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A new mode of swimming in singly flagellated Pseudomonas aeruginosa.

ABSTRACT: SignificanceThe monotrichous Pseudomonas aeruginosa was usually thought to swim in a pattern of "run and reverse" (possibly with pauses in between), where straight runs alternated with reverses with angular changes of swimming direction near 180°. Here, by simultaneously tracking the cell swimming and the morphology of its flagellum, we discovered a swimming mode in P. aeruginosa-the wrap mode, during which the flagellar filament wrapped around the cell body and induced large fluctuation of the body orientation. The wrap mode randomized swimming direction, resulting in a broad distribution of angular changes over 0 to 180° with a peak near 90°. This allowed the bacterium to explore the environment more efficiently, which we confirmed by stochastic simulations of P. aeruginosa chemotaxis.

SUBMITTER: Tian M

PROVIDER: S-EPMC9168846 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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A new mode of swimming in singly flagellated Pseudomonas aeruginosa.

Tian Maojin M Wu Zhengyu Z Zhang Rongjing R Yuan Junhua J

Proceedings of the National Academy of Sciences of the United States of America 20220329 14

SignificanceThe monotrichous Pseudomonas aeruginosa was usually thought to swim in a pattern of "run and reverse" (possibly with pauses in between), where straight runs alternated with reverses with angular changes of swimming direction near 180°. Here, by simultaneously tracking the cell swimming and the morphology of its flagellum, we discovered a swimming mode in P. aeruginosa-the wrap mode, during which the flagellar filament wrapped around the cell body and induced large fluct ...[more]

PMID: 35349348

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Project description:UnlabelledPseudomonas aeruginosais an opportunistic human pathogen that has long been known to chemotax. More recently, it has been established that chemotaxis is an important factor in the ability ofP. aeruginosato make biofilms. Genes that allowP. aeruginosato chemotax are homologous with genes in the paradigmatic model organism for chemotaxis,Escherichia coli However,P. aeruginosais singly flagellated andE. colihas multiple flagella. Therefore, the regulation of counterclockwise/clockwise flagellar motor bias that allowsE. colito efficiently chemotax by runs and tumbles would lead to inefficient chemotaxis byP. aeruginosa, as half of a randomly oriented population would respond to a chemoattractant gradient in the wrong sense. HowP. aeruginosaregulates flagellar rotation to achieve chemotaxis is not known. Here, we analyze the swimming trajectories of single cells in microfluidic channels and the rotations of cells tethered by their flagella to the surface of a variable-environment flow cell. We show thatP. aeruginosachemotaxes by symmetrically increasing the durations of both counterclockwise and clockwise flagellar rotations when swimming up the chemoattractant gradient and symmetrically decreasing rotation durations when swimming down the chemoattractant gradient. Unlike the case forE. coli, the counterclockwise/clockwise bias stays constant forP. aeruginosa We describeP. aeruginosa's chemotaxis using an analytical model for symmetric motor regulation. We use this model to do simulations that show that, givenP. aeruginosa's physiological constraints on motility, its distinct, symmetric regulation of motor switching optimizes chemotaxis.ImportanceChemotaxis has long been known to strongly affect biofilm formation by the opportunistic human pathogenP. aeruginosa, whose essential chemotaxis genes have homologues inE. coli, which achieves chemotaxis by biasing the relative probability of counterclockwise and clockwise flagellar rotation. However, the physiological difference between multiflagellatedE. coliand singly flagellatedP. aeruginosaimplies that biased motor regulation should preventP. aeruginosapopulations from chemotaxing efficiently. Here, we used experiments, analytical modeling, and simulations to demonstrate thatP. aeruginosauses unbiased, symmetric regulation of the flagellar motor to maximize its chemotaxis efficiency. This mode of chemotaxis was not previously known and demonstrates a new variant of a paradigmatic signaling system in an important human pathogen.

Project description:Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections often characterized by robust neutrophilic infiltration. Neutrophils provide the first line of defense against P. aeruginosa. Aside from their defense conferred by phagocytic activity, neutrophils also release neutrophil extracellular traps (NETs) to immobilize bacteria. Although NET formation is an important antimicrobial process, the details of its mechanism are largely unknown. The identity of the main components of P. aeruginosa responsible for triggering NET formation is unclear. In this study, our focus was to identify the main bacterial factors mediating NET formation and to gain insight into the underlying mechanism. We found that P. aeruginosa in its exponential growth phase promoted strong NET formation in human neutrophils while its NET-inducing ability dramatically decreased at later stages of bacterial growth. We identified the flagellum as the primary component of P. aeruginosa responsible for inducing NET extrusion as flagellum-deficient bacteria remained seriously impaired in triggering NET formation. Purified P. aeruginosa flagellin, the monomeric component of the flagellum, does not stimulate NET formation in human neutrophils. P. aeruginosa-induced NET formation is independent of the flagellum-sensing receptors TLR5 and NLRC4 in both human and mouse neutrophils. Interestingly, we found that flagellar motility, not flagellum binding to neutrophils per se, mediates NET release induced by flagellated bacteria. Immotile, flagellar motor-deficient bacterial strains producing paralyzed flagella did not induce NET formation. Forced contact between immotile P. aeruginosa and neutrophils restored their NET-inducing ability. Both the motAB and motCD genetic loci encoding flagellar motor genes contribute to maximal NET release; however the motCD genes play a more important role. Phagocytosis of P. aeruginosa and superoxide production by neutrophils were also largely dependent upon a functional flagellum. Taken together, the flagellum is herein presented for the first time as the main organelle of planktonic bacteria responsible for mediating NET release. Furthermore, flagellar motility, rather than binding of the flagellum to flagellum-sensing receptors on host cells, is required for P. aeruginosa to induce NET release.

Project description:Motility is an important virulence trait for many bacterial pathogens, allowing them to position themselves in appropriate locations at appropriate times. The motility structures type IV pili and flagella are also involved in sensing surface contact, which modulates pathogenicity. In Pseudomonas aeruginosa, the PilS-PilR two-component system (TCS) regulates expression of the type IV pilus (T4P) major subunit PilA, while biosynthesis of the single polar flagellum is regulated by a hierarchical system that includes the FleSR TCS. Previous studies of Geobacter sulfurreducens and Dichelobacter nodosus implicated PilR in regulation of non-T4P-related genes, including some involved in flagellar biosynthesis. Here we used transcriptome sequencing (RNA-seq) analysis to identify genes in addition to pilA with changes in expression in the absence of pilR Among the genes identified were 10 genes whose transcription increased in the pilA mutant but decreased in the pilR mutant, despite both mutants lacking T4P and pilus-related phenotypes. The products of these inversely dysregulated genes, many of which were hypothetical, may be important for virulence and surface-associated behaviors, as mutants had altered swarming motility, biofilm formation, type VI secretion system expression, and pathogenicity in a nematode model. Further, the PilSR TCS positively regulated transcription of fleSR, and thus many genes in the FleSR regulon. As a result, pilSR deletion mutants had defects in swimming motility that were independent of the loss of PilA. Together, these data suggest that in addition to controlling T4P expression, PilSR could have a broader role in the regulation of P. aeruginosa motility and surface sensing behaviors.IMPORTANCE Surface appendages such as type IV pili and flagella are important for establishing surface attachment and infection in a host in response to appropriate cues. The PilSR regulatory system that controls type IV pilus expression in Pseudomonas aeruginosa has an established role in expression of the major pilin PilA. Here we provide evidence supporting a new role for PilSR in regulating flagellum-dependent swimming motility in addition to pilus-dependent twitching motility. Further, even though both pilA and pilR mutants lack PilA and pili, we identified sets of genes downregulated in the pilR mutant and upregulated in a pilA mutant as well as genes downregulated only in a pilR mutant, independent of pilus expression. This finding suggests that change in the inner membrane levels of PilA is only one of the cues to which PilR responds to modulate gene expression. Identification of PilR as a regulator of multiple motility pathways may make it an interesting therapeutic target for antivirulence compounds.

Project description:Pyocin S5 (PyoS5) is a potent protein bacteriocin that eradicates the human pathogen Pseudomonas aeruginosa in animal infection models, but its import mechanism is poorly understood. Here, using crystallography, biophysical and biochemical analyses, and live-cell imaging, we define the entry process of PyoS5 and reveal links to the transport mechanisms of other bacteriocins. In addition to its C-terminal pore-forming domain, elongated PyoS5 comprises two novel tandemly repeated kinked 3-helix bundle domains that structure-based alignments identify as key import domains in other pyocins. The central domain binds the lipid-bound common polysaccharide antigen, allowing the pyocin to accumulate on the cell surface. The N-terminal domain binds the ferric pyochelin transporter FptA while its associated disordered region binds the inner membrane protein TonB1, which together drive import of the bacteriocin across the outer membrane. Finally, we identify the minimal requirements for sensitizing Escherichia coli toward PyoS5, as well as other pyocins, and suggest that a generic pathway likely underpins the import of all TonB-dependent bacteriocins across the outer membrane of Gram-negative bacteria.IMPORTANCE Bacteriocins are toxic polypeptides made by bacteria to kill their competitors, making them interesting as potential antibiotics. Here, we reveal unsuspected commonalities in bacteriocin uptake pathways, through molecular and cellular dissection of the import pathway for the pore-forming bacteriocin pyocin S5 (PyoS5), which targets Pseudomonas aeruginosa In addition to its C-terminal pore-forming domain, PyoS5 is composed of two tandemly repeated helical domains that we also identify in other pyocins. Functional analyses demonstrate that they have distinct roles in the import process. One recognizes conserved sugars projected from the surface, while the other recognizes a specific outer membrane siderophore transporter, FptA, in the case of PyoS5. Through engineering of Escherichia coli cells, we show that pyocins can be readily repurposed to kill other species. This suggests basic ground rules for the outer membrane translocation step that likely apply to many bacteriocins targeting Gram-negative bacteria.

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A new mode of swimming in singly flagellated Pseudomonas aeruginosa.

Publications

A new mode of swimming in singly flagellated <i>Pseudomonas aeruginosa</i>.

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