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Our study reveals a key molecular mechanism that controls the interaction between T4aP and EPS, which is a critical process for bacterial social motility, biofilm formation, predation, and other collective behaviors. We identify a specific aromatic residue (W146) in the major pilin PilA that mediates direct binding to glucosamine-containing polysaccharides, thereby linking T4aP function and extracellular matrix recognition. This finding provides new insight into how bacteria use glycans to coordinate group behaviors within microbial communities. These results open potential strategies for controlling biofilm-related processes, such as disrupting infections or guiding beneficial microbial assemblies in environmental and industrial settings.

The ability of bacteria to sense and respond to contact with surfaces is important for triggering changes in secondary messenger levels and gene expression, leading to the formation of biofilms and increased production of virulence factors. For Pseudomonas aeruginosa , the expression of functional type IVa pili is important for the accumulation of cyclic AMP (cAMP) following surface contact. Deletion of the PilT retraction ATPase paralog PilU leads to loss of pilus-mediated twitching motility but also high intracellular levels of cAMP, a phenotype mimicking that of surface-adapted cells. Here, we isolated twitching suppressors of a pilU deletion mutant that mapped to the pilin subunit PilA or pilus-tip adhesin PilY1 and showed that for most, elevated cAMP levels did not decrease when motility was restored. Twitching was dependent on functional PilT, and complementation with PilU further increased twitching for most mutants. These data show that in permissive contexts, PilU is not required for twitching motility, providing new insights into mechanisms of bacterial surface sensing and evolution of type IVa pilus motor function.

Uropathogenic E. coli (UPEC), which cause urinary tract infections (UTI), utilize type 1 pili, a chaperone usher pathway (CUP) pilus, to cause UTI and colonize the gut. The pilus rod, comprised of repeating FimA subunits, provides a structural scaffold for displaying the tip adhesin, FimH. We solved the 4.2 Å resolution structure of the type 1 pilus rod using cryo-electron microscopy. Residues forming the interactive surfaces that determine the mechanical properties of the rod were maintained by selection based on a global alignment of fimA sequences. We identified mutations that did not alter pilus production in vitro but reduced the force required to unwind the rod. UPEC expressing these mutant pili were significantly attenuated in bladder infection and intestinal colonization in mice. This study elucidates an unappreciated functional role for the molecular spring-like property of type 1 pilus rods in host-pathogen interactions and carries important implications for other pilus-mediated diseases. Escherichia coli, or E. coli for short, is a type of bacteria commonly found in the guts of people and animals. Certain types of E. coli can cause urinary tract infections (UTIs): they travel from the digestive tract up to the bladder (and sometimes to the kidneys) where they provoke painful symptoms. To cause the infection, the bacteria must become solidly attached to the lining of the bladder; otherwise they will get flushed out whenever urine is expelled. Pili are hair-like structures that cover a bacterium and allow it to attach to surfaces. E. coli has many different types of pili, but one seems particularly important in UTIs: type 1 pili. These pili are formed of subunits that assemble into a long coil-shaped rod, which is tipped by adhesive molecules that can stick to body surfaces. The current hypothesis is that the pili act as shock absorbers: when the bladder empties, the pili’s coil-like structure can unwind into a flexible straight fiber. This would take some of the forces off the adhesive molecules that are attached to the bladder, and help the bacteria to remain in place when urine flows out. However, the exact structure of type 1 pili is still unclear, and the essential role of their coil-like shape unconfirmed. Here, Spaulding, Schreiber, Zheng et al. use a microscopy method called cryo-EM to reveal the structure of the type 1 pili at near atomic-level, and identify the key units necessary for their coiling properties. The experiments show that pili with certain mutations in these units unwind much more easily when the bacteria carrying them are ‘tugged on’ with molecular tweezers. The bacteria with mutant pili are also less able to cause UTIs in mice. The coiling ability of the type 1 pili is therefore essential for E. coli to invade and colonize the bladder. Every year, over 150 million people worldwide experience a UTI; for 25% of women, the infection regularly returns. Antibiotics usually treat the problem but bacteria are becoming resistant to these drugs. New treatments could be designed if scientists understand what roles pili play in the infection mechanisms.

Surface sensing allows bacteria to colonize surfaces and form biofilms, with wide-ranging implications for bacterial survival, ecology, and human health. In Caulobacter crescentus , tight adherence (Tad) pili play an important role in surface sensing and attachment, however the molecular mechanisms of pilus-mediated mechanosensing remain unknown. Here, we demonstrate that CpaL, a potential pilus tip mechanosensory protein, could be a major regulatory element controlling Tad pilus-mediated surface attachment and colonization in C. crescentus . Specifically, CpaL plays a regulatory role in holdfast synthesis upon surface contact. By identifying CpaL as a key player in surface recognition, our work offers valuable insights into the mechanisms of bacterial adhesion.

Kingella kingae is an emerging pediatric pathogen and a leading cause of osteoarticular infections in children 6 months to 4 years of age. To establish infection, K. kingae relies on T4P, dynamic surface structures that mediate host cell adherence, motility, and DNA uptake. T4P are expressed by a wide range of bacterial pathogens beyond K. kingae , including Pseudomonas aeruginosa , Neisseria gonorrhoeae , Neisseria meningitidis , and Legionella pneumophila , among others. The type IV pilus is composed of pilin subunits, including a major pilin that displays significant antigenic diversity and low-abundance minor pilins that are highly conserved. This study demonstrates the importance of eight minor pilins in K. kingae virulence properties. Given the conservation of minor pilins across diverse bacterial species, targeting minor pilin complexes may provide a foundation for a new class of broad-spectrum antivirulence therapies that prevent bacterial colonization and disease.

ABSTRACT Bacteria and archaea rely on appendages called type IV pili (T4P) to participate in diverse behaviors including surface sensing, biofilm formation, virulence, protein secretion and motility across surfaces. T4P are broadly distributed fibers that dynamically extend and retract, and this dynamic activity is essential for their function in broad processes. Despite the essentiality of dynamics in T4P function, little is known about the role of these dynamics and molecular mechanisms controlling them. Recent advances in microscopy have yielded insight into the role of T4P dynamics in their diverse functions and recent structural work has expanded what is known about the inner workings of the T4P motor. This review discusses recent progress in understanding the function, regulation, and mechanisms of T4P dynamics. Dynamic and widely distributed appendages called type IV pili are essential to diverse microbial lifestyles.

Although the F-specific ssRNA phage MS2 has long had paradigm status, little is known about penetration of the genomic RNA (gRNA) into the cell. The phage initially binds to the F-pilus using its maturation protein (Mat), and then theMat-bound gRNA is released from the viral capsid and somehow crosses the bacterial envelope into the cytoplasm. To address the mechanics of this process, we fluorescently labeled the ssRNA phage MS2 to track F-pilus dynamics during infection. We discovered that ssRNA phage infection triggers the release of F-pili from host cells, and that higher multiplicity of infection (MOI) correlates with detachment of longer F-pili. We also report that entry of gRNA into the host cytoplasm requires the F-plasmid–encoded coupling protein, TraD, which is located at the cytoplasmic entrance of the F-encoded type IV secretion system (T4SS). However, TraD is not essential for pilus detachment, indicating that detachment is triggered by an early step of MS2 engagement with the F-pilus or T4SS. We propose a multistep model in which the ssRNA phage binds to the F-pilus and through pilus retraction engages with the distal end of the T4SS channel at the cell surface. Continued pilus retraction pulls the Mat-gRNA complex out of the virion into the T4SS channel, causing a torsional stress that breaks the mature F-pilus at the cell surface. We propose that phage-induced disruptions of F-pilus dynamics provides a selective advantage for infecting phages and thus may be prevalent among the phages specific for retractile pili.

Recent characterization of the obligate episymbiont Saccharibacteria (TM7) belonging to the candidate phyla radiation (CPR) has expanded the extent of microbial diversity. However, the episymbiotic lifestyle of TM7 is still underexploited due to the deficiency of cultivated representatives. Here, we describe gene-targeted TM7 cultivation guided by repurposing epicPCR (emulsion, paired isolation, and concatenation PCR) to capture in situ TM7‒host associations. Using this method, we obtained a novel Saccharibacteria isolate TM7i and its host Leucobacter aridicollis J1 from Cicadae Periostracum, the castoff shell of cicada. Genomic analyses and microscopic characterizations revealed that TM7i could bind to J1 through twitching-like motility mediated by type IV pili (T4P). We further showed that the inhibition of T4P extrusion suppressed the motility and host adherence of TM7i, resulting in its reduced growth. However, the inactivation of T4P had little effect on the growth of TM7i that had already adhered to J1, suggesting the essential role of T4P in host recognition by TM7i. By capturing CPR‒host association and elaborating the T4P-dependent episymbiotic association mechanism, our studies shed light on the distinct yet widespread lifestyle of CPR bacteria.

Bacteria can move across surfaces using type IV pili (T4P), which undergo cycles of extension, adhesion, and retraction. The T4P localization pattern varies between species; however, the underlying mechanisms are largely unknown. In the rod-shaped Myxococcus xanthus cells, T4P localize at the leading cell pole. As cells reverse their direction of movement, T4P are disassembled at the old leading pole and then form at the new leading pole. Thus, cells can form T4P at both poles but engage only one pole at a time in T4P formation. Here, we address how this T4P unipolarity is realized. We demonstrate that the small Ras-like GTPase MglA stimulates T4P formation in its GTP-bound state by direct interaction with the tetratricopeptide repeat (TPR) domain-containing protein SgmX. SgmX, in turn, is important for polar localization of the T4P extension ATPase PilB. The cognate MglA GTPase activating protein (GAP) MglB, which localizes mainly to the lagging cell pole, indirectly blocks T4P formation at this pole by stimulating the conversion of MglA-GTP to MglA-GDP. Based on these findings, we propose a model whereby T4P unipolarity is accomplished by stimulation of T4P formation at the leading pole by MglA-GTP and SgmX and indirect inhibition of T4P formation at the lagging pole by MglB due to its MglA GAP activity. During reversals, MglA, SgmX, and MglB switch polarity, thus laying the foundation for T4P formation at the new leading pole and inhibition of T4P formation at the new lagging pole.

As the use of phages to treat antibiotic-resistant pathogens such as Pseudomonas aeruginosa increases, it is important to understand the potential outcomes of phage exposure. Most therapeutic P. aeruginosa phages use lipopolysaccharides or type IV pili (T4P) as primary receptors. Studying the properties of strains resistant to T4P-targeting phages can guide the design of phage cocktails to mitigate treatment resistance. We show that depending on the mutation, some phage-resistant strains can revert to wild-type sequences, emphasizing the importance of combining diverse phages to suppress resurgence. By characterizing mutations that confer resistance, we can better understand whether pilus structural or regulatory components are more likely to be lost. Using phages to select for the loss of pilus function represents an unbiased approach to identify new mutations in pilus-related proteins, shedding light on understudied components. Building a database of such mutations will help guide strategies to target and disarm this key P. aeruginosa virulence factor.