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PubMed2006

Contribution of penicillin-binding protein homologs to antibiotic resistance, cell morphology, and virulence of Listeria monocytogenes EGDe.

Seven open reading frames, annotated as potential penicillin-binding-protein-encoding genes (lmo0441, lmo0540, lmo1438, lmo1892, lmo2039, lmo2229, and lmo2754), were targeted for insertional mutagenesis in Listeria monocytogenes EGDe. These genes were found to contribute in various degrees to beta-lactam resistance, cell morphology, or the virulence potential of this organism.

Amino Acid Sequence↗
PubMed2003

Antimicrobial characterization of human beta-defensin 3 derivatives.

Human beta-defensin 3 (hBD3) is a highly basic 45-amino-acid protein that acts both as an antimicrobial agent and as a chemoattractant molecule. Although the nature of its antimicrobial activity is largely electrostatic, the importance of the molecular structure on this activity is poorly understood. Two isoforms of hBD3 were synthesized: the first with native disulfide linkages and the second with nonnative linkages. In a third synthetic peptide, all cysteine residues were replaced with alpha-aminobutyric acid, creating a completely linear peptide. A series of six small, linear peptides corresponding to regions of hBD3 with net charges ranging from +4 to +8 (at pH 7) and lengths ranging from 9 to 20 amino acids were also synthesized. The linear full-length peptide showed the highest microbicidal activity against Escherichia coli and Staphylococcus aureus, while all three full-length forms showed equal activity against Candida albicans. The linear peptide also showed high activity against Enterococcus faecium and Pseudomonas aeruginosa. Peptides corresponding to the C terminus showed higher activities when tested against E. coli, with the most active peptides being the most basic. However, only the peptide corresponding to the N terminus of hBD3 showed any activity against S. aureus and C. albicans. Further, N-terminal deletion mutants of native hBD3 showed diminished activities against S. aureus. Thus, the antimicrobial properties of hBD3 derivatives are determined by both charge and structure.

Amino Acid Sequence↗
PubMed1991

Detection of luciferase gene sequence in nonluminescent Vibrio cholerae by colony hybridization and polymerase chain reaction.

Bioluminescence is a trait observed among approximately 10% of Vibrio cholerae isolates. We have demonstrated that not only do some strains of V. cholerae produce low levels of light, undetectable by the human eye, but the luciferase gene sequence is present in strains of V. cholerae which emit no detectable light, evidenced by hybridization with a luciferase DNA probe. Comparisons of the amino acid sequences of luciferase enzymes of marine species have shown that these proteins have diverged to the point where they have only short regions of amino acid identity. The polymerase chain reaction method of DNA amplification with oligonucleotide primers based on these regions was used to isolate a region of the luxA gene from both luminescent and nonluminescent V. cholerae strains. The nucleotide sequence of this region was determined and reveals that nonluminescent V. cholerae have 99.7% nucleotide sequence similarity in this region with the luminescent biovar V. cholerae bv. albensis as well as significant similarity to other species of bioluminescent bacteria, a finding that is in accord with the hypothesis that these species have a common luminescent ancestor, most probably from the marine environment.

Amino Acid Sequence↗
PubMed2001

Changes in activity and community structure of methane-oxidizing bacteria over the growth period of rice.

The activity and community structure of methanotrophs in compartmented microcosms were investigated over the growth period of rice plants. In situ methane oxidation was important only during the vegetative growth phase of the plants and later became negligible. The in situ activity was not directly correlated with methanotrophic cell counts, which increased even after the decrease in in situ activity, possibly due to the presence of both vegetative cells and resting stages. By dividing the microcosms into two soil and two root compartments it was possible to locate methanotrophic growth and activity, which was greatest in the rhizoplane of the rice plants. Molecular analysis by denaturing gradient gel electrophoresis and fluorescent in situ hybridization (FISH) with family-specific probes revealed the presence of both families of methanotrophs in soil and root compartments over the whole season. Changes in community structure were detected only for members of the Methylococcaceae and could be associated only with changes in the genus Methylobacter and not with changes in the dominance of different genera in the family Methylococcaceae. For the family Methylocystaceae stable communities in all compartments for the whole season were observed. FISH analysis revealed evidence of in situ dominance of the Methylocystaceae in all compartments. The numbers of Methylococcaceae cells were relatively high only in the rhizoplane, demonstrating the importance of rice roots for growth and maintenance of methanotrophic diversity in the soil.

Colony Count, Microbial↗
PubMed2002

Temporal changes in archaeal diversity and chemistry in a mid-ocean ridge subseafloor habitat.

The temporal variation in archaeal diversity in vent fluids from a midocean ridge subseafloor habitat was examined using PCR-amplified 16S rRNA gene sequence analysis and most-probable-number (MPN) cultivation techniques targeting hyperthermophiles. To determine how variations in temperature and chemical characteristics of subseafloor fluids affect the microbial communities, we performed molecular phylogenetic and chemical analyses on diffuse-flow vent fluids from one site shortly after a volcanic eruption in 1998 and again in 1999 and 2000. The archaeal population was divided into particle-attached (>3-microm-diameter cells) and free-living fractions to test the hypothesis that subseafloor microorganisms associated with active hydrothermal systems are adapted for a lifestyle that involves attachment to solid surfaces and formation of biofilms. To delineate between entrained seawater archaea and the indigenous subseafloor microbial community, a background seawater sample was also examined and found to consist only of Group I Crenarchaeota and Group II Euryarchaeota, both of which were also present in vent fluids. The indigenous subseafloor archaeal community consisted of clones related to both mesophilic and hyperthermophilic Methanococcales, as well as many uncultured Euryarchaeota, some of which have been identified in other vent environments. The particle-attached fraction consistently showed greater diversity than the free-living fraction. The fluid and MPN counts indicate that while culturable hyperthermophiles represent less than 1% of the total microbial community, the subseafloor at new eruption sites does support a hyperthermophilic microbial community. The temperature and chemical indicators of the degree of subseafloor mixing appear to be the most important environmental parameters affecting community diversity, and it is apparent that decreasing fluid temperatures correlated with increased entrainment of seawater, decreased concentrations of hydrothermal chemical species, and increased incidence of seawater archaeal sequences.

Archaea↗
PubMed2003

Population dynamics of Vibrio fischeri during infection of Euprymna scolopes.

The luminous bacterium Vibrio fischeri colonizes a specialized light-emitting organ within its squid host, Euprymna scolopes. Newly hatched juvenile squid must acquire their symbiont from ambient seawater, where the bacteria are present at low concentrations. To understand the population dynamics of V. fischeri during colonization more fully, we used mini-Tn7 transposons to mark bacteria with antibiotic resistance so that the growth of their progeny could be monitored. When grown in culture, there was no detectable metabolic burden on V. fischeri cells carrying the transposon, which inserts in single copy in a specific intergenic region of the V. fischeri genome. Strains marked with mini-Tn7 also appeared to be equivalent to the wild type in their ability to infect and multiply within the host during coinoculation experiments. Studies of the early stages of colonization suggested that only a few bacteria became associated with symbiotic tissue when animals were exposed for a discrete period (3 h) to an inoculum of V. fischeri cells equivalent to natural population levels; nevertheless, all these hosts became infected. When three differentially marked strains of V. fischeri were coincubated with juvenile squid, the number of strains recovered from an individual symbiotic organ was directly dependent on the size of the inoculum. Further, these results indicated that, when exposed to low numbers of V. fischeri, the host may become colonized by only one or a few bacterial cells, suggesting that symbiotic infection is highly efficient.

Animal Structures↗
PubMed2003

Incidence and diversity of microorganisms within the walls of an active deep-sea sulfide chimney.

A large, intact sulfide chimney, designated Finn, was recovered from the Mothra Vent Field on the Juan de Fuca Ridge in 1998. Finn was venting 302 degrees C fluids on the seafloor and contained complex mineralogical zones surrounding a large open central conduit. Examination of microorganisms within these zones, followed by community analysis with oligonucleotide probes, showed that there were variations in the abundance and diversity of eubacteria and archaea from the exterior to the interior of the chimney. The microbial abundance based upon epifluorescence microscopy and quantitative fatty acid analyses varied from >10(8) cells/g of sulfide 2 to 10 cm within the chimney wall to <10(5) cells/g in interior zones. Direct microscopic observation indicated that microorganisms were attached to mineral surfaces throughout the structure. Whole-cell hybridization results revealed that there was a transition from a mixed community of eubacteria and archaea near the cool exterior of the chimney to primarily archaea near the warm interior. Archaeal diversity was examined in three zones of Finn by cloning and sequencing of the 16S rRNA gene. The majority of sequences from the exterior of the chimney were related to marine group I of the Crenarchaeota and uncultured Euryarchaeota from benthic marine environments. In contrast, clone libraries from interior regions of the chimney contained sequences closely related to methanogens, Thermococcales, and Archaeoglobales, in addition to uncultured crenarchaeal phylotypes obtained from deep subsurface sites. These observations of microbial communities within an active hydrothermal chimney provide insight into the microbial ecology within such structures and may facilitate follow-up exploration into expanding the known upper temperature limits of life.

Archaea↗
PubMed2004

Diversity of nontuberculoid Mycobacterium species in biofilms of urban and semiurban drinking water distribution systems.

Nontuberculous mycobacteria (NTM) are ubiquitous and have been isolated from a variety of environmental sources, including water. Various NTM were isolated from biofilms in drinking water distribution systems in two urban and two semiurban areas in South Africa. Most of the isolates belonged to opportunistic pathogenic species of the NTM group, but none belonged to the Mycobacterium avium complex.

Biofilms↗
PubMed2005

Bacterial community dynamics and polycyclic aromatic hydrocarbon degradation during bioremediation of heavily creosote-contaminated soil.

Bacterial community dynamics and biodegradation processes were examined in a highly creosote-contaminated soil undergoing a range of laboratory-based bioremediation treatments. The dynamics of the eubacterial community, the number of heterotrophs and polycyclic aromatic hydrocarbon (PAH) degraders, and the total petroleum hydrocarbon (TPH) and PAH concentrations were monitored during the bioremediation process. TPH and PAHs were significantly degraded in all treatments (72 to 79% and 83 to 87%, respectively), and the biodegradation values were higher when nutrients were not added, especially for benzo(a)anthracene and chrysene. The moisture content and aeration were determined to be the key factors associated with PAH bioremediation. Neither biosurfactant addition, bioaugmentation, nor ferric octate addition led to differences in PAH or TPH biodegradation compared to biodegradation with nutrient treatment. All treatments resulted in a high first-order degradation rate during the first 45 days, which was markedly reduced after 90 days. A sharp increase in the size of the heterotrophic and PAH-degrading microbial populations was observed, which coincided with the highest rates of TPH and PAH biodegradation. At the end of the incubation period, PAH degraders were more prevalent in samples to which nutrients had not been added. Denaturing gradient gel electrophoresis analysis and principal-component analysis confirmed that there was a remarkable shift in the composition of the bacterial community due to both the biodegradation process and the addition of nutrients. At early stages of biodegradation, the alpha-Proteobacteria group (genera Sphingomonas and Azospirillum) was the dominant group in all treatments. At later stages, the gamma-Proteobacteria group (genus Xanthomonas), the alpha-Proteobacteria group (genus Sphingomonas), and the Cytophaga-Flexibacter-Bacteroides group (Bacteroidetes) were the dominant groups in the nonnutrient treatment, while the gamma-Proteobacteria group (genus Xathomonas), the beta-Proteobacteria group (genera Alcaligenes and Achromobacter), and the alpha-Proteobacteria group (genus Sphingomonas) were the dominant groups in the nutrient treatment. This study shows that specific bacterial phylotypes are associated both with different phases of PAH degradation and with nutrient addition in a preadapted PAH-contaminated soil. Our findings also suggest that there are complex interactions between bacterial species and medium conditions that influence the biodegradation capacity of the microbial communities involved in bioremediation processes.

Bacteria↗
PubMed1998

Acquired thermotolerance and temperature-induced protein accumulation in the extremely thermophilic bacterium Rhodothermus obamensis.

Temperature-induced changes in thermotolerance and protein composition were examined in heat-shocked cells and high-temperature-grown cells of the extremely thermophilic bacterium Rhodothermus obamensis. The survival at temperatures superoptimal for growth (90 and 95 degrees C) was enhanced in both heat-shocked cells and high-temperature-grown cells relative to that of cells grown at optimal temperatures. In a comparison of protein composition using two-dimensional gel electrophoresis, putative heat shock proteins (HSPs) and high-temperature growth-specific proteins (HGPs) were detected. N-terminal amino acid sequence analysis revealed that the putative HSPs were quite similar to the ATP-binding subunits of ABC transporters and the HGPs were proteins corresponding to domains II and III of elongation factor Tu. These results suggested that this extreme thermophile has developed temperature-induced responses that include increased survival under hyperthermal conditions, changes in protein composition, and also the production of novel HSPs.

Amino Acid Sequence↗
PubMed1996

Increased MCP-1, RANTES, and MIP-1alpha in bronchoalveolar lavage fluid of allergic asthmatic patients.

Chemokines are cytokines that induce chemotaxis of inflammatory cells. We studied the presence of chemokines in bronchoalveolar lavage fluid (BALF) obtained from nine allergic asthmatic patients and six nonsmoking normal individuals. The cells were pelleted, and ribonucleic acid (RNA) was extracted by using RNAzol B. BALF was assayed for monocyte chemoattractant protein-1 (MCP-1), regulated upon activation in normal T cells, expressed, probably secreted (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha) and interleukin-8 (IL-8) by enzyme-linked immunosorbent assay (ELISA). The levels of MCP-1, RANTES, and MIP-1alpha were significantly higher in the asthma patients than in the control subjects (p<0.04). The concentrations of RANTES and MCP-1 correlated with the lymphocyte count in the BAL specimens (r = 0.61 and 0.68, respectively). BALF showed eosinophil chemotactic activity in vitro that was blocked by anti-RANTES and anti-MCP-3 antibodies. The total cellular RNA was reverse-transcribed and the complementary deoxyribonucleic acid (cDNA) was amplified with the polymerase chain reaction (PCR) for MCP-1, MCP-3, RANTES, MIP-1alpha, IL-8, and beta-actin. We found that messenger ribonucleic acids (mRNAs) for MCP-1, MCP-3, RANTES, MIP-1alpha, and IL-8 were produced by BAL cells from most asthmatic and normal subjects. We conclude that chemokines are produced in the airways, and that an increased recovery of MCP-1, RANTES, and MIP-1alpha is observed in allergic asthmatic patients.

Adolescent↗
PubMed1995

Early pulmonary inflammation in infants with cystic fibrosis.

The mechanisms underlying the initiation of lung disease and early respiratory morbidity in cystic fibrosis (CF) are poorly understood. By identifying infants with CF through a statewide neonatal screening program, we investigated whether airway inflammation was present in these infants, with the goal of furthering our understanding of the early events in this lung disease. Bronchoalveolar lavage fluid (BALF) from 16 infants with CF (mean age, 6 mo) and 11 disease control infants (mean age, 12 mo) was examined for the following inflammatory parameters: (1) neutrophil count; (2) activity of free neutrophil elastase; (3) elastase/alpha 1-antiprotease inhibitor complexes; and (4) the level of interleukin-8 (IL-8). We also quantified the spontaneous level of expression of IL-8 mRNA transcripts by airway macrophages. Each index of airway inflammation was increased in the BALF of infants with CF as compared with control infants. In addition, both the number of neutrophils and IL-8 levels were increased in infants with CF who had negative cultures (n = 7) for common bacterial CF-related pathogens, as well as for common respiratory viruses and fungi at the time of bronchoalveolar lavage (BAL). These findings suggest that airway inflammation is already present in infants with CF who are as young as 4 wks. Furthermore, although many different cell types (e.g., epithelial cells) may express IL-8, airway macrophages appear to be a source of this chemokine, and may thus play a prominent role in early neutrophil influx into the lung.

Base Sequence↗
PubMed2000

Disturbed peripheral B lymphocyte homeostasis in systemic lupus erythematosus.

In patients with active systemic lupus erythematosus (SLE), a marked B lymphocytopenia was identified that affected CD19(+)/CD27(-) naive B cells more than CD19(+)/CD27(+) memory B cells, leading to a relative predominance of CD27-expressing peripheral B cells. CD27(high)/CD38(+)/CD19(dim)/surface Ig(low)/CD20(-)/CD138(+) plasma cells were found at high frequencies in active but not inactive SLE patients. Upon immunosuppressive therapy, CD27(high) plasma cells and naive CD27(-) B cells were markedly decreased in the peripheral blood. Mutational analysis of V gene rearrangements of individual B cells confirmed that CD27(+) B cells coexpressing IgD were memory B cells preferentially using V(H)3 family members with multiple somatic mutations. CD27(high) plasma cells showed a similar degree of somatic hypermutation, but preferentially employed V(H)4 family members. These results indicate that there are profound abnormalities in the various B cell compartments in SLE that respond differently to immunosuppressive therapy.

ADP-ribosyl Cyclase↗
PubMed2000

Evidence for two subgroups of CD4-CD8- NKT cells with distinct TCR alpha beta repertoires and differential distribution in lymphoid tissues.

NKT cells are a subset of T lymphocytes that is mainly restricted by the nonclassical MHC class I molecule, CD1d, and that includes several subpopulations, in particular CD4+ and CD4-CD8- (DN) cells. In the mouse, differential distribution of these subpopulations as well as heterogeneity in the expression of various markers as a function of tissue localization have been reported. We have thus undertaken a detailed study of the DN NKT cell subpopulation. With a highly sensitive semiquantitative RT-PCR technique, its TCR repertoire was characterized in various tissues. We found that mouse DN NKT cells are a variable mixture of two subgroups, one bearing the invariant Valpha14 chain paired to rearranged Vbeta2, Vbeta7, Vbeta8.1, Vbeta8.2, or Vbeta8.3 beta-chains and the other exhibiting unskewed alpha- and beta-chains. The proportion of these subgroups varies from about 100:0 in thymus, 80:20 in liver, and 50:50 in spleen to 20:80% in bone marrow, respectively. Finally, further heterogeneity in the tissue-derived DN NKT cells was discovered by sequencing extensively Vbeta8.2-Jbeta2.5 rearrangements in individual mice. Despite a few recurrences in TCR sequences, we found that each population exhibits its own and broad TCRbeta diversity.

Amino Acid Sequence↗
PubMed2001

SIN-1, a nitric oxide donor, ameliorates experimental allergic encephalomyelitis in Lewis rats in the incipient phase: the importance of the time window.

NO is involved in the regulation of immune responses. The role of NO in the pathogenesis of experimental allergic encephalomyelitis (EAE) is controversial. In this study, 3-morpholinosydnonimine (SIN-1), an NO donor, was administered to Lewis rats on days 5-7 postimmunization, i.e., during the incipient phase of EAE. SIN-1 reduced clinical signs of EAE compared with those in PBS-treated control rats and was accompanied by reduced ED1(+) macrophages and CD4(+) T cell infiltration within the CNS. Blood mononuclear cells (MNC) obtained on day 14 postimmunization revealed that SIN-1 administration enhanced NO and IFN-gamma production by blood MNC and suppressed Ag- and mitogen-induced proliferative responses. MHC class II, B7-1 and B7-2 were down-regulated in SIN-1-treated EAE rats. Simultaneously, frequencies of apoptotic cells among blood MNC were increased. In vivo, SIN-1 is likely to behave as an NO donor. Administration of SIN-1 induced NO production, but did not affect superoxide and peroxynitrite formation. Enhanced NO production during the priming phase of EAE thus promotes apoptosis, down-regulates disease-promoting immune reactivities, and ameliorates clinical EAE, mainly through SIN-1-derived NO, without depending on NO synthase.

Adjuvants, Immunologic↗
PubMed2002

Vaccination with tumor peptide in CpG adjuvant protects via IFN-gamma-dependent CD4 cell immunity.

The low frequency of tumor Ag-specific T cells in vivo has made it challenging to directly measure their clonal sizes and cytokine signatures. We used a new generation ELISPOT approach to study the constitutive immunogenicity of the RMA tumor in syngeneic B6 mice and adjuvant-guided immunity against an MHC class II-restricted RMA peptide, H11.1. The RMA tumor was found to activate cells of the innate immune system and to induce a type 1 polarized, RMA-specific CD4 and CD8 T cell response. With clonal sizes approximately 10/10(6), the magnitude of this constitutively induced immune response did not suffice to control the tumor cell growth. In contrast, immunization with H11.1 peptide, using an immunostimulatory CpG oligonucleotide or CFA as adjuvant, engaged approximately 25- or approximately 10-fold higher clonal sizes of type 1 polarized CD4 cells, respectively. Therefore, the CpG oligonucleotide functioned as a stronger type 1 adjuvant and, unlike CFA, elicited protective immunity. The protection was IFN-gamma dependent, as it was not inducible in IFN-gamma knockout mice. Therefore, CpG adjuvant-guided induction of type 1 immunity against tumor Ags might be a promising subunit vaccination approach.

Adjuvants, Immunologic↗
PubMed2006

Adhesion- and degranulation-promoting adapter protein is required for efficient thymocyte development and selection.

Adhesion- and degranulation-promoting adapter protein (ADAP) is required in TCR-induced activation and proliferation of peripheral T cells. Loss of ADAP also impairs TCR-initiated inside-out activation of the integrin LFA-1 (CD11a/CD18, alphaLbeta2). In this study, we demonstrate that ADAP-deficient CD4/CD8 double-positive (DP) cells have a diminished ability to proliferate, and that these DP thymocytes up-regulate CD69 poorly in vivo. Moreover, in both MHC class I- and class II-restricted TCR transgenic models, loss of ADAP interferes with both positive and negative selection. ADAP deficiency also impairs the ability of transgene-bearing DP thymocytes to form conjugates with Ag-loaded presenting cells. These findings suggest that ADAP is critical for thymocyte development and selection.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Human immune responses to the highly repetitive Plasmodium falciparum antigen Pf332.

The B and T cell responses to EB200, a repetitive part of the Plasmodium falciparum antigen Pf332, were examined in malaria-exposed Senegalese adults. Most donors had high levels of antibodies to recombinant EB200 and 17 overlapping peptides spanning EB200. Taking proliferation and/or cytokine (interferon-gamma and interleukin-4) production as a measure of T cell activation, eight of the EB200-derived peptides induced responses in > 40% of the donors tested. There was no general association between the different types of T cell responses measured, emphasizing the importance of including multiple parameters when analyzing T cell responses and suggesting that EB200 induces functionally distinct T cell responses. The most efficient peptide for induction of proliferative responses was one previously shown to induce T cell responses in five different H-2 congenic mouse strains primed with EB200, suggesting that this is a universal T cell epitope. The presence of multiple B and T cell epitopes in EB200, widely recognized by humans, is important since EB200 has been shown to elicit protective antibody responses in monkeys and may be considered for inclusion in malaria subunit vaccines.

Adolescent↗