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1.
Int J Food Microbiol ; 320: 108524, 2020 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-32000116

RESUMEN

Salmonella is a bacterial pathogen frequently involved in human gastrointestinal infections including those associated with low-moisture foods such as dehydrated food powders/spices, vegetable seeds, and tree nuts. The survival/persistence of Salmonella on low moisture foods and in dry environments is enhanced by its ability in developing biofilms. This study was undertaken to identify the genetic determinants critical for Salmonella attachment and biofilm formation. E. coli SM10 lambda pir, with a kanamycin resistant marker on mini-Tn10 (mini-Tn10:lacZ:kanr), an ampicillin resistant marker on the mini-Tn10-bearing suicidal plasmid pLBT and a streptomycin sensitive marker on the SM10 chromosome, was used as a donor (ampr, kanr, streps), and three Salmonella strains (amps, kans, strepr) were used as recipients in a transposon mutagenesis study. The donor and each recipient were co-incubated overnight on tryptic soy agar at 37 °C, and mutant colonies (amps, kanr, strepr) were subsequently selected. A single-banded degenerate PCR product, amplified from each mutant genome using oligonucleotide primers derived from the end of min-Tn10 and restriction enzyme EcoR I- or Pst I-recognizing sequence, were analyzed using the Sanger sequencing technology. Acquired DNA sequences were compared to those deposited in the Genbank using BLAST search. Cells of Salmonella mutants accumulated either significantly more or less (P < 0.05) biofilms than their parent cells on polystyrene surface. Sequence analysis of degenerate PCR products revealed that the mini-Tn10 from pLBT had inserted into the cdg, trx, fadI or rxt on Salmonella chromosomes. Results of the research will likely help strategize future antimicrobial intervention for control of pathogen attachment and biofilm formation.


Asunto(s)
Adhesión Bacteriana/genética , Biopelículas/crecimiento & desarrollo , Salmonella enterica/genética , Escherichia coli/genética , Genes Bacterianos/genética , Humanos , Mutagénesis Insercional , Mutación , Salmonella enterica/fisiología
2.
Arch Microbiol ; 202(4): 835-842, 2020 May.
Artículo en Inglés | MEDLINE | ID: mdl-31865430

RESUMEN

Edwardsiella tarda (E. tarda) is distributed widely in a variety of hosts including humans, other mammals and fish, and it is worthwhile to notice that E. tarda -caused fish infections lead to the most important bacterial disease in fish. Considering Eha acting as a transcriptional regulator in E. tarda strain ET13 have been reported previously, to better understand its pathogenesis due to this, a type of cell of epithelial cell line (Caco-2) infection model for the pathogen was established in the laboratory. We focused on studying various parameters such as lactate dehydrogenase release (to measure cytotoxicity) and cell adhesions, both of which are related to the bacterial pathogenesis. Furthermore biofilm formation, hemolytic activity, and adhesion to Caco-2 cells were decreased in an E.tarda mutant strain with deletion in-frame isogenic gene eha (∆eha) compared to the wild-type and the complementary strain eha+ (an engineered construct of ∆eha expressing eha); Meanwhile, we found that hemolytic activity and biofilm formation were significantly enhanced in the strain eha+. Moreover, the ∆eha strain had attenuated pathogenicity in the zebrafish infection model. The data also demonstrated that the series of genes fimA, esrB, gadB, mukF, katB, and katG are regulated by eha based on a quantitative reverse transcription polymerase chain reaction tests and analysis. Thus our research data indicated that eha has an impact on hemolytic activity, biofilm formation, adhesion, and pathogenicity of pathogenic strain ET13 and plays an essential role in manifesting the virulence factors.


Asunto(s)
Biopelículas , Edwardsiella tarda/fisiología , Edwardsiella tarda/patogenicidad , Animales , Adhesión Bacteriana/genética , Proteínas Bacterianas/genética , Células CACO-2 , Edwardsiella tarda/genética , Edwardsiella tarda/metabolismo , Infecciones por Enterobacteriaceae/microbiología , Enfermedades de los Peces/microbiología , Hemólisis/genética , Humanos , Mutación , Eliminación de Secuencia , Virulencia/genética , Factores de Virulencia/genética , Pez Cebra
3.
BMC Genomics ; 20(1): 767, 2019 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-31640553

RESUMEN

BACKGROUND: Escherichia coli C forms more robust biofilms than other laboratory strains. Biofilm formation and cell aggregation under a high shear force depend on temperature and salt concentrations. It is the last of five E. coli strains (C, K12, B, W, Crooks) designated as safe for laboratory purposes whose genome has not been sequenced. RESULTS: Here we present the complete genomic sequence of this strain in which we utilized both long-read PacBio-based sequencing and high resolution optical mapping to confirm a large inversion in comparison to the other laboratory strains. Notably, DNA sequence comparison revealed the absence of several genes thought to be involved in biofilm formation, including antigen 43, waaSBOJYZUL for lipopolysaccharide (LPS) synthesis, and cpsB for curli synthesis. The first main difference we identified that likely affects biofilm formation is the presence of an IS3-like insertion sequence in front of the carbon storage regulator csrA gene. This insertion is located 86 bp upstream of the csrA start codon inside the - 35 region of P4 promoter and blocks the transcription from the sigma32 and sigma70 promoters P1-P3 located further upstream. The second is the presence of an IS5/IS1182 in front of the csgD gene. And finally, E. coli C encodes an additional sigma70 subunit driven by the same IS3-like insertion sequence. Promoter analyses using GFP gene fusions provided insights into understanding this regulatory pathway in E. coli. CONCLUSIONS: Biofilms are crucial for bacterial survival, adaptation, and dissemination in natural, industrial, and medical environments. Most laboratory strains of E. coli grown for decades in vitro have evolved and lost their ability to form biofilm, while environmental isolates that can cause infections and diseases are not safe to work with. Here, we show that the historic laboratory strain of E. coli C produces a robust biofilm and can be used as a model organism for multicellular bacterial research. Furthermore, we ascertained the full genomic sequence of this classic strain, which provides for a base level of characterization and makes it useful for many biofilm-based applications.


Asunto(s)
Biopelículas/crecimiento & desarrollo , Escherichia coli/genética , Genoma Bacteriano/genética , Adhesión Bacteriana/genética , Mapeo Cromosómico , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genes Reguladores/genética , Regiones Promotoras Genéticas , Estrés Salino/genética , Inversión de Secuencia , Temperatura , Factores de Transcripción/genética
4.
Microb Pathog ; 137: 103739, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31513896

RESUMEN

Helicobacter pylori infection can cause a wide range of digestive diseases. Gene hp0788 encodes an outer membrane protein HofF, which can reduce the bacterial adherence to the GES-1 cells and affect pathogenesis of H. pylori. In this study, the role of hp0788 in H. pylori infection was further analyzed. RNA-seq data showed that two genes (hp0523 and hp0539), located on the cagPAI, were down-regulated in Δ0788 mutant. The changes were confirmed through qRT-PCR, and the expression of these two genes will be almost recovered to the normal level in complemented strain. These two genes, hp0523 and hp0539, are known to be necessary for integrated T4SS, which related to CagA translocation and IL-8 induction. In H. pylori infected assay, lower amount of phosphorylated CagA and lower induction of IL-8 were both detected in GES-1 cells infected by Δ0788 mutant, compared with the wild type strain. Meanwhile, these reductions almost recovered to the wild-type level in complemented strain. These results reveal that there is a correlation between hp0788 disruption and CagA/IL-8 decline. Deletion of CagA-encoding gene (hp0547) in Δ0788 mutant was further constructed. The double deleted mutant shows lower IL-8-inducing capability than Δ0788 mutant, indicated the correlation between deficiency of CagA and reduced IL-8 production. These results together imply that disruption of hp0788 might affect the efficiency of T4SS and CagA injection, then weaken the induction of IL-8 in infected GES-1 cells.


Asunto(s)
Proteínas Bacterianas/genética , Células Epiteliales/microbiología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Gastropatías/microbiología , Antígenos Bacterianos/genética , Adhesión Bacteriana/genética , Línea Celular Tumoral , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/fisiología , Humanos , Interleucina-8/metabolismo
5.
Microb Pathog ; 137: 103748, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31521802

RESUMEN

Salmonellosis is a serious threat to human and animal health. Salmonella adhesion to the host cell is an initial and most crucial step in the pathogenesis of salmonellosis. Many factors are involved in the adhesion process of Salmonella infection. Fimbriae are one of the most important factors in the adhesion of Salmonella. The Salmonella fimbriae are assembled in three types of assembly pathways: chaperon-usher, nucleation-precipitation, and type IV fimbriae. These assembly pathways lead to multiple types of fimbriae. Salmonella fimbriae bind to host cell receptors to initiate adhesion. So far, many receptors have been identified, such as Toll-like receptors. However, several receptors that may be involved in the adhesive mechanism of Salmonella fimbriae are still un-identified. This review aimed to summarize the types of Salmonella fimbriae produced by different assembly pathways and their role in adhesion. It also enlisted previously discovered receptors involved in adhesion. This review might help readers to develop a comprehensive understanding of Salmonella fimbriae, their role in adhesion, and recently developed strategies to counter Salmonella infection.


Asunto(s)
Adhesinas Bacterianas/fisiología , Adhesión Bacteriana/fisiología , Fimbrias Bacterianas/fisiología , Salmonella/fisiología , Adhesinas Bacterianas/genética , Animales , Adhesión Bacteriana/genética , Proteínas Bacterianas/genética , Proteínas Fimbrias/genética , Proteínas Fimbrias/fisiología , Genes Bacterianos , Humanos , Salmonella/genética , Infecciones por Salmonella , Receptores Toll-Like
6.
J Microbiol Biotechnol ; 29(11): 1806-1816, 2019 Nov 28.
Artículo en Inglés | MEDLINE | ID: mdl-31546294

RESUMEN

Candida albicans is an opportunistic fungus possessing multiple virulence factors controlling pathogenicity. Cell wall proteins are the most important among these factors, being the first elements contacting the host. Ddr48 is a cell wall protein consisting of 212 amino acids. A DDR48 haploinsufficient mutant strain was previously found necessary for proper oxidative stress response and drug resistance. In this study, we aimed to further elucidate the role of Ddr48 by performing additional phenotypic characterization assays. A combinatory proteomic and bioinformatics approach was also undertaken to determine differentially expressed cell wall proteins. Results showed that the mutant strain exhibited a 10% decrease in adhesion mirrored by a 20% decrease in biofilm formation, and slight sensitivity to menadione, diamide, and SDS. Both strains showed similar hyphae formation, virulence, temperature tolerance, and calcofluor white and Congo red sensitivities. Furthermore, a total of 8 and 10 proteins were identified exclusively in the wild-type strain grown under filamentous and nonfilamentous conditions respectively. Results included proteins responsible for superoxide stress resistance (Sod4 and Sod6), adhesion (Als3, Hyr4, Pmt1, and Utr2), biofilm formation (Hsp90, Ece1, Rim9, Ipp1, and Pra1) and cell wall integrity (Utr2 and Pga4). The lack of detection of these proteins in the mutant strain correlates with the observed phenotypes.


Asunto(s)
Candida albicans/fisiología , Pared Celular/metabolismo , Proteínas Fúngicas/genética , Estrés Oxidativo/genética , Factores de Virulencia/genética , Adhesión Bacteriana/genética , Biopelículas/crecimiento & desarrollo , Candida albicans/genética , Candida albicans/metabolismo , Pared Celular/genética , Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/metabolismo , Hifa/genética , Hifa/metabolismo , Mutación , Fenotipo , Proteómica , Factores de Virulencia/metabolismo
7.
Vet Res ; 50(1): 69, 2019 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-31547880

RESUMEN

Haemophilus parasuis is part of the microbiota of the upper respiratory tract in swine. However, virulent strains can cause a systemic disease known as Glässer's disease. Several virulence factors have been described in H. parasuis including the virulence-associated trimeric autotransporters (VtaAs). VtaA2 is up-regulated during infection and is only found in virulent strains. In order to determine its biological function, the vtaA2 gene was cloned with its native promotor region in pACYC184, and the transformed Escherichia coli was used to perform functional in vitro assays. VtaA2 was found to have a role in attachment to plastic, mucin, BSA, fibronectin and collagen. As other VtaAs from H. parasuis, the passenger domain of VtaA2 contains collagen domains. In order to examine the contribution of the collagen repeats to VtaA2 function, a recombinant vtaA2 without the central collagen domains was obtained and named vtaA2OL. VtaA2OL showed similar capacity than VtaA2 to adhere to plastic, mucin, BSA, fibronectin and plasma but a reduced capacity to adhere to collagen, suggesting that the collagen domains of VtaA2 are involved in collagen attachment. No function in cell adhesion and invasion to epithelial alveolar cell line A549 or unspecific binding to primary alveolar macrophages was found. Likewise VtaA2 had no role in serum or phagocytosis resistance. We propose that VtaA2 mediates adherence to the host by binding to the mucin, found in the upper respiratory tract mucus, and to the extracellular matrix proteins, present in the connective tissue of systemic sites, such as the serosa.


Asunto(s)
Adhesión Bacteriana/genética , Infecciones por Haemophilus/veterinaria , Haemophilus parasuis/fisiología , Enfermedades de los Porcinos/microbiología , Factores de Virulencia/genética , Animales , Escherichia coli/genética , Infecciones por Haemophilus/microbiología , Porcinos , Virulencia/genética
8.
Infect Immun ; 87(12)2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31548319

RESUMEN

Antibodies are essential for immunity against Ehrlichia chaffeensis, and protective mechanisms involve blocking of ehrlichial attachment or complement and Fcγ-receptor-dependent destruction. In this study, we determined that major outer membrane protein 1 (OMP-19) hypervariable region 1 (HVR1)-specific human monoclonal antibodies (huMAbs) are protective through conventional extracellular neutralization and, more significantly, through a novel intracellular TRIM21-mediated mechanism. Addition of OMP-1-specific huMAb EHRL-15 (IgG1) prevented infection by blocking attachment/entry, a mechanism previously reported; conversely, OMP-1-specific huMAb EHRL-4 (IgG3) engaged intracellular TRIM21 and initiated an immediate innate immune response and rapid intracellular degradation of ehrlichiae. EHRL-4-TRIM21-mediated inhibition was significantly impaired in TRIM21 knockout THP-1 cells. EHRL-4 interacted with cytosolic Fc receptor TRIM21, observed by confocal microscopy and confirmed by co-immunoprecipitation. E. chaffeensis-EHRL-4-TRIM21 complexes caused significant upregulation of proinflammatory cytokine/chemokine transcripts and resulted in rapid (<30 min) nuclear accumulation of NF-κB and TRIM21 and ehrlichial destruction. We investigated the role of TRIM21 in the autophagic clearance of ehrlichiae in the presence of EHRL-4. Colocalization between EHRL-4-opsonized ehrlichiae, polyubiquitinated TRIM21, autophagy regulators (ULK1 and beclin 1) and effectors (LC3 and p62), and lysosome-associated membrane protein 2 (LAMP2) was observed. Moreover, autophagic flux defined by conversion of LC3I to LC3II and accumulation and degradation of p62 was detected, and EHRL-4-mediated degradation of E. chaffeensis was abrogated by the autophagy inhibitor 3-methyladenine. Our results demonstrate that huMAbs are capable of inhibiting E. chaffeensis infection by distinct effector mechanisms: extracellularly by neutralization and intracellularly by engaging TRIM21, which mediates a rapid innate immune response that mobilizes the core autophagy components, triggering localized selective autophagic degradation of ehrlichiae.


Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Ehrlichia chaffeensis/inmunología , Ribonucleoproteínas/genética , Adenina/análogos & derivados , Adenina/farmacología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Antígenos Bacterianos/genética , Autofagia/inmunología , Adhesión Bacteriana/genética , Sistemas CRISPR-Cas , Línea Celular Tumoral , Ehrlichia chaffeensis/genética , Técnicas de Inactivación de Genes , Humanos , Inmunidad Humoral/inmunología , FN-kappa B/genética , Células THP-1
9.
Infect Immun ; 87(12)2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31548326

RESUMEN

The type IV pilus (Tfp) of nontypeable Haemophilus influenzae (NTHI) mediates adherence, colonization, motility, and biofilm formation, and the major protein subunit, PilA, is a promising vaccine candidate. Thus, it is crucial to understand how Tfp expression is regulated within the microenvironments of the human nasopharynx, which NTHI colonizes asymptomatically, and the more distal regions of the respiratory tract where NTHI-induced diseases occur. Here, we examined the effects of coculture of NTHI with human airway epithelial cells and heme availability on Tfp expression at temperatures typical of the human nasopharynx (34°C) or warmer anatomical sites during infection (37°C). Tfp expression was estimated by pilA promoter activity, pilA gene expression, and relative abundances of PilA and pilin protein. The results revealed that at both temperatures, NTHI cocultured with airway epithelial cells demonstrated significantly greater expression of pilA, PilA/pilin protein, and likely, fully assembled Tfp than NTHI cultured on an abiotic surface. Because NTHI is a heme auxotroph, we hypothesized that availability of heme from host cells might be a signal for Tfp expression. Thereby, we cultured NTHI in iron-limited medium, and we observed that supplementation with heme significantly increased pilA promoter activity. Collectively, our data suggested that NTHI Tfp expression was stimulated by soluble factor(s) released by epithelial cells, which are present in all microenvironments of the respiratory tract. The expression of this target antigen under conditions that mimic the human airway strongly supports the rationale for the use of PilA as a vaccine immunogen to prevent NTHI-induced diseases of the respiratory tract.


Asunto(s)
Proteínas Fimbrias/biosíntesis , Proteínas Fimbrias/inmunología , Fimbrias Bacterianas/inmunología , Haemophilus influenzae/inmunología , Nasofaringe/inmunología , Adhesión Bacteriana/genética , Vacunas Bacterianas/inmunología , Células Cultivadas , Técnicas de Cocultivo , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Proteínas Fimbrias/genética , Fimbrias Bacterianas/metabolismo , Infecciones por Haemophilus/inmunología , Infecciones por Haemophilus/microbiología , Hemo/metabolismo , Humanos , Nasofaringe/microbiología , Regiones Promotoras Genéticas/genética , Sistema Respiratorio/citología
10.
BMC Vet Res ; 15(1): 319, 2019 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-31488137

RESUMEN

BACKGROUND: Small non-coding RNAs (sRNAs) regulate bacterial gene expression at the post-transcriptional level. STnc640 is a type of sRNA that was identified in Salmonella Typhimurium. RESULTS: In this study, STnc640 in Salmonella Enteritidis was confirmed to be an Hfq-dependent sRNA. TargetRNA software analysis showed that fimbrial genes fimA and bcfA were likely to be the target genes of STnc640. To investigate the target mRNAs and function of STnc640 in pathogenicity, we constructed the deletion mutant strain 50336△stnc640 and the complemented strain 50336△stnc640/pstnc640 in Salmonella Enteritidis 50336. The RT-qPCR results showed that the mRNA level of fimA was decreased, while bcfA was unchanged in 50336△stnc640 compared with that in the wild type (WT) strain. The adhesion ability of 50336△stnc640 to Caco-2 cells was increased compared to the 50336 WT strain. The virulence of 50336△stnc640 was enhanced in a one-day-old chicken model of S. Enteritidis disease as determined by quantifying the 50% lethal dose (LD50) of the bacterial strains. CONCLUSIONS: The results demonstrate that STnc640 contributes to the virulence of Salmonella Enteritidis.


Asunto(s)
Antígenos Bacterianos/genética , Proteínas Fimbrias/genética , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano/fisiología , ARN Pequeño no Traducido/fisiología , Salmonella enteritidis/genética , Salmonella enteritidis/patogenicidad , Animales , Adhesión Bacteriana/genética , Células CACO-2 , Pollos , Femenino , Humanos , Masculino , Enfermedades de las Aves de Corral/virología , Salmonelosis Animal/virología , Eliminación de Secuencia , Virulencia/genética
11.
mBio ; 10(5)2019 09 10.
Artículo en Inglés | MEDLINE | ID: mdl-31506311

RESUMEN

Bacterial adhesion is accompanied by altered gene expression, leading to "emergent" properties of biofilm bacteria that are alien to planktonic ones. With the aim of revealing the role of environmental adhesion forces in emergent biofilm properties, genes in Streptococcus mutans UA159 and a quorum-sensing-deficient mutant were identified that become expressed after adhesion to substratum surfaces. Using atomic force microscopy, adhesion forces of initial S. mutans colonizers on four different substrata were determined and related to gene expression. Adhesion forces upon initial contact were similarly low across different substrata, ranging between 0.2 and 1.2 nN regardless of the strain considered. Bond maturation required up to 21 s, depending on the strain and substratum surface involved, but stationary adhesion forces also were similar in the parent and in the mutant strain. However, stationary adhesion forces were largest on hydrophobic silicone rubber (19 to 20 nN), while being smallest on hydrophilic glass (3 to 4 nN). brpA gene expression in thin (34 to 48 µm) 5-h S. mutans UA159 biofilms was most sensitive to adhesion forces, while expression of gbpB and comDE expressions was weakly sensitive. ftf, gtfB, vicR, and relA expression was insensitive to adhesion forces. In thicker (98 to 151 µm) 24-h biofilms, adhesion-force-induced gene expression and emergent extracellular polymeric substance (EPS) production were limited to the first 20 to 30 µm above a substratum surface. In the quorum-sensing-deficient S. mutans, adhesion-force-controlled gene expression was absent in both 5- and 24-h biofilms. Thus, initial colonizers of substratum surfaces sense adhesion forces that externally trigger emergent biofilm properties over a limited distance above a substratum surface through quorum sensing.IMPORTANCE A new concept in biofilm science is introduced: "adhesion force sensitivity of genes," defining the degree up to which expression of different genes in adhering bacteria is controlled by the environmental adhesion forces they experience. Analysis of gene expression as a function of height in a biofilm showed that the information about the substratum surface to which initially adhering bacteria adhere is passed up to a biofilm height of 20 to 30 µm above a substratum surface, highlighting the importance and limitations of cell-to-cell communication in a biofilm. Bacteria in a biofilm mode of growth, as opposed to planktonic growth, are responsible for the great majority of human infections, predicted to become the number one cause of death in 2050. The concept of adhesion force sensitivity of genes provides better understanding of bacterial adaptation in biofilms, direly needed for the design of improved therapeutic measures that evade the recalcitrance of biofilm bacteria to antimicrobials.


Asunto(s)
Adhesión Bacteriana/genética , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Streptococcus mutans/genética , Adhesión Bacteriana/fisiología , Proteínas Bacterianas/metabolismo , Liasas de Carbono-Azufre/genética , Matriz Extracelular de Sustancias Poliméricas , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía de Fuerza Atómica , Mutación , Percepción de Quorum/genética , Streptococcus mutans/metabolismo
12.
Commun Biol ; 2: 322, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31482141

RESUMEN

There is an urgent need to understand the global epidemiological landscape of carbapenem-resistant Escherichia coli (CREC). Here we provide combined genomic and phenotypic characterization of the emergence of a CREC clone from the ST410 lineage. We show that the clone expands with a single plasmid, within which there is frequent switching of the carbapenemase gene type between bla NDM and bla OXA-181 with no impact on plasmid stability or fitness. A search for clone-specific traits identified unique alleles of genes involved in adhesion and iron acquisition, which have been imported via recombination. These recombination-derived allelic switches had no impact on virulence in a simple infection model, but decreased efficiency in binding to abiotic surfaces and greatly enhanced fitness in iron limited conditions. Together our data show a footprint for evolution of a CREC clone, whereby recombination drives new alleles into the clone which provide a competitive advantage in colonizing mammalian hosts.


Asunto(s)
Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Escherichia coli/genética , Adhesión Bacteriana/efectos de los fármacos , Adhesión Bacteriana/genética , Biopelículas/efectos de los fármacos , Enterobacteriaceae Resistentes a los Carbapenémicos/efectos de los fármacos , Enterobacteriaceae Resistentes a los Carbapenémicos/patogenicidad , Células Clonales , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Escherichia coli/patogenicidad , Genes Bacterianos , Hierro/farmacología , Pruebas de Sensibilidad Microbiana , Filogenia , Plásmidos/genética , Polimorfismo de Nucleótido Simple/genética , Recombinación Genética/genética
13.
Microbiology ; 165(10): 1061-1074, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31436522

RESUMEN

In radiating populations of Pseudomonas fluorescens SBW25, adaptive wrinkly spreader (WS) mutants are able to gain access to the air-liquid (A-L) interface of static liquid microcosms and achieve a significant competitive fitness advantage over other non-biofilm-forming competitors. Aerotaxis and flagella-based swimming allows SBW25 cells to move into the high-O2 region located at the top of the liquid column and maintain their position by countering the effects of random cell diffusion, convection and disturbance (i.e. physical displacement). However, wild-type cells showed significantly lower levels of enrichment in this region compared to the archetypal WS, indicating that WS cells employ an additional mechanism to transfer to the A-L interface where displacement is no longer an issue and a biofilm can develop at the top of the liquid column. Preliminary experiments suggest that this might be achieved through the expression of an as yet unidentified surface active agent that is weakly associated with WS cells and alters liquid surface tension, as determined by quantitative tensiometry. The effect of physical displacement on the colonization of the high-O2 region and A-L interface was reduced through the addition of agar or polyethylene glycol to increase liquid viscosity, and under these conditions the competitive fitness of the WS was significantly reduced. These observations suggest that the ability to transfer to the A-L interface from the high-O2 region and remain there without further expenditure of energy (through, for example, the deployment of flagella) is a key evolutionary innovation of the WS, as it allows subsequent biofilm development and significant population increase, thereby affording these adaptive mutants a competitive fitness advantage over non-biofilm-forming competitors located within the liquid column.


Asunto(s)
Biopelículas/crecimiento & desarrollo , Pseudomonas fluorescens/fisiología , Adhesión Bacteriana/genética , Evolución Biológica , Ambiente , Flagelos/genética , Aptitud Genética , Oxígeno/metabolismo , Fenotipo , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/crecimiento & desarrollo , Tensión Superficial , Taxia , Viscosidad
14.
J Biol Chem ; 294(40): 14499-14511, 2019 10 04.
Artículo en Inglés | MEDLINE | ID: mdl-31439670

RESUMEN

Growth of the cholera bacterium Vibrio cholerae in a biofilm community contributes to both its pathogenicity and survival in aquatic environmental niches. The major components of V. cholerae biofilms include Vibrio polysaccharide (VPS) and the extracellular matrix proteins RbmA, RbmC, and Bap1. To further elucidate the previously observed overlapping roles of Bap1 and RbmC in biofilm architecture and surface attachment, here we investigated the structural and functional properties of Bap1. Soluble expression of Bap1 was possible only after the removal of an internal 57-amino-acid-long hydrophobic insertion sequence. The crystal structure of Bap1 at 1.9 Å resolution revealed a two-domain assembly made up of an eight-bladed ß-propeller interrupted by a ß-prism domain. The structure also revealed metal-binding sites within canonical calcium blade motifs, which appear to have structural rather than functional roles. Contrary to results previously observed with RbmC, the Bap1 ß-prism domain did not exhibit affinity for complex N-glycans, suggesting an altered role of this domain in biofilm-surface adhesion. Native polyacrylamide gel shift analysis did suggest that Bap1 exhibits lectin activity with a preference for anionic or linear polysaccharides. Our results suggest a model for V. cholerae biofilms in which Bap1 and RbmC play dominant but differing adhesive roles in biofilms, allowing bacterial attachment to diverse environmental or host surfaces.


Asunto(s)
Proteínas Bacterianas/ultraestructura , Cólera/enzimología , Proteínas de la Matriz Extracelular/ultraestructura , Conformación Proteica , Vibrio cholerae/enzimología , Secuencia de Aminoácidos/genética , Amiloide/química , Adhesión Bacteriana/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión/genética , Biopelículas , Cólera/genética , Cólera/microbiología , Cristalografía por Rayos X , Proteínas de la Matriz Extracelular/química , Regulación Bacteriana de la Expresión Génica/genética , Lectinas/química , Metales/química , Polisacáridos/química , Vibrio cholerae/química , Vibrio cholerae/genética , Vibrio cholerae/patogenicidad , Factores de Virulencia/genética
15.
mBio ; 10(4)2019 08 13.
Artículo en Inglés | MEDLINE | ID: mdl-31409683

RESUMEN

Nontuberculous mycobacteria (NTM) are emerging opportunistic pathogens that colonize household water systems and cause chronic lung infections in susceptible patients. The ability of NTM to form surface-attached biofilms in the nonhost environment and corded aggregates in vivo is important to their ability to persist in both contexts. Underlying the development of these multicellular structures is the capacity of mycobacterial cells to adhere to one another. Unlike most other bacteria, NTM spontaneously and constitutively aggregate in vitro, hindering our ability to understand the transition between planktonic and aggregated cells. While culturing a model NTM, Mycobacterium smegmatis, in rich medium, we fortuitously discovered that planktonic cells accumulate after ∼3 days of growth. By providing selective pressure for bacteria that disperse earlier, we isolated a strain with two mutations in the oligopeptide permease operon (opp). A mutant lacking the opp operon (Δopp) disperses earlier than wild type (WT) due to a defect in nutrient uptake. Experiments with WT M. smegmatis revealed that growth as aggregates is favored when carbon is replete, but under conditions of low available carbon relative to available nitrogen, M. smegmatis grows as planktonic cells. By adjusting carbon and nitrogen sources in defined medium, we tuned the cellular C/N ratio such that M. smegmatis grows either as aggregates or as planktonic cells. C/N-mediated aggregation regulation is widespread among NTM with the possible exception of rough-colony Mycobacterium abscessus isolates. Altogether, we show that NTM aggregation is a controlled process that is governed by the relative availability of carbon and nitrogen for metabolism.IMPORTANCE Free-living bacteria can assemble into multicellular structures called biofilms. Biofilms help bacteria tolerate multiple stresses, including antibiotics and the host immune system. Nontuberculous mycobacteria are a group of emerging opportunistic pathogens that utilize biofilms to adhere to household plumbing and showerheads and to avoid phagocytosis by host immune cells. Typically, bacteria regulate biofilm formation by controlling expression of adhesive structures to attach to surfaces and other bacterial cells. Mycobacteria harbor a unique cell wall built chiefly of long-chain mycolic acids that confers hydrophobicity and has been thought to cause constitutive aggregation in liquid media. Here we show that aggregation is instead a regulated process dictated by the balance of available carbon and nitrogen. Understanding that mycobacteria utilize metabolic cues to regulate the transition between planktonic and aggregated cells reveals an inroad to controlling biofilm formation through targeted therapeutics.


Asunto(s)
Adhesión Bacteriana , Carbono/metabolismo , Nitrógeno/metabolismo , Micobacterias no Tuberculosas/fisiología , Adhesión Bacteriana/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Medios de Cultivo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mutación , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/fisiología , Micobacterias no Tuberculosas/crecimiento & desarrollo , Micobacterias no Tuberculosas/metabolismo , Operón
16.
Vet Res ; 50(1): 43, 2019 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-31164171

RESUMEN

Riemerella anatipestifer is a major pathogenic agent of duck septicemic and exudative diseases. Genetic analyses suggest that this pathogen has a novel protein secretion system, known as the "type IX secretion system" (T9SS). We previously reported that deletion of the AS87_RS08465 gene significantly reduced the bacterial virulence of the R. anatipestifer strain Yb2, but the mechanism remained unclear. The AS87_RS08465 gene is predicted to encode the gliding motility protein GldM (GldM) protein, a key component of the T9SS complex. In this study, Western blotting analysis demonstrated that R. anatipestifer GldM was localized to the cytomembrane. Further study revealed that the adhesion and invasion capacities of the mutant strain RA2281 (designated Yb2ΔgldM) in Vero cells and the bacterial loads in the blood of infected ducks were significantly reduced. RNA-Seq and PCR analyses showed that six genes were upregulated and five genes were downregulated in the mutant strain Yb2ΔgldM and that these genes were mainly involved in the secretion of proteins. Yb2ΔgldM was also found to be defective in gliding motility and protein secretion. Liquid chromatography-tandem mass spectrometry analysis revealed that nine of the proteins had a conserved T9SS C-terminal domain and were differentially secreted by Yb2ΔgldM compared to Yb2. The complementation strain cYb2ΔgldM recovered the adhesion and invasion capacities in Vero cells and the bacterial loads in the blood of infected ducks as well as the bacterial gliding motility and most protein secretion in the mutant strain Yb2ΔgldM to the levels of the wild-type strain Yb2. Taken together, these results indicate that R. anatipestifer GldM is associated with T9SS and is important in bacterial virulence.


Asunto(s)
Adhesión Bacteriana/genética , Expresión Génica , Riemerella/genética , Riemerella/patogenicidad , Sistemas de Secreción Tipo IV/genética , Mutación , Péptido Hidrolasas/biosíntesis , Riemerella/enzimología , Sistemas de Secreción Tipo IV/metabolismo , Virulencia/genética , Factores de Virulencia/genética
17.
Microbes Environ ; 34(3): 244-251, 2019 Sep 25.
Artículo en Inglés | MEDLINE | ID: mdl-31189768

RESUMEN

The thermophilic hydrogenotrophic methanogen, Methanothermobacter sp. CaT2, which possesses an extracellular sugar layer, commonly aggregates by itself or with other microorganisms. To elucidate the molecular mechanisms responsible for this aggregation, the aggregation-defective mutant, CLA160, was isolated. Optical and electron microscopy observations revealed that the mutant exhibited a significant reduction in aggregation. Genomic sequencing showed that CLA160 has a single point mutation, causing a nonsense mutation in MTCT_1020, which encodes a hypothetical protein. Motif and domain analyses indicated that the hypothetical protein bears two membrane-spanning segments at the N- and C-terminal regions and a large middle repeat-containing region. The results of a bioinformatic analysis suggested that the first middle region (RII) of the protein or the whole structure is responsible for the function of the product of MTCT_1020 in the aggregation of CaT2. A treatment with proteinase K suppressed sedimentation in CaT2, indicating a reduction in aggregation, with almost no effect on sedimentation in CLA160. The addition of Ca2+ or Mg2+ ions enhanced sedimentation in CaT2, whereas a DNase treatment had no effect on sedimentation in either strain. These results suggest that the hypothetical protein encoded by MTCT_1020 plays a key role as a membrane-bound adhesion protein in the aggregation of CaT2, which is enhanced by the addition of Ca2+ or Mg2+ ions.


Asunto(s)
Adhesión Bacteriana/genética , Proteínas Bacterianas/genética , Methanobacteriaceae/genética , Adhesión Bacteriana/efectos de los fármacos , Proteínas Bacterianas/química , Cationes Bivalentes/farmacología , Endopeptidasa K/farmacología , Genoma Bacteriano/genética , Calor , Metano/metabolismo , Methanobacteriaceae/clasificación , Methanobacteriaceae/ultraestructura , Mutación , Filogenia , Dominios Proteicos , Análisis de Secuencia de ADN
18.
Nat Microbiol ; 4(9): 1545-1557, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31182799

RESUMEN

How bacteria colonize surfaces and how they distinguish the individuals around them are fundamental biological questions. Type IV pili are a widespread and multipurpose class of cell surface polymers. Here we directly visualize the DNA-uptake pilus of Vibrio cholerae, which is produced specifically during growth on its natural habitat-chitinous surfaces. As predicted, these pili are highly dynamic and retract before DNA uptake during competence for natural transformation. Interestingly, DNA-uptake pili can also self-interact to mediate auto-aggregation. This capability is conserved in disease-causing pandemic strains, which typically encode the same major pilin subunit, PilA. Unexpectedly, however, we discovered that extensive strain-to-strain variability in PilA (present in environmental isolates) creates a set of highly specific interactions, enabling cells producing pili composed of different PilA subunits to distinguish between one another. We go on to show that DNA-uptake pili bind to chitinous surfaces and are required for chitin colonization under flow, and that pili capable of self-interaction connect cells on chitin within dense pili networks. Our results suggest a model whereby DNA-uptake pili function to promote inter-bacterial interactions during surface colonization. Moreover, they provide evidence that type IV pili could offer a simple and potentially widespread mechanism for bacterial kin recognition.


Asunto(s)
Quitina/metabolismo , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Vibrio cholerae/fisiología , Adhesión Bacteriana/genética , ADN Bacteriano/metabolismo , Proteínas Fimbrias/genética , Fimbrias Bacterianas/genética , Variación Genética , Humanos , Transformación Bacteriana , Vibrio cholerae/genética , Vibrio cholerae/metabolismo , Vibrio cholerae/patogenicidad
19.
Elife ; 82019 06 10.
Artículo en Inglés | MEDLINE | ID: mdl-31180327

RESUMEN

The second messenger signaling molecule cyclic diguanylate monophosphate (c-di-GMP) drives the transition between planktonic and biofilm growth in many bacterial species. Pseudomonas aeruginosa has two surface sensing systems that produce c-di-GMP in response to surface adherence. Current thinking in the field is that once cells attach to a surface, they uniformly respond by producing c-di-GMP. Here, we describe how the Wsp system generates heterogeneity in surface sensing, resulting in two physiologically distinct subpopulations of cells. One subpopulation has elevated c-di-GMP and produces biofilm matrix, serving as the founders of initial microcolonies. The other subpopulation has low c-di-GMP and engages in surface motility, allowing for exploration of the surface. We also show that this heterogeneity strongly correlates to surface behavior for descendent cells. Together, our results suggest that after surface attachment, P. aeruginosa engages in a division of labor that persists across generations, accelerating early biofilm formation and surface exploration.


Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , GMP Cíclico/análogos & derivados , Pseudomonas aeruginosa/metabolismo , Adhesión Bacteriana/genética , Adhesión Bacteriana/fisiología , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , GMP Cíclico/metabolismo , Regulación Bacteriana de la Expresión Génica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiología , Percepción de Quorum/genética
20.
Microb Pathog ; 133: 103547, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-31112774

RESUMEN

Death from infectious diseases has caused concerns about increases in the resistance of pathogens, impelling researchers to create novel therapeutic solutions. The management of intestinal tract problems has been the advance use of probiotics in medicine. The aim of this study was evaluate the physicochemical cell surface and adhesion properties of recombinant Lacotococcus lactis NZ1330 containing Ama r 2 gene, followed by the assessment of the antagonistic activity of this strain against the Escherichia coli causing urinary tract infection (UTI) in humans. For this purpose, cloning and expression of Ama r 2 gene were done. Afterwards, acid and bile resistance, which are the primary characteristics of any probiotic, were evaluated. The r-L. lactis NZ1330 was examined for the physicochemical properties of cell surfaces and the adhesion properties against Escherichia coli. Furthermore, the potential of the recombinant strain to adhere to adenocarcinoma intestinal cell line, Caco-2 cells, as well as the antagonistic properties of r-L. lactis NZ1330 against E. coli was investigated. r-L. lactis NZ1330 was capable of surviving at low pH and different concentrations of bile salts. 40.1% hydrophobicity, 36.5% auto-aggregation and 14.4% co-aggregation were observed for this strain. The adhesion level of r-L. lactis NZ1330 was 5.7% which was also confirmed by scanning electron microscopy (SEM). r-L. lactis NZ1330 was able to compete, inhibit and displace the adhesion of Escherichia coli to Caco-2 cells. r-L. lactis NZ1330 was considered to be a reliable probiotic alternative by showing these desirable properties. Results revealed that Ama r 2 gene expression had no effect on the positive probiotic properties of L. lactis NZ1330, proving this strain could be a suitable probiotic host for the expression of this allergen.


Asunto(s)
Antibiosis , Adhesión Bacteriana , Escherichia coli/crecimiento & desarrollo , Escherichia coli/patogenicidad , Lactococcus lactis/genética , Lactococcus lactis/fisiología , Infecciones Urinarias/microbiología , Adhesión Bacteriana/genética , Ácidos y Sales Biliares , Células CACO-2 , Agregación Celular , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Interacciones Microbiota-Huesped , Humanos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Intestinos/microbiología , Probióticos/farmacología , Transformación Bacteriana
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