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1.
Nucleic Acids Res ; 48(5): 2348-2356, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31960057

RESUMO

Gene gain by horizontal gene transfer is a major pathway of genome innovation in bacteria. The current view posits that acquired genes initially need to be silenced and that a bacterial chromatin protein, H-NS, plays a role in this silencing. However, we lack direct observation of the early fate of a horizontally transferred gene to prove this theory. We combine sequencing, flow cytometry and sorting, followed by microscopy to monitor gene expression and its variability after large-scale random insertions of a reporter gene in a population of Escherichia coli bacteria. We find that inserted promoters have a wide range of gene-expression variability related to their location. We find that high-expression clones carry insertions that are not correlated with H-NS binding. Conversely, binding of H-NS correlates with silencing. Finally, while most promoters show a common level of extrinsic noise, some insertions show higher noise levels. Analysis of these high-noise clones supports a scenario of switching due to transcriptional interference from divergent ribosomal promoters. Altogether, our findings point to evolutionary pathways where newly-acquired genes are not necessarily silenced, but may immediately explore a wide range of expression levels to probe the optimal ones.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteínas de Fímbrias/genética , Regulação Bacteriana da Expressão Gênica , Mutagênese Insercional , Regiões Promotoras Genéticas , Cromatina/química , Cromatina/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismo , Dosagem de Genes , Inativação Gênica , Transferência Genética Horizontal , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Transgenes
2.
Res Vet Sci ; 128: 308-314, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31901569

RESUMO

As most pathogens invade the bodies through the mucosa, it is crucial to develop vaccines that induce mucosal immunity. To this end, we generated a safe and effective vaccine candidate that displayed fimbrial protein 987P of enterotoxigenic Escherichia coli (ETEC) on the surface of Lactobacillus casei (L.casei) CICC 6105 by using poly-γ-glutamate synthetase A (PgsA) as an anchoring matrix. After gavage inoculation of the recombinant strain pLA-987P/L.casei into specific-pathogen-free (SPF) BALB/c mice, high levels of mucosal immunoglobulin A (IgA) were induced in fecal samples, intestine and lung lavage fluids and systemic immunoglobulin G of IgG subclasses (IgG1, IgG2b, and IgG2a) was produced in serum. T-cell proliferation assays showed the stimulation index (SI) of the groups immunized with pLA-987P/L.casei to be significantly higher than that of the control group. The recombinant L.casei promoted T cells to produce both Th1 and Th2 cytokines, while the number of splenic IL-4 Spot forming cells (SFC) exceeded the number of IFN-γ SFC by 2.26-fold (P < .01). >83.3% of the vaccinated mice were protected from challenge with a lethal dose of virulent strain C83916. These results indicate that the recombinant L.casei expressing ETEC 987P fimbrial protein could elicit a protective immune response against ETEC 987P infection effectively.


Assuntos
Adesinas de Escherichia coli/imunologia , Escherichia coli Enterotoxigênica/imunologia , Vacinas contra Escherichia coli/biossíntese , Proteínas de Fímbrias/imunologia , Lactobacillus casei/imunologia , Microrganismos Geneticamente Modificados/imunologia , Adesinas de Escherichia coli/genética , Administração Oral , Animais , Antígenos Heterófilos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/imunologia , Vacinas contra Escherichia coli/imunologia , Proteínas de Fímbrias/genética , Imunidade Humoral , Imunidade nas Mucosas , Imunogenicidade da Vacina , Lactobacillus casei/genética , Camundongos , Camundongos Endogâmicos BALB C , Transformação Bacteriana/genética , Transformação Bacteriana/imunologia , Vacinação/métodos
3.
Vet Microbiol ; 239: 108445, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31767071

RESUMO

Avian pathogenic E. coli (APEC) and human uropathogenic E. coli (UPEC) harbour common virulence factors in spite of being associated with disease in different hosts. APEC strains have been shown to have zoonotic potential. In contrast, it is not known whether UPEC strains can cause infection in immunologically competent hens. The objective of the current study was to compare the ability of the well-characterized UPEC strain, UTI89, and the APEC strain, F149H1S2, to infect human and avian cells in culture and to cause salpingitis in an infection model in adult laying hens. In vitro characterization showed that the strains grew equally well in human urine, and both were able to infect human intestinal (Int407) and bladder (J82) epithelial cell lines, and they survived in avian macrophages (HD11) to the same extent. Groups of adult birds were inoculated with 108 bacteria directly into the oviduct using a surgical procedure. After an infection period of 48 h, bacterial load in the oviduct was determined by dilution series, and pathology was determined based on gross lesions and histological observations. Similar counts of UPEC UTI89 (ST95) and the APEC strain F149H1S2 (ST117) were obtained from tissues of infected birds, and salpingitis as evaluated by clinical score and histopathology was observed to a similar extent after infection with the two strains. Together, the results showed that UPEC UTI89 and APEC F149H1S2 have a similar potential for causing salpingitis in laying hens in the model used. No infection differences were observed between the UPEC UTI89 wild type and a mutant strain with knock-out of the well-known virulence gene, fimH, (UPEC UTI89ΔfimH), showing that the salpingitis model is not suitable for the detection of all UPEC virulence factors.


Assuntos
Infecções por Escherichia coli/veterinária , Escherichia coli/patogenicidade , Doenças das Aves Domésticas/microbiologia , Infecções do Sistema Genital/veterinária , Adesinas de Escherichia coli/genética , Animais , Linhagem Celular , Galinhas , Infecções por Escherichia coli/microbiologia , Proteínas de Fímbrias/genética , Humanos , Mutação , Doenças das Aves Domésticas/patologia , Infecções do Sistema Genital/microbiologia , Infecções do Sistema Genital/patologia , Especificidade da Espécie , Fatores de Virulência/genética
4.
EMBO J ; 38(22): e102145, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31609039

RESUMO

Type IV pili (TFP) are multifunctional micrometer-long filaments expressed at the surface of many prokaryotes. In Neisseria meningitidis, TFP are crucial for virulence. Indeed, these homopolymers of the major pilin PilE mediate interbacterial aggregation and adhesion to host cells. However, the mechanisms behind these functions remain unclear. Here, we simultaneously determined regions of PilE involved in pilus display, auto-aggregation, and adhesion by using deep mutational scanning and started mining this extensive functional map. For auto-aggregation, pili must reach a minimum length to allow pilus-pilus interactions through an electropositive cluster of residues centered around Lys140. For adhesion, results point to a key role for the tip of the pilus. Accordingly, purified pili interacting with host cells initially bind via their tip-located major pilin and then along their length. Overall, these results identify functional domains of PilE and support a direct role of the major pilin in TFP-dependent aggregation and adhesion.


Assuntos
Aderência Bacteriana , Agregação Celular , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/fisiologia , Mutação , Neisseria meningitidis/fisiologia , Proteínas de Fímbrias/química , Regulação Bacteriana da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Mutagênese Sítio-Dirigida
5.
mSphere ; 4(5)2019 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-31578246

RESUMO

Gene diversification is a common mechanism pathogens use to alter surface structures to aid in immune avoidance. Neisseria gonorrhoeae uses a gene conversion-based diversification system to alter the primary sequence of the gene encoding the major subunit of the pilus, pilE Antigenic variation occurs when one of the nonexpressed 19 silent copies donates part of its DNA sequence to pilE We have developed a method using Pacific Biosciences (PacBio) amplicon sequencing and custom software to determine pilin antigenic variation frequencies. The program analyzes 37 variable regions across the strain FA1090 1-81-S2 pilE gene and can be modified to determine sequence variation from other starting pilE sequences or other diversity generation systems. Using this method, we measured pilin antigenic variation frequencies for various derivatives of strain FA1090 and showed we can also analyze pilin antigenic variation frequencies during macrophage infection.IMPORTANCE Diversity generation systems are used by many unicellular organism to provide subpopulations of cell with different properties that are available when needed. We have developed a method using the PacBio DNA sequencing technology and a custom computer program to analyze the pilin antigenic variation system of the organism that is the sole cause of the sexually transmitted infection, gonorrhea.


Assuntos
Variação Antigênica , Proteínas de Fímbrias/genética , Neisseria gonorrhoeae/genética , Análise de Sequência de DNA/métodos , Proteínas de Fímbrias/imunologia , Gonorreia/microbiologia , Humanos , Software , Células U937
6.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31570556

RESUMO

The Porphyromonas gingivalis strain ATCC 33277 (33277) and 381 genomes are nearly identical. However, strain 33277 displays a significantly diminished capacity to stimulate host cell Toll-like receptor 2 (TLR2)-dependent signaling and interleukin-1ß (IL-1ß) production relative to 381, suggesting that there are strain-specific differences in one or more bacterial immune-modulatory factors. Genomic sequencing identified a single nucleotide polymorphism in the 33277 fimB allele (A→T), creating a premature stop codon in the 33277 fimB open reading frame relative to the 381 fimB allele. Gene exchange experiments established that the 33277 fimB allele reduces the immune-stimulatory capacity of this strain. Transcriptome comparisons revealed that multiple genes related to carboxy-terminal domain (CTD) family proteins, including the gingipains, were upregulated in 33277 relative to 381. A gingipain substrate degradation assay demonstrated that cell surface gingipain activity is higher in 33277, and an isogenic mutant strain deficient for the gingipains exhibited an increased ability to induce TLR2 signaling and IL-1ß production. Furthermore, 33277 and 381 mutant strains lacking CTD cell surface proteins were more immune-stimulatory than the parental wild-type strains, consistent with an immune-suppressive role for the gingipains. Our data show that the combination of an intact fimB allele and limited cell surface gingipain activity in P. gingivalis 381 renders this strain more immune-stimulatory. Conversely, a defective fimB allele and high-level cell surface gingipain activity reduce the capacity of P. gingivalis 33277 to stimulate host cell innate immune responses. In summary, genomic and transcriptomic comparisons identified key virulence characteristics that confer divergent host cell innate immune responses to these highly related P. gingivalis strains.


Assuntos
Proteínas de Fímbrias/genética , Proteínas de Fímbrias/imunologia , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/imunologia , Infecções por Bacteroidaceae/imunologia , Infecções por Bacteroidaceae/microbiologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Imunidade Inata/genética , Imunidade Inata/imunologia , Interleucina-1beta/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Transdução de Sinais/imunologia , Células THP-1 , Receptor 2 Toll-Like/metabolismo
7.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31548326

RESUMO

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.


Assuntos
Proteínas de Fímbrias/biossíntese , Proteínas de Fímbrias/imunologia , Fímbrias Bacterianas/imunologia , Haemophilus influenzae/imunologia , Nasofaringe/imunologia , Aderência Bacteriana/genética , Vacinas Bacterianas/imunologia , Células Cultivadas , Técnicas de Cocultura , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/metabolismo , Infecções por Haemophilus/imunologia , Infecções por Haemophilus/microbiologia , Heme/metabolismo , Humanos , Nasofaringe/microbiologia , Regiões Promotoras Genéticas/genética , Sistema Respiratório/citologia
8.
Microbiology ; 165(11): 1245-1250, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31486760

RESUMO

In Salmonella enterica serovar Typhimurium, the RcsCDB regulatory system controls the expression of genes involved in synthesis of colanic acid, formation of flagella and virulence. Here, we show that activation of the RcsCDB system downregulates expression of std, an operon that encodes fimbriae involved in Salmonella attachment to the mucus layer in the large intestine. Bioinformatic analysis predicts the existence of an RcsB-binding site located 180 bp upstream to the +1 transcription start site of the std promoter, and electrophoretic mobility shift assays confirm that RcsB binds the std promoter region in vitro. This study adds RcsB to the list of regulators of std transcription and provides an example of modulation of fimbriae synthesis by a signal transduction system.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Salmonella typhimurium/metabolismo , Transdução de Sinais , Aderência Bacteriana , Proteínas de Bactérias/genética , Sítios de Ligação , Mutação , Óperon , Regiões Promotoras Genéticas , Salmonella typhimurium/genética , Transcrição Genética
9.
BMC Vet Res ; 15(1): 319, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31488137

RESUMO

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.


Assuntos
Antígenos de Bactérias/genética , Proteínas de Fímbrias/genética , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/fisiologia , Pequeno RNA não Traduzido/fisiologia , Salmonella enteritidis/genética , Salmonella enteritidis/patogenicidade , Animais , Aderência Bacteriana/genética , Células CACO-2 , Galinhas , Feminino , Humanos , Masculino , Doenças das Aves Domésticas/virologia , Salmonelose Animal/virologia , Deleção de Sequência , Virulência/genética
10.
PLoS Genet ; 15(9): e1008393, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31525185

RESUMO

Type IV pili are dynamic cell surface appendages found throughout the bacteria. The ability of these structures to undergo repetitive cycles of extension and retraction underpins their crucial roles in adhesion, motility and natural competence for transformation. In the best-studied systems a dedicated retraction ATPase PilT powers pilus retraction. Curiously, a second presumed retraction ATPase PilU is often encoded immediately downstream of pilT. However, despite the presence of two potential retraction ATPases, pilT deletions lead to a total loss of pilus function, raising the question of why PilU fails to take over. Here, using the DNA-uptake pilus and mannose-sensitive haemagglutinin (MSHA) pilus of Vibrio cholerae as model systems, we show that inactivated PilT variants, defective for either ATP-binding or hydrolysis, have unexpected intermediate phenotypes that are PilU-dependent. In addition to demonstrating that PilU can function as a bona fide retraction ATPase, we go on to make the surprising discovery that PilU functions exclusively in a PilT-dependent manner and identify a naturally occurring pandemic V. cholerae PilT variant that renders PilU essential for pilus function. Finally, we show that Pseudomonas aeruginosa PilU also functions as a PilT-dependent retraction ATPase, providing evidence that the functional coupling between PilT and PilU could be a widespread mechanism for optimal pilus retraction.


Assuntos
Adenosina Trifosfatases/genética , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/fisiologia , Fímbrias Bacterianas/fisiologia , Lectina de Ligação a Manose/genética , Lectina de Ligação a Manose/metabolismo , Proteínas Motores Moleculares/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/metabolismo
11.
Vet Microbiol ; 235: 301-309, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31383317

RESUMO

We have previously demonstrated that prophage phiv142-3 enhances the colonization ability of avian pathogenic Escherichia coli (APEC) strain DE142. However, the mechanism of this action remains unknown. In this study, we demonstrate that deletion of phiv142-3 orf20 leads to a decrease in the survival ability in chicken serum, adhesion, and ability to invade DF-1 cells of mutant strain DE142Δorf20 compared with that of wild type (WT). Avian infection assays showed that bacterial loads in lungs and hearts of chickens challenged with the mutant are decreased to 7% and 8.3% compared with those challenged with the WT. The number of flagella and I fimbriae of the mutant are decreased and the mutant exhibits filamentation. However, protein ORF20 shows no adhesion ability to DF-1 cells in adherence inhibition experiments, indicating that it does not directly participate in adhesion. qRT-PCR revealed that the deletion of orf20 leads to reduction in the expression of nine genes related to the exportation of flagellar protein and two I-fimbriae-related genes (fimA and fimH), but does not affect genes related to the synthesis of flagella and other adhesins. Compared with the WT, the transcription level of the cell-division-associated genes minC and minD was increased 1.4-fold and 2.5-fold in mutant DE142Δorf20, respectively, indicating that orf20 affects the morphology of DE142 by regulating expression of minC and minD. Thus, our study revealed that orf20 in prophage phiv142-3 played a role in flagellar exportation, cell morphology, and I fimbriae synthesis.


Assuntos
Aderência Bacteriana , Escherichia coli/fisiologia , Escherichia coli/virologia , Fímbrias Bacterianas/metabolismo , Flagelos/metabolismo , Prófagos/genética , Adesinas Bacterianas/genética , Animais , Carga Bacteriana , Galinhas/microbiologia , Escherichia coli/genética , Escherichia coli/patogenicidade , Proteínas de Escherichia coli/genética , Proteínas de Fímbrias/genética , Proteínas de Membrana/genética , Mutação , Fatores de Virulência/genética
13.
PLoS Pathog ; 15(7): e1007915, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31329635

RESUMO

Expression of ABO and Lewis histo-blood group antigens by the gastrointestinal epithelium is governed by an α-1,2-fucosyltransferase enzyme encoded by the Fut2 gene. Alterations in mucin glycosylation have been associated with susceptibility to various bacterial and viral infections. Salmonella enterica serovar Typhimurium is a food-borne pathogen and a major cause of gastroenteritis. In order to determine the role of Fut2-dependent glycans in Salmonella-triggered intestinal inflammation, Fut2+/+ and Fut2-/- mice were orally infected with S. Typhimurium and bacterial colonization and intestinal inflammation were analyzed. Bacterial load in the intestine of Fut2-/- mice was significantly lower compared to Fut2+/+ mice. Analysis of histopathological changes revealed significantly lower levels of intestinal inflammation in Fut2-/- mice compared to Fut2+/+ mice and measurement of lipocalin-2 level in feces corroborated histopathological findings. Salmonella express fimbriae that assist in adherence of bacteria to host cells thereby facilitating their invasion. The std fimbrial operon of S. Typhimurium encodes the π-class Std fimbriae which bind terminal α(1,2)-fucose residues. An isogenic mutant of S. Typhimurium lacking Std fimbriae colonized Fut2+/+ and Fut2-/- mice to similar levels and resulted in similar intestinal inflammation. In vitro adhesion assays revealed that bacteria possessing Std fimbriae adhered significantly more to fucosylated cell lines or primary epithelial cells in comparison to cells lacking α(1,2)-fucose. Overall, these results indicate that Salmonella-triggered intestinal inflammation and colonization are dependent on Std-fucose interaction.


Assuntos
Fímbrias Bacterianas/metabolismo , Fucose/metabolismo , Salmonella typhimurium/patogenicidade , Animais , Aderência Bacteriana , Colite/etiologia , Colite/metabolismo , Colite/microbiologia , Feminino , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/genética , Fucosiltransferases/deficiência , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Interações entre Hospedeiro e Microrganismos , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos CBA , Camundongos Knockout , Óperon , Salmonelose Animal/etiologia , Salmonelose Animal/metabolismo , Salmonelose Animal/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/fisiologia
14.
Appl Microbiol Biotechnol ; 103(18): 7317-7324, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31359104

RESUMO

Long polar fimbria (LPF) is one of the few fimbrial adhesins of enterohemorrhagic Escherichia coli (E. coli) O157:H7 associated with colonization on host intestine, and both two types of LPF (including LPF1 and LPF2) play essential roles during the bacterial infection process. Though the fimbriae had been well studied in intestinal pathogenic E. coli strains, new evidences from our research revealed that it might be the key virulence for bovine mastitis pathogenic E. coli (MPEC) as well. This article summarizes the current knowledge on the LPF in E. coli, focusing on its genetic characteristics, prevalence, expression regulation, and adherence mechanism in different pathotypes of E. coli strains.


Assuntos
Aderência Bacteriana , Escherichia coli O157/genética , Escherichia coli O157/patogenicidade , Proteínas de Escherichia coli/fisiologia , Proteínas de Fímbrias/fisiologia , Fímbrias Bacterianas/fisiologia , Animais , Bovinos , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Feminino , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Humanos , Intestinos/microbiologia , Mastite Bovina/microbiologia , Virulência
15.
Environ Sci Pollut Res Int ; 26(26): 27338-27352, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31325090

RESUMO

Vibrio cholerae is a leading waterborne pathogen worldwide. Continuous monitoring of V. cholerae contamination in aquatic products and identification of risk factors are crucial for assuring food safety. In this study, we determined the virulence, antimicrobial susceptibility, heavy metal tolerance, and genetic diversity of 400 V. cholerae isolates recovered from commonly consumed freshwater fish (Aristichthys nobilis, Carassius auratus, Ctenopharyngodon idellus, and Parabramis pekinensis) collected in July and August of 2017 in Shanghai, China. V. cholerae has not been previously detected in the half of these fish species. The results revealed an extremely low occurrence of pathogenic V. cholerae carrying the major virulence genes ctxAB (0.0%), tcpA (0.0%), ace (0.0%), and zot (0.0%). However, high incidence of virulence-associated genes was observed, including the RTX toxin gene cluster (rtxA-D) (83.0-97.0%), hlyA (87.8%), hapA (95.0%), and tlh (76.0%). Meanwhile, high percentages of resistance to antimicrobial agents streptomycin (65.3%), ampicillin (44.5%), and rifampicin (24.0%) were observed. Approximately 30.5% of the isolates displayed multidrug resistant (MDR) phenotypes with 42 resistance profiles, which were significantly different among the four fish species (MARI, P = 0.001). Additionally, tolerance of isolates to heavy metals Hg2+ (49.3%), Zn2+ (30.3%), and Pb2+ (12.0%) was observed. The enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR)-based fingerprinting of the 400 V. cholerae isolates revealed 328 ERIC-genotypes, which demonstrated a large degree of genomic variation among the isolates. Overall, the results of this study support the need for food safety risk assessment of aquatic products.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Produtos Pesqueiros/microbiologia , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/patogenicidade , Animais , Toxinas Bacterianas/genética , China , Farmacorresistência Bacteriana/genética , Proteínas de Fímbrias/genética , Água Doce , Variação Genética , Genótipo , Metais Pesados/toxicidade , Oligopeptídeos/genética , Vibrio cholerae/genética , Vibrio cholerae/isolamento & purificação , Virulência/genética , Fatores de Virulência/genética
16.
mBio ; 10(3)2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186316

RESUMO

Natural competence is the term used to describe the uptake of "naked" extracellular DNA by bacteria; it plays a significant role in horizontal genetic exchange. It is associated with type IV pili, and specialized competence pili mediate DNA uptake. Here, we show that the crystal structure of a competence-associated protein from Thermus thermophilus, ComZ, consists of a type II secretion pseudopilin-like domain, with a large ß-solenoid domain inserted into the ß-sheet of the pilin-like fold. ComZ binds with high affinity to another competence-associated pilin, PilA2, which lies adjacent to the comZ gene in the genome. The crystal structure of PilA2 revealed a similar type II secretion pseudopilin-like fold, with a small subdomain; docking simulations predicted that PilA2 binds between the pseudopilin-like and ß-solenoid domains of ComZ. Electrophoretic shift analysis and DNase protection studies were used to show that ComZ alone and the ComZ/PilA2 complex are able to bind DNA. Protection against reductive dimethylation was used in combination with mass spectrometry and site-directed mutagenesis to identify two lysine residues in ComZ which are involved in DNA binding. They are located between the two domains in ComZ, on the opposite side from the predicted PilA2 binding site. These results suggest a model in which PilA2 assists ComZ in forming the competence pilus tip and DNA binds to the side of the fiber. The results demonstrate how a type IV pilin can be adapted to a specific function by domain insertion and provide the first structural insights into a tip-located competence pilin.IMPORTANCE Thermus thermophilus is a thermophilic bacterium which is capable of natural transformation, the uptake of external DNA with high efficiency. DNA uptake is thought to be mediated by a competence-associated pilus, which binds the DNA substrate and mediates its transfer across the outer membrane and periplasm. Here, we describe the structural and functional analysis of two pilins which are known to be essential for DNA uptake, ComZ and PilA2. ComZ adopts an unusual structure, incorporating a large ß-solenoid domain into the pilin structural framework. We argue on structural grounds that this structure cannot readily be accommodated into the competence pilus fiber unless it is at the tip. We also show that ComZ binds DNA and identify two lysine residues which appear to be important for DNA binding. These results suggest a model in which ComZ and PilA2 form a tip-associated DNA receptor which mediates DNA uptake.


Assuntos
Competência de Transformação por DNA , Proteínas de Fímbrias/química , Fímbrias Bacterianas/química , Receptores de Superfície Celular/química , Thermus thermophilus/genética , Sítios de Ligação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Domínios Proteicos , Receptores de Superfície Celular/genética
17.
Nat Microbiol ; 4(9): 1545-1557, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31182799

RESUMO

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.


Assuntos
Quitina/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Vibrio cholerae/fisiologia , Aderência Bacteriana/genética , DNA Bacteriano/metabolismo , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Variação Genética , Humanos , Transformação Bacteriana , Vibrio cholerae/genética , Vibrio cholerae/metabolismo , Vibrio cholerae/patogenicidade
18.
PLoS Pathog ; 15(6): e1007671, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31181116

RESUMO

Pathogens rely on a complex virulence gene repertoire to successfully attack their hosts. We were therefore surprised to find that a single fimbrial gene reconstitution can return the virulence-attenuated commensal strain Escherichia coli 83972 to virulence, defined by a disease phenotype in human hosts. E. coli 83972pap stably reprogrammed host gene expression, by activating an acute pyelonephritis-associated, IRF7-dependent gene network. The PapG protein was internalized by human kidney cells and served as a transcriptional agonist of IRF-7, IFN-ß and MYC, suggesting direct involvement of the fimbrial adhesin in this process. IRF-7 was further identified as a potent upstream regulator (-log (p-value) = 61), consistent with the effects in inoculated patients. In contrast, E. coli 83972fim transiently attenuated overall gene expression in human hosts, enhancing the effects of E. coli 83972. The inhibition of RNA processing and ribosomal assembly indicated a homeostatic rather than a pathogenic end-point. In parallel, the expression of specific ion channels and neuropeptide gene networks was transiently enhanced, in a FimH-dependent manner. The studies were performed to establish protective asymptomatic bacteriuria in human hosts and the reconstituted E. coli 83972 variants were developed to improve bacterial fitness for the human urinary tract. Unexpectedly, P fimbriae were able to drive a disease response, suggesting that like oncogene addiction in cancer, pathogens may be addicted to single super-virulence factors.


Assuntos
Adesinas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Adesinas de Escherichia coli/genética , Linhagem Celular , Escherichia coli/genética , Escherichia coli/patogenicidade , Feminino , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Humanos , Fator Regulador 7 de Interferon/metabolismo , Interferon beta/metabolismo , Rim/metabolismo , Rim/microbiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo
19.
J Appl Microbiol ; 127(2): 459-471, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31087803

RESUMO

AIMS: The aim of this study was to assess anti-biofilm and antimicrobial effects of auranofin, an anti-rheumatic agent, on uropathogenic Escherichia coli (UPEC) biofilm formation. METHODS AND RESULTS: The minimum inhibitory concentration and biofilm inhibition concentration of auranofin against UPEC ranged from 24 to 32 µg ml-1 . Biofilm eradication concentration and XTT reduction concentration of auranofin were found to be at 112 µg ml-1 . Confocal laser scanning microscopy results confirmed that biofilm was inhibited by auranofin. These results indicate that auranofin possesses potent anti-biofilm and antimicrobial activities against UPEC. Effects of auranofin on type 1 fimbriae gene (fimH) and response regulator gene (rpoS) to stress were explored using quantitative real time-polymerase chain reaction. In addition, combination of auranofin and tetracycline showed synergistic effect. CONCLUSIONS: These data indicate that auranofin has inhibitory effect on biofilm formation and synergistic effect on UPEC infection when it is combined with tetracycline. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study strongly suggest that auranofin is a promising alternative anti-biofilm and antimicrobial agent to prevent UPEC biofilm formation in UTIs. Auranofin already approved for human use have the advantage of being able to be put into clinical use relatively quickly.


Assuntos
Antibacterianos/farmacologia , Auranofina/farmacologia , Biofilmes/efeitos dos fármacos , Escherichia coli Uropatogênica/efeitos dos fármacos , Adesinas de Escherichia coli/genética , Adesinas de Escherichia coli/metabolismo , Reposicionamento de Medicamentos , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Humanos , Tetraciclina/farmacologia , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/isolamento & purificação , Escherichia coli Uropatogênica/fisiologia
20.
PLoS One ; 14(5): e0217369, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31125361

RESUMO

Multivalent proteins or protein dendrimers are useful for clinical and biotechnological applications. However, assembly of chemically defined protein dendrimers is a challenging endeavor. In the past, majority of protein dendrimers have been developed on branched lysine scaffolds and are usually limited to a valency of two to four. The naturally occurring cyclodextrin (CD) scaffold composed of 6-8 glucose units offers the possibility of expanding the valency. Here we have adapted a chemoenzymatic-click strategy for displaying heptavalent peptides and large proteins on the ß-cyclodextrin (ß-CD) scaffold. We demonstrate that recombinant proteins (engineered with a LPXTG pentapeptide motif at the carboxy terminus), labeled with an alkyne moiety by sortase-mediated ligation, can be easily clicked on to the azide-derivatized ß-cyclodextrin through the Huisgen cycloaddition reaction yielding a well-defined heptavalent display of proteins.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Química Click/métodos , Reação de Cicloadição/métodos , Ciclodextrinas/química , Cisteína Endopeptidases/metabolismo , Peptídeos/química , Sequência de Aminoácidos , Aminoaciltransferases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cisteína Endopeptidases/genética , Dendrímeros/síntese química , Dendrímeros/química , Proteínas de Fímbrias/química , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Modelos Moleculares , Biossíntese Peptídica , Peptídeos/síntese química , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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