Cleavage of Streptococcus pneumoniae ribosomal protein L27 by the Prp protease

Mukherjee, A.; Nasef, M. O.; Lindstrom, P. M.; Chembilikandy, V.; Orihuela, C. J.; Dokland, T.

doi:10.1101/2025.08.01.668195

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bioRxiv · 10.1101/2025.08.01.668195

Cleavage of Streptococcus pneumoniae ribosomal protein L27 by the Prp protease

Mukherjee, A.·Nasef, M. O.·Lindstrom, P. M.·Chembilikandy, V.·Orihuela, C. J.·Dokland, T.

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Abstract

Streptococcus pneumoniae is one of the most important human respiratory pathogens worldwide. The increase in antibiotic resistance in S. pneumoniae and other pathogens is a significant public health concern. The streptococcal 70S ribosome is a prime target for antibiotics. Ribosomal protein L27 reaches into the peptidyl transferase center with its extended N-terminus and may be involved in the translation process. We have shown that L27 in Firmicutes, including staphylococci and streptococci, has an additional 9-12 amino acid N-terminal extension compared to Gram-negative organisms like Escherichia coli. The extension is cleaved by a protease called Prp that is absent from organisms that lack the extension. In S. aureus, Prp and the N-terminal extension of L27 are essential. Here, we have characterized the cleavage of L27 by Prp in S. pneumoniae. Prp forms dimers that efficiently cleave L27 in vitro. An inactive form of Prp (PrpC34S) binds to L27 without cleaving, whereas L27 with a mutation (F12A) of the cleavage site does not bind Prp. Overexpression of PrpC34S in vivo is detrimental to S. pneumoniae growth. Surprisingly, a S. pneumoniae {Delta}prp strain was viable, apparently due to cleavage of L27 by another, unknown protease. Unlike in S. aureus, a mutant strain lacking the N-terminal extension of L27 was viable, but showed impaired growth. Our study sheds light on a process that could be exploited for novel antibiotics, but emphasizes important differences between streptococci and staphylococci. HIGHLIGHTSO_LIRibosomal protein L27 in S. pneumoniae is N-terminally processed by Prp protease C_LIO_LIA S. pneumoniae {Delta}prp mutant is viable, but exhibits impaired growth C_LIO_LICleavage of L27 is required for viability, but the N-terminal extension is not essential C_LIO_LIIn the absence of Prp, L27 is processed by another protease. C_LIO_LIThere are distinct differences in the role of Prp between S. aureus and S. pneumoniae. C_LI

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Mukherjee, A., Nasef, M. O., Lindstrom, P. M., Chembilikandy, V., Orihuela, C. J., Dokland, T.. 2025-08-01. Cleavage of Streptococcus pneumoniae ribosomal protein L27 by the Prp protease. https://doi.org/10.1101/2025.08.01.668195

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