Functional Diversity of Homologous P1B-ATPases in Metal Homeostasis and Host-Microbe Interaction

Patel, Sarju

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Functional Diversity of Homologous P1B-ATPases in Metal Homeostasis and Host-Microbe Interaction

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Copper and iron are trace elements that form an indispensable part of many proteins and are crucial for the well-being of all cells. At the same time, the intracellular levels of these metals require careful regulation, as excess or deficiency may be lethal. P1B-ATPases are key players in metal homeostasis. They belong to the superfamily of P-type ATPases, transmembrane proteins present in virtually all life forms, are responsible for solute translocation across biological membranes. The goal of this thesis is to improve our understanding of the structural and functional roles of P1B-ATPases in metal homeostasis by focusing on the host-microbe interaction. The thesis first describes the importance of Cu+ distribution in the outcome of host-microbe interaction. Copper is an important element in host–microbe interactions, acting both as a catalyst in enzymes and as a potential toxin. Cu+-ATPases drive cytoplasmic Cu+ efflux and protect bacteria against metal overload. Many pathogenic and symbiotic bacteria contain multiple Cu+-ATPase genes within particular genetic environments, suggesting alternative roles for each resulting protein. This hypothesis was tested by characterizing five homologous Cu+-ATPases present in the symbiotic organism Sinorhizobium meliloti. Mutation of each gene led to different phenotypes and abnormal nodule development in the alfalfa host. Distinct responses were detected in free-living S. meliloti mutant strains exposed to metal and redox stresses. Differential gene expression was detected under Cu+, oxygen or nitrosative stress. These observations suggest that CopA1a maintains the cytoplasmic Cu+ quota and its expression is controlled by Cu+ levels. CopA1b is also regulated by Cu+ concentrations and is required during symbiosis for bacteroid maturation. CopA2-like proteins, FixI1 and FixI2, are necessary for the assembly of two different cytochrome c oxidases at different stages of bacterial life. CopA3 is a phylogenetically distinct Cu+-ATPase that does not contribute to Cu+ tolerance. It is regulated by redox stress and required during symbiosis. We postulated a model where non-redundant homologous Cu+-ATPases, operating under distinct regulation, transport Cu+ to different target proteins. In its second part, the thesis describes the novel Fe2+-ATPases and their influence in the host-microbe interaction. Little is known about iron efflux transporters in bacterial systems. Recently, the participation of Bacillus subtilis PfeT, a P1B4-ATPase, in cytoplasmic Fe2+ efflux has been proposed. We report here the distinct roles of mycobacterial P1B4-ATPases in the homeostasis of Co2+ and Fe2+. Mutation of Mycobacterium smegmatis ctpJ affects the homeostasis of both ions. Alternatively, a M. tuberculosis ctpJ mutant is more sensitive to Co2+ than Fe2+, while mutation of the homologous M. tuberculosis ctpD leads to Fe2+ sensitivity but no alterations in Co2+ homeostasis. In vitro, the three enzymes are activated by both Fe2+ and Co2+ and bind one equivalent of either ion at their transport site. However, equilibrium binding affinities and activity kinetics show that M. tuberculosis CtpD has higher affinity for Fe2+ and twice the Fe2+ stimulated activity than the CtpJs. These parameters are accompanied by a lower activation by and affinity for Co2+. Analysis of Fe2+ and Co2+ binding to CtpD by X-ray spectroscopy shows that both ions are coordinated by 5-6 O/N atoms with similar geometry. Mutagenesis studies suggest the involvement of invariant Ser, His and Glu in metal coordination. Interestingly, replacement of Cys in the conserved CPS sequence at the metal binding pocket leads to a large reduction in Fe2+ but not Co2+ binding affinity. We propose that CtpJ ATPases participate in the control of steady state Fe2+ levels. CtpD, required for M. tuberculosis virulence, is a high affinity Fe2+ transporter involved in the rapid response to iron dyshomeostasis generated upon redox stress. These studies provide significant insights into the metal selectivity, regulation, transport kinetics and functional diversity of homologous P1B-ATPases in Cu+ and Fe2+ homeostasis. Moreover, these biochemical characterizations can be integrated with the structural-functional analysis to elucidate the complex metal distribution networks.

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