MLVA polymorphism of Salmonella enterica subspecies isolated from humans, animals, and food in Cambodia

<p>Abstract</p> <p>Background</p> <p><it>Salmonella </it>(<it>S</it>.) <it>enterica </it>is the main cause of salmonellosis in humans and animals. The epidemiology of this infection involves large geographical distances, and strains related to an episode of salmonellosis therefore need to be reliably discriminated. Due to the limitations of serotyping, molecular genotyping methods have been developed, including multiple loci variable number of tandem repeats (VNTR) analysis (MLVA). In our study, 11 variable number tandem-repeats markers were selected from the <it>S. enterica </it>Typhimurium LT2 genome to evaluate the genetic diversity of 206 <it>S. enterica </it>strains collected in Cambodia between 2001 and 2007.</p> <p>Findings</p> <p>Thirty one serovars were identified from three sources: humans, animals and food. The markers were able to discriminate all strains from 2 to 17 alleles. Using the genotype phylogeny repartition, MLVA distinguished 107 genotypes clustered into two main groups: <it>S. enterica </it>Typhi and other serovars. Four serovars (Derby, Schwarzengrund, Stanley, and Weltevreden) were dispersed in 2 to 5 phylogenic branches. Allelic variations within <it>S. enterica </it>serovars was represented using the minimum spanning tree. For several genotypes, we identified clonal complexes within the serovars. This finding supports the notion of endemo-epidemic diffusion within animals, food, or humans. Furthermore, a clonal transmission from one source to another was reported. Four markers (STTR3, STTR5, STTR8, and Sal20) presented a high diversity index (DI > 0.80).</p> <p>Conclusions</p> <p>In summary, MLVA can be used in the typing and genetic profiling of a large diversity of <it>S. enterica </it>serovars, as well as determining the epidemiological relationships of the strains with the geography of the area.</p

On demand utilization of phosphoribosyl pyrophosphate by downstream anabolic pathways

International audienceThe pentose phosphate pathway (PPP) is critical for anabolism and biomass production. Here we show that the essential function of PPP in yeast is the synthesis of phosphoribosyl pyrophosphate (PRPP) catalyzed by PRPP-synthetase. Using combinations of yeast mutants, we found that a mildly decreased synthesis of PRPP affects biomass production, resulting in reduced cell size, while a more severe decrease ends up affecting yeast doubling time. We establish that it is PRPP itself that is limiting in invalid PRPP-synthetase mutants and that the resulting metabolic and growth defect can be bypassed by proper supplementation of the medium with ribose-containing precursors or by the expression of bacterial or human PRPPsynthetase. In addition, using documented pathologic human hyperactive forms of PRPPsynthetase, we show that intracellular PRPP as well as its derived products can be increased in both human and yeast cells and we describe the ensuing metabolic and physiological consequences. Finally, we found that PRPP consumption appears to take place "on demand" by the various PRPP-utilizing pathways, as shown by blocking or increasing the flux in specific PRPP-consuming metabolic routes. Overall, our work reveals important similarity between human and yeast for both synthesis and consumption of PRPP

het-B allorecognition in Podospora anserina is determined by pseudo-allelic interaction of genes encoding a HET and lectin fold domain protein and a PII-like protein.

Filamentous fungi display allorecognition genes that trigger regulated cell death (RCD) when strains of unlike genotype fuse. Podospora anserina is one of several model species for the study of this allorecognition process termed heterokaryon or vegetative incompatibility. Incompatibility restricts transmission of mycoviruses between isolates. In P. anserina, genetic analyses have identified nine incompatibility loci, termed het loci. Here we set out to clone the genes controlling het-B incompatibility. het-B displays two incompatible alleles, het-B1 and het-B2. We find that the het-B locus encompasses two adjacent genes, Bh and Bp that exist as highly divergent allelic variants (Bh1/Bh2 and Bp1/Bp2) in the incompatible haplotypes. Bh encodes a protein with an N-terminal HET domain, a cell death inducing domain bearing homology to Toll/interleukin-1 receptor (TIR) domains and a C-terminal domain with a predicted lectin fold. The Bp product is homologous to PII-like proteins, a family of small trimeric proteins acting as sensors of adenine nucleotides in bacteria. We show that although the het-B system appears genetically allelic, incompatibility is in fact determined by the non-allelic Bh1/Bp2 interaction while the reciprocal Bh2/Bp1 interaction plays no role in incompatibility. The highly divergent C-terminal lectin fold domain of BH determines recognition specificity. Population studies and genome analyses indicate that het-B is under balancing selection with trans-species polymorphism, highlighting the evolutionary significance of the two incompatible haplotypes. In addition to emphasizing anew the central role of TIR-like HET domains in fungal RCD, this study identifies novel players in fungal allorecognition and completes the characterization of the entire het gene set in that species

Multiple-locus variable-number tandem-repeat analysis of S<it>treptococcus pneumoniae</it> and comparison with multiple loci sequence typing

Abstract Background Streptococcus pneumoniae infections remain a major cause of morbidity and mortality worldwide. The diversity of pneumococci was first evidenced by serotyping of their capsular polysaccharides, responsible of virulence, resolving into more than 93 serotypes. Molecular tools have been developed to track the emergence and the spread of resistant, hyper virulent or non-vaccine type clones, particularly DNA-based methods using genetic polymorphism. Pulsed-Field Gel Electrophoresis analysis (PFGE) and Multiple Loci Sequence Typing (MLST) are the most frequently used genotyping techniques for S. pneumoniae. MLST is based on sequence comparison of housekeeping genes clustering isolates within sequence types. The availability of genome sequence data from different S. pneumoniae strains facilitated the search for other class of genetic markers as polymorphic DNA sequences for a Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA). This study aims at confirming the relevance of MLVA of S. pneumoniae, comparing MLST and MLVA performances when discriminating subgroups of strains belonging to the same Sequence Type (ST), and defining a restricted but universal set of MLVA markers that has at least the same discriminatory power as MLST for S. pneumoniae by applying marker sets used by different authors on 331 isolates selected in UK. Results A minimum spanning tree was built including the serotypes distribution and comparing MLVA and MLST results. 220 MLVA types were determined grouped in 10 Sequence Types (ST). MLVA differentiated ST162 in two clonal complexes. A minimal set was defined: ms 25 and ms37, ms17, ms19, ms33, ms39, and ms40 including two universal markers. The selection was based on MLVA markers with a Diversity Index >0.8 and a selection of others depending of the population tested and the aim of the study. This set of 7 MLVA markers yields strain clusters similar to those obtained by MLST. Conclusions MLVA can discriminate relevant subgroups among strains belonging to the same ST. MLVA offers the possibility to deduce the ST from the MLVA Type. It permits to investigate local outbreaks or to track the worldwide spread of clones and the emergence of variants.</p

Comparison of the genetic and domain architectures of the different incompatibility systems in <i>Podospora anserina</i>.

Diagram illustrates the different genetic architectures underlying incompatibility. Genetically, systems are categorized into non-allelic and allelic systems. Antagonistic incompatibility partners are represented in blue and red. For each system, the type of RCD execution domain is given (HET, HeLo, Patatin, GSDM) while systems involving NLRs are marked. For s/S, the system involves an NLR (NWD2) but this NLR is not directly involved in the incompatibility reaction (hence the parentheses). See text for details and references.</p

Oligonucleotides.

Filamentous fungi display allorecognition genes that trigger regulated cell death (RCD) when strains of unlike genotype fuse. Podospora anserina is one of several model species for the study of this allorecognition process termed heterokaryon or vegetative incompatibility. Incompatibility restricts transmission of mycoviruses between isolates. In P. anserina, genetic analyses have identified nine incompatibility loci, termed het loci. Here we set out to clone the genes controlling het-B incompatibility. het-B displays two incompatible alleles, het-B1 and het-B2. We find that the het-B locus encompasses two adjacent genes, Bh and Bp that exist as highly divergent allelic variants (Bh1/Bh2 and Bp1/Bp2) in the incompatible haplotypes. Bh encodes a protein with an N-terminal HET domain, a cell death inducing domain bearing homology to Toll/interleukin-1 receptor (TIR) domains and a C-terminal domain with a predicted lectin fold. The Bp product is homologous to PII-like proteins, a family of small trimeric proteins acting as sensors of adenine nucleotides in bacteria. We show that although the het-B system appears genetically allelic, incompatibility is in fact determined by the non-allelic Bh1/Bp2 interaction while the reciprocal Bh2/Bp1 interaction plays no role in incompatibility. The highly divergent C-terminal lectin fold domain of BH determines recognition specificity. Population studies and genome analyses indicate that het-B is under balancing selection with trans-species polymorphism, highlighting the evolutionary significance of the two incompatible haplotypes. In addition to emphasizing anew the central role of TIR-like HET domains in fungal RCD, this study identifies novel players in fungal allorecognition and completes the characterization of the entire het gene set in that species.</div

Incompatibility phenotype of chimeric <i>Bh</i> and <i>Bp</i> alleles.

Upper panels, ΔB strains (lacking the entire B1 haplotype) were transformed with either Bh1 or the chimeric Bh2-derived alleles as given and individual transformants were assayed in barrage test in confrontation to B2 (lines of B2 tester strains are marked with an asterisk). The domain diagram of Bh1 and the chimeric alleles are recalled. Lower panels, ΔB strains (lacking the entire B1 haplotype) were transformed with either Bp2 or the chimeric Bp1-derived alleles as given and individual transformants were assayed in barrage test in confrontation to B1 (lines of B1 tester strains are marked with an asterisk). The domain diagram of Bh1 and the chimeric allele are recalled. On each plate, the upper three implants are positive control for incompatibility, that is B1 strains in the upper panels and B2 strains in the lower panels. (PDF)</p

<i>Bh1</i> and <i>Bp2</i> control <i>het-B</i> incompatibility.

(A) Strains as given were confronted in all combinations on solid corn meal agar medium and after 5 days at 26°C the confrontation zone was imaged using a binocular lens (image size is 4.3x4.3 mm). Full test plates are given in S3 Fig. Under the strain designation, the constitution at Bh and Bp is recalled. On the top right part of the diagram, the barrage results are repeated graphically (/, for compatibility; //, for incompatibility) to improve readability. Note that ΔBh1 and ΔBp2 are compatible with all testers and that ΔBh2 and ΔBp1 retain the same incompatibility spectrum as the B1 and B2 strains from which they derive. (B) Transformation efficiency of the Bp1 and Bp2 alleles in different het-B genetic backgrounds. Note that Bp2 leads to a strong reduction in transformation efficiency in the B1 background but not in ΔBh1. Results are quadruplicates with standard deviations. (C) Interpretative model of het-B incompatibility interactions, het-B incompatibility is asymmetric and determined solely by the Bh1/Bp2 interaction, the reciprocal Bh2/Bp1 interaction is compatible. Het-B2 incompatibility type is determined by Bp2 and het-B1 incompatibility type by Bh1.</p

Genotypes and strain information of the Wageningen Collection and other members of the <i>P</i>. <i>anserina</i> species complex.

Genotypes and strain information of the Wageningen Collection and other members of the P. anserina species complex.</p