PrsA2 belongs to a family of secretion chaperones found across Gram-positive bacteria. Its day job begins at the moment a protein is pushed out of the bacterial cell. Newly secreted proteins emerge in an unfolded, precarious state, and in the periplasmic space or at the cell surface they face the challenge of folding correctly in an environment very different from the inside of the cell. PrsA2 contains a peptidyl prolyl isomerase domain, an enzyme module that accelerates the slow interconversion of proline residues between structural states, together with a foldase domain that helps client proteins reach their mature, active conformations. Among its most important clients is listeriolysin O, the pore-forming toxin that allows Listeria to escape from the membrane-bound vacuoles it is trapped in after entry into host cells. Without properly folded LLO, the bacterium cannot break free into the nutrient-rich cytoplasm where it replicates, and the entire infection strategy collapses.

What has drawn the attention of the Cahoon laboratory and colleagues is a structural feature of PrsA2 that does not fit the tidy picture of folded domains and catalytic pockets. At the carboxyl terminus of the protein sits a tail of just ten amino acids, dominated by serine residues. This tail belongs to a growing category of protein segments known as intrinsically disordered regions, or IDRs. Unlike conventional domains, IDRs never settle into a single stable three-dimensional arrangement. Instead, they behave as flexible, dynamic chains that can adopt many different conformations, a property that makes them ideal platforms for transient, condition-dependent interactions with other molecules. In chaperone proteins in particular, disordered regions often serve as the contact points where clients are captured and guided toward their folded states.

To test whether this serine-rich tail actually matters, the research team constructed mutant strains of Listeria monocytogenes in which the C-terminal tail was disrupted, and specifically in which the serine residues were altered. The consequences for virulence were dramatic. In a mouse septicemic infection model, a laboratory infection in which bacteria are introduced into the bloodstream, the tail mutants showed a marked loss of the ability to cause disease. A ten-residue flexibly disordered appendage, in other words, is a load-bearing component of one of medicine’s most formidable bacterial pathogens. The finding illustrates a theme that recurs across modern microbiology: proteins cannot be judged solely by their folded domains, because the floppy regions between and at the ends of those domains often carry functions of their own.

Hunting for the mechanistic explanation, the authors traced the virulence defect back to the bacterium’s most famous weapon. Secreted listeriolysin O activity was reduced in the tail mutant strains, meaning that the toxin leaving the cell was less effective at doing its job of punching holes in host membranes. Because LLO maturation is the central step that unlocks intracellular replication, this single biochemical shortfall is sufficient to account for much of the observed loss of virulence. The result places the PrsA2 C-tail squarely upstream of the toxin pipeline: it does not encode the toxin, but it is needed for the toxin to arrive at the host membrane in a competent, folded, pore-forming state.

The study then moved from bacterial genetics to molecular biophysics to ask how, at the level of individual molecules, the tail exerts its influence. Through a combination of biochemical and biophysical assays, the researchers established that the C-tail is required for PrsA2 to interact with and properly fold the LLO toxin. This is the signature function of an intrinsically disordered region: the serine-rich tail appears to act as a flexible recognition element, engaging the client protein and enabling the catalytic domains of PrsA2 to carry out their folding work. Deleting or mutating the tail does not destroy the enzyme domains themselves, but it breaks the handoff between recognition and catalysis, and the client protein is left to misfold or remain inactive.

The implications extend beyond toxin biology. The team found that the PrsA2 C-tail is also critical for survival under stress conditions, and, strikingly, for resistance to antibiotics that target the bacterial cell wall. Cell-wall active agents, which include familiar drug classes such as beta-lactams, are mainstays of treatment for serious Gram-positive infections. The new findings suggest that in Listeria the folding machinery that maintains the cell envelope and its associated secreted proteins is not merely a housekeeping system but a determinant of how well the bacterium tolerates these drugs. A chaperone feature that supports envelope integrity can therefore influence susceptibility to the very compounds designed to breach that envelope.

This dual role, virulence factor on one hand and stress-and-antibiotic survival factor on the other, makes PrsA2 a compelling subject for both basic and translational interest. Chaperones like PrsA2 have long been appreciated as potential antimicrobial targets because they sit at a bottleneck through which many secreted virulence proteins must pass. Inhibiting such a protein could in principle disarm an entire arsenal of toxins and enzymes at once, rather than targeting one toxin at a time. The discovery that a small disordered tail controls much of this functionality sharpens the picture of what such an inhibitor would need to disrupt, and it raises the possibility that the tail itself, or the interactions it mediates, could be exploited for new therapeutic strategies.

There is also a broader evolutionary dimension. PrsA homologs are well conserved across Gram-positive bacteria, a group that includes not only Listeria but also Staphylococcus aureus, Streptococcus species, and Bacillus anthracis, organisms responsible for some of the most consequential infectious diseases in human history. By characterizing the Listeria PrsA2 C-tail in detail, the study provides a template for asking whether serine-rich or otherwise disordered termini perform analogous roles in these related organisms. If the pattern holds, it would suggest a shared architectural principle in Gram-positive secretion biology, and a common vulnerability that could be targeted across a wide range of pathogens.

For the field of molecular microbiology, the work adds to a mounting body of evidence that intrinsically disordered regions deserve the same rigorous attention as structured domains. The ten serine-rich residues at the end of PrsA2 would be easy to overlook in a structural model, invisible to most crystallographic approaches and unremarkable in sequence. Yet removing them cripples a toxin, weakens a pathogen in a living host, and undermines resistance to frontline antibiotics. As Kumar, Agbavor, Alonzo, and Cahoon demonstrate, the flexible margins of bacterial proteins are not decorative. They are functional terrain, and understanding them may prove essential both to explaining how Listeria monocytogenes succeeds as a pathogen and to devising new ways to stop it.

Subject of Research: The role of the serine-rich C-terminal tail of the secretion chaperone PrsA2 in Listeria monocytogenes virulence, toxin folding, and antibiotic resistance

Article Title: The serine-rich C-terminal tail of the Listeria monocytogenes secretion chaperone PrsA2 is critical for bacterial virulence and resistance to cell-wall active antibiotics

Article References: Kumar, A. H., Agbavor, C., Alonzo 3rd, F., & Cahoon, L. A. (2026). The serine-rich C-terminal tail of the Listeria monocytogenes secretion chaperone PrsA2 is critical for bacterial virulence and resistance to cell-wall active antibiotics. PLOS Pathogens, 22(10), e1014676. https://doi.org/10.1371/journal.ppat.1014676

Image Credits: AI Generated

DOI: 10.1371/journal.ppat.1014676

Keywords: Listeria monocytogenes, PrsA2, chaperone, intrinsically disordered region, listeriolysin O, virulence, PPIase, antibiotic resistance, secretion, Gram-positive bacteria, PLOS Pathogens, cell-wall antibiotics