Lack of PPARγ in myeloid cells confers resistance to Listeria monocytogenes infection

The peroxisomal proliferator-activated receptor γ (PPARγ) is a nuclear receptor that controls inflammation and immunity. Innate immune defense against bacterial infection appears to be compromised by PPARγ. The relevance of PPARγ in myeloid cells, that organize anti-bacterial immunity, for the outcome of immune responses against intracellular bacteria such as Listeria monocytogenes in vivo is unknown. We found that Listeria monocytogenes infection of macrophages rapidly led to increased expression of PPARγ. This prompted us to investigate whether PPARγ in myeloid cells influences innate immunity against Listeria monocytogenes infection by using transgenic mice with myeloid-cell specific ablation of PPARγ (LysMCre×PPARγ(flox/flox)). Loss of PPARγ in myeloid cells results in enhanced innate immune defense against Listeria monocytogenes infection both, in vitro and in vivo. This increased resistance against infection was characterized by augmented levels of bactericidal factors and inflammatory cytokines: ROS, NO, IFNγ TNF IL-6 and IL-12. Moreover, myeloid cell-specific loss of PPARγ enhanced chemokine and adhesion molecule expression leading to improved recruitment of inflammatory Ly6C(hi) monocytes to sites of infection. Importantly, increased resistance against Listeria infection in the absence of PPARγ was not accompanied by enhanced immunopathology. Our results elucidate a yet unknown regulatory network in myeloid cells that is governed by PPARγ and restrains both listeriocidal activity and recruitment of inflammatory monocytes during Listeria infection, which may contribute to bacterial immune escape. Pharmacological interference with PPARγ activity in myeloid cells might represent a novel strategy to overcome intracellular bacterial infection.     

Effect of Bacillus Calmette-Guérin infection on delayed footpad reaction to Listeria monocytogenes

The role of peritoneal macrophages induced by Bacillus Calmette-Guérin (BCG) in the induction of immune responses to Listeria monocytogenes was studied in mice. The peritoneal macrophages from mice treated with BCG 14 days previously contained a high proportion of Ia-bearing macrophages (approximately 56%) and the cells showed not only a high level of listericidal activity but also a strong ability for presentation of listerial antigen to Listeria-immune T cells. An intraperitoneal inoculation with a low dose of Listeria, which can induce the maximal level of delayed footpad reaction (DFR) and positive migration inhibitory activity of macrophages in untreated mice, did not induce a detectable level of such responses in BCG-treated mice. The bacterial growth at an early stage of infection was suppressed by scavenger macrophages in these mice. On the other hand, BCG-treated mice showed the early development of DFR and macrophage migration inhibitory activity after an inoculation with a high dose of Listeria. It is revealed in transfer experiments that Listeria-pulsed peritoneal exudate cells induced by BCG elicited the highest level of DFR and positive migration inhibition of macrophages in normal mice at the earlier period of injection compared with Listeria-pulsed resident peritoneal cells. These results suggested that the increased activities of macrophages acting as scavenger cells and as antigen-presenting cells play important roles in the modification of immune responses to Listeria in BCG-treated mice.     

Production of IFN-β during Listeria monocytogenes infection is restricted to monocyte/macrophage lineage

The family of type I interferons (IFN), which consists of several IFN-α and one IFN-β, are produced not only after stimulation by viruses, but also after infection with non-viral pathogens. In the course of bacterial infections, these cytokines could be beneficial or detrimental. IFN-β is the primary member of type I IFN that initiates a cascade of IFN-α production. Here we addressed the question which cells are responsible for IFN-β expression after infection with the intracellular pathogen Listeria monocytogenes by using a genetic approach. By means of newly established reporter mice, maximum of IFN-β expression was observed at 24 hours post infection in spleen and, surprisingly, 48 hours post infection in colonized cervical and inguinal lymph nodes. Colonization of lymph nodes was independent of the type I IFN signaling, as well as bacterial dose and strain. Using cell specific reporter function and conditional deletions we could define cells expressing LysM as the major IFN-β producers, with cells formerly defined as Tip-DCs being the highest. Neutrophilic granulocytes, dendritic cells and plasmacytoid dendritic cells did not significantly contribute to type I IFN production.     

Zur Begeißelung von Listeria monocytogenes

Zusammenfassung Bei kulturellen, lichtmikroskopischen, elektronenmikroskopischen und serologischen Untersuchungen an L. monocytogenes-Stämmen wurden frühere Angaben von Griffin u. Robbins (1944) über den Begeißelungstyp sowie die von Leifson u. Palen (1955) beobachteten Geißelanomalien bestätigt. Bei dem als unbeweglich und geißellos beschriebenen Stamm B-5 wurden jedoch bei Zimmertemperatur unter geeigneten Züchtungsbedingungen vereinzelt Geißeln beobachtet. Der Stamm war schwach beweglich und ergab mit Listeria-H-Faktorenserum eine deutliche Geißelangglutination. Bisher erwiesen sich über 1000 geprüfte L. monocytogenes-Stämme als beweglich, so daß kein, Anlaß besteht, mit dem Vorkommen unbeweglicher Stämme dieser Art zu rechnen, vorausgesetzt, daß die Untersuchungen mit optimaler Methodik bei Züchtungstemperaturen zwischen 20–30°C vorgenommen werden.Med.-parasitologische Abteilung, Leiter: Prof. Dr. Piekarski.     

Effect of glucose on Listeria monocytogenes biofilm formation,and assessment of the biofilm’s sanitation tolerance

Listeria monocytogenes is an important cause of human foodborne infections and its ability to form biofilms is a serious concern to the food industry. To reveal the effect of glucose conditions on biofilm formation of L. monocytogenes, 20 strains were investigated under three glucose conditions (0.1, 1.0, and 2.0% w v^–1) by quantifying the number of cells in the biofilm and observing the biofilm structure after incubation for 24, 72, and 168 h. In addition, the biofilms were examined for their sensitivity to sodium hypochlorite. It was found that high concentrations of glucose reduced the number of viable cells in the biofilms and increased extracellular polymeric substance production. Moreover, biofilms formed at a glucose concentration of 1.0 or 2.0% were more resistant to sodium hypochlorite than those formed at a glucose concentration of 0.1%. This knowledge can be used to help design the most appropriate sanitation strategy.     

Development and validation of qualitative SYBR®Green Real-Time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes

A combination of four qualitative SYBR®Green qPCR screening assays targeting two levels of discrimination: Listeria genus (except Listeria grayi) and Listeria monocytogenes, is presented. These assays have been developed to be run simultaneously using the same polymerase chain reaction (PCR) programme. The paper also proposes a new validation procedure to specifically validate qPCR assays applied to food microbiology according to two guidelines: the ISO 22118 norm and the “Definition of minimum performance requirements for analytical methods of GMO testing”. The developed assays target the iap, prs and hlyA genes that belong to or neighbour the virulence cluster of Listeria spp. The selected primers were designed to amplify short fragments (60 to 103 bp) in order to obtain optimal PCR efficiency (between 97 and 107 % efficiency). The limit of detection of the SYBR®Green qPCR assays is two to five copies of target genes per qPCR reaction. These assays are highly accurate (98.08 and 100 % accuracy for the Listeria spp. and L. monocytogenes assays, respectively).     

Intracellular Accumulation of High Levels of γ-Aminobutyrate by Listeria monocytogenes 10403S in Response to Low pH: Uncoupling of γ-Aminobutyrate Synthesis from Efflux in a Chemically Defined Medium

It is well established that the glutamate decarboxylase (GAD) system is central to the survival of Listeria monocytogenes at low pH, both in acidic foods and within the mammalian stomach. The accepted model proposes that under acidic conditions extracellular glutamate is transported into the cell in exchange for an intracellular γ-aminobutyrate (GABA_i). The glutamate is then decarboxylated to GABA_i, a reaction that consumes a proton, thereby helping to prevent acidification of the cytoplasm. In this study, we show that glutamate supplementation had no influence on either growth rate at pH 5.0 or survival at pH 2.5 when L. monocytogenes 10403S was grown in a chemically defined medium (DM). In response to acidification, cells grown in DM failed to efflux GABA, even when glutamate was added to the medium. In contrast, in brain heart infusion (BHI), the same strain produced significant extracellular GABA (GABA_e) in response to acidification. In addition, high levels of GABA_i (>80 mM) were found in the cytoplasm in response to low pH in both growth media. Medium-swap and medium-mixing experiments revealed that the GABA efflux apparatus was nonfunctional in DM, even when glutamate was present. It was also found that the GadT2D2 antiporter/decarboxylase system was transcribed poorly in DM-grown cultures while overexpression of gadD1T1 and gadD3 occurred in response to pH 3.5. Interestingly, BHI-grown cells did not respond with upregulation of any of the GAD system genes when challenged at pH 3.5. The accumulation of GABA_i in cells grown in DM in the absence of extracellular glutamate indicates that intracellular glutamate is the source of the GABA_i. These results demonstrate that GABA production can be uncoupled from GABA efflux, a finding that alters the way we should view the operation of bacterial GAD systems.The capacity to produce γ-aminobutyric acid (GABA) through glutamate decarboxylation is commonly found in both Gram-negative and Gram-positive bacterial genera (10, 12). In several cases, this reaction has been shown to be critical for bacteria to survive potentially lethal acidic environments (15, 18, 20). It is generally held that the hydrogen ion consumed during the decarboxylation reaction helps to prevent excessive acidification of the cytoplasm, thereby protecting the cells against acidic environments. The GABA produced in the reaction is removed from the cell through the activity of an antiporter that exchanges a GABA molecule for an extracellular glutamate (Glu) molecule (6, 12).In Listeria monocytogenes, the Gram-positive food-borne pathogen that was the focus of the present study, the glutamate decarboxylase (GAD) system has been shown to play an essential role in acid tolerance (8, 9). Mutants compromised in their ability to catalyze this decarboxylation reaction survive poorly both in acidic foods (8) and gastric juice (9). The GAD system in most L. monocytogenes strains is encoded by a total of five genes. There are three genes, designated gadD1, gadD2, and gadD3, that encode distinct glutamate decarboxylase enzymes and two genes, designated gadT1 and gadT2, that encode two Glu-GABA antiporters. These genes are organized at three separate genetic loci: gadD1T1, gadT2D2, and gadD3 (11). The decarboxylase/antiporter system encoded by gadT2D2 plays a central role in allowing survival under extreme acidic conditions; mutants lacking either the GadT2 antiporter or the GadD2 decarboxylase are highly sensitive to low pH (9, 11). In contrast, the GadD1/GadT1 decarboxylase/antiporter system appears to be more important for growth under moderately acidic conditions (11). The genes encoding this system are absent from most serotype 4 strains, and this generally correlates with a reduced ability of these strains to grow well at low pH (11). The role of GadD3 is less clear since it has not been possible to generate a deletion mutant lacking the corresponding gene (9).Although the activity of the decarboxylase is generally thought to be coupled directly to the antiporter activity (i.e., the efflux of GABA is coupled to the supply of Glu) there is little direct evidence for this, even in bacteria where the system has been very well characterized. Most studies of the bacterial GAD system have used complex growth media when studying acid tolerance and GABA production (7, 8, 15). In the present study, we sought to determine whether extracellular Glu is a requirement for the production of GABA in L. monocytogenes. To do this, we have used a chemically defined growth medium (DM) that supports the growth of L. monocytogenes but does not include Glu. The results indicate that cells cultured in this medium do not produce extracellular GABA (GABA_e) in response to low pH but are capable of accumulating substantial pools of intracellular GABA (GABA_i). We establish that some component of complex medium is indispensable for efficient efflux of GABA. Surprisingly, supplementation of the DM with Glu failed to stimulate the extracellular release of GABA. We show that the GadD2/GadT2 decarboxylase/antiporter system is not transcribed when cells are grown in DM and suggest that this accounts for much of the difference in GABA production between cells cultured in DM and complex growth medium.     

The Timing of IFNβ Production Affects Early Innate Responses to Listeria monocytogenes and Determines the Overall Outcome of Lethal Infection

Dendritic cells (DCs) and natural killer (NK) cells are essential components of the innate immunity and play a crucial role in the first phase of host defense against infections and tumors. Listeria monocytogenes (Lm) is an intracellular pathogen that colonizes the cytosol of eukaryotic cells. Recent findings have shown Lm specifically in splenic CD8a(+) DCs shortly after intravenous infection. We examined gene expression profiles of mouse DCs exposed to Lm to elucidate the molecular mechanisms underlying DCs interaction with Lm. Using a functional genomics approach, we found that Lm infection induced a cluster of late response genes including type I IFNs and interferon responsive genes (IRGs) in DCs. Type I INFs were produced at the maximal level only at 24 h post infection indicating that the regulation of IFNs in the context of Lm infection is delayed compared to the rapid response observed with viral pathogens. We showed that during Lm infection, IFNγ production and cytotoxic activity were severely impaired in NK cells compared to E. coli infection. These defects were restored by providing an exogenous source of IFNβ during the initial phase of bacterial challenge. Moreover, when treated with IFNβ during early infection, NK cells were able to reduce bacterial titer in the spleen and significantly improve survival of infected mice. These findings show that the timing of IFNβ production is fundamental to the efficient control of the bacterium during the early innate phase of Lm infection.     

Amplified fragment length polymorphism analysis of Listeria monocytogenes by Experion™ automated microfluidic electrophoresis

Fifty Listeria monocytogenes strains were genotyped by sAFLP and PCR products were separated by agarose gel and automated chip-based microfluidic electrophoresis. A high congruency of results was observed comparing the two techniques, although for some cultures a better separation of sAFLP fragments was achieved with microfluidic system, which proved to be a highly reliable and reproducible tool to improve the molecular typing of L. monocytogenes, requiring lower volumes of samples and reducing significantly analysis time.     

Rapid, transient, and proportional activation of σ(B) in response to osmotic stress in Listeria monocytogenes

The osmotic activation of sigma B (σ(B)) in Listeria monocytogenes was studied by monitoring expression of four known σ(B)-dependent genes, opuCA, lmo2230, lmo2085, and sigB. Activation was found to be rapid, transient, and proportional to the magnitude of the osmotic stress applied, features that underpin the adaptability of this pathogen.     

Identification of phosphatidylinositol-specific phospholipase C activity in Listeria monocytogenes: a novel type of virulence factor?

A phospholipase C which cleaves phosphatidylinositol and glycosylphosphatidylinositol (GPI) anchors was identified in Listeria monocytogenes. This 36 kDa protein is encoded by the gene plcA, and is homologous to the Bacillus cereus, Bacillus thuringiensis and eukaryotic phosphatidylinositol-specific phospholipases C (PI-PLC). Expression of the plcA gene in Escherichia coli correlates with the appearance of PI-PLC activity in the cells. In Listeria monocytogenes, the activity is secreted to the culture medium. PI-PLC activity was only found in the two pathogenic species of the genus Listeria, namely L. monocytogenes and L. ivanovii. PI-PLC activity was lost and virulence decreased when the plcA gene was disrupted in the chromosome. This suggests that the PI-PLC of L. monocytogenes might be involved in virulence.     

Virulence Phenotyping and Molecular Characterization of a Low-pathogenicity Isolate of Listeria monocytogenes from Cow＇s Milk

A low-pathogenicity isolate of Listeria monocytogenes from cow＇s milk, as screened in mouse and chicken embryonated egg models, was examined for virulence-related phenotypic traits. Corresponding virulence genes （iap, prfA, picA, hly, mpl, actA, plcB, InlA and lnlB） were compared with L. monocytogenes reference strains 10403S and EGD to elucidate the possible molecular mechanisms of low virulence. Although L. monocytogenes H4 exhibited similar patterns to strain 10403S in terms of hemolytic activity, in vitro growth and invasiveness and even had higher adhesiveness, faster intracellular growth and higher phospholipase activity in vitro, it was substantially less virulent than the strain 10403S in mouse and chicken embryo models （50% lethal dose： 10^8.14 VS. 10^5.49 and 10^6.73 VS. 10^1.9, respectively）. The genes prfA, picA and mpl were homologous among L. monocytogenes strains H4, 10403S and EGD （〉98%）. Genes iap, hly, plcB, lnlA and lnlB of L. monocytogenes 10403S had higher homology to those of strain EGD （〉98%） than isolate H4. The homology of the gene hly between strain 10403S and isolate H4 was 96.9% at the nucleotide level, but 98.7% at the amino acid level. The actA gene of isolate H4 had deletions of 105 nucleotides corresponding to 35 amino acid deletions falling within the proline-rich region. Taken together, this study presents some clues as to reduced virulence to mice and chicken embryos of the isolate H4 probably as a result of deletion mutations of actA.     

The β2 Integrin Mac-1 Induces Protective LC3-Associated Phagocytosis of Listeria monocytogenes

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