Caenorhabditis elegans as a model for Candida albicans-Pseudomonas aeruginosa co-infection and infection induced prostaglandin production
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Date
2018-05
Authors
Mokoena, Nthabiseng Zelda
Journal Title
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Publisher
University of the Free State
Abstract
The discovery of substantial commonality between microbial pathogenesis in mammals and
invertebrate model hosts, such as the nematode Caenorhabditis elegans, has provided the
foundation for genetic analysis of microbial virulence factors in live animal models. In most
cases, Candida albicans yeast cells inhabit the human intestines, yet this opportunistic
pathogen can led to host tissues invasion, causing life-threatening infections in
immunocompromised hosts. Given the importance of this fungus to human health and its coexistence
with other pathogenic microbes, particularly bacteria, such as emerging Gramnegative
Pseudomonas aeruginosa, thus we used C. elegans as an infection model to study
interactions between C. albicans and P. aeruginosa. Our goal was to firstly, successfully
propagate and monitor the life cycle of C. elegans at 15 °C. Secondly, for this reason, we
established a liquid medium assay using C. elegans model for C. albicans or P. aeruginosa
monomicrobial infections. We demonstrate that the C. albicans yeast form establishes an
intestinal infection in C. elegans, while the hyphal form is not required to efficiently kill the
nematode. Furthermore, investigating mutants and genetically engineered C. albicans strains,
we proved that hyphal formation is indeed not required for full virulence in this system. Thirdly
we demonstrated that polymicrobial interactions are more virulent to C. elegans than
monomicrobial species. We also aimed to understand the genetic mechanisms of virulence
observed in C. elegans in vitro, since it was shown that not only does C. albicans and P.
aeruginosa kill the nematode C. elegans, but also that C. albicans and P. aeruginosa virulence
factors required for mammalian pathogenesis might also be required for efficient killing of C.
elegans. Here our in vitro results suggested that there are multiple virulence factors of P.
aeruginosa that may cause virulence, including pyoverdine, pyocyanin and swarming motility.
Another factor that contributes to virulence is the hydrolytic enzyme production, known to
facilitate pathogenicity of bacteria, protozoa, and pathogenic yeasts. Our results demonstrated
that although C. albicans and P. aeruginosa possess a wide range of hydrolytic enzymes,
proteinases are more predominantly associated with virulence. Furthermore, when comparing
the effect of infection on the microbial burden of specific pathogens, from monomicrobial
infections, it is clear that the virulence observed in killing of nematodes was not due to number
of cells but rather specific virulence factors of the different strains. Surprisingly, from
polymicrobial infections, we see that for both P. aeruginosa strains, co-infection resulted in an
increased microbial burden. This is due to the fact that virulence of co-infection is strongly
influenced by microbial burden and that this is dependent on the specific strains in the coinfection.
For further understanding of the influence of virulence that underlie susceptibility to
this pathogens, we used this pathogen model system to further evaluate the influence of infection towards the nematodes fatty acid composition. Total lipids of C. elegans were
extracted using chloroform and methanol [2:1 ratio (v/v)]. Fatty acids composition of the
extracted total lipids was converted to their corresponding fatty acids methyl esters (FAMEs)
and analysed by gas chromatography (GC). From the nematodes feeding on control E. coli
OP50, we identified twenty-three different fatty acids ranging from 12 to 22 carbons in length,
with 35 % being saturated, while 65 % being unsaturated. We then only focused on major
unsubstituted long chain fatty acids (LCFAs), with margaric acid (17:0) being the predominant
saturated fatty acid, comprising of an average of 24 % total major fatty acids. Through this
process, after C. albicans and P. aeruginosa infection, we identified three fatty acids that have
varying degrees of influence in C. elegans, namely linoleic acid (18:2n6), eicosapentaenoic
acid (20:5n3) and docosenoic acid (22:1n9). Therefore, we observed changes in fatty acid
profile being strain dependent, however there is no clear correlation between production of
fatty acid and virulence. We further extended the usage of C. elegans infection models to
investigate the influence of signalling molecules called prostaglandins, on infection. Here we
show that only the co-infection synthesize prostaglandin E2. Together, these results expand
the use of C. elegans in the field of polymicrobial pathogenesis and provide further evidence
of the likely importance of polymicrobial interactions. Since there is an urgent need for
development of new antimicrobial agents, C. elegans which is known to evaluate different
chemical compounds libraries could be used to solve some of the main obstacles in current
antimicrobial discovery.
Description
Keywords
Caenorhabditis elegans, Infection model, C. albicans, P. aeruginosa, Virulence, Infections, Dissertation (M.Sc. (Microbiology))--University of the Free State, 2018