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To define the genetic determinants of this adaptation, we performed a transposon screen in a lipid-rich, acidic medium that mimics the host environment and supports robust\n                  <jats:italic>M. tuberculosis<\/jats:italic>\n                  growth. This screen identified\n                  <jats:italic>ldtB<\/jats:italic>\n                  , encoding an L,D-transpeptidase, as essential for growth and survival under acid stress. Loss of LdtB decreased 3\u20133 peptidoglycan cross-linking, disrupted cell wall architecture, and impaired intrabacterial pH homeostasis, resulting in increased susceptibility to cell wall\u2013active antibiotics. Notably,\n                  <jats:italic>M. tuberculosis<\/jats:italic>\n                  lacking LdtB displayed heightened sensitivity to meropenem within macrophages, suggesting that targeting this enzyme could potentiate \u03b2-lactam efficacy during infection. These findings establish LdtB as a key mediator linking peptidoglycan homeostasis to acid stress resistance and underscores the importance of\n                  <jats:italic>in vitro<\/jats:italic>\n                  culture models that recapitulate the host microenvironment for uncovering new\n                  <jats:italic>in vivo<\/jats:italic>\n                  active therapeutic targets.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Teaser<\/jats:title>\n                  <jats:p>\n                    Disabling a key peptidoglycan cross-linking enzyme compromises\n                    <jats:italic>M. tuberculosis<\/jats:italic>\n                    survival under acidic stress and antibiotic exposure.\n                  <\/jats:p>\n                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