scholarly journals Modified techniques in quantification of intracellular Listeria monocytogenes in vitro infection

2020 ◽  
Vol 17 (4) ◽  
pp. 767-774
Author(s):  
Tran Thanh Thao ◽  
Nguyen Thi Ha
Keyword(s):  
Listeria Monocytogenes ◽  
Cell Biology ◽  
Large Scale ◽  
Secondary Infection ◽  
Host Cells ◽  
Infection Period ◽  
Colony Forming Unit ◽  
Infection Assay ◽  
Raw264.7 Macrophages

The demand for reliable methods for the quantification of intracellular bacteria is growing. Among modern methods such as PCR and flow cytometry, traditional methods including colony forming unit assay and immune-fluorescence are still the two most commonly techniques worldwide. In colony forming unit assay, there are variations among publications, making data results inconsistent across studies. The aim of this paper is to evaluate available techniques and develop improved protocols for the quantification of intracellular Listeria monocytogenes (LM) in vitro infection assay. This study has suggested different uptake time for phagocytic and non-phagocytic cells. Specifically, uptake time was determined at 0.5 hour after infection for RAW264.7 macrophages and 2 hours for L929 fibroblast host cells. To efficiently remove extracellular bacteria during infection period, gentamicin at high and low concentrations was used during the infection assay. High concentration of gentamicin was used to kill extracellular bacteria while low concentration of gentamicin was used to prevent secondary infection of host cells during the infection period. To obtain a more accurate number of alive LM from a large scale experiment, phosphate-buffered saline/PBS should be used rather than mili-Q (mQ) water to lyse the host cell as mQ water can kill additional bacteria unexpectedly. In immune-fluorescence, LM can be visualized by using either the LM expressing green fluorescence protein (GFP) or antibody against LM. To observed GFP signal, cells should be fixed with paraformaldehyde as methanol will rapidly dim the GFP signal. Findings from this study will benefit researchers engaged in both basic cell biology and infectious diseases.

Cellular and immunological basis of the host-parasite relationship during infection withNeospora caninum

Parasitology ◽  
2006 ◽  
Vol 133 (3) ◽  
pp. 261-278 ◽  
Author(s):  
A. HEMPHILL ◽  
N. VONLAUFEN ◽  
A. NAGULESWARAN
Keyword(s):  
Host Cell ◽  
Cell Biology ◽  
Neospora Caninum ◽  
Host Cells ◽  
Term Survival ◽  
Prevention Of Infection ◽  
Parasite Relationship ◽  
Potential Applications

Neospora caninumis an apicomplexan parasite that is closely related toToxoplasma gondii, the causative agent of toxoplasmosis in humans and domestic animals. However, in contrast toT. gondii, N. caninumrepresents a major cause of abortion in cattle, pointing towards distinct differences in the biology of these two species. There are 3 distinct key features that represent potential targets for prevention of infection or intervention against disease caused byN. caninum. Firstly, tachyzoites are capable of infecting a large variety of host cellsin vitroandin vivo. Secondly, the parasite exploits its ability to respond to alterations in living conditions by converting into another stage (tachyzoite-to-bradyzoite orvice versa). Thirdly, by analogy withT. gondii, this parasite has evolved mechanisms that modulate its host cells according to its own requirements, and these must, especially in the case of the bradyzoite stage, involve mechanisms that ensure long-term survival of not only the parasite but also of the host cell. In order to elucidate the molecular and cellular bases of these important features ofN. caninum, cell culture-based approaches and laboratory animal models are being exploited. In this review, we will summarize the current achievements related to host cell and parasite cell biology, and will discuss potential applications for prevention of infection and/or disease by reviewing corresponding work performed in murine laboratory infection models and in cattle.

scholarly journals Expression of Truncated Internalin A Is Involved in Impaired Internalization of Some Listeria monocytogenes Isolates Carried Asymptomatically by Humans

2003 ◽  
Vol 71 (3) ◽  
pp. 1217-1224 ◽  
Author(s):  
Maïwenn Olier ◽  
Fabrice Pierre ◽  
Sandrine Rousseaux ◽  
Jean-Paul Lemaître ◽  
André Rousset ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Surface Protein ◽  
Human Infection ◽  
Host Cells ◽  
Genetic Lineage ◽  
Chick Embryos ◽  
Asymptomatic Carriage ◽  
High Degree ◽  
Internalin A

ABSTRACT Fourteen human carriage Listeria monocytogenes isolates were compared to sporadic and epidemic-associated human strains in order to ascertain the pathogenic behavior of these unrecognized asymptomatic strains. Experimental infection of 14-day-old chick embryos revealed that the majority of the carriage strains were attenuated for virulence. Of the 10 attenuated carriage strains, 5 were affected in their invasion capacities in vitro. Western blot analysis with antibody directed against InlA, the surface protein implicated in the internalization in host cells, allowed correlation between the ability of the carriage strains to enter Caco-2 cells and InlA expression. Indeed, these five carriage strains produced truncated forms of InlA. Four of the five truncated forms of InlA had an apparent molecular mass of 47 kDa. In order to assess the existence of a genetic lineage, partial sequences of inlA gene of these four strains were compared and revealed that they had a high degree of sequence conservation at the gene (99.86%) and amino acid (100%) levels. Comparison of their nucleotide sequences with that of the corresponding segment of inlA from EGD-e and Scott A strains, taken as epidemic references, showed more divergence. Taken together, these observations suggest the presence of specific traits that characterize L. monocytogenes strains isolated during asymptomatic carriage. Some of these traits could provide some explanations about the determinants that make them unable to cause systemic human infection.

scholarly journals Detoxification of methylglyoxal by the glyoxalase system is required for glutathione availability and virulence activation in Listeria monocytogenes

2021 ◽  
Vol 17 (8) ◽  
pp. e1009819
Author(s):  
Andrea Anaya-Sanchez ◽  
Ying Feng ◽  
John C. Berude ◽  
Daniel A. Portnoy
Keyword(s):  
Listeria Monocytogenes ◽  
Transcriptional Activation ◽  
Protein Glycation ◽  
Host Cells ◽  
Intracellular Pathogen ◽  
Cell Monolayers ◽  
Food Borne ◽  
Virulence Regulator ◽  
Allosteric Activator

Listeria monocytogenes is a Gram-positive, food-borne pathogen that lives a biphasic lifestyle, cycling between the environment and as a facultative intracellular pathogen of mammals. Upon entry into host cells, L. monocytogenes upregulates expression of glutathione synthase (GshF) and its product, glutathione (GSH), which is an allosteric activator of the master virulence regulator PrfA. Although gshF mutants are highly attenuated for virulence in mice and form very small plaques in host cell monolayers, these virulence defects can be fully rescued by mutations that lock PrfA in its active conformation, referred to as PrfA*. While PrfA activation can be recapitulated in vitro by the addition of reducing agents, the precise biological cue(s) experienced by L. monocytogenes that lead to PrfA activation are not known. Here we performed a genetic screen to identify additional small-plaque mutants that were rescued by PrfA* and identified gloA, which encodes glyoxalase A, a component of a GSH-dependent methylglyoxal (MG) detoxification system. MG is a toxic byproduct of metabolism produced by both the host and pathogen, which if accumulated, causes DNA damage and protein glycation. As a facultative intracellular pathogen, L. monocytogenes must protect itself from MG produced by its own metabolic processes and that of its host. We report that gloA mutants grow normally in broth, are sensitive to exogenous MG and severely attenuated upon IV infection in mice, but are fully rescued for virulence in a PrfA* background. We demonstrate that transcriptional activation of gshF increased upon MG challenge in vitro, and while this resulted in higher levels of GSH for wild-type L. monocytogenes, the glyoxalase mutants had decreased levels of GSH, presumably due to the accumulation of the GSH-MG hemithioacetal adduct. These data suggest that MG acts as a host cue that leads to GSH production and activation of PrfA.

scholarly journals Cell-based meat: the need to assess holistically

2020 ◽  
Vol 98 (8) ◽  
Author(s):  
Cameron Faustman ◽  
Deb Hamernik ◽  
Michael Looper ◽  
Steven A Zinn
Keyword(s):  
Knowledge Base ◽  
Muscle Tissue ◽  
Manufacturing Process ◽  
Cell Biology ◽  
Large Scale ◽  
New Product ◽  
Subsequent Research ◽  
Potential Alternative ◽  
Proof Of Principle

Abstract Proof-of-principle for large-scale engineering of edible muscle tissue, in vitro, was established with the product’s introduction in 2013. Subsequent research and commentary on the potential for cell-based meat to be a viable food option and potential alternative to conventional meat have been significant. While some of this has focused on the biology and engineering required to optimize the manufacturing process, a majority of debate has focused on cultural, environmental, and regulatory considerations. Animal scientists and others with expertise in muscle and cell biology, physiology, and meat science have contributed to the knowledge base that has made cell-based meat possible and will continue to have a role in the future of the new product. Importantly, the successful introduction of cell-based meat that looks and tastes like conventional meat at a comparable price has the potential to displace and/or complement conventional meat in the marketplace.

scholarly journals Comparison of Widely Used Listeria monocytogenes Strains EGD, 10403S, and EGD-e Highlights Genomic Differences Underlying Variations in Pathogenicity

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
Author(s):  
Christophe Bécavin ◽  
Christiane Bouchier ◽  
Pierre Lechat ◽  
Cristel Archambaud ◽  
Sophie Creno ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Cell Biology ◽  
Virulence Genes ◽  
Constitutive Expression ◽  
Nucleotide Polymorphisms ◽  
Pathogenic Potential ◽  
Bacterial Gene ◽  
Content Type

ABSTRACTFor nearly 3 decades, listeriologists and immunologists have used mainly three strains of the same serovar (1/2a) to analyze the virulence of the bacterial pathogenListeria monocytogenes. The genomes of two of these strains, EGD-e and 10403S, were released in 2001 and 2008, respectively. Here we report the genome sequence of the third reference strain, EGD, and extensive genomic and phenotypic comparisons of the three strains. Strikingly, EGD-e is genetically highly distinct from EGD (29,016 single nucleotide polymorphisms [SNPs]) and 10403S (30,296 SNPs), and is more related to serovar 1/2c than 1/2a strains. We also found that while EGD and 10403S strains are genetically very close (317 SNPs), EGD has a point mutation in the transcriptional regulator PrfA (PrfA*), leading to constitutive expression of several major virulence genes. We generated an EGD-e PrfA* mutant and showed that EGD behaves like this strainin vitro, with slower growth in broth and higher invasiveness in human cells than those of EGD-e and 10403S. In contrast, bacterial counts in blood, liver, and spleen during infection in mice revealed that EGD and 10403S are less virulent than EGD-e, which is itself less virulent than EGD-e PrfA*. Thus, constitutive expression of PrfA-regulated virulence genes does not appear to provide a significant advantage to the EGD strain during infectionin vivo, highlighting the fact thatin vitroinvasion assays are not sufficient for evaluating the pathogenic potential ofL. monocytogenesstrains. Together, our results pave the way for deciphering unexplained differences or discrepancies in experiments using differentL. monocytogenesstrains.IMPORTANCEOver the past 3 decades,Listeriahas become a model organism for host-pathogen interactions, leading to critical discoveries in a broad range of fields, including bacterial gene regulation, cell biology, and bacterial pathophysiology. Scientists studyingListeriause primarily three pathogenic strains: EGD, EGD-e, and 10403S. Despite many studies on EGD, it is the only one of the three strains whose genome has not been sequenced. Here we report the sequence of its genome and a series of important genomic and phenotypic differences between the three strains, in particular, a critical mutation in EGD’s PrfA, the main regulator ofListeriavirulence. Our results show that the three strains display differences which may play an important role in the virulence differences observed between the strains. Our findings will be of critical relevance to listeriologists and immunologists who have used or may useListeriaas a tool to study the pathophysiology of listeriosis and immune responses.

scholarly journals Production of Membrane Vesicles in Listeria monocytogenes Cultured with or without Sub-Inhibitory Concentrations of Antibiotics and Their Innate Immune Responses In Vitro

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 415
Author(s):  
Jung-Hwa Woo ◽  
Shukho Kim ◽  
Taewon Lee ◽  
Je-Chul Lee ◽  
Ji-Hyun Shin
Keyword(s):  
Listeria Monocytogenes ◽  
Inflammatory Responses ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Host Cells ◽  
Proteinase K ◽  
Tnf Α ◽  
Food Borne Illness ◽  
Proteinase K Treatment

Listeriosis is a food-borne illness caused by Listeria monocytogenes. Ampicillin (AMP) alone or in combination with gentamicin (GEN) is the first-line treatment option. Membrane vesicle (MV) production in L. monocytogenes under antibiotic stress conditions and pathologic roles of these MVs in hosts have not been reported yet. Thus, the aim of this study was to investigate the production of MVs in L. monocytogenes cultured with sub-minimum inhibitory concentrations (MICs) of AMP, GEN, or trimethoprim/sulfamethoxazole (SXT) and determine pathologic effects of these MVs in colon epithelial Caco-2 cells. L. monocytogenes cultured in tryptic soy broth with 1/2 MIC of AMP, GEN, or SXT produced 6.0, 2.9, or 1.5 times more MV particles, respectively, than bacteria cultured without antibiotics. MVs from L. monocytogenes cultured with AMP (MVAMP), GEN (MVGEN), or SXT (MVSXT) were more cytotoxic to Caco-2 cell than MVs obtained from cultivation without antibiotics (MVTSB). MVAMP induced more expression of tumor necrosis factor (TNF)-α gene than MVTSB, MVGEN and MVSXT, whereas MVTSB induced more expression of interleukin (IL)-1β and IL-8 genes than other MVs. Expression of pro-inflammatory cytokine genes by L. monocytogenes MVs was significantly inhibited by proteinase K treatment of MVs. In conclusion, antibiotic stress can trigger the biogenesis of MVs in L. monocytogenes and MVs produced by L. monocytogenes exposed to sub-MIC of AMP can induce strong pro-inflammatory responses by expressing TNF-α gene in host cells, which may contribute to the pathology of listeriosis.

scholarly journals The redox-responsive transcriptional regulator Rex represses fermentative metabolism and is required for Listeria monocytogenes pathogenesis

2021 ◽  
Vol 17 (8) ◽  
pp. e1009379
Author(s):  
Cortney R. Halsey ◽  
Rochelle C. Glover ◽  
Maureen K. Thomason ◽  
Michelle L. Reniere
Keyword(s):  
Listeria Monocytogenes ◽  
Bacterial Infections ◽  
Transcriptional Regulator ◽  
Host Cells ◽  
Bacterial Survival ◽  
Gi Tract ◽  
Fermentative Metabolism ◽  
Peripheral Organs ◽  
Redox Responsive

The Gram-positive bacterium Listeria monocytogenes is the causative agent of the foodborne disease listeriosis, one of the deadliest bacterial infections known. In order to cause disease, L. monocytogenes must properly coordinate its metabolic and virulence programs in response to rapidly changing environments within the host. However, the mechanisms by which L. monocytogenes senses and adapts to the many stressors encountered as it transits through the gastrointestinal (GI) tract and disseminates to peripheral organs are not well understood. In this study, we investigated the role of the redox-responsive transcriptional regulator Rex in L. monocytogenes growth and pathogenesis. Rex is a conserved canonical transcriptional repressor that monitors the intracellular redox state of the cell by sensing the ratio of reduced and oxidized nicotinamide adenine dinucleotides (NADH and NAD+, respectively). Here, we demonstrated that L. monocytogenes Rex represses fermentative metabolism and is therefore required for optimal growth in the presence of oxygen. We also show that in vitro, Rex represses the production of virulence factors required for survival and invasion of the GI tract, as a strain lacking rex was more resistant to acidified bile and invaded host cells better than wt. Consistent with these results, Rex was dispensable for colonizing the GI tract and disseminating to peripheral organs in an oral listeriosis model of infection. However, Rex-dependent regulation was required for colonizing the spleen and liver, and L. monocytogenes lacking the Rex repressor were nearly sterilized from the gallbladder. Taken together, these results demonstrated that Rex functions as a repressor of fermentative metabolism and suggests a role for Rex-dependent regulation in L. monocytogenes pathogenesis. Importantly, the gallbladder is the bacterial reservoir during listeriosis, and our data suggest redox sensing and Rex-dependent regulation are necessary for bacterial survival and replication in this organ.

scholarly journals Comparative Phenotypic, Molecular, and Virulence Characterization of Vibrio parahaemolyticus O3:K6 Isolates

2002 ◽  
Vol 68 (6) ◽  
pp. 2901-2909 ◽  
Author(s):  
P. S. Marie Yeung ◽  
Micaela C. Hayes ◽  
Angelo DePaola ◽  
Charles A. Kaysner ◽  
Laura Kornstein ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Epithelial Cells ◽  
Vibrio Parahaemolyticus ◽  
Large Scale ◽  
Genetic Relationships ◽  
Host Cells ◽  
Pathogenic Potential ◽  
Lactate Dehydrogenase Activity ◽  
Cytotoxicity Studies

ABSTRACT Historically, Vibrio parahaemolyticus infections have been characterized by sporadic cases caused by multiple, diverse serotypes. However, since 1996, V. parahaemolyticus serotype O3:K6 strains have been associated with several large-scale outbreaks of illness, suggesting the emergence of a “new” group of organisms with enhanced virulence. We have applied three different molecular subtyping techniques to identify an appropriate method for differentiating O3:K6 isolates from other serotypes. Pulsed-field gel electrophoresis (PFGE) following NotI digestion differentiated seven closely related subtypes among O3:K6 and related strains, which were distinct from PFGE patterns for non-O3:K6 isolates. Ribotyping and tdh sequencing were less discriminatory than PFGE, but further confirmed close genetic relationships among recent O3:K6 isolates. In vitro adherence and cytotoxicity studies with human epithelial cells showed that O3:K6 isolates exhibited statistically higher levels of adherence and cytotoxicity to host cells than non-O3:K6 isolates. Epithelial cell cytotoxicity patterns were determined with a lactate dehydrogenase release assay. At 3 h postinfection, high relative cytotoxicities (>50% maximum lactate dehydrogenase activity) were found among a greater proportion of recently isolated O3:K6 and closely related strains (75%) than among the non-O3:K6 isolates (23%). A statistically significant relationship between adherence and cytotoxicity suggests that the pathogenic potential of some isolates may be associated with increased adherence to epithelial cells. Our findings suggest that enhanced adherence and cytotoxicity may contribute to the apparent unique pathogenic potential of V. parahaemolyticus O3:K6 strains.

scholarly journals Listeria monocytogenes PrsA2 Is Required for Virulence Factor Secretion and Bacterial Viability within the Host Cell Cytosol

2010 ◽  
Vol 78 (11) ◽  
pp. 4944-4957 ◽  
Author(s):  
Francis Alonzo ◽  
Nancy E. Freitag
Keyword(s):  
Listeria Monocytogenes ◽  
Host Cell ◽  
Host Cells ◽  
Broth Culture ◽  
Cell Infection ◽  
Bacterial Viability ◽  
Cell Cytosol ◽  
Critical Requirement ◽  
Host Cell Cytosol

ABSTRACT In the course of establishing its replication niche within the cytosol of infected host cells, the facultative intracellular bacterial pathogen Listeria monocytogenes must efficiently regulate the secretion and activity of multiple virulence factors. L. monocytogenes encodes two predicted posttranslocation secretion chaperones, PrsA1 and PrsA2, and evidence suggests that PrsA2 has been specifically adapted for bacterial pathogenesis. PrsA-like chaperones have been identified in a number of Gram-positive bacteria, where they are reported to function at the bacterial membrane-cell wall interface to assist in the folding of proteins translocated across the membrane; in some cases, these proteins have been found to be essential for bacterial viability. In this study, the contributions of PrsA2 and PrsA1 to L. monocytogenes growth and protein secretion were investigated in vitro and in vivo. Neither PrsA2 nor PrsA1 was found to be essential for L. monocytogenes growth in broth culture; however, optimal bacterial viability was found to be dependent upon PrsA2 for L. monocytogenes located within the cytosol of host cells. Proteomic analyses of prsA2 mutant strains in the presence of a mutationally activated allele of the virulence regulator PrfA revealed a critical requirement for PrsA2 activity under conditions of PrfA activation, an event which normally takes place within the host cell cytosol. Despite a high degree of amino acid similarity, no detectable degree of functional overlap was observed between PrsA2 and PrsA1. Our results indicate a critical requirement for PrsA2 under conditions relevant to host cell infection.

scholarly journals A novel C-terminal mutation resulting in constitutive activation of the Listeria monocytogenes central virulence regulatory factor PrfA

Microbiology ◽  
2011 ◽  
Vol 157 (11) ◽  
pp. 3138-3149 ◽  
Author(s):  
Bobbi Xayarath ◽  
Jennifer I. Smart ◽  
Kimberly J. Mueller ◽  
Nancy E. Freitag
Keyword(s):  
Listeria Monocytogenes ◽  
Dna Binding ◽  
Binding Affinity ◽  
Mammalian Cells ◽  
Host Cells ◽  
Ecological Niches ◽  
Bacterial Gene ◽  
Constitutive Activation ◽  
Dna Binding Affinity

The environmental bacterium Listeria monocytogenes survives and replicates in a variety of diverse ecological niches that range from the soil to the cytosol of infected mammalian cells. The ability of L. monocytogenes to replicate within an infected host requires the expression of a number of secreted bacterial gene products whose expression is regulated by the transcriptional activator PrfA. PrfA becomes activated following bacterial entry into host cells; however, the mechanism by which this activation occurs remains unknown. Here we describe a novel C-terminal mutation that results in the high-level constitutive activation of PrfA and yet, in contrast with other described prfA* activation mutations, only modestly increases PrfA DNA binding affinity. L. monocytogenes strains containing the prfA P219S mutation exhibited high levels of PrfA-dependent virulence gene expression, were hyperinvasive in tissue culture models of infection, were fully motile and were hypervirulent in mice. In contrast with PrfA G145S and other mutationally activated PrfA proteins, the PrfA P219S protein readily formed homodimers and did not exhibit a dramatic increase in its DNA-binding affinity for target promoters. Interestingly, the prfA P219S mutation is located adjacent to the prfA K220 residue that has been previously reported to contribute to PrfA DNA binding activity. prfA P219S therefore appears to constitutively activate PrfA via a novel mechanism which minimally affects PrfA DNA binding in vitro.

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