Listeria monocytogenes Infection of Bat Pipistrellus nathusii Epithelial cells Depends on the Invasion Factors InlA and InlB.
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ABSTRACT:
SUBMITTER: Povolyaeva O
PROVIDER: S-EPMC7690591 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Listeria monocytogenes is a facultative intracellular pathogen that infects a wide variety of cells, causing the life-threatening disease listeriosis. L. monocytogenes virulence factors include two surface invasins, InlA and InlB, known to promote bacterial uptake by host cells, and the secreted pore-forming toxin listeriolysin O (LLO), which disrupts the phagosome to allow bacterial proliferation in the cytosol. In addition, plasma membrane perforation by LLO has been shown to facilitate L. monocytogenes internalization into epithelial cells. In this work, we tested the host cell range and importance of LLO-mediated L. monocytogenes internalization relative to the canonical invasins, InlA and InlB. We measured the efficiencies of L. monocytogenes association with and internalization into several human cell types (hepatocytes, cytotrophoblasts, and endothelial cells) using wild-type bacteria and isogenic single, double, and triple deletion mutants for the genes encoding InlA, InlB and LLO. No role for InlB was detected in any tested cells unless the InlB expression level was substantially enhanced, which was achieved by introducing a mutation (prfA*) in the gene encoding the transcription factor PrfA. In contrast, InlA and LLO were the most critical invasion factors, although they act in a different manner and in a cell-type-dependent fashion. As expected, InlA facilitates both bacterial attachment and internalization in cells that express its receptor, E-cadherin. LLO promotes L. monocytogenes internalization into hepatocytes, but not into cytotrophoblasts and endothelial cells. Finally, LLO and InlA cooperate to increase the efficiency of host cell invasion by L. monocytogenes.
| S-EPMC6204736 | biostudies-literature
Project description:In this study, a number of Listeria monocytogenes strains of different origins were evaluated for in vitro invasion capacity for various human cell types (monocytic THP-1, enterocytic Caco-2, and hepatocytic HepG2 cells) and for expression levels of specific virulence genes. For THP-1 cells, no differences between clinical and nonclinical L. monocytogenes strains in invasion capacity or in production of the proinflammatory cytokine interleukin-8 (IL-8) were observed, whereas for the Caco-2 and HepG2 cells, significant differences in invasion capacity were noticed. On average, the clinical strains showed a significantly lower invasion capacity than the nonclinical L. monocytogenes strains. Furthermore, it was shown that the clinical strains induce lower IL-8 levels in HepG2 cells than do the nonclinical strains. This observation led us to study the mRNA expression levels of inlA, inlB, and ami, important virulence genes mediating adhesion and invasion of eukaryotic cells, by real-time reverse transcription-PCR for 27 clinical and 37 nonclinical L. monocytogenes strains. Significant differences in inlA and inlB expression were observed, with clinical strains showing a lower expression level than nonclinical strains. These observations were in accordance with in vitro invasion of Caco-2 and HepG2 cells, respectively. The results of this study indicate that differential expression levels of inlA and inlB possibly play a role in the virulence capacities of L. monocytogenes strains. The lower capacity of clinical strains to invade HepG2 cells and to induce IL-8 is possibly a mechanism of immune evasion used by specific L. monocytogenes strains.
| S-EPMC1489604 | biostudies-literature
Project description:The facultative intracellular pathogen Listeria monocytogenes causes listeriosis, a rare but life-threatening disease. Host cell entry begins with activation of the human receptor tyrosine kinase MET through the bacterial invasion protein InlB, which contains an internalin domain, a B-repeat, and three GW domains. The internalin domain is known to bind MET, but no interaction partner is known for the B-repeat. Adding the B-repeat to the internalin domain potentiates MET activation and is required to stimulate Madin-Darby canine kidney (MDCK) cell scatter. Therefore, it has been hypothesized that the B-repeat may bind a co-receptor on host cells. To test this hypothesis, we mutated residues that might be important for binding an interaction partner. We identified two adjacent residues in strand ?2 of the ?-grasp fold whose mutation abrogated induction of MDCK cell scatter. Biophysical analysis indicated that these mutations do not alter protein structure. We then tested these mutants in human HT-29 cells that, in contrast to the MDCK cells, were responsive to the internalin domain alone. These assays revealed a dominant negative effect, reducing the activity of a construct of the internalin domain and mutated B-repeat below that of the individual internalin domain. Phosphorylation assays of MET and its downstream targets AKT and ERK confirmed the dominant negative effect. Attempts to identify a host cell receptor for the B-repeat were not successful. We conclude that there is limited support for a co-receptor hypothesis and instead suggest that the B-repeat contributes to MET activation through low affinity homodimerization.
| S-EPMC5207255 | biostudies-literature
Project description:Septins are filament-forming GTPases implicated in several cellular functions, including cytokinesis. We previously showed that SEPT2, SEPT9, and SEPT11 colocalize with several bacteria entering into mammalian non-phagocytic cells, and SEPT2 was identified as essential for this process. Here, we investigated the function of SEPT11, an interacting partner of SEPT9 whose function is still poorly understood. In uninfected HeLa cells, SEPT11 depletion by siRNA increased cell size but surprisingly did not affect actin filament formation or the colocalization of SEPT9 with actin filaments. SEPT11 depletion increased Listeria invasion, and incubating SEPT11-depleted cells with beads coated with the Listeria surface protein InlB also led to increased entry as compared with control cells. Strikingly, as shown by fluorescence resonance energy transfer, the InlB-mediated stimulation of Met signaling remained intact in SEPT11-depleted cells. Taken together, our results show that SEPT11 is not required for the bacterial entry process and rather restricts its efficacy. Because SEPT2 is essential for the InlB-mediated entry of Listeria, but SEPT11 is not, our findings distinguish the roles of different mammalian septins.
| S-EPMC2670166 | biostudies-literature
Project description:Listeria monocytogenes can cause a severe invasive food-borne disease known as listeriosis, and large outbreaks of this disease occur occasionally. Based on molecular-subtype data, epidemic clone (EC) strains have been defined, including ECI and ECIa, which have caused listeriosis outbreaks on different continents. While a number of molecular-subtyping studies of outbreak strains have been reported, few comprehensive data sets of virulence-associated characteristics of these strains are available. We assembled a set of human clinical isolates from 15 outbreaks that occurred worldwide between 1975 and 2002. Initial characterization of these strains showed significant variation in the ability to invade human Caco-2 intestinal epithelial cells and HepG2 hepatic cells; four strains showed consistently reduced invasion in both cell lines. DNA sequencing of inlA, which encodes a protein required for efficient Caco-2 and HepG2 invasion, showed that none of the invasion-attenuated strains contained known virulence-attenuating mutations in inlA. Phylogenetic analyses of inlA sequences revealed a well-supported clade containing a fully invasive ECI strain and three invasion-attenuated ECI strains, along with a fully invasive ECIa strain and an invasion-attenuated ECIa strain. Of the four invasion-attenuated strains, one strain showed both reduced inlA transcript levels and impaired swarming, one strain showed reduced inlA transcript levels, and two strains showed reduced swarming. Overall, our data show that (i) L. monocytogenes strains from outbreaks vary significantly in invasion efficiency and (ii) different mechanisms may contribute to reduced invasion efficiency. Association between EC strains and listeriosis outbreaks may involve characteristics other than virulence phenotypes, including survival and growth in food-associated environments.
| S-EPMC2737929 | biostudies-literature
Project description:Septins are conserved GTPases that form filaments and are required for cell division. During interphase, septin filaments associate with cellular membrane and cytoskeleton networks, yet the functional significance of these associations have, to our knowledge, remained unknown. We recently discovered that different septins, SEPT2 and SEPT11, regulate the InlB-mediated entry of Listeria monocytogenes into host cells. Here we address the role of SEPT2 and SEPT11 in the InlB-Met interactions underlying Listeria invasion to explore how septins modulate surface receptor function. We observed that differences in InlB-mediated Listeria entry correlated with differences in Met surface expression caused by septin depletion. Using atomic force microscopy on living cells, we show that septin depletion significantly reduced the unbinding force of InlB-Met interaction and the viscosity of membrane tethers at locations where the InlB-Met interaction occurs. Strikingly, the same order of difference was observed for cells in which the actin cytoskeleton was disrupted. Consistent with a proposed role of septins in association with the actin cytoskeleton, we show that cell elasticity is decreased upon septin or actin inactivation. Septins are therefore likely to participate in anchorage of the Met receptor to the actin cytoskeleton, and represent a critical determinant in surface receptor function.
| S-EPMC3077699 | biostudies-literature
Project description:InlB, a surface-localized protein of Listeria monocytogenes, induces phagocytosis in non-phagocytic mammalian cells by activating Met, a receptor tyrosine kinase. InlB also binds glycosaminoglycans and the protein gC1q-R, two additional host ligands implicated in invasion. We present the structure of InlB, revealing a highly elongated molecule with leucine-rich repeats that bind Met at one end, and GW domains that dissociably bind the bacterial surface at the other. Surprisingly, the GW domains are seen to resemble SH3 domains. Despite this, GW domains are unlikely to act as functional mimics of SH3 domains since their potential proline-binding sites are blocked or destroyed. However, we do show that the GW domains, in addition to binding glycosaminoglycans, bind gC1q-R specifically, and that this binding requires release of InlB from the bacterial surface. Dissociable attachment to the bacterial surface via the GW domains may be responsible for restricting Met activation to a small, localized area of the host cell and for coupling InlB-induced host membrane dynamics with bacterial proximity during invasion.
| S-EPMC131055 | biostudies-literature
Project description:The intracellular pathogen Listeria monocytogenes is distinguished by its ability to invade and replicate within mammalian cells. Remarkably, of the 15 serovars within the genus, strains belonging to serovar 4b cause the majority of listeriosis clinical cases and outbreaks. The Listeria O-antigens are defined by subtle structural differences amongst the peptidoglycan-associated wall-teichoic acids (WTAs), and their specific glycosylation patterns. Here, we outline the genetic determinants required for WTA decoration in serovar 4b L. monocytogenes, and demonstrate the exact nature of the 4b-specific antigen. We show that challenge by bacteriophages selects for surviving clones that feature mutations in genes involved in teichoic acid glycosylation, leading to a loss of galactose from both wall teichoic acid and lipoteichoic acid molecules, and a switch from serovar 4b to 4d. Surprisingly, loss of this galactose decoration not only prevents phage adsorption, but leads to a complete loss of surface-associated Internalin B (InlB),the inability to form actin tails, and a virulence attenuation in vivo. We show that InlB specifically recognizes and attaches to galactosylated teichoic acid polymers, and is secreted upon loss of this modification, leading to a drastically reduced cellular invasiveness. Consequently, these phage-insensitive bacteria are unable to interact with cMet and gC1q-R host cell receptors, which normally trigger cellular uptake upon interaction with InlB. Collectively, we provide detailed mechanistic insight into the dual role of a surface antigen crucial for both phage adsorption and cellular invasiveness, demonstrating a trade-off between phage resistance and virulence in this opportunistic pathogen.
| S-EPMC6779246 | biostudies-literature
Project description:The host cytoskeleton is a major target for bacterial pathogens during infection. In particular, pathogens usurp the actin cytoskeleton function to strongly adhere to the host cell surface, to induce plasma membrane remodeling allowing invasion and to spread from cell to cell and disseminate to the whole organism. Keratins are cytoskeletal proteins that are the major components of intermediate filaments in epithelial cells however, their role in bacterial infection has been disregarded. Here we investigate the role of the major epithelial keratins, keratins 8 and 18 (K8 and K18), in the cellular infection by Listeria monocytogenes. We found that K8 and K18 are required for successful InlB/cMet-dependent L. monocytogenes infection, but are dispensable for InlA/E-cadherin-mediated invasion. Both K8 and K18 accumulate at InlB-mediated internalization sites following actin recruitment and modulate actin dynamics at those sites. We also reveal the key role of K8 and K18 in HGF-induced signaling which occurs downstream the activation of cMet. Strikingly, we show here that K18, and at a less extent K8, controls the expression of cMet and other surface receptors such TfR and integrin ?1, by promoting the stability of their corresponding transcripts. Together, our results reveal novel functions for major epithelial keratins in the modulation of actin dynamics at the bacterial entry sites and in the control of surface receptors mRNA stability and expression.
| S-EPMC5960701 | biostudies-literature
Project description:Listeria monocytogenes is an intracellular bacterial pathogen that invades epithelial cells by subverting two cellular receptors, E-cadherin and Met. We recently identified type II phosphatidylinositol 4-kinases alpha and beta (PI4KIIalpha and PI4KIIbeta) as being required for bacterial entry downstream of Met. In this work, we investigated whether tetraspanins CD9, CD63, and CD81, which figure among the few described molecular partners of PI4KIIalpha, function as molecular adaptors recruiting PI4KIIalpha to the bacterial entry site. We observed by fluorescence microscopy that CD9, CD63, and CD81 are expressed and detected at the cellular surface and also within intracellular compartments, particularly in the case of CD63. In resting cells, colocalization of tetraspanins and PI4KIIalpha is detectable only in restricted areas of the perinuclear region. Upon infection with Listeria, endogenous CD9, CD63, and CD81 were recruited to the bacterial entry site but did not colocalize strictly with endogenous PI4KIIalpha. Live-cell imaging confirmed that tetraspanins and PI4KIIalpha do not follow the same recruitment dynamics to the Listeria entry site. Depletion of CD9, CD63, and CD81 levels by small interfering RNA demonstrated that CD81 is required for bacterial internalization, identifying for the first time a role for a member of the tetraspanin family in the entry of Listeria into target cells. Moreover, depletion of CD81 inhibits the recruitment of PI4KIIalpha but not that of the Met receptor to the bacterial entry site, suggesting that CD81 may act as a membrane organizer required for the integrity of signaling events occurring at Listeria entry sites.
| S-EPMC2798227 | biostudies-literature