Pro-inflammatory programmed cell death.
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Apoptosis↗
Biomedical subjects
B T Cookson
Publications and source records attributed to B T Cookson.
Bacterial antigens elicit T cell responses via adaptive and transitional immune recognition.
T cells are a critical component of host immune responses against bacterial pathogens. T cell activation relies on recognition of antigen(s) derived from the bacteria, and this activation triggers potent biological effector mechanisms. Therefore, the characterization of antigens that are stimulatory for T cells provides insight into host-pathogen interactions and advances rational vaccine design. The adaptive immune response is defined by its ability to detect variable or unique single-gene products, whereas a 'transitional' immune system recognizes more conserved structures or products of multigene pathways. This transitional system functionally overlaps the canonical innate and adaptive immune responses. Antigen identification has relied upon biochemistry, genetics and expression cloning strategies. The development of computational approaches, fuelled by advances in immunology and genomic information, will facilitate the discovery of antigens and expand our understanding of both beneficial and pathological immune responses.
Animals↗
Polymorphic internal transcribed spacer region 1 DNA sequences identify medically important yeasts.
Species-specific polymorphisms in the noncoding internal transcribed spacer 2 (ITS2) region of the rRNA operon provide accurate identification of clinically significant yeasts. In this study, we tested the hypothesis that ITS1 noncoding regions contain diagnostically useful alleles. The length of ITS1 region PCR products amplified from 40 species (106 clinical strains, 5 reference strains, and 30 type strains) was rapidly determined with single-base precision by automated capillary electrophoresis. Polymorphisms in the PCR product length permitted 19 species to be distinguished by ITS1 alone, compared with 16 species distinguished by using only ITS2. However, combination of both ITS alleles permitted identification of 30 species (98% of clinical isolates). The remaining 10 species with PCR products of similar sizes contained unique ITS alleles distinguishable by restriction enzyme analysis. DNA sequence analysis of amplified ITS1 region DNA from 79 isolates revealed species-specific ITS1 alleles for each of the 40 pathogenic species examined. This provided identification of unusual clinical isolates, and 53 diagnostic ITS1 sequences were deposited in GenBank. Phylogenetic analyses based on ITS sequences showed a similar overall topology to 26S rRNA gene-based trees. However, different species with identical 26S sequences contained distinct ITS alleles that provided species identification with strong statistical support. Together, these data indicate that the analysis of ITS polymorphisms can reliably identify 40 species of clinically significant yeasts and that the capacity for identifying potentially new pathogenic species by using this database holds significant promise.
DNA, Fungal↗
Diversity within reference strains of Corynebacterium matruchotii includes Corynebacterium durum and a novel organism.
Corynebacterium matruchotii has been the subject of numerous dental pathogenesis studies. The purpose of the present study was to resolve concerns about diversity within the reference strains of C. matruchotii through analysis of seven strains procured from the American Type Culture Collection (ATCC). Analysis of whole-cell fatty acid profiles with the library generation software of Microbial ID Inc. revealed that three types of organisms have been deposited in the ATCC as C. matruchotii. These three groups of organisms were also distinguishable by DNA-DNA dot blot hybridization, by sequences of two hypervariable regions of the 16S rRNA gene, and by the pyrrolidonyl arylamidase test. These studies indicate that two C. matruchotii reference strains, ATCC 33449 and ATCC 33822, are members of the recently proposed species, Corynebacterium durum. The colonial morphology and biochemical reactions of the C. durum strains are more diverse than originally reported. Strain ATCC 43833 is unique and represents a novel species. In addition to the type strain, ATCC 14266, true members of the species C. matruchotii include ATCC strains 14265, 33806, and 43832 plus two reference strains, L2 and Richardson 13, which comprise the vast majority of strains used in dental pathogenesis research with this species.
Bacterial Typing Techniques↗
Tsukamurella strandjordae sp. nov., a proposed new species causing sepsis.
We have isolated a gram-positive, weakly acid-alcohol-fast, irregular rod-shaped bacterium from cultures of blood from a 5-year-old girl with acute myelogenous leukemia. This isolate was compared with 14 other strains including reference strains of Tsukamurella species by a polyphasic approach based on physiological and biochemical properties, whole-cell short-chain fatty acid and mycolic acid analyses, DNA-DNA hybridization, and sequencing of the 16S rRNA gene. This isolate represents a new taxon within the genus Tsukamurella for which we propose the name Tsukamurella strandjordae sp. nov. Our study also revealed that Tsukamurella paurometabola ATCC 25938 represents a misnamed Tsukamurella inchonensis isolate and confirms that Tsukamurella wratislaviensis belongs to the genus Rhodococcus.
Actinomycetales↗
Transmission of Histoplasma capsulatum by organ transplantation.
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Adult↗
Characterization of CD4+ T cell responses during natural infection with Salmonella typhimurium.
CD4+ T cells are important for resistance to infection with Salmonella typhimurium. However, the Ag specificity of this T cell response is unknown. Here, we demonstrate that a significant fraction of Salmonella-specific CD4+ T cells respond to the flagellar filament protein, FliC, and that this Ag has the capacity to protect naive mice from lethal Salmonella infection. To characterize this Ag-specific response further, we generated FliC-specific CD4+ T cell clones from mice that had resolved infection with an attenuated strain of Salmonella. These clones were found to respond to an epitope from a constant region of FliC, enabling them to cross-react with flagellar proteins expressed by a number of distinct Salmonella serovars.
Amino Acid Sequence↗
Salmonella induces macrophage death by caspase-1-dependent necrosis.
We provide evidence that Salmonella typhimurium kills phagocytes by an unusual proinflammatory mechanism of necrosis that is distinguishable from apoptosis. Infection stimulated a distinctly diffuse pattern of DNA fragmentation in macrophages, which contrasted with the marked nuclear condensation displayed by control cells undergoing chemically induced apoptosis. In apoptotic cells, DNA fragmentation and nuclear condensation result from caspase-3-mediated proteolysis; caspases also subvert necrotic cell death by cleaving and inactivating poly ADP-ribose polymerase (PARP). Caspase-3 was not activated during Salmonella infection, and PARP remained in its active, uncleaved state. Another hallmark of apoptosis is sustained membrane integrity during cell death; yet, infected macrophages rapidly lost membrane integrity, as indicated by simultaneous exposure of phosphatidylserine with the uptake of vital dye and the release of the cytoplasmic enzyme lactate dehydrogenase. During experimentally induced necrosis, lethal ion fluxes through the plasma membrane can be prevented by exogenous glycine; similarly, glycine completely blocked Salmonella-induced cytotoxicity. Finally, inhibition of the interleukin (IL)-1-converting enzyme caspase-1 blocked the death of infected macrophages, but not control cells induced to undergo apoptosis or necrosis. Thus, Salmonella-infected macrophages are killed by an unusual caspase-1-dependent mechanism of necrosis.
Apoptosis↗
Application of 16S rRNA gene sequencing to identify Bordetella hinzii as the causative agent of fatal septicemia.
We report on the first case of fatal septicemia caused by Bordetella hinzii. The causative organism exhibited a biochemical profile identical to that of Bordetella avium with three commercial identification systems (API 20E, API 20 NE, and Vitek GNI+ card). However, its cellular fatty acid profile was not typical for either B. avium or previously reported strains of B. hinzii. Presumptive identification of the patient's isolate was accomplished by traditional biochemical testing, and definitive identification was achieved by 16S rRNA gene sequence analysis. Phenotypic features useful in distinguishing B. hinzii from B. avium were production of alkali from malonate and resistance to several antimicrobial agents.
Aged↗
Pseudomembranous colitis caused by a toxin A(-) B(+) strain of Clostridium difficile.
We report a case of severe pseudomembranous colitis due to a toxin A(-) B(+) strain of Clostridium difficile in an immunosuppressed patient and discuss the implications for diagnostic testing in suspected C. difficile-associated diarrhea.
Bacterial Proteins↗
Identification of medically important yeasts using PCR-based detection of DNA sequence polymorphisms in the internal transcribed spacer 2 region of the rRNA genes.
Identification of medically relevant yeasts can be time-consuming and inaccurate with current methods. We evaluated PCR-based detection of sequence polymorphisms in the internal transcribed spacer 2 (ITS2) region of the rRNA genes as a means of fungal identification. Clinical isolates (401), reference strains (6), and type strains (27), representing 34 species of yeasts were examined. The length of PCR-amplified ITS2 region DNA was determined with single-base precision in less than 30 min by using automated capillary electrophoresis. Unique, species-specific PCR products ranging from 237 to 429 bp were obtained from 92% of the clinical isolates. The remaining 8%, divided into groups with ITS2 regions which differed by </=2 bp in mean length, all contained species-specific DNA sequences easily distinguishable by restriction enzyme analysis. These data, and the specificity of length polymorphisms for identifying yeasts, were confirmed by DNA sequence analysis of the ITS2 region from 93 isolates. Phenotypic and ITS2-based identification was concordant for 427 of 434 yeast isolates examined using sequence identity of >/=99%. Seven clinical isolates contained ITS2 sequences that did not agree with their phenotypic identification, and ITS2-based phylogenetic analyses indicate the possibility of new or clinically unusual species in the Rhodotorula and Candida genera. This work establishes an initial database, validated with over 400 clinical isolates, of ITS2 length and sequence polymorphisms for 34 species of yeasts. We conclude that size and restriction analysis of PCR-amplified ITS2 region DNA is a rapid and reliable method to identify clinically significant yeasts, including potentially new or emerging pathogenic species.
DNA, Fungal↗
Identification of a natural T cell epitope presented by Salmonella-infected macrophages and recognized by T cells from orally immunized mice.
Murine infection with Salmonella typhimurium provides models for typhoid fever and long-lasting protective immunity conferred by oral vaccination with viable attenuated bacteria. To further understand the role of T cells in these systems, we identified a bacterial Ag recognized by murine T cells responding to a Salmonella infection. From orally infected mice, we derived a CD4+ Ak-restricted T cell clone (7.4.8) the stimulatory Ag of which was provided by S. typhimurium or its flagella, but not by other salmonellae or S. typhimurium mutants unable to synthesize the flagellar filament protein FliC. We mapped antigenic activity to FliC hypervariable region VI using a generally applicable method of sequential C-terminal truncation of recombinant MalE-FliC fusion proteins. Residues 339-350 are the minimal FliC structure capable of stimulating 7.4.8 and represent the first reported Salmonella-specific epitope recognized by T cells from infected mice. T cells with this specificity are generated by oral immunization, reactivity can be recovered for at least 5 mo afterwards, and FliC is the dominant recall Ag for CD4+ T cells from protectively immunized C3H/HeJ mice. FliC 339-350 is presented by macrophages infected with viable S. typhimurium, and presentation, but not bacterial uptake, is greatly enhanced by pretreatment of macrophages with IFN-gamma. These data point to the importance of IFN-gamma-activated macrophages in the stimulation of T cells responding to facultative intracellular pathogens like S. typhimurium and provide a model system for studying Ag-specific T cell responses in murine salmonellosis.
Administration, Oral↗
Evaluation of an interactive computer program to teach gram-stain interpretation.
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Computer-Assisted Instruction↗
Organization of the human annexin V (ANX5) gene.
We characterized the region of human chromosome 4q26-q28 that contains the gene encoding annexin V (placental anticoagulant protein I), a member of a family of calcium-dependent phospholipid binding proteins. A total of 14.5 kb, containing 9 introns, could be directly amplified from genomic DNA; the remainder was characterized from genomic clones in phage lambda and a yeast artificial chromosome. The gene was mapped with restriction enzymes BamHI, EcoRI, HindIII, SacI, StuI, and XbaI; the transcribed region spans 28 kb and contains 13 exons (44 tp 530 bp in size) and 12 introns (0.23 to 8.8 kb in size). Several putative transcription factor binding sites are present in the 5'-region, but the promoter has no recognizable TATA box. This study will facilitate further analysis of the functions of annexin V and its role in disease.
Animals↗
Bacteremia caused by a novel Bordetella species, "B. hinzii".
Bordetella spp. cause respiratory tract diseases in warm-blooded animals. Only Bordetella bronchiseptica has been reported to cause bacteremia in humans, and this rare infection usually occurs with pneumonia in immunocompromised patients. We describe "Bordetella hinzii" bacteremia in an AIDS patient without a respiratory illness. Combining biochemical phenotyping with fatty acid analysis permitted preliminary identification of this previously undescribed pathogen; identity was confirmed by DNA-DNA hybridization. This report extends the spectrum of human infections caused by the bordetellae.
Adult↗
Structure and polymorphisms of the human annexin III (ANX3) gene.
A 75-kb region encompassing the human annexin III (ANX3) gene on chromosome 4q21 was characterized from directly amplified genomic DNA and from six genomic clones in phage lambda (lambda ANX3-1 to lambda ANX3-6). The gene was mapped with restriction enzymes BamHI, EcoRI, HindIII, SacI, and XbaI, and primers were developed for nine sequence-tagged sites throughout the gene. The transcribed region spans 58 kb and contains 12 introns ranging from 0.3 to 19.1 kb and 13 exons ranging from 53 to 374 bases. Northern blots showed a single mRNA species of approximately 1.7 kb in all tissues examined. Three intragenic polymorphisms were identified: a tandem repeat of the sequence TAAA with at least six alleles, a two-allele SalI polymorphism detectable by polymerase chain reaction, and a two-allele BglII polymorphism.
Amino Acid Sequence↗
Mutagenesis of Bordetella pertussis with transposon Tn5tac1: conditional expression of virulence-associated genes.
The Tn5tac1 transposon contains a strong outward-facing promoter, Ptac, a lacI repressor gene, and a selectable Kanr gene. Transcription from Ptac is repressed by the lacI protein unless an inducer (isopropyl-beta-D-thiogalactopyranoside [IPTG]) is present. Thus, Tn5tac1 generates insertion mutations in Escherichia coli with conditional phenotypes because it is polar on distal gene expression when IPTG is absent and directs transcription of these genes when the inducer is present. To test the usefulness of Tn5tac1 in Bordetella pertussis, a nonenteric gram-negative bacterial pathogen, we chose the bifunctional adenylate cyclase-hemolysin determinant as an easily scored marker to monitor insertional mutagenesis. Tn5tac1 delivered to B. pertussis on conjugal suicide plasmids resulted in Kanr exconjugants at a frequency of 10(-3) per donor cell, and nonhemolytic (Hly-) mutants were found among the Kanr colonies at a frequency of about 1%. Of eight independent Kanr Hly- mutants, two were conditional and exhibited an Hly+ phenotype only in the presence of IPTG. Using a new quantitative assay for adenylate cyclase based on high-pressure liquid chromatography, we found that enzymatic activity in these two strains was specifically induced at least 500-fold in a dose-dependent fashion over the range of 0 to 125 microM IPTG. These data show that Ptac serves as a promoter, lacI is expressed and is functional, and IPTG can induce Ptac transcription in B. pertussis. Adenylate cyclase expression in whole cells, culture supernatants, and cell extracts from these strains depended upon IPTG, suggesting that the insertions do not merely alter secretion of adenylate cyclase-hemolysin. Other virulence determinants under control of the vir locus are expressed normally, implying that these Tn5tac1 insertions specifically regulate adenylate cyclase-hemolysin expression. We conclude that Tn5tac1 insertion mutations permit sensitive, exogenous control over the expression of genes of interest, providing a useful tool for studying virulence and other important traits of diverse bacterial species.
Adenylyl Cyclases↗
Primary structure of the peptidoglycan-derived tracheal cytotoxin of Bordetella pertussis.
The etiological agent of whooping cough, Bordetella pertussis, destroys the ciliated epithelial cells lining the large airways of infected individuals. This cytopathology can be reproduced in respiratory epithelium by tracheal cytotoxin (TCT), a small peptidoglycan-related molecule purified from the culture supernatant of growing B. pertussis organisms. Using fast atom bombardment mass spectrometry, we analyzed the positive- and negative-ion spectra of the purified, biologically active material and assigned a mass of 921 daltons to TCT. Analysis of fragment ions in these spectra as well as the spectra of the methyl ester and acetylated derivatives of TCT unambiguously defined the primary structure of TCT as N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramylalanyl-gamma- glutamyldiaminopimelylalanine. TCT is therefore identical with the ciliostatic anhydropeptidoglycan monomer released by Neisseria gonorrhoeae and with the neurologically active slow-wave sleep-promoting factor FSu. These and other structurally related glycopeptides containing muramic acid thus form a family of molecules with remarkably diverse biological activities.
Bordetella pertussis↗