Acinetobacter baumannii is an important cause of nosocomial infections and has been associated with a wide variety of illnesses. The role that the metal zinc (Zn) plays in the pathogenesis of a number of bacteria has been established and subsequently the ability to control intracellular Zn is critical for full virulence. The precise role intracellular Zn storage plays via L31 ribosomal proteins and their effect on pathogenesis has been less well characterised. The L31 ribosomal proteins are found as two paralogous forms; C+ form/RpmE1 (containing the Zn-binding motif CxxC) and the C- form/RpmE2 (lacking CxxC) which act as a potential Zn storage system.
To study these proteins in A. baumannii ATCC 17978 two individual mutant strains (ΔrpmE1 and ΔrpmE2) were constructed by gene replacement via homologous recombination. The ΔrpmE1 and ΔrpmE2 ATCC 17978 mutants were subjected to growth analysis that showed they had a slower growth rate in Zn-rich media compared to the parent, which was more pronounced in Zn-limited media.
Genetic analysis of the mutants by q-RT PCR revealed increased expression of the Zn-uptake regulator (zur) and a high affinity Zn-uptake system (znuA), however, the relative up regulation was different between the ATCC 17978 mutants. The regulation of these genes is likely influenced by reduced intracellular zinc levels, which were determined via inductively coupled plasma mass spectrometry (ICP-MS) to be 30% lower in the mutants compared to the parental cells. Examination of survival under oxidative stress conditions showed that ΔrpmE1 mutant cells are more sensitive to oxidative killing possibly due to a lack of oxidative stress protection afforded by Zn. Surprisingly, ΔrpmE2 mutant cells are more robust in the oxidative stress test performing significantly better than parent cells.
These results indicate that L31 proteins play a role in maintaining cellular fitness under Zn-limited conditions and are important for Zn homeostasis.