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1.
Sci Rep ; 11(1): 13859, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226594

RESUMO

The emergence and spread of antimicrobial resistance have been of serious concern to human health and the management of bacterial infectious diseases. Effective treatment of these diseases requires the development of novel therapeutics, preferably free of side effects. In this regard, natural products are frequently conceived to be potential alternative sources for novel antibacterial compounds. Herein, we have evaluated the antibacterial activity of the epicarp extracts of the Malaysian cultivar of yellow rambutan fruit (Nephelium lappaceum L.) against six pathogens namely, Bacillus subtilis, methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella enterica. Among a series of solvent extracts, fractions of ethyl acetate and acetone have revealed significant activity towards all tested strains. Chemical profiling of these fractions, via HPLC, LC-MS and GC-MS, has generated a library of potentially bioactive compounds. Downstream virtual screening, pharmacological prediction, and receptor-ligand molecular dynamics simulation have eventually unveiled novel potential antibacterial compounds, which can be extracted for medicinal use. We report compounds like catechin, eplerenone and oritin-4-beta-ol to be computationally inhibiting the ATP-binding domain of the chaperone, DnaK of P. aeruginosa and MRSA. Thus, our work follows the objective to propose new antimicrobials capable of perforating the barrier of resistance posed by both the gram positives and the negatives.


Assuntos
Infecções Bacterianas/tratamento farmacológico , Produtos Biológicos/farmacologia , Extratos Vegetais/farmacologia , Sapindaceae/química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/patogenicidade , Infecções Bacterianas/microbiologia , Infecções Bacterianas/patologia , Produtos Biológicos/química , Farmacorresistência Bacteriana/efeitos dos fármacos , Frutas/química , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/patogenicidade , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Testes de Sensibilidade Microbiana , Extratos Vegetais/química , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/patogenicidade
2.
J Mol Biol ; 433(19): 167175, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34303721

RESUMO

Virulence-associated type III secretion systems (T3SS) are utilized by Gram negative bacterial pathogens for injection of effector proteins into eukaryotic host cells. The transmembrane export apparatus at the core of T3SS is composed of a unique helical complex of the hydrophobic proteins SctR, SctS, SctT, and SctU. These components comprise a number of highly conserved charged residues within their hydrophobic domains. The structure of the closed state of the core complex SctR5S4T1 revealed that several of these residues form inter- and intramolecular salt bridges, some of which have to be broken for pore opening. Mutagenesis of individual residues was shown to compromise assembly or secretion of both, the virulence-associated and the related flagellar T3SS. However, the exact role of these conserved charged residues in the assembly and function of T3SS remains elusive. Here we performed an in-depth mutagenesis analysis of these residues in the T3SS of Salmonella Typhimurium, coupled to blue native PAGE, in vivo photocrosslinking and luciferase-based secretion assays. Our data show that these conserved salt bridges are not critical for assembly of the respective protein but rather facilitate the incorporation of the following subunit into the assembling complex. Our data also indicate that these conserved charged residues are critical for type III-dependent secretion and reveal a functional link between SctSE44 and SctTR204 and the cytoplasmic domain of SctU in gating the T3SS injectisome. Overall, our analysis provides an unprecedented insight into the delicate requirements for the assembly and function of the machinery at the core of T3SS.


Assuntos
Salmonella enterica/metabolismo , Sistemas de Secreção Tipo III/química , Sistemas de Secreção Tipo III/metabolismo , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Mutação , Conformação Proteica , Domínios Proteicos , Salmonella enterica/genética , Salmonella enterica/patogenicidade , Sistemas de Secreção Tipo III/genética , Virulência
3.
Nucleic Acids Res ; 49(9): 5319-5335, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33939833

RESUMO

FinO-domain proteins represent an emerging family of RNA-binding proteins (RBPs) with diverse roles in bacterial post-transcriptional control and physiology. They exhibit an intriguing targeting spectrum, ranging from an assumed single RNA pair (FinP/traJ) for the plasmid-encoded FinO protein, to transcriptome-wide activity as documented for chromosomally encoded ProQ proteins. Thus, the shared FinO domain might bear an unusual plasticity enabling it to act either selectively or promiscuously on the same cellular RNA pool. One caveat to this model is that the full suite of in vivo targets of the assumedly highly selective FinO protein is unknown. Here, we have extensively profiled cellular transcripts associated with the virulence plasmid-encoded FinO in Salmonella enterica. While our analysis confirms the FinP sRNA of plasmid pSLT as the primary FinO target, we identify a second major ligand: the RepX sRNA of the unrelated antibiotic resistance plasmid pRSF1010. FinP and RepX are strikingly similar in length and structure, but not in primary sequence, and so may provide clues to understanding the high selectivity of FinO-RNA interactions. Moreover, we observe that the FinO RBP encoded on the Salmonella virulence plasmid controls the replication of a cohabitating antibiotic resistance plasmid, suggesting cross-regulation of plasmids on the RNA level.


Assuntos
Proteínas de Bactérias/metabolismo , Plasmídeos/genética , Pequeno RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/metabolismo , Salmonella enterica/genética , Ligantes , RNA Antissenso/metabolismo , Pequeno RNA não Traduzido/química , Salmonella enterica/metabolismo , Salmonella enterica/patogenicidade , Virulência
4.
Res Microbiol ; 172(4-5): 103836, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34029676

RESUMO

Salmonella enterica is a pathogen that induces self-limiting gastroenteritis and is of worldwide concern. Nisin, an antimicrobial peptide, has emerged as an alternative for the control of microbial growth but its effect on the virulence of pathogenic bacteria is not yet well-explored. This work aimed to evaluate the virulence of S. enterica in the presence of sub-inhibitory nisin using the experimental model Galleria mellonella. Sub-inhibitory concentrations of nisin of 11.72 and 46.88 µM did not affect the cellular viability of S. enterica but promoted changes in gene expression within 1 h of treatment, with increases of up to 3-fold of pagC, 1.8-fold of invA and 2.3-fold of invF. Larvae of G. mellonella inoculated with S. enterica combined with nisin at 46.88 µM presented mortality, and TL50 noticeably increased to 50% and 80% at 24 and 48 h post-infection, respectively. Defence responses, such as melanisation, nodulation, pseudopodia, immune response, and expression of defence proteins of the larvae G. mellonella were enhanced when the treatments with S. enterica were combined with 11.72 or 46.88 µM nisin. These results show an increase in virulence of S. enterica by sub-MIC concentration of nisin that needs to be explored.


Assuntos
Antibacterianos/administração & dosagem , Larva/microbiologia , Mariposas/microbiologia , Nisina/administração & dosagem , Salmonella enterica/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Nisina/farmacologia , Salmonella enterica/patogenicidade , Virulência
5.
Int J Food Microbiol ; 348: 109201, 2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-33930836

RESUMO

Salmonella enterica is frequently implicated in foodborne disease outbreaks associated with fresh-cut fruits. In the U.S., more than one third of fruit-related outbreaks have been linked to two S. enterica serotypes Newport and Typhimurium. Approximately 80% of fruit-related human salmonellosis cases were associated with tomatoes, cantaloupes and cucumbers. In this study, we investigated the population dynamics of S. Newport and S. Typhimurium on fresh-cut tomato, cantaloupe, cucumber and apple under short-term storage conditions. We further compared the transcriptomic profiles of a S. Newport strain on fresh-cut tomato and cantaloupe using high-throughput RNA-seq. We demonstrated that both S. enterica Newport and Typhimurium survived well on various fresh-cut fruit items under refrigeration storage conditions, independent of inoculation levels. However, S. enterica displayed variable survival behaviors on different types of fruits. For example, at 7 d storage, the population of S. enterica reduced less than 0.2 log (p > 0.05) on fresh-cut tomato and cantaloupe, in contrast to ~0.5 log (p < 0.05) on cucumber and apple. RNA-seq analysis suggested that S. enterica mediates its survival on fresh-cut fruits through differentially regulating genes involved in specific carbon utilization and metabolic pathways. Several known bacterial virulence factors (e.g., pag gene) were found to be differentially regulated on fresh-cut tomato and cantaloupe, suggesting a link between the events of food contamination and subsequent human infection. Findings from this study contribute to a better understanding of S. enterica survival mechanisms on fresh-cut produce.


Assuntos
Armazenamento de Alimentos/métodos , Doenças Transmitidas por Alimentos/microbiologia , Frutas/microbiologia , Infecções por Salmonella/transmissão , Salmonella enterica/crescimento & desenvolvimento , Contagem de Colônia Microbiana , Cucumis melo/microbiologia , Cucumis sativus/microbiologia , Surtos de Doenças , Metabolismo Energético/genética , Contaminação de Alimentos , Microbiologia de Alimentos , Humanos , Lycopersicon esculentum/microbiologia , Malus/microbiologia , Infecções por Salmonella/microbiologia , Salmonella enterica/genética , Salmonella enterica/patogenicidade , Sorogrupo , Transcriptoma
6.
Zoonoses Public Health ; 68(5): 402-412, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33655685

RESUMO

Reptiles are carriers of Salmonella and can intermittently shed bacteria in their faeces. Contact with snakes and lizards is a source of human salmonellosis. Here, two populations of reptiles, wild and captive were surveyed for Salmonella. One hundred thirty wild-caught reptiles were sampled for Salmonella including 2 turtle, 9 snake and 31 lizard species. Fifty-two of 130 (40%) animals were Salmonella positive: one of 5 (20%) turtles, 7 of 14 (50%) snakes and 44 of 111 (39.6%) lizards. One hundred twenty-two reptiles were sampled from a zoo collection including 1 turtle, 6 tortoise, 9 lizard, 14 snake and 1 crocodile species. Forty-two of 122 (34.4%) captive reptiles sampled were Salmonella positive. Salmonella was most commonly isolated from lizards and snakes. Fifteen serotypes were identified from zoo and 19 from wild-caught reptiles and most were members of subspecies enterica (I), salamae (II), arizonae (IIIa) or diarizonae (IIIb). Antimicrobial susceptibility testing was conducted on all Salmonella isolates; only two exhibited resistance, a Salmonella subsp. (II) ser. 21:z10 :z6 (Wandsbek) isolate cultured from a wild-caught reptile and a Salmonella Typhimurium DT120 isolated from a captive snake. The invasive capacity of reptile-associated Salmonella strains into cultured human intestinal epithelial (Caco2) and mouse macrophages cell lines (J774A.1) was also investigated. All isolates were invasive into both cell lines. Significant (P ≤ 0.001) variability in invasiveness into polarized Caco2 cells was observed. Salmonella Eastbourne exhibited the highest invasiveness into Caco2 cells and Salmonella Chester the lowest, with mean per cent recoveries of 19.99 ± 0.32 and 1.23 ± 0.30, respectively. Invasion into J774A.1 macrophages was also variable but was not significant. Salmonella subsp. II ser. 17:g,t:- (Bleadon) exhibited the highest invasiveness into J774A.1 with a mean per cent recovery of 10.19 ± 0.19. Thus, reptile-associated Salmonellae are likely to have different capacities to cause disease in humans.


Assuntos
Animais Selvagens , Animais de Zoológico , Antibacterianos/farmacologia , Répteis , Salmonelose Animal/microbiologia , Salmonella enterica/patogenicidade , Animais , Austrália/epidemiologia , Farmacorresistência Bacteriana , Salmonelose Animal/epidemiologia , Salmonella enterica/efeitos dos fármacos
7.
Lett Appl Microbiol ; 73(1): 54-63, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33765334

RESUMO

Lactic Acid Bacteria (LAB) regulate and maintain the stability of healthy microbial flora, inhibit the adhesion of pathogenic bacteria and promote the colonization of beneficial micro-organisms. The drug resistance and pathogenicity of Salmonella enteritis SE47 isolated from retail eggs were investigated. Meanwhile, Enterococcus faecalis L76 and Lactobacillus salivarius LAB35 were isolated from intestine of chicken. With SE47 as indicator bacteria, the diameters of L76 and LAB35 inhibition zones were 12 mm and 8·5 mm, respectively, by agar inhibition circle method, which indicated that both of them had inhibitory effect on Salmonella, and L76 had better antibacterial effect; two chicken-derived lactic acid bacteria isolates and Salmonella SE47 were incubated with Caco-2. The adhesion index of L76 was 17·5%, which was much higher than that of LAB35 (10·21%) and SE47 (4·89%), this experiment shows that the higher the bacteriostatic effect of potential probiotics, the stronger the adhesion ability; then Caco-2 cells were incubated with different bacteria, and the survival of Caco-2 cells was observed by flow cytometry. Compared with Salmonella SE47, the results showed that lactic acid bacteria isolates could effectively protect Caco-2 cells; finally, after different bacteria incubated Caco-2 cells, according to the cytokine detection kit, the RNA of Caco-2 cells was extracted and transcribed into cDNA, then detected by fluorescence quantitative PCR, the results showed that L76 could protect Caco-2 cells from the invasion of Salmonella SE47, with less cell membrane rupture and lower expression of MIF and TNF genes. Therefore, the lactic acid bacteria isolates can effectively inhibit the adhesion of Salmonella and protect the integrity of intestinal barrier.


Assuntos
Antibiose/fisiologia , Ovos/microbiologia , Lactobacillales/fisiologia , Infecções por Salmonella/microbiologia , Salmonella enterica/fisiologia , Animais , Células CACO-2 , Galinhas/microbiologia , Farmacorresistência Bacteriana/fisiologia , Enterococcus faecalis/isolamento & purificação , Enterococcus faecalis/fisiologia , Humanos , Lactobacillus salivarius/isolamento & purificação , Lactobacillus salivarius/fisiologia , Probióticos/isolamento & purificação , Probióticos/farmacologia , Salmonella enterica/patogenicidade
8.
Crit Rev Microbiol ; 47(4): 397-434, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33751923

RESUMO

Adaptation to environments with constant fluctuations imposes challenges that are only overcome with sophisticated strategies that allow bacteria to perceive environmental conditions and develop an appropriate response. The gastrointestinal environment is a complex ecosystem that is home to trillions of microorganisms. Termed microbiota, this microbial ensemble plays important roles in host health and provides colonization resistance against pathogens, although pathogens have evolved strategies to circumvent this barrier. Among the strategies used by bacteria to monitor their environment, one of the most important are the sensing and signalling machineries of two-component systems (TCSs), which play relevant roles in the behaviour of all bacteria. Salmonella enterica is no exception, and here we present our current understanding of how this important human pathogen uses TCSs as an integral part of its lifestyle. We describe important aspects of these systems, such as the stimuli and responses involved, the processes regulated, and their roles in virulence. We also dissect the genomic organization of histidine kinases and response regulators, as well as the input and output domains for each TCS. Lastly, we explore how these systems may be promising targets for the development of antivirulence therapeutics to combat antibiotic-resistant infections.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Infecções por Salmonella/microbiologia , Salmonella enterica/metabolismo , Salmonella enterica/patogenicidade , Animais , Proteínas de Bactérias/genética , Ecossistema , Humanos , Salmonella enterica/genética , Transdução de Sinais , Virulência
9.
Int J Food Microbiol ; 343: 109091, 2021 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-33639477

RESUMO

This study investigated the antimicrobial resistance determinants, virulence factors and identified serovars in 37 Salmonella enterica strains isolated from human stool and contaminated foods linked to outbreaks that occurred in Brazil over 7 years using whole genome sequencing (WGS). Phylogenetic analysis of selected serovars (S. Typhimurium, S. Infantis, S. London, and S. Johannesburg) was performed. Ten distinct serovars were identified and, 51% of the tested strains (n = 19) showed disagreement with the previous conventional serotyping. The antimicrobial resistance (AMR) determinants or plasmids varied among the strains. Resistome analysis revealed the presence of resistance genes to aminoglycosides [aac (6')-laa, aph (3″)-lb, aph (6)-ld, aadA1 and aadA2], sulfonamides (sul1), trimethoprin (dfrA8), fosfomycin (fosA7) and tetracyclines (tetA, tetB, tetC), as well as point mutations in parC (T57S) and gyrA (S83F). Plasmidome showed the presence of IncHI2, IncHI2A, IncFIB (S), IncFII (S), IncI1 and p0111 plasmids. Eight Salmonella pathogenicity islands and up to 102 stress and/or virulence genes were identified in the evaluated genomes. Virulence genes of K88 fimbrial adhesin were first reported in S. enterica (S. Pomona, S. Bredeney and S. Mbandaka strains). pilW gene was first identified in S. Pomona. Phylogenetic analysis showed that some serovars circulated in Brazil for decades, primarily within the poultry production chain. Findings highlighted the virulence and AMR determinants in strains that may lead to recurring food outbreaks.


Assuntos
Farmacorresistência Bacteriana/genética , Doenças Transmitidas por Alimentos/microbiologia , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/genética , Fatores de Virulência/genética , Adesinas Bacterianas/genética , Animais , Antibacterianos/farmacologia , Brasil , Fezes/microbiologia , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Genoma Bacteriano/genética , Ilhas Genômicas/genética , Humanos , Filogenia , Plasmídeos/genética , Aves Domésticas/microbiologia , Doenças das Aves Domésticas/microbiologia , Salmonella enterica/isolamento & purificação , Salmonella enterica/patogenicidade , Sorotipagem , Virulência/genética , Sequenciamento Completo do Genoma
10.
Nat Immunol ; 22(2): 216-228, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33462454

RESUMO

CD4+ effector lymphocytes (Teff) are traditionally classified by the cytokines they produce. To determine the states that Teff cells actually adopt in frontline tissues in vivo, we applied single-cell transcriptome and chromatin analyses to colonic Teff cells in germ-free or conventional mice or in mice after challenge with a range of phenotypically biasing microbes. Unexpected subsets were marked by the expression of the interferon (IFN) signature or myeloid-specific transcripts, but transcriptome or chromatin structure could not resolve discrete clusters fitting classic helper T cell (TH) subsets. At baseline or at different times of infection, transcripts encoding cytokines or proteins commonly used as TH markers were distributed in a polarized continuum, which was functionally validated. Clones derived from single progenitors gave rise to both IFN-γ- and interleukin (IL)-17-producing cells. Most of the transcriptional variance was tied to the infecting agent, independent of the cytokines produced, and chromatin variance primarily reflected activities of activator protein (AP)-1 and IFN-regulatory factor (IRF) transcription factor (TF) families, not the canonical subset master regulators T-bet, GATA3 or RORγ.


Assuntos
Bactérias/patogenicidade , Infecções Bacterianas/microbiologia , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD4-Positivos/parasitologia , Colo/microbiologia , Colo/parasitologia , Microbioma Gastrointestinal , Heligmosomatoidea/patogenicidade , Enteropatias Parasitárias/parasitologia , Animais , Bactérias/imunologia , Infecções Bacterianas/genética , Infecções Bacterianas/imunologia , Infecções Bacterianas/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Cromatina/genética , Cromatina/metabolismo , Citrobacter rodentium/imunologia , Citrobacter rodentium/patogenicidade , Colo/imunologia , Colo/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Heligmosomatoidea/imunologia , Interações Hospedeiro-Patógeno , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Enteropatias Parasitárias/genética , Enteropatias Parasitárias/imunologia , Enteropatias Parasitárias/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nematospiroides dubius/imunologia , Nematospiroides dubius/patogenicidade , Nippostrongylus/imunologia , Nippostrongylus/patogenicidade , Fenótipo , Salmonella enterica/imunologia , Salmonella enterica/patogenicidade , Análise de Célula Única , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Transcriptoma
11.
J Appl Microbiol ; 130(6): 2123-2131, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33150646

RESUMO

AIMS: This study compared the capacity of strains of Salmonella enterica serovars Enteritidis and Dublin isolated in Brazil to invade epithelial cells, to be internalized by and survive within macrophages, and to stimulate cytokine release in vitro. METHODS AND RESULTS: Both serovars infected 75 and 73% Caco-2 (human) and MDBK (bovine) epithelial cells respectively. Salmonella Dublin and S. Enteritidis (i) were internalized at the respective rates of 79·6 and 65·0% (P ≤ 0·05) by U937 (human) macrophages, and 70·4 and 66·9% by HD11 (chicken) macrophages; and (ii) multiplied at the respective rates of 3·2- and 2·7-fold within U937 cells, and 1·9- and 1·1-fold (P ≤ 0·05) within HD11 cells respectively. Seventy per cent of 10 S. Dublin strains stimulated IL-8 production, while 70% of S. Enteritidis strains enhanced production of IL-1ß, IL-6, IL-8, IL-10, IL-12p70 and TNF in Caco-2 cells. CONCLUSIONS: Compared with S. Enteritidis, S. Dublin had stronger ability to survive within macrophages and induced weak cytokine production, which may explain the higher incidence of invasive diseases caused by S. Dublin in humans. SIGNIFICANCE AND IMPACT OF THE STUDY: This study compared S. enterica serovars Enteritidis and Dublin to provide comparative data about the profile of the two serovars in cells from humans, the common host and their respective natural animal hosts and vice versa in order to check the differences between these two phylogenetically closely related serovars that share antigenic properties but present different phenotypic behaviours.


Assuntos
Citocinas/metabolismo , Células Epiteliais/microbiologia , Macrófagos/microbiologia , Infecções por Salmonella/imunologia , Infecções por Salmonella/microbiologia , Salmonella enterica/imunologia , Salmonella enterica/patogenicidade , Animais , Brasil , Células CACO-2 , Bovinos , Galinhas , Células Epiteliais/imunologia , Humanos , Macrófagos/imunologia , Viabilidade Microbiana , Sorogrupo , Células U937
12.
Methods Mol Biol ; 2182: 103-115, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32894490

RESUMO

Live cell fluorescence imaging is the method of choice to visualize dynamic cellular processes in time and space, such as adhesion to and invasion of polarized epithelial cells by Salmonella enterica sv. Typhimurium. Scanning electron microscopy provides highest resolution of surface structures of infected cells, providing ultrastructure of the apical side of host cells and infecting Salmonella. Combining both methods toward correlative light and scanning electron microscopy (CLSEM) enables new insights in adhesion and invasion mechanisms regarding dynamics over time, and high spatial resolution with precise time lines. To correlate fast live cell imaging of polarized monolayer cells with scanning electron microscopy, we developed a robust method by using gold mesh grids as convenient CLSEM carriers for standard microscopes. By this, we were able to unravel the morphology of the apical structures of monolayers of polarized epithelial cells at distinct time points during Salmonella infection.


Assuntos
Polaridade Celular/fisiologia , Células Epiteliais/microbiologia , Microscopia Eletrônica de Varredura/métodos , Infecções por Salmonella/microbiologia , Salmonella enterica/patogenicidade , Animais , Linhagem Celular , Cães , Células Madin Darby de Rim Canino
13.
Methods Mol Biol ; 2182: 117-126, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32894491

RESUMO

Salmonella enterica is a Gram-negative intracellular pathogen that causes a range of life-threatening diseases in humans and animals worldwide. In a systemic infection, the ability of Salmonella to survive/replicate in macrophages, particularly in the liver and spleen, is crucial for virulence. Transformed macrophage cell lines and primary macrophages prepared from mouse bone marrow are commonly used models for the study of Salmonella infection. However, these models raise technical or ethical issues that highlight the need for alternative methods. This chapter describes a technique for immortalizing early hematopoietic progenitor cells derived from wild-type or transgenic mice and using them to produce macrophages. It validates, through a specific example, the interest of this cellular approach for the study of Salmonella infection.


Assuntos
Células Precursoras de Granulócitos/microbiologia , Proteínas de Homeodomínio/metabolismo , Macrófagos/microbiologia , Infecções por Salmonella/microbiologia , Animais , Linhagem Celular Transformada/metabolismo , Linhagem Celular Transformada/microbiologia , Linhagem Celular Transformada/patologia , Linhagem Celular Tumoral , Células Precursoras de Granulócitos/metabolismo , Fígado/metabolismo , Fígado/microbiologia , Fígado/patologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infecções por Salmonella/metabolismo , Infecções por Salmonella/patologia , Salmonella enterica/patogenicidade , Baço/metabolismo , Baço/microbiologia , Baço/patologia , Virulência/genética
14.
Sci Rep ; 10(1): 21539, 2020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33299016

RESUMO

Salmonella enterica variants exhibit diverse host adaptation, outcome of infection, and associated risk to food safety. Analysis of the distribution of Salmonella enterica serovar Derby (S. Derby) subtypes in human and swine identified isolates with a distinct PFGE profile that were significantly under-represented in human infections, consistent with further host adaptation to swine. Here we show that isolates with this PFGE profile form a distinct phylogenetic sub-clade within S. Derby and exhibit a profound reduction in invasion of human epithelial cells, and a relatively small reduction in swine epithelial cells. A single missense mutation in hilD, that encodes the master-regulator of the Salmonella Pathogenicity Island 1 (SPI-1), was present in the adapted lineage. The missense mutation resulted in a loss of function of HilD that accounted for reduced invasion in human epithelial cells. The relatively small impact of the mutation on interaction with swine cells was consistent with an alternative mechanism of invasion in this pathogen-host combination.


Assuntos
Proteínas de Bactérias/genética , Infecções por Salmonella/genética , Salmonella enterica/genética , Fatores de Transcrição/genética , Animais , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Ilhas Genômicas/genética , Humanos , Mutação/genética , Filogenia , Salmonelose Animal/genética , Salmonella enterica/metabolismo , Salmonella enterica/patogenicidade , Sorogrupo , Suínos , Fatores de Transcrição/metabolismo , Fatores de Virulência/genética
15.
PLoS One ; 15(12): e0240949, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33290416

RESUMO

Salmonellosis and listeriosis together accounted for more than one third of foodborne illnesses in the United States and almost half the hospitalizations for gastrointestinal diseases in 2018 while tuberculosis afflicted over 10 million people worldwide causing almost 2 million deaths. Regardless of the intrinsic virulence differences among Listeria monocytogenes, Salmonella enterica and Mycobacterium tuberculosis, these intracellular pathogens share the ability to survive and persist inside the macrophage and other cells and thrive in iron rich environments. Interferon-gamma (IFN-γ) is a central cytokine in host defense against intracellular pathogens and has been shown to promote iron export in macrophages. We hypothesize that IFN-γ decreases iron availability to intracellular pathogens consequently limiting replication in these cells. In this study, we show that IFN-γ regulates the expression of iron-related proteins hepcidin, ferroportin, and ferritin to induce iron export from macrophages. Listeria monocytogenes, S. enterica, and M. tuberculosis infections significantly induce iron sequestration in human macrophages. In contrast, IFN-γ significantly reduces hepcidin secretion in S. enterica and M. tuberculosis infected macrophages. Similarly, IFN-γ-activated macrophages express higher ferroportin levels than untreated controls even after infection with L. monocytogenes bacilli; bacterial infection greatly down-regulates ferroportin expression. Collectively, IFN-γ significantly inhibits pathogen-associated intracellular iron sequestration in macrophages and consequently retards the growth of intracellular bacterial pathogens by decreasing iron availability.


Assuntos
Interferon gama/farmacologia , Ferro/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Transporte Biológico Ativo/efeitos dos fármacos , Proteínas de Transporte de Cátions/metabolismo , Ferritinas/metabolismo , Hepcidinas/antagonistas & inibidores , Hepcidinas/metabolismo , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Interações entre Hospedeiro e Microrganismos/imunologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Imunidade Inata , Interferon gama/imunologia , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/patogenicidade , Macrófagos/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/patogenicidade , Proteínas Recombinantes/farmacologia , Salmonella enterica/crescimento & desenvolvimento , Salmonella enterica/patogenicidade , Células THP-1
16.
PLoS One ; 15(9): e0238630, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32911523

RESUMO

Salmonella enterica serovar Gallinarum (S. Gallinarum) can cause fowl typhoid, a severe systemic disease responsible for considerable economic losses. Chicken pathogenicity test is the traditional method for assessing the virulence of S. Gallinarum. However, this method is limited by several factors, including ethical considerations, costs, and the need for specialized facilities. Hence, we established a chicken embryo lethality assay (ELA) model to determine the virulence of S. Gallinarum. Three virulent and three avirulent representative strains, which were confirmed by the chicken pathogenicity test, were used to perform the ELA. The most significant difference between the virulent and avirulent strains could be observed when 13-day-old embryos were inoculated via the AC route and incubated for 5 days. Based on a 50% embryo lethal dose (ELD50), isolates considered to be virulent had a Log10ELD50 of ≤ 4.0, moderately virulent strains had a Log10ELD50 of 4.0-6.1, and avirulent isolates had a Log10ELD50 of ≥ 6.1. Different abilities to invade the liver of embryos were found between the virulent and avirulent strains by a growth curve experiment in vitro. The maximum colony-forming units (CFU) of the virulent strain was about 10,000 times higher than that of the avirulent strain in the liver at 5 days post infection. The ELA results of 42 field strains showed that thirty-two strains (76.2%) were virulent, nine were moderately virulent (21.4%), and one strain was avirulent (2.4%). In conclusion, these results suggest that the ELA can be used as an alternative method to assess the virulence of S. Gallinarum, which will contribute to the study of virulence genes, virulence evolution, pathogenic mechanisms and vaccine development.


Assuntos
Modelos Biológicos , Óvulo/microbiologia , Salmonella enterica/patogenicidade , Sorogrupo , Animais , Bioensaio , Embrião de Galinha , Salmonella enterica/crescimento & desenvolvimento , Virulência
17.
Genomics ; 112(6): 4863-4874, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32898642

RESUMO

The G-quadruplex structure is a highly conserved drug target for preventing infection of several human pathogens. We tried to explore G-quadruplex forming motifs as promising drug targets in the genome of Salmonella enterica that causes enteric fever in humans. Herein, we report three highly conserved G-quadruplex motifs (SE-PGQ-1, 2, and 3) in the genome of Salmonella enterica. Bioinformatics analysis inferred the presence of SE-PGQ-1 in the regulatory region of mgtA, SE-PGQ-2 in ORF of entA, and SE-PGQ-3 in the promoter region of malE and malK genes. The G-quadruplex forming sequences were confirmed by biophysical and biomolecular techniques. Cellular studies affirm the inhibitory effect of G-quadruplex specific ligands on Salmonella enterica growth. Further, PCR inhibition, reporter based assay, and RT-qPCR assays emphasize the biological relevance of G-quadruplexes in these genes. Thus, this study confirmed the presence of G-quadruplex motifs in Salmonella enterica and characterized them as a promising drug target.


Assuntos
Quadruplex G , Proteínas Ligantes de Maltose/genética , Salmonella enterica/genética , Virulência/genética , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/genética , Transporte de Íons , Regiões Promotoras Genéticas , Salmonella enterica/patogenicidade
18.
mBio ; 11(5)2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963007

RESUMO

The interaction and communication between bacteria and their hosts modulate many aspects of animal physiology and behavior. Dauer entry as a response to chronic exposure to pathogenic bacteria in Caenorhabditis elegans is an example of a dramatic survival response. This response is dependent on the RNA interference (RNAi) machinery, suggesting the involvement of small RNAs (sRNAs) as effectors. Interestingly, dauer formation occurs after two generations of interaction with two unrelated moderately pathogenic bacteria. Therefore, we sought to discover the identity of C. elegans RNAs involved in pathogen-induced diapause. Using transcriptomics and differential expression analysis of coding and long and small noncoding RNAs, we found that mir-243-3p (the mature form of mir-243) is the only transcript continuously upregulated in animals exposed to both Pseudomonas aeruginosa and Salmonella enterica for two generations. Phenotypic analysis of mutants showed that mir-243 is required for dauer formation under pathogenesis but not under starvation. Moreover, DAF-16, a master regulator of defensive responses in the animal and required for dauer formation was found to be necessary for mir-243 expression. This work highlights the role of a small noncoding RNA in the intergenerational defensive response against pathogenic bacteria and interkingdom communication.IMPORTANCE Persistent infection of the bacterivore nematode C. elegans with bacteria such as P. aeruginosa and S. enterica makes the worm diapause or hibernate. By doing this, the worm closes its mouth, avoiding infection. This response takes two generations to be implemented. In this work, we looked for genes expressed upon infection that could mediate the worm diapause triggered by pathogens. We identify mir-243-3p as the only transcript commonly upregulated when animals feed on P. aeruginosa and S. enterica for two consecutive generations. Moreover, we demonstrate that mir-243-3p is required for pathogen-induced dauer formation, a new function that has not been previously described for this microRNA (miRNA). We also find that the transcriptional activators DAF-16, PQM-1, and CRH-2 are necessary for the expression of mir-243 under pathogenesis. Here we establish a relationship between a small RNA and a developmental change that ensures the survival of a percentage of the progeny.


Assuntos
Bactérias/patogenicidade , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Diapausa , MicroRNAs/genética , Animais , Caenorhabditis elegans/microbiologia , Regulação da Expressão Gênica no Desenvolvimento , Interações Hospedeiro-Patógeno/genética , Mutação , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Interferência de RNA , Salmonella enterica/genética , Salmonella enterica/patogenicidade , Transdução de Sinais , Regulação para Cima
19.
Int J Biol Macromol ; 163: 1798-1809, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32961194

RESUMO

Lignin particles (LPs) have gained prominence due to their biodegradability and bioactive properties. LP production at nano and micro scale produced from organosolv lignin and the understanding of size's effect on their properties is unexplored. This work aimed to produce and characterize lignin nanoparticles and microparticles using a green synthesis process, based on ethanol-solubilized lignin and water. Spherical shape LPs, with a mean size of 75 nm and 215 nm and with a low polydispersity were produced, as confirmed by transmission electron microscopy and dynamic light scattering. LPs thermal stability improved over raw lignin, and the chemical structure of lignin was not affected by the production method. The antimicrobial tests proved that LPs presented a bacteriostatic effect on Escherichiacoli and Salmonella enterica. Regarding the antioxidant potential, LPs had a good antioxidant activity that increased with the reaction time and LPs concentration. LPs also presented an antioxidant effect against intracellular ROS, reducing the intracellular ROS levels significantly. Furthermore, the LPs showed a low cytotoxic effect in Caco-2 cell line. These results showed that LPs at different scales (nano and micro) present biological properties and are safe to be used in different high value industrial sectors, such as biomedical, pharmaceutical and food.


Assuntos
Química Verde , Lignina/química , Microplásticos/síntese química , Nanopartículas/química , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Antioxidantes/síntese química , Antioxidantes/química , Antioxidantes/farmacologia , Células CACO-2 , Proliferação de Células/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/patogenicidade , Etanol/química , Humanos , Lignina/síntese química , Lignina/farmacologia , Microplásticos/química , Microplásticos/farmacologia , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/patogenicidade , Água/química
20.
Front Immunol ; 11: 1786, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32903436

RESUMO

Patients who survive sepsis display prolonged immune dysfunction and heightened risk of secondary infection. CD4 T cells support a variety of cells required for protective immunity, and perturbations to the CD4 T cell compartment can decrease overall immune system fitness. Using the cecal ligation and puncture (CLP) mouse model of sepsis, we investigated the impact of sepsis on endogenous Ag-specific memory CD4 T cells generated in C57BL/6 (B6) mice infected with attenuated Listeria monocytogenes (Lm) expressing the I-Ab-restricted 2W1S epitope (Lm-2W). The number of 2W1S-specific memory CD4 T cells was significantly reduced on day 2 after sepsis induction, but recovered by day 14. In contrast to the transient numerical change, the 2W1S-specific memory CD4 T cells displayed prolonged functional impairment after sepsis, evidenced by a reduced recall response (proliferation and effector cytokine production) after restimulation with cognate Ag. To define the extent to which the observed functional impairments in the memory CD4 T cells impacts protection to secondary infection, B6 mice were infected with attenuated Salmonella enterica-2W (Se-2W) 30 days before sham or CLP surgery, and then challenged with virulent Se-2W after surgery. Pathogen burden was significantly higher in the CLP-treated mice compared to shams. Similar reductions in functional capacity and protection were noted for the endogenous OVA323-specific memory CD4 T cell population in sepsis survivors upon Lm-OVA challenge. Our data collectively show CLP-induced sepsis alters the number and function of Ag-specific memory CD4 T cells, which contributes (in part) to the characteristic long-lasting immunoparalysis seen after sepsis.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Coinfecção/imunologia , Imunidade Celular , Memória Imunológica , Sepse/imunologia , Animais , Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/microbiologia , Ceco/microbiologia , Ceco/cirurgia , Proliferação de Células , Coinfecção/metabolismo , Coinfecção/microbiologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Patógeno , Ligadura , Listeria monocytogenes/imunologia , Listeria monocytogenes/patogenicidade , Listeriose/imunologia , Listeriose/metabolismo , Listeriose/microbiologia , Ativação Linfocitária , Camundongos Endogâmicos C57BL , Punções , Infecções por Salmonella/imunologia , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologia , Salmonella enterica/imunologia , Salmonella enterica/patogenicidade , Sepse/metabolismo , Sepse/microbiologia
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