Fig. 1
PpKzl1 and PpKzl2 alignments with Lutzomyia longipalpis Kazal domains.
PpKzl1 (GenBank ID: EU045342) (A) and PpKzl2 (GenBank ID: JX171681) (B) are
both similar to putative proteins in L. longipalpis with Kazal-type domains:
LlKzl1 (GenBank ID: ABV60319) and LlKzl2 (contig 69116). Conserved residues are
in black and similar residues are in grey. Predicted signal peptides are
underlined, asterisks mark predicted P1 residues, conserved cysteines are
marked (C) and gaps are indicated by dashes.
Fig. 2:
PpKzl1 and PpKzl2 expression in adult females post-blood meal (PBM). PpKzl1
and PpKzl2 mRNA expression levels are regulated after a blood meal. A: PpKzl1
is up-regulated 24 h and 48 h PBM with highest expression at 48 h PBM. By 72 h
expression is down-regulated to levels similar to 0 h; B: PpKzl2 is
up-regulated 24 h, 48 h and 72 h PBM. Expression is highest at 48 h and
decreases between 48-72 h PBM. Values are the mean fold change of five or more
individual midguts with standard error of the mean. Expression was calibrated
to 0 h expression levels. Analysis used ANOVA t test with the Bonferroni
correction for multi-comparisons. *: p < 0.05; **: p < 0.01; ***: p <
0.001.
Fig. 3:
PpKzl1 and PpKzl2 expression in larval stages and pupa. A: PpKzl1 was
expressed in all larval stages and pupae. PpKzl1 expression was not
significantly different when compared between larval stages; B: PpKzl2 was also
expressed in all larval stages and pupae at similar expression levels. Five or
more individuals were pooled for each developmental stage and this was repeated
for a total of three replicates. Values are the mean fold change with standard
error of the mean. Expression was calibrated to expression levels in eggs.
ANOVA t test with the Bonferroni correction for multi-comparisons was used for
statistical analysis. L: larval stage; P: pupa.
Fig. 4:
PpKzl1 and PpKzl2 expression in adult females infected with Leishmania
major . Temporal expression profiles 24 h, 48 h and 72 h post-infective blood
meal (I) (▲) and post-non-infected blood meal (NI) (◊). Eight individual
midguts were assayed for each infected and non-infected time point. PpKzl1 and
PpKzl2 expression was not significantly different 24 h, 48 h and 72 h I when
compared to NI control groups. Bars are the mean fold change of eight
individual midguts. Expression was calibrated to expression in NI controls.
Statistical analysis used two-tailed unpaired t tests and two-tailed
Mann-Whitney U tests for parametric and nonparametric comparisons respectively
(p < 0.05).
Fig. 5:
rPpKzl2 enzyme inhibition activity. Activity was measured at increasing
concentrations of both rPpKzl2 and substrate. Reactions were fit with
Michaelis-Menten non-linear regression and apparent maximum velocity (V max )
values were used to calculate residual activity. Inhibition of α-chymotrypsin
activity was observed with decreasing V max . Activity of 0.25 µM
α-chymotrypsin was reduced to 9.4%. Residual activity of 2 µM trypsin was
reduced to 63.9%. Activity of 0.05 µM α-thrombin in the presence of rPpKzl2
was reduced to 33.5%. Reactions were run in triplicate and each graph
represents one of two replicates of each experiment.
pKzl1 multiple sequence alignment. Phlebotomus papatasi
PpKzl1 (GenBank ID: EU045342), Culicoides
sonorensis (GenBank ID: AAV84258), Drosophila
yakuba _a (GenBank ID: XP_002088400), Anopheles
darlingi _a (GenBank ID: ACI30143), Lutzomyia
longipalpis (Gen-Bank ID: ABV60319), Aedes aegypti
AaTi (GenBank ID: ABF18209), Culex quinquefasciatus (GenBank
ID: XP_001868221), Ochlerotatus
triseriatus salivary (GenBank ID: ACU30983), Aedes
albopictu s (GenBank ID: AAV90671), Triatoma
infestans infestin domain 4 (GenBank ID: AAK57342),
Anopheles gambiae _a (GenBank ID: XP_001230687),
An. gambiae _b (GenBank ID: XP_317819), An.
gambiae _c (GenBank ID: EAA12788), Drosophila
yakuba _b (GenBank ID: XP_002088399), D. yakuba _c
(GenBank ID: XP_002088401), D. yakuba _d (GenBank ID:
XP_002088397), Drosophila pseudoobscura pseudoobscura _a
(GenBank ID: XP_001356962), D. pseudoobscura pseudoobscura _b
(GenBank ID: XP_001356963) and An. darlingi _b (GenBank ID:
ACI30165). Asterisk means the predicted P1 residue, gaps are indicated by
dashes, conserved cysteines are in black and residues with more than 50%
conserved identity are in shades of grey.
PpKzl2 multiple sequence alignment. Phlebotomus papatasi
PpKzl2 (GenBank ID: JX171681), Anopheles darlingi (GenBank ID:
ACI30205), Drosophila mojavensis (GenBank ID: XP_002000106),
Culex quinquefasciatus (GenBank ID: XP_001842298),
Aedes aegypti (GenBank ID: XP_001658905), Manduca
sexta (GenBank ID: AAF16698), Nasonia vitripennis
(GenBank ID: XP_001600330), Bombyx mori (GenBank ID:
NP_001040250), Bombus terrestris (GenBank ID: XP_003401213),
Drosophila pseudoobscura pseudoobscura (GenBank ID:
XP_001359513), Drosophila persimilis (GenBank ID:
XP_002017393), Tribolium castaneum (GenBank ID: XP_974370),
Drosophila willistoni (GenBank ID: XP_002070657),
Drosophila grimshawi (GenBank ID: XP_001994337) ,
Solenopsis invicta (GenBank ID: ADC34234), Drosophila
virilis (GenBank ID: XP_002053264), Acromyrmex
echinatior (GenBank ID: EGI69242), Harpegnathos
saltator (GenBank ID: EFN89909), H. saltator 2
(GenBank ID: EFN81812) , Bombus impatiens (GenBank ID:
XP_003486913), Apis florae (GenBank ID: XP_003692060),
Camponotus floridanus (GenBank ID: EFN62548) and
Lutzomyia longipalpis (contig 69116). Asterisk means the
predicted P1 residue, gaps are indicated by dashes, conserved cysteines are in
black and residues with more than 50% conserved identity are in shades of
grey.
PpKzl1 and PpKzl2 expression in
Phlebotomus papatasi males and not in eggs.
Reverse-transcriptase polymerase chain reaction (PCR) was carried out in 20 µL
reactions with cDNA from one whole male and a pool of 10 eggs on an Eppendorf
Mastercycler gradient. Reactions were prepared with 10 µL of 2X GoTaq PCR
master mix (Promega, Madison, WI, USA), 0.2 µM forward and reverse primers, 1
µL cDNA and molecular grade water (Invitrogen, Carlsbad, CA, USA) to a total
volume of 20 µL. Reactions were carried out as follows: 95ºC/1 min followed by
26 cycles of 94ºC/30 s, 56.5ºC/30 s and 72ºC/1 min and a final step at 72ºC/5
min. The primers PpKzl111, PpKzl859 and PpTub148 specific for P.
papatasi Pp Kzl1 , PpKzl2 and
β-tubulin were used for PCR. PCR fragments (10 µL) were separated on a 1.5%
agarose gel stained with ethidium bromide. Primers for PpTub148 forward:
GCGATGACTCCTTCAACAC and reverse: GTGATCAATTGTTCGGGATG.
Michaelis-Menten non-linear regression of rPpKzl2 inhibition. Initial
velocity (V) over substrate concentration (S) was fit with Michaelis-Menten
non-linear regression for each concentration of rPpKzl2. A reduction in maximum
velocity and kinetic constant values was observed with increasing rPpKzl2 when
compared to the fit of 0 nM rPpKzl2. BAPNA: Na-Benzoyl-D,L-arginine
4-nitroanilide hydrochloride; S-2238:
H-D-Phenylalanyl-L-pipecolyl-Larginine-p-nitroaniline dihydrochloride;
Suc-AAPF-pNA: N-Succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine
4-nitroanilide.
PpKzl1 and PpKzl2 expression 72-144 h
post-blood meal (PBM). Temporal analysis with semi-quantitative reverse
transcriptase-polymerase chain reaction (PCR) indicated that
PpKzl1 and PpKzl2 transcript expression
remains constant 72-144 h PBM. Time points were pools of five midguts from
female sandflies. PCR was carried out in 20 µL reactions on an Eppendorf
Mastercycler gradient. Reactions were prepared with 10 µL of 2X GoTaq PCR
master mix (Promega, Madison, WI, USA), 0.2 µM forward and reverse primers, 1
µL cDNA and molecular grade water (Invitrogen, Carlsbad, CA, USA) to a total
volume of 20 µL. Reactions were carried out as follows: 95ºC/1 min followed by
26 cycles of 94ºC/30 s, 56.5ºC/30 s and 72ºC/1 min and a final step at 72ºC/5
min. The primers PpKzl111, PpKzl859 and PpTub148 specific for
Phlebotomus
papatasi Pp Kzl1 , PpKzl2
and β-tubulin were used for PCR. PCR fragments (10 µL) were separated on a 1.5%
agarose gel stained with ethidium bromide alongside 0.5 µg of exACTGene cloning
DNA Ladder (Fisher, Scientific, Pittsburgh, PA, USA). Intensities of PCR
fragments were standardised to β-tubulin and compared with known quantities of
the reference ladder using Total Lab 100 software (BioSystematica, Sarnau,
UK).