Please enable JavaScript.
Coggle requires JavaScript to display documents.
GENOMIC PALEOMICROBIOLOGY - Coggle Diagram
GENOMIC PALEOMICROBIOLOGY
plagues have affected humankind profoundly and intensely, culturally, religiously, biologically etc.
plague
symptoms
fever
headache
chills
weakness
swollen painful lymph nodes (buboes)
blackening/death of tissue - gangrene in extremities
abdominal pain
diarrhoea
bleeding from orifices & under skin
Yersinia pestis
3 major human pandemics
justinian
antiqua
E/C africa via Egypt to the med region
AD 541-542
afflicted the eastern Roman Empire - especially its capital Constantinople and eastern mediterranean refion
named after the eastern roman emperor Justinian I, who was in power at the time of the initial outbreak - he contracted the disease yet survived
outbreak considered by many historians as one of the defining moments in European ancient history
some scholars believe outbreak precipitated downfall of the roman empire
no reliable info on the number of fatalities exists - estimate it may have killed 25 million across the world - 1/4 of the world population at that time
Y pestis?
new insights - 2013
provenance of samples is a key issue
few reliable samples exist for analysis
housing developers uncovered a series of mass graves whilst doffing up farmland outside munich
carbon dating of bone fragments placed the remains within the timeframe of the Justinian plage - 4th century AD (± ~100 years)
an adult and child are buried together and skeletal remains intact
often a sign of sudden death and of infectious disease
samples analysed much the same as for Black Death samples
genome sequence
- 2014
tooth pulp from two separate skeletal samples
both generated a genome of y pestis
sequences compared against database of genomes from 131 sequenced y pestis strains from later pandemics and constructed a maximum likelihood phylogenetic tree for analysis
the y pestis lineages that caused the plague of Justinian and Black Death 800 years later were independent emergences from rodents into human beings. the authors believe that the later emergence of Black Death seems to have been caused by a separate event
strains appear to have originated in china before radiating into Europe
black death
medievalis
Central Asia via Caspian Sea to Europe, along silk routes
modern
orientalis (all current strains)
Yunnan region of china via Hong Kong through shipping globally
distinction mainly based on minor metabolic traits
bacteria acquired by humans via flea bites, rodents most likely the main host
bubonic
most common form
entry - bite of infected rat flea
spread lymphatic and systemic
disease, buboes (black hemorrhagic lymph nodes), pneumonia, internal organ hemorrhage
exit (highly contagious), nose, mouth, eyes
.
spread via lymphatics within macrophages
cell lysis in lymph nodes, liver and spleen, bacteria multiply extracellularly
very high bacterial loads spread to blood and other sites
overwhelming infection leads to multi-organ failure
pneumonic
much worse
much more virulent
much higher death rate
entry - nose, mouth, eyes
disease - pneumonia (usually 100% mortality)
exit (highly contagious), nose, mouth, eyes
septicaemia
Y. pestis
evolved from
Y. pseudotuberculosis
(enteropathogen) relatively recently (5-50,000 years?)
enteropathogen causes gastroenteritis, enteric disease
still exists in rodents across the world
parkhill et al. 2001
key findings
loss of many genes associated with ancestral enteropathogenic niche
enterotoxins and adhesins
numerous present as pseudogenes
large numbers of insertion sequence elements
contribute to gene loss/inactivation
transposons
gene loss streamlines reproduction
more efficient
faster
acquisition of insect toxin homologues
adaptation to insect vector host?
several plasmids, some common to other yersinia, some specific to pestis carrying virulence determinants
many clues as to the basis for pathogenicity/infection biology
Y. pestis
evolution from
Y. pseudotuberculosis
key gene gain and gene loss events in the evolution of mammalian pathogenesis in the
Yersinia
genus
non pathogenic yersinia acquires virulence plasmid
pYV
makes virulent
pathogenic yersinia acquires chromosome genes
hms
(for biofilm formation) and
HPI insect toxins
from bacteria in soil or animal gut
Y pseudotuberculosis
acquires plasmid
pFra
(encoding phospholipase D for survival in flea) from salmonella or other enterobacteria
Y pseudotuberculosis
(pFra) Pre-pestis 1 - flea transmission possible but inefficient
acquires plasmid
pPst
(encoding plasminogen activator for dissemination primary pneumonia in mammalian host). from bacteria in rat or flea gut. present in multiple copies
flea transmission more frequence
Y pseudotuberculosis
pFra pPst - Pre-pestis 2
2 more items...
acquisition of plasmids changes disease ecology and allows it to spread
the Black Death
a profound event in human history
believed to have been caused by the bacterium
Yersinia pestis
did
Y. pestis
cause Justinian and Black Death plagues?
high mortality rates and clinical manifestations differ from moden
Y pestis
if the same bug, why do we not see modern pandemics?
some PCR-based detections in ancient samples are inconsistent
contamination?
arrives in England in the summer of 1348
killed ~50% of the UK population over the next year
aka 32 million people today
resulted in unprecedented political, economic, cultural, social and religious impacts
bubonic plague swept across the UK several times over the last 1000 years, in particular during the 14th and 17th century
WHY DID SO MANY DIE?
PALEOMICROBIOLOGY
can't culture microbes from ancient samples, can we study their genomes instead?
major technical issues
DNA degraded - short fragments
pathogen DNA tiny proportion of total
direct sequencing of pathogen DNA not possible
unlike larger organisms bacteria very rarely leave fossils
DNA from ancient samples
if we find the skeleton of a known plague victim we can examine teeth
teeth have a good blood supply - pulp may have bacterial DNA remnants
molars are favoured samples
large
often intact
enamel used for radiating C14
pulp extracted
blood supply
pathogen DNA from systemic infections
DNA extracted
prevention of contamination in paleomicrobiology
sterility is paramount
risk
environmental flora
hand-borne flora
environmental flora
positive control
previous amplicons
cross-contamination (carry-over)
procedure
sample collection -> DNA extraction -> PCR
preventative measures
wearing gloves
scraping external surface
cleaning with filtered compressed air
cleansing with sterile water
ultraviolet irradiation
using dedicated, controlled room
wearing gloves
using primers only once (suicide pCR)
avoiding positive control
running 1 negative control for 3 samples
sequencing all the amplicons
an issue of crucial importance for palaeomicrobiological studies
microbial flora from the burial site
from the laboratory
accepted guidelines for aDNA labs
genomic makeup of
Y. pestis
- causative agent of bubonic plague
first genome sequenced in early 2001
20 genomes of modern Y pestis strains now sequenced
highly similar: 2700 positions differences in total
the PCP-1 plasmid
y pestis specific
high copy number (eg 100x more than the chromosome)
fishing out pathogen DNA
pPCP1 plasmid - found only in Y pestis, sequence is cut up into short (specific) sequences
pPCP1 backbone synthesised 'bait' DNA
hybridised with extracted DNA
samples eluted and sequenced using NGS
enrichment greatly increases pathogen yield
0.0005% Y.pestis before array enrichment
60% Y.pestis after array enrichment
sequencing the Black Death genome
phylogenetics
Black Death strain sits at the root of an evolutionary tree that comprises 17 contemporary strains of y pestis
indicates that the Black Death strain progenitor of the forms of y pestis that infects humans today
almost identical to modern bubonic plague strain
no unique traits obvious in the medieval organism
perceived increased virulence of the disease during the Black Death may not have been due to bacterial phenotype
genetically monomorphic
not really changed since medieval times
what caused the high mortality of Black Death?
malnutrition and host susceptibility
poor weather conditions and subsequent crop failures in the preceding years
pneumonic versus bubonic plague
pneumonic plague is transmitted via sneezing/inhalation of bacteria in droplets, so
person-person transmission
and has a 90-95% case fatality rate!
bubonic plage is transmitted via flea bites, but has about a ~45% case fatality rate if untreated
was Black Death due to pneumonic plague?
other pathogens/co-infections
have been implicated, perhaps as contributing factors
smallpox, typhoid and respiratory ailments
plague samples are being mines fro additional pathogenic agents
no treatments available
no effective public health measures
new genomics technologies
: tuberculosis in a mummy
alternative to hybridisation capture and PCR methods is metagenomics: open-ended sequencing of DNA recovered from uncultured samples without target-specific amplification or enrichmnet
approach used on
Mycobacterium tuberculosis
in a sample obtained from a Hungarian mummy
DNA was extracted from lung tissue and sequenced in a single Illumina MiSeq run
5.5 million paired-end reads were obtained with less than 1% aligned to the human genome and 8% to the
M. tuberculosis
reference strain - average per-sample depth of 32x coverage
Eighteenth-century genomes show that mixed infections were common at time of peak tuberculosis in Europe
Metagenomic analysis of tuberculosis in a mummy
phylogenetics
evolutionary context
comparative genomics
use only common DNA, recombination problematic
use SNPs to infer phylogenetic tree, depicting evolutionary relationships among samples
identify SNPs that describe the genetic variation among population
molecular clock
dna and protein sequences evolve at a rate that is relatively constant over time and among different organisms
can be calibrated using temporal information from aDNA, as samples can be dated
inaccurate bc rates can change due to selection pressures
multiple SNPs at the same site due to recombination can be an issue