Clinical and molecular phenotype of Aicardi-Goutières syndrome

Aicardi-Goutières syndrome (AGS) is a genetic encephalopathy whose clinical features mimic those of acquired in utero viral infection. AGS exhibits locus heterogeneity, with mutations identified in genes encoding the 3′→5′ exonuclease TREX1 and the three subunits of the RNASEH2 endonuclease complex. To define the molecular spectrum of AGS, we performed mutation screening in patients, from 127 pedigrees, with a clinical diagnosis of the disease. Biallelic mutations in TREX1, RNASEH2A, RNASEH2B, and RNASEH2C were observed in 31, 3, 47, and 18 families, respectively. In five families, we identified an RNASEH2A or RNASEH2B mutation on one allele only. In one child, the disease occurred because of a de novo heterozygous TREX1 mutation. In 22 families, no mutations were found. Null mutations were common in TREX1, although a specific missense mutation was observed frequently in patients from northern Europe. Almost all mutations in RNASEH2A, RNASEH2B, and RNASEH2C were missense. We identified an RNASEH2C founder mutation in 13 Pakistani families. We also collected clinical data from 123 mutation-positive patients. Two clinical presentations could be delineated: an early-onset neonatal form, highly reminiscent of congenital infection seen particularly with TREX1 mutations, and a later-onset presentation, sometimes occurring after several months of normal development and occasionally associated with remarkably preserved neurological function, most frequently due to RNASEH2B mutations. Mortality was correlated with genotype; 34.3% of patients with TREX1, RNASEH2A, and RNASEH2C mutations versus 8.0% RNASEH2B mutation-positive patients were known to have died (P = .001). Our analysis defines the phenotypic spectrum of AGS and suggests a coherent mutation-screening strategy in this heterogeneous disorder. Additionally, our data indicate that at least one further AGS-causing gene remains to be identified. © 2007 by The American Society of Human Genetics. All rights reserved.

New insights into telomeric DNA sequence (TTAGGG)n location in bat chromosomes

Molossidae species, Cynomops abrasus (2n = 34, fundamental number, FN = 64), Eumops auripendulus (2n = 42, FN = 62), Molossus rufus (2n = 48, FN = 64), Molossops temminckii (2n = 48, FN = 64), and Nyctinomops laticaudatus (2n = 48, FN = 64), and Phyllostomidae species, Phyllostomus discolor (2n = 32, FN = 60), have karyotypes with different chromosome and fundamental numbers, different localization of constitutive heterochromatin, and different numbers and location of nucleolar organizer regions (NORs). Fluorescence in situ hybridization with a human probe of the telomeric sequence (TTAGGG)n produced fluorescent signals in telomeric regions of the six bat species' chromosomes; in E. auripendulus, pericentromeric signals were also observed in the acrocentric and subtelocentric chromosomes. A relationship between telomeric sequences and NORs, and between telomeric sequences and constitutive heterochromatin was detected in chromosomes bearing NORs in C. abrasus, M. temminckii, N. laticaudatus, and P. discolor. No interstitial signal was observed in the meta- or submetacentric chromosomes of these species. ©FUNPEC-RP.

Detection of Paracoccidioides spp. in environmental aerosol samples

Taking into account that paracoccidioidomycosis infection occurs by inhalation of the asexual conidia produced by Paracoccidioides spp. in its saprobic phase, this work presents the collection of aerosol samples as an option for environmental detection of this pathogen, by positioning a cyclonic air sampler at the entrance of armadillo burrows. Methods included direct culture, extinction technique culture and Nested PCR of the rRNA coding sequence, comprising the ITS1-5.8S-ITS2 region. In addition, we evaluated one armadillo (Dasypus novemcinctus) as a positive control for the studied area. Although the pathogen could not be isolated by the culturing strategies, the aerosol sampling associated with molecular detection through Nested PCR proved the best method for discovering Paracoccidioides spp. in the environment. Most of the ITS sequences obtained in this investigation proved to be highly similar with the homologous sequences of Paracoccidioides lutzii from the GenBank database, suggesting that this Paracoccidioides species may not be exclusive to mid-western Brazil as proposed so far. © 2013 ISHAM.

PRP8 intein in cryptic species of Histoplasma capsulatum: Evolution and phylogeny

The PRP8 intein is the most widespread intein among the Kingdom Fungi. This genetic element occurs within the prp8 gene, and is transcribed and translated simultaneously with the gene. After translation, the intein excises itself from the Prp8 protein by an autocatalytic splicing reaction, subsequently joining the N and C terminals of the host protein, which retains its functional conformation. Besides the splicing domain, some PRP8 inteins also have a homing endonuclease (HE) domain which, if functional, makes the intein a mobile element capable of becoming fixed in a population. This work aimed to study (1) The occurrence of this intein in Histoplasma capsulatum isolates (n=. 99) belonging to different cryptic species collected in diverse geographical locations, and (2) The functionality of the endonuclease domains of H. capsulatum PRP8 inteins and their phylogenetic relationship among the cryptic species. Our results suggest that the PRP8 intein is fixed in H. capsulatum populations and that an admixture or a probable ancestral polymorphism of the PRP8 intein sequences is responsible for the apparent paraphyletic pattern of the LAmA clade which, in the intein phylogeny, also encompasses sequences from LAmB isolates. The PRP8 intein sequences clearly separate the different cryptic species, and may serve as an additional molecular typing tool, as previously proposed for other fungi genus, such as Cryptococcus and Paracoccidioides. © 2013 Elsevier B.V.

Candida albicans: genotyping methods and clade related phenotypic characteristics

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evolution of species diversity in the genus Chamaecostus (Costaceae): molecular phylogenetics and morphometric approaches

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

The genome sequence of the plant pathogen Xylella fastidiosa: The Xylella fastidiosa consortium of the organization for nucleotide sequencing and analysis, Sao Paulo, Brazil

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis - a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and 'two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.

Molecular support for the establishment of the new genus Laurenciella within the Laurencia complex (Ceramiales, Rhodophyta)

Currently, five genera are assigned to red seaweeds of the Laurencia complex worldwide: Chondrophycus, Laurencia s.s., Osmundea, Palisada and Yuzurua. The genera are segregated on the basis of morphological characters, especially the reproductive traits, and molecular sequences of the plastid-encoded gene rbcL. Four of the genera have been resolved as monophyletic, but not Laurencia s.s. In this study based on an rbcL gene phylogeny we show the presence of a sixth lineage within the Laurencia complex, viz., Laurencia marilzae plus two unidentified species of Laurencia from Brazil. The phylogenetic position of this group, combined with the high genetic divergence from Laurencia s.s. (8.2-11%), strongly support the establishment of a sixth genus for the complex, proposed here as Laurenciella gen. nov. This new taxon differs from Laurencia s.s. and from the other genera of the complex by molecular sequence data, but is indistinguishable from Laurencia s.s. by the usual morphological features.

Epidemiological and functional characterization of Streptococcus pneumoniae pili

Streptococcus pneumoniae is an important life threatening human pathogen causing agent of invasive diseases such as otitis media, pneumonia, sepsis and meningitis, but is also a common inhabitant of the respiratory tract of children and healthy adults. Likewise most streptococci, S. pneumoniae decorates its surface with adhesive pili, composed of covalently linked subunits and involved in the attachment to epithelial cells and virulence. The pneumococcal pili are encoded by two genomic regions, pilus islet 1 (PI-1), and pilus islet-2 (PI-2), which are present in about 30% and 16% of the pneumococcal strains, respectively. PI-1 exists in three clonally related variants, whereas PI-2 is highly conserved. The presence of the islets does not correlate with the serotype of the strains, but with the genotype (as determined by Multi Locus Sequence Typing). The prevalence of PI-1 and PI-2 positive strains is similar in isolates from invasive disease and carriage. To better dissect a possible association between PIs presence and disease we evaluated the distribution of the two PIs in a panel of 113 acute otitis media (AOM) clinical isolates from Israel. PI-1 was present in 30.1% (N=34) of the isolates tested, and PI-2 in 7% (N=8). We found that 50% of the PI-1 positive isolates belonged to the international clones Spain9V-3 (ST156) and Taiwan19F-14 (ST236), and that PI-2 was not present in the absence of Pl-1. In conclusion, there was no correlation between PIs presence and AOM, and, in general, the observed differences in PIs prevalence are strictly dependent upon regional differences in the distribution of the clones. Finally, in the AOM collection the prevalence of PI-1 was higher among antibiotic resistant isolates, confirming previous indications obtained by the in silico analysis of the MLST database collection. Since the pilus-1 subunits were shown to confer protection in mouse models of infection both in active and passive immunization studies, and were regarded as potential candidates for a new generation of protein-based vaccines, the functional characterization was mainly focused on S. pneumoniae pilus -1 components. The pneumococcal pilus-1 is composed of three subunits, RrgA, RrgB and RrgC, each stabilized by intra-molecular isopeptide bonds and covalently polymerized by means of inter-molecular isopeptide bonds to form an extended fibre. The pilus shaft is a multimeric structure mainly composed by the RrgB backbone subunit. The minor ancillary proteins are located at the tip and at the base of the pilus, where they have been proposed to act as the major adhesin (RrgA) and as the pilus anchor (RrgC), respectively. RrgA is protective in in vivo mouse models, and exists in two variants (clades I and II). Mapping of the sequence variability onto the RrgA structure predicted from X-ray data showed that the diversity was restricted to the “head” of the protein, which contains the putative binding domains, whereas the elongated “stalk” was mostly conserved. To investigate whether this variability could influence the adhesive capacity of RrgA and to map the regions important for binding, two full-length protein variants and three recombinant RrgA portions were tested for adhesion to lung epithelial cells and to purified extracellular matrix (ECM) components. The two RrgA variants displayed similar binding abilities, whereas none of the recombinant fragments adhered at levels comparable to those of the full-length protein, suggesting that proper folding and structural arrangement are crucial to retain protein functionality. Furthermore, the two RrgA variants were shown to be cross-reactive in vitro and cross-protective in vivo in a murine model of passive immunization. Taken together, these data indicate that the region implicated in adhesion and the functional epitopes responsible for the protective ability of RrgA may be conserved and that the considerable level of variation found within the “head” domain of RrgA may have been generated by immunologic pressure without impairing the functional integrity of the pilus.

Emergence of linezolid-resistant Staphylococcus aureus after prolonged treatment of cystic fibrosis patients in Cleveland, Ohio

Linezolid (LZD)-resistant Staphylococcus aureus (LRSA) isolates were monitored from 2000 to 2009 in Cleveland, OH. LRSA first emerged in 2004 only in cystic fibrosis (CF) patients, with 11 LRSA-infected CF patients being identified by 2009. LRSA was isolated from 8 of 77 CF patients with S. aureus respiratory tract infection treated with LZD from 2000 to 2006. Analysis of clinical data showed that the 8 CF patients with LRSA received more LZD courses (18.8 versus 5.9; P = 0.001) for a longer duration (546.5 versus 211.9 days; P < 0.001) and had extended periods of exposure to LZD (83.1 versus 30.1 days/year; P < 0.001) than the 69 with LZD-susceptible isolates. Five LRSA isolates included in the clinical analysis (2000 to 2006) and three collected in 2009 were available for molecular studies. Genotyping by repetitive extrapalindromic PCR and pulsed-field gel electrophoresis revealed that seven of these eight LRSA strains from unique patients were genetically similar. By multilocus sequence typing, all LRSA isolates were included in clonal complex 5 (seven of sequence type 5 [ST5] and one of ST1788, a new single-locus variant of ST5). However, seven different variants were identified by spa typing. According to the Escherichia coli numbering system, seven LRSA isolates contained a G2576T mutation (G2603T, S. aureus numbering) in one to four of the five copies of domain V of the 23S rRNA genes. One strain also contained a mutation (C2461T, E. coli numbering) not previously reported. Two strains, including one without domain V mutations, possessed single amino acid substitutions (Gly152Asp or Gly139Arg) in the ribosomal protein L3 of the peptidyltransferase center, substitutions not previously reported in clinical isolates. Emergence of LRSA is a serious concern for CF patients who undergo prolonged courses of LZD therapy.

Source attribution of human Campylobacter isolates by MLST and fla-typing and association of genotypes with quinolone resistance

Campylobacteriosis is the most frequent zoonosis in developed countries and various domestic animals can function as reservoir for the main pathogens Campylobacter jejuni and Campylobacter coli. In the present study we compared population structures of 730 C. jejuni and C. coli from human cases, 610 chicken, 159 dog, 360 pig and 23 cattle isolates collected between 2001 and 2012 in Switzerland. All isolates had been typed with multi locus sequence typing (MLST) and flaB-typing and their genotypic resistance to quinolones was determined. We used complementary approaches by testing for differences between isolates from different hosts with the proportion similarity as well as the fixation index and by attributing the source of the human isolates with Bayesian assignment using the software STRUCTURE. Analyses were done with MLST and flaB data in parallel and both typing methods were tested for associations of genotypes with quinolone resistance. Results obtained with MLST and flaB data corresponded remarkably well, both indicating chickens as the main source for human infection for both Campylobacter species. Based on MLST, 70.9% of the human cases were attributed to chickens, 19.3% to cattle, 8.6% to dogs and 1.2% to pigs. Furthermore we found a host independent association between sequence type (ST) and quinolone resistance. The most notable were ST-45, all isolates of which were susceptible, while for ST-464 all were resistant.

Dissemination of methicillin-resistant Staphylococcus aureus USA300 sequence type 8 lineage in Latin America.

BACKGROUND: Methicillin-resistant Staphylococus aureus (MRSA) is an important nosocomial and community-associated (CA) pathogen. Recently, a variant of the MRSA USA300 clone emerged and disseminated in South America, causing important clinical problems. METHODS: S. aureus isolates were prospectively collected (2006-2008) from 32 tertiary hospitals in Colombia, Ecuador, Peru, and Venezuela. MRSA isolates were subjected to antimicrobial susceptibility testing and pulsed-field gel electrophoresis and were categorized as health care-associated (HA)-like or CA-like clones on the basis of genotypic characteristics and detection of genes encoding Panton-Valentine leukocidin and staphylococcal cassette chromosome (SCC) mec IV. In addition, multilocus sequence typing of representative isolates of each major CA-MRSA pulsotype was performed, and the presence of USA300-associated toxins and the arcA gene was investigated for all isolates categorized as CA-MRSA. RESULTS: A total of 1570 S. aureus were included; 651 were MRSA (41%)--with the highest rate of MRSA isolation in Peru (62%) and the lowest in Venezuela (26%)--and 71%, 27%, and 2% were classified as HA-like, CA-like, and non-CA/HA-like clones, respectively. Only 9 MRSA isolates were confirmed to have reduced susceptibility to glycopeptides (glycopeptide-intermediate S. aureus phenotype). The most common pulsotype (designated ComA) among the CA-like MRSA strains was found in 96% of isolates, with the majority (81%) having a < or =6-band difference with the USA300-0114 strain. Representative isolates of this clone were sequence type 8; however, unlike the USA300-0114 strain, they harbored a different SCCmec IV subtype and lacked arcA (an indicator of the arginine catabolic mobile element). CONCLUSION: A variant CA-MRSA USA300 clone has become established in South America and, in some countries, is endemic in hospital settings.

Detailed analysis of sequence changes occurring during vlsE antigenic variation in the mouse model of Borrelia burgdorferi infection.

Lyme disease Borrelia can infect humans and animals for months to years, despite the presence of an active host immune response. The vls antigenic variation system, which expresses the surface-exposed lipoprotein VlsE, plays a major role in B. burgdorferi immune evasion. Gene conversion between vls silent cassettes and the vlsE expression site occurs at high frequency during mammalian infection, resulting in sequence variation in the VlsE product. In this study, we examined vlsE sequence variation in B. burgdorferi B31 during mouse infection by analyzing 1,399 clones isolated from bladder, heart, joint, ear, and skin tissues of mice infected for 4 to 365 days. The median number of codon changes increased progressively in C3H/HeN mice from 4 to 28 days post infection, and no clones retained the parental vlsE sequence at 28 days. In contrast, the decrease in the number of clones with the parental vlsE sequence and the increase in the number of sequence changes occurred more gradually in severe combined immunodeficiency (SCID) mice. Clones containing a stop codon were isolated, indicating that continuous expression of full-length VlsE is not required for survival in vivo; also, these clones continued to undergo vlsE recombination. Analysis of clones with apparent single recombination events indicated that recombinations into vlsE are nonselective with regard to the silent cassette utilized, as well as the length and location of the recombination event. Sequence changes as small as one base pair were common. Fifteen percent of recovered vlsE variants contained "template-independent" sequence changes, which clustered in the variable regions of vlsE. We hypothesize that the increased frequency and complexity of vlsE sequence changes observed in clones recovered from immunocompetent mice (as compared with SCID mice) is due to rapid clearance of relatively invariant clones by variable region-specific anti-VlsE antibody responses.

Molecular basis for class V beta-tubulin effects on microtubule assembly and paclitaxel resistance.

Vertebrates produce at least seven distinct beta-tubulin isotypes that coassemble into all cellular microtubules. The functional differences among these tubulin isoforms are largely unknown, but recent studies indicate that tubulin composition can affect microtubule properties and cellular microtubule-dependent behavior. One of the isotypes whose incorporation causes the largest change in microtubule assembly is beta5-tubulin. Overexpression of this isotype can almost completely destroy the microtubule network, yet it appears to be required in smaller amounts for normal mitotic progression. Moderate levels of overexpression can also confer paclitaxel resistance. Experiments using chimeric constructs and site-directed mutagenesis now indicate that the hypervariable C-terminal region of beta5 plays no role in these phenotypes. Instead, we demonstrate that two residues found in beta5 (Ser-239 and Ser-365) are each sufficient to inhibit microtubule assembly and confer paclitaxel resistance when introduced into beta1-tubulin; yet the single mutation of residue Ser-239 in beta5 eliminates its ability to confer these phenotypes. Despite the high degree of conservation among beta-tubulin isotypes, mutations affecting residue 365 demonstrate that amino acid substitutions can be context sensitive; i.e. an amino acid change in one isotype will not necessarily produce the same phenotype when introduced into a different isotype. Modeling studies indicate that residue Cys-239 of beta1-tubulin is close to a highly conserved Cys-354 residue suggesting the possibility that disulfide formation could play a significant role in the stability of microtubules formed with beta1- but not with beta5-tubulin.

Genotypes and antibiotic resistances of Campylobacter jejuni isolates from cattle and pigeons in dairy farms.

Campylobacter jejuni is the most common food-borne zoonotic pathogen causing human gastroenteritis worldwide and has assumed more importance in Italy following the increased consumption of raw milk. Our objectives were to get an overview of genotypes and antibiotic resistances in C. jejuni isolated from milk, cattle feces, and pigeons in dairy herds of Northern Italy. flaB-typing was applied to 78 C. jejuni isolates, previously characterized by Multi-Locus Sequence Typing, and genotypic resistances towards macrolides and quinolones based on point mutations in the 23S rRNA and gyrA genes, respectively, were determined. flaB-typing revealed 22 different types with one of them being novel and was useful to further differentiate strains with an identical Sequence Type (ST) and to identify a pigeon-specific clone. Macrolide resistance was not found, while quinolone resistance was detected in 23.3% of isolates. A relationship between specific genotypes and antibiotic resistance was observed, but was only significant for the Clonal Complex 206. Our data confirm that pigeons do not play a role in the spread of C. jejuni among cattle and they are not responsible for milk contamination. A relevant number of bulk milk samples were contaminated by C. jejuni resistant to quinolones, representing a possible source of human resistant strains.