Ly6G+ neutrophils are dispensable for defense against systemic Listeria monocytogenes infection

Listeria monocytogenes is a facultative intracellular bacterium that causes systemic infections in immunocompromised hosts. Early recruitment of myeloid cells, including inflammatory monocytes and neutrophils, to sites of L. monocytogenes infection is essential for the control of infection and host survival. Because previous experimental studies used depleting or blocking Abs that affected both inflammatory monocytes and neutrophils, the relative contributions of these cell populations to defense against L. monocytogenes infection remain incompletely defined. In this article, we used highly selective depletion strategies to either deplete inflammatory monocytes or neutrophils from L. monocytogenes-infected mice and demonstrate that neutrophils are dispensable for early and late control of infection. In contrast, inflammatory monocytes are essential for bacterial clearance during the innate and adaptive phases of the immune response to L. monocytogenes infection.     

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.     

Deferoxamine increases the susceptibility of beta-thalassemic, iron-overloaded mice to infection with Listeria monocytogenes.

The effect of the iron chelator deferoxamine (DFO) on resistance to infection with Listeria monocytogenes in mice with a condition analogous to human beta-thalassemia was studied. Intraperitoneal injection of 10 mg DFO resulted in significantly increased mortality when given one, three and six days before infection with L. monocytogenes (for all three time points, p less than 0.02). There were no significant differences in hematocrit, plasma iron, or splenic iron content between the two groups of mice during these time periods. In addition, splenic counts of L. monocytogenes were not significantly higher in DFO-treated compared to saline-treated mice three days after infection. Moreover, background C57Bl/6J mice were not more susceptible to Listeria infection after receiving DFO than were saline-treated controls. In conclusion, acute administration of DFO increases the susceptibility of beta-thalassemic mice to L. monocytogenes. The effect is not seen in background mice and suggests that DFO increases susceptibility to Listeria infection only in animals with iron overload.     

Analysis of the role of OpuC, an osmolyte transport system, in salt tolerance and virulence potential of Listeria monocytogenes

The success of Listeria monocytogenes as a food-borne pathogen owes much to its ability to survive a variety of stresses, both in the external environment prior to ingestion and subsequently within the animal host. Growth at high salt concentrations and low temperatures is attributed mainly to the accumulation of organic solutes such as glycine betaine and carnitine. We utilized a novel system for generating chromosomal mutations (based on a lactococcal pWVO1-derived Ori(+) RepA(-) vector, pORI19) to identify a listerial OpuC homologue. Mutating the operon in two strains of L. monocytogenes revealed significant strain variation in the observed activity of OpuC. Radiolabeled osmolyte uptake studies, together with growth experiments in defined media, linked OpuC to carnitine and glycine betaine uptake in Listeria. We also investigated the role of OpuC in contributing to the growth and survival of Listeria in an animal (murine) model of infection. Altering OpuC resulted in a significant reduction in the ability of Listeria to colonize the upper small intestine and cause subsequent systemic infection following peroral inoculation.     

Multieffector-functional immune responses of HMBPP-specific Vγ2Vδ2 T cells in nonhuman primates inoculated with Listeria monocytogenes ΔactA prfA*

Although Listeria monocytogenes can induce systemic infection causing spontaneous abortion, septicemia, and meningitis, studies have not been performed to investigate human anti-L. monocytogenes immune responses, including those of Ag-specific Vγ2Vδ2 T cells, a dominant human γδ T cell subset. L. monocytogenes is the only pathogen known to possess both the mevalonate and non-mevalonate isoprenoid biosynthesis pathways that produce metabolic phosphates or phosphoantigens activating human Vγ2Vδ2 T cells, making it interesting to explore in vivo anti-L. monocytogenes immune responses of Vγ2Vδ2 T cells. In this study, we demonstrated that subclinical systemic L. monocytogenes infection of rhesus macaques via parenteral inoculation or vaccination with an attenuated Listeria strain induced multieffector-functional immune responses of phosphoantigen-specific Vγ2Vδ2 T cells. Subclinical systemic infection and reinfection with attenuated L. monocytogenes uncovered the ability of Vγ2Vδ2 T cells to mount expansion and adaptive or recall-like expansion. Expanded Vγ2Vδ2 T cells could traffic to and accumulate in the pulmonary compartment and intestinal mucosa. Expanded Vγ2Vδ2 T cells could evolve into effector cells producing IFN-γ, TNF-α, IL-4, IL-17, or perforin after L. monocytogenes infection, and some effector Vγ2Vδ2 T cells could coproduce IL-17 and IFN-γ, IL-4 and IFN-γ, or TNF-α and perforin. Surprisingly, in vivo-expanded Vγ2Vδ2 T effector cells in subclinical L. monocytogenes infection could directly lyse L. monocytogenes-infected target cells and inhibit intracellular L. monocytogenes bacteria. Thus, we present the first demonstration, to our knowledge, of multieffector-functional Vγ2Vδ2 T cell responses against L. monocytogenes.     

Sigma B在单核细胞增生李斯特菌耐受环境压力胁迫中的作用

摘要：Sigma B（σB）因子在单核细胞增生李斯特菌（Listeria monocytogenes）的压力应答和毒力调控中起着重要的作用。研究发现单核细胞增生李斯特菌的致病力与耐受环境条件的胁迫息息相关。在压力存在的情况下能启动一些基因的表达,以增强细菌对环境的耐受性,这些基因包括与耐受渗透压、酸碱性环境、氧化、极端温度和宿主体内胆碱等环境压力相关的基因。本文综述了σB因子在上述几种环境压力胁迫中的作用,为深入了解该菌的生理特征、探讨食品的最佳生产和保藏条件、防止细菌的感染等方面提供新的理论依据。     

Connatal listeriosis--a case report and the possibilities of microbiological diagnosis

The third most frequent agent of perinatal bacterial meningitis is Listeria monocytogenes, in Hungary, its occurrence is, however, uncommon. This raises the possibility of diagnostical mistakes. A connatal listeriosis case validated microbiologically referred to in this report calls attention to Listeria, as a rare but relevant pathogen of neonatal infections. If clinical background suggests infection, the pathogenic role of L. monocytogenes should be taken into consideration. The etiological significance of the agent has to be verified by a competent clinical microbiology laboratory, since maternal listeriosis should be treated and the serious connatal manifestations should be prevented. Epidemiology of perinatal infection by L. monocytogenes, and its diagnostic tools especially the use of selective media are discussed.     

Listeria monocytogenes virulence proteins induce surface expression of Fas ligand on T lymphocytes

Virulence factors secreted by Listeria monocytogenes are known to interfere with host cellular signalling pathways. We investigated whether L. monocytogenes modulates T-cell receptor signalling by examining surface expression of proteins known to be upregulated on activated T cells. In vitro culture of murine splenocytes with L. monocytogenes resulted in a specific and dose-dependent upregulation of Fas ligand (FasL). Induction of FasL expression was also observed for pathogenic Listeria ivanovii but not for non-pathogenic Listeria innocua, indicating involvement of Listeria virulence protein(s). Examination of L. monocytogenes strains deficient in different virulence genes demonstrated that FasL upregulation was dependent on the expression of two secreted proteins: listeriolysin O (LLO) and phosphatidylcholine-preferring phospholipase C (PC-PLC). Treatment of cells with purified proteins demonstrated that LLO was sufficient for inducing FasL, while PC-PLC synergized with LLO for the induction of FasL expression. FasL-expressing cells induced by L. monocytogenes were capable of killing Fas-expressing target cells. Furthermore, L. monocytogenes infection results in upregulation of FasL on T cells in mice. These results describe a novel function for LLO and PC-PLC and suggest that L. monocytogenes may use these virulence factors to modulate the host immune response.     

李斯特菌侵染宿主细胞过程中磷脂酶D活性变化的初步研究

磷脂酶D(phospholipase,PLD)在感染和炎症反应中发挥重要作用,然而其在单核增生李斯特菌(简称李斯特菌)侵染非洲绿猴肾细胞(Vero)中是否被激活尚未见报道。对李斯特菌刺激下的Vero细胞内PLD的活性变化进行了初步研究。以李斯特菌、佛波醇PMA分别刺激正常细胞及高效表达PLD2-K758R功能缺陷蛋白腺病毒预感染的Vero细胞,研究Vero细胞内PLD活性变化。结果发现,与对照组相比(不给予刺激),在李斯特菌和佛波醇PMA刺激正常Vero细胞过程中,胞内PLD活性增强非常显著。mPLD2-K758R蛋白的过度表达对Vero细胞的PLD基础活性无影响,但对李斯特菌和PMA诱导的PLD激活有明显抑制作用。这初步表明,在李斯特菌诱导的Vero细胞对其吞噬过程中的确伴有PLD的激活,并且可能主要是PLD2亚型被激活,但此吞噬过程中PLD的激活机制及激活后PLD的具体功能尚有待进一步研究。     

Exploration of host-pathogen interactions using Listeria monocytogenes and Drosophila melanogaster

The facultative intracellular bacterial pathogen Listeria monocytogenes is capable of replicating within a broad range of host cell types and host species. We report here the establishment of the fruit fly Drosophila melanogaster as a new model host for the exploration of L. monocytogenes pathogenesis and host response to infection. Listeria monocytogenes was capable of establishing lethal infections in adult fruit flies and larvae with extensive bacterial replication occurring before host death. Bacteria were found in the cytosol of insect phagocytic cells, and were capable of directing host cell actin polymerization. Bacterial gene products necessary for intracellular replication and cell-to-cell spread within mammalian cells were similarly found to be required within insect cells, and although previous work has suggested that L. monocytogenes virulence gene expression requires temperatures above 30 degrees C, bacteria within insect cells were found to express virulence determinants at 25 degrees C. Mutant strains of Drosophila that were compromised for innate immune responses demonstrated increased susceptibility to L. monocytogenes infection. These data indicate L. monocytogenes infection of fruit flies shares numerous features of mammalian infection, and thus that Drosophila has the potential to serve as a genetically tractable host system that will facilitate the analysis of host cellular responses to L. monocytogenes infection.     

Lipoproteins of Listeria monocytogenes are critical for virulence and TLR2-mediated immune activation

Numerous cell surface components of Listeria influence and regulate innate immune recognition and virulence. Here, we demonstrate that lipidation of prelipoproteins in Listeria monocytogenes is required to promote NF-kappaB activation via TLR2. In HeLa cells transiently expressing TLR2, L. monocytogenes and Listeria innocua mutants lacking the prolipoprotein diacylglyceryl transferase (lgt) gene are unable to induce TLR2-dependent activation of NF-kappaB, a property intrinsic to their isogenic parental strains. TLR2-dependent immune recognition is directed to secreted, soluble lipoproteins as evidenced by the sensitivity of the response to lipoprotein lipase. Studies of bone marrow-derived macrophages of C57BL/6 wild-type and TLR2-deficient mice infected with wild-type and lgt mutant strains indicate that the absence of host TLR2 receptor signaling has consequences similar to those of the absence of the bacterial TLR2 ligand, i.e., a delay in cellular immune responses directed toward the bacterium. Infection studies with the wild-type and TLR2(-/-) mice indicated attenuation of the lgt deletion mutant in both mouse strains, implying multiple roles of lipoproteins during infection. Further characterization of the Delta lgt mutant indicated that it is impaired for both invasion and intracellular survival and exhibits increased susceptibility to cationic peptides. Our studies identify lipoproteins as the immunologically active ligand of TLR2 and assign a critical role for this receptor in the recognition of these bacteria during infection, but they also reveal the overall importance of the lipoproteins for the pathogenicity of Listeria.     

Listeria monocytogenes: a multifaceted model

The opportunistic intracellular pathogen Listeria monocytogenes has become a paradigm for the study of host-pathogen interactions and bacterial adaptation to mammalian hosts. Analysis of L. monocytogenes infection has provided considerable insight into how bacteria invade cells, move intracellularly, and disseminate in tissues, as well as tools to address fundamental processes in cell biology. Moreover, the vast amount of knowledge that has been gathered through in-depth comparative genomic analyses and in vivo studies makes L. monocytogenes one of the most well-studied bacterial pathogens.     

Early programming of T cell populations responding to bacterial infection

The duration of infection and the quantity of Ag presented in vivo are commonly assumed to influence, if not determine, the magnitude of T cell responses. Although the cessation of in vivo T cell expansion coincides with bacterial clearance in mice infected with Listeria monocytogenes, closer analysis suggests that control of T cell expansion and contraction is more complex. In this report, we show that the magnitude and kinetics of Ag-specific T cell responses are determined during the first day of bacterial infection. Expansion of Ag-specific T lymphocyte populations and generation of T cell memory are independent of the duration and severity of in vivo bacterial infection. Our studies indicate that the Ag-specific T cell response to L. monocytogenes is programmed before the peak of the innate inflammatory response and in vivo bacterial replication.     

Auto, a surface associated autolysin of Listeria monocytogenes required for entry into eukaryotic cells and virulence

Listeria monocytogenes is an opportunistic food-borne human and animal pathogen. Several surface proteins expressed by this intracellular pathogen are critical for the infectious process. By in silico analysis we compared the surface protein repertories of L. monocytogenes and of the non-pathogenic species Listeria innocua and identified a gene encoding a surface protein of L. monocytogenes absent in L. innocua. This gene that we named aut encodes a protein (Auto) of 572 amino acids containing a signal sequence, a N-terminal autolysin domain and a C-terminal cell wall-anchoring domain made up of four GW modules. We show here that the aut gene is expressed independently of the virulence gene regulator PrfA and encodes a surface protein with an autolytic activity. We provide evidence that Auto is required for entry of L. monocytogenes into cultured non-phagocytic eukaryotic cells. The low invasiveness of an aut deletion mutant correlates with its reduced virulence following intravenous inoculation of mice and oral infection of guinea pigs. During infection, the autolytic activity of Auto may also be critical. Auto appears thus as a novel type of L. monocytogenes virulence factor.     

单增李斯特菌生物膜及其形成机制的研究进展

单增李斯特菌(Lm)是重要的人兽共患食源性病原菌。Lm生物膜与其致病性和耐药性密切相关。影响Lm生物膜形成的关键因子有鞭毛糖蛋白、胞外基质和群体感应系统等。鞭毛糖蛋白能促进菌体聚集,从而直接影响生物膜的形成。胞外DNA参与Lm粘附和生物膜早期的形成,并与胞外多糖和胞外结合蛋白一起构成生物膜胞外基质。Lm的Agr群体感应系统正调控生物膜形成,是一种集合毒力因子、耐药因子和生物膜的整体水平调控网络体系。     

Biodiversity of the species Listeria monocytogenes and the genus Listeria

This review describes the Listeria monocytogenes genome sequences available today and their comparison with that of Listeria innocua and Listeria welshimeri by highlighting their characteristic features and common traits. The diversity present among them is analysed with emphasis on putative virulence and host-pathogen interaction related functions. Then large-scale studies comparing gene content of Listeria and how these studies contributed to typing applications will be discussed. Finally, evolutionary conclusions and future perspectives in Listeria genomics are presented.     

REALIS: postgenomic analysis of Listeria Monocytogenes

Listeria monocytogenes is a remarkably successful food-borne pathogen. It is capable a) of surviving and proliferating under conditions that exist within the food chain, such as at low temperatures, high salt and low pH and b) of colonizing animal host tissues after ingestion of contaminated food, causing opportunistic infections mainly, but not exclusively, in immunocompromised hosts. The ultimate goals of REALIS are two fold: Firstly, it aims to completely decipher all genes required for survival in and adaptation of Listeria monocytogenes to two very different environments, ie., the infected host and the external environment. Secondly, using genomics and postgenomic tools, REALIS seeks to precisely address fundamental questions regarding evolutionary relationships between pathogenic and non-pathogenic Listeria and to define qualities of particularly successful clonal pathovariants in causing disease. This project will provide both industry and health care managers with rational approaches to curbing food-borne contamination, minimising risks of infection and providing novel pharmacological approaches for halting the fulminant course of infection.     

Evaluation of a new chromogenic agar for the detection of Listeria in food

AIM: Rapid identification of Listeria in food is important in protecting consumers from infection. The development of chromogenic media such as agar Listeria according to Ottaviani and Agosti (ALOA) has allowed more rapid detection of Listeria monocytogenes, with presumptive identification of this pathogenic species after only 24 h of incubation. The aim of this study was to evaluate Oxoid chromogenic Listeria agar (OCLA) in comparison with ALOA and a traditional, nonchromogenic medium, Oxford agar. METHODS AND RESULTS: Media were compared using pure cultures, spiked food samples and naturally contaminated samples. Whilst development of typical colony morphology took 48 h on Oxford agar, Listeria spp. were frequently detected after 24 h of incubation on OCLA and ALOA. There was no significant difference in recovery between the two chromogenic media. CONCLUSIONS: Results indicate that OCLA gives equivalent recovery of Listeria spp. compared with ALOA. Whilst L. monocytogenes was frequently detected after 24 h of incubation, a 48-h incubation time was necessary to ensure detection of both L. monocytogenes and other Listeria spp. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that a commercially available chromogenic medium other than ALOA is appropriate for use in the international standard method. The commercial availability of more than one medium will facilitate the more widespread use of the method, thus increasing confidence in the ability to detect L. monocytogenes in food in the presence of other Listeria spp.