Zinc Acquisition Mechanisms Differ Between Environmental and Virulent Francisella Species

Francisella tularensis is a Tier 1 Select Agent with a high potential for lethality and no approved vaccine. A better understanding of Francisella nutritional requirements and virulence factors is required for the development of therapeutics. Zinc is an essential nutrient for bacterial growth. Because host cells can restrict pathogen access to zinc as an antimicrobial defense mechanism, intracellular pathogens such as Francisella must sense their environment and acquire zinc in response. While acquisition of zinc has been shown to be required for virulence of numerous intracellular pathogens, zinc uptake has not been characterized in Francisella. RNA sequencing of both Francisella novicida U112 and the virulent Francisella tularensis ssp. tularensis Schu S4 strains grown under zinc limitation identified a limited number of genes predicted to contribute to zinc uptake and homeostasis. In many bacteria, the conserved transcription factor Zur is a key regulator of zinc acquisition. An F. novicida zur homolog was identified and a transposon mutant in this gene was used to identify mechanisms of zinc uptake by RNA sequencing. Only five genes were identified by RNA sequencing, of which three were confirmed by quantitative RT-PCR as regulated by Zur and zinc limitation. One of these genes, FTN_0879, is predicted to encode a protein with similarity to the zupT family of zinc transporters, which are not typically regulated by Zur. Although a putative znuACB operon encoding a high affinity zinc transporter was identified in U112 and Schu S4, expression of this operon was not controlled by Zur or zinc concentration. Disruption of zupT but not znuA in U112 impaired growth under zinc limitation, suggesting that ZupT is the primary mechanism for zinc acquisition in these conditions.

Subsequent investigation in the virulent F. tularensis subsp. tularensis Schu S4 strain identified genetic differences that affect the importance of these genes in zinc uptake. In Schu S4, zupT is a pseudogene and attempts to delete znuA were unsuccessful, suggesting that it is essential in this strain. A reverse TetR repression system was utilized to knockdown expression of znuA in Schu S4, revealing that znuA is required for growth under zinc limitation and contributes to intracellular growth within macrophages. Despite growth defects in the znuA mutant strain, zinc-dependent immune responses were not induced during infection with wildtype Schu S4, and neither zinc sequestration nor supplementation decreased bacterial burden during infection. Overall, this work identifies genes necessary for adapting to zinc limitation and highlights nutritional differences between environmental and virulent Francisella strains.