Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the top three causes of opportunistic human infections. A major factor in its prominence as a pathogen is its intrinsic resistance to antibiotics and disinfectants. Here we report the complete sequence of P. aeruginosa strain PAO1. At 6.3 million base pairs, this is the largest bacterial genome sequenced, and the sequence provides insights into the basis of the versatility and intrinsic drug resistance of P. aeruginosa. Consistent with its larger genome size and environmental adaptability, P. aeruginosa contains the highest proportion of regulatory genes observed for a bacterial genome and a large number of genes involved in the catabolism, transport and efflux of organic compounds as well as four potential chemotaxis systems. We propose that the size and complexity of the P. aeruginosa genome reflect an evolutionary adaptation permitting it to thrive in diverse environments and resist the effects of a variety of antimicrobial substances.

Staphylococcus aureus genetic loci impacting growth and survival in multiple infection environments.

The Gram-positive bacterium Staphylococcus aureus infects diverse tissues and causes a wide spectrum of diseases, suggesting that it possesses a repertoire of distinct molecular mechanisms promoting bacterial survival in disparate in vivo environments. Signature-tag transposon mutagenesis screening of a 1520-member library identified numerous S. aureus genetic loci affecting growth and survival in four complementary animal infection models including mouse abscess, bacteraemia and wound and rabbit endocarditis. Of a total of 237 in vivo attenuated mutants identified by the murine models, less than 10% showed attenuation in all three models, emphasizing the advantage of screening in diverse disease environments. The largest gene class identified by these analyses encoded peptide and amino acid transporters, some of which were important for S. aureus survival in all animal infection models tested. The identification of staphylococcal loci affecting growth, persistence and virulence in multiple tissue environments provides insight into the complexities of human infection and on the molecular mechanisms that could be targeted by new antibacterial therapies.

VH usage and somatic hypermutation in peripheral blood B cells of patients with rheumatoid arthritis (RA).

The human antibody repertoire has been demonstrated to have a marked V-gene-dependent bias that is conserved between individuals. In RA patients, certain heavy chain V genes (VH) have been found to be preferentially used for encoding autoantibodies. To determine if such preferential use of VH genes in autoantibodies is associated with a general distortion of the V gene repertoire in RA patients, the VH composition of peripheral blood B cells was analysed among four RA patients and four age- and sex-matched healthy controls. Usage of individual VH genes (eight VH3 and three VH4 genes tested by hybridization with a set of gene-specific oligonucleotide probes) was highly biased among RA patients, but no evidence of a distortion in the bias was observed compared with healthy controls. However, the occurrence of somatic mutations in these VH genes (estimated by differential hybridization with motif-specific oligonucleotide probes targeted to CDR and FR of the tested genes, and by DNA sequence analysis) was strikingly different between patients and healthy subjects. The number of VH3 rearrangements that had accumulated somatic mutations and the number of mutations per rearrangement were significantly elevated in three of the four RA patients. A slight but not significant elevation in mutations among rearranged VH4 genes was also observed in these patients. These data suggest that although usage of individual VH genes among peripheral blood B cells is not affected by the disease, the autoimmune process may involve a significant fraction of the B cell compartment.

Immunoglobulin heavy chain variable region gene usage in bone marrow transplant recipients: lack of somatic mutation indicates a maturational arrest.

Many recipients of bone marrow transplant (BMT) make normal amounts of serum immunoglobulin but are deficient in generating specific antibody responses to exogenous stimuli. To determine if abnormal usage of VH genes contributes to this immunodeficiency, the usage of VH genes was determined in peripheral blood B cells of four BMT recipients, two of whom had developed chronic graft versus host disease. The pattern of usage of VH3 or VH4 genes assessed at either 90 days or approximately 1 year after transplant was similar to that observed in healthy subjects and was marked by the over utilization of two elements, one VH3 and one VH4. However, the repertoires of each of the four BMT recipients appeared to be less complex than the repertoires of healthy subjects. The differences were a consequence of the accumulation of somatic mutations among rearrangements in the controls but not in the BMT recipients. The failure to accumulate somatic mutations in rearranged VH genes is consistent with a defect in antigen driven B-cell responses. These results indicate the although the VH gene content of the repertoire has normalized by 90 days posttransplant, a maturational arrest in B-cell differentiation associated with antigen activation persists for at least 1 year after BMT.

Polymorphism and utilization of human VH Genes.

The human VH germline repertoire comprises approximately 100 elements, which can be grouped into seven families based on nucleotide sequence similarity. Members of different families are interspersed throughout the complex, with limited sets of alleles identified for most loci. Linkage disequilibrium between most elements is weak. Variation within the population can be attributed to differences in nucleotide sequence between allelic genes as well as to differences in the number of genes present. Gene number per haplotype varies as a result of the common occurrence of insertion/deletion polymorphisms, which may be small, involving a single element, or may be extensive, involving four or five elements. In some cases, such polymorphisms may involve duplication of a functional VH gene segment on some haplotypes and deletion of the gene on others. The resulting variation in germline composition of the VH locus may have profound effects on VH gene utilization.

Staphylococcal enterotoxin type A internal deletion mutants: serological activity and induction of T-cell proliferation.

Previous findings indicate that the N-terminal region of staphylococcal enterotoxin type A (SEA) is required for its ability to induce T-cell proliferation. To better localize internal peptides of SEA that are important for induction of murine T-cell proliferation, SEA mutants that had internal deletions in their N-terminal third were constructed. A series of unique restriction enzyme sites were first engineered into sea; only one of these changes resulted in an amino acid substitution (the aspartic acid residue at position 60 of mature SEA was changed to a glycine [D60G]). Because the D60G substitution had no discernible effect on serological or biological activity, the sea allele encoding this mutant SEA was used to construct a panel of mutant SEAs lacking residues 3 to 17, 19 to 23, 24 to 28, 29 to 49, 50 to 55, 56 to 59, 61 to 73, 68 to 74, or 74 to 85. Recombinant plasmids with the desired mutations were constructed in Escherichia coli and transferred to Staphylococcus aureus. Staphylococcal culture supernatants containing the mutant SEAs were examined. Western immunoblot analysis with polyclonal anti-SEA antiserum revealed that each of the recombinant S. aureus strains produced a mutant SEA of the predicted size. All the mutant SEAs exhibited increased sensitivity to monkey stomach lavage fluid in vitro, which is consistent with these mutants having conformations unlike that of wild-type SEA or the SEA D60G mutant. In general, deletion of internal peptides had a deleterious effect on the ability to induce T-cell proliferation; only SEA mutants lacking either residues 3 to 17 or 56 to 59 consistently produced a statistically significant increase in the incorporation of [3H]thymidine. In the course of this work, two monoclonal antibodies that had different requirements for binding to SEA in Western blots were identified. The epitope for one monoclonal antibody was contained within residues 108 to 230 of mature SEA. Binding of the other monoclonal antibody to SEA appeared to be dependent on the conformation of SEA.

The carboxyl-terminal region of staphylococcal enterotoxin type A is required for a fully active molecule.

Staphylococcal enterotoxin type A (SEA) gene (sea+) mutations were constructed by exonuclease III digestion or cassette mutagenesis. Five different sea mutations that had 1, 3, 7, 39, and 65 codons deleted from the 3' end of sea+ were identified and confirmed by restriction enzyme and nucleotide sequence analyses. Each of these sea mutations was constructed in Escherichia coli and transferred to Staphylococcus aureus by using the plasmid vector pC194. Culture supernatants from the parent S. aureus strain that lacked an enterotoxin gene (negative controls) and from derivatives that contained either sea+ (positive control) or a sea mutation were examined for in vitro sensitivity to degradation by monkey stomach lavage fluid, the ability to cause emesis when administered by an intragastric route to rhesus monkeys, and the ability to induce T-cell proliferation and by Western immunoblot analysis and a gel double-diffusion assay with polyclonal antibodies prepared against SEA. Altered SEAs corresponding to the predicted sizes were visualized by Western blot analysis of culture supernatants for each of the staphylococcal derivatives that contained a sea mutation. The altered SEA that lacked the C-terminal amino acid residue behaved like SEA in all of the assays performed. The altered SEA that lacked the three C-terminal residues of SEA caused T-cell proliferation but was not emetic; this altered SEA was degraded in vitro by monkey stomach lavage fluid and did not reach in the gel double diffusion assay. Altered SEAs that lacked 7, 39, or 65 carboxyl-terminal residues were degraded by stomach lavage fluid in vitro, did not produce an emetic response, and did not induce T-cell proliferation or form a visible reaction in the gel double-diffusion assay.