Effect of pyomelanin pigment production by Burkholderia cenocepacia on virulence and resistance to reactive nitrogen and oxygen species in vitro and in vivo. Público
Wu, Linda (Spring 2020)
Published
Abstract
Burkholderia cenocepacia is a Gram-negative bacilli that is known to infect patients with immunocompromising diseases, particularly those with cystic fibrosis (CF) and chronic granulomatous disease (CGD). Some strains of B. cenocepacia are known to naturally produce a pigment known as pyomelanin. Previous research on B. cenocepacia C5424 strain has shown a decrease in the resistance to oxidative stress when the pyomelanin pigment was absent. This project aims to investigate the role of pyomelanin presence on the resistance and virulence of the naturally pigmented strain J2315 and naturally non-pigmented strain K56-2.
To investigate the impact of pigmentation on the resistance of B. cenocepacia strains to oxidative stress, a non-pigmented isogenic strain was generated from the naturally pigmented strain J2315 utilizing an allelic exchange of a previously known gene in the pyomelanin production pathway. In the same manner, a pigmented isogenic strain was generated from the naturally non-pigmented strain K56-2. These isogenic mutant strains were developed prior to the start of this project by a former lab member of the Goldberg lab. These strains were subjected to oxidative stress with H2O2 and NO oxidants at various concentrations in vitro. The results reflected an inherent difference in the resistance of J2315 and K56-2 to oxidative stress. However, at particular concentrations of reactive oxygen and nitrogen species, the presence of the pigment demonstrated a slight protective advantage for the naturally non-pigmented K56-2 strain.
To determine the impact of pyomelanin on the virulence of B. cenocepacia strains in vivo, we performed an intratracheal infection in CGD mice models and observed the colonization levels and survival of the mice post-infection. We detected no significant differences between the colonization levels of the wild-type strains and their isogenic mutants, indicating that within the CGD mice model, the presence or absence of the pigment does not impact the virulence of the strain.
Table of Contents
Introduction…………………………………………………………………………1
Figure 1: Pyomelanin synthesis pathway ……………………………………3
Materials and Methods……………………………………………………………5
Table 1: Burkholderia cenocepacia strains used in this project.………5
Results and Discussion………………………………………………...…………7
Figure 2: Phenotypes of wild-type (J2315 and K56-2) and isogenic mutant strains (J2315::KhmgA and K56-2::JhmgA).…8
Figure 3: Growth curves of J2315::KhmgA, J2315, K56-2::JhmgA, and K56-2 treated with various concentrations of H2O2 over an 18 hours incubation period…10
Figure 4: Growth curves of J2315::KhmgA, J2315, K56-2::JhmgA, and K56-2 treated with various concentrations of SNP over a 20 hours growth period….………12
Figure 5: Survival of wild-type strains J2315, J2315::KhmgA, K56-2, K56-2::JhmgA and treated with 50 mM and100 mM H2O2 monitored over 1 hr.………………14
Figure 6: Survival of wild-type strains J2315, J2315::KhmgA, K56-2, K56-2::JhmgA and treated with 100 mM SNP monitored over 2 hrs……………………….………15
Figure 7: Bacterial load in lung homogenates of CGD mice infected intratracheally with WT J2315, and K56-2 and isogenic mutants J2315::KhmgA, and K56-2::JhmgA..17
Figure 8: Survival rates of CGD mice after intratracheal infection with wild-type J2315, K56-2, and isogenic mutants J2315::KhmgA, and K56-2::JhmgA ………………....18
Future Directions…………………………………………………………………………………….……21
References…………………………………………………………………………………………………21
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Effect of pyomelanin pigment production by Burkholderia cenocepacia on virulence and resistance to reactive nitrogen and oxygen species in vitro and in vivo. () | 2019-12-09 10:41:16 -0500 |
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