Biomedical subjects

M S Villanueva

Publications and source records attributed to M S Villanueva.

9 recordsLinked to original sources

PubMed1997

MHC class I antigen processing of Listeria monocytogenes proteins: implications for dominant and subdominant CTL responses.

Listeria monocytogenes (L. monocytogenes) secretes proteins associated with its virulence into the cytosol of infected cells. These secreted proteins are degraded by host cell proteasomes and processed into peptides that are bound by MHC class I molecules in the endoplasmic reticulum. We have found that the MHC class I antigen-processing pathway is very efficient at generating the epitopes that are presented to cytolytic T lymphocytes (CTL). Depending on which antigen is investigated, from 3 to 30% of degraded antigens are processed into nonamer peptides that are bound by MHC class I molecules. Surprisingly, neither the efficiency of epitope generation nor the absolute number of epitopes per infected cell determines the magnitude of the in vivo CTL response. One of the least prevalent epitopes, derived from an antigen that is virtually undetectable in infected cells, primes the immunodominant CTL response in L. monocytogenes-infected mice. Our studies suggest that immunodominant and subdominant T-cell responses cannot be predicted by the prevalence of antigens or epitopes alone, and that additional factors, yet to be determined, are involved.

Animals↗

PubMed1996

CTL epitope generation is tightly linked to cellular proteolysis of a Listeria monocytogenes antigen.

Listeria monocytogenes is a pathogenic intracellular bacterium that secretes proteins into the cytosol of host cells. A major secreted protein, p60, is processed by the host cell into the nonamer peptides p60 217-225 and p60 449-457, which are presented to CTL by H-2Kd MHC class I molecules. Herein, we use two membrane permeable peptide aldehyde protease inhibitors, LLnL and Z-LLF, to inhibit cytosolic proteolysis in L. monocytogenes-infected cells. These inhibitors, which have been shown to inhibit proteasomes, completely abrogate cytosolic p60 degradation. The effect of LLnL and Z-LLF on p60 epitope generation was determined by acid-eluting, HPLC-purifying, and quantifying p60 217-225 and p60 449-457 from infected cells. We show a direct linkage between p60 degradation and epitope generation. However, the two inhibitors have quantitatively different effects on the generation of the two epitopes. Our findings implicate proteasomes in the earliest stages of Ag degradation and suggest that different CTL epitopes can be generated by distinct proteolytic processes.

Amino Acid Sequence↗

PubMed1995

Listeriolysin is processed efficiently into an MHC class I-associated epitope in Listeria monocytogenes-infected cells.

Listeria monocytogenes is an intracellular pathogen that enters the cytoplasm of infected cells by secreting listeriolysin (LLO), a protein that destroys the phagosomal membrane. In infected mice, LLO is a major Ag detected by protective, MHC class I-restricted CTLs. Although the role of LLO in pathogenesis and host immunity is well established, its rate of intracellular synthesis has yet to be determined. Herein we show that cytosolic L. monocytogenes secrete LLO at a relatively low rate of approximately one molecule per bacterium per minute. Under extracellular labeling conditions, the rate of LLO secretion is approximately 50-fold higher. Intracellular LLO synthesis suffices, however, for the accumulation of 600 to 1000 H-2Kd-associated LLO 91-99 epitopes per cell. We calculate that between four and 11 LLO molecules are degraded for each LLO 91-99 epitope bound by H-2Kd. Our findings indicate that the antigenicity of LLO, with respect to MHC class I-restricted CTLs, cannot be attributed to high levels of intracellular secretion. Rather, LLO is a dominant Ag because it is rapidly degraded and very efficiently processed into an MHC class I-associated epitope.

Animals↗

PubMed1994

Infection with Listeria monocytogenes impairs sialic acid addition to host cell glycoproteins.

Listeria monocytogenes is a facultative intracellular bacterium that causes severe disease in neonates and immunocompromised adults. Although entry, multiplication, and locomotion of Listeria in the cytosol of infected cells are well described, the impact of such infection on the host cell is unknown. In this report, we investigate the effect of L. monocytogenes infection on MHC class I synthesis, processing, and intracellular trafficking. We show that L. monocytogenes infection interferes with normal processing of N-linked oligosaccharides on the major histocompatibility complex (MHC) class I heavy chain molecule, H-2Kd, resulting in a reduced sialic acid content. The glycosylation defect is more pronounced as the infection progresses and results from interference with the addition of sialic acid rather than its removal by a neuraminidase. The effect is found in two different cell lines and is not limited to MHC class I molecules since CD45, a surface glycoprotein, and LGP120, a lysosomal glycoprotein, are similarly affected by L. monocytogenes infection. The glycosylation defect is specific for infection by L. monocytogenes since neither Trypanosoma cruzi nor Yersinia enterocolitica, two other intracellular pathogens, reproduces the effect. The resultant hyposialylation of H-2Kd does not impair its surface expression in infected cells. Diminished sialic acid content of surface glycoproteins may enhance host-defense by increasing susceptibility to lysis and promoting clearance of Listeria-infected cells.

Animals↗

PubMed1994

Efficiency of MHC class I antigen processing: a quantitative analysis.

Listeria monocytogenes is an intracellular pathogen that secretes proteins into host cell cytosol. One such protein, the murein hydrolase p60, is processed by the host cell into the nonamer peptide p60 217-225 and presented to cytotoxic T lymphocytes by the H-2Kd MHC class I molecule. Using strains of L. monocytogenes that secrete different amounts of p60, we show that the rate of p60 217-225 production is proportional to the quantity of intracellular antigen. The appearance of p60 217-225 is coupled to the degradation of newly synthesized p60. By accounting for the rate of intracellular antigen secretion and degradation, we estimate that approximately 35 p60 molecules are degraded to produce one p60 217-225 epitope. These findings provide an estimate of the efficiency of antigen processing and shed light on the capacity of the MHC class I antigen processing pathway to accommodate foreign antigens.

Animals↗

PubMed1993

Trypanosomiasis of the central nervous system.

American (Chagas' disease) and African (sleeping sickness) trypanosomiasis are diseases that are endemic in parts of Latin American and Africa, respectively. Physicians in developed countries may occasionally see cases because of extensive travel and immigration from endemic countries. In addition, in American trypanosomiasis, transmission via contaminated blood transfusions poses significant risks of causing acute Chagas' disease in immunocompromised patients. Both forms of trypanosomiasis have neurologic manifestations. Chagas' disease can present as acute meningoencephalitis in children and some immunocompromised patients. Additionally, there are increasingly frequent reports of neurologic syndromes, including CNS mass lesions, in HIV-infected patients. African sleeping sickness has prominent CNS pathologic features in stage II disease. Proposed pathogenetic mechanisms and diagnostic and therapeutic considerations are reviewed in this article.

Animals↗

PubMed1992

Spliced leader RNA sequences of Trypanosoma rangeli are organized within the 5S rRNA-encoding genes.

The spliced leader RNA(SL RNA)-encoding genes of the salivarian New World trypanosome, Trypanosoma rangeli, are organized within the 5S rRNA tandem repeats. Each repeat contains genes encoding an SL RNA and a 5S rRNA in the same orientation of transcription. This SL-5S organization is also present in the African trypanosome, Trypanosoma vivax. A similar association of SL and 5S genes has been observed in some nematodes, but has not been described previously in trypanosomatids.

Animals↗

PubMed1991

A new member of a family of site-specific retrotransposons is present in the spliced leader RNA genes of Trypanosoma cruzi.

A new member of a family of site-specific retrotransposons is described in the New World trypanosome Trypanosoma cruzi. This element, CZAR (cruzi-associated retrotransposon), resembles two previously described retrotransposons found in the African trypanosome T. brucei gambiense and the mosquito trypanosomatid Crithidia fasciculata in specifically inserting between nucleotides 11 and 12 of the highly conserved 39-mer of the spliced leader RNA (SL-RNA) gene. CZAR is similar in overall organization to the other two SL-RNA-associated elements. It possesses two potential long open reading frames which resemble the gag and pol genes of retroviruses. In the pol open reading frame, all three elements contain similarly arranged endonuclease domains and share extensive amino acid homology in the reverse transcriptase region. All are associated with the SL-RNA gene locus and are present in low copy numbers. They do not appear to have 5' truncated versions. All three retrotransposons are otherwise quite distinct from one another, with no significant overall amino acid homology. The presence of such retroelements inserted into the identical site within SL-RNA gene sequences in at least three evolutionarily distant trypanosomatid species argues for a functional role. Because these elements appear to have a precise target site requirement for integration, we refer to them as SL siteposons.

Amino Acid Sequence↗

PubMed1984

Specific in vitro adenylylation of the simian virus 40 large tumor antigen.

Incubation of the simian virus 40 (SV40) large tumor antigen (T) from either transformed or lytically infected cells with adenosine [8-3H]-, [alpha-32P]-, or [alpha-[35S]thio]-triphosphate in the presence of Mg2+ resulted in its labeling as defined by the appearance of an intact, appropriately immunoreactive band in NaDodSO4/polyacrylamide gels. Radioactivity remained associated with the protein after boiling in buffer containing 3% NaDodSO4, and 2-mercaptoethanol as well as after heating in 0.1 M HCl, 0.1 M NH4OH, or hydroxylamine, but it was dissociated after incubation in 0.1 M NaOH at 37 degrees C. After limited boiling of gel-purified [alpha-32P] ATP + T complex in 5.6 M HCl, o-[32P]phosphoserine was released, and snake venom phosphodiesterase or 0.5 M piperidine treatment of such a complex resulted in the liberation of [alpha-32P]AMP. The reaction proceeded when either purified, soluble T or insoluble, specifically immunoprecipitated antigen was used as substrate. ATP and dATP were the preferred nucleotide substrates by comparison with the other six standard ribonucleoside or deoxynucleoside triphosphates. Partial tryptic digests of T + [alpha-32P]ATP complexes revealed the presence of a single labeled peptide of Mr approximately equal to 12 - 14 X 10(3), and after exhaustive digestion, there was a single radioactive spot in the fingerprint. These data indicate that T can be adenylylated at a specific seryl residue(s) in a limited portion of the protein surface. Furthermore, adenylylation appears to be reversible and to proceed by a pyrophosphorylytic mechanism, since the nucleotide was released from the protein following incubation of adenylylated T with Mg2+, sodium pyrophosphate, and poly(dT).

Adenosine Triphosphate↗