Department of Microbiology graduate students, postdocs and research scientists along with graduate students affiliated with Microbiology professors presented posters during the 2017 Health Sciences Research Week. Five microbiology graduate students presented on Tuesday, April 18th including Tiffany Borbón, Ashley Cooney, Kayley Janssen, Lauren Kinkead,  and Kai Rogers. Microbiology postdocs and research scientists Kristopher Heilmann, Jeffrey Kavanaugh, Fengyin Li, and Kyp Oxley presented the following day.

Tiffany Borbón | Gwendolyn Clay, Breanna Scorza, Alan Sariol, Yani Chen, Bayan Zhanbolat “Bacterial Co-infections in Murine Cutaneous Leishmaniasis
Ashley Cooney | Brajesh Singh, Ni Li, David A Stoltz, Paul B McCray, Patrick L Sinn “A piggyBac transposon/adenoviral viral-based hybrid vector system for improved in vivo CFTR delivery
Kayley H Janssen | Tom Cribbs, Deven Voelker, Barbara I Kazmierczak, Tim Yahr “Hfq inhibits Vfr translation to prevent expression of the Pseudomonas aeruginosa type III secretion system”
Lauren C Kinkead | Justin T Schwartz, Laura C Whitmore, Brandi B Ketelsen, Lee-Ann H Allen “Elucidation of the mechanism of apoptosis inhibition by Francisella tularensis”
Kai Rogers | Bethany Rhein, Natarajan Ayithan, Wendy Maury “The Effect of Macrophage M2 Polarization on Ebola Virus Infection”

The Department of Microbiology has named Tiffany Borbón and Ashley Cooney as recipients of the Usha Balakrishnan Prize for the best graduate student poster presentation at the 2017 Health Sciences Research Week. Congratulations Ashley and Tiffany!

Read the following selected research descriptions from the participants.

Microbiology graduate students

Tiffany Borbón   Bacterial Co-Infection in Murine Cutaneous Leishmaniasis   Tiffany Y. Borbón1, Gwendolyn Clay1, Breanna Scorza1, Alan Sariol1, Yani Chen1, Bayan Zhanbolat1, Fayyaz Sutterwala2, and Mary E. Wilson1,3

1University of Iowa, 2Cedars-Sinai Medical Center, 3Iowa City Veterans’ Affairs Medical Center

Cutaneous leishmaniasis (CL) is caused by Leishmania spp. leading to skin lesions and ulceration. In humans, treatment is often best initiated after ulceration. Furthermore, bacterial superinfection of CL lesions by organisms like Staphylococcus aureus is common. Immune responses associated with ulceration and their effects on CL disease are underexplored. We hypothesized bacteria present during CL activate inflammatory responses that contribute to outcome of parasitic infection.

We injected L. major, S. aureus, or both intradermally into ears of C57BL/6 mice. We monitored lesion volume, analyzed inflammation by histology, microbial burden and expression of immune mediators by qPCR, and ex vivo antigen stimulation of draining lymph nodes (LNs) by cytokine multiplex at 4 weeks post-infection (p.i.).

We observed two-fold larger lesions in co-infected ears compared to S. aureus- or L. major-infected ears. However, ears harvested 4 weeks p.i. showed no difference in microbial burdens. Histology revealed greater neutrophils in co-infected ears compared to L. major-infected ears, and many lymphocytes and histiocytes in both groups receiving L. major. Inflammatory gene expression transcripts were similar between L. major and co-infected groups. Leishmania antigen-stimulated LN cells from co-infected mice released over two-fold more IL-17A than LN cells from L. major- or S. aureus-infected mice. There was no detectable S. aureus DNA remaining in ears.

Overall, these data suggest co-infection with S. aureus leads to prolonged increase in inflammation, which does not alter L. major burden, but augments lesion pathology. These pathologic changes could be related to a more vigorous TH17 type response.

Ashley Cooney    PiggyBac transposon/adenoviral viral-based hybrid vector system for improved in vivo CFTR delivery  Ashley L. Cooney1,2, Brajesh Singh2, Ni Li2, David A. Stoltz3, Paul B. McCray Jr.1,2 and Patrick L. Sinn2

Departments of Microbiology1, Pediatrics2 and Internal Medicine3, Carver College of Medicine, The University of Iowa, Iowa City, IA 52242, USA

PURPOSE: The DNA transposon piggyBac is a potential therapeutic agent for multiple genetic diseases such as cystic fibrosis (CF). Recombinant piggyBac transposon and transposase are typically co-delivered by plasmid transfection; however, plasmid delivery is inefficient in somatic cells in vivo and is a barrier to the therapeutic application of transposon-based vector systems. Previously, we demonstrated that adenovirus (Ad) is an effective transposon delivery vehicle in mice. Furthermore, we show persistent correction of the chloride transport defect in well-differentiated primary cultures of human airway epithelial cells derived from CF patients.

METHODS: We delivered piggyBac/Ad vector expressing GFP and secreted luciferase to newborn (<7 day old) wild-type pigs and harvested tissues after 5 days. We quantified whole lung distribution by Real Time quantitative PCR and used fluorescence imaging to determine GFP localization. Additionally, we measured secreted luciferase in the broncheoalveolar lavage (BAL) and serum. In the CF pig studies, we delivered an Ad vector carrying cystic fibrosis transmembrane conductance regulator (CFTR) to newborn CF pigs and determined phenotypic correction by a variety of metrics including Ussing chamber analysis, bacterial killing and immunohistochemistry.

RESULTS: As a proof of principle, we optimized delivery of 1st generation Ad vector to the airways of newborn wild-type pigs. Five days following aerosolization, we observed wide spread reporter gene expression in the conducting airways and detected secreted luciferase in the BAL and serum. After delivery of Ad-CFTR to newborn CF pigs, we observed correction of chloride current in freshly excised tracheal explants.  Furthermore, Ad-CFTR rescued bacterial killing ability to wild-type levels.

CONCLUSIONS: Combining the emerging technologies of DNA transposon-based vectors with well-studied adenoviral delivery provides new tools for in vivo gene transfer and presents an exciting opportunity to increase the delivery efficiency for therapeutic genes such as CFTR.

Lauren Kinkead  Mechanisms of human neutrophil apoptosis inhibition by Francisella tularensis   Lauren Kinkead, Justin Schwartz, Adam Pickrum, Laura Whitmore, Brandi Ketelsen, Lee-Ann Allen   

Francisella tularensis is a Gram-negative, facultative intracellular bacterium and the causative agent of the zoonotic disease tularemia. Inhalation of as few as ten organisms can cause severe pneumonic disease with a mortality rate of up to 60% if infections are untreated. The pathogenicity of this organism is thought to be dependent on its ability to modulate host immune responses; it is noteworthy that neutrophils contribute to tissue destruction and disease severity. In previous work we demonstrated that F. tularensis inhibits neutrophil activation and escapes the phagosome to replicate in the cytosol. In addition, we recently demonstrated that F. tularensis prolongs human neutrophil lifespan by interfering the intrinsic, extrinsic, and phagocytosis-induced apoptotic pathways. How this prolongation occurs is incompletely defined, but our published data suggest secreted factors function in this process. Accordingly, we now show that media conditioned by either F. tularensis strain LVS or by LVS-infected neutrophils significantly delays neutrophil apoptosis in a dose-dependent manner. Additional biochemical fractionation studies indicate that LPS and capsule are neither necessary nor sufficient to delay neutrophil apoptosis and suggest that F. tularensis lipoproteins contribute to apoptosis inhibition dependent on the presence of TLR2. Prolonged neutrophil survival also requires PI3K and p38 MAPK activity; whether this is specifically linked to lipoproteins and TLR2 activation is under investigation. Therefore, our data provide new insight into the mechanisms by which F. tularensis manipulates neutrophil function and causes disease.

Kai Rogers   The Effect of Macrophage M2 Polarization on Ebola Virus Infection  Kai Rogers1, Bethany Rhein2, Natarajan Ayithan3, Wendy Maury1

1 Department of Microbiology, University of Iowa, Iowa City, IA 2 Department of Molecular Medicine, Mayo Clinic, Rochester, MN 3 Institute of Human Virology, University of Maryland, Baltimore, MD

Ebola virus (EBOV), a negative sense RNA virus and member of the Filoviridae family, is capable of causing tremendous morbidity and mortality. It is appreciated that cells of the innate immune system are important target populations in the initial stages and dissemination of EBOV infection. Our findings indicate the activation status of macrophages significantly impacts the susceptibility of these cells to EBOV infection. Ex vivo, M-CSF-matured resident peritoneal macrophages from C57BL/6 interferon α/β receptor (IFNAR) knockout mice were treated with well-established activation cytokines to elicit an anti-inflammatory M2a phenotype, which was confirmed by qRT-PCR for expression of appropriate marker genes. Cells were infected with a BSL-2 model virus (EBOV/rVSV) expressing GFP and infection was quantified at 24 hours by flow cytometry. We demonstrate that macrophages generated by IL4/IL13 treatment were significantly more susceptible to EBOV/rVSV infection than M-CSF-treated cells. We found that IL4/IL13-treated macrophages bound more EBOV/rVSV than M-CSF-treated cells, suggesting that the enhancement of infection of these cells may be due to enhanced receptor expression. To explore this, expression of C-type lectins and phosphatidylserine receptors that might mediate EBOV entry were assessed. We found RNA levels of SIGNR3 (a murine ortholog of human DC-SIGN) to be significantly higher in the IL4/13 treated macrophages than in M-CSF treated cells. Competitive inhibition studies using mannan reduced EBOV/rVSV entry in IL4/13-stimulated macrophages and ectopic SIGNR3 expression enhanced EBOV/rVSV infection, consistent with a role for  SIGNR3-dependent uptake in M2a macrophages. In vivo studies demonstrated that mice exposed to IL4/IL13 24 hours prior to infection showed greatly elevated morbidity and mortality compared to PBS-treated littermates. These results demonstrate that M2a polarization enhances infection at least in part by elevating expression of the C-type lectin, SIGNR3. Further, this is the first demonstration that murine SIGNR3 serves as a receptor for EBOV.

Microbiology postdocs and research scientists

Kristopher Heilmann and Jeffrey Kavanaugh   Disruption of Staphylococcus aureus biofilms through digestion of fibronectin binding protein A by cysteine protease, ScpA     Kristopher P. Heilmann, Jeffrey S. Kavanaugh and Alexander R. Horswill

The opportunistic pathogen Staphylococcus aureus causes simple skin infections and life-threatening ailments, such as sepsis, endocarditis, and osteomyelitis. Approximately 70% of injuries to active duty US service members are orthopedic related and have a high risk of complicating, chronic infections due to biofilms in which S. aureus attaches to and grows on medical implant material. Biofilm infections are intractable due to their inherent resistance to antimicrobial therapies and host immune defenses, but in vitro studies have shown S. aureus biofilms can be dispersed by proteases that are produced by S. aureus. We found that S. aureus biofilms are particularly sensitive to the cysteine protease Staphopain A (ScpA), and proteomic studies identified fibronectin binding proteins (FnBPAB) as potential ScpA targets. Biofilm assays confirmed reduced biofilm forming capacity for a FnBPA knockout mutant, that could be restored by complementation with FnBPA expressed from a plasmid. To further define the interaction between FnBPA and ScpA, a S. aureus strain was engineered to secrete a soluble, his-tagged, full-length version of FnBPA, and the FnBPA was purified and subjected to digestion with purified ScpA. SDS-PAGE analysis indicates multiple cleavage events, so N-terminal protein sequencing of the digestion products is underway in order to characterize the cleavage sites. Once the cleavage sites have been identified, the ability of truncated FnBPA’s to restore biofilm forming capacity to the FnBPA knockout mutant will be used to gauge which cleavage sites are critical for disruption of S. aureus biofilms.

Kyp Oxley   B lymphocyte antigen-presenting cell (APC) vaccines are effective in a mouse melanoma model.    Kyp L. Oxley1,2,  Brett M. Hanson1,  Laura L. Stunz1, Ashley N. Zani1 and Gail A. Bishop1,2.    Depts. of Microbiology & Internal Medicine, University of Iowa1 and VA Medical Center, Iowa City, IA2. 

Cellular vaccines employ APCs, typically dendritic cells (DCs), to deliver tumor antigens to patients that result in anti-tumor T cell responses. However, DCs are difficult to isolate and differentiate in vitro. B cell vaccines (Bvac) have advantages in addressing these challenges. Easily isolated in large numbers from blood, we demonstrated that in vitro-activated B cells are effective APCs, inducing strong antigen-specific CD8 T cell responses.

We applied the Bvac system to the B16 mouse melanoma model and demonstrated that Bvac presenting tumor cell lysates provide an effective antigen stimulus, resulting in prolonged survival compared to Bvac pulsed with the melanoma peptide TRP2. This finding demonstrates that known tumor antigenic peptides are not required, enhancing Bvac clinical applicability and versatility.

Additionally, Bvac upregulate extracellular receptors typical of extravasation and homing to secondary lymphoid organs. In migration experiments, Bvac migrated to the chemokines CCR19 and CCR21.  Concurrently, CD4 and CD8 T cells migrated into Bvac conditioned media. These findings indicate that B cell cancer vaccines are worth further exploration as a therapeutic option. 

The following graduate students who work in microbiology labs and presented are listed below:

Tina Arkee | Alicia Wallis, Gail A Bishop “TRAF3-mediated regulation of the T cell receptor complex”
Alan Sariol | Stanley Perlman “Regulatory T cell memory in MHV infection"  
Breanna M Scorza | Mark Wacker, Kelly Messingham, Peter Kim, Aloysius Klingelhutz, Janet Fairley “Differential Activation of Human Keratinocytes by Leishmania spp. Causing Localized or Disseminated Disease"
Stephanie L Silva-Del Toro | Micaela Fosnick, Laura Whitmore, Jenna McCracken, Lee-Ann Allen “Mechanisms of Neutrophil Nuclear Hypersegmentation”
Matthew D Strub | Paul B McCray “A CONNECTIVITY MAPPING APPROACH REVEALS SEVERAL NEW LEAD COMPOUNDS FOR CYSTIC FIBROSIS THERAPEUTICS”
Alicia Wallis | Zuoan Yi, Jon Houtman, Gail Bishop “TRAF3 regulates Csk and PTPN22 to enhance T cell receptor (TCR) signaling.”
Amy Whillock | Gail Bishop “The role of TRAF3 in BCR signaling”