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Klebsiella pneumoniae is an important pathogen causing difficult-to-treat urinary tract infections (UTIs). Over 1.5 million women per year suffer from recurrent UTI, reducing quality of life and causing substantial morbidity and mortality, especially in the hospital setting. Uropathogenic E. coli (UPEC) is the most prevalent cause of UTI. Like UPEC, K. pneumoniae relies on type 1 pili, tipped with the mannose-binding adhesin FimH, to cause cystitis. However, K. pneumoniae FimH is a poor binder of mannose, despite a mannose-binding pocket identical to UPEC FimH. FimH is composed of two domains that are in an equilibrium between tense (low-affinity) and relaxed (high-affinity) conformations. Substantial interdomain interactions in the tense conformation yield a low-affinity, deformed mannose-binding pocket, while domain-domain interactions are broken in the relaxed state, resulting in a high-affinity binding pocket. Using crystallography, we identified the structural basis by which domain-domain interactions direct the conformational equilibrium of K. pneumoniae FimH, which is strongly shifted toward the low-affinity tense state. Removal of the pilin domain restores mannose binding to the lectin domain, thus showing that poor mannose binding by K. pneumoniae FimH is not an inherent feature of the mannose-binding pocket. Phylogenetic analyses of K. pneumoniae genomes found that FimH sequences are highly conserved. However, we surveyed a collection of K. pneumoniae isolates from patients with long-term indwelling catheters and identified isolates that possessed relaxed higher-binding FimH variants, which increased K. pneumoniae fitness in bladder infection models, suggesting that long-term residence within the urinary tract may select for higher-binding FimH variants., Competing Interests: Competing interests statement:D.A.H. serves on the Board of Directors of BioVersys AG, Basel, Switzerland. S.J.H., J.W.J., and J.S.P. own company stock in Fimbrion Therapeutics, who has licensed the mannoside patents, and they may benefit if the company is successful in marketing mannosides, S.J.H., J.W.J. and J.S.P. are co-inventors on the issued patents US 10,273,260, US 9,957,289, and US 8,937,167 which cover the use of mannoside-based FimH ligand antagonists for the treatment of disease. J.W.J. is an inventor on the patent applications covering FIM1006, FIM1028, FIM1033, and FIM2065; WO2017156508 and US20200002303.

The adhesin FimH is expressed by commensal Escherichia coli and is implicated in urinary tract infections, where it mediates adhesion to mannosylated glycoproteins on urinary and intestinal epithelial cells in the presence of a high-shear fluid environment. The FimH-mannose bond exhibits catch behavior in which bond lifetime increases with force, because tensile force induces a transition in FimH from a compact native to an elongated activated conformation with a higher affinity to mannose. However, the lifetime of the activated state of FimH has not been measured under force. Here we apply multiplexed magnetic tweezers to apply a preload force to activate FimH bonds with yeast mannan, then we measure the lifetime of these activated bonds under a wide range of forces above and below the preload force. A higher fraction of FimH-mannan bonds were activated above than below a critical preload force, confirming the FimH catch bond behavior. Once activated, FimH detached from mannose with multi-state kinetics, suggesting the existence of two bound states with a 20-fold difference in dissociation rates. The average lifetime of activated FimH-mannose bonds was 1000 to 10,000 s at forces of 30-70 pN. Structural explanations of the two bound states and the high force resistance provide insights into structural mechanisms for long-lived, force-resistant biomolecular interactions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Background: In the present study, we aimed to determine the frequency of the csgA, fimH, mrkD, foc, papaGI, papGII and papGIII genes, to provide and to design fimbrial adhesin gene (FAG) patterns and profiles for the isolated uropathogenic Escherichia coli (UPEC) strains., Methods: The enrollment of 108 positive urine samples was performed during seven months, between January 2022 and July 2022. The UPEC strains were confirmed through the standard microbiological and biochemical tests. The antimicrobial susceptibility test was performed through the Kirby-Bauer disc diffusion method. Molecular screening of FAGs was done through the polymerase chain reaction technology. The statistical analyses including chi square and Fisher's exact tests were performed to interpret the obtained results in the present study., Results: As the main results, the antimicrobial resistance (AMR) patterns, multi- (MDR) and extensively drug-resistance (XDR) patterns and FAG patterns were designed and provided. fimH (93.3%), csgA (90.4%) and papG (37.5%) (papGII (30.8%)) genes were recognized as the top three FAGs, respectively. Moreover, the frequency of csgA-fimH gene profile was identified as the top FAG pattern (46.2%) among the others. The isolates bearing csgA-fimH gene profile were armed with a versatile of phenotypic AMR patterns. In the current study, 27.8%, 69.4% and 1.9% of the UPEC isolates were detected as extended-spectrum ß-lactamases (ESBLs) producers, MDR and XDR strains, respectively., Conclusions: In conclusion, detection, providing and designing of patterns and profiles in association with FAGs, AMR feature in UPEC strains give us an effective option to have a successful and influential prevention for both of UTIs initiation and AMR feature., (© 2024. The Author(s).)

During pilus assembly within the Gram-positive bacterial envelope, membrane-bound sortase enzymes sequentially crosslink specific pilus protein monomers through their cell wall sorting signals (CWSS), starting with a designated tip pilin, followed by the shaft made of another pilin, ultimately anchoring the fiber base pilin to the cell wall. To date, the molecular determinants that govern pilus tip assembly and the underlying mechanism remain unknown. Here, we addressed this in the model organism Actinomyces oris . This oral microbe assembles a pathogenically important pilus (known as type 2 fimbria) whose shafts, made of FimA pilins, display one of two alternate tip pilins-FimB or the coaggregation factor CafA-that share a markedly similar CWSS. We demonstrate that swapping the CWSS of CafA with that of FimB produces a functional hybrid, which localizes at the pilus tip and mediates polymicrobial coaggregation, whereas alanine-substitution of the conserved FLIAG motif within the CWSS hampers these processes. Remarkably, swapping the CWSS of the normal cell wall-anchored glycoprotein GspA with that of CafA promotes the assembly of hybrid GspA at the FimA pilus tip. Finally, exchanging the CWSS of the Corynebacterium diphtheriae shaft pilin SpaA with that of CafA leads to the FLIAG motif-dependent localization of the heterologous pilus protein SpaA at the FimA pilus tip in A. oris . Evidently, the CWSS and the FLIAG motif of CafA are both necessary and sufficient for its destination to the cognate pilus tip specifically assembled by a designated sortase in the organism., Importance: Gram-positive pili, whose precursors harbor a cell wall sorting signal (CWSS) needed for sortase-mediated pilus assembly, typically comprise a pilus shaft and a tip adhesin. How a pilin becomes a pilus tip, nevertheless, remains undetermined. We demonstrate here in Actinomyces oris that the CWSS of the tip pilin CafA is necessary and sufficient to promote pilus tip assembly, and this functional assembly involves a conserved FLIAG motif within the CWSS. This is evidenced by the fact that an A. oris cell-wall anchored glycoprotein, GspA, or a heterologous shaft pilin from Corynebacterium diphtheriae , SpaA, engineered to have the CWSS of CafA in place of their CWSS, localizes at the pilus tip in a process that requires the FLIAG motif. Our findings provide the molecular basis for sortase-catalyzed pilus tip assembly that is very likely employed by other Gram-positive bacteria and potential bioengineering applications to display antigens at controlled surface distance., Competing Interests: The authors declare no conflict of interest.

Bacterial conjugation is a process by which DNA is transferred unidirectionally from a donor cell to a recipient cell. It is the main means by which antibiotic resistance genes spread among bacterial populations. It is crucially dependent upon the elaboration of an extracellular appendage, termed "pilus," by a large double-membrane-spanning secretion system termed conjugative "type IV secretion system." Here we present the structure of the conjugative pilus encoded by the R388 plasmid. We demonstrate that, as opposed to all conjugative pili produced so far for cryoelectron microscopy (cryo-EM) structure determination, the conjugative pilus encoded by the R388 plasmid is greatly stimulated by the presence of recipient cells. Comparison of its cryo-EM structure with existing conjugative pilus structures highlights a number of important differences between the R388 pilus structure and that of its homologs, the most prominent being the highly distinctive conformation of its bound lipid., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Adherence to both cellular and abiotic surfaces is a crucial step in the interaction of bacterial pathogens and commensals with their hosts. Bacterial surface structures known as fimbriae or pili play a fundamental role in the early colonization stages by providing specificity or tropism. Among the various fimbrial families, the chaperone-usher family has been extensively studied due to its ubiquity, diversity, and abundance. This family is named after the components that facilitate their biogenesis. Type 1 fimbria and P pilus, two chaperone-usher fimbriae associated with urinary tract infections, have been thoroughly investigated and serve as prototypes that have laid the foundations for understanding the biogenesis of this fimbrial family. Additionally, the study of the mechanisms regulating their expression has also been a subject of great interest, revealing that the regulation of the expression of the genes encoding these structures is a complex and diverse process, involving both common global regulators and those specific to each operon., (© 2024. The Author(s).)

The bacterial tight adherence pilus system (TadPS) assembles surface pili essential for adhesion and colonisation in many human pathogens. Pilus dynamics are powered by the ATPase CpaF (TadA), which drives extension and retraction cycles in Caulobacter crescentus through an unknown mechanism. Here we use cryogenic electron microscopy and cell-based light microscopy to characterise CpaF mechanism. We show that CpaF assembles into a hexamer with C2 symmetry in different nucleotide states. Nucleotide cycling occurs through an intra-subunit clamp-like mechanism that promotes sequential conformational changes between subunits. Moreover, a comparison of the active sites with different nucleotides bound suggests a mechanism for bidirectional motion. Conserved CpaF residues, predicted to interact with platform proteins CpaG (TadB) and CpaH (TadC), are mutated in vivo to establish their role in pilus processing. Our findings provide a model for how CpaF drives TadPS pilus dynamics and have broad implications for how other ancient type 4 filament family members power pilus assembly., (© 2024. The Author(s).)

Currently, fresh, unprocessed food has become a relevant element of the chain of transmission of enteropathogenic infections. To survive on a plant surface and further spread the infections, pathogens like Salmonella have to attach stably to the leaf surface. Adhesion, driven by various virulence factors, including the most abundant fim operon encoding type 1 fimbriae, is usually an initial step of infection, preventing physical removal of the pathogen. Adhesion properties of Salmonella's type 1 fimbriae and its FimH adhesin were investigated intensively in the past. However, there is a lack of knowledge regarding its role in interaction with plant cells. Understanding the mechanisms and structures involved in such interaction may facilitate efforts to decrease the risk of contamination and increase fresh food safety. Here, we applied Salmonella genome site-directed mutagenesis, adhesion assays, protein-protein interactions, and biophysics methods based on surface plasmon resonance to unravel the role of FimH adhesin in interaction with spinach leaves. We show that FimH is at least partially responsible for Salmonella binding to spinach leaves, and this interaction occurs in a mannose-independent manner. Importantly, we identified a potential FimH receptor as endo-1,3-β-d-Glucanase and found that this interaction is strong and specific, with a dissociation constant in the nanomolar range. This research advances our comprehension of Salmonella's interactions with plant surfaces, offering insights that can aid in minimizing contamination risks and improving the safety of fresh, unprocessed foods., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Fimbriae are essential virulence factors for many bacterial pathogens. Fimbriae are extracellular structures that attach bacteria to surfaces. Thus, fimbriae mediate a critical step required for any pathogen to establish infection by anchoring a bacterium to host tissue. The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7encodes 16 fimbriae that may be important for EHEC to initiate infection and allow for productive expression of virulence traits important in later stages of infection, including a type III secretion system (T3SS) and Shiga toxin; however, the roles of most EHEC fimbriae are largely uncharacterized. Here, we provide evidence that two EHEC fimbriae, Yad and Yeh, modulate expression of diverse genes including genes encoding T3SS and Shiga toxin and that these fimbriae are required for robust colonization of the gastrointestinal tract. These findings reveal a significant and previously unappreciated role for fimbriae in bacterial pathogenesis as important determinants of virulence gene expression.IMPORTANCEFimbriae are extracellular proteinaceous structures whose defining role is to anchor bacteria to surfaces. This is a fundamental step for bacterial pathogens to establish infection in a host. Here, we show that the contributions of fimbriae to pathogenesis are more complex. Specifically, we demonstrate that fimbriae influence expression of virulence traits essential for disease progression in the intestinal pathogen enterohemorrhagic Escherichia coli . Gram-positive and Gram-negative bacteria express multiple fimbriae; therefore, these findings may have broad implications for understanding how pathogens use fimbriae, beyond adhesion, to initiate infection and coordinate gene expression, which ultimately results in disease., Competing Interests: The authors declare no conflict of interest.

Pili are bacterial surface structures important for surface adhesion. In the alphaproteobacterium Caulobacter crescentus, the global regulator CtrA activates transcription of roughly 100 genes, including pilA which codes for the pilin monomer that makes up the pilus filament. While most CtrA-activated promoters have a single CtrA-binding site at the - 35 position and are induced at the early to mid-predivisional cell stage, the pilA promoter has 3 additional upstream CtrA-binding sites and it is induced at the late predivisional cell stage. Reporter constructs where these additional sites were disrupted by deletion or mutation led to increased activity compared to the WT promoter. In synchronized cultures, these mutations caused pilA transcription to occur approximately 20 min earlier than WT. The results suggested that the site overlapping the - 35 position drives pilA gene expression while the other upstream CtrA-binding sites serve to reduce and delay expression. EMSA experiments showed that the - 35 Site has lower affinity for CtrA∼P compared to the other sites, suggesting binding site affinity may be involved in the delay mechanism. Mutating the upstream inhibitory CtrA-binding sites in the pilA promoter caused significantly higher numbers of pre-divisional cells to express pili, and phage survival assays showed this strain to be significantly more sensitive to pilitropic phage. These results suggest that pilA regulation evolved in C. crescentus to provide an ecological advantage within the context of phage infection., (© 2024. The Author(s).)

Sortase-dependent pili are long surface appendages that mediate attachment, colonization and biofilm formation in certain genera and species of Gram-positive bacteria. Ligilactobacillus ruminis is an autochthonous gut commensal that relies on sortase-dependent LrpCBA pili for host adherence and persistence. X-ray crystal structure snapshots of the backbone pilin LrpA were captured in two atypical bent conformations leading to a zigzag morphology in the LrpCBA pilus structure. Small-angle X-ray scattering and structural analysis revealed that LrpA also adopts the typical linear conformation, resulting in an elongated pilus morphology. Various conformational analyses and biophysical experiments helped to demonstrate that a hinge region located at the end of the flexible N-terminal domain of LrpA facilitates a new closure-and-twist motion for assembling dynamic pili during the assembly process and host attachment. Further, the incongruent combination of flexible domain-driven conformational dynamics and rigid isopeptide bond-driven stability observed in the LrpCBA pilus might also extend to the sortase-dependent pili of other bacteria colonizing a host.

The natural transformation system of the thermophilic bacterium Thermus thermophilus comprises at least 16 competence proteins. Recently we found that the outer membrane (OM) competence protein PilW interacts with the secretin channel, which guides type IV pili (T4P) and potential DNA transporter pseudopili through the OM. Here we have used biochemical techniques to study the interactions of cytoplasmic, inner membrane (IM) and OM components of the DNA transporter in T. thermophilus. We report that PilW is part of a heteropolymeric complex comprising of the cytoplasmic PilM protein, IM proteins PilN, PilO, PilC and the secretin PilQ. Co-purification studies revealed that PilO directly interacts with PilW. In vitro affinity co-purification studies using His-tagged PilC led to the detection of PilC-, PilW-, PilN- and PilO-containing complexes. PilO was identified as direct interaction partner of the polytopic IM protein PilC. PilC was also found to directly interact with the cytoplasmic T4P disassembly ATPase PilT1 thereby triggering PilT1 ATPase activity. This, together with the detection of heteropolymeric PilC complexes which contain PilT1 and the pilins PilA2, PilA4 and PilA5 is in line with the hypothesis that PilC connects the depolymerization ATPase to the base of the pili possibly allowing energy transduction for disassembly of the pilins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Many prokaryotes use swimming motility to move toward favorable conditions and escape adverse surroundings. Regulatory mechanisms governing bacterial flagella-driven motility are well-established; however, little is yet known about the regulation underlying swimming motility propelled by the archaeal cell surface structure, the archaella. Previous research showed that the deletion of the adhesion pilins (PilA1-6), subunits of the type IV pili cell surface structure, renders the model archaeon Haloferax volcanii non-motile. In this study, we used ethyl methanesulfonate mutagenesis and a motility assay to identify motile suppressors of the ∆ pilA [ 1-6 ] strain. Of the eight suppressors identified, six contain missense mutations in archaella biosynthesis genes, arlI and arlJ. In trans expression of arlI and arlJ mutant constructs in the respective multi-deletion strains ∆ pilA [ 1-6 ]∆ arlI and ∆ pilA [ 1-6 ]∆ arlJ confirmed their role in suppressing the ∆ pilA [ 1-6 ] motility defect. Additionally, three suppressors harbor co-occurring disruptive missense and nonsense mutations in cirA , a gene encoding a proposed regulatory protein. A deletion of cirA resulted in hypermotility, while cirA expression in trans in wild-type cells led to decreased motility. Moreover, quantitative real-time PCR analysis revealed that in wild-type cells, higher expression levels of arlI , arlJ , and the archaellin gene arlA1 were observed in motile early-log phase rod-shaped cells compared to non-motile mid-log phase disk-shaped cells. Conversely, ∆ cirA cells, which form rods during both early- and mid-log phases, exhibited similar expression levels of arl genes in both growth phases. Our findings contribute to a deeper understanding of the mechanisms governing archaeal motility, highlighting the involvement of ArlI, ArlJ, and CirA in pilin-mediated motility regulation.IMPORTANCEArchaea are close relatives of eukaryotes and play crucial ecological roles. Certain behaviors, such as swimming motility, are thought to be important for archaeal environmental adaptation. Archaella, the archaeal motility appendages, are evolutionarily distinct from bacterial flagella, and the regulatory mechanisms driving archaeal motility are largely unknown. Previous research has linked the loss of type IV pili subunits to archaeal motility suppression. This study reveals three Haloferax volcanii proteins involved in pilin-mediated motility regulation, offering a deeper understanding of motility regulation in this understudied domain while also paving the way for uncovering novel mechanisms that govern archaeal motility. Understanding archaeal cellular processes will help elucidate the ecological roles of archaea as well as the evolution of these processes across domains., Competing Interests: The authors declare no conflict of interest.

Type IV pili (T4P) represent one of the most common varieties of surface appendages in archaea. These filaments, assembled from small pilin proteins, can be many microns long and serve diverse functions, including adhesion, biofilm formation, motility, and intercellular communication. Here, we determine atomic structures of two distinct adhesive T4P from Saccharolobus islandicus via cryo-electron microscopy (cryo-EM). Unexpectedly, both pili were assembled from the same pilin polypeptide but under different growth conditions. One filament, denoted mono-pilus, conforms to canonical archaeal T4P structures where all subunits are equivalent, whereas in the other filament, the tri-pilus, the same polypeptide exists in three different conformations. The three conformations in the tri-pilus are very different from the single conformation found in the mono-pilus, and involve different orientations of the outer immunoglobulin-like domains, mediated by a very flexible linker. Remarkably, the outer domains rotate nearly 180° between the mono- and tri-pilus conformations. Both forms of pili require the same ATPase and TadC-like membrane pore for assembly, indicating that the same secretion system can produce structurally very different filaments. Our results show that the structures of archaeal T4P appear to be less constrained and rigid than those of the homologous archaeal flagellar filaments that serve as helical propellers., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Amongst the major types of archaeal filaments, several have been shown to closely resemble bacterial homologues of the Type IV pili (T4P). Within Sulfolobales, member species encode for three types of T4P, namely the archaellum, the UV-inducible pilus system (Ups) and the archaeal adhesive pilus (Aap). Whereas the archaellum functions primarily in swimming motility, and the Ups in UV-induced cell aggregation and DNA-exchange, the Aap plays an important role in adhesion and twitching motility. Here, we present a cryoEM structure of the Aap of the archaeal model organism Sulfolobus acidocaldarius. We identify the component subunit as AapB and find that while its structure follows the canonical T4P blueprint, it adopts three distinct conformations within the pilus. The tri-conformer Aap structure that we describe challenges our current understanding of pilus structure and sheds new light on the principles of twitching motility., (© 2024. The Author(s).)

Type IV pili are filamentous appendages found in most bacteria and archaea, where they can support functions such as surface adhesion, DNA uptake, aggregation, and motility. In most bacteria, PilT-family ATPases disassemble adhesion pili, causing them to rapidly retract and produce twitching motility, important for surface colonization. As archaea do not possess PilT homologs, it was thought that archaeal pili cannot retract and that archaea do not exhibit twitching motility. Here, we use live-cell imaging, automated cell tracking, fluorescence imaging, and genetic manipulation to show that the hyperthermophilic archaeon Sulfolobus acidocaldarius exhibits twitching motility, driven by retractable adhesion (Aap) pili, under physiologically relevant conditions (75 °C, pH 2). Aap pili are thus capable of retraction in the absence of a PilT homolog, suggesting that the ancestral type IV pili in the last universal common ancestor (LUCA) were capable of retraction., (© 2024. The Author(s).)

Pseudomonas aeruginosa is one of the most common nosocomial pathogens worldwide, known for its virulence, drug resistance, and elaborate sensor-response network. The primary challenge encountered by pathogens during the initial stages of infection is the immune clearance arising from the host. The resident macrophages of barrier organs serve as the frontline defense against these pathogens. Central to our understanding is the mechanism by which bacteria modify their behavior to circumvent macrophage-mediated clearance, ensuring their persistence and colonization. To successfully evade macrophage-mediated phagocytosis, bacteria must possess an adaptive response mechanism. Two-component systems provide bacteria the agility to navigate diverse environmental challenges, translating external stimuli into cellular adaptive responses. Here, we report that the well-documented histidine kinase, LadS, coupled to a cognate two-component response regulator, PA0034, governs the expression of a vital adhesin called chaperone-usher pathway pilus cupA. The LadS/PA0034 system is susceptible to interference from the reactive oxygen species likely to be produced by macrophages and further lead to a poor adhesive phenotype with scantily cupA pilus, impairing the phagocytosis efficiency of macrophages during acute infection. This dynamic underscores the intriguing interplay: as macrophages deploy reactive oxygen species to combat bacterial invasion, the bacteria recalibrate their exterior to elude these defenses., Importance: The notoriety of Pseudomonas aeruginosa is underscored by its virulence, drug resistance, and elaborate sensor-response network. Yet, the mechanisms by which P. aeruginosa maneuvers to escape phagocytosis during acute infections remain elusive. This study pinpoints a two-component response regulator, PA0034, coupled with the histidine kinase LadS, and responds to macrophage-derived reactive oxygen species. The macrophage-derived reactive oxygen species can impair the LadS/PA0034 system, resulting in reduced expression of cupA pilus in the exterior of P. aeruginosa . Since the cupA pilus is an important adhesin of P. aeruginosa , its deficiency reduces bacterial adhesion and changes their behavior to adopt a planktonic lifestyle, subsequently inhibiting the phagocytosis of macrophages by interfering with bacterial adhesion. Briefly, reactive oxygen species may act as environmental cues for the LadS/PA0034 system. Upon recognition, P. aeruginosa may transition to a poorly adhesive state, efficiently avoiding engulfment by macrophages., Competing Interests: The authors declare no conflict of interest.

Bacteria utilize type IV pili (T4P) to interact with their environment, where they facilitate processes including motility, adherence, and DNA uptake. T4P require multisubunit, membrane-spanning nanomachines for assembly. The tight adherence (Tad) pili are an Archaea-derived T4P subgroup whose machinery exhibits significant mechanistic and architectural differences from bacterial type IVa and IVb pili. Most Tad biosynthetic genes are encoded in a single locus that is widespread in bacteria due to facile acquisition via horizontal gene transfer. These loci experience extensive structural rearrangements, including the acquisition of novel regulatory or biosynthetic genes, which fine-tune their function. This has permitted their integration into many different bacterial lifestyles, including the Caulobacter crescentus cell cycle, Myxococcus xanthus predation, and numerous plant and mammalian pathogens and symbionts., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Bacterial adhesion plays a vital role in forming and shaping the structure of electroactive biofilms that are essential for the performance of bioelectrochemical systems (BESs). Type IV pili are known to mediate cell adhesion in many Gram-negative bacteria, but the mechanism of pili-mediated cell adhesion of Geobacter species on anode surface remains unclear. Herein, a minor pilin PilV2 was found to be essential for cell adhesion ability of Geobacter sulfurreducens since the lack of pilV2 gene depressed the cell adhesion capability by 81.2% in microplate and the anodic biofilm density by 23.1 % at -0.1 V and 37.7 % at -0.3 V in BESs. The less cohesiveness of mutant biofilms increased the charge transfer resistance and biofilm resistance, which correspondingly lowered current generation of the pilV2-deficient strain by up to 63.2 % compared with that of the wild-type strain in BESs. The deletion of pilV2 posed an insignificant effect on the production of extracellular polysaccharides, pili, extracellular cytochromes and electron shuttles that are involved in biofilm formation or extracellular electron transfer (EET) process. This study demonstrated the significance of pilV2 gene in cell adhesion and biofilm formation of G. sulfurreducens, as well as the importance of pili-mediated adhesion for EET of electroactive biofilm., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Oral cancer is among the most common cancer in the world. Tobacco, alcohol, and viruses have been regarded as a well- known risk factors of OCC however, 15% of OSCC cases occurred each year without these known risk factors. Recently a myriad of studies has shown that bacterial infections lead to cancer. Accumulated shreds of evidence have demonstrated the role of Porphyromonas gingivalis (P. gingivalis) in OSCC. The virulence factor FimA of P. gingivalis activates the oncogenic pathways in OSCC by upregulating various cytokines. It also led to the inactivation of a tumor suppressor protein p53. The present Insilico study uses High-Throughput Virtual Screening, molecular docking, and molecular dynamics techniques to find the potential compounds against the target protein FimA. The goal of this study is to identify the anti-cancer lead compounds retrieved from natural sources that can be used to develop potent drug molecules to treat P.gingivalis-related OSCC. The anticancer natural compounds library was screened to identify the potential lead compounds. Furthermore, these lead compounds were subjected to precise docking, and based on the docking score potential lead compounds were identified. The top docked receptor-ligand complex was subjected to molecular dynamics simulation. A study of this insilico finding provides potent lead molecules which help in the development of therapeutic drugs against the target protein FimA in OSCC., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The biphasic assembly of Gram-positive pili begins with the covalent polymerization of distinct pilins catalyzed by a pilus-specific sortase, followed by the cell wall anchoring of the resulting polymers mediated by the housekeeping sortase. In Actinomyces oris, the pilus-specific sortase SrtC2 not only polymerizes FimA pilins to assemble type 2 fimbriae with CafA at the tip, but it can also act as the anchoring sortase, linking both FimA polymers and SrtC1-catalyzed FimP polymers (type 1 fimbriae) to peptidoglycan when the housekeeping sortase SrtA is inactive. To date, the structure-function determinants governing the unique substrate specificity and dual enzymatic activity of SrtC2 have not been illuminated. Here, we present the crystal structure of SrtC2 solved to 2.10-Å resolution. SrtC2 harbors a canonical sortase fold and a lid typical for class C sortases and additional features specific to SrtC2. Structural, biochemical, and mutational analyses of SrtC2 reveal that the extended lid of SrtC2 modulates its dual activity. Specifically, we demonstrate that the polymerizing activity of SrtC2 is still maintained by alanine-substitution, partial deletion, and replacement of the SrtC2 lid with the SrtC1 lid. Strikingly, pilus incorporation of CafA is significantly reduced by these mutations, leading to compromised polymicrobial interactions mediated by CafA. In a srtA mutant, the partial deletion of the SrtC2 lid reduces surface anchoring of FimP polymers, and the lid-swapping mutation enhances this process, while both mutations diminish surface anchoring of FimA pili. Evidently, the extended lid of SrtC2 enables the enzyme the cell wall-anchoring activity in a substrate-selective fashion., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The pil gene cluster for Type IV pilus (Tfp) biosynthesis is commonly present and highly conserved in Streptococcus sanguinis . Nevertheless, Tfp-mediated twitching motility is less common among strains, and the factors determining twitching activity are not fully understood. Here, we analyzed the functions of three major pilin proteins (PilA1, PilA2, and PilA3) in the assembly and activity of Tfp in motile S. sanguinis CGMH010. Using various recombinant pilA deletion strains, we found that Tfp composed of different PilA proteins varied morphologically and functionally. Among the three PilA proteins, PilA1 was most critical in the assembly of twitching-active Tfp, and recombinant strains expressing motility generated more structured biofilms under constant shearing forces compared to the non-motile recombinant strains. Although PilA1 and PilA3 shared 94% identity, PilA3 could not compensate for the loss of PilA1, suggesting that the nature of PilA proteins plays an essential role in twitching activity. The single deletion of individual pilA genes had little effect on the invasion of host endothelia by S. sanguinis CGMH010. In contrast, the deletion of all three pilA genes or pilT , encoding the retraction ATPase, abolished Tfp-mediated invasion. Tfp- and PilT-dependent invasion were also detected in the non-motile S. sanguinis SK36, and thus, the retraction of Tfp, but not active twitching, was found to be essential for invasion.

Extracellular cytochrome filaments are proposed to serve as conduits for long-range extracellular electron transfer. The primary functional physiological evidence has been the reported inhibition of Geobacter sulfurreducens Fe(III) oxide reduction when the gene for the filament-forming cytochrome OmcS is deleted. Here we report that the OmcS-deficient strain from that original report reduces Fe(III) oxide as well as the wild-type, as does a triple mutant in which the genes for the other known filament-forming cytochromes were also deleted. The triple cytochrome mutant displayed filaments with the same 3 nm diameter morphology and conductance as those produced by Escherichia coli heterologously expressing the G. sulfurreducens PilA pilin gene. Fe(III) oxide reduction was inhibited when the pilin gene in cytochrome-deficient mutants was modified to yield poorly conductive 3 nm diameter filaments. The results are consistent with the concept that 3 nm diameter electrically conductive pili (e-pili) are required for G. sulfurreducens long-range extracellular electron transfer. In contrast, rigorous physiological functional evidence is lacking for cytochrome filaments serving as conduits for long-range electron transport., Importance: Unraveling microbial extracellular electron transfer mechanisms has profound implications for environmental processes and advancing biological applications. This study on Geobacter sulfurreducens challenges prevailing beliefs on cytochrome filaments as crucial components thought to facilitate long-range electron transport. The discovery of an OmcS-deficient strain's unexpected effectiveness in Fe(III) oxide reduction prompted a reevaluation of the key conduits for extracellular electron transfer. By exploring the impact of genetic modifications on G. sulfurreducens ' performance, this research sheds light on the importance of 3-nm diameter electrically conductive pili in Fe(III) oxide reduction. Reassessing these mechanisms is essential for uncovering the true drivers of extracellular electron transfer in microbial systems, offering insights that could revolutionize applications across diverse fields., Competing Interests: The authors declare no conflict of interest.

Type IVa pili (T4aP) are ubiquitous cell surface filaments important for surface motility, adhesion to surfaces, DNA uptake, biofilm formation, and virulence. T4aP are built from thousands of copies of the major pilin subunit and tipped by a complex composed of minor pilins and in some systems also the PilY1 adhesin. While major pilins of structurally characterized T4aP have lengths of <165 residues, the major pilin PilA of Myxococcus xanthus is unusually large with 208 residues. All major pilins have a conserved N-terminal domain and a variable C-terminal domain, and the additional residues of PilA are due to a larger C-terminal domain. We solved the structure of the M. xanthus T4aP (T4aP Mx ) at a resolution of 3.0 Å using cryo-EM. The T4aP Mx follows the structural blueprint of other T4aP with the pilus core comprised of the interacting N-terminal α1-helices, while the globular domains decorate the T4aP surface. The atomic model of PilA built into this map shows that the large C-terminal domain has more extensive intersubunit contacts than major pilins in other T4aP. As expected from these greater contacts, the bending and axial stiffness of the T4aP Mx is significantly higher than that of other T4aP and supports T4aP-dependent motility on surfaces of different stiffnesses. Notably, T4aP Mx variants with interrupted intersubunit interfaces had decreased bending stiffness, pilus length, and strongly reduced motility. These observations support an evolutionary scenario whereby the large major pilin enables the formation of a rigid T4aP that expands the environmental conditions in which the T4aP system functions., Competing Interests: Competing interests statement:The authors declare no competing interest.

The retractile type IV pilus (T4P) is important for virulence of the opportunistic human pathogen Pseudomonas aeruginosa . The single-stranded RNA (ssRNA) phage PP7 binds to T4P and is brought to the cell surface through pilus retraction. Using fluorescence microscopy, we discovered that PP7 detaches T4P, which impairs cell motility and restricts the pathogen's virulence. Using cryo-electron microscopy, mutagenesis, optical trapping, and Langevin dynamics simulation, we resolved the structure of PP7, T4P, and the PP7/T4P complex and showed that T4P detachment is driven by the affinity between the phage maturation protein and its bound pilin, plus the pilus retraction force and speed, and pilus bending. Pilus detachment may be widespread among other ssRNA phages and their retractile pilus systems and offers new prospects for antibacterial prophylaxis and therapeutics.

Acinetobacters pose a significant threat to human health, especially those with weakened immune systems. Type IV pili of acinetobacters play crucial roles in virulence and antibiotic resistance. Single-stranded RNA bacteriophages target the bacterial retractile pili, including type IV. Our study delves into the interaction between Acinetobacter phage AP205 and type IV pili. Using cryo-electron microscopy, we solve structures of the AP205 virion with an asymmetric dimer of maturation proteins, the native Acinetobacter type IV pili bearing a distinct post-translational pilin cleavage, and the pili-bound AP205 showing its maturation proteins adapted to pilin modifications, allowing each phage to bind to one or two pili. Leveraging these results, we develop a 20-kilodalton AP205-derived protein scaffold targeting type IV pili in situ, with potential for research and diagnostics., (© 2024. The Author(s).)

Urinary tract infections (UTIs), common bacterial infections in communities and medical facilities, are mainly mediated by FimH. The glycan sites of the uromodulin protein play a crucial role in protecting against UTIs by interacting with FimH. A bioinspired approach using glycan-FimH interactions may effectively reduce bacteria through an antiadhesive mechanism, thereby curbing bacterial resistance. However, typical antiadhesive therapy alone fails to address the excessive reactive oxygen species and inflammatory response during UTIs. To bridge this gap, antioxidant nanozymes with antiadhesive ability were developed as nanodecoys to counter bacteria and inflammation. Specifically, ultrasmall dextran-coated ceria (DEC) was engineered to address UTIs, with dextran blocking FimH adhesion and ceria exhibiting anti-inflammatory properties. DECs, metabolizable by the kidneys, reduced bacterial content in the urinary tract, mitigating inflammation and tissue damage. In murine models, DECs successfully treated acute UTIs, repeated infections, and catheter-related UTIs. This dual approach not only highlights the potential of nanozymes for UTIs but also suggests applicability to other FimH-induced infections in the lungs and bowels, marking a significant advancement in nanozyme-based clinical approaches.

Type IV pili (T4P) are prevalent, polymeric surface structures in pathogenic bacteria, making them ideal targets for effective vaccines. However, bacteria have evolved efficient strategies to evade type IV pili-directed antibody responses. Neisseria meningitidis are prototypical type IV pili-expressing Gram-negative bacteria responsible for life threatening sepsis and meningitis. This species has evolved several genetic strategies to modify the surface of its type IV pili, changing pilin subunit amino acid sequence, nature of glycosylation and phosphoforms, but how these modifications affect antibody binding at the structural level is still unknown. Here, to explore this question, we determine cryo-electron microscopy (cryo-EM) structures of pili of different sequence types with sufficiently high resolution to visualize posttranslational modifications. We then generate nanobodies directed against type IV pili which alter pilus function in vitro and in vivo. Cyro-EM in combination with molecular dynamics simulation of the nanobody-pilus complexes reveals how the different types of pili surface modifications alter nanobody binding. Our findings shed light on the impressive complementarity between the different strategies used by bacteria to avoid antibody binding. Importantly, we also show that structural information can be used to make informed modifications in nanobodies as countermeasures to these immune evasion mechanisms., (© 2024. The Author(s).)

Importance: Type 4 filaments (T4F) are nanomachines ubiquitous in prokaryotes, centered on filamentous polymers of type 4 pilins. T4F are exceptionally versatile and widespread virulence factors in bacterial pathogens. The mechanisms of filament assembly and the many functions they facilitate remain poorly understood because of the complexity of T4F machineries. This hinders the development of anti-T4F drugs. The significance of our research lies in characterizing the simplest known T4F-the Com pilus that mediates DNA uptake in competent monoderm bacteria-and showing that four protein components universally conserved in T4F are sufficient for filament assembly. The Com pilus becomes a model for elucidating the mechanisms of T4F assembly., Competing Interests: The authors declare no conflict of interest.

Importance: Type IV pili and type II secretion systems are members of the widespread type IV filament (T4F) superfamily of nanomachines that assemble dynamic and versatile surface fibers in archaea and bacteria. The assembly and retraction of T4 filaments with diverse surface properties and functions require the plasma membrane platform proteins of the GspF/PilC superfamily. Generally considered dimeric, platform proteins are thought to function as passive transmitters of the mechanical energy generated by the ATPase motor, to somehow promote insertion of pilin subunits into the nascent pilus fibers. Here, we generate and experimentally validate structural predictions that support the trimeric state of a platform protein PulF from a type II secretion system. The PulF trimers form selective proton or sodium channels which might energize pilus assembly using the membrane potential. The conservation of the channel sequence and structural features implies a common mechanism for all T4F assembly systems. We propose a model of the oligomeric PulF-PulE ATPase complex that provides an essential framework to investigate and understand the pilus assembly mechanism., Competing Interests: The authors declare no conflict of interest.

Bacterial chromosomal DNA is structured and compacted by proteins known as bacterial chromatin proteins (i.e., nucleoid-associated proteins or NAPs). DNA-dependent RNA polymerase (RNAP) must frequently interact with bacterial chromatin proteins because they often bind DNA genome-wide. In some cases, RNAP must overcome barriers bacterial chromatin proteins impose on transcription. One key bacterial chromatin protein in Escherichia coli that influences transcription is the histone-like nucleoid structuring protein, H-NS. H-NS binds to DNA and forms nucleoprotein filaments. To investigate the effect of H-NS filaments on RNAP elongation, we developed an in vitro transcription assay to monitor RNAP progression on a DNA template bound by H-NS. In this method, initiation and elongation by RNAP are uncoupled by first initiating transcription in the presence of only three ribonucleoside triphosphates (rNTPs) to halt elongation just downstream of the promoter. Before elongation is restarted by addition of the fourth NTP, an H-NS filament is formed on the DNA so that transcript elongation occurs on an H-NS nucleoprotein filament template. Here, we provide detailed protocols for performing in vitro transcription through H-NS filaments, analysis of the transcription products, and visualization of H-NS filament formation on DNA by electrophoretic mobility shift assay (EMSA). These methods enable insight into how H-NS affects RNAP transcript elongation and provide a starting point to determine effects of other bacterial chromatin proteins on RNAP elongation., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Within the highly diverse type four filament (TFF or T4F) superfamily, the machineries of type IVa pili (T4aP) and the type 2 secretion system (T2SS) in diderm bacteria exhibit a substantial sequence similarity despite divergent functions and distinct appearances: T4aP can extend micrometers beyond the outer membrane, whereas the endopili in the T2SS are restricted to the periplasm. The determination of the structure of individual components and entire filaments is crucial to understand how their structure enables them to serve different functions. However, the dynamics of these filaments poses a challenge for their high-resolution structure determination. This review presents different approaches that have been used to study the structure and dynamics of T4aP and T2SS endopili by means of integrative structural biology, cryo-electron microscopy (cryo-EM), and molecular dynamics simulations. Their conserved features and differences are presented. The non-helical stretch in the long-conserved N-terminal helix which is characteristic of all members of the TFF and the impact of calcium on structure, function, and dynamics of these filaments are discussed in detail., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

The Gram-positive bacterium Actinomyces oris expresses a unique cell wall-anchored fimbria comprised of the fimbrial shaft FimA and the tip fimbrillin CafA, whose gene is not genetically linked to the fimA locus, unlike many other fimbrial gene loci in Gram-positive bacteria. Mutational analyses of individual fimbrillins, FimA and CafA, in A. oris often rely on multi-copy plasmids that may alter the stoichiometry of fimbrillins in vivo, hence fimbrial assembly. Here, we provide a robust method for single-copy gene expression and mutagenesis in A. oris, using CafA as an experimental model. This method can be applied for single-copy gene editing in various bacterial systems., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Many species of pathogenic gram-positive bacteria display covalently crosslinked protein polymers (called pili or fimbriae) that mediate microbial adhesion to host tissues. These structures are assembled by pilus-specific sortase enzymes that join the pilin components together via lysine-isopeptide bonds. The archetypal SpaA pilus from Corynebacterium diphtheriae is built by the Cd SrtA pilus-specific sortase, which crosslinks lysine residues within the SpaA and SpaB pilins to build the shaft and base of the pilus, respectively. Here, we show that Cd SrtA crosslinks SpaB to SpaA via a K139(SpaB)-T494(SpaA) lysine-isopeptide bond. Despite sharing only limited sequence homology, an NMR structure of SpaB reveals striking similarities with the N-terminal domain of SpaA ( N SpaA) that is also crosslinked by Cd SrtA. In particular, both pilins contain similarly positioned reactive lysine residues and adjacent disordered AB loops that are predicted to be involved in the recently proposed "latch" mechanism of isopeptide bond formation. Competition experiments using an inactive SpaB variant and additional NMR studies suggest that SpaB terminates SpaA polymerization by outcompeting N SpaA for access to a shared thioester enzyme-substrate reaction intermediate., (© 2023 Wiley Periodicals LLC.)

Salmonella enterica serovar Enteritidis ( S . Enteritidis) is a zoonotic pathogen that can infect both humans and animals. Among the 13 types of fimbrial operons in S . Enteritidis, the highly conserved Peg fimbriae play a crucial role in the adhesion and invasion of S . Enteritidis into host cells but are not well studied. In this study, we identified the ATP synthase subunit alpha (ATPase α) as a ligand of Peg fimbriae using ligand blotting and mass spectrometry techniques. We confirmed the in vitro binding of ATPase α to the purified adhesion protein (PegD). Furthermore, we used siRNA to suppress the expression of ATPase α gene Atp5a1 in Leghorn male hepatoma (LMH) cells, which resulted in a significant reduction in the adhesion rate of S . Enteritidis to the cells ( P  < 0.05). The findings in this study provide insight into the mechanism of S . Enteritidis infection through Peg fimbriae and highlight the importance of ATPase α in the adhesion process. RESEARCH HIGHLIGHTS Ligand blotting was performed to screen the ligand of S. Enteritidis Peg fimbriae.Binding assay confirmed that ATPase α is the ligand of the Peg fimbriae.siRNA targeting ATPase α gene ( Atp5a1 ) significantly reduced S . Enteritidis adhesion.

Group A Streptococcus (GAS) is a globally important pathogen causing a broad range of human diseases. GAS pili are elongated proteins with a backbone comprised repeating T-antigen subunits, which extend from the cell surface and have important roles in adhesion and establishing infection. No GAS vaccines are currently available, but T-antigen-based candidates are in pre-clinical development. This study investigated antibody-T-antigen interactions to gain molecular insight into functional antibody responses to GAS pili. Large, chimeric mouse/human Fab-phage libraries generated from mice vaccinated with the complete T18.1 pilus were screened against recombinant T18.1, a representative two-domain T-antigen. Of the two Fab identified for further characterization, one (designated E3) was cross-reactive and also recognized T3.2 and T13, while the other (H3) was type-specific reacting with only T18.1/T18.2 within a T-antigen panel representative of the major GAS T-types. The epitopes for the two Fab, determined by x-ray crystallography and peptide tiling, overlapped and mapped to the N-terminal region of the T18.1 N-domain. This region is predicted to be buried in the polymerized pilus by the C-domain of the next T-antigen subunit. However, flow cytometry and opsonophagocytic assays showed that these epitopes were accessible in the polymerized pilus at 37°C, though not at lower temperature. This suggests that there is motion within the pilus at physiological temperature, with structural analysis of a covalently linked T18.1 dimer indicating "knee-joint" like bending occurs between T-antigen subunits to expose this immunodominant region. This temperature dependent, mechanistic flexing provides new insight into how antibodies interact with T-antigens during infection.

Urinary tract infection is among the most common infections worldwide, typically studied in animals and cell lines with limited uropathogenic strains. Here, we assessed diverse bacterial species in a human urothelial microtissue model exhibiting full stratification, differentiation, innate epithelial responses, and urine tolerance. Several uropathogens invaded intracellularly, but also commensal Escherichia coli , suggesting that invasion is a shared survival strategy, not solely a virulence hallmark. The E. coli adhesin FimH was required for intracellular bacterial community formation, but not for invasion. Other shared lifestyles included filamentation (Gram-negatives), chaining (Gram-positives), and hijacking of exfoliating cells, while biofilm-like aggregates were formed mainly with Pseudomonas and Proteus . Urothelial cells expelled invasive bacteria in Rab-/LC3-decorated structures, while highly cytotoxic/invasive uropathogens, but not commensals, disrupted host barrier function and strongly induced exfoliation and cytokine production. Overall, this work highlights diverse species-/strain-specific infection strategies and corresponding host responses in a human urothelial microenvironment, providing insights at the microtissue, cell, and molecular level.

The nature of alpha-D-mannose-natural aldohexose sugar, C-2 glucose epimer, whose intended use is for preventing urinary tract infections-in the interaction with E. coli is addressed in order to drive the issue of its regulatory classification as a medicinal product or medical device. PRISMA systematic review approach was applied; Delphi Panel method was used to target consensus on statements retrieved from evidence. Based on regulatory definitions and research evidence, the mechanism of D-mannose does not involve a metabolic or immunological action while there is uncertainty regarding the pharmacological action. Specific interaction between the product and the bacteria within the body occurs, but its nature is inert: it does not induce a direct response activating or inhibiting body processes. Moreover, the action of D-mannose takes place, even if inside the bladder, outside the epithelium on bacteria that have not yet invaded the urothelial tissue. Therefore, its mechanism of action is not directed to host structures but to structures (bacteria) external to the host's tissues. On the basis of current regulation, the uncertainty as regard a pharmacological action of alpha-D-mannose makes possible its medical device classification: new regulations and legal judgments can add further considerations. From a pharmacological perspective, research is driven versus synthetic mannosides: no further considerations are expected on alpha-D-mannose., (© 2023. The Author(s).)

Importance: Type IVa pili (T4aP) are widespread bacterial cell surface structures with important functions in motility, surface adhesion, biofilm formation, and virulence. Different bacteria have adapted different piliation patterns. To address how these patterns are established, we focused on the bipolar localization of the T4aP machine in the model organism Myxococcus xanthus by studying the localization of the PilQ secretin, the first component of this machine that assembles at the poles. Based on experiments using a combination of fluorescence microscopy, biochemistry, and computational structural analysis, we propose that PilQ, and specifically its AMIN domains, binds septal and polar peptidoglycan, thereby enabling polar Tgl localization, which then stimulates PilQ multimerization in the outer membrane. We also propose that the presence and absence of AMIN domains in T4aP secretins contribute to the different piliation patterns across bacteria., Competing Interests: The authors declare no conflict of interest.

There are few biophysical studies or structural characterizations of the type IV pilin system of extremophile bacteria, such as the acidophilic Acidithiobacillus thiooxidans. We set out to analyze their pili-comprising proteins, pilins, because these extracellular proteins are in constant interaction with protons of the acidic medium in which At. thiooxidans grows. We used the web server Operon Mapper to analyze and identify the cluster codified by the minor pilin of At. thiooxidans. In addition, we carried an in-silico characterization of such pilins using the VL-XT algorithm of PONDR® server. Our results showed that structural disorder prevails more in pilins of At. thiooxidans than in non-acidophilic bacteria. Further computational characterization showed that the pilins of At. thiooxidans are significantly enriched in hydroxy (serine and threonine) and amide (glutamine and asparagine) residues, and significantly reduced in charged residues (aspartic acid, glutamic acid, arginine and lysine). Similar results were obtained when comparing pilins from other Acidithiobacillus and other acidophilic bacteria from another genus versus neutrophilic bacteria, suggesting that these properties are intrinsic to pilins from acidic environments, most likely by maintaining solubility and stability in harsh conditions. These results give guidelines for the application of extracellular proteins of acidophiles in protein engineering., (© 2023. The Author(s), under exclusive licence to Springer Nature Japan KK, part of Springer Nature.)

At the transition to stationary phase, a subpopulation of Bacillus subtilis cells can enter the developmental state of competence, where DNA is taken up through the cell envelope, and is processed to single stranded DNA, which is incorporated into the genome if sufficient homology between sequences exists. We show here that the initial step of transport across the cell wall occurs via a true pilus structure, with an average length of about 500 nm, which assembles at various places on the cell surface. Once assembled, the pilus remains at one position and can be retracted in a time frame of seconds. The major pilin, ComGC, was studied at a single molecule level in live cells. ComGC was found in two distinct populations, one that would correspond to ComGC freely diffusing throughout the cell membrane, and one that is relatively stationary, likely reflecting pilus-incorporated molecules. The ratio of 65% diffusing and 35% stationary ComGC molecules changed towards more stationary molecules upon addition of external DNA, while the number of pili in the population did not strongly increase. These findings suggest that the pilus assembles stochastically, but engages more pilin monomers from the membrane fraction in the presence of transport substrate. Our data support a model in which transport of environmental DNA occurs through the entire cell surface by a dynamic pilus, mediating efficient uptake through the cell wall into the periplasm, where DNA diffuses to a cell pole containing the localized transport machinery mediating passage into the cytosol., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kilb et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Fluid flow is thought to prevent bacterial adhesion, but some bacteria use adhesins with catch bond properties to enhance adhesion under high shear forces. However, many studies on bacterial adhesion either neglect the influence of shear force or use shear forces that are not typically found in natural systems. In this study, we use microfluidics and single-cell imaging to examine how the human pathogen Pseudomonas aeruginosa interacts with surfaces when exposed to shear forces typically found in the human body (0.1 pN to 10 pN). Through cell tracking, we demonstrate that the angle between the cell and the surface predicts if a cell will depart the surface. We discover that at lower shear forces, type IV pilus retraction tilts cells away from the surface, promoting surface departure. Conversely, we show that higher shear forces counterintuitively enhance adhesion by counteracting type IV pilus retraction-dependent cell tilting. Thus, our results reveal that P. aeruginosa exhibits behavior reminiscent of a catch bond, without having a specific adhesin that is enhanced by force. Instead, P. aeruginosa couples type IV pilus dynamics and cell geometry to tune adhesion to its mechanical environment, which likely provides a benefit in dynamic host environments.

Bacterial colonization is mediated by fimbriae, which are thin hair-like appendages dispersed from the bacterial surface. The aggregative adherence fimbriae from enteroaggregative E. coli are secreted through the outer membrane and consist of polymerized minor and major pilin subunits. Currently, the understanding of the structural morphology and the role of the minor pilin subunit in the polymerized fimbriae are limited. In this study we use small-angle X-ray scattering to reveal the structural morphology of purified fimbriae in solution. We show that the aggregative fimbriae are compact arrangements of subunit proteins Agg5A + Agg3B which are assembled pairwise on a flexible string rather than extended in relatively straight filaments. Absence of the minor subunit leads to less compact fimbriae, but did not affect the length. The study provides novel insights into the structural morphology and assembly of the aggregative adherence fimbriae. Our study suggests that the minor subunit is not located at the tip of the fimbriae as previously speculated but has a higher importance for the assembled fimbriae by affecting the global structure., (© 2023. Springer Nature Limited.)

The pilus is an extracellular structural part that can be detected in some Streptococcus pneumoniae ( S. pneumoniae ) isolates (type I pili are found in approximately 30% of strains, while type II pili are found in approximately 20%). It is anchored to the cell wall by LPXTG-like motifs on the peptidoglycan. Two kinds of pili have been discovered, namely, pilus-1 and pilus-2. The former is encoded by pilus islet 1 (PI-1) and is a polymer formed by the protein subunits RrgA, RrgB and RrgC. The latter is encoded by pilus islet 2 (PI-2) and is a polymer composed mainly of the structural protein PitB. Although pili are not necessary for the survival of S. pneumoniae , they serve as the structural basis and as virulence factors that mediate the adhesion of bacteria to host cells and play a direct role in promoting the adhesion, colonization and pathogenesis of S. pneumoniae . In addition, as candidate antigens for protein vaccines, pili have promising potential for use in vaccines with combined immunization strategies. Given the current understanding of the pili of S. pneumoniae regarding the genes, proteins, structure, biological function and epidemiological relationship with serotypes, combined with the immunoprotective efficacy of pilins as protein candidates for vaccines, we here systematically describe the research status and prospects of S. pneumoniae pili and provide new ideas for subsequent vaccine research and development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Miao, Cui, Yan and Jiang.)

We report the synthesis and biological characterization of a novel class of multivalent glycoconjugates as hit compounds for the design of new antiadhesive therapies against urogenital tract infections (UTIs) caused by uropathogenic E. coli strains (UPEC). The first step of UTIs is the molecular recognition of high mannose N-glycan expressed on the surface of urothelial cells by the bacterial lectin FimH, allowing the pathogen adhesion required for mammalian cell invasion. The inhibition of FimH-mediated interactions is thus a validated strategy for the treatment of UTIs. To this purpose, we designed and synthesized d-mannose multivalent dendrons supported on a calixarene core introducing a significant structural change from a previously described family of dendrimers bearing the same dendrons units on a flexible pentaerythritol scaffold core. The new molecular architecture increased the inhibitory potency against FimH-mediated adhesion processes by about 16 times, as assessed by yeast agglutination assay. Moreover, the direct molecular interaction of the new compounds with FimH protein was assessed by on-cell NMR experiments acquired in the presence of UPEC cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Many bacterial surface proteins and carbohydrates are modified with phosphorylcholine (ChoP), which contributes to host mimicry and can also promote colonization and survival in the host. However, the ChoP biosynthetic pathways that are used in bacterial species that express ChoP have not been systematically studied. For example, the well-studied Lic-1 pathway is absent in some ChoP-expressing bacteria, such as Neisseria meningitidis and Neisseria gonorrhoeae. This raises a question as to the origin of the ChoP used for macromolecule biosynthesis in these species. In the current study, we used in silico analyses to identify the potential pathways involved in ChoP biosynthesis in genomes of the 26 bacterial species reported to express a ChoP-modified biomolecule. We used the four known ChoP biosynthetic pathways and a ChoP transferase as search terms to probe for their presence in these genomes. We found that the Lic-1 pathway is primarily associated with organisms producing ChoP-modified carbohydrates, such as lipooligosaccharide. Pilin phosphorylcholine transferase A (PptA) homologs were detected in all bacteria that express ChoP-modified proteins. Additionally, ChoP biosynthesis pathways, such as phospholipid N -methyltransferase (PmtA), phosphatidylcholine synthase (Pcs), or the acylation-dependent phosphatidylcholine biosynthesis pathway, which generate phosphatidylcholine, were also identified in species that produce ChoP-modified proteins. Thus, a major finding of this study is the association of a particular ChoP biosynthetic pathway with a cognate, target ChoP-modified surface factor; i.e., protein versus carbohydrate. This survey failed to identify a known biosynthetic pathway for some species that express ChoP, indicating that a novel ChoP biosynthetic pathway(s) may remain to be identified. IMPORTANCE The modification of bacterial surface virulence factors with phosphorylcholine (ChoP) plays an important role in bacterial virulence and pathogenesis. However, the ChoP biosynthetic pathways in bacteria have not been fully understood. In this study, we used in silico analysis to identify potential ChoP biosynthetic pathways in bacteria that express ChoP-modified biomolecules and found the association between a specific ChoP biosynthesis pathway and the cognate target ChoP-modified surface factor., Competing Interests: The authors declare no conflict of interest.

Multicellular communities of contiguous cells attached to solid surfaces called biofilms represent a common microbial strategy to improve resilience in adverse environments. 1 , 2 , 3 While bacterial biofilms have been under intense investigation, whether archaeal biofilms follow similar assembly rules remains unknown. 4 , 5 Haloferax volcanii is an extremely halophilic euryarchaeon that commonly colonizes salt crust surfaces. H. volcanii produces long and thin appendages called type IV pili (T4Ps). These play a role in surface attachment and biofilm formation in both archaea and bacteria. In this study, we employed biophysical experiments to identify the function of T4Ps in H. volcanii biofilm morphogenesis. H. volcanii expresses not one but six types of major pilin subunits that are predicted to compose T4Ps. Non-invasive imaging of T4Ps in live cells using interferometric scattering (iSCAT) microscopy reveals that piliation varies across mutants expressing single major pilin isoforms. T4Ps are necessary to secure attachment of single cells to surfaces, and the adhesive strength of pilin mutants correlates with their level of piliation. In flow, H. volcanii forms clonal biofilms that extend in three dimensions. Notably, the expression of PilA2, a single pilin isoform, is sufficient to maintain levels of piliation, surface attachment, and biofilm formation that are indistinguishable from the wild type. Furthermore, we discovered that fluid flow stabilizes biofilm integrity; as in the absence of flow, biofilms tend to lose cohesion and disperse in a density-dependent manner. Overall, our results demonstrate that T4P-surface and possibly T4P-T4P interactions promote biofilm formation and integrity and that flow is a key factor regulating archaeal biofilm formation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The type 2 secretion system (T2SS) is a bacterial nanomachine composed of an inner membrane assembly platform, an outer membrane pore and a dynamic endopilus. T2SS endopili are organized into a homo-multimeric body formed by the major pilin capped by a heterocomplex of four minor pilins. The first model of the T2SS endopilus was recently released, even if structural dynamics insights are still required to decipher the role of each protein in the full tetrameric complex. Here, we applied continuous-wave and pulse EPR spectroscopy using nitroxide-gadolinium orthogonal labelling strategies to investigate the hetero-oligomeric assembly of the minor pilins. Overall, our data are in line with the endopilus model even if they evidenced conformational flexibility and alternative orientations at local scale of specific regions of minor pilins. The integration of different labelling strategies and EPR experiments demonstrates the pertinence of this approach to investigate protein-protein interactions in such multiprotein heterocomplexes., (© 2023 Aix-Marseille Univ, CNRS. ChemBioChem published by Wiley-VCH GmbH.)

Dental plaque is a complex microbial biofilm community of many species and a major cause of oral infections and infectious endocarditis. Plaque development begins when primary colonizers attach to oral tissues and undergo coaggregation. Primary colonizers facilitate cellular attachment and inter-bacterial interactions through sortase-dependent pili (or fimbriae) extending out from their cell surface. Consequently, the sortase enzyme is viewed as a potential drug target for controlling biofilm formation and avoiding infection. Streptococcus sanguinis is a primary colonizing bacterium whose pili consist of three different pilin subunits that are assembled together by the pilus-specific (C-type) SsaSrtC sortase. Here, we report on the crystal structure determination of the recombinant wild-type and active-site mutant forms of SsaSrtC. Interestingly, the SsaSrtC structure exhibits an open-lid conformation, although a conserved DPX motif is lacking in the lid. Based on molecular docking and structural analysis, we identified the substrate-binding residues essential for pilin recognition and pilus assembly. We also demonstrated that while recombinant SsaSrtC is enzymatically active toward the five-residue LPNTG sorting motif peptide of the pilins, this activity is significantly reduced by the presence of zinc. We further showed that rutin and α-crocin are potential candidate inhibitors of the SsaSrtC sortase via structure-based virtual screening and inhibition assays. The structural knowledge gained from our study will provide the means to develop new approaches that target pilus-mediated attachment, thereby preventing oral biofilm growth and infection., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The bacterial fimbrial adhesin FimH is a remarkable and well-studied catch-bond protein found at the tip of E. coli type 1 pili, which allows pathogenic strains involved in urinary tract infections to bind high-mannose glycans exposed on human epithelia. The catch-bond behavior of FimH, where the strength of the interaction increases when a force is applied to separate the two partners, enables the bacteria to resist clearance when they are subjected to shear forces induced by urine flow. Two decades of experimental studies performed at the single-molecule level, as well as x-ray crystallography and modeling studies, have led to a consensus picture whereby force separates the binding domain from an inhibitor domain, effectively triggering an allosteric conformational change in the former. This force-induced allostery is thought to be responsible for an increased binding affinity at the core of the catch-bond mechanism. However, some important questions remain, the most challenging one being that the crystal structures corresponding to these two allosteric states show almost superimposable binding site geometries, which questions the molecular origin for the large difference in affinity. Using molecular dynamics with a combination of enhanced-sampling techniques, we demonstrate that the static picture provided by the crystal structures conceals a variety of binding site conformations that have a key impact on the apparent affinity. Crucially, the respective populations in each of these conformations are very different between the two allosteric states of the binding domain, which can then be related to experimental affinity measurements. We also evidence a previously unappreciated but important effect: in addition to the well-established role of the force as an allosteric regulator via domain separation, application of force tends to directly favor the high-affinity binding site conformations. We hypothesize that this additional "local" catch-bond effect could delay unbinding between the bacteria and the host cell before the "global" allosteric transition occurs, as well as stabilizing the complex even more once in the high-affinity allosteric state., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)