5-Oxoproline (5OP) is a poorly researched ubiquitous natural amino acid found in all life forms. We have previously shown that Salmonella enterica serovar Typhimurium (Salmonella) responds to 5OP exposure by reducing cyclic-di-GMP levels, and resultant cellulose dependent cellular aggregation in a YfeA and BcsA dependent manner. To understand if 5OP was specifically sensed by Salmonella we compared the interaction of Salmonella with 5OP to that of the chemically similar and biologically relevant molecule, l-proline. We show that l-proline but not 5OP can be utilized by Salmonella as a nutrient source. We also show that 5OP but not l-proline regulates cellulose dependent cellular aggregation. These results imply that 5OP is utilized by Salmonella as a specific signal. However, l-proline is a 5OP aggregation inhibitor implying that while it cannot activate the aggregation pathway by itself, it can inhibit 5OP dependent activation. We then show that in a l-proline transporter knockout mutant l-proline competition remain unaffected, implying sensing of 5OP is extracellular. Last, we identify a transcriptional effect of 5OP exposure, upregulation of the mgtCBR operon, known to be activated during host invasion. While mgtCBR is known to be regulated by both low pH and l-proline starvation, we show that 5OP regulation of mgtCBR is indirect through changes in pH and is not dependent on the 5OP chemical structure similarity to l-proline. We also show this response to be PhoPQ dependent. We further show that the aggregation response is independent of pH modulation, PhoPQ and MgtC and that the mgtCBR transcriptional response is independent of YfeA and BcsA. Thus, the two responses are mediated through two independent signaling pathways. To conclude, we show Salmonella responds to 5OP specifically to regulate aggregation and not specifically to regulate gene expression. When and where in the Salmonella life cycle does 5OP sensing takes place remains an open question. Furthermore, because 5OP inhibits c-di-GMP through the activation of an external sensor, and does not require an internalization step like many studied biofilm inhibitors, 5OP or derivatives might be developed into useful biofilm inhibitors.
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