Non-phagocytic Epithelial Cells Research Articles

Effect of nystatin on invasion of <i>Serratia grimesii</i> and <i>Serratia proteamaculans</i> bacteria into epithelial cells

BACKGROUND: Bacteria use various endocytic pathways during entering non-phagocytic cells. The involvement of caveolae/lipid rafts in bacterial invasion has been demonstrated for many bacterial pathogens. However, for bacteria of the genus Serratia, the involvement of membrane microdomains in the process of bacterial internalization has been poorly studied. AIM: To evaluate the involvement of caveolae/lipid rafts in the invasion of S. grimesii and S. proteamaculans bacteria into non-phagocytic epithelial Caco-2 and M-HeLa cells using nystatin. MATERIALS AND METHODS: M-HeLa and Caco-2 epithelial cells were incubated with 50 μM nystatin for 1 hour at 37 ºC, after which they were infected with the bacteria S. grimesii strain 30063 and S. proteamaculans strain 94, the multiplicity of infection was 100 bacteria per cell. The number of intracellular bacteria was assessed using gentamicin protection assay. The level of caveolin-1 in cells was visualized using confocal microscopy and Western blotting. The expression of Toll-like receptors genes were measured by real-time RT-PCR. RESULTS: Treatment of epithelial cells with nystatin reduces the internalization of S. grimesii and S. proteamaculans into M-HeLa cells by 30% and does not affect penetration into Caco-2 cells. At the same time, nystatin does not affect the redistribution / the integrity impairment of lipid rafts and does not lead to the cytoskeleton reorganization of eukaryotic cells. The addition of nystatin increases the level of caveolin-1 in M-HeLa cells (caveolin-1 is not expressed in Caco-2), which leads to a change plasma membrane fluidity. Nystatin promotes the secretion of proinflammatory cytokines interleukin-6 and interleukin-8 in both cell lines. Infection of M-HeLa cells pretreated with nystatin with the studied bacteria leads to an increase in the expression of tlr2 and tlr4 genes, but does not exceed the level of their expression in control samples. Therefore, it is impossible to speak unambiguously about the participation of Toll-like receptors in the invasion of Serratia bacteria. CONCLUSIONS: The results obtained suggest that the interaction of bacteria with eukaryotic cells induces the expression of caveolin-1, which leads to a change plasma membrane components mobility. This may be due to the fact that β1-integrin is involved in the invasion of the studied bacteria, which should be stabilized at the plasma membrane upon binding of the ligand due to the formation of a cholesterol- and sphingolipid-rich membrane microenvironment.

Shigella generates distinct IAM subpopulations during epithelial cell invasion to promote efficient intracellular niche formation

The facultative intracellular pathogen Shigella flexneri invades non-phagocytic epithelial gut cells. Through a syringe-like apparatus called type 3 secretion system, it injects effector proteins into the host cell triggering actin rearrangements leading to its uptake within a tight vacuole, termed the bacterial-containing vacuole (BCV). Simultaneously, Shigella induces the formation of large vesicles around the entry site, which we refer to as infection-associated macropinosomes (IAMs). After entry, Shigella ruptures the BCV and escapes into the host cytosol by disassembling the BCV remnants. Previously, IAM formation has been shown to be required for efficient BCV escape, but the molecular events associated with BCV disassembly have remained unclear. To identify host components required for BCV disassembly, we performed a microscopy-based screen to monitor the recruitment of BAR domain-containing proteins, which are a family of host proteins involved in membrane shaping and sensing (e.g. endocytosis and recycling) during Shigella epithelial cell invasion. We identified endosomal recycling BAR protein Sorting Nexin-8 (SNX8) localized to IAMs in a PI(3)P-dependent manner before BCV disassembly. At least two distinct IAM subpopulations around the BCV were found, either being recycled back to cellular compartments such as the plasma membrane or transitioning to become RAB11A positive “contact-IAMs” involved in promoting BCV rupture. The IAM subpopulation duality was marked by the exclusive recruitment of either SNX8 or RAB11A. Hindering PI(3)P production at the IAMs led to an inhibition of SNX8 recruitment at these compartments and delayed both, the step of BCV rupture time and successful BCV disassembly. Finally, siRNA depletion of SNX8 accelerated BCV rupture and unpeeling of BCV remnants, indicating that SNX8 is involved in controlling the timing of the cytosolic release. Overall, our work sheds light on how Shigella establishes its intracellular niche through the subversion of a specific set of IAMs.

Three-dimensional label-free visualization of the interactions of PM2.5 with macrophages and epithelial cells using optical diffraction tomography

Particulate matter ≤ 2.5 µm (PM2.5) poses health risks related to various diseases and infections. However, the interactions between PM2.5 and cells such as uptake and cell responses have not been fully investigated despite advances in bioimaging techniques, because the heterogeneous morphology and composition of PM2.5 make it challenging to employ labeling techniques, such as fluorescence. In this work, we visualized the interaction between PM2.5 and cells using optical diffraction tomography (ODT), which provides quantitative phase images by refractive index distribution. Through ODT analysis, the interactions of PM2.5 with macrophages and epithelial cells, such as intracellular dynamics, uptake, and cellular behavior, were successfully visualized without labeling techniques. ODT analysis clearly shows the behavior of phagocytic macrophages and nonphagocytic epithelial cells for PM2.5. Moreover, ODT analysis could quantitatively compare the accumulation of PM2.5 inside the cells. PM2.5 uptake by macrophages increased substantially over time, but uptake by epithelial cells increased only marginally. Our findings indicate that ODT analysis is a promising alternative approach to visually and quantitatively understanding the interaction of PM2.5 with cells. Therefore, we expect ODT analysis to be employed to investigate the interactions of materials and cells that are difficult to label.

Envelope-Fusion-Syncytium Formation in Microplitis bicoloratus bracovirus Maturation.

The viral envelope is essential for virus maturation. Virus-mediated syncytium formations are induced by viral envelope proteins that cause membrane fusion of the infected cells. Polydnaviridae (Polydnavirus) are enveloped viruses with multiple nucleocapsids, and virions mature in symbiotic parasitoid wasp ovaries. However, the mechanism governing the envelope packaging of multiple nucleocapsids remains unclear. In this study, we used transmission electron microscopy to examine the process whereby multiple nucleocapsids of Microplitis bicoloratus bracovirus are packaged into an envelope and observed envelope-fusion-syncytium formation in symbiotic wasp calyx cells during virus maturation. The virus maturation process in calyx cells comprised four stages: pre-virogenic stroma, virogenic stroma, assembly, and fusion. Each virus contained a single envelope with one nucleocapsid in the assembly stage; multiple envelopes then fused to form a viral envelope with multiple nucleocapsids (i.e., the envelope-fusion-syncytium) around the envelope fusion core in the fusion stage. The envelope-fusion-syncytium then stabilized the virions that were released into the lumen of the ovary across the calyx epithelial layer. The phagocytic calyx epithelial cells on the border of the calyx and ovary lumen cleared the majority of non-enveloped nucleocapsids. In contrast, non-phagocytic calyx epithelial cells with microvilli and a cuticular line between the ovary wall and the lumen remained intact in the ovary lumen. These results indicate that envelope-fusion-syncytium formation is important for packaging multiple nucleocapsids in bracovirus maturation.

Akt Inhibition Promotes Autophagy and Clearance of Group B Streptococcus from the Alveolar Epithelium.

Group B Streptococcus (GBS) is a gram-positive bacterium that is harmless for healthy individuals but may provoke invasive disease in young infants and immunocompromised hosts. GBS invades the epithelial barriers to enter the bloodstream, and thus strategies that enhance epithelial cell responses may hamper GBS invasion. In the present study, we sought to investigate whether the inhibition of Akt, a kinase that regulates host inflammatory responses and autophagy via suppression of mTOR, can enhance the response of non-phagocytic alveolar epithelial cells against GBS. Treatment of the alveolar epithelial cell line A549 with the Akt inhibitor MK-2206 resulted in the enhanced production of reactive oxygen species and inflammatory mediators in response to GBS. Additionally, Akt inhibition via MK-2206 resulted in elevated LC3II/I ratios and increased autophagic flux in alveolar epithelial cells. Importantly, the inhibition of Akt promoted GBS clearance both in alveolar epithelial cells in vitro and in lung tissue in vivo in a murine model of GBS pneumonia. The induction of autophagy was essential for GBS clearance in MK-2206 treated cells, as knockdown of ATG5, a critical component of autophagy, abrogated the effect of Akt inhibition on GBS clearance. Our findings highlight the role of Akt kinase inhibition in promoting autophagy and GBS clearance in the alveolar epithelium. The inhibition of Akt may serve as a promising measure to strengthen epithelial barriers and prevent GBS invasion in susceptible hosts.

Foscarnet-Type Inorganic-Organic Hybrid Nanoparticles for Effective Antiviral Therapy.

[ZrO]2+[(FCN)0.4(OH)0.8]2- and Gd3+[FCN]3- inorganic-organic hybrid nanoparticles (IOH-NPs) are novel saline antiviral nanocarriers with foscarnet (FCN) as a drug anion. FCN as a pyrophosphate analogue serves as a prototype of a viral DNA polymerase inhibitor. FCN is used for the treatment of herpesvirus infections, including the drug-resistant cytomegalovirus (CMV) and herpes simplex viruses, HSV-1 and HSV-2. The novel [ZrO]2+[(FCN)0.4(OH)0.8]2- and Gd3+[FCN]3- IOH-NPs are characterized by aqueous synthesis, small size (20-30 nm), low material complexity, high biocompatibility, and high drug load (up to 44 wt % FCN per nanoparticle). The antiviral activity of the FCN-type IOH-NPs is probed for the human cytomegalovirus (HCMV). Moreover, the uptake of FCN-type IOH-NPs into vesicles, cytoplasm, and nuclei of nonphagocytic lung epithelial cells is evaluated. As a result, a promising antiviral activity of the FCN-type IOH-NPs that significantly outperforms freely dissolved FCN at the level of clinical formulations is observed, encouraging a future use of FCN-type IOH-NPs for the delivery of antivirals against respiratory viruses.

A large panel of chicken cells are invaded in vivo by Salmonella Typhimurium even when depleted of all known invasion factors.

Poultry are the main source of human infection by Salmonella. As infected poultry are asymptomatic, identifying infected poultry farms is difficult, thus controlling animal infections is of primary importance. As cell tropism is known to govern disease, our aim was therefore to identify infected host-cell types in the organs of chicks known to be involved in Salmonella infection and investigate the role of the three known invasion factors in this process (T3SS-1, Rck and PagN). Chicks were inoculated with wild-type or isogenic fluorescent Salmonella Typhimurium mutants via the intracoelomic route. Our results show that liver, spleen, gall bladder and aortic vessels could be foci of infection, and that phagocytic and non-phagocytic cells, including immune, epithelial and endothelial cells, are invaded in vivo in each organ. Moreover, a mutant defective for the T3SS-1, Rck and PagN remained able to colonize organs like the wild-type strain and invaded non-phagocytic cells in each organ studied. As the infection of the gall bladder had not previously been described in chicks, invasion of gall bladder cells was confirmed by immunohistochemistry and infection was shown to last several weeks after inoculation. Altogether, for the first time these findings provide insights into cell tropism of Salmonella in relevant organs involved in Salmonella infection in chicks and also demonstrate that the known invasion factors are not required for entry into these cell types.

Persistence of Burkholderia thailandensis E264 in lung tissue after a single binge alcohol episode.

BackgroundBinge drinking, an increasingly common form of alcohol use disorder, is associated with substantial morbidity and mortality; yet, its effects on the immune system’s ability to defend against infectious agents are poorly understood. Burkholderia pseudomallei, the causative agent of melioidosis can occur in healthy humans, yet binge alcohol intoxication is increasingly being recognized as a major risk factor. Although our previous studies demonstrated that binge alcohol exposure increased B. pseudomallei near-neighbor virulence in vivo and increased paracellular diffusion and intracellular invasion, no experimental studies have examined the extent to which bacterial and alcohol dosage play a role in disease progression. In addition, the temporal effects of a single binge alcohol dose prior to infection has not been examined in vivo.Principal findingsIn this study, we used B. thailandensis E264 a close genetic relative of B. pseudomallei, as useful BSL-2 model system. Eight-week-old female C57BL/6 mice were utilized in three distinct animal models to address the effects of 1) bacterial dosage, 2) alcohol dosage, and 3) the temporal effects, of a single binge alcohol episode. Alcohol was administered comparable to human binge drinking (≤ 4.4 g/kg) or PBS intraperitoneally before a non-lethal intranasal infection. Bacterial colonization of lung and spleen was increased in mice administered alcohol even after bacterial dose was decreased 10-fold. Lung and not spleen tissue were colonized even after alcohol dosage was decreased 20 times below the U.S legal limit. Temporally, a single binge alcohol episode affected lung bacterial colonization for more than 24 h after alcohol was no longer detected in the blood. Pulmonary and splenic cytokine expression (TNF-α, GM-CSF) remained suppressed, while IL-12/p40 increased in mice administered alcohol 6 or 24 h prior to infection. Increased lung and not intestinal bacterial invasion was observed in human and murine non-phagocytic epithelial cells exposed to 0.2% v/v alcohol in vitro.ConclusionsOur results indicate that the effects of a single binge alcohol episode are tissue specific. A single binge alcohol intoxication event increases bacterial colonization in mouse lung tissue even after very low BACs and decreases the dose required to colonize the lungs with less virulent B. thailandensis. Additionally, the temporal effects of binge alcohol alters lung and spleen cytokine expression for at least 24 h after alcohol is detected in the blood. Delayed recovery in lung and not spleen tissue may provide a means for B. pseudomallei and near-neighbors to successfully colonize lung tissue through increased intracellular invasion of non-phagocytic cells in patients with hazardous alcohol intake.

Three-dimensional reconstruction of leukocyte internalisation in the luminal uterine epithelium following mating

Following mating, leukocytes are recruited to the uterine epithelium where they phagocytose spermatozoa and mediate maternal immune tolerance as well as a mild inflammatory response. In this ultrastructural study we utilised array tomography, a high-resolution volume scanning electron microscopy approach to 3D reconstruct the cellular relationships formed by leukocytes recruited to the luminal uterine epithelium 12 h post-mating in the rat. We report that following mating, neutrophils and macrophages are internalised by the luminal uterine epithelium, with multiple leukocytes internalised via contortion through a small tunnel in the apical membrane into a large membrane-bound vacuole within the cytoplasm of luminal uterine epithelial cells (UECs).Once internalised within the UECs, recruited leukocytes appear to phagocytose material within the membrane-bound vacuole and most ultimately undergo a specialised cell death, including vacuolisation and loss of membrane integrity. As these observations involve ultrastructurally normal leukocytic cells internalised within non-phagocytic epithelial cells, these observations are consistent with the formation of cell-in-cell structures via entosis, rather than phagocytic engulfment by UECs.Although cell-in-cell structures have been reported in normal and pathological conditions elsewhere, the data collected herein represents the first evidence of the formation of cell-in-cell structures within the uterine epithelium as a novel component of the maternal inflammatory response to mating.

Arcanobacterium haemolyticum Utilizes Both Phospholipase D and Arcanolysin To Mediate Its Uptake into Nonphagocytic Cells

Arcanobacterium haemolyticum is an emerging human pathogen that causes pharyngitis and wound infections. A few studies have suggested that A. haemolyticum is able to induce its uptake into nonphagocytic epithelial cells, but the bacterial factors associated with host cell invasion and the host cell processes involved have yet to be studied. We investigated how two A. haemolyticum virulence factors, arcanolysin (ALN) and phospholipase D (PLD), affect the ability of the bacteria to adhere to and subsequently invade Detroit 562 pharyngeal epithelial cells. The sphingomyelinase activity of phospholipase D was necessary to increase bacterial adherence, while the absence of a functional arcanolysin had no effect on A. haemolyticum adherence but did lead to a decrease in A. haemolyticum invasion into Detroit 562 cells. Because of the known roles of cholesterol-dependent cytolysins in disrupting calcium gradients and inducing F-actin-mediated bacterial internalization, we sought to determine whether ALN and PLD played a similar role in the ability of A. haemolyticum to invade nonphagocytic cells. Elimination of extracellular calcium and inhibition of the Arp2/3 complex or F-actin polymerization also caused a decrease in the ability of A. haemolyticum to invade Detroit 562 cells. Overall, our findings suggest that A. haemolyticum utilizes phospholipase D primarily for adherence and utilizes arcanolysin primarily for invasion into Detroit 562 cells in a process dependent on extracellular calcium and F-actin polymerization. Our work marks the first insight into how the individual activities of arcanolysin and phospholipase D affect A. haemolyticum host-pathogen interactions using the biologically relevant Detroit 562 cell line.

Host lipid sensing promotes invasion of cells with pathogenic Salmonella

Pathogenic Salmonella species initiate infection by invading non-phagocytic intestinal epithelial cells (IEC). This invasion is brought about by a number of Salmonella invasion promoting molecules (Sips) encoded by the Salmonella Pathogenicity Island - 1 (SPI-1). Intracellular delivery of some of these molecules also brings about caspase-1 – mediated pyroptotic cell death that contributes to pathogen clearance. These molecules are secreted and delivered inside cells upon contact of Salmonella with one or more host signals whose identity has not been established. We show that lysophosphatidylcholine (LPC) released following activation of caspase-1 in Salmonella – infected cells and abundant in plasma amplifies production of Sips from this pathogen and promotes its cellular invasion. LPC brings about adenylate cyclase and cAMP receptor protein (CRP) - dependent de novo synthesis of SipC that is accompanied by its translocation to bacterial cell surface and release into the outside milieu. Treatment of Salmonella with LPC produces sustained induction of SPI - 1 transcriptional regulator, hilA. Our findings reveal a novel host lipid sensing - driven regulatory mechanism for Salmonella invasion.

PilY1 Promotes Legionella pneumophila Infection of Human Lung Tissue Explants and Contributes to Bacterial Adhesion, Host Cell Invasion, and Twitching Motility.

Legionnaires' disease is an acute fibrinopurulent pneumonia. During infection Legionella pneumophila adheres to the alveolar lining and replicates intracellularly within recruited macrophages. Here we provide a sequence and domain composition analysis of the L. pneumophila PilY1 protein, which has a high homology to PilY1 of Pseudomonas aeruginosa. PilY1 proteins of both pathogens contain a von Willebrand factor A (vWFa) and a C-terminal PilY domain. Using cellular fractionation, we assigned the L. pneumophila PilY1 as an outer membrane protein that is only expressed during the transmissive stationary growth phase. PilY1 contributes to infection of human lung tissue explants (HLTEs). A detailed analysis using THP-1 macrophages and A549 lung epithelial cells revealed that this contribution is due to multiple effects depending on host cell type. Deletion of PilY1 resulted in a lower replication rate in THP-1 macrophages but not in A549 cells. Further on, adhesion to THP-1 macrophages and A549 epithelial cells was decreased. Additionally, the invasion into non-phagocytic A549 epithelial cells was drastically reduced when PilY1 was absent. Complementation variants of a PilY1-negative mutant revealed that the C-terminal PilY domain is essential for restoring the wild type phenotype in adhesion, while the putatively mechanosensitive vWFa domain facilitates invasion into non-phagocytic cells. Since PilY1 also promotes twitching motility of L. pneumophila, we discuss the putative contribution of this newly described virulence factor for bacterial dissemination within infected lung tissue.

Two Small Molecules Block Oral Epithelial Cell Invasion by Porphyromons gingivalis.

Porphyromonas gingivalis is a keystone pathogen of periodontitis. One of its bacterial characteristics is the ability to invade various host cells, including nonphagocytic epithelial cells and fibroblasts, which is known to facilitate P. gingivalis adaptation and survival in the gingival environment. In this study, we investigated two small compounds, Alop1 and dynasore, for their role in inhibition of P. gingivalis invasion. Using confocal microscopy, we showed that these two compounds significantly reduced invasion of P. gingivalis and its outer membrane vesicles into human oral keratinocytes in a dose-dependent manner. The inhibitory effects of dynasore, a dynamin inhibitor, on the bacterial entry is consistent with the notion that P. gingivalis invasion is mediated by a clathrin-mediated endocytic machinery. We also observed that microtubule arrangement, but not actin, was altered in the host cells treated with Alop1 or dynasore, suggesting an involvement of microtubule in this inhibitory activity. This work provides an opportunity to develop compounds against P. gingivalis infection.

Host-pathogen reorganisation during host cell entry by Chlamydia trachomatis

Chlamydia trachomatis is obligate intracellular bacterial pathogen that remains a significant public health burden worldwide. A critical early event during infection is chlamydial entry into non-phagocytic host epithelial cells. Like other Gram-negative bacteria, C. trachomatis uses a type III secretion system (T3SS) to deliver virulence effector proteins into host cells. These effectors trigger bacterial uptake and promote bacterial survival and replication within the host cell. In this review, we highlight recent cryo-electron tomography that has provided striking insights into the initial interactions between Chlamydia and its host. We describe the polarised structure of extracellular C. trachomatis elementary bodies (EBs), and the supramolecular organisation of T3SS complexes on the EB surface, in addition to the changes in host and pathogen architecture that accompany bacterial internalisation and EB encapsulation into early intracellular vacuoles. Finally, we consider the implications for further understanding the mechanism of C. trachomatis entry and how this might relate to those of other bacteria and viruses.

A Mycobacterial Phosphoribosyltransferase Promotes Bacillary Survival by Inhibiting Oxidative Stress and Autophagy Pathways in Macrophages and Zebrafish

Mycobacterium tuberculosis employs various strategies to modulate host immune responses to facilitate its persistence in macrophages. The M. tuberculosis cell wall contains numerous glycoproteins with unknown roles in pathogenesis. Here, by using Concanavalin A and LC-MS analysis, we identified a novel mannosylated glycoprotein phosphoribosyltransferase, encoded by Rv3242c from M. tuberculosis cell walls. Homology modeling, bioinformatic analyses, and an assay of phosphoribosyltransferase activity in Mycobacterium smegmatis expressing recombinant Rv3242c (MsmRv3242c) confirmed the mass spectrometry data. Using Mycobacterium marinum-zebrafish and the surrogate MsmRv3242c infection models, we proved that phosphoribosyltransferase is involved in mycobacterial virulence. Histological and infection assays showed that the M. marinum mimG mutant, an Rv3242c orthologue in a pathogenic M. marinum strain, was strongly attenuated in adult zebrafish and also survived less in macrophages. In contrast, infection with wild type and the complemented ΔmimG:Rv3242c M. marinum strains showed prominent pathological features, such as severe emaciation, skin lesions, hemorrhaging, and more zebrafish death. Similarly, recombinant MsmRv3242c bacteria showed increased invasion in non-phagocytic epithelial cells and longer intracellular survival in macrophages as compared with wild type and vector control M. smegmatis strains. Further mechanistic studies revealed that the Rv3242c- and mimG-mediated enhancement of intramacrophagic survival was due to inhibition of autophagy, reactive oxygen species, and reduced activities of superoxide dismutase and catalase enzymes. Infection with MsmRv3242c also activated the MAPK pathway, NF-κB, and inflammatory cytokines. In summary, we show that a novel mycobacterial mannosylated phosphoribosyltransferase acts as a virulence and immunomodulatory factor, suggesting that it may constitute a novel target for antimycobacterial drugs.

Salmonella-host interactions - modulation of the host innate immune system.

Salmonella enterica (S. enterica) are Gram-negative bacteria that can invade a broad range of hosts causing both acute and chronic infections. This phenotype is related to its ability to replicate and persist within non-phagocytic host epithelial cells as well as phagocytic dendritic cells and macrophages of the innate immune system. Infection with S. enterica manifests itself through a broad range of clinical symptoms and can result in asymptomatic carriage, gastroenteritis, systemic disease such as typhoid fever and in severe cases, death (1). Exposure to S. enterica serovars Typhi and Paratyphi exhibits clinical symptoms including diarrhea, fatigue, fever, and temperature fluctuations. Other serovars such as the non-typhoidal Salmonella (NTS), of which there are over 2,500, are commonly contracted as, but not limited to, food-borne sources causing gastrointestinal symptoms, which include diarrhea and vomiting. The availability of complete genome sequences for many S. enterica serovars has facilitated research into the genetic determinants of virulence for this pathogen. This work has led to the identification of important bacterial components, including flagella, type III secretion systems, lipopolysaccharides, and Salmonella pathogenicity islands, all of which support the intracellular life cycle of S. enterica. Studies focusing on the host–pathogen interaction have provided insights into receptor activation of the innate immune system. Therefore, characterizing the host–S. enterica interaction is critical to understand the pathogenicity of the bacteria in a clinically relevant context. This review outlines salmonellosis and the clinical manifestations between typhoidal and NTS infections as well as discussing the host immune response to infection and the models that are being used to elucidate the mechanisms involved in Salmonella pathogenicity.

B cell subsets are activated and produce cytokines during early phases of Francisella tularensis LVS infection

Francisella tularensis, a facultative intracellular Gram-negative bacterium, causes the illness tularemia. The infection of mice with live vaccine strain is considered to be a model of human tularemia. F. tularensis infects predominantly such phagocytic cells as macrophages or neutrophils, but it also infects non-phagocytic hepatocytes, epithelial cells, and murine and human B cell lines. Based on work with the murine tularemia model, we report here that F. tularensis LVS infects peritoneal CD19+ cells – exclusively B-1a cells – early after intraperitoneal infection in vivo. The peritoneal and consequently spleen CD19+ cells are activated by the F. tularensis LVS infection to express the activation markers from MHC class II, CD25, CD54, CD69, and the co-stimulatory molecules CD80 and CD86. As early as 12 h post-infection, the peritoneal CD19+ cells produce IFN-γ, IL-1β, IL-4, IL-6, IL-12, IL-17, IL-23, and TNF-α. The spleen CD19+ cells respond to infection with some delay. Moreover, the F. tularensis infected A20 B cell line activates CD3+ spleen cells isolated from naïve mice. Thus, the data presented here suggest that B cells have all the attributes to actively participate in the induction and regulation of the adaptive immune response during early stages of F. tularensis infection.

IglC and PdpA are important for promoting Francisella invasion and intracellular growth in epithelial cells.

The highly infectious bacteria, Francisella tularensis, colonize a variety of organs and replicate within both phagocytic as well as non-phagocytic cells, to cause the disease tularemia. These microbes contain a conserved cluster of important virulence genes referred to as the Francisella Pathogenicity Island (FPI). Two of the most characterized FPI genes, iglC and pdpA, play a central role in bacterial survival and proliferation within phagocytes, but do not influence bacterial internalization. Yet, their involvement in non-phagocytic epithelial cell infections remains unexplored. To examine the functions of IglC and PdpA on bacterial invasion and replication during epithelial cell infections, we infected liver and lung epithelial cells with F. novicida and F. tularensis ‘Type B’ Live Vaccine Strain (LVS) deletion mutants (ΔiglC and ΔpdpA) as well as their respective gene complements. We found that deletion of either gene significantly reduced their ability to invade and replicate in epithelial cells. Gene complementation of iglC and pdpA partially rescued bacterial invasion and intracellular growth. Additionally, substantial LAMP1-association with both deletion mutants was observed up to 12 h suggesting that the absence of IglC and PdpA caused deficiencies in their ability to dissociate from LAMP1-positive Francisella Containing Vacuoles (FCVs). This work provides the first evidence that IglC and PdpA are important pathogenic factors for invasion and intracellular growth of Francisella in epithelial cells, and further highlights the discrete mechanisms involved in Francisella infections between phagocytic and non-phagocytic cells.

Modern Aspects on <i>Shigella</i> Pathogenecity and Vaccine Development - A Review

Shigella species are intracytosolic Gram-negative invasive enteropathogenic bacteria, causing the rupture, invasion and inflammatory destruction of the human colonic epithelium. They utilize the host cytoskeletal components to form propulsive actin tails. The so-called invasive phenotype of Shigella is linked to expression of a type III secretory system (TTSS) injecting effector proteins into the epithelial cell membrane and cytoplasm, thereby inducing local but massive changes in the cell cytoskeleton that lead to bacterial internalization into non-phagocytic intestinal epithelial cells. The molecular and cellular bases of this invasive phenotype essentially encompass crossing of the epithelial lining, apoptotic killing of macrophages, entry into epithelial cells, and escape into the cytoplasm, followed by cell-to-cell spread. Intracellular colonization is likely to protect the micro-organisms from killing by humoral and cellular effectors of the innate immune response. Concurrently, the capacity of Shigella to reprogram invaded epithelial cells to produce proinflammatory mediators plays a major role in the strong inflammatory profile of the disease. This profile is likely to impact on the nature and quality of the adaptive response, which is dominated by humoral protection at the mucosal level. In recent years, a large amount of information has been generated regarding the host, pathogen and environmental factors that impact the pathogenesis of shigellosis at the cellular and molecular level. This review summarizes what is currently known about Shigella, detailing those factors that contribute to pathogenesis and examining the current progress in the development of a vaccine

Modern Aspects on <i>Shigella</i> Pathogenecity and Vaccine Development - A Review

Shigella species are intracytosolic Gram-negative invasive enteropathogenic bacteria, causing the rupture, invasion and inflammatory destruction of the human colonic epithelium. They utilize the host cytoskeletal components to form propulsive actin tails. The so-called invasive phenotype of Shigella is linked to expression of a type III secretory system (TTSS) injecting effector proteins into the epithelial cell membrane and cytoplasm, thereby inducing local but massive changes in the cell cytoskeleton that lead to bacterial internalization into non-phagocytic intestinal epithelial cells. The molecular and cellular bases of this invasive phenotype essentially encompass crossing of the epithelial lining, apoptotic killing of macrophages, entry into epithelial cells, and escape into the cytoplasm, followed by cell-to-cell spread. Intracellular colonization is likely to protect the micro-organisms from killing by humoral and cellular effectors of the innate immune response. Concurrently, the capacity of Shigella to reprogram invaded epithelial cells to produce proinflammatory mediators plays a major role in the strong inflammatory profile of the disease. This profile is likely to impact on the nature and quality of the adaptive response, which is dominated by humoral protection at the mucosal level. In recent years, a large amount of information has been generated regarding the host, pathogen and environmental factors that impact the pathogenesis of shigellosis at the cellular and molecular level. This review summarizes what is currently known about Shigella, detailing those factors that contribute to pathogenesis and examining the current progress in the development of a vaccine