Many functions of Pseudomonas aeruginosa type IVa pili, including twitching motility and surface sensing, depend on dynamic cycles of filament assembly and disassembly powered by the cytoplasmic ATPases PilB, PilT, and PilU. Deletion of pilT results in loss of twitching and pilus-specific bacteriophage susceptibility, while non-twitching pilU mutants remain susceptible to pilus-specific phages, indicating that they still produce retractable pili. pilU mutants have high basal levels of the secondary messenger cyclic AMP (cAMP) that normally increases following surface contact, suggesting aberrant surface sensing. To better understand PilU’s role in pilus biology, we solved its X-ray crystal structure and used phylogenetic analyses to identify conserved differences between PilT and PilU. Chemical mutagenesis followed by whole-genome sequencing was used to identify suppressors in the ΔpilU mutant background that restored twitching motility. The mutations mapped to the major pilin, PilA, or the pilus tip adhesin, PilY1. Both the position and nature of the substitutions in PilA impacted restoration of motility, and it was dependent on functional PilT. Complementation of most pilU suppressors with PilU in trans further increased motility, while the expression of wild-type PilA in trans decreased motility in a dose-dependent manner. Notably, cAMP levels remained elevated in most twitching pilU suppressor mutants, showing that surface sensing and motility can be uncoupled. Together, our data suggest that the bacterial response to surfaces reflects a complex interaction of PilU function with specific alleles of PilY1 and PilA that together modulate pilus dynamics and function. IMPORTANCE 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.

ABSTRACT Type IV pili (T4P) are important virulence factors that allow bacteria to adhere to and rapidly colonize their hosts. T4P are primarily composed of major pilins that undergo cycles of extension and retraction and minor pilins that initiate pilus assembly. Bacteriophages use T4P as receptors and exploit pilus dynamics to infect their hosts. Some bacteria encode pilin accessory proteins that post-translationally glycosylate major pilins to evade phage binding. TfpY is an accessory protein of unknown function that is widespread and structurally conserved among T4P-expressing bacteria. Here, we use Pseudomonas aeruginosa as a model to characterize the functional role of TfpY and its homologues in pilus assembly. TfpY expression is required for optimal pilus assembly and function; however, it does not provide phage defence, unlike previously characterized accessory proteins. TfpY can cross-complement twitching in strains expressing heterologous P. aeruginosa pilins, suggesting TfpY and its homologues play a common role in pilus assembly. We showed that TfpY likely interacts with the major pilin and specific minor pilins but is not incorporated into the pilus itself. We propose that TfpY, along with the minor pilins at the pilus tip, primes pilus assembly. We identified two unique gain-of-function mutations in T4P regulatory genes that non-specifically restore twitching in tfpY mutants by increasing levels of cAMP and expression of T4P components. This study enhances our understanding of the complex functional and regulatory relationships between pilin and accessory proteins. IMPORTANCE Type IV pili are surface filaments that enable versatile pathogens, like Pseudomonas aeruginosa , to adhere to and colonize surfaces. Pili are composed of diverse proteins called pilins, which serve as host receptors for phages. P. aeruginosa uses specific accessory proteins to glycosylate pilins to evade phage infection. Here, we show that TfpY is a conserved accessory protein that does not mediate phage defence. Instead, we propose a mechanism where TfpY facilitates efficient pilus assembly and function. A better understanding of TfpY function will provide insight into how its associated pilins have evolved to resist phage infection in the absence of post-translational modification, how some phages overcome this barrier to infection, and how this can guide the design of phage-based therapeutics.

The PA7-clade (or group 3) of Pseudomonas aeruginosa is now recognized as a distinct species, Pseudomonas paraeruginosa. We report here the genomic sequences of six new strains of P. paraeruginosa: Zw26 (the first complete genome of a cystic fibrosis isolate of P. paraeruginosa), draft genomes of four burn and wound strains from Argentina very closely related to PA7, and of Pa5196, the strain in which arabinosylation of type IV pili was documented. We compared the genomes of 82 strains of P. paraeruginosa and confirmed that the species is divided into two sub-clades. Core genomes are very similar, while most differences are found in "regions of genomic plasticity" (RGPs). Several genomic deletions were identified, and most are common to the CR1 sub-clade that includes Zw26 and Pa5196. All strains lack the type 3 secretion system (T3SS) and instead use an alternative virulence strategy involving an exolysin, a characteristic shared with group 5 P. aeruginosa. All strains tend to be multiresistant like PA7, with a significant proportion of carbapenem-resistant strains, either oprD mutants or carrying carbapenemase genes. Although P. paraeruginosa is still relatively rare, it has a worldwide distribution. Its multiresistance and its alternative virulence strategy need to be considered in future therapeutic development.IMPORTANCEPseudomonas aeruginosa is an important opportunistic pathogen causing respiratory infections, notably in cystic fibrosis, and burn and wound infections. Our study reports six new genomes of Pseudomonas paraeruginosa, a new species recently reported as distinct from P. aeruginosa. The number of sequenced genomes of P. paraeruginosa is only about 1% that of P. aeruginosa. We compare the genomic content of nearly all strains of P. paraeruginosa in GenBank, highlighting the differences in core and accessory genomes, antimicrobial resistance genes, and virulence factors. This novel species is very similar in environmental spectrum to P. aeruginosa but is notably resistant to last-line antibiotics and uses an alternative virulence strategy based on exolysin-this strategy being shared with some P. aeruginosa outliers.

BACKGROUND:Antibiotic use during the coronavirus disease 2019 (Covid-19) pandemic was common in the outpatient setting, but was not supported by guidelines. We sought to evaluate the role of this antibiotic use on downstream antibiotic resistance. METHODS:We performed a population-wide cohort study of all nonhospitalized adults 66 years of age or older in Ontario, Canada, from January 1, 2020, to June 30, 2021, with a first identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated the relationship between outpatient peri-Covid-19 antibiotic exposure (within a period of 7 days before or after index SARS-CoV-2 reporting) and downstream isolation of an antibiotic-resistant organism from clinical culture within 6 months. We calculated adjusted odds ratios of the association between peri-Covid-19 prescribing and antibiotic-resistant organism detection, as well as the adjusted attributable fractions of downstream antibiotic-resistant organisms. RESULTS:Of the 53,533 eligible individuals included, 8228 (15%) were prescribed a peri-Covid-19 antibiotic, and 1477 (3%) had a downstream antibiotic-resistant organism identified. The adjusted odds ratio for the presence of any antibiotic-resistant organism with peri-Covid-19 antibiotic use was 1.24 (95% confidence interval [CI], 1.09 to 1.41), while the adjusted odds ratio for the presence of gram-negative antibiotic-resistant organisms was 1.27 (95% CI, 1.11 to 1.46) and for gram-positive antibiotic-resistant organisms it was 1.02 (95% CI, 0.70 to 1.48). Among all individuals who received an antibiotic within 7 days of SARS-CoV-2 diagnosis, the attributable fraction of downstream antimicrobial resistance related to peri-Covid-19 antibiotic use was 17% (95% CI, 7 to 26%). Among all individuals with a SARS-CoV-2 diagnosis, the population-attributable fraction of downstream antimicrobial resistance related to peri-Covid-19 antibiotic use was 4% (95% CI, 2 to 7%). CONCLUSIONS:Peri-Covid-19 antibiotic use was associated with downstream antimicrobial resistance, and particularly the presence of gram-negative antibiotic-resistant organisms. (Funded by the Canadian Institutes of Health Research Operating Grant [grant number 179461] and others).

The type IV pilus family uses PilT/VirB11-like ATPases to rapidly assemble and disassemble pilin subunits. Among these, the tight adherence (Tad) pilus performs both functions using a single bifunctional ATPase, CpaF. Here, we determine three conformationally distinct structures of CpaF hexamers with varying nucleotide occupancies by cryo-electron microscopy. Analysis of these structures suggest ATP binding and hydrolysis expand and rotate the hexamer pore clockwise while subsequent ADP release contracts the ATPase. Truncation of the intrinsically disordered region of CpaF in Caulobacter crescentus equally reduces pilus extension and retraction events observed using fluorescence microscopy, but does not reduce ATPase activity. AlphaFold3 modeling suggests that CpaF and other motors of the type IV filament superfamily employ conserved secondary structural features to engage their respective platform proteins. From these data, we propose that CpaF uses a clockwise, rotary mechanism of catalysis to assemble a right-handed, helical Tad pilus, a process broadly applicable to other single motor systems.

In Pseudomonas aeruginosa, retractable protein filaments called type IV pili (T4P) facilitate surface adherence, sensing, and directional movement known as twitching motility. T4P are necessary for the bacteria to engage in surface-associated behaviors, including establishing acute infections. Pilus extension is driven by the hexameric ATPase, PilB, at the base of the T4P nanomachine in coordination with various protein regulatory effectors. The cyclic-di-GMP binding protein, FimX, works with PilB to mediate normal extension processes, though how this effector controls pilus assembly remains unclear. To explore the role of FimX in T4P function, we leveraged the significant ΔfimX twitching motility deficit to screen for mutants capable of overcoming this phenotype. We identified suppressor mutations that increase twitching in a ΔfimX background, mapping primarily to cyclic-AMP homeostatic machinery or to PilB, the FimX target. Distinct suppressor mutations in PilB increased ATP hydrolysis in vitro and the activity of each suppressor was subject to modulation by FimX. Using microscopy to monitor the extension dynamics of fluorescently labelled T4P, we showed that ΔfimX mutants produce slow-to-extend, short pili, a phenotype that is rescued by mutations enhancing PilB ATP hydrolysis and/or re-introduction of FimX. Together, these data may imply FimX normally acts as a regulator of PilB activity in cells, potentially enabling P. aeruginosa to fine-tune pilus extension dynamics in response to environmental cues.

Chronic urinary tract infections are persistent bacterial infections with the potential to drive antibiotic resistance. Like other persistent bacterial infections, intracellular bacterial reservoirs and biofilm formation hinder the clearance of pathogens despite long courses of antibiotic therapy. New strategies for treatment of these persistent infections are needed. Here we describe the results of an open-label individual patient study using bacteriophage therapy to treat a chronic urinary tract infection in an immunocompetent 72-year-old woman without underlying urolithiasis or indwelling devices. We co-administered HP3.1, HP3, and ES19 bacteriophages with demonstrated in vitro activity via bladder instillation, orally, and as a topical formulation. The primary outcome was safety and tolerability of the treatment. Adverse events were monitored through daily symptom logs and laboratory analysis of blood samples obtained throughout the study. We found that the treatment was safe and well tolerated with no serious adverse events reported. No adverse events were deemed related to the study material. The secondary outcomes were clinical and microbiological efficacy as monitored through daily symptom logs and standard urine culture, respectively. Two weeks after initial clinical improvement, her condition relapsed and culture was consistent with previous isolates. Treatment with ertapenem following bacteriophage therapy led to sustained clinical and microbiologic cure. Exploratory analysis through whole genome sequencing of pre- and post-treatment isolates identified mutations in genes associated with adhesion and evasion that may influence virulence and promote clearance. These results inform expanded randomized clinical trials and support the growing literature that bacteriophage therapies are safe and effective.

Type IVa pili (T4aP) are important virulence factors for many bacterial pathogens. Previous studies suggested that the retraction ATPase, PilT, modulates pathogenicity due to its critical role in pilus dynamics and twitching motility. Here we use a Caenorhabditis elegans slow-killing model to show that hyperpiliation, not loss of pilus retraction, reduces virulence of Pseudomonas aeruginosa strains PAK and PA14. Hyperactivating point mutations in the P. aeruginosa PilSR two-component system that controls transcription of the major pilin gene, pilA, increased levels of surface pili to the same extent as deleting pilT, without impairing twitching motility. These functionally hyperpiliated PilSR mutants had significant defects in pathogenicity that were rescued by deleting pilA or through disruption of hyperpiliation via deletion of the type III secretion system needle-length regulator, PscP. Hyperpiliated pilT deletion or pilO point mutants showed similar PilA-dependent impairments in virulence, validating the phenotype. Together, our data support a model where a surfeit of pili reduces virulence, potentially through the prevention of effective engagement of contact-dependent virulence factors. These findings suggest that the role of T4aP retraction in virulence should be revised. IMPORTANCE Pseudomonas aeruginosa is a major contributor to hospital-acquired infections and particularly problematic due to its intrinsic resistance to many front-line antibiotics. Strategies to combat this and other important pathogens include the development of anti-virulence therapeutics. We show that the pathogenicity of P. aeruginosa is impaired when the amount of T4aP expressed on the cell surface increases, independent of the bacteria's ability to twitch. We propose that having excess T4aP on the cell surface may physically interfere with productive engagement of the contact-dependent type III secretion toxin delivery system. A better understanding of how T4aP modulate interaction of bacteria with target cells will improve the design of therapeutics targeting components involved in the regulation of T4aP expression and function to reduce the clinical burden of P. aeruginosa and other T4aP-expressing bacteria.

ABSTRACT Peptidoglycan (PG) is an important bacterial macromolecule that confers cell shape and structural integrity, and is a key antibiotic target. Its synthesis and turnover are carefully coordinated with other cellular processes and pathways. Despite established connections between the biosynthesis of PG and the outer membrane, or PG and DNA replication, links between PG and folate metabolism remain comparatively unexplored. Folate is an essential cofactor for bacterial growth and is required for the synthesis of many important metabolites. Here we show that inhibition of folate synthesis in the important Gram-negative pathogen Pseudomonas aeruginosa has downstream effects on PG metabolism and integrity that can manifest as the formation of a subpopulation of round cells that can undergo explosive lysis. Folate inhibitors potentiated β-lactams by perturbation of PG recycling, reducing expression of the AmpC β-lactamase. Supporting this mechanism, folate inhibitors also synergized with fosfomycin, an inhibitor of MurA, the first committed step in PG synthesis that can be bypassed by PG recycling. These insights led to the design of a dual-active inhibitor that overcomes NDM-1 metallo-β lactamase-mediated meropenem resistance and synergizes with the folate inhibitor, trimethoprim. We show that folate and PG metabolism are intimately connected, and targeting this connection can overcome antibiotic resistance in Gram-negative pathogens.IMPORTANCETo combat the alarming global increase in superbugs amid the simultaneous scarcity of new drugs, we can create synergistic combinations of currently available antibiotics or chimeric molecules with dual activities, to minimize resistance. Here we show that older anti-folate drugs synergize with specific cell wall biosynthesis inhibitors to kill the priority pathogen, Pseudomonas aeruginosa. Anti-folate drugs caused a dose-dependent loss of rod cell shape followed by explosive lysis, and synergized with β-lactams that target D,D-carboxypeptidases required to tailor the cell wall. Anti-folates impaired cell wall recycling and subsequent downstream expression of the chromosomally encoded β-lactamase, AmpC, which normally destroys β-lactam antibiotics. Building on the anti-folate-like scaffold of a metallo-β-lactamase inhibitor, we created a new molecule, MLLB-2201, that potentiates β-lactams and anti-folates and restores meropenem activity against metallo-β-lactamase-expressing Escherichia coli. These strategies are useful ways to tackle the ongoing rise in dangerous bacterial pathogens.

To cause infection, bacterial pathogens must overcome host immune factors and barriers to nutrient acquisition. Reproducing these aspects of host physiology in vitro has shown great promise for antibacterial drug discovery. When used as a bacterial growth medium, human serum replicates several aspects of the host environment, including innate immunity and iron limitation. We previously reported that a high-throughput chemical screen using serum as the growth medium enabled the discovery of novel growth inhibitors overlooked by conventional screens. Here, we report that a subset of compounds from this high-throughput serum screen display an unexpected growth enhancing phenotype and are enriched for synthetic siderophores. We selected 35 compounds of diverse chemical structure and quantified their ability to enhance bacterial growth in human serum. We show that many of these compounds chelate iron, suggesting they were acting as siderophores and providing iron to the bacteria. For two different pharmacophores represented among these synthetic siderophores, conjugation to the β-lactam antibiotic ampicillin imparted iron-dependent enhancement in antibacterial activity. Conjugation of the most potent growth-enhancing synthetic siderophore with the monobactam aztreonam produced MLEB-22043, a broad-spectrum antibiotic with significantly improved activity against Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa. This synthetic siderophore-monobactam conjugate uses multiple TonB-dependent transporters for uptake into P. aeruginosa. Like aztreonam, MLEB-22043 demonstrated activity against metallo-β-lactamase expressing bacteria, and, when combined with the β-lactamase inhibitor avibactam, was active against clinical strains coexpressing the NDM-1 metallo-β-lactamase and serine β-lactamases. Our work shows that human serum is an effective bacterial growth medium for the high-throughput discovery of synthetic siderophores, enabling the development of novel Trojan Horse antibiotics.

Bacteria invest significant resources into the continuous creation and tailoring of their essential protective peptidoglycan (PG) cell wall. Several soluble PG biosynthesis products in the periplasm are transported to the cytosol for recycling, leading to enhanced bacterial fitness. GlcNAc-1,6-anhydroMurNAc and peptide variants are transported by the essential major facilitator superfamily importer AmpG in Gram-negative pathogens including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Accumulation of GlcNAc-1,6-anhydroMurNAc-pentapeptides also results from β-lactam antibiotic induced cell wall damage. In some species, these products upregulate the β-lactamase AmpC, which hydrolyzes β-lactams to allow for bacterial survival and drug-resistant infections. Here, we have used cryo-electron microscopy and chemical synthesis of substrates in an integrated structural, biochemical, and cellular analysis of AmpG. We show how AmpG accommodates the large GlcNAc-1,6-anhydroMurNAc peptides, including a unique hydrophobic vestibule to the substrate binding cavity, and characterize residues involved in binding that inform the mechanism of proton-mediated transport.

Biofilms are surface-associated communities of bacteria that grow in a self-produced matrix of polysaccharides, proteins, and extracellular DNA (eDNA). Sub-minimal inhibitory concentrations (sub-MIC) of antibiotics induce biofilm formation, potentially as a defensive response to antibiotic stress. However, the mechanisms behind sub-MIC antibiotic-induced biofilm formation are unclear. We show that treatment of Pseudomonas aeruginosa with multiple classes of sub-MIC antibiotics with distinct targets induces biofilm formation. Further, addition of exogenous eDNA or cell lysate failed to increase biofilm formation to the same extent as antibiotics, suggesting that the release of cellular contents by antibiotic-driven bacteriolysis is insufficient. Using a genetic screen for stimulation-deficient mutants, we identified the outer membrane porin OprF and the ECF sigma factor SigX as important. Similarly, loss of OmpA – the Escherichia coli OprF homolog – prevented sub-MIC antibiotic stimulation of E. coli biofilms. Our screen also identified the periplasmic disulfide bond-forming enzyme DsbA and a predicted cyclic-di-GMP phosphodiesterase encoded by PA2200 as essential for biofilm stimulation. The phosphodiesterase activity of PA2200 is likely controlled by a disulfide bond in its regulatory domain, and folding of OprF is influenced by disulfide bond formation, connecting the mutant phenotypes. Addition of reducing agent dithiothreitol prevented sub-MIC antibiotic biofilm stimulation. Finally, activation of a c-di-GMP-responsive promoter follows treatment with sub-MIC antibiotics in the wild-type but not an oprF mutant. Together, these results show that antibiotic-induced biofilm formation is likely driven by a signaling pathway that translates changes in periplasmic redox state into elevated biofilm formation through increases in c-di-GMP.

Infections of the urinary tract are commonAU : PleaseconfirmthatallheadinglevUrinary tract infections (UTIs) rank near the top of the long list of medical issues that can affect humans.They are the most common reason for outpatient visits in the United States (US), and clinics around the world see millions of cases annually.UTIs can be broadly categorized as uncomplicated infections that are usually straightforward to resolve, versus complicated UTIs that because of the pathogen, anatomy or health status of the patient, and/or presence of medical devices such as urinary catheters or stents, are more challenging to resolve.Complicated UTIs are associated with formation of antibiotic-tolerant bacterial or fungal biofilm communities on medical devices during prolonged use, and risk is correlated with duration of device placement [1].Although uncomplicated UTIs are relatively simple to treat, they can be recurrent, defined as 2 or more symptomatic infections in 6 months, or 3 in 12 months, often with the same pathogen [2].If inadequately treated, both types of UTIs can progress to severe disease, including kidney damage and sepsis, with the urinary tract being a major source of bloodstream infections [3,4]. Uncomplicated UTIs disproportionately affect womenMore than half of adult women will have an uncomplicated UTI at some point in their lives, and risk is correlated with increasing age, with a prevalence of approximately 20% in those over 65 years [2].Between 20% and 30% of women will experience a recurrence of their initial UTI within 3 to 4 months [5].These numbers are likely an underestimate, because UTIs are not reportable in most jurisdictions.Women are more susceptible than men due to specific anatomical and physiological differences [6].In women, the urethral opening is closer to the anus, a primary source of bacteria that can cause uncomplicated UTIs.The shorter female urethra means bacteria have less distance to travel from the external opening to reach the bladder.Sexual activity can introduce bacteria into the urethra, and women are more likely to get UTIs after intercourse than men [7].Changes in function of the urogenital tract related to pregnancy, menopause, or use of diaphragms or spermicidal agents for birth control can increase the risk for infection [8].For both men and women, conditions that prevent the bladder from emptying completely-including prolapsed uterus or bladder, the presence of kidney or bladder stones, or medical devices-can lead to urine retention and increased infection risk.Elderly adults or people with diseases such as diabetes, in whom immune function is decreased, are also more susceptible to UTIs [4].

Background: Chlorhexidine gluconate (CHG) and povidone-iodine (PI) are commonly used to prevent prosthetic joint infection (PJI) during total joint replacement; however, their effective concentrations and impact on biofilms are not well defined. Aim: To determine: (1) the in-vitro minimum inhibitory concentration of CHG and PI against model PJI-causing organisms and clinical isolates; (2) their impact on biofilm formation; (3) whether there is a synergistic benefit to combining the two solutions; and (4) whether adding the antibiotic vancomycin impacts antiseptic activity. Methods: We measured in-vitro growth and biofilm formation of Staphylococcus epidermidis, , meticillin-sensitive and meticillin-resistant Staphylococcus aureus, , Escherichia coli, , Pseudomonas aeruginosa and Candida albicans, , as well as recent clinical isolates, in the presence of increasing concentrations of CHG and/or PI. Checkerboard assays were used to measure potential synergy of the solutions together and with vancomycin. Findings: CHG and PI inhibited growth and biofilm formation of all model organisms tested at concentrations of 0.0004% and 0.33% or lower, respectively; highly dilute concentrations paradoxically increased biofilm formation. The solutions did not synergize with one another and acted independently of vancomycin. Conclusion: CHG and PI are effective at lower concentrations than typically used, establishing baselines to support further clinical trials aimed at optimizing wound disinfection. There is no synergistic advantage to using both in combination. Vancomycin is effective at inhibiting the growth of S. epidermidis and S. aureus; ; however, it stimulates P. aeruginosa biofilm production, suggesting in the rare case of P. aeruginosa PJI, it could exacerbate infection. (c) 2024 The Authors. Published by Elsevier Ltd on behalf of The Healthcare Infection Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

The type IV pilus family uses PilT-like ATPases to rapidly assemble and disassemble pilin subunits. Among them, the tight adherence (Tad) pilus performs both functions using a single bi-functional ATPase, CpaF. How ATP catalysis by CpaF facilitates both assembly and disassembly of the Tad pilus remains unclear. Here, we determined electron cryo-microscopy structures of CpaF in three distinct conformations and nucleotide occupancies. Packing unit analyses revealed differences in nucleotide coordination in the active sites, as well as synchronized domain movements during the catalytic cycle. Alphafold3 modeling demonstrated that CpaF and other motors within the type IV filament superfamily use a similar binding interface to engage their respective platform proteins. From these data, we propose that CpaF employs a clockwise, rotary mechanism of catalysis, which couples chemical energy to mechanical force during the biogenesis of Tad pili, a process broadly applicable to other single motor systems. ### Competing Interest Statement The authors have declared no competing interest.

Type IV pili (T4P) produced by the pathogen Pseudomonas aeruginosa play a pivotal role in adhesion, surface motility, biofilm formation, and infection in humans. Despite the significance of T4P as a potential therapeutic target, key details of their dynamic assembly and underlying molecular mechanisms of pilus extension and retraction remain elusive, primarily due to challenges in isolating intact T4P machines from the bacterial cell envelope. Here, we combine cryo-electron tomography with subtomogram averaging and integrative modelling to resolve in-situ architectural details of the dynamic T4P machine in P. aeruginosa cells. The T4P machine forms 7-fold symmetric cage-like structures anchored in the cell envelope, providing a molecular framework for the rapid exchange of major pilin subunits during pilus extension and retraction. Our data suggest that the T4P adhesin PilY1 forms a champagne-cork-shaped structure, effectively blocking the secretin channel in the outer membrane whereas the minor-pilin complex in the periplasm appears to contact PilY1 via the central pore of the secretin gate. These findings point to a hypothetical model where the interplay between the secretin protein PilQ and the PilY1-minor-pilin priming complex is important for optimizing conformations of the T4P machine in P. aeruginosa, suggesting a gate-keeping mechanism that regulates pilus dynamics. Type IV pili (T4P) enable bacteria to sense, move, and adhere to surfaces. Here, the authors solve the structure of the T4P machine in Pseudomonas aeruginosa, revealing the localization and regulatory role of its tip adhesin, PilY1.

The gold standard for the isolation and characterization of bacteriophages (phages), the plaque assay, has remained almost unchanged for over 100 years. The need for improvements to its scalability has been driven home by successes with personalized phage therapy requiring large phage libraries and rapid sensitivity testing. Using a robotic pinning platform, we miniaturized plaque assays from bacterial lawns to micro-colonies from 100 nl of inoculant, increasing throughput by >1000 fold without compromising sensitivity. A comparable manual workflow with one quarter the throughput maintained the same sensitivity. These micro-plaque assays can replace plaque assays as a new gold standard in phage biology. As proof of principle, we used our technique to isolate and de-replicate 21 unique Pseudomonas aeruginosa phages from a single environmental sample. We then demonstrated – using the same assay – that of 17 multi-drug resistant clinical P. aeruginosa strains , 15 were susceptible to infection by one or more of the 21 phages tested. Our method allows rapid isolation and de-replication of phages, as well as enabling screening of large phage libraries against bacterial isolates of interest.

Urinary tract infections (UTIs) are a problem worldwide, affecting almost half a billion people each year. Increasing antibiotic resistance and limited therapeutic options have led to the exploration of alternative therapies for UTIs, including bacteriophage (phage) therapy. This systematic review aims at evaluating the efficacy of phage therapy in treating UTIs. We employed a comprehensive search strategy for any language, any animal, and any publication date. A total of 55 in vivo and clinical studies were included. Of the studies, 22% were published in a non-English language, 32.7% were before the year 1996, and the rest were after 2005. The results of this review suggest that phage therapy for UTIs can be effective; more than 72% of the included articles reported microbiological and clinical improvements. On the other hand, only 5 randomized controlled trials have been completed, and case reports and case series information were frequently incomplete for analysis. Overall, this comprehensive systematic review identifies preliminary evidence supporting the potential of phage therapy as a safe and viable option for the treatment of UTIs.

Exposure of Escherichia coli to sub-inhibitory antibiotics stimulates biofilm formation through poorly characterized mechanisms. Using a high-throughput Congo Red binding assay to report on biofilm matrix production, we screened ~4000 E. coli K12 deletion mutants for deficiencies in this biofilm stimulation response. We screened using three different antibiotics to identify core components of the biofilm stimulation response. Mutants lacking acnA, nuoE, or lpdA failed to respond to sub-MIC cefixime and novobiocin, implicating central metabolism and aerobic respiration in biofilm stimulation. These genes are members of the ArcA/B regulon-controlled by a respiration-sensitive two-component system. Mutants of arcA and arcB had a 'pre-activated' phenotype, where biofilm formation was already high relative to wild type in vehicle control conditions, and failed to increase further with the addition of sub-MIC cefixime. Using a tetrazolium dye and an in vivo NADH sensor, we showed spatial co-localization of increased metabolic activity with sub-lethal concentrations of the bactericidal antibiotics cefixime and novobiocin. Supporting a role for respiratory stress, the biofilm stimulation response to cefixime and novobiocin was inhibited when nitrate was provided as an alternative electron acceptor. Deletion of a gene encoding part of the machinery for respiring nitrate abolished its ameliorating effects, and nitrate respiration increased during growth with sub-MIC cefixime. Finally, in probing the generalizability of biofilm stimulation, we found that the stimulation response to translation inhibitors, unlike other antibiotic classes, was minimally affected by nitrate supplementation, suggesting that targeting the ribosome stimulates biofilm formation in distinct ways. By characterizing the biofilm stimulation response to sub-MIC antibiotics at a systems level, we identified multiple avenues for design of therapeutics that impair bacterial stress management.

Peptidoglycan is an important bacterial macromolecule that confers cell shape and structural integrity, and a key antibiotic target. The synthesis and turnover of peptidoglycan are carefully coordinated with other cellular processes and pathways. Although there are established connections between peptidoglycan and DNA replication or outer membrane biosynthesis, connections between peptidoglycan and folate metabolism are comparatively unexplored. Folate is an essential cofactor for bacterial growth and required for the synthesis of many important metabolites. Here we show that inhibition of folate synthesis in the important Gram-negative pathogen Pseudomonas aeruginosa has downstream effects on peptidoglycan metabolism and integrity. Folate inhibitors reduced expression of the AmpC β-lactamase through perturbation of peptidoglycan recycling, potentiating the activity of β-lactams normally cleaved by that resistance enzyme. Folate inhibitors also synergized with fosfomycin, which inhibits MurA - the first committed step in peptidoglycan synthesis - resulting in dose-dependent formation of round cells that underwent explosive lysis.The insights from this work were used to design a dual-active inhibitor that overcomes NDM-1-mediated meropenem resistance and synergizes with the folate inhibitor, trimethoprim. This work shows that folate and peptidoglycan metabolism are intimately connected and offers new opportunities to exploit this relationship in strategies to overcome antibiotic resistance in Gram-negative pathogens.