Author: Betty Suryawati

Suryawati, Betty, 2017 THE ROLE OF L31 PROTEINS IN ACINETOBACTER BAUMANNII ZINC HOMEOSTASIS, Flinders University, School of Biological Sciences

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Acinetobacter baumannii is an important nosocomial pathogen associated with a wide variety of illnesses. Its role as a significant human pathogen has been well established. The ability to cause infections is not only multifactorial but also requires a number of cofactors; one of these factors is the metal ion zinc (Zn2+). The role that Zn2+ plays in the pathogenesis of a number of bacteria has been well established and the ability to obtain and control the intracellular concentration of Zn2+ is critical for full bacterial virulence. In some bacteria the L31 proteins play a role in this Zn2+ homeostasis. Two paralagous genes encode two different L31 ribosomal proteins, RpmE1 and RpmE2, where one protein contains the Zn2+-binding motif (RpmE1, C+) and the other does not (RpmE2, C- form). The ability of the bacteria to alternate between these two L31 forms effectively increases the availability of Zn2+ during growth in Zn2+-limited media, allowing for Zn2+ to be available for use in metabolically critical cell functions. In A. baumannii, little is known about the role of Zn2+ and if it can assist in colonisation of host niches. Additionally, how RpmE1 and RpmE2 function has not been elucidated. This projects aims to examine the effect of Zn2+ limitation on A. baumannii and investigate the precise role the L31 ribosomal proteins play in A. baumannii intracellular Zn2+ and in virulence.

The effect of Zn2+ deficiency on A. baumannii phenotypes and global gene expression responses were examined. The results revealed that Zn2+ limitation affected bacterial growth, biofilm formation, and susceptibility to antibiotics. The RNA-seq transcriptomic analysis revealed that A. baumannii showed a strong transcriptional response to Zn2+ limitation, predominantly via increased expression of Zn2+-acquisition mechanisms and other ion transporters and receptors. Genes previously known to contain a Zur-binding site were up-regulated, however many genes which do not contain this motif were also affected indicating that these are Zn2+ dependant, including genes involved in bacterial virulence. Moreover, many genes were also differentially expressed under these conditions involving diverse processes other than Zn2+ acquisition, such as carbohydrate, lipid, secondary metabolites biosynthesis, transport, and catabolism, and genes involve in energy production and conversion.

To investigate the role of L31 proteins in A. baumannii ATCC 17978 two individual deletion mutants (ΔrpmE1 and ΔrpmE2) were constructed. The effects of the inactivation of these genes were examined at a phenotypic and global transcriptional level. The ΔrpmE1 mutant showed changed in multiple bacterial phenotypes, including a slight growth defect in Zn2+-limited medium, a reduction in motility, an impairment in biofilm formation and an increased sensitivity to oxidative stress. The ΔrpmE2 mutant also showed similar changes in phenotypes as except that the motility was not significantly affected. In addition, when grown in Zn2+-limited medium, the the levels of intracellular Zn2+ in both mutant strains were 25% lower compared to the parent.

The unique effect of the deletion of rpmE1 when cells were grown in Zn2+-replete conditions was the reduced expression of the Zur global transcription repressor involved in Zn2+ homeostasis. Zur negatively regulates genes that have a Zur-binding site which include rpmE2, A1S_0146, A1S_0452, A1S_0453, A1S_2829, A1S_0391, A1S_3411, and A1S_3412. In contrast, in ΔrpmE2 most of the genes encoding ribosomal proteins were highly up-regulated. The RNA-seq analysis revealed that there were a large number of shared genes differentially expressed in both mutants. This included genes involved in; a Type VI secretion system (A1S_1292-1310), lipid metabolism (A1S_1699-1705), carbohydrate metabolism (A1S_1880-1894) and the csuABABCDE cluster (A1S_2213-2218). All of these showed decreased expression in both Zn2+-replete and Zn2+-limited conditions. Thus, these alterations show an association between these ribosomal proteins beyond sharing the same function as a part of the ribosomal complex.

In conclusion, the transcriptional changes in response to the deletion of L31 ribosomal proteins showed pleiotropic effects. The data from phenotypic assays, inductively coupled plasma mass spectrometry, and RNA-seq analysis revealed that rpmE1 and rpmE2 play significant roles in A. baumannii ATCC 17978, not only in response to Zn2+ limitation, but also in various bacterial phenotype and virulence features.

Keywords: L31 proteins, RpmE1, RpmE2, Zinc, Acinetobacter baumannii, Homeostasis, RNA-seq, Virulence

Subject: Biological Sciences thesis

Thesis type: Doctor of Philosophy
Completed: 2017
School: School of Biological Sciences
Supervisor: Prof. Melissa Brown