INVESTIGADORES
FARBER Marisa Diana
congresos y reuniones científicas
Título:
MLST scheme for Anaplasma marginale reveals pathogen strong adaptation to a very specific cell type
Autor/es:
GUILLEMI E; RUYBAL P; ZIMMER P.; PRINCIPI D.; FRUTOS R.; WILCOWSKY S; FARBER M
Lugar:
Zaragoza
Reunión:
Conferencia; Seventh Ticks and Tick-borne Pathogens International Conference; 2011
Institución organizadora:
TTP7: Ticks and Tick-borne pathogens International Conference
Resumen:
A highly reproducible and
discriminative typing system is essential for better understanding of Anaplasma
marginale epidemiology, in order
to analyze the population
structure, design and apply strategic control measures and monitor the
performance of live vaccines.
The multilocus sequence typing
(MLST) is a highly discriminatory method for isolate characterization on the basis of 7 housekeeping genes sequenced fragments. For each
gene fragment, the different sequences are assigned as distinct alleles, and
each isolate is defined as a particular haplotype (the allelic profile or
sequence type -ST-) by the combination of alleles at each of the seven loci. Due
to the scarce knowledge status of Anaplasma
marginale diversity in Argentina, the aim of this study was to develop the MLST strategy to
discriminate between A. marginale isolates in order to
study the genetic diversity and estimate the population structure of the pathogen among assorted enzootic
regions.
Fourteen annotated genes coding for highly conserved proteins were pre-selected based
on reported bacterial MLST schemes. Primers were designed using
Primer3 program in order to amplify an internal fragment of 500 to 700-bp. Seven genes (dnaA, ftsz, groEl, lipA, recA, secY and sucB) were selected
based on: number of single nucleotide polymorphic sites
(SNPs), genome distribution, absence of selective
pressure (dN / dS <1) and specificity of primers. PCR amplifications were performed
and amplicons were sequenced using the Big Dye Terminator v3.1 kit from Applied
Biosystems and analyzed on an ABI 3130XL genetic analyzer from the same
supplier. Raw trace files were FASTA-converted and contigs were
assembled, aligned and assigned to a defined ST using an in-house-developed
pipeline (MLST-pipeline) available at http://bioinformatica.inta.gov.ar.
Genetic variation among these
7 gene sequences was evaluated using as refereneces the available annotated
genomes from A. marginale strains (Mississippi,
Puerto Rico, Florida and Virginia), 5 reference strains
from Argentina (Mercedes, Salta, Quitilipi, Rosalí and Virasoro), 2 isolates from E.E.U.U ( from South Idaho and Oklahoma) and 1 from
South Africa. We also analysed 26 samples from naturally infected
animals from the argentinean enzootic area. Phylogenetic
analyses were performed using Maximum Likelihood (ML) method for each locus
separately and for the concatenated sequences using PhyML (version 2.4.4) with
500 bootstrap replicates using orthologous genes from the closely related
Rickettsiale Ehrlichia ruminantium as
outgroup.
Phylogenetic
analysis showed that in the tree from concatenated sequences the foreign reference
strains were clustered separately (bootstrap = 1) with a longer distance on an
external branch for the African strain. The argentinean strains clustered together
in a main group; however, seven of them branched separately within this group
with strong bootstraps supporting the segregation whereas the remaining strains
were grouped within a single subcluster. To further investigate if the
topology observed for the concatenated sequences was supported by the
distribution of the various haplotypes, a similar phylogenetic analysis was
conducted on each locus considered separately. Tree topologies were different
than those observed for the concatenated sequences, with different topologies
for each locus. The locus yielding the topology resembling the most that of the
concatenated genes was dnaA. DNA
polymorphism analysis reflected very low number of haplotypes or alleles, with
the exception of dnaA and recA which were significantly more
diverse. Recombination events, accompanied by low linkage disequilibrium
figures, were detected for dnA, recA
and sucB using several functions from
the DnaSP 5.00.02 package.
The
most striking features, however, were the almost complete absence of singletons
and non-synonymous substitutions, indicative of negative selection and absence
of population expansion; this seems to be the result of a very strong
adaptation to a very specific cell type and cell environment.