Transcriptional response of Leptospira interrogans to iron limitation and characterization of a PerR homolog

Abstract

Leptospirosis is a globally significant zoonosis caused by Leptospira spp. Iron is essential for growth of most bacterial species. Since iron availability is low in the host, pathogens have evolved complex iron acquisition mechanisms to survive and establish infection. In many bacteria, expression of iron uptake and storage proteins is regulated by Fur. L. interrogans encodes four predicted Fur homologs; we have constructed a mutation in one of these, la1857. We conducted microarray analysis to identify iron-responsive genes and to study the effects of la1857 mutation on gene expression. Under iron-limiting conditions, 43 genes were upregulated and 49 genes were downregulated in the wild type. Genes encoding proteins with predicted involvement in inorganic ion transport and metabolism (including TonB-dependent proteins and outer membrane transport proteins) were overrepresented in the upregulated list, while 54% of differentially expressed genes had no known function. There were 16 upregulated genes of unknown function which are absent from the saprophyte L. biflexa and which therefore may encode virulence-associated factors. Expression of iron-responsive genes was not significantly affected by mutagenesis of la1857, indicating that LA1857 is not a global regulator of iron homeostasis. Upregulation of heme biosynthetic genes and a putative catalase in the mutant suggested that LA1857 is more similar to PerR, a regulator of the oxidative stress response. Indeed, the la1857 mutant was more resistant to peroxide stress than the wild type. Our results provide insights into the role of iron in leptospiral metabolism and regulation of the oxidative stress response, including genes likely to be important for virulence.

FIG. 1.

Percentages of genes which were up- or downregulated under iron limitation and across the L. interrogans sv Lai genome in each COG category. The COG functional categories are as follows: information storage and processing (11% of coding sequences in L. interrogans serovar Lai genome) (includes J, translation; K, transcription; L, replication, recombination, and repair); cellular processes and signaling (20% of coding sequences in the serovar Lai genome) (includes D, cell cycle control, cell division, and chromosome partitioning; V, defense mechanisms; T, signal transduction mechanisms; M, cell wall, membrane, or envelope biogenesis; N, cell motility; U, intracellular trafficking, secretion, and vesicular transport; O, posttranslational modification, protein turnover, and chaperones); metabolism (19% of coding sequences in the serovar Lai genome) (includes C, energy production and conversion; G, carbohydrate transport and metabolism; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; P, inorganic ion transport and metabolism; Q, secondary metabolite biosynthesis, transport, and catabolism); poorly characterized (50% of coding sequences in the serovar Lai genome) (includes R, general function prediction only; S, function unknown; and −, not in COGs). The asterisk indicates that genes predicted to encode inorganic ion transport and metabolic proteins were overrepresented among genes upregulated by low-iron conditions (Fisher's exact test, P < 10⁻⁵).

FIG. 2.

Phylogenetic analysis of L. interrogans Fur homologs and other Fur family proteins generated by neighborhood joining with 100 bootstrap replicates and rooted at the midpoint. Branch support values are shown as percentages.

FIG. 3.

Alignment of amino acid sequences of LA1857 and B. subtilis PerR showing identical residues (*), conserved residues (:), and semiconserved residues (.). Residues shown to be important for divalent metal binding (Fe²⁺ or Mn²⁺) in B. subtilis PerR are also present in LA1857 (boxed).

FIG. 4.

Minimum bactericidal concentrations of hydrogen peroxide and cumene hydroperoxide for the LA1857 mutant (Mut) and wild type (WT). Three biological replicates were tested in triplicate, yielding identical results. The LA1857 mutant was 8-fold more resistant to killing by hydrogen peroxide, whereas there was no difference in resistance to cumene hydroperoxide.