Presented by Roxana Hickey at the Gordon Research Conference on Microbial Population Biology, July 2015

1. Focusing the diversity of Gardnerella vaginalis
through the lens of ecotypes
ACKNOWLEDGMENTS
We are grateful to Sam Hunter, Matt Settles, Matt Pennell, Michael France,
and Benji Oswald for their insight and assistance. This work was supported
by funds from the National Institute of General Medical Sciences of the
National Institutes of Health under award number P30 GM103324.
REFERENCES
1
Gardner & Dukes (1955) Am J Obstet Gynecol 2
Wiggins et al.
(2001) Sex Transm Inf 3
Moncla et al. (2015) PLoS ONE 4
LeBlond-
Bourget et al. (1996) Int J Syst Bact 5
Ahmed et al. (2012) J Bact
6
Balashov et al. (2015) J Med Microbiol
MORE INFORMATION
Forneylab:http://people.ibest.uidaho.edu/~lforney/
roxana.hickey@gmail.com lforney@uidaho.edu
Roxana Hickey1,2, Omar Cornejo3, Haruo Suzuki4 & Larry Forney1,2
1
Department of Biological Sciences & 2
Institute for Bioinformatics and Evolutionary Studies, University of Idaho, Moscow, ID, USA; 3
School of
Biological Sciences, Washington State University, WA, USA; 4
Biopathway Analysis Center, Yamaguchi University, Japan
RESULTS
CONCLUSIONS
METHODS
INTRODUCTION
FIGURE 3. Genome clusters of G. vaginalis based on protein family repertoire. A)
Dendrogram constructed from the average linkage hierarchical clustering of genomes
based on the Bray-Curtis dissimilarity matrix of count data for 2,399 protein families. B)
Counts of select protein families with putative virulence potential. C) Nugent scores and
D) health status of women G. vaginalis isolates were collected from. NR=not reported.
CLADE 1
CLADE 4
CLADE 5
CLADE 3
CLADE 2
FIGURE 4. Primary concordance tree based on 664 single-copy
core genes of G. vaginalis. Bayesian concordance analysis was
performed with BUCKy software, and branch labels indicate
concordance factors as the percentage of gene trees that contain
a given clade.
GARDNERELLA VAGINALIS HAS AN OPEN
PANGENOME AND SMALL CORE GENOME
FIGURE 2. Number of protein families in the pangenome of G.
vaginalis. Accumulation rarefaction curves were calculated based
on the presence or absence of protein families and singleton
coding genes using the “specaccum” function in the vegan R
package with 100 bootstrap permutations of randomized sample
order.
694 CORE PROTEIN FAMILIES =
55% OF EACH GENOME, 29% OF SHARED GENOME
2,392 PROTEIN FAMILIES IN 2+ GENOMES
1,495 SINGLETON CODING SEQUENCES
FOUR GENOME CLUSTERS FROM SHARED PROTEIN
REPERTOIRE DIFFER IN PUTATIVE VIRULENCE FACTORS
PHYLOGENETIC CONCORDANCE IN THE
CORE GENOME SUGGESTS FIVE CLADES
GREATER CONSISTENCY IN GENOMIC CHARACTERISTICS
AND LARGER CORE GENOMES WITHIN CLADES
CORE
901
CORE
886
CORE
998
CORE
969
CORE
1,204
FIGURE 5. Variation in genome size and
GC composition among five clades of G.
vaginalis.
FIGURE 6. Core, accessory and unique
protein family counts within each clade of
G. vaginalis.
CLADES DIFFER IN NUTRITIONAL
CAPABILITIES AND PUTATIVE VIRULENCE
FACTORS, ESPECIALLY GLYCOSIDASES
35 G. vaginalis genomes
PATRIC DATABASE
Cluster coding gene sequences
into protein families
ORTHOMCL 70% AA SIMILARITY
Identify core, accessory &
unique protein families
Determine SEQUENCE
CLUSTERS from concordance
in core gene phylogenies
CLUSTAL + MRBAYES + BUCKY
Determine GENOME
CLUSTERS from comparison
of total protein repertoire
HIERARCHICAL CLUSTERING
Evaluate putative ecological differences
with functional enrichment analysis
PROTEIN FAMILIES, GO CATEGORIES, KEGG PATHWAYS
ODDS RATIO >> FISHER’S EXACT TEST >> FDR ADJUSTMENT
CAN THE ECOTYPE CONCEPT CONNECT
ECOLOGICAL SIMILARITY WITH EVOLUTIONARY
RELATEDNESS OF STRAINS OF G. VAGINALIS?
ECOTYPE: set of strains representing an evolutionary lineage
within a species that are both ECOLOGICALLY DISTINCT and
GENETICALLY SIMILAR as the result of common ancestry
‣ Gardnerella vaginalis originally classified as
Haemophilus vaginalis in 19551
and proposed as the
etiological agent of bacterial vaginosis (BV)
๏ BV characterized by discharge, itching, odor and elevated
risks to STIs and preterm birth
!
!
!
!
!
!
!
‣ Degradation of mucus via glycosidases (e.g. sialidase)
proposed as a virulence mechanism,2,3
but genetic and
phenotypic diversity complicate efforts to link specific
strains to BV
‣ Gardnerella is nested within the Bifidobacterium spp.,4
which are generally regarded as
beneficial to humans
FIGURE 1. Maximum-
likelihood 16S rRNA
gene phylogeny of 18
strains of G. vaginalis
and 20 strains of
Bifidobacterium spp.
u n d e r t h e G T R +
g a m m a m o d e l o f
sequence evolution
with 1,000 bootstrap
replicates.
Bifidobacterium vs.
Gardnerella 16S rRNA
91-95% identical
!
G. vaginalis strains
99-100% identical
MUCH ADO ABOUT GARDNERELLA VAGINALIS
‣ Genomic and phylogenetic analysis supports existence of genetically
cohesive ecotypes with distinct functional characteristics
‣ Recapitulated and added to clades determined previously on 17 genomes5
‣ Added functional information to clade-BV associations determined by
Balashov et al. using clade-specific qPCR6
๏ Clades 1,3 associated with BV; clade 4 common in both healthy and BV
B V
G A R D N E R E L L A
VA G I N A L I S
9 3 - 1 0 0 % B V C A S E S
B V
!
!
!
G . VA G I N A L I S
4 0 - 6 0 % H E A LT H Y
W O M E N
THE
PROBLEM
CLADE 1
!
BAD?
CLADE 2
!
RISKY?
CLADE 3
!
BAD?
CLADE 4
!
GOOD?
CLADE 5
!
???
ADDITIONAL
TYPES?
CLADE 1
Galactose metabolism:
α-galactosidase *
β-galactosidase *
α-glucosidase *
5C sugar metabolism
ABC transporters, permeases *
Zeta toxin *
CLADE 2
!
Sialidase (2X)
4 orthologs, 2 unique *
!
Thiamine biosynthesis
!
ECF transporter *
CLADE 3
!
Polyketide sugar
unit biosynthesis
CLADE 4
!
Thiamine
biosynthesis
!
Sialidase
CLADE 5
!
!
Sialidase
OR > 1
absent
unique*
DIFFERENT DEGREES OF VIRULENCE?
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