Biology subjects

Burrows, L. L.

Publications and source records attributed to Burrows, L. L..

At least 19 recordsLinked to original sources

bioRxiv2026

Structural diversification of phage tail fibres enables recognition of diverse type IV pili

Viruses must recognize receptors on host surfaces to initiate infection, but these receptors can evolve rapidly, posing a fundamental challenge to viral persistence. The type IV pilus of Pseudomonas aeruginosa is an ideal model system to study virus-receptor coevolution because its major pilin subunit PilA exhibits extensive sequence and chemical diversity while remaining essential for phage attachment. Here, we combined large-scale comparative genomics with structural and functional analyses to determine how pilus-dependent phages maintain infectivity despite extensive receptor diversification. Pilin variation was concentrated at solvent-exposed regions, altering filament surface chemistry while preserving key subunit-subunit interfaces. Despite this variation, phages recognized divergent pilins more effectively than polyclonal antisera. However, phages differed markedly in their sensitivity to receptor perturbation: some required electrostatic and structural compatibility, whereas others tolerated substantial receptor modification, including the presence of post-translational glycosylation. Comparisons of AlphaFold3 structural models revealed two distinct tail fibre architectures associated with those phenotypes. Phages encoding tail fibres with conserved receptor-binding domains were more sensitive to receptor perturbation within a given pilin background, while those with structurally diversified binding regions infected strains expressing highly divergent pilins. Together, these findings suggest that modular diversification of receptor-binding proteins provides a structural route by which phages accommodate receptor evolution. SIGNIFICANCEUnderstanding the determinants of viral host range is essential for explaining how viruses persist in the face of receptor diversification. Using pilus-dependent bacteriophages that bind diverse type IV pilins in Pseudomonas aeruginosa, we show that differences in tail fibre architecture are associated with distinct sensitivities to receptor variation. Phages with structurally conserved receptor-binding regions are sensitive to receptor perturbation, whereas those with diversified binding architectures remain infectious across strains expressing highly divergent pilins. Our results identify a structural correlate of receptor recognition breadth and reveal how receptor-binding protein architecture contributes to viral host range and adaptation. These data can inform the selection or engineering of broad-host range phages for therapeutic applications.

microbiology↗

bioRxiv2026

A broad-spectrum phage-encoded mechanism to disarm bacterial type IV filaments

Phages can modify host cell physiology to thwart competitors. The Pseudomonas aeruginosa-specific phage DMS3 encodes Aqs1, a protein inhibitor of type IV pilus (T4P) function to prevent host cell recognition by other phages that leverage these filaments for infection. Aqs1 disrupts T4P by binding to the hexameric ATPase PilB, required to power pilus filament extension, though several mechanistic details remain unclear. We show that Aqs1 has broad-spectrum activity and can disrupt T4P function in a variety of Gram negative bacteria. This protein inhibits PilB by binding to a solvent-exposed hydrophobic patch on the N2-domain, distal to the active site. Binding destabilizes the hexamer, preventing PilB accumulation at T4P machines. Aqs1 likely disrupts PilB oligomerization by displacing a flexible linker segment between the PilB N1- and N2-domains required for inter-subunit contact. Together, the Aqs1 mode of action provides a design template for broad-spectrum inhibitors of diverse bacterial virulence factors. SignificanceThe phage-encoded protein Aqs1 disables type IV pilus (T4P) production in Pseudomonas aeruginosa by targeting the hexameric ATPase responsible for assembling pilus fibers. We show that despite originating from a P. aeruginosa-specific phage, Aqs1 can also disable T4 ATPase-dependent phenotypes across other pathogenic bacteria and homologous systems. Mechanistically, Aqs1 binds to a conserved patch on the PilB N2-domain away from the active site. Binding here breaks apart the PilB oligomer, preventing it from acting on T4P machines. Aqs1 binding at the N2-domain patch likely displaces a flexible PilB linker segment that binds to this site to stabilize the hexamer. Our work highlights a novel and conserved PilB allosteric site which is exploited by the phage-encoded protein Aqs1 to disrupt diverse T4 systems in multiple bacteria.

microbiology↗

bioRxiv2026

Vacidobactin A: An anti-Pseudomonas aeruginosa siderophore

Multidrug-resistant (MDR) Pseudomonas aeruginosa poses a significant clinical challenge due to its poorly permeable outer membrane, efflux systems, biofilm formation, and rapid acquisition of resistance genes. The lack of new treatments for P. aeruginosa infections underscores the necessity for innovative therapeutic solutions. Iron uptake is essential for bacterial survival, making it a promising target for the development of new antimicrobials. Iron-chelating siderophores are vital for bacterial iron acquisition, important agents for direct antimicrobial action, adjuvants to enhance the effectiveness of currently available antibiotics, and components of prodrugs that facilitate the transport of covalently linked antibiotics into the cell. Here, we report the anti-pseudomonal activity of vacidobactin A, a siderophore produced by the soil bacterium Variovorax paradoxus, identified through a screen of natural product extracts targeting a clinical MDR strain of P. aeruginosa. Vacidobactin A inhibits P. aeruginosa growth by limiting iron availability, particularly in strains that do not produce pyoverdine, their native siderophore. Expression of a TonB-dependent transporter sourced from the vacidobactin producer in a P. aeruginosa pyoverdine and pyochelin-null mutant restored its ability to acquire iron and grow in the presence of vacidobactin. Additionally, vacidobactin A synergized with thiostrepton, which hijacks pyoverdine receptors to enter the cell and inhibit protein synthesis. This study supports the therapeutic potential of targeting P. aeruginosa iron acquisition pathways and leveraging siderophores as adjuvants to enhance the efficacy of existing antimicrobials. These findings, along with recent advancements in siderophore-based research and combination therapies, offer innovative strategies to combat antibiotic-resistant infections. IMPORTANCEMultidrug-resistant Pseudomonas aeruginosa is a critical priority pathogen for which new therapeutic strategies are urgently needed. Iron acquisition is essential for P. aeruginosa survival and virulence, yet remains underexploited as a drug target. Here, we demonstrate that vacidobactin A, a siderophore produced by Variovorax paradoxus, suppresses P. aeruginosa growth by limiting iron availability, particularly in strains deficient in pyoverdine production. We further show that vacidobactin A enhances the activity of thiostrepton, an antibiotic that exploits siderophore uptake pathways. These findings highlight iron competition as a source of anti-pseudomonal agents and support the development of siderophore-based therapeutics and adjuvant strategies. Targeting iron acquisition networks offers a mechanistically distinct approach to combat antibiotic resistance and expands the repertoire of vulnerabilities that can be leveraged against this highly drug-resistant pathogen.

microbiology↗

bioRxiv2026

Reversible biological motion with unidirectional catalysis through inversion of ATPase orientation

Biological and engineered machines generally achieve reversibility through regulated switches in the directionality of fixed-orientation motors. Bacterial Tad pilus nanomachines extend and retract pili using a single motor with a unidirectional catalytic mechanism, a capability with no precedent. Using AlphaFold3 modeling, comparative structural analyses, and pilus activity assays, we find that the Tad motor ATPase CpaF achieves bidirectionality through physical inversion; alternating which face of the ATPase toroid engages the platform complex. The two orientations contact the pilus machinery in mutually exclusive extension- or retraction-specific configurations that drive reversible pilus dynamics using the same unidirectional catalytic cycle. Retraction requires conserved C-terminal residues in CpaF whose nucleotide-driven motions oppose those of the extension interface. Thus, nature has adopted a solution for reversible movement not yet conceived by human engineering.

microbiology↗

bioRxiv2026

BAY 11-7082 potentiates select β-lactams to inhibit growth of methicillin-resistant Staphylococcus aureus.

{beta}-lactams are an important class of antibiotics that target penicillin-binding proteins (PBPs) essential to bacterial cell wall synthesis. They are used to treat serious bacterial infections, including those caused by the versatile pathogen Staphylococcus aureus. Some strains carry the resistance gene, mecA, encoding a {beta}-lactam-insensitive PBP2A that allows for peptidoglycan synthesis in the presence of {beta}-lactams like methicillin, limiting treatment options. We previously identified BAY 11-7082 as having antibacterial activity against methicillin-resistant S. aureus (MRSA) and showed that it re-sensitizes MRSA to {beta}-lactams like penicillin G, making BAY 11-7082 and its analogues promising candidates for the development of an antibiotic adjuvant. Although the direct antibacterial mechanism of BAY 11-7082 remains undefined, here we aimed to better understand how it potentiates {beta}-lactam activity. We tested BAY 11-7082 in combination with a variety of {beta}-lactams and identified a subset that were synergistic in MRSA but not methicillin-susceptible S. aureus, suggesting they may directly or indirectly impact the function of PBP2A. Electron and fluorescence microscopy studies revealed that unlike other {beta}-lactam adjuvants, such as those that target the biosynthesis of wall teichoic acids (WTAs), BAY 11-7082 failed to impact cell division, disrupt PBP2 localization, or activate a sensitive reporter of cell wall damage. However, the production of WTA was necessary for BAY 11-7082-{beta}-lactam synergy. Together these data suggest its mechanism of {beta}-lactam potentiation is distinct from known compounds, making BAY 11-7082 a useful tool to better understand the complexity of resistance in MRSA.

microbiology↗

bioRxiv2025

The repertoire of resistance mutations selected by a Pseudomonas aeruginosa type IV pilus-targeting lytic bacteriophage

P. aeruginosa type IV pili (T4P) are complex surface-exposed nanomachines used for twitching motility and biofilm formation. Exposure to bacteriophages that use T4P as their primary receptor can select for resistance mutations that reduce or abolish pilus expression. Since T4P are important virulence factors, such loss can affect pathogenicity and fitness, desirable outcomes for phage therapy. Here, we evaluated the repertoire of mutations in P. aeruginosa PAO1 exposed to the pilus-specific lytic phage PO4 and their ability to revert after removal of phage. Resistant isolates ranged from hyperpiliated to bald, but none retained twitching motility. We focused on isolates that still expressed surface pili, including one with an internal 4-amino acid duplication in the essential prepilin peptidase/methyltransferase, PilD. This duplication, distal to the predicted active sites, delayed prepilin processing. Accumulation of unprocessed subunits suppressed expression of new prepilins via inhibition of PilSR two-component system activity, restricting availability of functional subunits and conferring phage resistance. Introduction of a PilS point mutation that makes cells insensitive to accumulation of unprocessed pilins restored motility and phage susceptibility. Of the mutants tested, only those with a duplication in pilD recovered wild-type motility following removal of phage pressure, likely through slip-strand mispairing as a codon-modified variant did not revert. This work shows that resistance to pilus-specific phages does not require loss of pilus expression, and certain mutations can allow bacteria to regain pilus function. Characterizing spontaeous mutations selected by phages can help to define the function and vulnerabilities of the type IV pilus system. SIGNIFICANCEAs use of phages to treat antibiotic-resistant pathogens such as Pseudomonas aeruginosa increases, it is important to understand potential outcomes of phage exposure. Most therapeutic P. aeruginosa phages use lipopolysaccharides (LPS) or T4P as primary receptors. Studying the properties of strains resistant to T4P-targeting phages can guide the design of phage cocktails to mitigate treatment resistance. We show that depending on the mutation, some phage-resistant strains can revert to wild-type sequences, emphasizing the importance of combining diverse phages to supress resurgence. By characterizing mutations that confer resistance, we can better understand whether pilus structural or regulatory components are more likely to be lost. Using phages to select for loss of pilus function represents an unbiased approach to identify new mutations in pilus-related proteins, shedding light on understudied components. Building a database of such mutations will help guide strategies to target and disarm this key P. aeruginosa virulence factor.

microbiology↗

bioRxiv2025

Twitching motility suppressors reveal a role for FimX in type IV pilus extension dynamics

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 {Delta}fimX twitching motility deficit to screen for mutants capable of overcoming this phenotype. We identified suppressor mutations that increase twitching in {Delta}fimX 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 this activity was subject to modulation by FimX. Using microscopy to monitor the extension dynamics of fluorescently labelled T4P, we showed that {Delta}fimX 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 implicate FimX as a regulator of PilB enzymatic function, potentially enabling P. aeruginosa to fine-tune pilus extension dynamics in response to environmental cues. SummaryType IV pili enable Pseudomonas aeruginosa to attach to surfaces, move (twitch), and form biofilms. Pilus extension is powered by the motor protein PilB, which is regulated by other factors, including FimX, a protein that binds cyclic-di-GMP. Although FimX is important for twitching, how it influences PilB was unclear. We deleted fimX, which severely reduces motility, and searched for mutants that regained movement. We identified two types: some had mutations in PilB that increased its ATPase activity, allowing it to function without FimX, while others affected the cyclic-AMP signaling pathway and increased overall production of pilus components, showing that motility can also be improved through changes in quantity versus quality. Our results suggest that FimX normally fine-tunes PilB enzymatic activity, enabling dynamic control of pilus extension in response to surface signals. This work helps explain how P. aeruginosa adapts to different environments, a process crucial for infection and biofilm development.

microbiology↗

bioRxiv2024

Coordinated conformational changes in the Tad pilus ATPase CpaF facilitate a rotary mechanism of catalysis

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 bifunctional 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.

microbiology↗

bioRxiv2024

Micro-plaque assays: A high-throughput method to detect, isolate, and characterize bacteriophages

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.

microbiology↗

bioRxiv2023

Metabolic connections between folate and peptidoglycan pathways in Pseudomonas aeruginosa inform rational design of a dual-action inhibitor

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 {beta}-lactamase through perturbation of peptidoglycan recycling, potentiating the activity of {beta}-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.

microbiology↗

bioRxiv2023

A genetic screen identifies a role for oprF in Pseudomonas aeruginosa biofilm stimulation by subinhibitory antibiotics

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, indicating a potential 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 to find stimulation-deficient mutants, we identified the outer membrane porin OprF and the extracytoplasmic function sigma factor SigX as important for the phenotype. Similarly, loss of OmpA - the Escherichia coli OprF homologue - prevented sub-MIC antibiotic stimulation of E. coli biofilms. The C-terminal PG-binding domain of OprF was dispensable for biofilm stimulation. 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 the reducing agent dithiothreitol prevented sub-MIC antibiotic biofilm stimulation. Finally, we show that 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 signalling pathway that translates changes in periplasmic redox state into elevated biofilm formation through increases in c-di-GMP. SignificanceBacterial biofilms cause significant treatment challenges in clinical settings due to their ability to resist antibiotic killing. Paradoxically, sub-inhibitory levels of a number of chemically distinct antibiotics, with different modes of action, can stimulate biofilm formation of multiple bacterial species. In this work we used the biofilm forming opportunistic pathogen Pseudomonas aeruginosa to look for mutants blind to sub-inhibitory antibiotic exposure, failing to demonstrate biofilm stimulation in response to 3 different antibiotics. We identified key players in this response, including the outer membrane porin OprF and the sigma factor SigX. Notably, the hits from our mutant screen connect changes in periplasmic redox state to elevated biofilm formation via c-di-GMP signaling. By understanding these underlying mechanisms, we can better strategize interventions against biofilm-mediated antibiotic resistance in P. aeruginosa and other bacterial species.

microbiology↗

bioRxiv2023

Nutrient Limitation Sensitizes Pseudomonas aeruginosa to Vancomycin

Traditional antibacterial screens rely on growing bacteria in nutrient-replete conditions which are not representative of the natural environment or sites of infection. Instead, screening in more physiologically relevant conditions may reveal novel activity for existing antibiotics. Here, we screened a panel of antibiotics reported to lack activity against the opportunistic Gram-negative bacterium, Pseudomonas aeruginosa, under low-nutrient conditions, and discovered that the glycopeptide vancomycin inhibited growth of P. aeruginosa at low micromolar concentrations through its canonical mechanism of action, disruption of peptidoglycan cross-linking. Spontaneous vancomycin-resistant mutants had activating mutations in the sensor kinase of the two-component CpxSR system, which induced cross-resistance to almost all classes of y-lactams, including the siderophore antibiotic cefiderocol. Other mutations that conferred vancomycin resistance mapped to WapR, an -1,3-rhamnosyltransferase involved in lipopolysaccharide core biosynthesis. A WapR P164T mutant had a modified LPS profile compared to wild type that was accompanied by increased susceptibility to select bacteriophages. We conclude that screening in nutrient-limited conditions can reveal novel activity for existing antibiotics and lead to discovery of new and impactful resistance mechanisms. For Table of Contents Use Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/536232v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1119ebeorg.highwire.dtl.DTLVardef@1ccd6c0org.highwire.dtl.DTLVardef@126f3deorg.highwire.dtl.DTLVardef@54a9c0_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

A single extracellular loop of FoxA controls ligand specificity, uptake, and signaling in Pseudomonas aeruginosa

The outer membrane of Gram-negative bacteria prevents many antibiotics from reaching intracellular targets. However, some antimicrobials can take advantage of iron import transporters to cross this barrier. We showed previously that the thiopeptide antibiotic, thiocillin, exploits the nocardamine (ferrioxamine E) xenosiderophore transporter, FoxA, of the opportunistic pathogen Pseudomonas aeruginosa for uptake. Here we show that FoxA also transports the xenosiderophore bisucaberin and describe at 2.5 [A] resolution the first crystal structure of bisucaberin bound to FoxA. Bisucaberin is distinct from other siderophores because it forms a 3:2 rather than 1:1 siderophore-iron complex. Mutations in a single extracellular loop of FoxA differentially affected nocardamine, thiocillin, and bisucaberin binding, uptake, and signal transduction. These results show that in addition to modulating ligand binding, the extracellular loops of siderophore transporters are of fundamental importance for controlling ligand uptake and its regulatory consequences, which has implications for the development of siderophore-antibiotic conjugates to treat difficult infections.

microbiology↗

bioRxiv2022

Pseudomonas aeruginosa FpvB is a high-affinity transporter for xenosiderophores ferrichrome and ferrioxamine B

Iron is essential for many biological functions in bacteria but its poor solubility is a limiting factor for growth. Bacteria produce siderophores, soluble natural products that bind iron with high affinity, to overcome this challenge. Siderophore-iron complexes return to the cell through specific outer-membrane transporters. The opportunistic pathogen Pseudomonas aeruginosa makes multiple transporters that recognize its own siderophores, pyoverdine and pyochelin, and xenosiderophores produced by other bacteria, which gives it a competitive advantage. Some antibiotics exploit these transporters to bypass the membrane to reach their intercellular targets - including the thiopeptide antibiotic, thiostrepton (TS), which uses the pyoverdine transporters FpvA and FpvB to cross the outer membrane. Here, we assessed TS susceptibility in the presence of various siderophores and discovered that ferrichrome and ferrioxamine B antagonized TS uptake via FpvB. Unexpectedly, we found that FpvB transports ferrichrome and ferrioxamine B with higher affinity than pyoverdine. Site-directed mutagenesis of FpvB coupled with competitive growth inhibition and affinity label quenching studies suggested that the siderophores and antibiotic share a common binding site in an aromatic pocket formed by the plug and barrel domains, but have differences in their binding mechanism and molecular determinants for uptake. This work describes an alternative uptake pathway for ferrichrome and ferrioxamine B in P. aeruginosa and emphasizes the promiscuity of siderophore transporters, with implications for Gram-negative antibiotic development via the Trojan horse approach.

microbiology↗

bioRxiv2022

Central metabolism is a key player in E. coli biofilm stimulation by sub-MIC antibiotics

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. Mutants lacking acnA, nuoE, or lpdA failed to respond to sub-MIC 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 antibiotics. Supporting a role for respiratory stress, the biofilm stimulation response was inhibited when nitrate was provided as an alternative electron acceptor. Deletion of genes encoding the nitrate respiratory machinery abolished its effects, and nitrate respiration increased during growth with sub-MIC antibiotics. In probing the generalizability of biofilm stimulation, we found that the stimulation response to translation inhibitors was minimally affected by nitrate supplementation. Finally, using a metabolism-sensitive dye, we showed spatial co-localization of increased respiration with sub-MIC bactericidal antibiotics. 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.

microbiology↗

bioRxiv2021

Exploration of 1 BAY 11-7082 as a novel antibiotic

Exposure of the Gram-negative pathogen Pseudomonas aeruginosa to sub-inhibitory concentrations of antibiotics increases formation of biofilms. We exploited this phenotype to identify molecules with potential antimicrobial activity in a biofilm-based high-throughput screen. The anti-inflammatory compound BAY 11-7082 induced dose-dependent biofilm stimulation, indicative of antibacterial activity. We confirmed that BAY 11-7082 inhibits growth of P. aeruginosa and other priority pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). We synthesized 27 structural analogues, including a series based on the related scaffold 3-(phenylsulfonyl)-2-pyrazinecarbonitrile (PSPC), 10 of which displayed increased anti-Staphylococcal activity. Because the parent molecule inhibits the NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome, we measured the ability of select analogues to reduce IL-1{beta} production in mammalian macrophages, identifying minor differences in the structure-activity relationship for the anti-inflammatory and antibacterial properties of this scaffold. Although we could evolve stably resistant MRSA mutants with cross resistance to BAY 11-7082 and PSPC, their lack of shared mutations suggested that the two molecules could have multiple targets. Finally, we showed that BAY 11-7082 and its analogues potentiate the activity of penicillin G against MRSA, suggesting that this scaffold may serve as an interesting starting point for the development of antibiotic adjuvants. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/462244v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1387110org.highwire.dtl.DTLVardef@86f1ddorg.highwire.dtl.DTLVardef@170edaborg.highwire.dtl.DTLVardef@103a36d_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2021

Hyperpiliation reduces Pseudomonas aeruginosa pathogenicity by impeding type III secretion

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 by interfering with function of the contact-dependent type III secretion system (T3SS). 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 by increasing the length of T3SS needles via deletion of the 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 prevents effective engagement of contact-dependent virulence factors. These findings suggest that the role of T4aP retraction in virulence should be revised. SIGNIFICANCEPseudomonas 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 development of anti-virulence therapeutics. We show that the pathogenicity of P. aeruginosa is impaired when the amount of type IVa pili (T4aP) expressed on the cell surface increases, independent of the bacterias ability to twitch. We propose that having excess T4aP on the cell surface can 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 regulation of T4aP expression and function, to reduce the clinical burden of P. aeruginosa and other T4aP-expressing bacteria.

microbiology↗

bioRxiv2020

TsaP binds PilQ and is involved in cyclic di-GMP signaling

The type IV pilus machinery is a multi-protein complex that polymerizes and depolymerizes a pilus fibre used for attachment, twitching motility, phage adsorption, natural competence, protein secretion, and surface-sensing. An outer membrane secretin pore is required for passage of the pilus fibre out of the cell. Herein, the structure of the tetradecameric secretin, PilQ, from the Pseudomonas aeruginosa type IVa pilus system was determined to 4.3 [A] and 4.4 [A] resolution in the presence and absence of C7 symmetric spokes, respectively. The heptameric spokes were found to be the two tandem C-terminal domains of TsaP. TsaP forms a belt around PilQ and while the protein is not essential for twitching motility, over-expression of TsaP triggers a signal cascade upstream of PilY1 leading to cyclic di-GMP up-regulation. These results resolve the identity of the spokes identified with Proteobacterial PilQ homologs and may reveal a new component of the surface-sensing cyclic di-GMP signal cascade. IMPACT STATEMENTThe type IV pilus is critical for bacterial virulence. The co-structure of the pilus secretin PilQ and TsaP is determined. Characterization of TsaP implicates it in surface-sensing signal transduction.

microbiology↗