosol by proteasomal degradation and translocated into the endoplasmic reticulum by the peptide transporter TAP. Empty MHC class I molecules associate with TAP prior to their acquisition of peptides, a process which is assisted and controlled by a series of chaperones. The first part of this review summarizes our current knowledge of this assembly pathway and describes recent observations that tapasin functions as an endoplasmic reticulum retention molecule for empty MHC class I molecules. To defeat the presentation of virus-derived peptides, several DNA viruses have devised strategies to interfere with MHC class I assembly. Although these evasion strategies have evolved independently and differ mechanistically they often target the same step in this pathway. We compare escape mechanisms of different viruses with particular emphasis on the retention of newly synthesized MHC class I molecules in the endoplasmic reticulum and the inhibition of peptide transport by viral proteins.

Interaction of murine MHC class I molecules with tapasin and TAP enhances peptide loading and involves the heavy chain alpha3 domain.

In human cells the association of MHC class I molecules with TAP is thought to be mediated by a third protein termed tapasin. We now show that tapasin is present in murine TAP-class I complexes as well. Furthermore, we demonstrate that a mutant H-2Dd molecule that does not interact with TAP due to a Glu to Lys mutation at residue 222 of the H chain (Dd(E222K)) also fails to bind to tapasin. This finding supports the view that tapasin bridges the association between class I and TAP and implicates residue 222 as a site of contact with tapasin. The inability of Dd(E222K) to interact with tapasin and TAP results in impaired peptide loading within the endoplasmic reticulum. However, significant acquisition of peptides can still be detected as assessed by the decay kinetics of cell surface Dd(E222K) molecules and by the finding that prolonged viral infection accumulates sufficient target structures to stimulate T cells at 50% the level observed with wild-type Dd. Thus, although interaction with tapasin and TAP enhances peptide loading, it is not essential. Finally, a cohort of Dd(E222K) molecules decays more rapidly on the cell surface compared with wild-type Dd molecules but much more slowly than peptide-deficient molecules. This suggests that some of the peptides obtained in the absence of an interaction with tapasin and TAP are suboptimal, suggesting a peptide-editing function for tapasin/TAP in addition to their role in enhancing peptide loading.

Retention of empty MHC class I molecules by tapasin is essential to reconstitute antigen presentation in invertebrate cells.

Presentation of antigen-derived peptides by major histocompatibility complex (MHC) class I molecules is dependent on an endoplasmic reticulum (ER) resident glycoprotein, tapasin, which mediates their interaction with the transporter associated with antigen processing (TAP). Independently of TAP, tapasin was required for the presentation of peptides targeted to the ER by signal sequences in MHC class I-transfected insect cells. Tapasin increased MHC class I peptide loading by retaining empty but not peptide-containing MHC class I molecules in the ER. Upon co-expression of TAP, this retention/release function of tapasin was sufficient to reconstitute MHC class I antigen presentation in insect cells, thus defining the minimal non-housekeeping functions required for MHC class I antigen presentation.

Primary intracerebellar osteosarcoma arising within an epidermoid cyst.

The authors report a case of a primary extraskeletal osteosarcoma arising within an epidermoid cyst in the parenchyma of the cerebellum in a 64-year-old woman. On initial presentation, the tumor involved the midline cerebellum without attachment to the surrounding dura mater or calvarium. Complete medical and radiologic evaluation failed to reveal a primary skeletal or other extraskeletal osteosarcoma. To our knowledge, this is the first reported case of a primary extraskeletal osteosarcoma within the cerebellum. Osteosarcoma as a primary brain tumor is exceedingly rare, and only three cases (all occurring within the cerebral hemispheres) have been reported previously. The histogenesis of primary sarcomas of the brain is not evident. The associated finding of an epidermoid cyst suggests the tumor originated from a teratoma.

Sequence, linkage to H2-K, and function of mouse tapasin in MHC class I assembly.

Assembly of major histocompatibility complex (MHC) class I molecules in human cells is dependent on the accessory protein tapasin, which mediates their interaction with the transporters associated with antigen processing (TAP) and thereby ensures efficient peptide binding. Analysis of a mouse tapasin complementary DNA defined a conserved polypeptide sharing sequences diagnostic of a transmembrane protein related to the immunoglobulin superfamily, and an endoplasmic reticulum retention motif. The mouse tapasin gene was mapped about 70 kilobases from H2-K at the centromeric end of the mouse MHC. Expression of mouse tapasin in a tapasin-deficient human mutant cell line restored the normal assembly and expression of class I alleles. Thus, tapasin is a structurally and functionally conserved component of the MHC class I antigen processing pathway. Its genetic linkage to the class I and TAP subunit genes in the MHC may be of significance in the coordinate expression and functional coadaptation of the diverse gene products.

Translation of the flagellar gene fliO of Salmonella typhimurium from putative tandem starts.

The flagellar gene fliO of Salmonella typhimurium can be translated from an AUG codon that overlaps the termination codon of fliN (K. Ohnishi et al., J. Bacteriol. 179:6092-6099, 1997). However, it had been concluded on the basis of complementation analysis that in Escherichia coli a second start codon 60 bp downstream was the authentic one (J. Malakooti et al., J. Bacteriol. 176:189-197, 1994). This raised the possibility of tandem translational starts, such as occur for the chemotaxis gene cheA; this possibility was increased by the existence of a stem-loop sequence covering the second start, a feature also found with cheA. Protein translated from the first start codon was detected regardless of whether the second start codon was present; it was also detected when the stem-loop structure was disrupted or deleted. Translation from the second start codon, either as the natural one (GUG) or as AUG, was not detected when the first start and intervening sequence were intact. Nor was it detected when the first codon was attenuated (by conversion of AUGAUG to AUAAUA; in S. typhimurium there is a second, adjacent, AUG) or eliminated (by conversion to CGCCGC); disruption of the stem-loop structure still did not yield detectable translation from the second start. When the entire sequence up to the second start was deleted, translation from the second start was detected provided the natural codon GUG had been converted to AUG. A fliO null mutant could be fully complemented in swarm assays whenever the first start and intervening sequence were present, regardless of the state of the second start. Reasonably good complementation occurred when the first start and intervening sequence were absent provided the second start was intact, either as AUG or as GUG; thus translation from the GUG codon must have been occurring even though protein levels were too low to be detected. The translated intervening sequence is rather divergent between S. typhimurium and E. coli and corresponds to a substantial cytoplasmic domain prior to the sole transmembrane segment, which is highly conserved; the sequence following the second start begins immediately prior to that transmembrane segment. The significance of the data for FliO is discussed and compared to the equivalent data for CheA. Attention is also drawn to the fact that given an optimal ribosome binding site, AUA can serve as a fairly efficient start codon even though it seldom if ever appears to be used in nature.

The FliO, FliP, FliQ, and FliR proteins of Salmonella typhimurium: putative components for flagellar assembly.

The flagellar genes fliO, fliP, fliQ, and fliR of Salmonella typhimurium are contiguous within the fliLMNOPQR operon. They are needed for flagellation but do not encode any known structural or regulatory components. They may be involved in flagellar protein export, which proceeds by a type III export pathway. The genes have been cloned and sequenced. The sequences predict proteins with molecular masses of 13,068, 26,755, 9,592, and 28,933 Da, respectively. All four gene products were identified experimentally; consistent with their high hydrophobic residue content, they segregated with the membrane fraction. From N-terminal amino acid sequence analysis, we conclude that fliO starts immediately after fliN rather than at a previously proposed site downstream. FliP existed in two forms, a 25-kDa form and a 23-kDa form. N-terminal amino acid analysis of the 23-kDa form demonstrated that it had undergone cleavage of a signal peptide--a rare process for prokaryotic cytoplasmic membrane proteins. Site-directed mutation at the cleavage site resulted in impaired processing, which reduced, but did not eliminate, complementation of a fliP mutant in swarm plate assays. A cloned fragment encoding the mature form of the protein could also complement the fliP mutant but did so even more poorly. Finally, when the first transmembrane span of MotA (a cytoplasmic membrane protein that does not undergo signal peptide cleavage) was fused to the mature form of FliP, the fusion protein complemented very weakly. Higher levels of synthesis of the mutant proteins greatly improved function. We conclude that, for insertion of FliP into the membrane, cleavage is important kinetically but not absolutely required.

Physiological and biochemical analyses of FlgH, a lipoprotein forming the outer membrane L ring of the flagellar basal body of Salmonella typhimurium.

The FlgH protein of Salmonella typhimurium, from which the outer membrane L ring of the flagellar basal body is constructed, has a consensus motif (LTG C) for lipoylation and signal peptide cleavage. We have confirmed the previous finding (M. Homma, K. Ohnishi, T. Iino, and R. M. Macnab, J. Bacteriol. 169:3617-3624, 1987) that it is synthesized in precursor form and processed to a mature form with an apparent molecular mass of ca. 25 kDa. flgH alleles with an in-frame deletion or a 3' truncation still permitted processing. The deletion permitted partial restoration of motility in complementation tests, whereas the truncation did not. Globomycin, an antibiotic which inhibits signal peptide cleavage of prolipoproteins, caused accumulation of precursor forms of FlgH. When cells transformed with a plasmid containing the flgH gene were grown in the presence of [3H]palmitate, a 25-kDa protein doublet was found to be radiolabeled; its identity as FlgH was confirmed by shifts in mobility when the internally deleted and truncated alleles of the gene were used. Hook-basal body complexes from cells grown in the presence of [3H]palmitate demonstrated that FlgH incorporated into flagellar structure was also labeled. An in-frame fusion between the leader sequence of the periplasmic protein PeIB and the mature FlgH sequence, with the putative N-terminal cysteine replaced by glycine, resulted in production of a fusion protein that was processed to its mature form. With a low-copy-number plasmid, the ability of this pelB-flgH fusion to complement a flgH mutant was poor, but with a high-copy-number plasmid, it was comparable to that of the wild type. Although lacking the N-terminal cysteine and therefore being incapable of lipoylation via a thioether linkage, the mutant protein still incorporated [3H]palmitate at low levels, perhaps through acylation of the N-terminal alpha-amino group. We conclude that FlgH is a lipoprotein and that under normal physiological conditions the lipoyl modification is necessary for FlgH to function properly as the L-ring protein of the flagellar basal body. We suggest that the N terminus of FlgH is responsible for anchoring the basal body in the outer membrane and that the C terminus may be responsible for binding to the P ring to form the L,P-ring complex.

Comparative analysis of flagellin sequences from Escherichia coli strains possessing serologically distinct flagellar filaments with a shared complex surface pattern.

Escherichia coli morphotype E flagellar filaments have a characteristic surface pattern of short-pitch loops when examined by electron microscopy. Seven of the 50 known E. coli H (flagellar antigen) serotypes (H1, H7, H12, H23, H45, H49, and H51) produce morphotype E filaments. Polymerase chain reaction was used to amplify flagellin structural (fliC) genes from E. coli strains producing morphotype E flagellar filaments and from strains with flagellar filaments representing other morphotypes. A single DNA fragment was obtained from each strain, and the size of the amplified DNA correlated with the molecular mass of the corresponding flagellin protein. This finding and hybridization data suggest that these bacteria are monophasic. fliC genes from three E. coli serotypes (H1, H7, and H12) possessing morphotype E flagellar filaments were sequenced in order to assess the contribution of conserved flagellin primary sequence to the characteristic filament architecture. The H1 and H12 fliC sequences were identical in length (1,788 bp), while the H7 fliC sequence was shorter (1,755 bp). The deduced molecular masses of the FliC proteins were 60,857 Da (H1), 59,722 Da (H7), and 60,978 Da (H12). The H1, H7, and H12 flagellins demonstrated 98 to 99% identity over the amino-terminal region (190 amino acid residues) and 89% (H7) to 99% (H1 and H12) identity in the carboxy-terminal region (100 amino acid residues). The complete primary amino acid sequences for H1 and H12 flagellins differed by only 10 amino acids, accounting for previously reported serological cross-reactions. However, the central region of H7 flagellin had only 38% identity with H1 and H12 flagellins. The characteristic morphology of morphotype E flagellar filaments is therefore not dependent on a highly conserved primary sequence within the exposed central region. Comparison of morphotype E E. coli flagellins with those from E. coli K-12, Serratia marcescens, and several Salmonella serovars supported the established concept of highly conserved terminal regions flanking a variable central region.

Monoclonal antibodies against serotype specific and conserved epitopes in morphotype E Escherichia coli flagellins.

Monoclonal antibodies (mAbs) were used to examine the interrelationships between morphologically identical flagellar filaments from Escherichia coli H serotype strains belonging to morphotype E. Serotype specific mAbs recognised epitopes exposed on the surface of flagellar filaments from H1, H7, H23, H49 and H51, but were inaccessible to immunolabelling in H45. Several mAbs which recognised conserved epitopes were also examined. mAb 7-56.1 recognised an epitope present in all morphotype E flagellins but not expressed on the filament surface. Similarly, mAb 1-5.1 recognised an internal epitope shared only by serotypes H1 and H12. Serotype H23 expressed a surface epitope which was present but not surface exposed in H7, H1 and H45 filaments.

A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing.

The lipopolysaccharide (LPS) O-antigen side chains of Klebsiella serotype O1 have been studied by using mutants selected by resistance to a Klebsiella bacteriophage designated O1-A. Two classes of LPS mutants were identified. The major group (90%) synthesized rough LPS. The remaining 10% of the mutants produced a novel LPS profile that lacked the highest-molecular-weight O-substituted molecules (HMW-LPS) but still produced lower-molecular-weight O-substituted species (LMW-LPS). By using antisera raised against mutant Klebsiella strains and antiserum specific for Pasteurella haemolytica serotype 4, it was demonstrated that HMW-LPS and LMW-LPS contain shared epitopes. HMW-LPS also contained an epitope absent in LMW-LPS. This unique epitope was recognized by a monoclonal antibody (O1-52.6) and appears to be responsible for the serological cross-reaction between the O antigens of Klebsiella O1 and Escherichia coli O19. This HMW-LPS epitope was present in eight other Klebsiella O1 isolates which were examined. Electron microscopy demonstrated that HMW-LPS excluded overlying capsular polysaccharide for a distance of 25 to 40 nm. The distance was reduced to 10 to 18 nm in strains which synthesized only LMW-LPS and to zero in rough LPS strains. The HMW-LPS of Klebsiella O1 was shown to be an important virulence determinant, since this molecule was responsible for the resistance of the bacterium to nonspecific, complement-mediated serum killing.

Mutants of Escherichia coli O9:K30 with altered synthesis and expression of the capsular K30 antigen.

Escherichia coli K30 produces a thermostable group I capsular polysaccharide. Two classes of mutants were isolated with defects in the synthesis or expression of capsule. The most common mutant phenotype was acapsular (K-), with no K-antigen synthesized. A second class of mutants, termed Ki or intermediate forms, produced colonies which were indistinguishable from those of acapsular forms yet K-antigenicity was expressed. Previous studies had demonstrated that E. coli strains that produce K30 antigen synthesize a lipopolysaccharide (LPS) fraction that is recognised by monoclonal antibodies against the K30 antigen. Synthesis of this LPS fraction was not affected in Ki forms. The results of morphological examination, LPS analysis and phage sensitivity studies are consistent with the interpretation that the defect in Ki strains results from an inability to polymerize the K30 antigen. Using plasmid pULB113 (RP4::mini-Mu), mutations resulting in both K- and Ki phenotypes were localized near the his region of the chromosome.