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1.
PLoS Comput Biol ; 17(7): e1009140, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34292935

RESUMO

The metabolic capabilities of the species and the local environment shape the microbial interactions in a community either through the exchange of metabolic products or the competition for the resources. Cells are often arranged in close proximity to each other, creating a crowded environment that unevenly reduce the diffusion of nutrients. Herein, we investigated how the crowding conditions and metabolic variability among cells shape the dynamics of microbial communities. For this, we developed CROMICS, a spatio-temporal framework that combines techniques such as individual-based modeling, scaled particle theory, and thermodynamic flux analysis to explicitly incorporate the cell metabolism and the impact of the presence of macromolecular components on the nutrients diffusion. This framework was used to study two archetypical microbial communities (i) Escherichia coli and Salmonella enterica that cooperate with each other by exchanging metabolites, and (ii) two E. coli with different production level of extracellular polymeric substances (EPS) that compete for the same nutrients. In the mutualistic community, our results demonstrate that crowding enhanced the fitness of cooperative mutants by reducing the leakage of metabolites from the region where they are produced, avoiding the resource competition with non-cooperative cells. Moreover, we also show that E. coli EPS-secreting mutants won the competition against the non-secreting cells by creating less dense structures (i.e. increasing the spacing among the cells) that allow mutants to expand and reach regions closer to the nutrient supply point. A modest enhancement of the relative fitness of EPS-secreting cells over the non-secreting ones were found when the crowding effect was taken into account in the simulations. The emergence of cell-cell interactions and the intracellular conflicts arising from the trade-off between growth and the secretion of metabolites or EPS could provide a local competitive advantage to one species, either by supplying more cross-feeding metabolites or by creating a less dense neighborhood.


Assuntos
Biologia Computacional/métodos , Interações Microbianas/fisiologia , Microbiota/fisiologia , Modelos Biológicos , Escherichia coli/metabolismo , Escherichia coli/fisiologia , Salmonella enterica/metabolismo , Salmonella enterica/fisiologia , Análise Espaço-Temporal
2.
Sci Rep ; 11(1): 15296, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315964

RESUMO

The microbial diversity and quantitative dynamics during the insect's development stages constitute recently developed putative tools in forensic and medical studies. Meanwhile, little is known on the role of insects in spreading foodborne pathogenic bacteria and on the impact of these pathogens on the overall insects and feeding substrate microbiome composition. Here, we provide the first characterization of the bacterial communities harbored in adult and immature stages of Lucilia sericata, one of the first colonizers of decomposed human remains, in the presence of the foodborne pathogen Salmonella enterica using 16S rRNA Illumina sequencing and qPCR. The pathogen transmission from the wild adults to the second generation was observed, with a 101.25× quantitative increase. The microbial patterns from both insect and liver samples were not influenced by the artificial introduction of this pathogenic foodborne bacteria, being dominated by Firmicutes and Proteobacteria. Overall, our results provided a first detailed overview of the insect and decomposed substrate microbiome in the presence of a human pathogen, advancing the knowledge on the role of microbes as postmortem interval estimators and the transmission of pathogenic bacteria.


Assuntos
Dípteros/microbiologia , Comportamento Alimentar , Microbiota , Salmonella enterica/metabolismo , Animais
3.
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
4.
Commun Biol ; 4(1): 646, 2021 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-34059784

RESUMO

The flagellar protein export apparatus switches substrate specificity from hook-type to filament-type upon hook assembly completion, thereby initiating filament assembly at the hook tip. The C-terminal cytoplasmic domain of FlhA (FlhAC) serves as a docking platform for flagellar chaperones in complex with their cognate filament-type substrates. Interactions of the flexible linker of FlhA (FlhAL) with its nearest FlhAC subunit in the FlhAC ring is required for the substrate specificity switching. To address how FlhAL brings the order to flagellar assembly, we analyzed the flhA(E351A/W354A/D356A) ΔflgM mutant and found that this triple mutation in FlhAL increased the secretion level of hook protein by 5-fold, thereby increasing hook length. The crystal structure of FlhAC(E351A/D356A) showed that FlhAL bound to the chaperone-binding site of its neighboring subunit. We propose that the interaction of FlhAL with the chaperon-binding site of FlhAC suppresses filament-type protein export and facilitates hook-type protein export during hook assembly.


Assuntos
Proteínas de Bactérias/metabolismo , Flagelos/metabolismo , Proteínas de Membrana/metabolismo , Salmonella enterica/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Flagelos/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Chaperonas Moleculares/genética , Mutação/genética , Ligação Proteica , Transporte Proteico/genética , Especificidade por Substrato
5.
Int J Food Microbiol ; 351: 109268, 2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34098467

RESUMO

The increase in multidrug-resistant Salmonella enterica and its spread from food to humans are considered a serious public health concern worldwide. Little is currently known about the prevalence of extended-spectrum ß-lactamase (ESBL)-producing S. enterica in fish in Africa. Therefore, this study aimed to investigate the existence of ESBL-producing S. enterica in retail fish in Egypt. In total, 200 fish samples were collected randomly from various retail fish markets in Egypt. S. enterica were detected in 19 (9.5%; 95% CI: 5.8-14.4) of the fish samples analyzed. Of the 19 non-repetitive S. enterica isolates, 18 were serologically categorized into eight S. enterica serovars and a non-typable serovar. All 19 S. enterica isolates (100%) showed multidrug-resistant phenotypes to at least three classes of antimicrobials, and 11 (57.9%) exhibited an ESBL-resistant phenotype and harbored at least one ESBL-encoding gene. The ESBL-producing S. enterica serovars were as follows: Kentucky (3 isolates; 15.8%), Enteritidis (2 isolates; 10.5%), Typhimurium (2 isolates; 10.5%), and 1 isolate (5.3%) each of Infantis, Virchow, Paratyphi B, and Senftenberg. The identified ß-lactamase-encoding genes included ESBL-encoding genes blaCTX-M-3, blaCTX-M-14, blaCTX-M-15, blaSHV-1, blaSHV-2 and blaSHV-12; the AmpC ß-lactamase-encoding gene blaCMY-2; and the narrow-spectrum ß-lactamase-encoding genes blaTEM-1 and blaOXA-1. All S. enterica isolates were negative for carbapenemase-encoding genes. Molecular analysis of plasmid transferability and replicon typing revealed that most plasmids (with ß-lactamase-encoding genes) were transferrable, and the most common incompatibility groups were IncI1, IncA/C, IncHI1, and IncN. To the best of our knowledge, this is the first report for molecular characterization of ESBL-producing S. enterica in fish in Egypt. The occurrence of ESBL-producing S. enterica in retail fish constitutes a potential public health threat with the possibility of transmission of these strains with resistance genes to humans. Such transmission would exacerbate the resistance to an important class of antibiotics commonly used in hospitals to treat typhoid and non-typhoidal Salmonella infections.


Assuntos
Peixes/microbiologia , Salmonelose Animal/microbiologia , Salmonella enterica/metabolismo , beta-Lactamases/metabolismo , Animais , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Egito/epidemiologia , Humanos , Plasmídeos/genética , Prevalência , Saúde Pública , Salmonelose Animal/epidemiologia , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/genética , Salmonella enterica/isolamento & purificação , beta-Lactamases/genética
6.
Int J Mol Sci ; 22(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34066237

RESUMO

CsgA is an aggregating protein from bacterial biofilms, representing a class of functional amyloids. Its amyloid propensity is defined by five fragments (R1-R5) of the sequence, representing non-perfect repeats. Gate-keeper amino acid residues, specific to each fragment, define the fragment's propensity for self-aggregation and aggregating characteristics of the whole protein. We study the self-aggregation and secondary structures of the repeat fragments of Salmonella enterica and Escherichia coli and comparatively analyze their potential effects on these proteins in a bacterial biofilm. Using bioinformatics predictors, ATR-FTIR and FT-Raman spectroscopy techniques, circular dichroism, and transmission electron microscopy, we confirmed self-aggregation of R1, R3, R5 fragments, as previously reported for Escherichia coli, however, with different temporal characteristics for each species. We also observed aggregation propensities of R4 fragment of Salmonella enterica that is different than that of Escherichia coli. Our studies showed that amyloid structures of CsgA repeats are more easily formed and more durable in Salmonella enterica than those in Escherichia coli.


Assuntos
Amiloide/química , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Salmonella enterica/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Agregados Proteicos , Conformação Proteica , Salmonella enterica/genética , Salmonella enterica/crescimento & desenvolvimento , Homologia de Sequência
7.
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
8.
Commun Biol ; 4(1): 464, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846530

RESUMO

The FliH2FliI complex is thought to pilot flagellar subunit proteins from the cytoplasm to the transmembrane export gate complex for flagellar assembly in Salmonella enterica. FliI also forms a homo-hexamer to hydrolyze ATP, thereby activating the export gate complex to become an active protein transporter. However, it remains unknown how this activation occurs. Here we report the role of a positively charged cluster formed by Arg-26, Arg-27, Arg-33, Arg-76 and Arg-93 of FliI in flagellar protein export. We show that Arg-33 and Arg-76 are involved in FliI ring formation and that the fliI(R26A/R27A/R33A/R76A/R93A) mutant requires the presence of FliH to fully exert its export function. We observed that gain-of-function mutations in FlhB increased the probability of substrate entry into the export gate complex, thereby restoring the export function of the ∆fliH fliI(R26A/R27A/R33A/R76A/R93A) mutant. We suggest that the positive charge cluster of FliI is responsible not only for well-regulated hexamer assembly but also for substrate entry into the gate complex.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/genética , Flagelos/metabolismo , Proteínas de Membrana/genética , Salmonella enterica/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Filogenia , Transporte Proteico , Salmonella enterica/metabolismo , Alinhamento de Sequência
9.
mBio ; 12(2)2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33849975

RESUMO

Methylation of cytosine in DNA at position C5 increases the rate of C→T mutations in bacteria and eukaryotes. Methylation at the N4 position, employed by some restriction-modification systems, is not known to increase the mutation rate. Here, I report that a Salmonella enterica Type III restriction-modification system that includes a cytosine-N4 methyltransferase causes an enormous increase in the rate of mutation of the methylated cytosines, which occur at the overlined C in the motif CACC̅GT Mutations consist mainly of C→A transversions, the rate of which is increased ∼500-fold by the restriction-modification system. The rate of C→T transitions is also increased and somewhat exceeds that at C5-methylated cytosines in Dcm sites. Two other Salmonella N4 methyltransferases investigated do not have such dramatic effects, although in one case there is a modest increase in C→A mutations along with an increase in C→T mutations. The sensitivity of the C→A rate to orientation with respect to both DNA replication and transcription is higher at hypermutable sites than at other cytosines, suggesting a fundamental mechanistic difference between hypermutation and ordinary mutation.IMPORTANCE Mutation produces the raw material for adaptive evolution but also imposes a burden because most mutations are deleterious. The rate of mutation at a particular site is affected by a variety of factors. In both prokaryotes and eukaryotes, methylation of C at the C5 position, a naturally occurring DNA modification, greatly increases the rate of C→T mutation. A distinct C modification that occurs in prokaryotes, methylation at N4, is not known to increase mutation rate. Here, I report that a bacterial restriction-modification system, found in some Salmonella bacteria, increases the rate of C→A mutation by a factor of 500 at sites that it methylates at N4. This rate increase is much greater than that caused by C5 methylation. Although fewer than 1 in 1,600 positions analyzed are methylation sites, over 10% of all mutations occur at these sites. Like other examples of extremely high mutation rate, whether naturally occurring or the result of laboratory mutation, this phenomenon may shed light on the mechanism of mutation in general.


Assuntos
Citosina/metabolismo , Metilação de DNA , Metiltransferases/metabolismo , Mutação , Salmonella enterica/genética , Sequência de Bases , Salmonella enterica/enzimologia , Salmonella enterica/metabolismo , Especificidade por Substrato
10.
Nucleic Acids Res ; 49(8): 4705-4724, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33849070

RESUMO

Antisense peptide nucleic acids (PNAs) inhibiting mRNAs of essential genes provide a straight-forward way to repurpose our knowledge of bacterial regulatory RNAs for development of programmable species-specific antibiotics. While there is ample proof of PNA efficacy, their target selectivity and impact on bacterial physiology are poorly understood. Moreover, while antibacterial PNAs are typically designed to block mRNA translation, effects on target mRNA levels are not well-investigated. Here, we pioneer the use of global RNA-seq analysis to decipher PNA activity in a transcriptome-wide manner. We find that PNA-based antisense oligomer conjugates robustly decrease mRNA levels of the widely-used target gene, acpP, in Salmonella enterica, with limited off-target effects. Systematic analysis of several different PNA-carrier peptides attached not only shows different bactericidal efficiency, but also activation of stress pathways. In particular, KFF-, RXR- and Tat-PNA conjugates especially induce the PhoP/Q response, whereas the latter two additionally trigger several distinct pathways. We show that constitutive activation of the PhoP/Q response can lead to Tat-PNA resistance, illustrating the utility of RNA-seq for understanding PNA antibacterial activity. In sum, our study establishes an experimental framework for the design and assessment of PNA antimicrobials in the long-term quest to use these for precision editing of microbiota.


Assuntos
Oligonucleotídeos Antissenso/farmacologia , Ácidos Nucleicos Peptídicos/farmacologia , Peptídeos/química , RNA Mensageiro/metabolismo , Salmonella enterica/efeitos dos fármacos , Estresse Fisiológico/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Farmacorresistência Bacteriana/genética , Testes de Sensibilidade Microbiana , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Ácidos Nucleicos Peptídicos/metabolismo , Peptídeos/metabolismo , Peptídeos/farmacologia , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA-Seq , Salmonella enterica/genética , Salmonella enterica/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologia
11.
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
12.
Nat Commun ; 12(1): 1546, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33750771

RESUMO

Many bacterial pathogens rely on virulent type III secretion systems (T3SSs) or injectisomes to translocate effector proteins in order to establish infection. The central component of the injectisome is the needle complex which assembles a continuous conduit crossing the bacterial envelope and the host cell membrane to mediate effector protein translocation. However, the molecular principles underlying type III secretion remain elusive. Here, we report a structure of an active Salmonella enterica serovar Typhimurium needle complex engaged with the effector protein SptP in two functional states, revealing the complete 800Å-long secretion conduit and unraveling the critical role of the export apparatus (EA) subcomplex in type III secretion. Unfolded substrates enter the EA through a hydrophilic constriction formed by SpaQ proteins, which enables side chain-independent substrate transport. Above, a methionine gasket formed by SpaP proteins functions as a gate that dilates to accommodate substrates while preventing leaky pore formation. Following gate penetration, a moveable SpaR loop first folds up to then support substrate transport. Together, these findings establish the molecular basis for substrate translocation through T3SSs and improve our understanding of bacterial pathogenicity and motility.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Transporte Proteico/fisiologia , Salmonella typhimurium/metabolismo , Sistemas de Secreção Tipo III/química , Sistemas de Secreção Tipo III/metabolismo , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Microscopia Crioeletrônica , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Salmonella enterica/metabolismo , Salmonella typhimurium/genética , Sistemas de Secreção Tipo III/genética
13.
J Dairy Sci ; 104(1): 198-210, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33189289

RESUMO

While the increase in thermal resistance of microorganisms at reduced water activity is demonstrated for low-moisture food products, the effect of storage time on the thermal resistance of microorganisms in low-moisture foods is not well established. As low-moisture foods are stored for long periods and are used as ingredients, cross-contamination can occur at any time period before the lethality step. Therefore, this study was designed to investigate the effect of storage time (30, 60, and 90 d) on the thermal resistance of Salmonella and Enterococcus faecium NRRL B-2354 in milk powders at a low water activity of 0.10 (conservative level). In this study, 2 milk powders, whole milk powder (WMP) and nonfat dry milk (NFDM), were inoculated with a 5-serotype Salmonella cocktail or E. faecium and equilibrated to a water activity of 0.10. The thermal resistance of Salmonella and E. faecium in WMP and NFDM were determined at different storage times (30, 60, and 90 d) at 85°C. The storage time had no effect on the thermal inactivation kinetics of Salmonella within 90 d of storage at 85°C. In the second part of this study, isothermal treatments were also conducted at higher temperatures (90 and 95°C) to evaluate the suitability of E. faecium as a surrogate for Salmonella in milk powders. The D-values of Salmonella in WMP with 30 d of storage at 85, 90, and 95°C were 7.98, 3.35, and 1.68 min. The corresponding values for E. faecium were 16.96, 7.90, and 4.16 min. Higher D-values of E. faecium indicates that it is a conservative surrogate. Similar results were found for NFDM. In general, D-values of both microorganisms are slightly higher in NFDM than WMP. Two primary models (log-linear and Weibull) were compared for their goodness-of-fit. The Weibull model was found to be more appropriate than the log-linear model. This study provides valuable information for establishing process validation for the pasteurization of milk powders.


Assuntos
Enterococcus faecium/metabolismo , Microbiologia de Alimentos , Leite/microbiologia , Salmonella enterica/metabolismo , Animais , Cinética , Leite/química , Pasteurização , Pós , Temperatura
14.
Methods Mol Biol ; 2182: 67-82, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32894488

RESUMO

Salmonella enterica is an invasive, facultative intracellular pathogen with a highly sophisticated intracellular lifestyle. Invasion and intracellular proliferation are dependent on the translocation of effector proteins by two distinct type III secretion systems (T3SS) into the host cell. To unravel host-pathogen interactions, dedicated imaging techniques visualizing Salmonella effector proteins during the infection are essential. Here we describe a new approach utilizing self-labeling enzyme (SLE) tags as a universal labeling tool for tracing effector proteins. This method is able to resolve the temporal and spatial dynamics of effector proteins in living cells. The method is applicable to conventional confocal fluorescence microscopy, but also to tracking and localization microscopy (TALM), and super-resolution microscopy (SRM) of single molecules, allowing the visualization of effector proteins beyond the optical diffraction limit.


Assuntos
Proteínas de Bactérias/metabolismo , Salmonella enterica/metabolismo , Linhagem Celular Tumoral , Células HeLa , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Transporte Proteico/fisiologia , Sistemas de Secreção Tipo III/metabolismo
15.
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
16.
Int J Mol Sci ; 21(22)2020 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-33187094

RESUMO

Many bacteria possess multiple chemosensory pathways that are composed of homologous signaling proteins. These pathways appear to be functionally insulated from each other, but little information is available on the corresponding molecular basis. We report here a novel mechanism that contributes to pathway insulation. We show that, of the four CheB paralogs of Pseudomonas aeruginosa PAO1, only CheB2 recognizes a pentapeptide at the C-terminal extension of the McpB (Aer2) chemoreceptor (KD = 93 µM). McpB is the sole chemoreceptor that stimulates the Che2 pathway, and CheB2 is the methylesterase of this pathway. Pectobacterium atrosepticum SCRI1043 has a single CheB, CheB_Pec, and 19 of its 36 chemoreceptors contain a C-terminal pentapeptide. The deletion of cheB_Pec abolished chemotaxis, but, surprisingly, none of the pentapeptides bound to CheB_Pec. To determine the corresponding structural basis, we solved the 3D structure of CheB_Pec. Its structure aligned well with that of the pentapeptide-dependent enzyme from Salmonella enterica. However, no electron density was observed in the CheB_Pec region corresponding to the pentapeptide-binding site in the Escherichia coli CheB. We hypothesize that this structural disorder is associated with the failure to bind pentapeptides. Combined data show that CheB methylesterases can be divided into pentapeptide-dependent and independent enzymes.


Assuntos
Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/fisiologia , Células Quimiorreceptoras/metabolismo , Quimiotaxia/fisiologia , Escherichia coli/metabolismo , Metiltransferases/metabolismo , Pectobacterium/metabolismo , Pseudomonas aeruginosa/metabolismo , Salmonella enterica/metabolismo , Transdução de Sinais/fisiologia
17.
J Bacteriol ; 202(22)2020 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-32900833

RESUMO

YggS (COG0325) is a pyridoxal 5'-phosphate (PLP)-binding protein proposed to be involved in homeostasis of B6 vitamers. In Salmonella enterica, lack of yggS resulted in phenotypes that were distinct and others that were similar to those of a yggS mutant of Escherichia coli Like other organisms, yggS mutants of S. enterica accumulate endogenous pyridoxine 5'-phosphate (PNP). Data herein show that strains lacking YggS accumulated ∼10-fold more PLP in growth medium than a parental strain. The deoxyxylulose 5-phosphate-dependent biosynthetic pathway for PLP and the PNP/pyridoxamine 5'-phosphate (PMP) oxidase credited with interconverting B6 vitamers were replaced with a single PLP synthase from Saccharomyces cerevisiae The impact of a yggS deletion on the intracellular and extracellular levels of B6 vitamers in this restructured strain supported a role for PdxH in PLP homeostasis and led to a general model for YggS function in PLP-PMP cycling. Our findings uncovered broader consequences of a yggS mutation than previously reported and suggest that the accumulation of PNP is not a direct effect of lacking YggS but rather a downstream consequence.IMPORTANCE Pyridoxal 5'-phosphate (PLP) is an essential cofactor for enzymes in all domains of life. Perturbations in PLP or B6 vitamer content can be detrimental, notably causing B6-dependent epilepsy in humans. YggS homologs are broadly conserved and have been implicated in altered levels of B6 vitamers in multiple organisms. The biochemical activity of YggS, expected to be conserved across domains, is not yet known. Herein, a simplified heterologous pathway minimized metabolic variables and allowed the dissection of this system to generate new metabolic knowledge that will be relevant to understanding YggS.


Assuntos
Proteínas de Bactérias/metabolismo , Carbono-Nitrogênio Liases/metabolismo , Proteínas de Transporte/metabolismo , Fosfato de Piridoxal/análogos & derivados , Proteínas de Saccharomyces cerevisiae/metabolismo , Salmonella enterica/metabolismo , Vitamina B 6/metabolismo , Proteínas de Bactérias/genética , Vias Biossintéticas/genética , Carbono-Nitrogênio Liases/genética , Proteínas de Transporte/genética , Homeostase , Mutação , Fosfato de Piridoxal/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Salmonella enterica/genética
18.
Nucleic Acids Res ; 48(21): 11857-11867, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-32954419

RESUMO

Expression of Salmonella enterica loci harboring undermethylated GATC sites at promoters or regulatory regions was monitored by single cell analysis. Cell-to-cell differences in expression were detected in ten such loci (carA, dgoR, holA, nanA, ssaN, STM1290, STM3276, STM5308, gtr and opvAB), with concomitant formation of ON and OFF subpopulations. The ON and OFF subpopulation sizes varied depending on the growth conditions, suggesting that the population structure can be modulated by environmental control. All the loci under study except STM5308 displayed altered patterns of expression in strains lacking or overproducing Dam methylase, thereby confirming control by Dam methylation. Bioinformatic analysis identified potential binding sites for transcription factors OxyR, CRP and Fur, and analysis of expression in mutant backgrounds confirmed transcriptional control by one or more of such factors. Surveys of gene expression in pairwise combinations of Dam methylation-dependent loci revealed independent switching, thus predicting the formation of a high number of cell variants. This study expands the list of S. enterica loci under transcriptional control by Dam methylation, and underscores the relevance of the DNA adenine methylome as a source of phenotypic heterogeneity.


Assuntos
Proteínas de Bactérias/genética , Metilação de DNA , Regulação Bacteriana da Expressão Gênica , Salmonella enterica/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , Fatores de Transcrição/genética , Adenina/metabolismo , Proteínas de Bactérias/metabolismo , Biologia Computacional/métodos , Heterogeneidade Genética , Loci Gênicos , Genótipo , Fenótipo , Salmonella enterica/metabolismo , Análise de Célula Única/métodos , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética
19.
J Microbiol Biotechnol ; 30(10): 1516-1524, 2020 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-32807755

RESUMO

Climate change is expected to affect not only availability and quality of water, the valuable resource of human life on Earth, but also ultimately public health issue. A six-year monitoring (total 20 times) of Escherichia coli O157, Salmonella enterica, Legionella pneumophila, Shigella sonnei, Campylobacter jejuni, and Vibrio cholerae was conducted at five raw water sampling sites including two lakes, Hyundo region (Geum River) and two locations near Water Intake Plants of Han River (Guui region) and Nakdong River (Moolgeum region). A total 100 samples of 40 L water were tested. Most of the targeted bacteria were found in 77% of the samples and at least one of the target bacteria was detected (65%). Among all the detected bacteria, E. coli O157 were the most prevalent with a detection frequency of 22%, while S. sonnei was the least prevalent with a detection frequency of 2%. Nearly all the bacteria (except for S. sonnei) were present in samples from Lake Soyang, Lake Juam, and the Moolgeum region in Nakdong River, while C. jejuni was detected in those from the Guui region in Han River. During the six-year sampling period, individual targeted noxious bacteria in water samples exhibited seasonal patterns in their occurrence that were different from the indicator bacteria levels in the water samples. The fact that they were detected in the five Korea's representative water environments make it necessary to establish the chemical and biological analysis for noxious bacteria and sophisticated management systems in response to climate change.


Assuntos
Microbiologia da Água , Recursos Hídricos , Campylobacter jejuni/isolamento & purificação , Campylobacter jejuni/metabolismo , Fenômenos Químicos , Escherichia coli O157/isolamento & purificação , Escherichia coli O157/metabolismo , Legionella pneumophila/isolamento & purificação , Legionella pneumophila/metabolismo , República da Coreia , Rios/microbiologia , Salmonella enterica/isolamento & purificação , Salmonella enterica/metabolismo , Shigella sonnei/isolamento & purificação , Shigella sonnei/metabolismo , Vibrio cholerae/isolamento & purificação , Vibrio cholerae/metabolismo , Qualidade da Água
20.
J Microbiol Biotechnol ; 30(11): 1729-1738, 2020 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-32830190

RESUMO

Salmonellosis is a form of gastroenteritis caused by Salmonella infection. The main transmission route of salmonellosis has been identified as poorly cooked meat and poultry products contaminated with Salmonella. However, in recent years, the number of outbreaks attributed to contaminated raw produce has increased dramatically. To understand how Salmonella adapts to produce, transcriptomic analysis was conducted on Salmonella enterica serovar Virchow exposed to fresh-cut radish greens. Considering the different Salmonella lifestyles in contact with fresh produce, such as motile and sessile lifestyles, total RNA was extracted from planktonic and epiphytic cells separately. Transcriptomic analysis of S. Virchow cells revealed different transcription profiles between lifestyles. During bacterial adaptation to fresh-cut radish greens, planktonic cells were likely to shift toward anaerobic metabolism, exploiting nitrate as an electron acceptor of anaerobic respiration, and utilizing cobalamin as a cofactor for coupled metabolic pathways. Meanwhile, Salmonella cells adhering to plant surfaces showed coordinated upregulation in genes associated with translation and ribosomal biogenesis, indicating dramatic cellular reprogramming in response to environmental changes. In accordance with the extensive translational response, epiphytic cells showed an increase in the transcription of genes that are important for bacterial motility, nucleotide transporter/metabolism, cell envelope biogenesis, and defense mechanisms. Intriguingly, Salmonella pathogenicity island (SPI)-1 and SPI-2 displayed up- and downregulation, respectively, regardless of lifestyles in contact with the radish greens, suggesting altered Salmonella virulence during adaptation to plant environments. This study provides molecular insights into Salmonella adaptation to plants as an alternative environmental reservoir.


Assuntos
Contaminação de Alimentos , Salmonella enterica/genética , Salmonella enterica/metabolismo , Transcriptoma , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Ilhas Genômicas/genética , Estilo de Vida , Raphanus/microbiologia , Infecções por Salmonella , Análise de Sequência de RNA , Virulência/genética
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