RESUMEN
The tad (tight adherence) locus of Aggregatibacter actinomycetemcomitans includes genes for the biogenesis of Flp pili, which are necessary for bacterial adhesion to surfaces, biofilm formation, and pathogenesis. Although studies have elucidated the functions of some of the Tad proteins, little is known about the regulation of the tad locus in A. actinomycetemcomitans. A promoter upstream of the tad locus was previously identified and shown to function in Escherichia coli. Using a specially constructed reporter plasmid, we show here that this promoter (tadp) functions in A. actinomycetemcomitans. To study expression of the pilin gene (flp-1) relative to that of tad secretion complex genes, we used Northern hybridization analysis and a lacZ reporter assay. We identified three terminators, two of which (T1 and T2) can explain flp-1 mRNA abundance, while the third (T3) is at the end of the locus. T1 and T3 have the appearance and behavior of intrinsic terminators, while T2 has a different structure and is inhibited by bicyclomycin, indicating that T2 is probably Rho dependent. To help achieve the appropriate stoichiometry of the Tad proteins, we show that a transcriptional-termination cascade is important to the proper expression of the tad genes. These data indicate a previously unreported mechanism of regulation in A. actinomycetemcomitans and lead to a more complete understanding of its Flp pilus biogenesis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Pasteurellaceae/genética , Pasteurellaceae/metabolismo , Regiones Promotoras Genéticas/fisiología , Transcripción Genética/fisiología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Cable pili are unique peritrichous adherence organelles expressed by certain strains of the opportunistic human pathogen Burkholderia cenocepacia. Cable pili have been proposed to facilitate binding to human epithelial cells and mucin, and may play a role in the ability of B. cenocepacia to colonise the respiratory tract of compromised hosts. In this study, a genetic approach was undertaken to assess the role of cable pili in mediating adherence as well as bacterial cell-cell interactions. The cblA gene, encoding the major pilin subunit, was insertionally inactivated, and the resulting mutant was shown to be blocked in CblA expression and in cable pilus morphogenesis. Although non-piliated, the cblA mutant was not defective in adherence to either porcine mucin or to cultured A549 human respiratory epithelial cells. Microscopic and flow cytometric analyses of B. cenocepacia cultures revealed that cable pilus expression facilitated the formation of diffuse cell networks, whereas disruption of cable pilus biogenesis enhanced autoaggregation and the formation of compact cell aggregates. Autoaggregation was observed both in culture and during B. cenocepacia infection of A549 epithelial cell monolayers. These findings indicate that cable pilus expression plays an important role in mediating B. cenocepacia cell-cell interactions, and that both cable pilus-dependent and cable pilus-independent mechanisms may contribute to B. cenocepacia adherence to cellular and acellular surfaces.
Asunto(s)
Adhesión Bacteriana , Burkholderia/genética , Burkholderia/fisiología , Fimbrias Bacterianas/genética , Adhesión Bacteriana/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Burkholderia/ultraestructura , Línea Celular , Células Epiteliales/microbiología , Proteínas Fimbrias/biosíntesis , Proteínas Fimbrias/genética , Fimbrias Bacterianas/fisiología , Expresión Génica , Genes Bacterianos/fisiología , Humanos , Mucinas/metabolismo , Mutagénesis InsercionalRESUMEN
Pseudomonas aeruginosa is a leading cause of hospital-associated infections in the seriously ill, and the primary agent of chronic lung infections in cystic fibrosis patients. A major obstacle to effective control of P. aeruginosa infections is its intrinsic resistance to most antibiotic classes, which results from chromosomally encoded drug-efflux systems and multiple acquired resistance mechanisms selected by years of aggressive antibiotic therapy. These factors demand new strategies and drugs to prevent and treat P. aeruginosa infections. Herein, we describe a monoclonal antibody (mAb) selection strategy on whole P. aeruginosa cells using single-chain variable fragment phage libraries derived from healthy individuals and patients convalescing from P. aeruginosa infections. This approach enabled identification of mAbs that bind three distinct epitopes on the product of the Psl. This exopolysaccharide is important for P. aeruginosa attachment to mammalian cells, and for the formation and maintenance of biofilms produced by nonmucoid and mucoid P. aeruginosa isolates. Functional screens revealed that mAbs to one epitope exhibit superior activity in opsonophagocytic killing and cell attachment assays, and confer significant protection in multiple animal models. Our results indicate that Psl is an accessible serotype-independent surface feature and promising novel protective antigen for preventing P. aeruginosa infections. Furthermore, our mAb discovery strategy holds promise for application to other bacterial pathogens.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Polisacáridos Bacterianos/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/uso terapéutico , Adhesión Bacteriana/efectos de los fármacos , Adhesión Bacteriana/inmunología , Línea Celular Tumoral , Humanos , Estimación de Kaplan-Meier , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Viabilidad Microbiana/efectos de los fármacos , Viabilidad Microbiana/inmunología , Mutación , Biblioteca de Péptidos , Neumonía/tratamiento farmacológico , Neumonía/inmunología , Neumonía/microbiología , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/clasificación , Pseudomonas aeruginosa/genética , Serotipificación , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunologíaRESUMEN
The Tad (tight adherence) macromolecular transport system, which is present in many bacterial and archaeal species, represents an ancient and major new subtype of type II secretion. The tad genes are present on a genomic island named the widespread colonization island (WCI), and encode the machinery that is required for the assembly of adhesive Flp (fimbrial low-molecular-weight protein) pili. The tad genes are essential for biofilm formation, colonization and pathogenesis in the genera Aggregatibacter (Actinobacillus), Haemophilus, Pasteurella, Pseudomonas, Yersinia, Caulobacter and perhaps others. Here we review the structure, function and evolution of the Tad secretion system.
Asunto(s)
Actinobacteria/patogenicidad , Adhesión Bacteriana , Proteínas Bacterianas , Islas Genómicas , Bacterias Gramnegativas/patogenicidad , Infecciones por Actinobacillus/microbiología , Actinobacteria/genética , Actinobacteria/crecimiento & desarrollo , Actinobacteria/metabolismo , Aggregatibacter actinomycetemcomitans/genética , Aggregatibacter actinomycetemcomitans/crecimiento & desarrollo , Aggregatibacter actinomycetemcomitans/metabolismo , Aggregatibacter actinomycetemcomitans/patogenicidad , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Flagelos/metabolismo , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Gramnegativas/metabolismo , Humanos , Periodontitis/microbiología , Ratas , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
The tad locus of Actinobacillus actinomycetemcomitans encodes genes for the biogenesis of Flp pili, which allow the bacterium to adhere tenaciously to surfaces and form strong biofilms. Although tad (tight adherence) loci are widespread among bacterial and archaeal species, very little is known about the functions of the individual components of the Tad secretion apparatus. Here we characterize the mechanism by which the pre-Flp1 prepilin is processed to the mature pilus subunit. We demonstrate that the tadV gene encodes a prepilin peptidase that is both necessary and sufficient for proteolytic maturation of Flp1. TadV was also found to be required for maturation of the TadE and TadF pilin-like proteins, which we term pseudopilins. Using site-directed mutagenesis, we show that processing of pre-Flp1, pre-TadE, and pre-TadF is required for biofilm formation. Mutation of a highly conserved glutamic acid residue at position +5 of Flp1, relative to the cleavage site, resulted in a processed pilin that was blocked in assembly. In contrast, identical mutations in TadE or TadF had no effect on biofilm formation, indicating that the mechanisms by which Flp1 pilin and the pseudopilins function are distinct. We also determined that two conserved aspartic acid residues in TadV are critical for function of the prepilin peptidase. Together, our results indicate that the A. actinomycetemcomitans TadV protein is a member of a novel subclass of nonmethylating aspartic acid prepilin peptidases.
Asunto(s)
Aggregatibacter actinomycetemcomitans/enzimología , Proteínas Bacterianas/metabolismo , Endopeptidasas/metabolismo , Proteínas Fimbrias/metabolismo , Aggregatibacter actinomycetemcomitans/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Secuencia Conservada , Análisis Mutacional de ADN , Endopeptidasas/química , Endopeptidasas/genética , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis Sitio-Dirigida , Mutación , Operón , Unión Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
Cable pili are peritrichous organelles expressed by certain strains of Burkholderia cenocepacia, believed to facilitate colonization of the lower respiratory tract in cystic fibrosis patients. The B. cenocepacia cblBACDS operon encodes the structural and accessory proteins required for the assembly of cable pili, as well as a gene designated cblS, predicted to encode a hybrid sensor kinase protein of bacterial two-component signal transduction systems. In this study we report the identification of two additional genes, designated cblT and cblR, predicted to encode a second hybrid sensor kinase and a response regulator, respectively. Analyses of the deduced amino acid sequences of the cblS and cblT gene products revealed that both putative sensor kinases have transmitter and receiver domains and that the cblT gene product has an additional C-terminal HPt domain. Mutagenesis of the cblS, cblT, or cblR gene led to a block in expression of CblA, the major pilin subunit, and a severe decrease in cblA transcript abundance. Using transcriptional fusion analyses, the decrease in the abundance of the cblA transcript in the cblS, cblT, and cblR mutants was shown to be due to a block in transcription from the cblB-proximal promoter, located upstream of the cblBACDS operon. Furthermore, ectopic expression of either cblS or cblR in wild-type B. cenocepacia strain BC7 led to a significant increase, while ectopic expression of cblT resulted in a dramatic decrease, in abundance of the CblA major pilin and the cblA transcript. Our results demonstrate that the B. cenocepacia cblS, cblT, and cblR genes are essential for cable pilus expression and that their effect is exerted at the level of transcription of the cblBACDS operon. These findings are consistent with the proposed function of the cblSTR gene products as a multicomponent signal transduction pathway controlling the expression of cable pilus biosynthetic genes in B. cenocepacia.
Asunto(s)
Proteínas Bacterianas/genética , Complejo Burkholderia cepacia/metabolismo , Fimbrias Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Transducción de Señal , Proteínas Bacterianas/metabolismo , Complejo Burkholderia cepacia/genética , Complejo Burkholderia cepacia/ultraestructura , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/ultraestructura , Humanos , Microscopía Electrónica , Datos de Secuencia Molecular , Mutación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Análisis de Secuencia de ADN , Transcripción GenéticaRESUMEN
Burkholderia cenocepacia is an important member of the Burkholderia cepacia complex, a group of closely related bacteria that inhabits a wide variety of environmental niches in nature and that also colonizes the lungs of compromised humans. Certain strains of B. cenocepacia express peritrichous adherence organelles known as cable pili, thought to be important in the colonization of the lower respiratory tract. The genetic locus required for cable pilus biogenesis is comprised of at least five genes, designated cblB, cblA, cblC, cblD, and cblS. In this study a transcriptional analysis of cbl gene expression was undertaken. The principal promoter, located upstream of the cbl locus, was identified and characterized. By using lacZ transcriptional fusions, the effects of multiple environmental cues on cbl gene expression were examined. High osmolarity, temperature of 37 degrees C, acidic pH, and low iron bioavailability were found to induce cbl gene expression. Northern hybridization analysis of the cbl locus identified a single, stable transcript corresponding to cblA, encoding the major pilin subunit. Transcriptional fusion studies combined with reverse transcription-PCR analysis indicated that the stable cblA transcript is the product of an mRNA processing event. This event may ensure high levels of expression of the major pilin, relative to other components of the assembly pathway. Our findings lend further insight into the control of cable pilus biogenesis in B. cenocepacia and provide evidence for regulation of cbl gene expression on both the transcriptional and posttranscriptional levels.
Asunto(s)
Burkholderia/genética , Fimbrias Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Transcripción Genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Clonación Molecular , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cloruro de Sodio/farmacología , TemperaturaRESUMEN
Burkholderia cepacia is an important opportunistic human pathogen that affects immunocompromised individuals, particularly cystic fibrosis (CF) patients. Colonization of the lungs of a CF patient by B. cepacia can lead not only to a decline in respiratory function but also to an acute systemic infection, such as bacteremia. We have previously demonstrated that a CF clinical isolate of B. cepacia, strain J2315, can invade and survive within cultured respiratory epithelial cells. In order to further characterize the mechanisms of invasion of B. cepacia, we screened a transposon-generated mutant library of strain J2315 for mutants defective in invasion of A549 respiratory epithelial cells. Here we describe isolation and characterization of a nonmotile mutant of B. cepacia with reduced invasiveness due to disruption of fliG, which encodes a component of the motor-switch complex of the flagellar basal body. We also found that a defined null mutation in fliI, a gene encoding a highly conserved ATPase required for protein translocation via the flagellar type III secretion system, also resulted in loss of motility and a significant reduction in invasion. Both mutants lacked detectable intracellular flagellin and failed to export detectable amounts of flagellin into culture supernatants, suggesting that disruption of fliG and fliI impaired flagellar biogenesis. The reduction in invasion did not appear to be due to defective adherence of the flagellar mutants to A549 cells, suggesting that functional flagella and motility are required for full invasiveness of B. cepacia. Our findings indicate that flagellum-mediated motility may facilitate penetration of host epithelial barriers by B. cepacia, contributing to establishment of infection and systemic spread of the organism.
Asunto(s)
Burkholderia cepacia/fisiología , Flagelos/fisiología , Adhesión Bacteriana , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Humanos , Pulmón/microbiología , Movimiento , MutaciónRESUMEN
Type III secretion systems are utilized by a number of gram-negative bacterial pathogens to deliver virulence-associated proteins into host cells. Using a PCR-based approach, we identified homologs of type III secretion genes in the gram-negative bacterium Burkholderia cepacia, an important pulmonary pathogen in immunocompromised patients and patients with cystic fibrosis. One of the genes, designated bscN, encodes a member of a family of ATP-binding proteins believed to generate energy driving virulence protein secretion. Genetic dissection of the regions flanking the bscN gene revealed a locus consisting of at least 10 open reading frames, predicted to encode products with significant homology to known type III secretion proteins in other bacteria. A defined null mutation was generated in the bscN gene, and the null strain and wild-type parent strain were examined by use of a murine model of B. cepacia infection. Quantitative bacteriological analysis of the lungs and spleens of infected C57BL/6 mice revealed that the bscN null strain was attenuated in virulence compared to the parent strain, with significantly lower bacterial recovery from the lungs and spleens at 3 days postinfection. Moreover, histopathological changes, including an inflammatory cell infiltrate, were more pronounced in the lungs of mice infected with the wild-type parent strain than in those of mice infected with the isogenic bscN mutant. These results implicate type III secretion as an important determinant in the pathogenesis of B. cepacia.