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1.
Food Microbiol ; 87: 103351, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31948609

RESUMO

The objective of this work was to investigate the effect of stress conditions frequently encountered in food-associated environments on virulence-associated characteristics of eight strains of Listeria monocytogenes. Strains were grown at low (11 °C, cold stress) and optimal (37 °C) temperatures and in high NaCl concentrations (6% NaCl, 11 °C; cold-osmotic stress) and tested for their ability to invade the human intestinal epithelial Caco-2 cells. Results demonstrate that the correlation between exposure to cold stress and increased invasion phenotype is strain-dependent as strains investigated exhibited different behaviours, i.e. exposure to cold stress conditions resulted in a significant increase of invasion levels in five out of the eight strains tested, when compared to growth under optimal conditions. On the other hand, when these cold-adapted cells were subsequently submitted to high salt concentrations and low temperature, their enhanced ability to invade Caco-2 was lost. Surprisingly, saturated fatty acids (SFA) and branched chain fatty acids (BCFA) decreased when L. monocytogenes were exposed to stress conditions as opposed to what has been observed in other studies, therefore highlighting that further studies will need to deepen in the understanding of the lipid metabolism of these strains. The effect of stress conditions on the survival of three selected L. monocytogenes strains through an in vitro gastrointestinal (GI) tract digestion model was further investigated. The exposure to cold-osmotic stress increased the survival of one strain through the GI tract.


Assuntos
Listeria monocytogenes/fisiologia , Listeriose/microbiologia , Adaptação Fisiológica , Células CACO-2 , Temperatura Baixa , Contagem de Colônia Microbiana , Humanos , Listeria monocytogenes/química , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/patogenicidade , Pressão Osmótica , Cloreto de Sódio/metabolismo , Estresse Fisiológico , Virulência
2.
Food Microbiol ; 87: 103381, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31948622

RESUMO

Indirect impedance has been used for the detection and enumeration of bacteria, however there is limited data regarding the ability of the method to measure growth and inhibition of microorganisms in food in response to preservatives. The aim of this study was to evaluate the suitability of the technique to determine maximum growth rates of Listeria innocua (used as a surrogate for Listeria monocytogenes) in complex food matrices to which multiple preservative factors had been applied and assess the suitability of the data for use in predictive microbiology. Growth of L. innocua in laboratory medium (BHI broth) and two food matrices (zucchini purée and béarnaise sauce) under varying conditions of pH (5 & 5.3), water activity (0.93, 0.96 & 0.98) and acetic and propionic acid concentration (0, 1 & 2 mM) was monitored by the conductimetric Rapid Automated Bacterial Impedance Technology (R.A.B.I.T) system by means of CO2 emission for up to 120 h. Growth rates of L. innocua were determined for several conditions across the three test matrices and a good correlation between detection times and initial inoculum level was observed in most cases (R2 ≥ 0.82). However, growth of L. innocua was not detected in a large number of conditions and comparison of growth rates determined by indirect impedance to those determined by plate counts indicated that in general, the R.A.B.I.T. system under-estimated growth. This study demonstrates that there are limitations associated with the technology, and as a result the system may be unsuitable for measuring microbial growth rates in complex food matrices under the environmental conditions tested and within the time duration of the study.


Assuntos
Contagem de Colônia Microbiana/métodos , Técnicas Eletroquímicas/métodos , Microbiologia de Alimentos/métodos , Listeria/química , Listeria/crescimento & desenvolvimento , Dióxido de Carbono/análise , Dióxido de Carbono/metabolismo , Impedância Elétrica , Contaminação de Alimentos/análise , Concentração de Íons de Hidrogênio , Listeria/metabolismo , Listeria monocytogenes/química , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/metabolismo , Água/análise , Água/metabolismo
3.
Food Microbiol ; 86: 103310, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31703859

RESUMO

The objective of this study was to develop a qPCR method for specific enumeration of viable Listeria monocytogenes in food processing facilities and heat treated products. Primers specific for L. monocytogenes were designed to amplify a short (199 bp) or long (1561 bp) fragment of the listeriolysin (hly) gene. The short- and long-amplicon qPCR methods with and without propidium monoazide (PMA) treatment of the cells were tested for their ability to discriminate between viable (no heat) and heat-killed cells (90 °C, 10 min). The PMA-qPCR methods were subsequently used to assess the survival of L. monocytogenes during desiccation (33% RH, 15 °C) on stainless steel surfaces for ten days with and without prior biofilm formation. The long-amplicon qPCR method had a limit of quantification (LOQ) of 1.32 log CFU/reaction (efficiency 92%, R2 = 0.991), while the LOQ for the short-amplicon qPCR method was 1.44 log CFU/reaction (efficiency 102%, R2 = 0.991). PMA was essential for detection of viable cells, and the long-amplicon PMA-qPCR significantly (p < 0.05) reduced the signal from heat-killed cells compared to the short-amplicon method. L. monocytogenes survival during desiccation without biofilm formation was accurately enumerated with the long-amplicon PMA-qPCR method. However, when L. monocytogenes had formed biofilm prior to desiccation, the long-amplicon PMA-qPCR accurately measured the log fold inactivation but underestimated the number of viable cells even with use of an optimized DNA extraction method. This long-amplicon PMA-qPCR method can aid in the detection and enumeration of viable L. monocytogenes cells to further the understanding of its survival and persistence in food processing facilities. The developed method was demonstrated to work on both heat and desiccation treated cells and highlights the importance of amplicon size in viability-qPCR.


Assuntos
Antibacterianos/farmacologia , Azidas/farmacologia , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/crescimento & desenvolvimento , Propídio/análogos & derivados , Reação em Cadeia da Polimerase em Tempo Real/métodos , Primers do DNA/genética , DNA Bacteriano/genética , Dessecação , Temperatura Alta , Listeria monocytogenes/química , Listeria monocytogenes/genética , Viabilidade Microbiana/efeitos dos fármacos , Propídio/farmacologia
4.
Food Microbiol ; 86: 103315, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31703881

RESUMO

Non-thermal food processing and replacement of chemical additives by natural antimicrobials are promising trends in the food industry. The objective of the present work was to evaluate the effect of a process which combines mild high hydrostatic pressure - HHP (200 and 300 MPa, 5 min, 10 °C), phage Listex™ P100 and the bacteriocin pediocin PA-1 as a new non-thermal process for destruction of Listeria monocytogenes (104 CFU mL-1 or 107 CFU mL-1) in milk. For inoculum levels of 104 CFU mL-1, HHP combined with phage P100 eliminated L. monocytogenes immediately after pressurization. When L. monocytogenes was inoculated at levels of 107 CFU mL-1, a synergistic effect between phage P100, pediocin PA-1 and HHP (300 MPa) on the inactivation of L. monocytogenes was observed during storage of milk at 4 °C. For non-pressure treated samples inoculated with phage or pediocin or both, L. monocytogenes counts decreased immediately after biocontrol application, but regrowth was observed in a few samples during storage. Phage particles were stable during refrigerated storage for seven days while pediocin PA-1 remained stable only during three days. Further studies will have to be performed to validate the findings of this work in specific applications (e.g. production of raw milk cheese).


Assuntos
Bacteriófagos/fisiologia , Conservação de Alimentos/métodos , Conservantes de Alimentos/farmacologia , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/virologia , Leite/microbiologia , Pediocinas/farmacologia , Animais , Bovinos , Contagem de Colônia Microbiana , Conservação de Alimentos/instrumentação , Pressão Hidrostática , Listeria monocytogenes/química , Listeria monocytogenes/crescimento & desenvolvimento
5.
Nat Struct Mol Biol ; 26(10): 946-954, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31582852

RESUMO

The ClpXP machinery is a two-component protease complex that performs targeted protein degradation in bacteria and mitochondria. The complex consists of the AAA+ chaperone ClpX and the peptidase ClpP. The hexameric ClpX utilizes the energy of ATP binding and hydrolysis to engage, unfold and translocate substrates into the catalytic chamber of tetradecameric ClpP, where they are degraded. Formation of the complex involves a symmetry mismatch, because hexameric AAA+ rings bind axially to the opposing stacked heptameric rings of the tetradecameric ClpP. Here we present the cryo-EM structure of ClpXP from Listeria monocytogenes. We unravel the heptamer-hexamer binding interface and provide novel insight into the ClpX-ClpP cross-talk and activation mechanism. Comparison with available crystal structures of ClpP and ClpX in different states allows us to understand important aspects of the complex mode of action of ClpXP and provides a structural framework for future pharmacological applications.


Assuntos
Proteínas de Bactérias/ultraestrutura , Endopeptidase Clp/ultraestrutura , Listeria monocytogenes/ultraestrutura , Proteínas de Bactérias/química , Microscopia Crioeletrônica , Endopeptidase Clp/química , Ativação Enzimática , Listeria monocytogenes/química , Listeriose/microbiologia , Modelos Moleculares , Multimerização Proteica , Proteólise
6.
Mikrochim Acta ; 186(7): 401, 2019 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-31183576

RESUMO

A method is described for single-step detection of V. parahaemolyticus in seafood via aptamer-based SERS. A gold-coated polydimethylsiloxane (PDMS) film was used for the enhancement of Raman scattering. The Raman reporter 4-mercaptobenzoic acid was linked to aptamer modified gold nanoparticles (AuNPs) served as a signalling probe. The negatively charged signalling probe was assembled onto the cysteamine-modified Au-PDMS film through electrostatic adsorption. On addition of V. parahaemolyticus, it will be bound by the aptamer as a biorecognition element, and this leads to the dissociation of the signalling probe from the Au-PDMS film. Hence, the Raman signal (at 1592 cm-1) decreases. The assay has a wide linear response that covers the 1.2 × 102 to 1.2 × 106 cfu·mL-1 V. parahaemolyticus concentration range. The detection limit is 12 cfu·mL-1. The method was successfully applied to the determination of V. parahaemolyticus in oyster and salmon samples. Graphical abstract Schematic presentation of a surface-enhanced Raman spectroscopic method for single step detection of Vibrio parahaemolyticus using gold coated polydimethylsiloxane as the active substrate and aptamer modified gold nanoparticles. This solid substrate simplified the analysis procedures and enhanced the sensitivity.


Assuntos
Aptâmeros de Nucleotídeos/química , Dimetilpolisiloxanos/química , Ouro/química , Nanopartículas Metálicas/química , Vibrio parahaemolyticus/química , Sequência de Bases , Benzoatos/química , Técnicas Biossensoriais/métodos , Cisteamina/química , Escherichia coli/química , Limite de Detecção , Listeria monocytogenes/química , Salmonella typhimurium/química , Sensibilidade e Especificidade , Análise Espectral Raman , Staphylococcus aureus/química , Compostos de Sulfidrila/química , Vibrio parahaemolyticus/isolamento & purificação
7.
PLoS One ; 14(4): e0215017, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30970033

RESUMO

Listeria monocytogenes is a foodborne pathogen that causes listeriosis and can lead to serious clinical problems, such as sepsis and meningitis, in immunocompromised patients and neonates. Due to a growing number of antibiotic-resistant L. monocytogenes strains, listeriosis can steadily become refractory to antibiotic treatment. To develop novel therapeutics against listeriosis, the drug resistance mechanism of L. monocytogenes needs to be determined. The transcription factor LftR from L. monocytogenes regulates the expression of a putative multidrug resistance transporter, LieAB, and belongs to the PadR-2 subfamily of the PadR family. Despite the functional significance of LftR, our molecular understanding of the transcriptional regulatory mechanism for LftR and even for the PadR-2 subfamily is highly limited. Here, we report the crystal structure of LftR, which forms a dimer and protrudes two winged helix-turn-helix motifs for DNA recognition. Structure-based mutational and comparative analyses showed that LftR interacts with operator DNA through a LftR-specific mode as well as a common mechanism used by the PadR family. Moreover, the LftR dimer harbors one intersubunit cavity in the center of the dimeric structure as a putative ligand-binding site. Finally, conformational flexibilities in the LftR dimer and in the cavity suggest that a ligand-induced regulatory mechanism would be used by the LftR transcription factor.


Assuntos
Proteínas de Bactérias/química , Listeria monocytogenes/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Humanos , Ligantes , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
8.
Int J Med Microbiol ; 309(3-4): 199-212, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30962079

RESUMO

Membrane vesicles (MVs) are produced by various Gram positive and Gram negative pathogenic bacteria and play an important role in virulence. In this study, the membrane vesicles (MVs) of L. monocytogenes were isolated from the culture supernatant. High-resolution electron microscopy and dynamic light scattering analysis revealed that L. monocytogenes MVs are spherical with a diameter of 200 to 300 nm in size. Further, comprehensive proteomic analyses of MVs and whole cells of L. monocytogenes were performed using LC/MS/MS. A total of 1355 and 312 proteins were identified in the L. monocytogenes cells and MVs, respectively. We identified that 296 proteins are found in both whole cells, and MV proteome and 16 proteins were identified only in the MVs. Also, we have identified the virulence factors such as listeriolysin O (LLO), internalin B (InlB), autolysin, p60, NLP/P60 family protein, UPF0356 protein, and PLC-A in MVs. Computational prediction of host-MV interactions revealed a total of 1841 possible interactions with the host involving 99 MV proteins and 1513 host proteins. We elucidated the possible pathway that mediates internalization of L. monocytogenes MV to host cells and the subsequent pathogenesis mechanisms. The in vitro infection assays showed that the purified MVs could induce cytotoxicity in Caco-2 cells. Using endocytosis inhibitors, we demonstrated that MVs are internalized via actin-mediated endocytosis. These results suggest that L. monocytogenes MVs can interact with host cell and contribute to the pathogenesis of L. monocytogenes during infection.


Assuntos
Proteínas de Bactérias/metabolismo , Vesículas Extracelulares/metabolismo , Interações Hospedeiro-Patógeno , Listeria monocytogenes/patogenicidade , Fatores de Virulência/metabolismo , Actinas/metabolismo , Células CACO-2 , Sobrevivência Celular , Endocitose , Vesículas Extracelulares/química , Vesículas Extracelulares/ultraestrutura , Humanos , Listeria monocytogenes/química , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Proteômica , Sorogrupo , Virulência
9.
Nat Chem ; 11(5): 463-469, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31011175

RESUMO

Staphylococci secrete autoinducing peptides (AIPs) as signalling molecules to regulate population-wide behaviour. AIPs from non-Staphylococcus aureus staphylococci have received attention as potential antivirulence agents to inhibit quorum sensing and virulence gene expression in the human pathogen Staphylococcus aureus. However, only a limited number of AIP structures from non-S. aureus staphylococci have been identified to date, as the minute amounts secreted in complex media render it difficult. Here, we report a method for the identification of AIPs by exploiting their thiolactone functionality for chemoselective trapping and enrichment of the compounds from the bacterial supernatant. Standard liquid chromatography mass spectrometry analysis, guided by genome sequencing data, then readily provides the AIP identities. Using this approach, we confirm the identity of five known AIPs and identify the AIPs of eleven non-S. aureus species, and we expect that the method should be extendable to AIP-expressing Gram-positive bacteria beyond the Staphylococcus genus.


Assuntos
Proteínas de Bactérias/análise , Depsipeptídeos/análise , Staphylococcus/química , Sequência de Aminoácidos , Proteínas de Bactérias/síntese química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Cisteína/química , Depsipeptídeos/síntese química , Depsipeptídeos/isolamento & purificação , Depsipeptídeos/farmacologia , Limite de Detecção , Listeria monocytogenes/química , Estrutura Molecular , Percepção de Quorum/efeitos dos fármacos , Staphylococcus/metabolismo
10.
J Pharm Biomed Anal ; 169: 181-187, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-30877929

RESUMO

A new, facile, low-cost, and highly sensitive method for detection of Listeria monocytogenes involving a combination of asymmetric polymerase chain reaction (aPCR) and rolling circle amplification (RCA) had been developed. The aPCR-RCA processes were not new but components of the processes made the assay useful. Twenty-one thymine (21-T) tagged forward primer generated universal twenty-one adenine (21-A) aPCR amplicons after aPCR amplification. A poly-T sequence dumbbell-like RCA template was produced through the blunt-end ligation activity of T4 DNA ligase. After the mixture of aPCR amplicons and dumbbell-like RCA template, the RCA reaction would initiate when the addition of phi29 DNA polymerase, then a large number of G-quadruplex sequences were produced which allowed the intercalation of Thioflavin T (3,6-dimethyl-2-(4-dimethylaminophenyl) benzo-thiazolium cation, THT) for easy fluorescence detection. Under the optimal conditions, the assay showed a limit of detection (LOD) of 4.8 × 101 CFU/mL in pure culture and 4.0 × 102 CFU/g in spiked lettuce homogenates. By changing the aPCR primer, the aPCR-RCA method developed in this study had a potential to detect other bacteria without the design an RCA template for each bacterium.


Assuntos
Bioensaio/métodos , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Listeria monocytogenes/química , Reação em Cadeia da Polimerase/métodos , Benzotiazóis/química , Primers do DNA/química , Primers do DNA/genética , Fluorescência , Quadruplex G , Limite de Detecção , Listeria monocytogenes/genética
11.
J Appl Microbiol ; 126(5): 1496-1507, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30761711

RESUMO

AIM: To investigate the use of a light scattering sensor, BActerial Rapid Detection using Optical scattering Technology (BARDOT) coupled with a multipathogen selective medium, Salmonella, Escherichia and Listeria (SEL), for concurrent detection of the three major foodborne pathogens in a single assay. METHODS AND RESULTS: BARDOT was used to detect and distinguish the three major pathogens, Salmonella enterica, Shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes from food based on colony scatter signature patterns on SEL agar (SELA). Multiple strains of three test pathogens were grown on SELA, and BARDOT was used to generate colony scatter image libraries for inclusive (SEL Library) and exclusive (non-SEL Library) bacterial group. These pathogens were further differentiated using the SEL scatter image library. Raw chicken and hotdog samples were artificially inoculated with pathogens (100 CFU per 25 g each), and enriched in SEL broth at 37°C for 18 h and colonies were grown on SELA for 11-22 h before screening with BARDOT. The BARDOT sensor successfully detected and differentiated Salmonella, STEC and Listeria on SELA with high classification accuracy 92-98%, 91-98% and 83-98% positive predictive values (PPV) respectively; whereas the nontarget strains showed only 0-13% PPV. BARDOT-identified colonies were further confirmed by multiplex PCR targeting inlB gene of L. monocytogenes, stx2 of STEC and sefA of S. enterica serovar Enteritidis. CONCLUSIONS: The results show that BARDOT coupled with SELA can efficiently screen for the presence of three major pathogens simultaneously in a test sample within 29-40 h. SIGNIFICANCE AND IMPACT OF THE STUDY: This innovative SELA-BARDOT detection platform can reduce turnaround time and economic burden on food industries by offering a label-free, noninvasive on-plate multipathogen screening technology for reducing microbial food safety and public health concerns.


Assuntos
Escherichia coli , Microbiologia de Alimentos/métodos , Listeria monocytogenes , Salmonella enterica , Espalhamento de Radiação , Animais , Galinhas , Escherichia coli/química , Escherichia coli/isolamento & purificação , Luz , Listeria monocytogenes/química , Listeria monocytogenes/isolamento & purificação , Carne/microbiologia , Salmonella enterica/química , Salmonella enterica/isolamento & purificação
12.
Folia Microbiol (Praha) ; 64(4): 567-577, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30661218

RESUMO

The formation of a hardly removable biofilm in food processing and clinical settings calls for a deeper understanding of composition of the matrix that protects the biofilm cells, as the crucial matrix component is extracellular DNA (eDNA), participating in adhesion, aggregation and penetration reduction, yet serving as a horizontal gene transfer reservoir. Therefore, we evaluated eDNA release from the biofilm of two pathogens, Listeria monocytogenes and Staphylococcus aureus, with respect to their origin under different culturing condition. Primarily, the biofilms were observed by confocal laser scanning microscopy (CLSM) under conditions mimicking the food processing environment and human body. The eDNA was quantitatively characterised based on its area by IMARIS. Next, the eDNA content and biofilm formation were quantified by spectrophotometry. Data from both sets of experiments were statistically evaluated. The eDNA release varied between the microorganism, culturing conditions and the origin of strains. Independent of the method used, the clinical strains of S. aureus released more eDNA than the food related strains at 37 °C. eDNA content can be crucial discriminating matrix component between food related and clinical strains. Deeper understanding of the eDNA role in such a phenomenon could facilitate the design of effective strategy for biofilm disruption.


Assuntos
Biofilmes , Espaço Extracelular/microbiologia , Listeria monocytogenes/genética , Listeriose/microbiologia , Staphylococcus aureus/genética , Transporte Biológico , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Humanos , Listeria monocytogenes/química , Listeria monocytogenes/fisiologia , Microscopia Confocal , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/química , Staphylococcus aureus/fisiologia
13.
Anat Rec (Hoboken) ; 301(12): 2095-2102, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30312532

RESUMO

Enteropathogenic Escherichia coli (EPEC), Salmonella typhimurium, and Listeria monocytogenes usurp the actin cytoskeleton for their attachment, internalization and transport within and amongst infected cells. To try to gain a greater understanding of the molecular components utilized by these microbes during their infections we previously concentrated actin-rich structures generated during EPEC infections (called pedestals) and identified the heat shock cognate 70 protein (Hsc70) as a potential candidate. This multifunctional protein classically acts as a chaperone for the proper folding of a variety of proteins and is involved in uncoating clathrin from coated pits. Here we demonstrated that Hsc70 is recruited to actin structures generated during EPEC, Listeria and Salmonella infections, but not to the same location as clathrin. Anat Rec, 301:2095-2102, 2018. © 2018 Wiley Periodicals, Inc.


Assuntos
Actinas/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Listeria monocytogenes/metabolismo , Actinas/análise , Animais , Proteínas de Choque Térmico HSC70/análise , Células HeLa , Humanos , Listeria monocytogenes/química
14.
Anat Rec (Hoboken) ; 301(12): 2103-2111, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30312538

RESUMO

The ingestion of enteropathogenic Escherichia coli (EPEC), Listeria monocytogenes, or Salmonella enterica serovar Typhimurium leads to their colonization of the intestinal lumen, which ultimately causes an array of ailments ranging from diarrhea to bacteremia. Once in the intestines, these microbes generate various actin-rich structures to attach, invade, or move within the host intestinal epithelial cells. Although an assortment of actin-associated proteins has been identified to varying degrees at these structures, the localization of many actin stabilizing proteins have yet to be analyzed. Here, we examined the recruitment of the actin-associated proteins, calponin 1 and 2 at EPEC pedestals, L. monocytogenes actin clouds, comet tails and listeriopods, and S. Typhimurium membrane ruffles. In other systems, calponins are known to bind to and stabilize actin filaments. In EPEC pedestals, calponin 1 was recruited uniformly throughout the structures while calponin 2 was enriched at the apical tip. During L. monocytogenes infections, calponin 1 was found through all the actin-rich structures generated by the bacteria, while calponin 2 was only present within actin-rich structures formed by L. monocytogenes near the host cell membrane. Finally, both calponins were found within S. Typhimurium-generated membrane ruffles. Taken together, we have shown that although calponin 1 is recruited to actin-rich structures formed by the three bacteria, calponin 2 is specifically recruited to only membrane-bound actin-rich structures formed by the bacteria. Thus, our findings suggest that calponin 2 is a novel marker for membrane-bound actin structures formed by pathogenic bacteria. Anat Rec, 301:2103-2111, 2018. © 2018 Wiley Periodicals, Inc.


Assuntos
Actinas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Escherichia coli Enteropatogênica/metabolismo , Listeria monocytogenes/metabolismo , Proteínas dos Microfilamentos/metabolismo , Salmonella enterica/metabolismo , Actinas/análise , Células CACO-2 , Proteínas de Ligação ao Cálcio/análise , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Escherichia coli Enteropatogênica/química , Humanos , Listeria monocytogenes/química , Proteínas dos Microfilamentos/análise , Salmonella enterica/química
15.
J Am Chem Soc ; 140(38): 11926-11930, 2018 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-30196699

RESUMO

Many dynamic biological processes are regulated by protein-protein interactions and protein localization. Experimental techniques to probe such processes with temporal and spatial precision include photoactivatable proteins and chemically induced dimerization (CID) of proteins. CID has been used to study several cellular events, especially cell signaling networks, which are often reversible. However, chemical dimerizers that can be both rapidly activated and deactivated with high spatiotemporal resolution are currently limited. Herein, we present a novel chemical inducer of protein dimerization that can be rapidly turned on and off using single pulses of light at two orthogonal wavelengths. We demonstrate the utility of this molecule by controlling peroxisome transport and mitotic checkpoint signaling in living cells. Our system highlights and enhances the spatiotemporal control offered by CID. This tool addresses biological questions on subcellular levels by controlling protein-protein interactions.


Assuntos
Proteínas de Bactérias/metabolismo , Cumarínicos/química , Indicadores e Reagentes/química , Trimetoprima/química , Proteínas de Bactérias/química , Cumarínicos/toxicidade , Desenho de Drogas , Escherichia coli/enzimologia , Células HeLa , Humanos , Indicadores e Reagentes/toxicidade , Cinetocoros/metabolismo , Listeria monocytogenes/química , Mitocôndrias/metabolismo , Peroxissomos/metabolismo , Multimerização Proteica , Rhodococcus/enzimologia , Trimetoprima/toxicidade , Raios Ultravioleta
16.
J Biol Chem ; 293(35): 13626-13635, 2018 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-29976754

RESUMO

Listeria monocytogenes causes listeriosis, a potentially fatal food-borne disease. The condition is especially harmful to pregnant women. Listeria outbreaks can originate from diverse foods, highlighting the need for novel strategies to improve food safety. The first step in Listeria invasion is internalization of the bacteria, which is mediated by the interaction of the internalin family of virulence factors with host cell receptors. A crucial interaction for Listeria invasion of the placenta, and thus a target for therapeutic intervention, is between internalin B (InlB) and the receptor c-Met. Single-domain antibodies (VHH, also called nanobodies, or sdAbs) from camel heavy-chain antibodies are a novel solution for preventing Listeria infections. The VHH R303, R330, and R326 all bind InlB with high affinity; however, the molecular mechanism behind their mode of action was unknown. We demonstrate that despite a high degree of sequence and structural diversity, the VHH bind a single epitope on InlB. A combination of gentamicin protection assays and florescent microscopy establish that InlB-specific VHH inhibit Listeria invasion of HeLa cells. A high-resolution X-ray structure of VHH R303 in complex with InlB showed that the VHH binds at the c-Met interaction site on InlB, thereby acting as a competitive inhibitor preventing bacterial invasion. These results point to the potential of VHH as a novel class of therapeutics for the prevention of listeriosis.


Assuntos
Proteínas de Bactérias/metabolismo , Listeria monocytogenes/efeitos dos fármacos , Listeriose/prevenção & controle , Proteínas de Membrana/metabolismo , Anticorpos de Domínio Único/farmacologia , Proteínas de Bactérias/química , Cristalografia por Raios X , Células HeLa , Humanos , Listeria monocytogenes/química , Listeria monocytogenes/metabolismo , Listeriose/metabolismo , Listeriose/microbiologia , Proteínas de Membrana/química , Modelos Moleculares , Conformação Proteica , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-met/metabolismo , Anticorpos de Domínio Único/química , Fatores de Virulência/química , Fatores de Virulência/metabolismo
17.
Int J Food Microbiol ; 283: 7-13, 2018 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-29933230

RESUMO

Traditionally, predictive growth models for food pathogens are developed based on experiments in broth media, resulting in models which do not incorporate the influence of food microstructure. The use of model systems with various microstructures is a promising concept to get more insight into the influence of food microstructure on microbial dynamics. By means of minimal variation of compositional and physicochemical factors, these model systems can be used to study the isolated effect of certain microstructural aspects on microbial growth, survival and inactivation. In this study, the isolated effect on microbial growth dynamics of Listeria monocytogenes of two food microstructural aspects and one aspect influenced by food microstructure were investigated, i.e., the nature of the food matrix, the presence of fat droplets, and microorganism growth morphology, respectively. To this extent, fish-based model systems with various microstructures were used, i.e., a liquid, a second more viscous liquid system containing xanthan gum, an emulsion, an aqueous gel, and a gelled emulsion. Growth experiments were conducted at 4 and 10 °C, both using homogeneous and surface inoculation (only for the gelled systems). Results regarding the influence of the growth morphology indicated that the lag phase of planktonic cells in the liquid system was similar to the lag phase of submerged colonies in the xanthan system. The lag phase of submerged colonies in each gelled system was considerably longer than the lag phase of surface colonies on these respective systems. The maximum specific growth rate of planktonic cells in the liquid system was significantly lower than for submerged colonies in the xanthan system at 10 °C, while no significant differences were observed at 4 °C. The maximum cell density was higher for submerged colonies than for surface colonies. The nature of the food matrix only exerted an influence on the maximum specific growth rate, which was significantly higher in the viscous systems than in the gelled systems. The presence of a small amount of fat droplets improved the growth of L. monocytogenes at 4 °C, resulting in a shorter lag phase and a higher maximum specific growth rate. The obtained results could be useful in the determination of a set of suitable microstructural parameters for future predictive models that incorporate the influence of food microstructure on microbial dynamics.


Assuntos
Peixes/microbiologia , Listeria monocytogenes/crescimento & desenvolvimento , Animais , Contagem de Colônia Microbiana , Meios de Cultura/metabolismo , Microbiologia de Alimentos , Conservação de Alimentos/métodos , Cinética , Listeria monocytogenes/química , Listeria monocytogenes/isolamento & purificação , Listeria monocytogenes/metabolismo , Modelos Biológicos , Temperatura Ambiente
18.
J Bacteriol ; 200(13)2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29661862

RESUMO

Listeria monocytogenes is a food pathogen capable of growing at a broad temperature range from 50°C to refrigerator temperatures. A key requirement for bacterial activity and growth at low temperatures is the ability to adjust the membrane lipid composition to maintain cytoplasmic membrane fluidity. In this study, we confirmed earlier findings that the extents of fatty acid profile adaptation differed between L. monocytogenes strains. We were able to demonstrate for isolates from food that growth rates at low temperatures and resistance to freeze-thaw stress were not impaired by a lower adaptive response of the fatty acid composition. This indicated the presence of a second adaptation mechanism besides temperature-regulated fatty acid synthesis. For strains that showed weaker adaptive responses in their fatty acid profiles to low growth temperature, we could demonstrate a significantly higher concentration of isoprenoid quinones. Three strains even showed a higher quinone concentration after growth at 6°C than at 37°C, which is contradictory to the reduced respiratory activity at lower growth temperatures. Analyses of the membrane fluidity in vivo by measuring generalized polarization and anisotropy revealed modulation of the transition phase. Strains with increased quinone concentrations showed an expanded membrane transition phase in contrast to strains with pronounced adaptations of fatty acid profiles. The correlation between quinone concentration and membrane transition phase expansion was confirmed by suppression of quinone synthesis. A reduced quinone concentration resulted in a narrower transition phase. Expansion of the phase transition zone by increasing the concentration of non-fatty acid membrane lipids is discussed as an additional mechanism improving adaptation to temperature shifts for L. monocytogenes strains.IMPORTANCEListeria monocytogenes is a foodborne pathogen with an outstanding temperature range for growth. The ability for growth at temperatures close to the freezing point constitutes a serious contamination potential for cold stored food. The only known mechanism of the species for adaptation of membrane fluidity is modification of the membrane fatty acid composition. We were able to demonstrate that, at least for some strains, this adaptation mechanism is supported by regulation of the menaquinone concentration. The increase of this neutral membrane lipid is correlated with fluidization of the membrane under low-temperature conditions and therefore represents a fatty acid-independent mechanism for adaptation to low temperatures.


Assuntos
Membrana Celular/química , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/metabolismo , Quinonas/metabolismo , Terpenos/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Temperatura Baixa , Ácidos Graxos/metabolismo , Listeria monocytogenes/química , Listeria monocytogenes/genética , Fluidez de Membrana
19.
J Biol Chem ; 293(24): 9265-9276, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29666193

RESUMO

Listeria monocytogenes (Lm) is a facultative intracellular bacterial pathogen and the causative agent of listeriosis, a rare but fatal disease. During infection, Lm can traverse several physiological barriers; it can cross the intestine and placenta barrier and, in immunocompromised individuals, the blood-brain barrier. With the recent plethora of sequenced genomes available for Lm, it is clear that the complete repertoire of genes used by Lm to interact with its host remains to be fully explored. Recently, we focused on secreted Lm proteins because they are likely to interact with host cell components. Here, we investigated a putatively secreted protein of Lm, Lmo1656, that is present in most sequenced strains of Lm but absent in the nonpathogenic species Listeria innocua. lmo1656 gene is predicted to encode a small, positively charged protein. We show that Lmo1656 is secreted by Lm Furthermore, deletion of the lmo1656 gene (Δlmo1656) attenuates virulence in mice infected orally but not intravenously, suggesting that Lmo1656 plays a role during oral listeriosis. We identified sorting nexin 6 (SNX6), an endosomal sorting component and BAR domain-containing protein, as a host cell interactor of Lmol656. SNX6 colocalizes with WT Lm during the early steps of infection. This colocalization depends on Lmo1656, and RNAi of SNX6 impairs infection in infected tissue culture cells, suggesting that SNX6 is utilized by Lm during infection. Our results reveal that Lmo1656 is a novel secreted virulence factor of Lm that facilitates recruitment of a specific member of the sorting nexin family in the mammalian host.


Assuntos
Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Listeria monocytogenes/fisiologia , Listeriose/metabolismo , Nexinas de Classificação/metabolismo , Fatores de Virulência/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Feminino , Listeria monocytogenes/química , Listeriose/microbiologia , Camundongos Endogâmicos BALB C , Fatores de Virulência/química
20.
Food Microbiol ; 69: 18-24, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28941900

RESUMO

A central composite design was implemented to study the effect of three factors on HHP-induced L. monocytogenes inactivation in Spanish chorizo sausage, in order to increase its effectiveness: product aw (0.79-0.92), pressure intensities (349-600 MPa, at 18 °C) and holding time (0-12.53 min). Response surface methodology was implemented with backward stepwise regression to generate a model that best fitted to the experimental data. All the three factors studied significantly influenced HHP inactivation of L. monocytogenes (p < 0.05). Pathogen reductions increased as the pressure and duration of HHP treatments rose. Low values of aw seemed to exert a protective effect on L. monocytogenes and pressures below 400 MPa did not lead to significant pathogen reductions. The model was validated with independent published data. Accuracy and bias factors were also determined to evaluate the performance of the developed model, which was considered acceptable for prediction purposes. The model generated represents a mathematical tool that will help food manufacturers to improve the efficacy of HHP processing of chorizo sausage and observe the regulatory authority's specifications regarding L. monocytogenes levels while maintaining food safety.


Assuntos
Contaminação de Alimentos/prevenção & controle , Manipulação de Alimentos/métodos , Conservação de Alimentos/métodos , Listeria monocytogenes/crescimento & desenvolvimento , Produtos da Carne/microbiologia , Animais , Contaminação de Alimentos/análise , Manipulação de Alimentos/instrumentação , Conservação de Alimentos/instrumentação , Inocuidade dos Alimentos , Pressão Hidrostática , Listeria monocytogenes/química , Modelos Biológicos , Suínos
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