Iron Limitation in Klebsiella pneumoniae Defines New Roles for Lon Protease in Homeostasis and Degradation by Quantitative Proteomics

Abstract

Nutrient adaptation is key in limiting environments for the promotion of microbial growth and survival. In microbial systems, iron is an essential component for many cellular processes, and bioavailability varies greatly among different conditions. In the bacterium, Klebsiella pneumoniae, the impact of iron limitation is known to alter transcriptional expression of iron-acquisition pathways and influence secretion of ironbinding siderophores, however, a comprehensive view of iron limitation at the protein level remains to be defined. Here, we apply a mass-spectrometry-based quantitative proteomics strategy to profile the global impact of iron limitation on the cellular proteome and extracellular environment (secretome) of K. pneumoniae. Our data define the impact of iron on proteins involved in transcriptional regulation and emphasize the modulation of a vast array of proteins associated with iron acquisition, transport, and binding. We also identify proteins in the extracellular environment associated with conventional and non-conventional modes of secretion, as well as vesicle release. In particular, we demonstrate a new role for Lon protease in promoting iron homeostasis outside of the cell. Characterization of a Lon protease mutant in K. pneumoniae validates roles in bacterial growth, cell division, and virulence, and uncovers novel degradation candidates of Lon protease associated with improved iron utilization strategies in the absence of the enzyme. Overall, we provide evidence of unique connections between Lon and iron in a bacterial system and suggest a new role for Lon protease in the extracellular environment during nutrient limitation.

Zinc limitation in Klebsiella pneumoniae profiled by quantitative proteomics influences transcriptional regulation and cation transporter-associated capsule production

“…Each condition included four biological replicates. Samples were grown until cells reached mid-log phase, upon which, total proteome analysis was performed as previously described [ 9 ]. Briefly, cells were collected, washed twice with phosphate-buffered saline (PBS), and resuspended in cold 100 mM Tris-HCl (pH 8.5).…”

“…For secretome analysis, the culture supernatant was filtered through a 0.22 μM syringe filter and then performed as previously described [ 9 ]. For each sample, 500 μl of filtered supernatant was treated with DTT, IAA, followed by digestion using LysC and trypsin.…”

“…Gene deletion mutant was complemented by construction of vector containing the full-length copy of chaB under the control of an arabinose-inducible promoter as previously described [ 9 ]. The gene was cloned to produce a FLAG-tagged ChaB fusion protein upon expression.…”

“…Previous studies investigating the relationship between K. pneumoniae and transition metal availability focused primarily on iron limitation. These studies found an iron-associated impact on bacterial growth, capsule development, and protease secretion [ 7 – 9 ]. Moreover, K. pneumoniae produces siderophores, iron-chelating molecules that can acquire free iron from the environment or sequester it from host proteins, to regulate intracellular iron levels during infection [ 10 – 12 ].…”

Zinc limitation in Klebsiella pneumoniae profiled by quantitative proteomics influences transcriptional regulation and cation transporter-associated capsule production

“…Previous studies investigating the relationship between K. pneumoniae and transition metal availability focused primarily on iron limitation. These studies found an iron-associated impact on bacterial growth, capsule development, and protease secretion [7][8][9] . Moreover, K. pneumoniae produces siderophores, iron-chelating molecules that can acquire free iron from the environment or acquire it from host proteins, to regulate intracellular iron levels during infection [10][11][12] .…”