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The bacterium harbors the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, which provide adaptive immunity against invading foreign nucleic acids. Recent studies suggest that certain CRISPR-Cas systems can regulate endogenous genes, influencing bacterial virulence. However, their role in regulating pathogenicity in\n                      <jats:italic toggle=\"yes\">K. pneumoniae<\/jats:italic>\n                      remains poorly understood. This study investigates the regulatory role of the type I-E* CRISPR-Cas system in a hypervirulent\n                      <jats:italic toggle=\"yes\">K. pneumoniae<\/jats:italic>\n                      strain, focusing on its impact on histidine metabolism and pathogenicity. Transcriptome analyses identified differentially expressed genes (DEGs) between the\n                      <jats:italic toggle=\"yes\">casABECD<\/jats:italic>\n                      -deletion and wild-type strains, including significant upregulation of the histidine utilization (Hut) operon and downregulation of biofilm-related genes. These molecular changes resulted in enhanced histidine metabolic activity, reduced biofilm formation, attenuated virulence in A549 lung epithelial cells, and improved survival of\n                      <jats:italic toggle=\"yes\">Galleria mellonella<\/jats:italic>\n                      , as validated through phenotypic and virulence assays. Our bioinformatic analysis indicated that the CRISPR-Cas system in\n                      <jats:italic toggle=\"yes\">K. pneumoniae<\/jats:italic>\n                      targets the\n                      <jats:italic toggle=\"yes\">hutT<\/jats:italic>\n                      sequence, which is part of the Hut operon. Furthermore, the overexpression of\n                      <jats:italic toggle=\"yes\">hutT<\/jats:italic>\n                      mitigated CRISPR-Cas-mediated repression of the Hut operon, as observed in virulence assays, while simultaneous deletion of\n                      <jats:italic toggle=\"yes\">hutH<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">casABECD<\/jats:italic>\n                      restored the reduced virulence in the \u0394\n                      <jats:italic toggle=\"yes\">casABECD<\/jats:italic>\n                      strain. Additionally, deletion of\n                      <jats:italic toggle=\"yes\">casABECD<\/jats:italic>\n                      significantly enhances the growth of the strain in medium with histidine as the sole carbon source, highlighting the intricate regulatory role of the CRISPR-Cas system in metabolic adaptation. Collectively, these findings uncover a novel role for the CRISPR-Cas system in regulating metabolic pathways and virulence in hypervirulent\n                      <jats:italic toggle=\"yes\">K. pneumoniae<\/jats:italic>\n                      .\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>\n                        <jats:bold>IMPORTANCE<\/jats:bold>\n                      <\/jats:title>\n                      <jats:p>\n                        Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are primarily recognized for their roles in adaptive immunity against foreign genetic elements in bacteria. However, emerging evidence indicates that these systems can also regulate endogenous genes, thereby influencing bacterial physiology and virulence. In this study, we demonstrate that the type I-E* CRISPR-Cas system in\n                        <jats:italic toggle=\"yes\">Klebsiella pneumoniae<\/jats:italic>\n                        targets the\n                        <jats:italic toggle=\"yes\">hutT<\/jats:italic>\n                        gene, a critical component of the histidine utilization (Hut) pathway. This targeting potentially impacts\n                        <jats:italic toggle=\"yes\">hutT<\/jats:italic>\n                        transcription and alters the expression of other\n                        <jats:italic toggle=\"yes\">hut<\/jats:italic>\n                        genes, ultimately enhancing bacterial virulence. Our findings reveal a previously unrecognized regulatory mechanism through which CRISPR-Cas systems facilitate metabolic adaptation and pathogenicity in\n                        <jats:italic toggle=\"yes\">K. pneumoniae<\/jats:italic>\n                        . This study broadens our understanding of the multifaceted roles of CRISPR-Cas systems in bacterial physiology and pathobiology, with implications for clinically relevant pathogens.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/msphere.00215-25","type":"journal-article","created":{"date-parts":[[2025,5,19]],"date-time":"2025-05-19T09:00:39Z","timestamp":1747645239000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":4,"title":["Type I-E* CRISPR-Cas of\n                    <i>Klebsiella pneumoniae<\/i>\n                    upregulates bacterial virulence by targeting endogenous histidine utilization 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