Evaluation of Anti-Elongation Factor-Tu (EF-Tu) Antibodies as a Potential Immunotherapeutic Approach to Combat Pseudomonas aeruginosa Infections in vivo Open Access

Abstract

Pseudomonas aeruginosais a Gram-negative bacteria that is ubiquitous in the environment. It is the primary causative agent of nosocomial (hospital-acquired) infections, causing mortality in critically ill and immunocompromised patients, those with burns, surgical wounds, and in people with cystic fibrosis (CF). Treatment of P. aeruginosainfections can be extremely challenging due to its intrinsic antibiotic resistance and its remarkable ability to acquire more resistance, leading to the emergence of multidrug-resistant (MDR) P. aeruginosastrains.

Vaccination against P. aeruginosawould help reduce the need for antibiotics and hence overcome many of the problems associated with antibiotic resistance, yet there is no licensed vaccine available for human use. It was recently discovered that one of P. aeruginosa’s essential proteins, elongation factor Tu (EF-Tu), possesses moonlighting (multifunctional) activities. Although primarily known as a GTPase that aids in protein translation, when it is surface-exposed and post-translationally modified by a methyltransferase called EftM, EF-Tu has been shown to mediate adherence between the bacteria and the host cell.

Our lab has previously shown using a mouse model of infection, that when PAO1, a commonly used laboratory strain, lacks eftM, the gene responsible for EftM production, there is significantly less bacterial adherence in both the nasal cavities and lungs, indicating that EF-Tu plays a role during the initial stages of pathogenesis. EF-Tu has been identified as an immunogenic antigen in a large number of bacterial pathogens. Antibodies against EF-Tu have been found to be induced in infections several organisms including Burkholderia sppand Staphylococcus aureus. Because of this, EF-Tu has been suggested to be a viable vaccine candidate for a number of different microbes. Here, we explore the possibility of using recombinant EF-Tu as a potential vaccine candidate for P. aeruginosa. We will determine whether antibodies to P. aeruginosaEF-Tu can provide protection both actively and passively in murine respiratory models of infection.

Table of Contents

Introduction....................................................................................................................................1

Material and Methods...................................................................................................................4

            Figure 1: Process for electroporating P. aeruginosaand expressing EF-Tu ......................6

            Figure 2: EF-Tu purification steps.......................................................................................7

Results and Discussion.................................................................................................................11

            Figure 3: SDS-PAGE samples of purified fractions stained with SimplyBlue SafeStain.12

            Figure 4: Western immunoblot of reaction of consecutive antisera with purified modified 

                        or modified protein................................................................................................13

            Figure 5: Serum antibody response in BALB/c mice following subcutaneous 

                        immunization with P. aeruginosa rEF-Tu.............................................................14

            Figure 6: Bacterial load and survival rate of BALB/c mice immunized with rEF-Tu after 

                        intranasal challenge with P. aeruginosa PA103....................................................16

            Figure 7: Comparison of anti-rEF-Tu serum IgG and IgA from mice immunized with

                        unmodified or modified rEF-Tu before and after rEF-Tu + CpG vaccination......18

            Figure 8: Nasal wash and lung colonization following passive immunization.................20

                                                                                                                                                

References.....................................................................................................................................22

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Microbiology
Keyword	EF-Tu microbiology vaccine
Committee Chair / Thesis Advisor	Joanna B. Goldberg, Emory University
Committee Members	Jens Wrammert, Emory University Levi Morran, Emory University

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