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Microfluidic chip-enabled proteome profiling of bacterial infection of macrophages reveals new pathogenic regulators of immune evasion.

ABSTRACT: Microfluidic chip enabled cell sorting of non-phagocytosed K. pneumoniae cells from BALB/c macrophages during co-culture. These cell populations were analyzed to determine differences in phagocytosed K. pneumoniae versus non-phagocytosed at the protein level. This resulted in the identification of several iron-associated proteins associated with K. pneumoniae that evaded phagocytosis. Evidence in the literature indicated that secreted products from macrophages (i.e., cytokines) significantly increases expression of iron acquisition genes in bacteria, prompting our investigation (and the contents of our submission) into the changes in K. pneumoniae at the proteome-level when exposed to secretion products of macrophages exposed to pathogens (i.e., K. pneumoniae) versus unexposed, with specific emphasis on iron-associated proteins. It was confirmed that pathogen-exposed macrophage supernatant (i.e., activated macrophages induced higher production of iron-associated proteins in exposed K. pneumoniae compared to non-activated macrophages.

INSTRUMENT(S):

ORGANISM(S): Klebsiella Pneumoniae

TISSUE(S): Cell Suspension Culture, Macrophage

SUBMITTER: Chelsea Reitzel

LAB HEAD: Jennifer Geddes-McAlister

PROVIDER: PXD051800 | Pride | 2026-03-24

REPOSITORIES: Pride

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			Action	DRS
	MD015P-DMEM-so0301-1-Try-V1.raw	Raw
	MD015P-DMEM-so0301-2-Try-V1.raw	Raw
	MD015P-DMEM-so0301-3-Try-V1.raw	Raw
	MD015P-DMEM-so0301-4-Try-V1.raw	Raw
	MD015P-MO-so0301-1-Try-V2.raw	Raw

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Project description:The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen, whose virulence requires proliferation inside host cells, including macrophages. Although some Francisella determinants of intracellular growth have been identified, much remains to be understood about the pathogenesis of this organism. In particular, how Francisella responds to its intracellular environment could provide clues about its intracellular biology and reveal pathogenic determinants based on their intracellular expression profiles. Here we have performed a global transcriptional profiling of the highly virulent F. tularensis subsp. tularensis Schu S4 strain during its intracellular cycle within primary murine macrophages. Phagocytosed bacteria rapidly responded to their intracellular environment and progressively altered their transcriptional profile over time. Differential gene expression profiles were revealed that correlated with specific intracellular locations of the bacteria. Upregulation of general and oxidative stress response genes was a hallmark of the early phagosomal and late endosomal stages, while induction of a subset of transport and metabolic genes characterized the cytosolic replication stages. Expression of the Francisella Pathogenicity Island (FPI) genes, the functions of which are associated with intracellular proliferation, increased during the intracellular cycle. Similarly, 27 chromosomal loci putatively encoding hypothetical, secreted, outer membrane proteins or transcriptional regulators were identified as upregulated during the intracellular cycle. In-frame deletion of FTT0383, the Schu S4 ortholog of fevR, of FTT0369c or FTT1676 abolished the ability of Schu S4 to either survive or proliferate intracellularly, demonstrating that bacterial factors of intracellular pathogenesis can be identified based on their intracellular expression profile. In conclusion, establishing the intracellular transcriptome of Francisella has revealed important aspects of its intracellular biology and identified novel virulence determinants of this pathogen. Keywords: Time series Intracellular cycle within primary murine macrophages using the time series of zero, one, two, four, eight, twelve, sixteen and twenty-four hours

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Microfluidic chip-enabled proteome profiling of bacterial infection of macrophages reveals new pathogenic regulators of immune evasion.

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