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1.
Antimicrob Agents Chemother ; 64(3)2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-31907180

RESUMO

Pseudomonas aeruginosa is a multidrug-resistant nosocomial pathogen. We showed previously that thiostrepton (TS), a Gram-positive thiopeptide antibiotic, is imported via pyoverdine receptors and synergizes with iron chelator deferasirox (DSX) to inhibit the growth of P. aeruginosa and Acinetobacter baumannii clinical isolates. A small number of P. aeruginosa and A. baumannii isolates were resistant to the combination, prompting us to search for other compounds that could synergize with TS against those strains. From literature surveys, we selected 14 compounds reported to have iron-chelating activity, plus one iron analogue, and tested them for synergy with TS. Doxycycline (DOXY), ciclopirox olamine (CO), tropolone (TRO), clioquinol (CLI), and gallium nitrate (GN) synergized with TS. Individual compounds were bacteriostatic, but the combinations were bactericidal. Our spectrophotometric data and chrome azurol S agar assay confirmed that the chelators potentiate TS activity through iron sequestration rather than through their innate antimicrobial activities. A triple combination of TS plus DSX plus DOXY had the most potent activity against P. aeruginosa and A. baumannii isolates. One P. aeruginosa clinical isolate was resistant to the triple combination but susceptible to a triple combination containing higher concentrations of CLI, CO, or DOXY. All A. baumannii isolates were susceptible to the triple combinations. Our data reveal a diverse set of compounds with dual activity as antibacterial agents and TS adjuvants, allowing combinations to be tailored for resistant clinical isolates.

2.
Nat Commun ; 10(1): 5198, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31729381

RESUMO

Type IV pilus-like systems are protein complexes that polymerize pilin fibres. They are critical for virulence in many bacterial pathogens. Pilin polymerization and depolymerization are powered by motor ATPases of the PilT/VirB11-like family. This family is thought to operate with C2 symmetry; however, most of these ATPases crystallize with either C3 or C6 symmetric conformations. The relevance of these conformations is unclear. Here, we determine the X-ray structures of PilT in four unique conformations and use these structures to classify the conformation of available PilT/VirB11-like family member structures. Single particle electron cryomicroscopy (cryoEM) structures of PilT reveal condition-dependent preferences for C2, C3, and C6 conformations. The physiologic importance of these conformations is validated by coevolution analysis and functional studies of point mutants, identifying a rare gain-of-function mutation that favours the C2 conformation. With these data, we propose a comprehensive model of PilT function with broad implications for PilT/VirB11-like family members.

3.
Artigo em Inglês | MEDLINE | ID: mdl-31262758

RESUMO

Pseudomonas aeruginosa is a biofilm-forming opportunistic pathogen and is intrinsically resistant to many antibiotics. In a high-throughput screen for molecules that modulate biofilm formation, we discovered that the thiopeptide antibiotic thiostrepton (TS), which is considered to be inactive against Gram-negative bacteria, stimulated P. aeruginosa biofilm formation in a dose-dependent manner. This phenotype is characteristic of exposure to antimicrobial compounds at subinhibitory concentrations, suggesting that TS was active against P. aeruginosa Supporting this observation, TS inhibited the growth of a panel of 96 multidrug-resistant (MDR) P. aeruginosa clinical isolates at low-micromolar concentrations. TS also had activity against Acinetobacter baumannii clinical isolates. The expression of Tsr, a 23S rRNA-modifying methyltransferase from TS producer Streptomyces azureus, in trans conferred TS resistance, confirming that the drug acted via its canonical mode of action, inhibition of ribosome function. The deletion of oligopeptide permease systems used by other peptide antibiotics for uptake failed to confer TS resistance. TS susceptibility was inversely proportional to iron availability, suggesting that TS exploits uptake pathways whose expression is increased under iron starvation. Consistent with this finding, TS activity against P. aeruginosa and A. baumannii was potentiated by the FDA-approved iron chelators deferiprone and deferasirox and by heat-inactivated serum. Screening of P. aeruginosa mutants for TS resistance revealed that it exploits pyoverdine receptors FpvA and FpvB to cross the outer membrane. We show that the biofilm stimulation phenotype can reveal cryptic subinhibitory antibiotic activity, and that TS has activity against select multidrug-resistant Gram-negative pathogens under iron-limited growth conditions, similar to those encountered at sites of infection.

4.
Microbiol Spectr ; 7(2)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30825300

RESUMO

Type IV pilus (T4P)-like systems have been identified in almost every major phylum of prokaryotic life. They include the type IVa pilus (T4aP), type II secretion system (T2SS), type IVb pilus (T4bP), Tad/Flp pilus, Com pilus, and archaeal flagellum (archaellum). These systems are used for adhesion, natural competence, phage adsorption, folded-protein secretion, surface sensing, swimming motility, and twitching motility. The T4aP allows for all of these functions except swimming and is therefore a good model system for understanding T4P-like systems. Recent structural analyses have revolutionized our understanding of how the T4aP machinery assembles and functions. Here we review the structure and function of the T4aP.


Assuntos
Proteínas de Fímbrias/química , Fímbrias Bacterianas/química , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Flagelos/química , Flagelos/metabolismo , Transporte Proteico , Sistemas de Secreção Tipo II
5.
Front Microbiol ; 9: 1630, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30072980

RESUMO

Type IV pili (T4P) are surface-exposed protein fibers that play key roles in the bacterial life cycle via surface attachment/adhesion, biofilm formation, motility, and development. The order Myxococcales (myxobacteria) are members of the class Deltaproteobacteria and known for their large genome size and complex social behaviors, including gliding motility, fruiting body formation, biofilm production, and prey hunting. Myxococcus xanthus, the best-characterized member of the order, relies on the appropriate expression of 17 type IVa (T4aP) genes organized in a single cluster plus additional genes (distributed throughout the genome) for social motility and development. Here, we compared T4aP genes organization within the myxobacteria to understand their evolutionary origins and diversity. We found that T4aP genes are organized as large clusters in suborder Cystobacterineae, whereas in other two suborders Sorangiineae and Nannocystineae, these genes are dispersed throughout the genome. Based on the genomic organization, the phylogeny of conserved proteins, and synteny studies among 28 myxobacterial and 66 Proteobacterial genomes, we propose an evolutionary model for the origin of myxobacterial T4aP genes independently from other orders in class Deltaproteobacteria. Considering a major role for T4P, this study further proposes the origins and evolution of social motility in myxobacteria and provides a foundation for understanding how complex-behavioral traits, such as gliding motility, multicellular development, etc., might have evolved in this diverse group of complex organisms.

6.
mBio ; 9(4)2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-30042200

RESUMO

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.


Assuntos
Proteínas de Bactérias/genética , Flagelos/fisiologia , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/genética , Fatores de Transcrição/genética , Regulação para Baixo , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Movimento , Ativação Transcricional , Regulação para Cima
7.
Curr Opin Microbiol ; 45: 164-169, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30053750

RESUMO

Biofilms are a typical mode of growth for most microorganisms and provide them with a variety of survival benefits. Biofilms can pose medical and industrial challenges due to their increased tolerance of antimicrobials and disinfectants. Exposure of bacteria to subinhibitory concentrations of those compounds can further exacerbate the problem, as they provoke physiological changes that lead to increased biofilm production and potential therapeutic failure. The protected niche of a biofilm provides conditions that promote selection for persisters and resistant mutants. In this review we discuss our current understanding of the mechanisms underlying biofilm stimulation in response to subinhibitory antimicrobials, and how we might exploit this 'anti-antibiotic' phenotype to treat biofilm-related infections and discover new compounds.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Fenômenos Fisiológicos Bacterianos/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Animais , Bactérias/genética , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Humanos , Testes de Sensibilidade Microbiana
8.
PLoS Pathog ; 14(5): e1007074, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29775484

RESUMO

Type IV pili are expressed by a wide range of prokaryotes, including the opportunistic pathogen Pseudomonas aeruginosa. These flexible fibres mediate twitching motility, biofilm maturation, surface adhesion, and virulence. The pilus is composed mainly of major pilin subunits while the low abundance minor pilins FimU-PilVWXE and the putative adhesin PilY1 prime pilus assembly and are proposed to form the pilus tip. The minor pilins and PilY1 are encoded in an operon that is positively regulated by the FimS-AlgR two-component system. Independent of pilus assembly, PilY1 was proposed to be a mechanosensory component that-in conjunction with minor pilins-triggers up-regulation of acute virulence phenotypes upon surface attachment. Here, we investigated the link between the minor pilins/PilY1 and virulence. pilW, pilX, and pilY1 mutants had reduced virulence towards Caenorhabditis elegans relative to wild type or a major pilin mutant, implying a role in pathogenicity that is independent of pilus assembly. We hypothesized that loss of specific minor pilins relieves feedback inhibition on FimS-AlgR, increasing transcription of the AlgR regulon and delaying C. elegans killing. Reporter assays confirmed that FimS-AlgR were required for increased expression of the minor pilin operon upon loss of select minor pilins. Overexpression of AlgR or its hyperactivation via a phosphomimetic mutation reduced virulence, and the virulence defects of pilW, pilX, and pilY1 mutants required FimS-AlgR expression and activation. We propose that PilY1 and the minor pilins inhibit their own expression, and that loss of these proteins leads to FimS-mediated activation of AlgR that suppresses expression of acute-phase virulence factors and delays killing. This mechanism could contribute to adaptation of P. aeruginosa in chronic lung infections, as mutations in the minor pilin operon result in the loss of piliation and increased expression of AlgR-dependent virulence factors-such as alginate-that are characteristic of such infections.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Fímbrias/fisiologia , Pseudomonas aeruginosa/patogenicidade , Transativadores/metabolismo , Alelos , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Clonagem Molecular , Proteínas de Fímbrias/genética , Expressão Gênica , Mutação/genética , Óperon/fisiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiologia , Transativadores/genética , Técnicas do Sistema de Duplo-Híbrido , Virulência
9.
ACS Infect Dis ; 4(7): 1041-1047, 2018 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-29771109

RESUMO

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen, designated by the World Health Organization as a critical priority for development of new therapeutics due to high levels of intrinsic and acquired antibiotic resistance. Other challenges include its versatility (it can persist in the environment and most strains are capable of causing disease in compromised hosts), robust efflux mechanisms that limit drug penetration, and the propensity to form antimicrobial-tolerant biofilms. Novel therapeutics in development to prevent or treat P. aeruginosa infections include vaccines, biologics such as antimicrobial peptides and therapeutic antibodies, virulence inhibitors, antimicrobials with novel targets, antibody-drug conjugates, resistance inhibitor-antibiotic or antibiotic-potentiator combinations, and bacteriophages or phage-derived lysins.


Assuntos
Antibacterianos/farmacologia , Desenvolvimento de Medicamentos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/uso terapêutico , Descoberta de Drogas , Farmacorresistência Bacteriana , Sinergismo Farmacológico , Quimioterapia Combinada , Metabolismo Energético/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Genoma Bacteriano , Humanos , Infecções por Pseudomonas/tratamento farmacológico , Fagos de Pseudomonas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/virologia , Fatores de Virulência
10.
ACS Infect Dis ; 4(6): 936-943, 2018 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-29342354

RESUMO

Type IVa pili (T4aP) are long, thin surface filaments involved in attachment, motility, biofilm formation, and DNA uptake. They are important virulence factors for many bacteria, including Pseudomonas aeruginosa, an opportunistic pathogen and common cause of hospital-acquired infections. Each helical filament contains thousands of monomers of the major pilin subunit, PilA. Each P. aeruginosa strain expresses one of five phylogenetically distinct major pilins, which vary in sequence and the nature of their associated accessory protein(s). Here, we present the backbone resonance assignment of the C-terminal domain of the group III PilA from strain PA14, a highly virulent, globally distributed clone. Secondary structure probabilities calculated from chemical shifts were in excellent agreement with previous homology modeling using a group V pilin structural template. The analysis revealed that the distal segment of the αß loop had high microsecond-millisecond dynamics compared with other loop regions. Shortening of this segment by internal deletion abrogated pilus assembly in a dominant negative manner, suggesting a potential role in pilin polymerization. Pilin conformations that support optimal interactions of both the conserved hydrophobic N-termini in the pilus core and hydrophilic loops creating the filament surface may be necessary to produce stable filaments.


Assuntos
Proteínas de Fímbrias/química , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/fisiologia , Modelos Moleculares , Conformação Proteica , Pseudomonas aeruginosa/fisiologia , Proteínas de Fímbrias/genética , Espectroscopia de Ressonância Magnética , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
11.
Nat Microbiol ; 3(1): 47-52, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29133883

RESUMO

Since phages present a major challenge to survival in most environments, bacteria express a battery of anti-phage defences including CRISPR-Cas, restriction-modification and abortive infection systems 1-4 . Such strategies are effective, but the phage genome-which encodes potentially inhibitory gene products-is still allowed to enter the cell. The safest way to preclude phage infection is to block initial phage adsorption to the cell. Here, we describe a cell-surface modification that blocks infection by certain phages. Strains of the opportunistic pathogen Pseudomonas aeruginosa express one of five different type IV pilins (T4P) 5 , two of which are glycosylated with O-antigen units 6 or polymers of D-arabinofuranose 7-9 . We propose that predation by bacteriophages that use T4P as receptors selects for strains that mask potential phage binding sites using glycosylation. Here, we show that both modifications protect P. aeruginosa from certain pilus-specific phages. Alterations to pilin sequence can also block phage infection, but glycosylation is considered less likely to create disadvantageous phenotypes. Through construction of chimeric phages, we show that specific phage tail proteins allow for infection of strains with glycosylated pili. These studies provide insight into first-line bacterial defences against predation and ways in which phages circumvent them, and provide a rationale for the prevalence of pilus glycosylation in nature.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Fagos de Pseudomonas/fisiologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/virologia , Arabinose/análogos & derivados , Arabinose/metabolismo , Proteínas de Bactérias/genética , Proteínas de Fímbrias/genética , Glicosilação , Especificidade de Hospedeiro , Modelos Moleculares , Mutação , Fagos de Pseudomonas/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/ultraestrutura , Proteínas da Cauda Viral/genética
12.
J Bacteriol ; 199(16)2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28583947

RESUMO

FimV is a Pseudomonas aeruginosa inner membrane hub protein that modulates levels of the second messenger, cyclic AMP (cAMP), through the activation of adenylate cyclase CyaB. Although type IVa pilus (T4aP)-dependent twitching motility is modulated by cAMP levels, mutants lacking FimV are twitching impaired, even when exogenous cAMP is provided. Here we further define FimV's cAMP-dependent and -independent regulation of twitching. We confirmed that the response regulator of the T4aP-associated Chp chemotaxis system, PilG, requires both FimV and the CyaB regulator, FimL, to activate CyaB. However, in cAMP-replete backgrounds-lacking the cAMP phosphodiesterase CpdA or the CheY-like protein PilH or expressing constitutively active CyaB-pilG and fimV mutants failed to twitch. Both cytoplasmic and periplasmic domains of FimV were important for its cAMP-dependent and -independent roles, while its septal peptidoglycan-targeting LysM motif was required only for twitching motility. Polar localization of the sensor kinase PilS, a key regulator of transcription of the major pilin, was FimV dependent. However, unlike its homologues in other species that localize flagellar system components, FimV was not required for swimming motility. These data provide further evidence to support FimV's role as a key hub protein that coordinates the polar localization and function of multiple structural and regulatory proteins involved in P. aeruginosa twitching motility.IMPORTANCEPseudomonas aeruginosa is a serious opportunistic pathogen. Type IVa pili (T4aP) are important for its virulence, because they mediate dissemination and invasion via twitching motility and are involved in surface sensing, which modulates pathogenicity via changes in cAMP levels. Here we show that the hub protein FimV and the response regulator of the Chp system, PilG, regulate twitching independently of their roles in the modulation of cAMP synthesis. These functions do not require the putative scaffold protein FimL, proposed to link PilG with FimV. PilG may regulate asymmetric functioning of the T4aP system to allow for directional movement, while FimV appears to localize both structural and regulatory elements-including the PilSR two-component system-to cell poles for optimal function.


Assuntos
AMP Cíclico/metabolismo , Proteínas de Fímbrias/metabolismo , Locomoção , Pseudomonas aeruginosa/fisiologia , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes , Pseudomonas aeruginosa/metabolismo
13.
Nat Commun ; 8: 15091, 2017 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-28474682

RESUMO

Type IVa pili are protein filaments essential for virulence in many bacterial pathogens; they extend and retract from the surface of bacterial cells to pull the bacteria forward. The motor ATPase PilB powers pilus assembly. Here we report the structures of the core ATPase domains of Geobacter metallireducens PilB bound to ADP and the non-hydrolysable ATP analogue, AMP-PNP, at 3.4 and 2.3 Å resolution, respectively. These structures reveal important differences in nucleotide binding between chains. Analysis of these differences reveals the sequential turnover of nucleotide, and the corresponding domain movements. Our data suggest a clockwise rotation of the central sub-pores of PilB, which through interactions with PilC, would support the assembly of a right-handed helical pilus. Our analysis also suggests a counterclockwise rotation of the C2 symmetric PilT that would enable right-handed pilus disassembly. The proposed model provides insight into how this family of ATPases can power pilus extension and retraction.


Assuntos
Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/ultraestrutura , Adenilil Imidodifosfato/metabolismo , Proteínas de Bactérias/ultraestrutura , Proteínas de Fímbrias/ultraestrutura , Fímbrias Bacterianas/metabolismo , Proteínas Motores Moleculares/ultraestrutura , Oxirredutases/ultraestrutura , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Fímbrias/metabolismo , Geobacter , Modelos Moleculares , Proteínas Motores Moleculares/metabolismo , Nucleotídeos/metabolismo , Oxirredutases/metabolismo , Virulência
14.
mBio ; 8(1)2017 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-28143978

RESUMO

Type IVa pili (T4aP) are ubiquitous microbial appendages used for adherence, twitching motility, DNA uptake, and electron transfer. Many of these functions depend on dynamic assembly and disassembly of the pilus by a megadalton-sized, cell envelope-spanning protein complex located at the poles of rod-shaped bacteria. How the T4aP assembly complex becomes integrated into the cell envelope in the absence of dedicated peptidoglycan (PG) hydrolases is unknown. After ruling out the potential involvement of housekeeping PG hydrolases in the installation of the T4aP machinery in Pseudomonas aeruginosa, we discovered that key components of inner (PilMNOP) and outer (PilQ) membrane subcomplexes are recruited to future sites of cell division. Midcell recruitment of a fluorescently tagged alignment subcomplex component, mCherry-PilO, depended on PilQ secretin monomers-specifically, their N-terminal PG-binding AMIN domains. PilP, which connects PilO to PilQ, was required for recruitment, while PilM, which is structurally similar to divisome component FtsA, was not. Recruitment preceded secretin oligomerization in the outer membrane, as loss of the PilQ pilotin PilF had no effect on localization. These results were confirmed in cells chemically blocked for cell division prior to outer membrane invagination. The hub protein FimV and a component of the polar organelle coordinator complex-PocA-were independently required for midcell recruitment of PilO and PilQ. Together, these data suggest an integrated, energy-efficient strategy for the targeting and preinstallation-rather than retrofitting-of the T4aP system into nascent poles, without the need for dedicated PG-remodeling enzymes. IMPORTANCE: The peptidoglycan (PG) layer of bacterial cell envelopes has limited porosity, representing a physical barrier to the insertion of large protein complexes involved in secretion and motility. Many systems include dedicated PG hydrolase components that create space for their insertion, but the ubiquitous type IVa pilus (T4aP) system lacks such an enzyme. Instead, we found that components of the T4aP system are recruited to future sites of cell division, where they could be incorporated into the cell envelope during the formation of new poles, eliminating the need for PG hydrolases. Targeting depends on the presence of septal PG-binding motifs in specific components, as removal of those motifs causes delocalization. This preinstallation strategy for the T4aP assembly system would ensure that both daughter cells are poised to extrude pili from new poles as soon as they separate from one another.


Assuntos
Divisão Celular , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Pseudomonas aeruginosa/fisiologia , Transporte Proteico
15.
Structure ; 24(10): 1778-1787, 2016 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-27705815

RESUMO

Type IVa pili (T4aP) function as bacterial virulence factors. T4aP pass through the outer membranes of Gram-negative bacteria via homo-oligomeric secretins. We present a 7.4 Å cryoelectron microscopy structure of the Pseudomonas aeruginosa PilQ secretin. Peripheral and internal features show that the secretin is composed of 14 subunits with C7 symmetry. The channel is a ribbed cylinder with central peripheral spokes and a central gate closed on the periplasmic side. The structure suggests that during pilus extrusion, the central gate is displaced to the interior walls and that no additional conformational changes are required, as the internal diameter can accommodate the pilus. The N1 domain was resolved, while the N0 and the N-terminal ß-domains proposed to bind peptidoglycan were absent in class average images and the final 3D map, indicating a high flexibility. These data provide the highest-resolution structure to date of a T4aP secretin.


Assuntos
Proteínas de Fímbrias/química , Peptidoglicano/metabolismo , Pseudomonas aeruginosa/metabolismo , Microscopia Crioeletrônica , Proteínas de Fímbrias/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Pseudomonas aeruginosa/química
16.
J Biol Chem ; 291(38): 19923-38, 2016 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-27474743

RESUMO

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and is resistant to many antibiotics. Type IV pili (T4P) are among the key virulence factors used by P. aeruginosa for host cell attachment, biofilm formation, and twitching motility, making this system a promising target for novel therapeutics. Point mutations in the conserved PilMNOP alignment subcomplex were previously shown to have distinct effects on assembly and disassembly of T4P, suggesting that it may function in a dynamic manner. We introduced mutations encoding Cys substitutions into pilN and/or pilO on the chromosome to maintain normal stoichiometry and expression levels and captured covalent PilNO heterodimers, as well as PilN and PilO homodimers, in vivo Most covalent PilN or PilO homodimers had minimal functional impact in P. aeruginosa, suggesting that homodimers are a physiologically relevant state. However, certain covalent homo- or heterodimers eliminated twitching motility, suggesting that specific PilNO configurations are essential for T4P function. These data were verified using soluble N-terminal truncated fragments of PilN and PilO Cys mutants, which purified as a mixture of homo- and heterodimers at volumes consistent with a tetramer. Deletion of genes encoding alignment subcomplex components, PilM or PilP, but not other T4P components, including the motor ATPases PilB or PilT, blocked in vivo formation of disulfide-bonded PilNO heterodimers, suggesting that both PilM and PilP influence the heterodimer interface. Combined, our data suggest that T4P function depends on rearrangements at PilN and PilO interfaces.


Assuntos
Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Mutação de Sentido Incorreto , Multimerização Proteica , Pseudomonas aeruginosa/metabolismo , Substituição de Aminoácidos , Cisteína/genética , Cisteína/metabolismo , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Pseudomonas aeruginosa/genética
17.
J Bacteriol ; 198(16): 2263-74, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27297880

RESUMO

UNLABELLED: FimV is a Pseudomonas aeruginosa inner membrane protein that regulates intracellular cyclic AMP (cAMP) levels-and thus type IV pilus (T4P)-mediated twitching motility and type II secretion (T2S)-by activating the adenylate cyclase CyaB. Its cytoplasmic domain contains three predicted tetratricopeptide repeat (TPR) motifs separated by an unstructured region: two proximal to the inner membrane and one within the "FimV C-terminal domain," which is highly conserved across diverse homologs. Here, we present the crystal structure of the FimV C terminus, FimV861-919, containing a TPR motif decorated with solvent-exposed, charged side chains, plus a C-terminal capping helix. FimV689, a truncated form lacking this C-terminal motif, did not restore wild-type levels of twitching or surface piliation compared to the full-length protein. FimV689 failed to restore wild-type levels of the T4P motor ATPase PilU or T2S, suggesting that it was unable to activate cAMP synthesis. Bacterial two-hybrid analysis showed that TPR3 interacts directly with the CyaB activator, FimL. However, FimV689 failed to restore wild-type motility in a fimV mutant expressing a constitutively active CyaB (fimV cyaB-R456L), suggesting that the C-terminal motif is also involved in cAMP-independent functions of FimV. The data show that the highly conserved TPR-containing C-terminal domain of FimV is critical for its cAMP-dependent and -independent functions. IMPORTANCE: FimV is important for twitching motility and cAMP-dependent virulence gene expression in P. aeruginosa FimV homologs have been identified in several human pathogens, and their functions are not limited to T4P expression. The C terminus of FimV is remarkably conserved among otherwise very diverse family members, but its role is unknown. We provide here biological evidence for the importance of the C-terminal domain in both cAMP-dependent (through FimL) and -independent functions of FimV. We present X-ray crystal structures of the conserved C-terminal domain and identify a consensus sequence for the C-terminal TPR within the conserved domain. Our data extend our knowledge of FimV's functionally important domains, and the structures and consensus sequences provide a foundation for studies of FimV and its homologs.


Assuntos
Proteínas de Bactérias/metabolismo , Sequência Conservada/fisiologia , AMP Cíclico/metabolismo , Pseudomonas aeruginosa/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cristalografia por Raios X , AMP Cíclico/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Modelos Moleculares , Filogenia , Conformação Proteica , Pseudomonas aeruginosa/genética , Sistemas de Secreção Tipo II
18.
Proc Natl Acad Sci U S A ; 113(21): 6017-22, 2016 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-27162347

RESUMO

Type IV pili are important virulence factors for many pathogens, including Pseudomonas aeruginosa Transcription of the major pilin gene-pilA-is controlled by the PilS-PilR two-component system in response to unknown signals. The absence of a periplasmic sensing domain suggested that PilS may sense an intramembrane signal, possibly PilA. We suggest that direct interactions between PilA and PilS in the inner membrane reduce pilA transcription when PilA levels are high. Overexpression in trans of PilA proteins with diverse and/or truncated C termini decreased native pilA transcription, suggesting that the highly conserved N terminus of PilA was the regulatory signal. Point mutations in PilA or PilS that disrupted their interaction prevented autoregulation of pilA transcription. A subset of PilA point mutants retained the ability to interact with PilS but could no longer decrease pilA transcription, suggesting that interaction between the pilin and sensor kinase is necessary but not sufficient for pilA autoregulation. Furthermore, PilS's phosphatase motif was required for the autoregulation of pilA transcription, suggesting that under conditions where PilA is abundant, the PilA-PilS interaction promotes PilR dephosphorylation and thus down-regulation of further pilA transcription. These data reveal a clever bacterial inventory control strategy in which the major subunit of an important P. aeruginosa virulence factor controls its own expression.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Fímbrias/biossíntese , Regulação Bacteriana da Expressão Gênica/fisiologia , Regiões Promotoras Genéticas/fisiologia , Pseudomonas aeruginosa/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética/fisiologia , Proteínas de Bactérias/genética , Proteínas de Fímbrias/genética , Mutação , Fosforilação/fisiologia , Pseudomonas aeruginosa/genética , Fatores de Transcrição/genética
19.
Future Med Chem ; 8(9): 975-92, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27228070

RESUMO

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and is resistant to most antibiotics. With therapeutic options against P. aeruginosa dwindling, and the lack of new antibiotics in advanced developmental stages, strategies for preserving the effectiveness of current antibiotics are urgently required. ß-Lactam antibiotics are important agents for treating P. aeruginosa infections, thus, adjuvants that potentiate the activity of these compounds are desirable for extending their lifespan while new antibiotics - or antibiotic classes - are discovered and developed. In this review, we discuss recent research that has identified exploitable targets of cell-wall metabolism for the design and development of compounds that hinder resistance and potentiate the activity of antipseudomonal ß-lactams.


Assuntos
Antibacterianos/farmacologia , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Descoberta de Drogas , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/química , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/metabolismo
20.
J Biol Chem ; 291(21): 11003-15, 2016 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-27022027

RESUMO

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that expresses type IVa pili. The pilus assembly system, which promotes surface-associated twitching motility and virulence, is composed of inner and outer membrane subcomplexes, connected by an alignment subcomplex composed of PilMNOP. PilM binds to the N terminus of PilN, and we hypothesize that this interaction causes functionally significant structural changes in PilM. To characterize this interaction, we determined the crystal structures of PilM and a PilM chimera where PilM was fused to the first 12 residues of PilN (PilM·PilN(1-12)). Structural analysis, multiangle light scattering coupled with size exclusion chromatography, and bacterial two-hybrid data revealed that PilM forms dimers mediated by the binding of a novel conserved motif in the N terminus of PilM, and binding PilN abrogates this binding interface, resulting in PilM monomerization. Structural comparison of PilM with PilM·PilN(1-12) revealed that upon PilN binding, there is a large domain closure in PilM that alters its ATP binding site. Using biolayer interferometry, we found that the association rate of PilN with PilM is higher in the presence of ATP compared with ADP. Bacterial two-hybrid data suggested the connectivity of the cytoplasmic and inner membrane components of the type IVa pilus machinery in P. aeruginosa, with PilM binding to PilB, PilT, and PilC in addition to PilN. Pull-down experiments demonstrated direct interactions of PilM with PilB and PilT. We propose a working model in which dynamic binding of PilN facilitates functionally relevant structural changes in PilM.


Assuntos
Proteínas de Fímbrias/química , Proteínas de Fímbrias/metabolismo , Pseudomonas aeruginosa/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação/genética , Cristalografia por Raios X , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/química , Fímbrias Bacterianas/classificação , Fímbrias Bacterianas/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidade
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