frankel, g. | bioRxiv Science

bioRxiv2026

PgaR is a positive regulator of the pgaABCD biosynthetic operon in Klebsiella pneumoniae

The biosynthetic locus encoding the exopolysaccharide poly-N-acetyl-glucosamine (PNAG) is widely conserved across bacteria, including the WHO critical-priority pathogen Klebsiella pneumoniae (Kp). In Kp, PNAG synthesis is mediated by the pgaABCD operon, yet its lineage-specific regulation remains incompletely defined. Using a comparative genomics approach to interrogate the pgaABCD locus across the high-risk clonal Kp complex 258 (CC258) lineage, we identified a previously uncharacterised positive transcriptional regulator located immediately upstream of pgaA, which we designate pgaR. Phylogenetic analysis revealed recurrent evolutionary events affecting this regulatory region, including repeated deletion or truncation of pgaR and a G>A substitution upstream of the pgaR start codon. Functional characterisation demonstrated that loss of pgaR abolishes pgaABCD expression and PNAG production, whereas the upstream G>A substitution drives PNAG hyper-production. In vitro, Kp produce extensive extracellular PNAG networks under static growth conditions, consistent with a role in biofilm architecture. Despite this, PNAG expression was dispensable in murine pneumonia and peritonitis models, while PNAG hyper-production significantly attenuated virulence and disease severity, indicating a fitness cost associated with sustained overexpression. Collectively, we discovered PgaR as a novel gene regulator of the pgaABCD operon. We show a previously unrecognised lineage-specific layer of PNAG regulation in Kp and demonstrate that opposing PNAG phenotypes: loss and hyper-production, have independently and repeatedly emerged among clinical CC258 isolates, highlighting dynamic selection acting on biofilm-associated traits in this high-risk pathogen. ImportanceThe exopolysaccharide poly-N-acetyl-glucosamine (PNAG) is widely conserved in bacteria, including the WHO critical-priority pathogen Klebsiella pneumoniae. However, how PNAG production is regulated in high-risk lineages has remained unclear. Here, we identify PgaR as a previously unrecognised positive regulator of the pgaABCD operon in clonal complex 258, a globally disseminated and drug-resistant lineage. We show that natural genetic variation within this regulatory region leads to strikingly different PNAG phenotypes: complete loss of production or hyper-production. While PNAG contributes to extracellular matrix formation in vitro, it is dispensable for virulence in murine infection models, and sustained overproduction imposes a fitness cost. The repeated and independent emergence of both loss- and gain-of-function variants among clinical isolates reveals dynamic evolutionary pressures acting on biofilm-associated traits. These findings uncover a lineage-specific layer of PNAG regulation and highlight how modulation of surface polysaccharide expression shapes pathogen fitness and adaptation.

microbiology↗

bioRxiv2024

Cryo-EM structure of the conjugation H-pilus reveals the cyclic nature of the TrhA pilin

Conjugation, the major driver of the spread of antimicrobial resistance genes, relies on a conjugation pilus for DNA transfer. Conjugative pili, such as the F-pilus, are dynamic tubular structures, composed of a polymerized pilin, that mediate the initial donor-recipient interactions, a process known as mating pair formation (MPF). IncH are low-copy-number plasmids, traditionally considered broad host range, which are found in bacteria infecting both humans and animals. The reference IncHI1 plasmid R27, isolated from Salmonella enterica serovar Typhi, encodes the conjugative H-pilus subunit TrhA containing 74 residues after cleavage of the signal sequence. Here, we show that the H-pilus forms long filamentous structures that mediate MPF, and describe its cryo electron-microscopic (cryo-EM) structure at 2.2 [A] resolution. Like the F pilus, the H-pilin subunits form helical assemblies with phospholipid molecules at a stochiometric ratio of 1:1. While there were previous reports that the T-pilus from Agrobacterium tumefaciens was composed of cyclic subunits, three recent cryo-EM structures of the T-pilus found no such cyclization. Here, we report that the H-pilin is cyclic, with a covalent bond connecting the peptide backbone between the N- and C-termini. Both the cryo-EM map and mass spectrometry revealed cleavage of the last five residues of the pilin, followed by cyclization via condensation of the amine and carboxylate residues. The cyclic nature of the pilin could stabilize the pilus and may explain the high incidence of IncH plasmid dissemination. SignificanceA major medical challenge is the spread of bacteria which are resistant to antibiotics. The resistance genes are spread via mobilized DNA, mainly via a process named conjugation. During conjugation, a resistant bacterium (donor), transfers the resistance DNA to another bacterium (recipient) in a contact-dependent manner. The initial donor-recipient interaction is mediated by a hollow filament expressed by the donor, named the conjugation pilus, that binds the recipient. This pilus is built via polymerization of a small protein subunit, pilin. Here, we report the atomic structure of the H-pilus, whose pilin subunit has an unusual cyclic structure where the N- and C-termini of the protein are covalently linked by a peptide bond.

microbiology↗