1) The document discusses a study that aims to investigate the role of host cell microRNA-155 in elucidating the mechanisms by which Leishmania parasites evade the immune system and cause leishmaniasis. 2) The study hypothesizes that infecting Leishmania species manipulate host miR-155 expression to target SMAD2, preventing apoptosis in macrophages and dendritic cells in order to cause disease. 3) The study involves three aims: 1) determining if miR-155 directly targets the human SMAD2 gene, 2) assessing miR-155 targeting of SMAD2 during Leishmania infection, and 3) investigating the effects on SMAD2 protein levels and TGF-β

1. National Science Foundation Graduate Research Fellowship Program Connor Ratycz
Background: The spectrum of molecular mechanisms Leishmania parasites utilize to suppress
leishmanicidal events of host immune cells in order to evade immune destruction and cause
disease in humans remain largely unknown. Caused by Leishmania parasites, leishmaniasis is a
diverse group of neglected tropical diseases with symptoms ranging from cutaneous legions to
fatal visceral pathologies. Leishmaniasis remains a global problem as 12 million people are
affected worldwide, with additional hundreds of millions at risk1
. Once inoculated through the
bite of a sand fly, the parasites actively invade host macrophages (MC) and dendritic cells (DC)
where they replicate, evading anti-parasitic responses and travel to the periphery to cause further
infection2
. Since little is known about Leishmania pathogenesis, this study aims to investigate the
role of host cell microRNA-155 to elucidate the mechanisms of immune evasion of Leishmania
and understand the immunopathology of leishmaniasis.
MicroRNAs (miRs) are endogenous non-coding small RNAs (~22 nucleotides) that comprise
a prominent class of gene regulatory factors that regulate target messenger RNA (mRNA)
expression. miRs regulate expression of at least 30% of human genes, and it is believed each
miR can target 100 transcripts and a single mRNA can be regulated by multiple miRs3
. Several
pathogens have been shown to manipulate host cell miR profiles as a strategy to evade the
immune response4,5
. It has been observed that Leishmania parasites initiate changes in various
miRs expression levels upon MC infection, including miR-1556
. Expressed in MC and DC, miR-
155 is an important regulator of immunity as it has been shown to play a role in innate and
adaptive immune responses. Moreover, miR-155 has been shown regulate the transforming
growth factor (TGF)- β pathway by targeting SMAD2, which affects the ability of MC and DC
to respond to TGF- β stimulation8
. TGF- β, a down-regulator of the immune response via
inducing apoptosis, is produced by Leishmania-infected MC. Paradoxically, despite increased
levels of TGF- β, Leishmania infection inhibits MC death10,11
. Therefore, I hypothesize that
infecting parasite species (L. major or L. donovani) manipulate host miR-155 expression to
target SMAD2, preventing apoptosis in MC and DC to cause disease in humans.
Aim 1: Determine if miR-155 Directly Targets Human SMAD2 in U937 Monocyte Lines:
Using a bioinformatics database, miR-155 was predicted to target SMAD2 at two sites near the
end of its 3’ untranslated region (UTR)12
. To test the miR-155 target potential to the 3’ UTR of
the SMAD2 transcript, I will use Light Switch 3’UTR luciferase reporter plasmids (SwitchGear
Genomics). The reporter constructs will be transfected via the Amaxa Cell Line Nucleofector Kit
C specific for U937 cell line which is available in the McDowell lab. The U937 cells will be co-
transfected with miR-155 mimic and a normalizing vector, an empty vector, or a plasmid
containing the miR-155 target sequence of SMAD2. Using a luminometer, fluorescence output
will be quantified to determine specific activity of miR-155 to the SMAD2 target sequence. Due
to the two target sites on the SMAD2 transcript, I anticipate that U937 cells co-transfected with
miR-155 mimic and the 3’UTR target vector will have a diminished fluorescence intensity
compared to the controls, indicating miR-155 target specificity.
Aim 2: miR-155 Target Reporters During Leishmania spp. Infections: After validation of
miR-155 targeting of SMAD2 transcripts, I will assess miR-155 specificity to SMAD2 in the

2. National Science Foundation Graduate Research Fellowship Program Connor Ratycz
context of Leishmania infection. I will co-transfect U937 cells with 3’UTR Lenti GoCLones and
miR-155 mimics, using previously confirmed targets of miR-155 as positive controls and non-
targets as negative controls. I will also use human monocyte-derived dendritic cells (hMDDC)
only transfected with the GoClone and infect with L. major or L. donovani in vitro to evaluate
parasite-influenced SMAD2 targeting.
Aim 3: Investigate SMAD2 Protein Level and TGF-β-dependent Gene Expression: I
anticipate my findings in Aims 1 and 2 to support the hypothesis that miR-155 targets SMAD2 in
the context of Leishmania infection, which indicates the TGF-β signaling pathway is negatively
regulated by miR-155. To determine the effects of increased levels of miR-155, I will use U937
cells integrated with a lentiviral vector system to induce miR-155 transgene expression via
doxycycline. After induction of miR-155 over time, the SMAD2 protein levels and mRNA will
be quantified via Western blot analysis and RT-qPCR, respectively. I will also investigate how
SMAD2 targeting by miR-155 can modulate TGF- β/SMAD2-dependent genes. To assess this
phenomenon, I will use U937 cells with or without overexpression of miR-155 stimulated with
or without TGF- β and assess the expression of TGF- β-dependent genes involved in immunity
(IL-1 β, IL-4 receptor, pro-apoptosis). As a control, I plan to use SMAD2 as the protein remains
unaffected by TGF- β stimulation. RNA will be extracted and the gene expression levels will be
assessed via RT-qPCR.
Intellectual Merit and Broader Impacts: Elucidating how pathogens adjust the host cell
microenvironment for immune evasion is crucial for a better understanding of host-pathogen
interactions for the development of efficient therapies. To date, few studies have examined the
expression of host miRs in connection with Leishmania infection biology. Therefore, this study
will use a novel approach of examining how Leishmania parasites modulate miR-155 levels to
target TGF- β genes, preventing apoptosis in infected MC and DC. Such information will
provide a deeper understanding of immune regulation of Leishmania uncovering potential new
targets for clinical therapies which may reveal universal insights into intracellular infections.
Neglected diseases remain a barrier of many economically developing nations to compete in
a global marketplace. Approximately 1.5 million new cases of cutaneous and ~500,000 of
visceral leishmaniasis occur annually, primarily in low and middle income nations1
. Although
leishmaniasis treatments exist, the disease causes severe morbidity and loss of labor potential in
infected individuals and treatment is costly and toxic. Furthermore, sand flies, the insect vector
of Leishmania parasites, have the potential to expand into non-endemic ranges, spreading the
diseases to immunologically naive populations as a result of global climate change. Thus, a
deeper knowledge of leishmaniasis immunobiology for effective clinical interventions is a
necessity. This work, funded by the NSF, will provide a solid foundation for completing my
dissertation and will allow me to continue to fulfill my potential as a researcher. 1
CDC. (2013)
http://www.cdc.gov/parasites/leishmaniasis/biology.html. 2
Kaye et al. (2011) Nat. Rev. Micro. 9, 604-615. 3
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Katiyar-Agarwal et al. (2010) Ann. Rev.
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Lemaire et al. (2013) PLoS Negl. Trop. Dis. 7,10. 7
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Barral et al. (1993) Proc. Nat. Acad. Sci.90, 3. 9
Moore et al. (1994) J. Imm. 152,6. 10
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Leuk. Bio. 76,1. 11
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