RESUMEN
Feral swine are invasive in the United States and a reservoir for infectious diseases. The increase in feral swine population and the geographic range are a concern for the spread of zoonotic diseases to humans and livestock. Feral swine could contribute to the spread of Coxiella burnetii, the causative agent of human Q fever. In this study, we characterized the seroprevalence of C. burnetii in feral swine populations of Hawai'i and Texas, which have low and high rates of human Q fever, respectively. Seropositivity rates were as high as 0.19% and 6.03% in Hawai'i and Texas, respectively, indicating that feral swine cannot be ruled out as a potential reservoir for disease transmission and spread. In Texas, we identified the overlap between seropositivity of feral swine and human Q fever incidence. These results indicate that there is a potentially low but detectable risk of C. burnetii exposure associated with feral swine populations in Hawai'i and Texas.
RESUMEN
Burkholderia pseudomallei (Bp), causing a highly fatal disease called melioidosis, is a facultative intracellular pathogen that attaches and invades a variety of cell types. We previously identified BP1026B_I0091 as a surface attachment protein (Sap1) and an essential virulence factor, contributing to Bp pathogenesis in vitro and in vivo. The expression of sap1 is regulated at different stages of Bp intracellular lifecycle by unidentified regulator(s). Here, we identified SapR (BP1026B_II1046) as a transcriptional regulator that activates sap1, using a high-throughput transposon mutagenesis screen in combination with Tn-Seq. Consistent with phenotypes of the Δsap1 mutant, the ΔsapR activator mutant exhibited a significant reduction in Bp attachment to the host cell, leading to subsequent decreased intracellular replication. RNA-Seq analysis further revealed that SapR regulates sap1. The regulation of sap1 by SapR was confirmed quantitatively by qRT-PCR, which also validated the RNA-Seq data. SapR globally regulates genes associated with the bacterial membrane in response to diverse environments, and some of the genes regulated by SapR are virulence factors that are required for Bp intracellular infection (e.g., type III and type VI secretion systems). This study has identified the complex SapR regulatory network and its importance as an activator of an essential Sap1 attachment factor.
RESUMEN
Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a disease endemic to the tropics. Melioidosis manifests in various ways ranging from acute skin lesions to pneumonia and, in rare cases, infection of the central nervous system. Bp is a facultative intracellular pathogen and it can infect various cell types. The Bp intracellular lifecycle has been partially elucidated and is highly complex. Herein, we have identified a transcriptional regulator, BP1026B_II1198, that is differentially expressed as Bp transits through host cells. A deletion mutant of BP1026B_II1198 was attenuated in RAW264.7 cell and BALB/c mouse infection. To further characterize the function of this transcriptional regulator, we endeavored to determine the regulon of BP1026B_II1198. RNA-seq analysis showed the global picture of genes regulated while ChIP-seq analysis identified two specific BP1026B_II1198 binding regions on chromosome II. We investigated the transposon mutants of these genes controlled by BP1026B_II1198 and confirmed that these genes contribute to pathogenesis in RAW264.7 murine macrophage cells. Taken together, the data presented here shed light on the regulon of BP1026B_II1198 and its role during intracellular infection and highlights an integral portion of the highly complex regulation network of Bp during host infection.
Asunto(s)
Proteínas Bacterianas/genética , Burkholderia pseudomallei/patogenicidad , Regulación Bacteriana de la Expresión Génica , Melioidosis/microbiología , Proteínas Represoras/genética , Animales , Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/genética , Secuenciación de Inmunoprecipitación de Cromatina , Modelos Animales de Enfermedad , Humanos , Ratones , Mutación , Células RAW 264.7 , RNA-Seq , Regulón , Proteínas Represoras/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Prokaryotic cell transcriptomics has been limited to mixed or sub-population dynamics and individual cells within heterogeneous populations, which has hampered further understanding of spatiotemporal and stage-specific processes of prokaryotic cells within complex environments. Here we develop a 'TRANSITomic' approach to profile transcriptomes of single Burkholderia pseudomallei cells as they transit through host cell infection at defined stages, yielding pathophysiological insights. We find that B. pseudomallei transits through host cells during infection in three observable stages: vacuole entry; cytoplasmic escape and replication; and membrane protrusion, promoting cell-to-cell spread. The B. pseudomallei 'TRANSITome' reveals dynamic gene-expression flux during transit in host cells and identifies genes that are required for pathogenesis. We find several hypothetical proteins and assign them to virulence mechanisms, including attachment, cytoskeletal modulation, and autophagy evasion. The B. pseudomallei 'TRANSITome' provides prokaryotic single-cell transcriptomics information enabling high-resolution understanding of host-pathogen interactions.
Asunto(s)
Proteínas Bacterianas/genética , Burkholderia pseudomallei/genética , Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Genes Bacterianos/genética , Factores de Virulencia/genética , Animales , Burkholderia pseudomallei/citología , Burkholderia pseudomallei/patogenicidad , Línea Celular Tumoral , Membrana Celular/microbiología , Citoplasma/microbiología , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Melioidosis/microbiología , Ratones , Ratones Endogámicos BALB C , Células RAW 264.7 , Análisis de la Célula Individual/métodos , Vacuolas/microbiología , Virulencia/genéticaRESUMEN
It is generally believed that the Pseudomonas aeruginosa biofilm matrix itself acts as a molecular sieve or sink that contributes to significant levels of drug resistance, but it is becoming more apparent that multidrug efflux pumps induced during biofilm growth significantly enhance resistance levels. We present here a novel transcriptional regulator, PA3898, which controls biofilm formation and multidrug efflux pumps in P. aeruginosa A mutant of this regulator significantly reduced the ability of P. aeruginosa to produce biofilm in vitro and affected its in vivo fitness and pathogenesis in Drosophila melanogaster and BALB/c mouse lung infection models. Transcriptome analysis revealed that PA3898 modulates essential virulence genes/pathways, including multidrug efflux pumps and phenazine biosynthesis. Chromatin immunoprecipitation sequencing (ChIP-seq) identified its DNA binding sequences and confirmed that PA3898 directly interacts with promoter regions of four genes/operons, two of which are mexAB-oprM and phz2 Coimmunoprecipitation revealed a regulatory partner of PA3898 as PA2100, and both are required for binding to DNA in electrophoretic mobility shift assays. PA3898 and PA2100 were given the names MdrR1 and MdrR2, respectively, as novel repressors of the mexAB-oprM multidrug efflux operon and activators for another multidrug efflux pump, EmrAB. The interaction between MdrR1 and MdrR2 at the promoter regions of their regulons was further characterized via localized surface plasmon resonance and DNA footprinting. These regulators directly repress the mexAB-oprM operon, independent of its well-established MexR regulator. Mutants of mdrR1 and mdrR2 caused increased resistance to multiple antibiotics in P. aeruginosa, validating the significance of these newly discovered regulators.
Asunto(s)
Proteínas Bacterianas/genética , Pseudomonas aeruginosa/fisiología , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Biopelículas , Drosophila melanogaster , Regulación Bacteriana de la Expresión Génica , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Mutación , Fenazinas/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/patogenicidad , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Natural competency requires uptake of exogenous DNA from the environment and the integration of that DNA into recipient bacteria can be used for DNA-repair or genetic diversification. The Burkholderia genus is unique in that only some of the species and strains are naturally competent. We identified and characterized two genes, comE and crp, from naturally competent B. pseudomallei 1026b that play a role in DNA uptake and catabolism. Single-copies of rhamnose-inducible comE and crp genes were integrated into a Tn7 attachment-site in non-naturally competent Burkholderia including pathogens B. pseudomallei K96243, B. cenocepacia K56-2, and B. mallei ATCC23344. Strains expressing comE or crp were assayed for their ability to uptake and catabolize DNA. ComE and Crp allowed non-naturally competent Burkholderia species to catabolize DNA, uptake exogenous gfp DNA and express GFP. Furthermore, we used synthetic comE and crp to expand the utility of the λ-red recombineering system for genetic manipulation of non-competent Burkholderia species. A newly constructed vector, pKaKa4, was used to mutate the aspartate semialdehyde dehydrogenase (asd) gene in four B. mallei strains, leading to the complete attenuation of these tier-1 select-agents. These strains have been excluded from select-agent regulations and will be of great interest to the field.
Asunto(s)
Burkholderia pseudomallei/genética , Genes Bacterianos/genética , Animales , Aspartato-Semialdehído Deshidrogenasa/genética , Línea Celular , Reparación del ADN/genética , ADN Bacteriano/genética , Técnicas Genéticas , Vectores Genéticos/genética , Ratones , Ratones Endogámicos BALB C , Células RAW 264.7RESUMEN
Gene regulation network in Pseudomonas aeruginosa is complex. With a relatively large genome (6.2 Mb), there is a significant portion of genes that are proven or predicted to be transcriptional regulators. Many of these regulators have been shown to play important roles in biofilm formation and maintenance. In this study, we present a novel transcriptional regulator, PA1226, which modulates biofilm formation and virulence in P. aeruginosa. Mutation in the gene encoding this regulator abolished the ability of P. aeruginosa to produce biofilms in vitro, without any effect on the planktonic growth. This regulator is also essential for the in vivo fitness and pathogenesis in both Drosophila melanogaster and BALB/c mouse lung infection models. Transcriptome analysis revealed that PA1226 regulates many essential virulence genes/pathways, including those involved in alginate, pili, and LPS biosynthesis. Genes/operons directly regulated by PA1226 and potential binding sequences were identified via ChIP-seq. Attempts to confirm the binding sequences by electrophoretic mobility shift assay led to the discovery of a co-regulator, PA1413, via co-immunoprecipitation assay. PA1226 and PA1413 were shown to bind collaboratively to the promoter regions of their regulons. A model is proposed, summarizing our finding on this novel dual-regulation system.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/fisiología , Pseudomonas aeruginosa/patogenicidad , Factores de Transcripción/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Modelos Animales de Enfermedad , Drosophila melanogaster , Perfilación de la Expresión Génica , Humanos , Enfermedades Pulmonares/microbiología , Masculino , Ratones , Ratones Endogámicos BALB C , Pseudomonas aeruginosa/genética , Factores de Transcripción/genética , VirulenciaRESUMEN
Burkholderia spp. are genetically and physiologically diverse. Some strains are naturally transformable and capable of DNA catabolism. Burkholderia pseudomallei (Bp) strains 1026b and K96243 and B. thailandensis strain E264 are able to utilize DNA as a sole carbon source for growth, while only strains 1026b and E264 are naturally transformable. In this study, we constructed low-copy broad-host-range fosmid library, containing Bp strain 1026b chromosomal DNA fragments, and employed a novel positive selection approach to identify genes responsible for DNA uptake and DNA catabolism. The library was transferred to non-competent Bp K96243 and B. cenocepacia (Bc) K56-2, harboring chromosomally-inserted FRT-flanked sacB and pheS counter-selection markers. The library was incubated with DNA encoding Flp recombinase, followed by counter-selection on sucrose and chlorinated phenylalanine, to select for clones that took up flp-DNA, transiently expressed Flp, and excised the sacB-pheS cassette. Putative clones that survived the counter-selection were subsequently incubated with gfp-DNA and bacteria were visualized via fluorescent microscopy to confirm natural competency. Fosmid sequencing identified several 1026b genes implicated in DNA uptake, which were validated using chromosomal mutants. One of the naturally competent clones selected in Bc K56-2 enabled Bc, Bp and B. mallei to utilize DNA as a sole carbon source, and all fosmids were used to successfully create mutations in non-naturally-competent B. mallei and Bp strains.
Asunto(s)
Burkholderia pseudomallei/genética , ADN Bacteriano/metabolismo , Biblioteca de Genes , Genes Bacterianos , Plásmidos/genética , Alelos , Cromosomas Bacterianos/genética , Células Clonales , Reproducibilidad de los Resultados , Especificidad de la Especie , Transformación GenéticaRESUMEN
Bacterial cooperative associations and dynamics in biofilm microenvironments are of special interest in recent years. Knowledge of localized gene-expression and corresponding bacterial behaviors within the biofilm architecture at a global scale has been limited, due to a lack of robust technology to study limited number of cells in stratified layers of biofilms. With our recent pioneering developments in single bacterial cell transcriptomic analysis technology, we generated herein an unprecedented spatial transcriptome map of the mature in vitro Pseudomonas aeruginosa biofilm model, revealing contemporaneous yet altered bacterial behaviors at different layers within the biofilm architecture (i.e., surface, middle and interior of the biofilm). Many genes encoding unknown functions were highly expressed at the biofilm-solid interphase, exposing a critical gap in the knowledge of their activities that may be unique to this interior niche. Several genes of unknown functions are critical for biofilm formation. The in vivo importance of these unknown proteins was validated in invertebrate (fruit fly) and vertebrate (mouse) models. We envisage the future value of this report to the community, in aiding the further pathophysiological understanding of P. aeruginosa biofilms. Our approach will open doors to the study of bacterial functional genomics of different species in numerous settings.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Animales , Proteínas Bacterianas/genética , Drosophila/genética , Ratones , Mutación , Transcriptoma , Factores de Virulencia/genéticaRESUMEN
The fatty acid (FA) degradation pathway of Pseudomonas aeruginosa, an opportunistic pathogen, was recently shown to be involved in nutrient acquisition during BALB/c mouse lung infection model. The source of FA in the lung is believed to be phosphatidylcholine, the major component of lung surfactant. Previous research indicated that P. aeruginosa has more than two fatty acyl-CoA synthetase genes (fadD; PA3299 and PA3300), which are responsible for activation of FAs using ATP and coenzyme A. Through a bioinformatics approach, 11 candidate genes were identified by their homology to the Escherichia coli FadD in the present study. Four new homologues of fadD (PA1617, PA2893, PA3860, and PA3924) were functionally confirmed by their ability to complement the E. coli fadD mutant on FA-containing media. Growth phenotypes of 17 combinatorial fadD mutants on different FAs, as sole carbon sources, indicated that the four new fadD homologues are involved in FA degradation, bringing the total number of P. aeruginosa fadD genes to six. Of the four new homologues, fadD4 (PA1617) contributed the most to the degradation of different chain length FAs. Growth patterns of various fadD mutants on plant-based perfumery substances, citronellic and geranic acids, as sole carbon and energy sources indicated that fadD4 is also involved in the degradation of these plant-derived compounds. A decrease in fitness of the sextuple fadD mutant, relative to the ΔfadD1D2 mutant, was only observed during BALB/c mouse lung infection at 24 h.
Asunto(s)
Ácidos Grasos/metabolismo , Redes y Vías Metabólicas , Pseudomonas aeruginosa/metabolismo , Animales , Coenzima A Ligasas/genética , Coenzima A Ligasas/metabolismo , Ratones , Mutación , Fenotipo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crecimiento & desarrollo , Terpenos/metabolismo , Factores de Tiempo , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
We have made significant improvements to a broad-host-range system for the cloning and manipulation of large bacterial genomic regions based on site-specific recombination between directly repeated oriT sites during conjugation. Using two suicide capture vectors carrying flanking homology regions, oriT sites are recombined on either side of the target region. Using a broad-host-range conjugation helper plasmid, the region between the oriT sites is conjugated into an Escherichia coli recipient strain, where it is circularized and maintained as a chimeric mini-F vector. The cloned target region is functionalized in multiple ways to accommodate downstream manipulation. The target region is flanked with Gateway attB sites for recombination into other vectors and by rare 18-bp I-SceI restriction sites for subcloning. The Tn7-functionalized target can also be inserted at a naturally occurring chromosomal attTn7 site(s) or maintained as a broad-host-range plasmid for complementation or heterologous expression studies. We have used the oriTn7 capture technique to clone and complement Burkholderia pseudomallei genomic regions up to 140 kb in size and have created isogenic Burkholderia strains with various combinations of genomic islands. We believe this system will greatly aid the cloning and genetic analysis of genomic islands, biosynthetic gene clusters, and large open reading frames.