Immune-Complexed Adenovirus Induce AIM2-Mediated Pyroptosis in Human Dendritic Cells

Human adenoviruses (HAdVs) are nonenveloped proteinaceous particles containing a linear double-stranded DNA genome. HAdVs cause a spectrum of pathologies in all populations regardless of health standards. Following repeat exposure to multiple HAdV types, we develop robust and long-lived humoral and cellular immune responses that provide life-long protection from de novo infections and persistent HAdV. How HAdVs, anti-HAdV antibodies and antigen presenting cells (APCs) interact to influence infection is still incompletely understood. In our study, we used physical, pharmacological, biochemical, fluorescence and electron microscopy, molecular and cell biology approaches to dissect the impact of immune-complexed HAdV (IC-HAdV) on human monocyte-derived dendritic cells (MoDCs). We show that IC-HAdV generate stabilized complexes of ~200 nm that are efficiently internalized by, and aggregate in, MoDCs. By comparing IC-HAdV, IC-empty capsid, IC-Ad2ts1 (a HAdV-C2 impaired in endosomal escape due to a mutation that impacts protease encapsidation) and IC-AdL40Q (a HAdV-C5 impaired in endosomal escape due to a mutation in protein VI), we demonstrate that protein VI-dependent endosomal escape is required for the HAdV genome to engage the DNA pattern recognition receptor AIM2 (absent in melanoma 2). AIM2 engagement induces pyroptotic MoDC death via ASC (apoptosis-associated speck protein containing a caspase activation/recruitment domain) aggregation, inflammasome formation, caspase 1 activation, and IL-1β and gasdermin D (GSDMD) cleavage. Our study provides mechanistic insight into how humoral immunity initiates an innate immune response to HAdV-C5 in human professional APCs.     

The Tyrosine Kinase Btk Regulates the Macrophage Response to Listeria monocytogenes Infection

In this study we investigated the role of Bruton''s tyrosine kinase (Btk) in the immune response to the Gram-positive intracellular bacterium Listeria monocytogenes (Lm). In response to Lm infection, Btk was activated in bone marrow-derived macrophages (BMMs) and Btk ^−/− BMMs showed enhanced TNF-α, IL-6 and IL-12p40 secretion, while type I interferons were produced at levels similar to wild-type (wt) BMMs. Although Btk-deficient BMMs displayed reduced phagocytosis of E. coli fragments, there was no difference between wt and Btk ^−/− BMMs in the uptake of Lm upon infection. Moreover, there was no difference in the response to heat-killed Lm between wt and Btk ^−/− BMMs, suggesting a role for Btk in signaling pathways that are induced by intracellular Lm. Finally, Btk ^−/− mice displayed enhanced resistance and an increased mean survival time upon Lm infection in comparison to wt mice. This correlated with elevated IFN-γ and IL-12p70 serum levels in Btk ^−/− mice at day 1 after infection. Taken together, our data suggest an important regulatory role for Btk in macrophages during Lm infection.     

Type I interferon signaling regulates the composition of inflammatory infiltrates upon infection with Listeria monocytogenes

Type I IFN is key to the immune response to viral pathogens, however its role in bacterial infections is less well understood. Mice lacking the type I IFN receptor (IFNAR-/-) demonstrate enhanced resistance to infection with Listeriamonocytogenes. We have now determined that following infection with Listeria, the composition of innate cells recruited to the peritoneal cavity of IFNAR-/- mice reflects an increase in the frequency of neutrophils and a decrease in monocyte frequency compared to WT controls. These differences in inflammatory infiltrates could not be attributed to distinct bone marrow composition prior to infection or to level of apoptosis. We also observed no differences in neutrophil oxidative burst. However, blocking CXCR2 prevented enhanced neutrophil influx and hampered bacterial clearance. Taken together, these studies highlight a novel mechanism by which type I interferon signaling regulates the immune response to Listeria, through negative regulation of chemokines driving neutrophil recruitment.     

Inhibition of Listeria monocytogenes growth by the lactoperoxidase-thiocyanate-H2O2 antimicrobial system

The lactoperoxidase-thiocyanate-H2O2 system (LP system), consisting of lactoperoxidase (0.37 U/ml), KSCN (0.3 mM), and H2O2 (0.3 mM), delayed but did not prevent growth of L. monocytogenes Scott A at 5, 10, 20, and 30 degrees C in broth and at 20 degrees C in milk. The net lag periods determined spectrophotometrically varied inversely with temperature and were shorter at 5 and 10 degrees C for cultures from shaken versus from statically grown inocula. Lag periods for cultures from shaken and statically grown inocula, respectively, were 73 and 98 h at 5 degrees C, 22 and 32 h at 10 degrees C, both 8.9 h at 20 degrees C, and both 2.8 h at 30 degrees C. After the lag periods, the maximum specific growth rates were similar for each of the three treatments (complete LP system, H2O2 alone, or control broth) at 5, 10, and 20 degrees C and were 0.06 to 0.08, 0.09 to 0.1, and 0.32 to 0.36/h, respectively. At 20 degrees C in sterile reconstituted skim milk, the LP system restricted growth of Scott A, with log CFU counts per ml at 0, 36, and 68 h being 5.7, 6.4 and 7.9 (versus 5.7, 9.8, and 11.2 for controls). Possible explanations for the decreased lag times observed for cultures from aerobically grown inocula are discussed.     

Involvement of Listeria monocytogenes in the abortive disease

Listeria monocytogenes, an intracellular facultative germ that causes the invasion, sometimes fatal, in susceptible hosts is a food borne pathogen with ubiquitary spread that has generated a public health problem for such risk groups as: pregnant women, foetuses, new borns. 504 women with abortive disease were serologically investigated in 1999 for serotype 1a circulating in Romania. The most affected age group proved to be that in the range of 20-30 yrs: 378 (75%) cases. 107 (21.23%) female patients had the diagnostic titer (> or = 1/320): among these, 38 (7.53%) had miscarriages in the IVth-VIIIth month and 18 (3.57%) gave birth to dead foetuses; during pregnancy, 10 (1.98%) female patients received treatment with Ampicillin and 2 (0.39%) treatment with Erythromycin. In the age group > 31 yrs, the 1/320 titer was noticed in 21 (4.16%) female patients but among these only 4 (0.79%) had a history of miscarriage in the final pregnancy months; they were administered Ampicillin during pregnancy. Although there is no clear-cut evidence, our results point to the conclusion that these female patients were contaminated with Listeria monocytogenes.     

Listeria monocytogenes Triggers the Cell Surface Expression of Gp96 Protein and Interacts with Its N Terminus to Support Cellular Infection

Listeria monocytogenes is an intracellular food-borne pathogen causing listeriosis in humans. This bacterium deploys an arsenal of virulence factors that act in concert to promote cellular infection. Bacterial surface proteins are of primary importance in the process of host cell invasion. They interact with host cellular receptors, inducing/modulating specific cellular responses. We previously identified Vip, a Listeria surface protein covalently attached to the bacterial cell wall acting as a key virulence factor. We have shown that Vip interacts with Gp96 localized at the surface of host cells during invasion and that this interaction is critical for a successful infection in vivo. To better understand the importance of Vip-Gp96 interaction during infection, we aimed to characterize this interaction at the molecular level. Here we demonstrate that, during infection, L. monocytogenes triggers the cellular redistribution of Gp96, inducing its exposure at the cell surface. Upon infection, Gp96 N-terminal domain is exposed to the extracellular milieu in L2071 fibroblasts and interacts with Vip expressed by Listeria. We identified Gp96 (Asp¹–Leu¹⁷⁰) as sufficient to interact with Vip; however, we also showed that the region Tyr¹⁷⁹–Leu³⁹⁰ of Gp96 is important for the interaction. Our findings unravel the Listeria-induced surface expression of Gp96 and the topology of its insertion on the plasma membrane and improve our knowledge on the Vip-Gp96 interaction during Listeria infection.     

Phagocytes produce prostaglandin E2 in response to cytosolic Listeria monocytogenes

Listeria monocytogenes is an intracellular bacterium that elicits robust CD8⁺ T-cell responses. Despite the ongoing development of L. monocytogenes-based platforms as cancer vaccines, our understanding of how L. monocytogenes drives robust CD8⁺ T-cell responses remains incomplete. One overarching hypothesis is that activation of cytosolic innate pathways is critical for immunity, as strains of L. monocytogenes that are unable to access the cytosol fail to elicit robust CD8⁺ T-cell responses and in fact inhibit optimal T-cell priming. Counterintuitively, however, activation of known cytosolic pathways, such as the inflammasome and type I IFN, lead to impaired immunity. Conversely, production of prostaglandin E₂ (PGE₂) downstream of cyclooxygenase-2 (COX-2) is essential for optimal L. monocytogenes T-cell priming. Here, we demonstrate that vacuole-constrained L. monocytogenes elicit reduced PGE₂ production compared to wild-type strains in macrophages and dendritic cells ex vivo. In vivo, infection with wild-type L. monocytogenes leads to 10-fold increases in PGE₂ production early during infection whereas vacuole-constrained strains fail to induce PGE₂ over mock-immunized controls. Mice deficient in COX-2 specifically in Lyz2⁺ or CD11c⁺ cells produce less PGE₂, suggesting these cell subsets contribute to PGE₂ levels in vivo, while depletion of phagocytes with clodronate abolishes PGE₂ production completely. Taken together, this work demonstrates that optimal PGE₂ production by phagocytes depends on L. monocytogenes access to the cytosol, suggesting that one reason cytosolic access is required to prime CD8⁺ T-cell responses may be to facilitate production of PGE₂.     

Production of IL-12 and IL-18 in human dendritic cells upon infection by Listeria monocytogenes

Dendritic cells (DCs) are major antigen-presenting cells of the immune system, which need to be activated in order to initiate an immune response. Here, we describe the immunostimulatory effects on human monocyte-derived DCs observed upon infection with Listeria monocytogenes or after treatment with listerial lipoteichoic acid (LTA) and lipopolysaccharide (LPS), respectively. All stimuli caused upregulation of costimulatory molecules, induced T-cell proliferative responses and secretion of cytokines in vitro. Infection of DCs with L. monocytogenes induced release of interleukin (IL)-12 and IL-18. In contrast treatment with purified listerial LTA yielded high levels of IL-18 release, but only minimal IL-12 production. Treatment of DCs with LPS conversely induced significant amounts of IL-12 production, but no IL-18. The release of both stimulating cytokines IL-12 and IL-18 upon infection with entire bacteria suggests that attenuated strains of L. monocytogenes may be a valuable tool for subunit vaccine delivery.     

Inhibition of Listeria monocytogenes growth by the lactoperoxidase-thiocyanate-H2O2 antimicrobial system.

The lactoperoxidase-thiocyanate-H2O2 system (LP system), consisting of lactoperoxidase (0.37 U/ml), KSCN (0.3 mM), and H2O2 (0.3 mM), delayed but did not prevent growth of L. monocytogenes Scott A at 5, 10, 20, and 30 degrees C in broth and at 20 degrees C in milk. The net lag periods determined spectrophotometrically varied inversely with temperature and were shorter at 5 and 10 degrees C for cultures from shaken versus from statically grown inocula. Lag periods for cultures from shaken and statically grown inocula, respectively, were 73 and 98 h at 5 degrees C, 22 and 32 h at 10 degrees C, both 8.9 h at 20 degrees C, and both 2.8 h at 30 degrees C. After the lag periods, the maximum specific growth rates were similar for each of the three treatments (complete LP system, H2O2 alone, or control broth) at 5, 10, and 20 degrees C and were 0.06 to 0.08, 0.09 to 0.1, and 0.32 to 0.36/h, respectively. At 20 degrees C in sterile reconstituted skim milk, the LP system restricted growth of Scott A, with log CFU counts per ml at 0, 36, and 68 h being 5.7, 6.4 and 7.9 (versus 5.7, 9.8, and 11.2 for controls). Possible explanations for the decreased lag times observed for cultures from aerobically grown inocula are discussed.     

QuiC2 represents a functionally distinct class of dehydroshikimate dehydratases identified in Listeria species including Listeria monocytogenes

Many Listeria species including L. monocytogenes contain the pathway for the biosynthesis of protocatechuate from shikimate and quinate. The qui1 and qui2 operons within these Listeria spp. encode enzymes for this pathway. The diversion of shikimate pathway intermediates in some Listeria species to produce protocatechuate suggests an important biological role for this compound to these organisms. A total of seven ORFs, including quiC2, were identified within qui1 and qui2, however only three proteins encoded by the operons have been functionally annotated. The final step in Listeria's protocatechuate biosynthesis involves the conversion of dehydroshikimate by a dehydroshikimate dehydratase (DSD). In this study, we demonstrate that QuiC2 functions as a DSD in Listeria spp. through biochemical and structural analyses. Moreover, we show that QuiC2 forms a phylogenetic cluster distinct from other functionally annotated DSDs. The individual phylogenetic clusters of DSD are represented by enzymes that produce protocatechuate for distinct biological processes. Similarly, QuiC2 is expected to produce protocatechuate for a novel biological process. We postulate that protocatechuate produced by DSDs found within the QuiC2 phylogenetic cluster provides an ecological niche for representative organisms.     

Life of Listeria monocytogenes in the host cells' cytosol

In the past decades impressive knowledge has been accumulated concerning the basic mechanisms of interactions between intracellular bacteria and their host cells. Comparatively little is known on the metabolic requirements necessary for efficient replication of these bacteria within their specific host cell compartments. Recent developments in functional genomics have led to more extensive studies of the metabolic aspects that may be crucial for understanding the pathogenesis of intracellular bacteria. Here we summarize our present knowledge on the physiology of L. monocytogenes with emphasis on those parts that seem to be important for its ability to replicate in the cytosol of mammalian host cells.     

Limitation in the detection of Listeria monocytogenes in food in the presence of competing Listeria innocua

Aims: Detectability of Listeria monocytogenes at 10⁰ CFU per food sample in the presence of Listeria innocua using standard microbiological detection was evaluated and compared with the real‐time PCR‐based method. Methods and Results: Enrichment in half‐Fraser broth followed by subculture in Fraser broth according to EN ISO 11290‐1 was used. False‐negative detection of 10⁰ CFU L. monocytogenes was obtained in the presence of 10¹ CFU L. innocua per sample using the standard detection method in contrast to more than 10⁵ CFU L. innocua per sample using real‐time PCR. Identification of L. monocytogenes on the chromogenic medium by the standard procedure was impossible if L. innocua was able to overgrow L. monocytogenes by more than three orders of magnitude after the enrichment in model samples. These results were confirmed using naturally contaminated food samples. Conclusions: Standard microbiological method was insufficient for the reliable detection of 10⁰ CFU L. monocytogenes in the presence of more than 10⁰ CFU of L. innocua per sample. On the other hand, if the growth of L. monocytogenes was sufficient to reach the concentration equal to the detection limit of PCR, the amount of the other microflora present in the food sample including L. innocua was not relevant for success of the PCR detection of L. monocytogenes. Significance and Impact of the Study: After the enrichment, the PCR detection is more convenient than the standard one as PCR detection is not compromised by other present microflora.     

Modeling the Growth of Listeria monocytogenes in Soft Blue-White Cheese

The aim of this study was to develop a predictive model simulating growth over time of the pathogenic bacterium Listeria monocytogenes in a soft blue-white cheese. The physicochemical properties in a matrix such as cheese are essential controlling factors influencing the growth of L. monocytogenes. We developed a predictive tertiary model of the bacterial growth of L. monocytogenes as a function of temperature, pH, NaCl, and lactic acid. We measured the variations over time of the physicochemical properties in the cheese. Our predictive model was developed based on broth data produced in previous studies. New growth data sets were produced to independently calibrate and validate the developed model. A characteristic of this tertiary model is that it handles dynamic growth conditions described in time series of temperature, pH, NaCl, and lactic acid. Supplying the model with realistic production and retail conditions showed that the number of L. monocytogenes cells increases 3 to 3.5 log within the shelf life of the cheese.