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Phosphoproteome modulation by zinc and iron in Klebsiella pneumoniae reveals new virulence mechanisms and antibacterial targets

ABSTRACT: Klebsiella pneumoniae is a Gram-negative bacterium causing 10-20% mortality in immunocompromised individuals and is known to rapidly develop resistance towards existing antibiotics. This underscores a need for novel antimicrobials or strategies for resensitization. Bacterial adaptation to iron and zinc, which are key nutrients linked to growth, division, and virulence, offers promising targets for antibiotic intervention. In this thesis, mass spectrometry-based phosphoproteomics was used to profile K. pneumoniae in iron/zinc-replete and -limited media, identifying 574 phosphopeptides from 358 phosphoproteins. Two candidates, SbmA and TolQ, were selected for disruption based on hypothesized roles in iron acquisition and membrane stabilization, respectively. Through iron quantification assays, growth curves, electron microscopy, antibiotic susceptibility testing and infection assays, we identified a previously uncharacterized role for SbmA in iron acquisition and identified reduced growth, viability, increased β-lactam susceptibility, and enhanced immune clearance for ΔtolQ. High-throughput drug screening revealed two compounds that target SbmA or TolQ, reducing bacterial growth and enhance immune clearance. Together, these findings highlight phosphoproteomics as a powerful tool for identifying nutrient-regulated targets and position TolQ and SbmA as promising candidates for antimicrobial development.

INSTRUMENT(S):

ORGANISM(S): Klebsiella Pneumoniae Subsp. Pneumoniae

SUBMITTER: Chelsea Reitzel

LAB HEAD: Jennifer Geddes-McAlister

PROVIDER: PXD072239 | Pride | 2026-04-15

REPOSITORIES: Pride

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			Action	DRS
	MD015L-phospho-so0611-Fe1-Try-V1.raw	Raw
	MD015L-phospho-so0611-Fe2-Try-V1.raw	Raw
	MD015L-phospho-so0611-Fe3-Try-V1.raw	Raw
	MD015L-phospho-so0611-Fe4-Try-V1.raw	Raw
	MD015L-phospho-so0611-LM1-Try-V1.raw	Raw

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Project description:Iron is an essential cofactor for a wide range of cellular processes. Previous studies have shown that siderophore-mediated uptake and intracellular handling of iron are crucial for virulence of Aspergillus fumigatus. Here we show that the bzip-type transcription factor HapX plays a crucial role in the transcriptional remodeling required for adaption to iron starvation in this opportunistic fungal pathogen. HapX was found to be interconnected in a negative feed-back loop with the previously identified iron regulator SreA: SreA repressed expression of hapX during iron sufficiency and HapX repressed sreA during iron starvation. Genome-wide transcriptional profiling and analysis of selected metabolites (protophorphyrine IX, siderophores and amino acids) indicated extensive metabolic remodeling in response to iron starvation. HapX was found to participate in both, repression and activation of genes during iron starvation. HapX was in particular required for repression of iron-dependent and mitochondrial-localized activities including respiration, TCA cycle, amino acid metabolism, iron-sulfur-cluster biosynthesis and heme biosynthesis. Pathways positively affected by HapX included production of siderophores and the ribotoxin and major allergen AspF1. Analysis of the free amino acid pool revealed HapX-dependent coordination of the production of siderophores with the supply of its precursor ornithine. Consistent with the hapX expression pattern, HapX-deficiency was deleterious with respect to growth rate and conidiation during iron depleted but not iron-replete conditions. HapX-deficiency caused significant attenuation of virulence in a murine model aspergillosis underlining that A. fumigatus faces iron starvation in the host and that the HapX-dependent metabolic reprogramming is therefore crucial for virulence. A. fumigatus 293 and hapX mutants were grown in the presence and absence of iron and in cultures shifted from no iron to iron-containing conditions after 1 h incubation. Hybridizations were performed with biological replicates for wt vs hapX +/- iron. For the iron-shift experiments, there were biological replicates for wt in both conditions and for hapX in -iron but there was only a single biological sample for hapX iron-shift sample. All hybs were performed with flip-dye pairs.

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Phosphoproteome modulation by zinc and iron in Klebsiella pneumoniae reveals new virulence mechanisms and antibacterial targets

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