Dataset Information

The type IV pilus assembly ATPase PilB functions as a signaling protein to regulate exopolysaccharide production in Myxococcus xanthus.

ABSTRACT: Myxococcus xanthus possesses a form of surface motility powered by the retraction of the type IV pilus (T4P). Additionally, exopolysaccharide (EPS), the major constituent of bacterial biofilms, is required for this T4P-mediated motility in M. xanthus as the putative trigger of T4P retraction. The results here demonstrate that the T4P assembly ATPase PilB functions as an intermediary in the EPS regulatory pathway composed of the T4P upstream of the Dif signaling proteins in M. xanthus. A suppressor screen isolated a pilB mutation that restored EPS production to a T4P^- mutant. An additional PilB mutant variant, which is deficient in ATP hydrolysis and T4P assembly, supports EPS production without the T4P, indicating PilB can regulate EPS production independently of its function in T4P assembly. Further analysis confirms that PilB functions downstream of the T4P filament but upstream of the Dif proteins. In vitro studies suggest that the nucleotide-free form of PilB assumes the active signaling conformation in EPS regulation. Since M. xanthus PilB possesses conserved motifs with high affinity for c-di-GMP binding, the findings here suggest that c-di-GMP can regulate both motility and biofilm formation through a single effector in this surface-motile bacterium.

SUBMITTER: Black WP

PROVIDER: S-EPMC5544727 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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The type IV pilus assembly ATPase PilB functions as a signaling protein to regulate exopolysaccharide production in Myxococcus xanthus.

Black Wesley P WP Wang Lingling L Jing Xing X Saldaña Rafael Castañeda RC Li Feng F Scharf Birgit E BE Schubot Florian D FD Yang Zhaomin Z

Scientific reports 20170804 1

Myxococcus xanthus possesses a form of surface motility powered by the retraction of the type IV pilus (T4P). Additionally, exopolysaccharide (EPS), the major constituent of bacterial biofilms, is required for this T4P-mediated motility in M. xanthus as the putative trigger of T4P retraction. The results here demonstrate that the T4P assembly ATPase PilB functions as an intermediary in the EPS regulatory pathway composed of the T4P upstream of the Dif signaling proteins in M. xanthus. A suppress ...[more]

PMID: 28779124

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Project description:With the pressing antibiotic resistance pandemic, antivirulence has been increasingly explored as an alternative strategy against bacterial infections. The bacterial type IV pilus (T4P) is a well-documented virulence factor and an attractive target for small molecules for antivirulence purposes. The PilB ATPase is essential for T4P biogenesis because it catalyzes the assembly of monomeric pilins into the polymeric pilus filament. Here, we describe the identification of two PilB inhibitors by a high-throughput screen (HTS) in vitro and their validation as effective inhibitors of T4P assembly in vivo. We used Chloracidobacterium thermophilum PilB as a model enzyme to optimize an ATPase assay for the HTS. From a library of 2,320 compounds, benserazide and levodopa, two approved drugs for Parkinson's disease, were identified and confirmed biochemically to be PilB inhibitors. We demonstrate that both compounds inhibited the T4P-dependent motility of the bacteria Myxoccocus xanthus and Acinetobacter nosocomialis. Additionally, benserazide and levodopa were shown to inhibit A. nosocomialis biofilm formation, a T4P-dependent process. Using M. xanthus as a model, we showed that both compounds inhibited T4P assembly in a dose-dependent manner. These results suggest that these two compounds are effective against the PilB protein in vivo. The potency of benserazide and levodopa as PilB inhibitors both in vitro and in vivo demonstrate potentials of the HTS and its two hits here for the development of anti-T4P chemotherapeutics. IMPORTANCE Many bacterial pathogens use their type IV pilus (T4P) to facilitate and maintain an infection in a human host. Small-molecule inhibitors of the production or assembly of the T4P are promising for the treatment and prevention of infections by these bacteria, especially in our fight against antibiotic-resistant pathogens. Here, we report the development and implementation of a method to identify anti-T4P chemicals from compound libraries by high-throughput screen. This led to the identification and validation of two T4P inhibitors both in the test tubes and in bacteria. The discovery and validation pipeline reported here as well as the confirmation of two anti-T4P inhibitors provide new venues and leads for the development of chemotherapeutics against antibiotic-resistant infections.

Project description:Myxococcus xanthus arranges into two morphologically distinct biofilms depending on its nutritional status, i.e., coordinately spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. A secreted polysaccharide, referred to as exopolysaccharide (EPS), is a structural component of both biofilms and is also important for type IV pilus-dependent motility and fruiting body formation. Here, we characterize the biosynthetic machinery responsible for EPS biosynthesis using bioinformatics, genetics, heterologous expression, and biochemical experiments. We show that this machinery constitutes a Wzx/Wzy-dependent pathway dedicated to EPS biosynthesis. Our data support that EpsZ (MXAN_7415) is the polyisoprenyl-phosphate hexose-1-phosphate transferase responsible for the initiation of the repeat unit synthesis. Heterologous expression experiments support that EpsZ has galactose-1-P transferase activity. Moreover, MXAN_7416, renamed WzxEPS, and MXAN_7442, renamed WzyEPS, are the Wzx flippase and Wzy polymerase responsible for translocation and polymerization of the EPS repeat unit, respectively. In this pathway, EpsV (MXAN_7421) also is the polysaccharide copolymerase and EpsY (MXAN_7417) the outer membrane polysaccharide export (OPX) protein. Mutants with single in-frame deletions in the five corresponding genes had defects in type IV pilus-dependent motility and a conditional defect in fruiting body formation. Furthermore, all five mutants were deficient in type IV pilus formation, and genetic analyses suggest that EPS and/or the EPS biosynthetic machinery stimulates type IV pilus extension. Additionally, we identify a polysaccharide biosynthesis gene cluster, which together with an orphan gene encoding an OPX protein make up a complete Wzx/Wzy-dependent pathway for synthesis of an unknown polysaccharide.IMPORTANCE The secreted polysaccharide referred to as exopolysaccharide (EPS) has important functions in the social life cycle of M. xanthus; however, little is known about how EPS is synthesized. Here, we characterized the EPS biosynthetic machinery and showed that it makes up a Wzx/Wzy-dependent pathway for polysaccharide biosynthesis. Mutants lacking a component of this pathway had reduced type IV pilus-dependent motility and a conditional defect in development. These analyses also suggest that EPS and/or the EPS biosynthetic machinery is important for type IV pilus formation.

Project description:The regulation of biofilm and motile states as alternate bacterial lifestyles has been studied extensively in flagellated bacteria, where the second messenger cyclic-di-GMP (cdG) plays a crucial role. However, much less is known about the mechanisms of such regulation in motile bacteria without flagella. The bacterial type IV pilus (T4P) serves as a motility apparatus that enables Myxococcus xanthus to move on solid surfaces. PilB, the T4P assembly ATPase, is, therefore, required for T4P-dependent motility in M. xanthus. Interestingly, T4P is also involved in the regulation of exopolysaccharide as the biofilm matrix material in this bacterium. A newly discovered cdG-binding domain, MshEN, is conserved in the N-terminus of PilB (PilBN) in M. xanthus and other bacteria. This suggests that cdG may bind to PilB to control the respective outputs that regulate biofilm development and T4P-powered motility. In this study, we aimed to validate M. xanthus PilB as a cdG effector protein. We performed a systematic mutational analysis of its cdG-binding domain to investigate its relationship with motility, piliation, and biofilm formation. Excluding those resulting in low levels of PilB protein, all other substitution mutations in PilBN resulted in pilB mutants with distinct and differential phenotypes in piliation and biofilm levels in M. xanthus. This suggests that the PilBN domain plays dual roles in modulating motility and biofilm levels, and these two functions of PilB can be dependent on and independent of each other in M. xanthus. IMPORTANCE The regulation of motility and biofilm by cyclic-di-GMP in flagellated bacteria has been extensively investigated. However, our knowledge regarding this regulation in motile bacteria without flagella remains limited. Here, we aimed to address this gap by investigating a non-flagellated bacterium with motility powered by bacterial type-IV pilus (T4P). Previous studies hinted at the possibility of Myxococcus xanthus PilB, the T4P assembly ATPase, serving as a cyclic-di-GMP effector involved in regulating both motility and biofilm. Our findings strongly support the hypothesis that PilB directly interacts with cyclic-di-GMP to act as a potential switch to promote biofilm formation or T4P-dependent motility. These results shed light on the bifurcation of PilB functions and its pivotal role in coordinating biofilm formation and T4P-mediated motility.

Dataset Information

The type IV pilus assembly ATPase PilB functions as a signaling protein to regulate exopolysaccharide production in Myxococcus xanthus.

Publications

The type IV pilus assembly ATPase PilB functions as a signaling protein to regulate exopolysaccharide production in Myxococcus xanthus.

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