Lmo0171, a novel internalin-like protein, determines cell morphology of Listeria monocytogenes and its ability to invade human cell lines.
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ABSTRACT:
SUBMITTER: Stachowiak R
PROVIDER: S-EPMC4293459 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Listeria monocytogenes (Lm) uses InlA to invade the tips of the intestinal villi, a location at which cell extrusion generates a transient defect in epithelial polarity that exposes the receptor for InlA, E-cadherin, on the cell surface. As the dying cell is removed from the epithelium, the surrounding cells reorganize to form a multicellular junction (MCJ) that Lm exploits to find its basolateral receptor and invade. By examining individual infected villi using 3D-confocal imaging, we uncovered a novel role for the second major invasin, InlB, during invasion of the intestine. We infected mice intragastrically with isogenic strains of Lm that express or lack InlB and that have a modified InlA capable of binding murine E-cadherin and found that Lm lacking InlB invade the same number of villi but have decreased numbers of bacteria within each infected villus tip. We studied the mechanism of InlB action at the MCJs of polarized MDCK monolayers and find that InlB does not act as an adhesin, but instead accelerates bacterial internalization after attachment. InlB locally activates its receptor, c-Met, and increases endocytosis of junctional components, including E-cadherin. We show that MCJs are naturally more endocytic than other sites of the apical membrane, that endocytosis and Lm invasion of MCJs depends on functional dynamin, and that c-Met activation by soluble InlB or hepatocyte growth factor (HGF) increases MCJ endocytosis. Also, in vivo, InlB applied through the intestinal lumen increases endocytosis at the villus tips. Our findings demonstrate a two-step mechanism of synergy between Lm's invasins: InlA provides the specificity of Lm adhesion to MCJs at the villus tips and InlB locally activates c-Met to accelerate junctional endocytosis and bacterial invasion of the intestine.
| S-EPMC2869327 | biostudies-literature
Project description:Listeria monocytogenes (Lm) is an invasive foodborne pathogen that leads to severe central nervous system and maternal-fetal infections. Lm ability to actively cross the intestinal barrier is one of its key pathogenic properties. Lm crosses the intestinal epithelium upon the interaction of its surface protein internalin (InlA) with its host receptor E-cadherin (Ecad). InlA-Ecad interaction is species-specific, does not occur in wild-type mice, but does in transgenic mice expressing human Ecad and knock-in mice expressing humanized mouse Ecad. To study listeriosis in wild-type mice, InlA has been "murinized" to interact with mouse Ecad. Here, we demonstrate that, unexpectedly, murinized InlA (InlA(m)) mediates not only Ecad-dependent internalization, but also N-cadherin-dependent internalization. Consequently, InlA(m)-expressing Lm targets not only goblet cells expressing luminally-accessible Ecad, as does Lm in humanized mice, but also targets villous M cells, which express luminally-accessible N-cadherin. This aberrant Lm portal of entry results in enhanced innate immune responses and intestinal barrier damage, both of which are not observed in wild-type Lm-infected humanized mice. Murinization of InlA therefore not only extends the host range of Lm, but also broadens its receptor repertoire, providing Lm with artifactual pathogenic properties. These results challenge the relevance of using InlA(m)-expressing Lm to study human listeriosis and in vivo host responses to this human pathogen.
| S-EPMC3667765 | biostudies-literature
Project description:Listeria monocytogenes expresses surface proteins covalently anchored to the peptidoglycan by sortase enzymes. Inactivation of srtA attenuates Listeria virulence in mice (H. Bierne, S. K. Mazmanian, M. Trost, M. G. Pucciarelli, G. Liu, P. Dehoux, L. Jansch, F. Garcia-del Portillo, O. Schneewind, and P. Cossart, Mol. Microbiol. 43:869-881, 2002). We show here that an srtA mutant is more attenuated than an internalin mutant in orally infected guinea pigs and transgenic mice expressing human E-cadherin (hEcad mice), indicating the involvement of other SrtA substrates, LPXTG proteins, in food-borne listeriosis. Data recently generated with a listerial DNA macroarray identified two LPXTG protein-encoding genes present in the genomes of L. monocytogenes strains and absent from all other Listeria species, inlI (lmo0333) and inlJ (lmo2821). They also revealed two other LPXTG protein-encoding genes, ORF29 and ORF2568, present only in a subclass of L. monocytogenes serovars, including the epidemic serovar 4b. We report here that an inlJ deletion mutant, in contrast to inlI and ORF29 mutants, is significantly attenuated in virulence after intravenous infection of mice or oral inoculation of hEcad mice. Interestingly, a DeltaORF2568 strain showed a slight increase in virulence. inlJ encodes a leucine-rich repeat (LRR) protein that is structurally related to the listerial invasion factor internalin. However, the consensus sequence of the InlJ LRR defines a novel subfamily of cysteine-containing LRRs in bacteria. In conclusion, this postgenomic approach identified InlJ as a new virulence factor among the proteins belonging to the internalin family in L. monocytogenes.
| S-EPMC1230919 | biostudies-literature
Project description:In order to clarified characteristics and function of internalin G (inlG) in Listeria monocytogenes ATCC®19111 (1/2a) (LM), the immune protection of the inlG was evaluated in mice, the homologous recombination was used to construct inlG deletion strains, and their biological characteristics were studied by the transcriptomics analysis. As a result, the immunization of mice with the purified protein achieved a protective effect against bacterial infection. The deletion strain LM-AinlG was successfully constructed with genetic stability. The mouse infection test showed that the virulence of LM was decreased after the deletion of the inlG gene. The deletion strain showed enhanced adhesion to and invasion of Caco-2 cells. Compared to the wild strain, 18 genes were up-regulated, and 24 genes were down-regulated in the LM-AinlG. This study has laid a foundation for further research on the function of inlG and the pathogenesis of LM. In this study, immunization of mice with the purified inlG protein achieved a protective effect against Listeria monocytogenes infection. The virulence of LM-ΔinlG was decreased by mouse infection. However, the adhesion and invasion ability to Caco-2 cell were enhanced. Compared to the wild strain, 18 genes were up-regulated, and 24 genes were down-regulated in the LM-ΔinlG. This study has laid a foundation for further study of the function of the inlG and the listeriosis.
| S-EPMC9152910 | biostudies-literature
Project description:Internalin A (InlA), a protein required for Listeria monocytogenes virulence, is encoded by the inlA gene, which is only found in pathogenic strains of this genus. One of the best ways to detect and confirm the pathogenicity of the strain is the detection of one of the virulence factors produced by the microorganism. This paper focuses on the design of an electrochemical genosensor used to detect the inlA gene in Listeria strains without labelling the target DNA. The electrochemical sensor was obtained by immobilising an inlA gene probe (single-stranded oligonucleotide) on the surfaces of screen-printed gold electrodes (Au-SPEs) by means of a mercaptan-activated self-assembled monolayer (SAM). The hybridisation reaction occurring on the electrode surface was electrochemically transduced by differential pulse voltammetry (DPV) using methylene blue (MB) as an indicator. The covalently immobilised single-stranded DNA was able to selectively hybridise to its complementary DNA sequences in solution to form double-stranded DNA on the gold surface. A significant decrease of the peak current of the voltammogram (DPV) upon hybridisation of immobilised ssDNA was recorded. Whole DNA samples of L. monocytogenes strains could be discriminated from other nonpathogenic Listeria species DNA with the inlA gene DNA probe genosensor.
| S-EPMC3618917 | biostudies-literature
Project description:The effects of environmental stress exposure on Listeria monocytogenes growth and virulence-associated characteristics were investigated. Specifically, we measured the effects of temperature (7 or 37 degrees C), pH (5.5 or 7.4), the presence of salt and organic acids (375 mM NaCl, 8.45 mM sodium diacetate [SD], 275 mM sodium lactate [SL], or a combination of NaCl, SD, and SL), and deletion of sigB, which encodes a key stress response regulator, on the ability of L. monocytogenes to grow, invade Caco-2 cells, and survive exposure to synthetic gastric fluid (pH 2.5 or 4.5). Our results indicate that (i) L. monocytogenes log-phase generation times and maximum cell numbers are not dependent on the alternative sigma factor sigmaB in the presence of NaCl and organic acids at concentrations typically found in foods; (ii) growth inhibition of L. monocytogenes through the addition of organic acids is pH dependent; (iii) the ability of L. monocytogenes to invade Caco-2 cells is affected by growth phase, temperature, and the presence of salt and organic acids, with the highest relative invasion capabilities observed for cells grown with SL or NaCl at 37 degrees C and pH 7.4; (iv) growth of L. monocytogenes in the presence of NaCl, SD, or SL reduces its ability to survive exposure to gastric fluid; and (v) exposure of L. monocytogenes to gastric fluid reduces the enhanced invasiveness caused by growth in the presence of NaCl or SL. These findings suggest that virulence-associated characteristics that determine the L. monocytogenes infectious dose are likely to be affected by food-specific properties (e.g., pH or the presence of salt or organic acid).
| S-EPMC1538756 | biostudies-literature
Project description:Hepatocyte growth factor (HGF) is critical for tissue homeostasis and repair in many organs including the lung, heart, kidney, liver, nervous system, and skin. HGF is a heterodimeric protein containing 20 disulfide bonds distributed among an amino-terminal hairpin, four kringle domains, and a serine protease-like domain. Due to its complex structure, recombinant production of HGF in prokaryotes requires denaturation and refolding, processes that are impractical for large-scale manufacture. Thus, pharmaceutical quantities of HGF are not available despite its potential applications. A fragment of the Listeria monocytogenes internalin B protein from amino acids 36-321 (InlB₃₆₋₃₂₁) was demonstrated to bind to and partially activate the HGF receptor Met. InlB₃₆₋₃₂₁ has a stable β-sheet structure and is easily produced in its native conformation by Escherichia coli. We cloned InlB₃₆₋₃₂₁ (1×InlB₃₆₋₃₂₁) and engineered a head-to-tail repeat of InlB₃₆₋₃₂₁ with a linker peptide (2×InlB₃₆₋₃₂₁); 1×InlB₃₆₋₃₂₁ and 2×InlB₃₆₋₃₂₁ were purified from E. coli. Both 1× and 2×InlB₃₆₋₃₂₁ activated the Met tyrosine kinase. We subsequently compared signal transduction of the two proteins in primary lung endothelial cells. 2×InlB₃₆₋₃₂₁ activated ERK1/2, STAT3, and phosphatidylinositol 3-kinase/Akt pathways, whereas 1×InlB₃₆₋₃₂₁ activated only STAT3 and ERK1/2. The 2×InlB₃₆₋₃₂₁ promoted improved motility compared with 1×InlB₃₆₋₃₂₁ and additionally stimulated proliferation equivalent to full-length HGF. Both the 1× and 2×InlB₃₆₋₃₂₁ prevented apoptosis by the profibrotic peptide angiotensin II in cell culture and ex vivo lung slice cultures. The ease of large-scale production and capacity of 2×InlB₃₆₋₃₂₁ to mimic HGF make it a potential candidate as a pharmaceutical agent for tissue repair.
| S-EPMC3006264 | biostudies-literature
Project description:The Listeria monocytogenes genome contains genes encoding several internalins and internalin-like proteins. As L. monocytogenes is present in many environments and can infect numerous, diverse host species, the environmental temperature was hypothesized to be a signal that might affect internalin gene transcription. A subgenomic microarray was used to investigate temperature-dependent transcription of 24 members of the internalin gene family in L. monocytogenes 10403S. The levels of internalin gene transcripts for cells grown at 37 degrees C were compared to the levels of transcripts for cells grown at 16, 30, and 42 degrees C using competitive microarray hybridization, and the results were confirmed by performing quantitative reverse transcriptase PCR for 14 internalin genes. Based on these studies, the internalin genes can be grouped into the following five temperature-dependent categories: (i) four sigma(B)-dependent internalin genes (inlC2, inlD, lmo0331, and lmo0610) with the highest levels of transcripts at 16 degrees C and generally the lowest levels of transcripts at 37 degrees C; (ii) three partially PrfA-dependent internalin genes (inlA, inlB, and inlC) with the lowest levels of transcripts at 16 degrees C and the highest levels of transcripts at 37 and 42 degrees C; (iii) four genes (inlG, inlJ, lmo0514, and lmo1290) with the lowest levels of transcripts at 16 degrees C and the highest levels of transcripts at 30 and/or 37 degrees C; (iv) one gene (lmo0327) with the highest levels of transcripts at 16 degrees C and low levels of transcripts at higher temperatures; and (v) 12 internalin genes with no differences in the levels of transcripts at the temperatures used in this study. The temperature-dependent transcription patterns suggest that the relative importance of different internalins varies by environment, which may provide insight into the specific functions of these proteins.
| S-EPMC1892884 | biostudies-literature
Project description:The Listeria monocytogenes genome contains more than 20 genes that encode cell surface-associated internalins. To determine the contributions of the alternative sigma factor sigma(B) and the virulence gene regulator PrfA to internalin gene expression, a subgenomic microarray was designed to contain two probes for each of 24 internalin-like genes identified in the L. monocytogenes 10403S genome. Competitive microarray hybridization was performed on RNA extracted from (i) the 10403S parent strain and an isogenic Delta sigB strain; (ii) 10403S and an isogenic Delta prfA strain; (iii) a (G155S) 10403S derivative that expresses the constitutively active PrfA (PrfA*) and the Delta prfA strain; and (iv) 10403S and an isogenic Delta sigB Delta prfA strain. Sigma(B)- and PrfA-dependent transcription of selected genes was further confirmed by quantitative reverse-transcriptase polymerase chain reaction. For the 24 internalin-like genes examined, (i) both sigma(B) and PrfA contributed to transcription of inlA and inlB, (ii) only sigma(B) contributed to transcription of inlC2, inlD, lmo0331, and lmo0610; (iii) only PrfA contributed to transcription of inlC and lmo2445; and (iv) neither sigma(B) nor PrfA contributed to transcription of the remaining 16 internalin-like genes under the conditions tested.
| S-EPMC2688707 | biostudies-literature
Project description:BackgroundEcological, biochemical and genetic resemblance as well as clear differences of virulence between L. monocytogenes and L. innocua make this bacterial clade attractive as a model to examine evolution of pathogenicity. This study was attempted to examine the population structure of L. innocua and the microevolution in the L. innocua-L. monocytogenes clade via profiling of 37 internalin genes and multilocus sequence typing based on the sequences of 9 unlinked genes gyrB, sigB, dapE, hisJ, ribC, purM, gap, tuf and betL.ResultsL. innocua was genetically monophyletic compared to L. monocytogenes, and comprised four subgroups. Subgroups A and B correlated with internalin types 1 and 3 (except the strain 0063 belonging to subgroup C) and internalin types 2 and 4 respectively. The majority of L. innocua strains belonged to these two subgroups. Subgroup A harbored a whole set of L. monocytogenes-L. innocua common and L. innocua-specific internalin genes, and displayed higher recombination rates than those of subgroup B, including the relative frequency of occurrence of recombination versus mutation (rho/theta) and the relative effect of recombination versus point mutation (r/m). Subgroup A also exhibited a significantly smaller exterior/interior branch length ratio than expected under the coalescent model, suggesting a recent expansion of its population size. The phylogram based on the analysis with correction for recombination revealed that the time to the most recent common ancestor (TMRCA) of L. innocua subgroups A and B were similar. Additionally, subgroup D, which correlated with internalin type 5, branched off from the other three subgroups. All L. innocua strains lacked seventeen virulence genes found in L. monocytogenes (except for the subgroup D strain L43 harboring inlJ and two subgroup B strains bearing bsh) and were nonpathogenic to mice.ConclusionsL. innocua represents a young species descending from L. monocytogenes and comprises four subgroups: two major subgroups A and B, and one atypical subgroup D serving as a link between L. monocytogenes and L. innocua in the evolutionary chain. Although subgroups A and B appeared at approximately the same time, subgroup A seems to have experienced a recent expansion of the population size with higher recombination frequency and effect than those of subgroup B, and might represent the possible evolutionary direction towards adaptation to environments. The evolutionary history in the L. monocytogenes-L. innocua clade represents a rare example of evolution towards reduced virulence of pathogens.
| S-EPMC2867954 | biostudies-literature