Characterization of the Klebsiella pneumoniae Secretome Using Size-Exclusion Chromatography and Raman Spectroscopy

The secretome of ESKAPE pathogens, including Klebsiella pneumoniae, comprises a diverse array of bioactive molecules that govern virulence, antibiotic resistance, and the establishment of an immunosuppressive microenvironment. However, the high chemical complexity of the secretome impedes the identification of key metabolites mediating pathogenesis. In this study, we profiled the metabolite composition of cell-free K. pneumoniae supernatant using a combined approach of chromatographic fractionation and Raman spectroscopy. Chromatographic separation enabled the resolution of the complex secretome and revealed fractions with distinct biochemical signatures. A key finding was the identification of Fraction 3, characterized by a unique metabolic profile: it was enriched in nucleic acid fragments, peptides containing tyrosine and methionine, polysaccharides, and stress-response metabolites (e.g., citrate), while notably lacking markers of tryptophan and sterol-like lipids. These spectral signatures suggest a potential role for Fraction 3 metabolites in intercellular communication, biofilm formation, and protection against oxidative stress. The remaining fractions also exhibited distinct biochemical profiles, defined by unique profiles of lipids, nucleotides, and amino acids. Collectively, these data underscore the critical role of specific K. pneumoniae secreted metabolites to pathogen survival and host immune modulation. The combined approach effectively resolves functionally relevant secretome fractions, offering new avenues for identifying diagnostic and therapeutic targets for multidrug-resistant infections.