Evolutionary genomics of Staphylococcus aureus: Insights into the origin of methicillin-resistant strains and the toxic shock syndrome epidemic

An emerging theme in medical microbiology is that extensive variation exists in gene content among strains of many pathogenic bacterial species. However, this topic has not been investigated on a genome scale with strains recovered from patients with well-defined clinical conditions. Staphylococcus aureus is a major human pathogen and also causes economically important infections in cows and sheep. A DNA microarray representing >90% of the S. aureus genome was used to characterize genomic diversity, evolutionary relationships, and virulence gene distribution among 36 strains of divergent clonal lineages, including methicillin-resistant strains and organisms causing toxic shock syndrome. Genetic variation in S. aureus is very extensive, with ≈22% of the genome comprised of dispensable genetic material. Eighteen large regions of difference were identified, and 10 of these regions have genes that encode putative virulence factors or proteins mediating antibiotic resistance. We find that lateral gene transfer has played a fundamental role in the evolution of S. aureus. The mec gene has been horizontally transferred into distinct S. aureus chromosomal backgrounds at least five times, demonstrating that methicillin-resistant strains have evolved multiple independent times, rather than from a single ancestral strain. This finding resolves a long-standing controversy in S. aureus research. The epidemic of toxic shock syndrome that occurred in the 1970s was caused by a change in the host environment, rather than rapid geographic dissemination of a new hypervirulent strain. DNA microarray analysis of large samples of clinically characterized strains provides broad insights into evolution, pathogenesis, and disease emergence.

Molecular markers reveal that population structure of the human pathogen Candida albicans exhibits both clonality and recombination.

The life history of Candida albicans presents an enigma: this species is thought to be exclusively asexual, yet strains show extensive phenotypic variation. To address the population genetics of C. albicans, we developed a genetic typing method for codominant single-locus markers by screening randomly amplified DNA for single-strand conformation polymorphisms. DNA fragments amplified by arbitrary primers were initially screened for single-strand conformation polymorphisms and later sequenced using locus-specific primers. A total of 12 single base mutations and insertions were detected from six out of eight PCR fragments. Patterns of sequence-level polymorphism observed for individual strains detected considerable heterozygosity at the DNA sequence level, supporting the view that most C. albicans strains are diploid. Population genetic analyses of 52 natural isolates from Duke University Medical Center provide evidence for both clonality and recombination in C. albicans. Evidence for clonality is supported by the presence of several overrepresented genotypes, as well as by deviation of genotypic frequencies from random (Hardy-Weinberg) expectations. However, tests for nonrandom association of alleles across loci reveal less evidence for linkage disequilibrium than expected for strictly clonal populations. Although C. albicans populations are primarily clonal, evidence for recombination suggests that sexual reproduction or some other form of genetic exchange occurs in this species.

Rad51 expression and localization in B cells carrying out class switch recombination.

Rad51 is a highly conserved eukaryotic homolog of the prokaryotic recombination protein RecA, which has been shown to function in both recombinational repair of DNA damage and meiotic recombination in yeast. In primary murine B cells cultured with lipopolysaccharide (LPS) to stimulate heavy chain class switch recombination, Rad51 protein levels are dramatically induced. Immunofluorescent microscopy shows that anti-Rad51 antibodies stain foci that are localized within the nuclei of switching B cells. Immunohistochemical analysis of splenic sections shows that clusters of cells that stain brightly with anti-Rad51 antibodies are evident within several days after primary immunization and that Rad51 staining in vivo is confined to B cells that are switching from expression of IgM to IgG antibodies. Following switch recombination, B cells populate splenic germinal centers, where somatic hypermutation and clonal proliferation occur. Germinal center B cells are not stained by anti-Rad51 antibodies. Rad51 expression is therefore not coincident with somatic hypermutation, nor does Rad51 expression correlate simply with cell proliferation. These data suggest that Rad51, or a highly related member of the conserved RecA family, may function in class switch recombination.

Transgenic expression of PML/RARalpha impairs myelopoiesis.

The translocation found in acute promyelocytic leukemia rearranges the promyelocytic leukemia gene (PML) on chromosome 15 with the retinoic acid receptor alpha (RARalpha) on chromosome 17. This yields a fusion transcript, PML/RARalpha, a transcription factor with reported dominant negative functions in the absence of hormone. Clinical remissions induced with all-trans retinoic acid (RA) treatment in acute promyelocytic leukemia are linked to PML/RARalpha expression in leukemic cells. To evaluate the PML/RARalpha role in myelopoiesis, transgenic mice expressing PML/RARalpha were engineered. A full-length PML/RARalpha cDNA driven by the CD11b promoter was expressed in transgenic mice. Expression was confirmed in the bone marrow with a reverse transcription PCR assay. Basal total white blood cell and granulocyte counts did not appreciably differ between PML/RARalpha transgenic and control mice. Cell sorter analysis of CD11b+ bone marrow cells revealed similar CD11b+ populations in transgenic and control mice. However, in vitro clonal growth assays performed on peripheral blood from transgenic versus control mice revealed a marked reduction of myeloid progenitors, especially in those responding to granulocyte/ macrophage colony-stimulating factor. Granulocyte/macrophage colony-stimulating factor and kit ligand cotreatment did not overcome this inhibition. Impaired myelopoiesis in vivo was shown by stressing these mice with sublethal irradiation. Following irradiation, PML/RARalpha transgenic mice, as compared with controls, more rapidly depressed peripheral white blood cell and granulocyte counts. As expected, nearly all control mice (94.4%) survived irradiation, yet this irradiation was lethal to 45.8% of PML/RARalpha transgenic mice. Lethality was associated with more severe leukopenia in transgenic versus control mice. Retinoic acid treatment of irradiated PML/RARalpha mice enhanced granulocyte recovery. These data suggest that abnormal myelopoiesis due to PML/RARalpha expression is an early event in oncogenic transformation.

The control of hematopoiesis and leukemia: from basic biology to the clinic.

Hematopoiesis gives rise to blood cells of different lineages throughout normal life. Abnormalities in this developmental program lead to blood cell diseases including leukemia. The establishment of a cell culture system for the clonal development of hematopoietic cells made it possible to discover proteins that regulate cell viability, multiplication and differentiation of different hematopoietic cell lineages, and the molecular basis of normal and abnormal blood cell development. These regulators include cytokines now called colony-stimulating factors (CSFs) and interleukins (ILs). There is a network of cytokine interactions, which has positive regulators such as CSFs and ILs and negative regulators such as transforming growth factor beta and tumor necrosis factor (TNF). This multigene cytokine network provides flexibility depending on which part of the network is activated and allows amplification of response to a particular stimulus. Malignancy can be suppressed in certain types of leukemic cells by inducing differentiation with cytokines that regulate normal hematopoiesis or with other compounds that use alternative differentiation pathways. This created the basis for the clinical use of differentiation therapy. The suppression of malignancy by inducing differentiation can bypass genetic abnormalities that give rise to malignancy. Different CSFs and ILs suppress programmed cell death (apoptosis) and induce cell multiplication and differentiation, and these processes of development are separately regulated. The same cytokines suppress apoptosis in normal and leukemic cells, including apoptosis induced by irradiation and cytotoxic cancer chemotherapeutic compounds. An excess of cytokines can increase leukemic cell resistance to cytotoxic therapy. The tumor suppressor gene wild-type p53 induces apoptosis that can also be suppressed by cytokines. The oncogene mutant p53 suppresses apoptosis. Hematopoietic cytokines such as granulocyte CSF are now used clinically to correct defects in hematopoiesis, including repair of chemotherapy-associated suppression of normal hematopoiesis in cancer patients, stimulation of normal granulocyte development in patients with infantile congenital agranulocytosis, and increase of hematopoietic precursors for blood cell transplantation. Treatments that decrease the level of apoptosis-suppressing cytokines and downregulate expression of mutant p53 and other apoptosis suppressing genes in cancer cells could improve cytotoxic cancer therapy. The basic studies on hematopoiesis and leukemia have thus provided new approaches to therapy.

Human V delta 2+ gamma delta T-cell tolerance to foreign antigens of Toxoplasma gondii.

Little is known about the mechanisms involved in human gammadelta T-cell tolerance to self or to foreign antigens. Patients with congenital toxoplasmosis offer a unique opportunity to examine Vdelta2+ gammadelta T-cell tolerance. Analysis of gammadelta T cells in patients with congenital toxoplasmosis revealed evidence for anergy of these cells with or without clonal Vdelta2+ gammadelta T-cell expansion in the acute phase of the Toxoplasma infection. T cells in general were unresponsive and did not proliferate upon exposure to mitogens or to Toxoplasma lysate antigens or in response to live Toxoplasma-infected cells when the congenitally infected infants were 1 month of age, and they exhibited selective anergy to Toxoplasma lysate antigens and live Toxoplasma-infected cells when the infants were aged 5 months. During the chronic phase of congenital toxoplasmosis in the patients who were more than I year of age, the repertoires of the gammadelta T-cell receptors were found to be within normal ranges. In addition, in the chronic phase, the gammadelta T cells proliferated and secreted gamma-interferon in response to exposure to live Toxoplasmia-infected cells. By contrast, alphabeta T cells remained anergic. Vdelta2+ gammadelta T cells have been considered to undergo extrathymic maturation and thus to be subject to development of peripheral tolerance. Our findings indicate that Vdelta2+ gammadelta T-cell tolerance was lost in these infected infants earlier than alphabeta T-cell tolerance. These findings suggest that gammadelta T cells play a role in protection against Toxoplasma gondii in the chronic phase when congenitally infected children are more than 1 year of age, especially in those in whom alphabeta T cells continue to exhibit deficits in specific immune responses to Toxoplasma antigens.

Variant antigens and endothelial receptor adhesion in Plasmodium falciparum.

Parasite-derived proteins expressed on the surface of erythrocytes infected with Plasmodium falciparum are important virulence factors, since they mediate binding of infected cells to diverse receptors on vascular endothelium and are targets of a protective immune response. They are difficult to study because they undergo rapid clonal antigenic variation in vitro, which precludes the derivation of phenotypically homogeneous cultures. Here we have utilized sequence-specific proteases to dissect the role of defined antigenic variants in binding to particular receptors. By selection of protease-resistant subpopulations of parasites on defined receptors we (i) confirm the high rate of antigenic variation in vitro; (ii) demonstrate that a single infected erythrocyte can bind to intercellular adhesion molecule 1, CD36, and thrombospondin; (iii) show that binding to intercellular adhesion molecule 1 and CD36 are functions of the variant antigen; and (iv) suggest that binding to thrombospondin may be mediated by other components of the infected erythrocyte surface.

Enhanced and accelerated lymphoproliferation in Fas-null mice.

Fas is a 45-kDa membrane protein that transduces an apoptotic signal. The mouse lymphoproliferation (lpr) mutation is a leaky mutation of Fas. In this study, we examined lymphocyte development in Fas-null mice generated by gene targeting. The Fas-/- mice progressively accumulated abnormal T cells (Thy1+, B220+, CD4-, and CD8-) and developed lymphadenopathy and splenomegaly, which were much more accelerated and pronounced than those in lpr mice. In addition, the Fas-null mice showed lymphocytosis, accompanied by lymphocytic infiltration in the lungs and liver. The number of apparently normal B cells also increased, and large amounts of immunoglobulins, including anti-DNA antibodies, were produced. Thymic clonal deletion, assessed by deletion of T cells reactive to mouse endogenous superantigens, was apparently normal in the Fas-/- mice, whereas the peripheral clonal deletion of mature T cells against a bacterial superantigen was impaired. These results suggested that Fas plays a decisive role in peripheral clonal deletion but not in negative selection in the thymus.

Cell cycling and patterned cell proliferation in the wing primordium of Drosophila.

The pattern of cell proliferation in the Drosophila imaginal wing primordium is spatially and temporally heterogeneous. Direct visualization of cells in S, G2, and mitosis phases of the cell cycle reveals several features invariant throughout development. The fraction of cells in the disc in the different cell cycle stages is constant, the majority remaining in G1. Cells in the different phases of the cell cycle mainly appear in small synchronic clusters that are nonclonally derived but result from changing local cell-cell interactions. Cluster synchronization occurs before S and in the G2/M phases. Rates of cell division are neither constant nor clonal features. Cell cycle progression is linear rather than concentric. Clusters appear throughout the disc but with symmetries related to presumptive wing patterns, compartment boundaries, and vein clonal restrictions.

Selective pressure of a quinoxaline nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) on HIV-1 replication results in the emergence of nucleoside RT-inhibitor-specific (RT Leu-74-->Val or Ile and Val-75-->Leu or Ile) HIV-1 mutants.

The quinoxaline nonnucleoside RT inhibitor (NNRTI) (S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3,4- dihydroquinoxaline-2(1H)-thione (HBY 097) was used to select for drug-resistant HIV-1 variants in vitro. The viruses first developed mutations affecting the NNRTI-binding pocket, and five of six strains displayed the RT G190-->E substitution, which is characteristic for HIV-1 resistance against quinoxalines. In one variant, a new mutant (G190-->Q) most likely evolved from preexisting G190-->E mutants. The negative charge introduced by the G190-->E substitution was maintained at that site of the pocket by simultaneous selection for V179-->D together with G190-->Q. After continued exposure to the drug, mutations at positions so far known to be specific for resistance against nucleoside RT inhibitors (NRTIs) (L74-->V/I and V75-->L/I) were consistently detected in all cultures. The inhibitory activities of the cellular conversion product of 2',3'-dideoxyinosine (ddI, didanosine), 2',3'-dideoxyadenosine (ddA) and of 2',3'-didehydro-3'-deoxythymidine (d4T, stavudine) against these late-passage viruses were shown to be enhanced with the L74-->V/I RT mutant virus as compared with the wild-type (wt) HIV-1MN isolate. Clonal analysis proved linkage of the codon 74 and codon 75 mutations to the NNRTI-specific mutations in all RT gene fragments. The nonnucleoside- and nucleoside-resistance mutation sites are separated by approximately 35 A. We propose that the two sites "communicate" through the template-primer which is situated in the DNA-binding cleft between these two sites. Quinoxalines cause high selective pressure on HIV-1 replication in vitro; however, the implication of these findings for the treatment of HIV-1 infection has yet to be determined.

Human naive and memory T lymphocytes differ in telomeric length and replicative potential.

The present study has assessed the replicative history and the residual replicative potential of human naive and memory T cells. Telomeres are unique terminal chromosomal structures whose length has been shown to decrease with cell division in vitro and with increased age in vivo for human somatic cells. We therefore assessed telomere length as a measure of the in vivo replicative history of naive and memory human T cells. Telomeric terminal restriction fragments were found to be 1.4 +/- 0.1 kb longer in CD4+ naive T cells than in memory cells from the same donors, a relationship that remained constant over a wide range of donor age. These findings suggest that the differentiation of memory cells from naive precursors occurs with substantial clonal expansion and that the magnitude of this expansion is, on average, similar over a wide range of age. In addition, when replicative potential was assessed in vitro, it was found that the capacity of naive cells for cell division was 128-fold greater as measured in mean population doublings than the capacity of memory cells from the same individuals. Human CD4+ naive and memory cells thus differ in in vivo replicative history, as reflected in telomeric length, and in their residual replicative capacity.

Hematopoietic and lung abnormalities in mice with a null mutation of the common beta subunit of the receptors for granulocyte-macrophage colony-stimulating factor and interleukins 3 and 5.

Gene targeting was used to create mice with a null mutation of the gene encoding the common beta subunit (beta C) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3; multi-CSF), and interleukin 5 (IL-5) receptor complexes (beta C-/- mice). High-affinity binding of GM-CSF was abolished in beta C-/- bone marrow cells, while cells from heterozygous animals (beta C+/- mice) showed an intermediate number of high-affinity receptors. Binding of IL-3 was unaffected, confirming that the IL-3-specific beta chain remained intact. Eosinophil numbers in peripheral blood and bone marrow of beta C-/- animals were reduced, while other hematological parameters were normal. In clonal cultures of beta C-/- bone marrow cells, even high concentrations of GM-CSF and IL-5 failed to stimulate colony formation, but the cells exhibited normal quantitative responsiveness to stimulation by IL-3 and other growth factors. beta C-/- mice exhibited normal development and survived to young adult life, although they developed pulmonary peribronchovascular lymphoid infiltrates and areas resembling alveolar proteinosis. There was no detectable difference in the systemic clearance and distribution of GM-CSF between beta C-/- and wild-type littermates. The data establish that beta C is normally limiting for high-affinity binding of GM-CSF and demonstrate that systemic clearance of GM-CSF is not mediated via such high-affinity receptor complexes.

[Leu5]enkephalin-encoding sequences are targets for a specific DNA-binding factor.

A DNA-binding factor with high affinity and specificity for the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes has been characterized. The factor has the highest affinity for the [Leu5]-enkephalin-encoding sequence in the dynorphin B-encoding region of the prodynorphin gene, has relatively high affinity for other [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes, but has no apparent affinity for similar DNA sequences coding for [Met5]-enkephalin in the prodynorphin or proopiomelanocortin genes. The factor has been named [Leu5]enkephalin-encoding sequence DNA-binding factor (LEF). LEF has a nuclear localization and is composed of three subunits of about 60, 70, and 95 kDa, respectively. The highest levels were observed in rat testis, cerebellum, and spleen and were generally higher in late embryonal compared to newborn or adult animals. LEF activity was also recorded in human clonal tumor cell lines. LEF inhibited the transcription of reporter genes in artificial gene constructs where a [Leu5]enkephalin-encoding DNA fragment had been inserted between the transcription initiation site and the coding region of the reporter genes. These observations suggest that the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes also have regulatory functions realized through interaction with a specific DNA-binding factor.

Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene.

Retrovirus-mediated gene transfer into hematopoietic cells may provide a means of treating both inherited and acquired diseases involving hematopoietic cells. Implementation of this approach for disorders resulting from mutations affecting the beta-globin gene (e.g., beta-thalassemia and sickle cell anemia), however, has been hampered by the inability to generate recombinant viruses able to efficiently and faithfully transmit the necessary sequences for appropriate gene expression. We have addressed this problem by carefully examining the interactions between retroviral and beta-globin gene sequences which affect vector transmission, stability, and expression. First, we examined the transmission properties of a large number of different recombinant proviral genomes which vary both in the precise nature of vector, beta-globin structural gene, and locus control region (LCR) core sequences incorporated and in the placement and orientation of those sequences. Through this analysis, we identified one specific vector, termed M beta 6L, which carries both the human beta-globin gene and core elements HS2, HS3, and HS4 from the LCR and faithfully transmits recombinant proviral sequences to cells with titers greater than 10(6) per ml. Populations of murine erythroleukemia (MEL) cells transduced by this virus expressed levels of human beta-globin transcript which, on a per gene copy basis, were 78% of the levels detected in an MEL-derived cell line, Hu11, which carries human chromosome 11, the site of the beta-globin locus. Analysis of individual transduced MEL cell clones, however, indicated that, while expression was detected in every clone tested (n = 17), the levels of human beta-globin treatment varied between 4% and 146% of the levels in Hu11. This clonal variation in expression levels suggests that small beta-globin LCR sequences may not provide for as strict chromosomal position-independent expression of beta-globin as previously suspected, at least in the context of retrovirus-mediated gene transfer.

Sex proportions of Haemoproteus blood parasites and local mate competition.

Recent genetic evidence suggests that parasitic protozoa often reproduce by "selfing," defined as sexual stages from a single, clonal lineage fertilizing each other. Selfing favors production of an excess of female over male progeny. We tested whether the proportion of male gametocytes of blood parasites of the genus Haemoproteus was affected by variables that could influence the probability of selfing. Proportions of male Haemoproteus gametocytes from 11 passerine host populations were not affected by the age of the parasites' avian hosts, date in season, sex of host, intensity of host's infection, or prevalence of parasites within host populations.