PLoS neglected tropical diseases,
Год журнала:
2022,
Номер
16(6), С. e0010510 - e0010510
Опубликована: Июнь 24, 2022
Leishmaniasis
is
a
parasitic
vector-borne
disease
caused
by
the
protistan
flagellates
of
genus
Leishmania
.
(Viannia)
guyanensis
one
most
common
causative
agents
American
tegumentary
leishmaniasis.
It
has
previously
been
shown
that
L
strains
carry
endosymbiotic
RNA
virus
1
(LRV1)
cause
more
severe
form
in
mouse
model
than
those
do
not.
The
presence
was
implicated
into
parasite’s
replication
and
spreading.
In
this
respect,
studying
molecular
mechanisms
cellular
control
viral
infection
great
medical
importance.
Here,
we
report
~30.5
Mb
high-quality
genome
assembly
LRV1-positive
M4147.
This
strain
turned
establishing
CRISPR-Cas9
system
ablating
gene
encoding
phosphatidate
phosphatase
2-like
(PAP2L)
protein.
orthologue
conspicuously
absent
from
an
unusual
member
family
Trypanosomatidae,
Vickermania
ingenoplastis
,
species
with
mostly
bi-flagellated
cells.
Our
analysis
PAP2L-null
showed
increase
number
cells
strikingly
resembling
V
likely
as
result
disruption
cell
cycle,
significant
accumulation
phosphatidic
acid,
increased
virulence
compared
to
wild
type
The
ability
to
analyze
the
function
of
all
genes
in
a
genome
is
highly
desirable,
yet
challenging
Leishmania
due
repetitive
genome,
limited
DNA
repair
mechanisms,
and
lack
RNA
interference
most
species.
While
our
introduction
cytosine
base
editor
(CBE)
demonstrated
potential
overcome
these
limitations
(Engstler
Beneke,
2023),
challenges
remained,
including
low
transfection
efficiency,
variable
editing
rates
across
species,
parasite
growth
effects,
competition
between
deleterious
non-deleterious
mutations.
Here,
we
present
an
optimized
approach
addressing
issues.
We
identified
T7
RNAP
promoter
variant
ensuring
high
species
without
compromising
growth.
A
revised
CBE
single-guide
RNAs
(sgRNAs)
scoring
system
was
developed
prioritize
STOP
codon
generation.
Additionally,
triple-expression
construct
created
for
stable
integration
sgRNA
expression
cassettes
into
safe
harbor
locus
using
AsCas12a
ultra-mediated
double-strand
breaks,
increasing
efficiency
by
~400-fold
1
transfectant
per
70
transfected
cells.
Using
this
improved
small-scale
proof-of-principle
pooled
screen,
successfully
confirmed
essential
fitness-associated
functions
CK1.2,
CRK2,
CRK3,
AUK1/AIRK,
TOR1,
IFT88,
IFT139,
IFT140,
RAB5A
mexicana
,
demonstrating
significant
improvement
over
previous
method.
Lastly,
show
utility
co-expressing
ultra,
RNAP,
hybrid
CRISPR
gene
replacement
within
same
cell
line.
Overall,
improvements
will
broaden
range
possible
applications
enable
variety
loss-of-function
screens
near
future.
The
movement
and
pathogenicity
of
trypanosomatid
species,
the
causative
agents
trypanosomiasis
leishmaniasis,
are
dependent
on
a
flagellum
that
contains
an
axoneme
dynein-bound
doublet
microtubules
(DMTs).
In
this
work,
we
present
cryo–electron
microscopy
structures
DMTs
from
two
Leishmania
tarentolae
Crithidia
fasciculata
,
at
resolutions
up
to
2.7
angstrom.
revealed
27
trypanosomatid-specific
microtubule
inner
proteins,
specialized
dynein-docking
complex,
presence
paralogous
proteins
enable
higher-order
periodicities
or
proximal-distal
patterning.
Leveraging
genetic
tractability
quantified
location
contribution
each
structure-identified
protein
swimming
behavior.
Our
study
shows
proper
B-tubule
closure
is
critical
for
flagellar
motility,
exemplifying
how
integrating
structural
identification
with
systematic
gene
deletion
can
dissect
individual
contributions
motility.
Molecular Microbiology,
Год журнала:
2025,
Номер
unknown
Опубликована: Апрель 9, 2025
ABSTRACT
Blastocrithidia
nonstop
is
a
protist
with
highly
unusual
nuclear
genetic
code,
in
which
all
three
standard
stop
codons
are
reassigned
to
encode
amino
acids,
UAA
also
serving
as
sole
termination
codon.
In
this
study,
we
demonstrate
that
parasitic
flagellate
amenable
manipulation,
enabling
gene
ablation
and
protein
tagging.
Using
preassembled
Cas9
ribonucleoprotein
complexes,
successfully
disrupted
tagged
the
non‐essential
encoding
catalase.
These
advances
establish
single‐celled
eukaryote
model
organism
for
investigating
malleability
evolution
of
code
eukaryotes.
ABSTRACT
Human
visceral
leishmaniasis
(HVL)
is
the
second
most
lethal
tropical
parasitic
disease.
Currently,
no
prophylactic
or
therapeutic
vaccines
exist
for
HVL.
Thus,
development
of
an
efficacious
vaccine
still
needed.
We
previously
performed
immunoproteomics
analysis
on
Leishmania
amazonensis
parasite
extracts
to
identify
immunodominant
antigens
recognized
by
sera
vaccinated
and
protected
mice.
Among
identified
antigens,
we
discovered
a
novel,
unstudied
repetitive
protein,
initially
annotated
in
genomes
as
kinetoplast-associated
protein-like
protein
from
infantum
(LinKAP),
containing
conserved
domains
(trichohyalin-plectin-homology
[TPH]
TolA)
that
are
associated
with
other
mitochondrial
proteins.
LinKAP
sequences
across
trypanosomatids,
including
Endotrypanum,
Leishmania,
Trypanosoma
species.
Using
differential
centrifugation
subcellular
structures,
showed
was
enriched
fractions
colocalizing
mNeonGreen
labeling
at
endogenous
locus
using
CRISPR-Cas9
confocal
microscopy
confirmed
mitochondrial-associated
but
not
specifically
colocalized
kDNA.
cloned
expressed
truncated
version
(rLinKAP),
part
(15)
several
amino
acid
repeats,
demonstrating
over
85%
homology
L.
infantum,
amazonensis,
braziliensis,
mexicana
An
adjuvanted
formulation
Poly
ICLC,
polyinosinic-polycytidylic
(Poly
I:C)
stabilized
carboxymethylcellulose
polylysine,
used
vaccinate
mice
hamsters
leishmaniasis.
Animals
immunized
rLinKAP
potent
cellular
humoral
response
significant
decrease
tissue
parasitism
when
challenged
.
also
tested
Following
vaccination,
antibody
responses
were
enhanced,
became
apparent.
Our
treatment
protocol
inhibited
splenic
burden
75%
treated
In
conclusion,
our
antigen
discovery
strategy
observed
protective
effect
highlight
promising
candidate
IMPORTANCE
A
previous
reverse
vaccinology
study
(LinKAP)
potential
new
target,
this
promastigotes.
Interestingly,
trichohyalin-plectin-homology
(TPH)
TolA
protein.
further
characterized
highly
among
trypanosomatids.
validated
(rLinKAP)
both
vaccine,
immunize
animals
against
(VL).
This
disease,
caused
parasite,
affects
populations
globally
lacks
effective
vaccines.
Identifying
its
preliminary
characterization
provides
perspectives
studying
role
parasite's
biology.
The Journal of Cell Biology,
Год журнала:
2024,
Номер
223(9)
Опубликована: Май 14, 2024
Two
sets
of
motor
proteins
underpin
motile
cilia/flagella
function.
The
axoneme-associated
inner
and
outer
dynein
arms
drive
sliding
adjacent
axoneme
microtubule
doublets
to
periodically
bend
the
flagellum
for
beating,
while
intraflagellar
transport
(IFT)
kinesins
dyneins
carry
IFT
trains
bidirectionally
along
axoneme.
Despite
assembling
cilia
flagella,
train
speeds
have
only
previously
been
quantified
in
immobilized
flagella—mechanical
immobilization
or
genetic
paralysis.
This
has
limited
investigation
interaction
between
flagellar
beating.
Here,
uniflagellate
Leishmania
parasites,
we
use
high-frequency,
dual-color
fluorescence
microscopy
visualize
movement
beating
flagella.
We
discovered
that
adhesion
flagella
a
microscope
slide
is
detrimental,
reducing
speed
increasing
stalling.
In
free
move,
not
strongly
dependent
on
beat
type;
however,
permanent
disruption
by
deletion
genes
necessary
formation
regulation
showed
an
inverse
correlation
frequency
speed.
PLoS Pathogens,
Год журнала:
2024,
Номер
20(7), С. e1012382 - e1012382
Опубликована: Июль 11, 2024
Liposomal
amphotericin
B
is
an
important
frontline
drug
for
the
treatment
of
visceral
leishmaniasis,
a
neglected
disease
poverty.
The
mechanism
action
(AmB)
thought
to
involve
interaction
with
ergosterol
and
other
ergostane
sterols,
resulting
in
disruption
integrity
key
functions
plasma
membrane.
Emergence
clinically
refractory
isolates
Leishmania
donovani
L
.
infantum
ongoing
issue
knowledge
potential
resistance
mechanisms
can
help
alleviate
this
problem.
Here
we
report
characterisation
four
independently
selected
clones
that
are
resistant
AmB.
Whole
genome
sequencing
revealed
three
moderately
clones,
was
due
solely
deletion
gene
encoding
C24-sterol
methyltransferase
(
SMT1
).
fourth,
hyper-resistant
clone
(>60-fold)
found
have
24
bp
both
alleles
putative
cytochrome
P450
reductase
(P450R1).
Metabolic
profiling
indicated
these
parasites
were
virtually
devoid
(0.2%
versus
18%
total
sterols
wild-type)
had
marked
accumulation
14-methylfecosterol
(75%
0.1%
14-alpha
methylcholestanes.
These
substrates
sterol
demethylase
CYP51
)
suggesting
enzyme
may
be
bona
fide
P450R
specifically
involved
electron
transfer
from
NADPH
during
catalysis.
Deletion
P450R1
wild-type
cells
phenocopied
metabolic
changes
observed
our
AmB
as
well
nulls.
Likewise,
addition
wild
type
restored
profiles
type.
Our
studies
indicate
essential
amastigote
viability,
thus
loss
unlikely
driver
clinical
resistance.
Nevertheless,
investigating
underpinning
provided
insights
refine
understanding
biosynthetic
pathway.
Nucleic Acids Research,
Год журнала:
2023,
Номер
51(10), С. 5177 - 5192
Опубликована: Март 25, 2023
Abstract
TbMex67
is
the
major
mRNA
export
factor
known
to
date
in
trypanosomes,
forming
part
of
docking
platform
within
nuclear
pore.
To
explore
its
role
co-transcriptional
export,
recently
reported
Trypanosoma
brucei,
pulse
labelling
nascent
RNAs
with
5-ethynyl
uridine
(5-EU)
was
performed
cells
depleted
and
complemented
a
dominant-negative
mutant
(TbMex67-DN).
RNA
polymerase
(Pol)
II
transcription
unaffected,
but
procyclin
loci,
which
encode
mRNAs
transcribed
by
Pol
I
from
internal
sites
on
chromosomes
6
10,
showed
increased
levels
5-EU
incorporation.
This
due
readthrough
transcription,
proceeded
beyond
procyclin-associated
genes
up
start
site
opposite
strand.
Complementation
TbMex67-DN
also
I-dependent
formation
R-loops
γ-histone
2A
foci.
The
DN
exhibited
reduced
localisation
binding
chromatin
compared
wild-type
TbMex67.
Together
interaction
remodelling
TbRRM1
II,
transcription-dependent
association
nucleoporins,
our
findings
support
for
connecting
T.
brucei.
In
addition,
stalls
specific
contexts,
thereby
limiting
R-loop
replication
stress.