Cytochalasin D induces increased actin synthesis in HEp-2 cells.

In HEp-2 cells treated with 0.2 to 2.0 microM cytochalasin D (CD) for 7.5 to 24 h there was a 20 to 50% relative increase in actin content (units of actin per microgram of total cell protein). This augmentation, which was concentration and time dependent, was prevented by treatment with cycloheximide during exposure to CD. A 15 to 20% increase in the relative rate of actin synthesis in CD-treated HEp-2 cells (0.2 to 2.0 microM CD) was detectable after 1 h of treatment and increased to 30 to 50% by 24 h. This increased rate of actin synthesis was apparently responsible for the higher actin content of CD-treated HEp-2 cells. The concentration dependence of these effects of CD on actin metabolism correlated with the pattern seen for CD-triggered changes in cellular morphology and the underlying rearrangements of the actin-containing cytoskeletal structures, suggesting that the effects on metabolism and morphology were interrelated. Since the rapidly occurring cytoskeletal reorganization preceded the effects of CD on actin metabolism, it is proposed that actin synthesis is induced by the cytoskeletal rearrangement resulting from exposure to CD.     

Non-Invasive Providencia alcalifaciens Strains Fail to Attach to HEp-2 Cells

We investigated the adherence properties of six P. alcalifaciens strains with previously characterized differential invasive capabilities in HEp-2 cells. Highly invasive strains were found to attach to HEp-2 cell monolayers within 2 h post-infection and in large numbers on the eukaryotic cell surfaces within 3 h post-infection. In contrast, weakly or non-invasive P. alcalifaciens strains were non-adherent to HEp-2 cells even at 3 h post-infection. Highly invasive isolates were found to weakly bind F-actin using the fluorescent actin staining assay although these strains were negative for Escherichia coli attachment and effacing gene (eaeA) of enteropathogenic E. coli (EPEC). These results suggest that the strain variation in the ability of P. alcalifaciens to invade HEp-2 cells previously noted by several investigators may be linked to expression of key adhesin(s) on the cell surface of invasive isolates. Received: 10 April 2001 / Accepted: 17 April 2001     

Synergy between type 1 fimbriae expression and C3 opsonisation increases internalisation of E. coli by human tubular epithelial cells

Background  

Bacterial infection of the urinary tract is a common clinical problem with E. coli being the most common urinary pathogen. Bacterial uptake into epithelial cells is increasingly recognised as an important feature of infection. Bacterial virulence factors, especially fimbrial adhesins, have been conclusively shown to promote host cell invasion. Our recent study reported that C3 opsonisation markedly increases the ability of E. coli strain J96 to internalise into human proximal tubular epithelial cells via CD46, a complement regulatory protein expressed on host cell membrane. In this study, we further assessed whether C3-dependent internalisation by human tubular epithelial cells is a general feature of uropathogenic E. coli and investigated features of the bacterial phenotype that may account for any heterogeneity.     

Identification of entero-aggregative Escherichia coli based on surface properties

Twenty-nine strains of Escherichia coli that adhere to HEp-2 cells with a'stacked brick' pattern (EAggEC), and four nonadherent control strains, wereexamined for the ability to hybridize with gene probes for aggregative (AA) and diffuse (DA)HEp-2 cell adhesion phenotypes. These strains were also tested for the ability to express an 18 kDa membrane-associated outer- membrane protein (MAP), to agglutinate erythrocytes, and toproduce a pellicle during broth culture. Thirteen of the 29 HEp-2 adherent strains of E. coli hybridized with the gene probes for both AA and DA, and expressed an 18 kDa outermembrane protein (OMP) which was antigenically related to the MAP expressed by strains of E. coli O126:H27. The strains that did not carry the additional DA genes did notexpress an 18 kDa OMP. Although strains of EAggEC share the ability to adhere to HEp-2 cellswith a stacked brick pattern, these strains exhibit a diverse range of physical and biochemicalproperties. From the results of this study, it was concluded that currently, the possession ofEAggEC genes or the ability to adhere to HEp-2 cells in a stacked brick formation, remain theonly reliable means of identifying EAggEC.     

Identification of invasin: a protein that allows enteric bacteria to penetrate cultured mammalian cells

Bacterial strains harboring the Yersinia pseudotuberculosis inv locus were analyzed in order to investigate the mechanism of host cell penetration by an invasive pathogen. The inv locus was found to be necessary for Y. pseudotuberculosis to enter HEp-2 cells and sufficient to convert E. coli into a microorganism able to penetrate cultured cells. Both E. coli and Y. pseudotuberculosis strains harboring inv mutations were defective for entry into HEp-2 cells. Furthermore, molecular clones containing inv, and little additional DNA, converted E. coli into a microorganism that was indistinguishable from the parental Yersinia strain with regard to the entry of cultured cells. Data from in vitro protein synthesis indicated that a 103 kd protein was synthesized from inv, saturating the coding capacity of the locus. The nucleotide sequence shows an open reading frame corresponding to a protein of similar size. This protein, called invasin, is necessary for the microorganisms to penetrate HEp-2 cells, and is compartmentalized on the outer surface of the bacterium.     

Apoptosis in HEp-2 cells infected with Ureaplasma diversum

Background

Bacterial pathogens have many strategies for infecting and persisting in host cells. Adhesion, invasion and intracellular life are important features in the biology of mollicutes. The intracellular location of Ureaplasma diversum may trigger disturbances in the host cell. This includes activation or inhibition of pro and anti-apoptotic factors, which facilitate the development of host damage. The aim of the present study was to associate U. diversum infection in HEp-2 cells and apoptosis induction. Cells were infected for 72hs with four U. diversum clinical isolates and an ATCC strain. The U. diversum invasion was analyzed by Confocal Laser Scanning Microscopy and gentamicin invasion assay. The apoptosis was evaluated using pro-apoptotic and anti-apoptotic gene expression, and FITC Annexin V/Dead Cell Apoptosis Kit.

Results

The number of internalized ureaplasma in HEp-2 cells increased significantly throughout the infection. The flow cytometry analysis with fluorochromes to detect membrane depolarization and gene expression for caspase 2, 3 and 9 increased in infected cells after 24 hours. However, after 72 hours a considerable decrease of apoptotic cells was observed.

Conclusions

The data suggests that apoptosis may be initially induced by some isolates in association with HEp-2 cells, but over time, there was no evidence of apoptosis in the presence of ureaplasma and HEp-2 cells. The initial increase and then decrease in apoptosis could be related to bacterial pathogen-associated molecular pattern (PAMPS). Moreover, the isolates of U. diversum presented differences in the studied parameters for apoptosis. It was also observed that the amount of microorganisms was not proportional to the induction of apoptosis in HEp-2 cells.     

A role for host cell exocytosis in InlB‐mediated internalisation of Listeria monocytogenes

The bacterial surface protein InlB mediates internalisation of Listeria monocytogenes into human cells through interaction with the host receptor tyrosine kinase, Met. InlB‐mediated entry requires localised polymerisation of the host actin cytoskeleton. Apart from actin polymerisation, roles for other host processes in Listeria entry are unknown. Here, we demonstrate that exocytosis in the human cell promotes InlB‐dependent internalisation. Using a probe consisting of VAMP3 with an exofacial green fluorescent protein tag, focal exocytosis was detected during InlB‐mediated entry. Exocytosis was dependent on Met tyrosine kinase activity and the GTPase RalA. Depletion of SNARE proteins by small interfering RNA demonstrated an important role for exocytosis in Listeria internalisation. Depletion of SNARE proteins failed to affect actin filaments during internalisation, suggesting that actin polymerisation and exocytosis are separable host responses. SNARE proteins were required for delivery of the human GTPase Dynamin 2, which promotes InlB‐mediated entry. Our results identify exocytosis as a novel host process exploited by Listeria for infection.     

Role of the cystic fibrosis transmembrane conductance regulator in internalization of Pseudomonas aeruginosa by polarized respiratory epithelial cells

Pseudomonas aeruginosa is an important human pathogen, producing lung infection in individuals with cystic fibrosis (CF), patients who are ventilated and those who are neutropenic. The respiratory epithelium provides the initial barrier to infection. Pseudomonas aeruginosa can enter epithelial cells, although the mechanism of entry and the role of intracellular organisms in its life cycle are unclear. We devised a model of infection of polarized human respiratory epithelial cells with P. aeruginosa and investigated the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in adherence, uptake and IL-8 production by human respiratory epithelial cells. We found that a number of P. aeruginosa strains could invade and replicate within cells derived from a patient with CF. Intracellular bacteria did not produce host cell cytotoxicity over a period of 24 h. When these cells were transfected with wild-type CFTR, uptake of bacteria was significantly reduced and release of IL-8 following infection enhanced. We propose that internalized P. aeruginosa may play an important role in the pathogenesis of infection and that, by allowing greater internalization into epithelial cells, mutant CFTR results in an increased susceptibility of bronchial infection with this microbe.     

Mycobacterium tuberculosis Mce3C promotes mycobacteria entry into macrophages through activation of β2 integrin‐mediated signalling pathway

Establishment of infection by facultative intracellular pathogen Mycobacterium tuberculosis (Mtb) requires adherence to and internalisation by macrophages. However, the effector molecules exploited by Mtb for entry into macrophages remain to be fully understood. The mammalian cell entry (Mce) proteins play an essential role in facilitating the internalisation of mycobacteria into mammalian cells. Here, we characterized Mtb Mce3C as a new mycobacterial surface protein that could promote mycobacterial adhesion to and invasion of macrophages in an RGD motif‐dependent manner. We then further demonstrated that β2 integrin was required for Mce3C‐mediated cell entry. In addition, we found that binding of Mce3C recruited β2 integrin‐dependent signalling adaptors and induced local actin rearrangement at the site of mycobacterial invasion. By using specific antibodies and pharmacological inhibitors, we further demonstrated the involvement of Src‐family tyrosine kinases, spleen tyrosine kinase, Vav, Rho, and Rho‐associated kinase in Mce3C‐mediated mycobacterial invasion. Our results reveal a novel mechanism by which Mtb Mce3C exploits integrin‐mediated signalling cascade for Mce, providing potential targets for the development of therapies against Mtb infection.     

Adherence Inhibition of <Emphasis Type="Italic">Cronobacter sakazakii</Emphasis> to Intestinal Epithelial Cells by Prebiotic Oligosaccharides

Cronobacter sakazakii is an opportunistic pathogen that has been implicated in meningitis, NEC, and sepsis in neonates. Colonization and subsequent infection and invasion of C. sakazakii require that the organism adheres to host cell surfaces. Agents that inhibit or block attachment of the pathogen to epithelial cells could be useful in reducing infections. The goal of this research was to assess the ability of prebiotic galactooligosaccharides (GOS) and polydextrose (PDX) to inhibit adherence of C. sakazakii 4603 to a HEp-2 human cell line. Adherence experiments were performed in the presence or absence of prebiotics using HEp-2 cells grown to confluency on glass coverslips. Prebiotics and bacteria were added and incubated for 3 h. Coverslips were washed, and adherence was determined by cultural and microscopic methods. When measured microscopically or by cultural methods, significant reductions in adherence (56 and 71%, respectively) of C. sakazakii were observed in the presence of GOS (16 mg/ml). Adherence inhibition also occurred (48%) when a GOS–PDX blend (8 mg/ml each) was tested, although PDX by itself had less effect. Similar results were also observed for Caco-2 cells and also for another strain of C. sakazakii (29004). These results suggest that GOS and PDX, alone and in combination, may have an anti-adhesive effect on C. sakazakii and directly inhibit the adherence to gastrointestinal epithelial cells.     

Relationship between organization of the actin cytoskeleton and the cell cycle in normal and adenovirus-infected rat cells.

Flow cytometry and staining with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin were used to investigate organization of the actin cytoskeleton in rat embryo cells at different stages of normal and adenovirus E1A-induced cell cycles. In uninfected cells in G0-G1 and S phases, actin was predominantly in the form of stress fibers. In G2, this organization changed to peripheral rings of thin filaments, while during mitosis, actin had a diffuse distribution. Infection of quiescent rat cells by adenovirus caused them to enter the cell cycle and replicate DNA and also caused disruption of stress fibers. Rapid disappearance of stress fibers and the appearance of peripheral rings of actin filaments began from 13 h after infection and closely followed synthesis of the E1A proteins. Infected cells began S phase at about 24 h after infection, and cells in G2 and mitosis were seen from 30 to 50 h. Thus, disruption of the actin cytoskeleton is an early effect of E1A and not an indirect consequence of the entry of infected cells into the cell cycle.     

A new host cell internalisation pathway for SadA‐expressing staphylococci triggered by excreted neurochemicals

Staphylococcus aureus is a facultative intracellular pathogen that invades a wide range of professional and nonprofessional phagocytes by triggering internalisation by interaction of surface‐bound adhesins with corresponding host cell receptors. Here, we identified a new concept of host cell internalisation in animal‐pathogenic staphylococcal species. This new mechanism exemplified by Staphylococcus pseudintermedius ED99 is not based on surface‐bound adhesins but is due to excreted small neurochemical compounds, such as trace amines (TAs), dopamine (DOP), and serotonin (SER), that render host cells competent for bacterial internalisation. The neurochemicals are produced by only one enzyme, the staphylococcal aromatic amino acid decarboxylase (SadA). Here, we unravelled the mechanism of how neurochemicals trigger internalisation into the human colon cell line HT‐29. We found that TAs and DOP are agonists of the α2‐adrenergic receptor, which, when activated, induces a cascade of reactions involving a decrease in the cytoplasmic cAMP level and an increase in F‐actin formation. The signalling cascade of SER follows a different pathway. SER interacts with 5HT receptors that trigger F‐actin formation without decreasing the cytoplasmic cAMP level. The neurochemical‐induced internalisation in host cells is independent of the fibronectin‐binding protein pathway and has an additive effect. In a sadA deletion mutant, ED99ΔsadA, internalisation was decreased approximately threefold compared with that of the parent strain, and treating S. aureus USA300 with TAs increased internalisation by approximately threefold.     

Helicobacter pylori infection interferes with epithelial Stat6-mediated interleukin-4 signal transduction independent of cagA,cagE, or VacA

Helicobacter pylori is a bacterial pathogen evolved to chronically colonize the gastric epithelium, evade immune clearance by the host, and cause gastritis, peptic ulcers, and even gastric malignancies in some infected humans. In view of the known ability of this bacterium to manipulate gastric epithelial cell signal transduction cascades, we determined the effects of H. pylori infection on epithelial IL-4-Stat6 signal transduction. HEp-2 and MKN45 epithelial cells were infected with H. pylori strains LC11 or 8823 (type 1; cagA(+)/cagE(+)/VacA(+)), LC20 (type 2; cagA(-), cagE(-), VacA(-)), and cagA, cagE, and vacA isogenic mutants of strain 8823, with some cells receiving subsequent treatment with the Th2 cytokine IL-4, a known Stat6 activator. Immunofluorescence showed a disruption of Stat6-induced nuclear translocation by IL-4 in LC11-infected HEp-2 cells. IL-4-inducible Stat6 DNA binding in HEp-2 and MKN45 cells was abrogated by infection, but MKN45 cell viability was unaffected. A decrease in IL-4-mediated Stat6 tyrosine phosphorylation in nuclear and whole cell lysates was also observed following infection with strains LC11 and LC20, while neither strain altered IL-4 receptor chain alpha or Janus kinase 1 protein expression. Furthermore, parental strain 8823 and its isogenic cagA, cagE, and vacA mutants also suppressed IL-4-induced Stat6 tyrosine phosphorylation to comparable degrees. Thus, H. pylori did not directly activate Stat6, but blocked the IL-4-induced activation of epithelial Stat6. This may represent an evolutionarily conserved strategy to disrupt a Th2 response and evade the host immune system, allowing for successful chronic infection.     

Calcium signalling during cell interactions with bacterial pathogens

The ability of a pathogenic microorganism to cause a disease is conditioned by its ability to colonise a given niche and implicates the expression of specific determinants, i.e. virulence factors, that allow the pathogen to adhere to or to invade epithelial cells. Diseases may be induced by bacteria that replicate extracellularly and alter the epithelial mucosa by producing toxins. Ca2+ signalling has been implicated in various steps of bacterial infection. Bacterial toxins can induce an increase in free cytosolic Ca2+ in host cells, itself required for the toxin-mediated effects. Such toxins, by diffusing in the extracellular media, can act at a distance from the site of infection and have a global effect on the integrity of the epithelium by promoting the expression of pro-inflammatory cytokines. Independent on toxins, bacteria can induce Ca2+ responses that play a role in cytoskeletal rearrangements required for cell binding or internalisation of the microorganism. In some instances, invasion of the epithelium may be followed by bacterial access to deeper tissue, dissemination to other organs, and sometimes persistence in host cells in a parasitic-like mode. Such strategies underline the pathogen abilities to control innate defence cells such as professional phagocytes, and may implicate the diversion of Ca(2+)-dependent cellular processes that normally result in killing of the ingested bacteria. Finally, bacterial pathogens can also induce the cell release of ATP, a Ca2+ agonist, that may expand bacterial cell signalling by a paracrine or autocrine route, leading to enhanced colonisation or enhanced host cell responses to the invading microorganism.     

Participation of the Cytoskeletal and Lysosomal Compartments in Campylobacter jejuni Invasion of Caco-2 cells, the Cellular Response by Morphometric Analysis and the Presence of Cytokine and Chemokine Transcripts

This study aimed to evaluate the participation of actin and tubulin in the process of internalisation, the interaction of bacterial phagosomes with lysosomes, the morphometric changes and the expression of inflammatory cytokines in Caco-2 cells infected with Campylobacter jejuni. Both actin and tubulin participated in the process of internalisation. Inside the cells, lysosomes fuse with phagosomes, which may lead to bacterial death because after 2 h, the bacteria were not detected by Transmission electron microscopy (TEM). There is increased expression of TGF-β3 during the early stages, and IL-8 was expressed after 60 min p.i. This work showed that C. jejuni invades and causes major morphometric changes in epithelial cells. In response, the cells increase their expression of cytokines that can lead to inflammation. The mechanisms of invasion are dependent on actin and tubulin, and once internalised, lysosomes fuse with phagosomes.     

Evidence for regulation of actin synthesis in cytochalasin D-treated HEp-2 cells

In HEp-2 cells treated with 0.2 or 2.0 μM cytochalasin D (CD), the relative rate of actin synthesis increased for about 12 h and then reached a plateau; this increase was suppressed by actinomycin D (AD). When CD was washed from cells which had been treated for 20 h, the elevated rate of actin synthesis declined to the control value within ca 4 h, as the actin-containing cytoskeletal components rearranged by CD recovered their normal morphology. Subsequently, actin synthesis was depressed below control values for a prolonged period; during recovery from 2 h treatment with CD, this depression was of much shorter duration. Re-addition of CD to cells after a 3 h recovery period again induced the cytoskeletal alterations characteristic of CD treatment but did not reverse the prior decline in the rate of actin synthesis. In HEp-2 cells treated with cycloheximide during exposure to CD for 20 h, the relative rate of actin synthesis measured after removal of cycloheximide was twofold higher than with CD alone and such cells exhibited a twofold slower decline in the rate of actin synthesis during recovery from CD in the continued presence of cycloheximide. These effects of cycloheximide, which resemble observations on “super-induction”, suggest that actin synthesis in CD-treated and recovering HEp-2 cells may be regulated by a repressor protein. The possibility that the proposed repressor protein is actin and that actin may thus be a feedback inhibitor of its own synthesis is discussed.     

Corynebacterium pseudodiphtheriticum isolated from relevant clinical sites of infection: a human pathogen overlooked in emerging countries

Aims:  To examine the occurrence of and to determine the antimicrobial susceptibility of Corynebacterium pseudodiphtheriticum among patients with bacterial infections at a teaching hospital.
Methods and Results:  A total of 113 Coryne. pseudodiphtheriticum strains identified by conventional biochemical methods and API-Coryne System were recovered from patients from different age groups: 65·48% adults (18 to ≤59 years old), 9·73% aged (≥60 years old); 14·15% infants (<18 years old); 4·42% newborns (0–7 days). Micro-organisms were mostly related to infections in the urinary (29·2%) and respiratory tracts (27·45%) and intravenous sites (18·6%). Clinical samples were obtained only from 32·7% patients (26 adults, four aged, four infants and three newborns) presenting at least one of the predisposing conditions: end-stage renal disease; renal transplant; AIDS and Mycobacterium tuberculosis infection; cancer, hepatic cirrhosis; haemodialysis and catheter use. Antimicrobial susceptibility tests identified multiresistant phenotypes. Most strains (>50%) were resistant to oxacillin, erythromycin and clindamycin.
Conclusions:  Despite significant differences in age and functional status of patients Coryne. pseudodiphtheriticum may be implicated as a cause of respiratory and nonrespiratory human infections.
Significance and Impact of the Study:  Data are valuable for practitioners indicating the occurrence of multiresistant phenotypes and the possibility of severe infections due to Coryne. pseudodiphtheriticum , a pathogen usually overlooked in emerging countries.     

Comparison of adherence to and penetration of a human laryngeal epithelial cell line by group A streptococci of various M protein types

Clinically isolated group A streptococci (GAS) of different M protein types were studied using aminoglycoside exclusion and [2,8-3H]adenine radiolabeled GAS assays to compare the abilities of different strains to adhere to and internalize within human laryngeal epithelial (HEp-2) cells. GAS isolated from patients with pharyngitis and GAS isolated from patients with more severe disease, such as necrotizing fasciitis, adhered to and penetrated HEp-2 cells equally well. M3, M4, M6, and M12 strains adhered to and were internalized within HEp-2 cells more than M1 strains. M18 GAS producing hyaluronic acid capsules were less adherent and less invasive than the M3, M4, M6, and M12 strains. An M3-producing GAS strain and its M protein-deficient isogenic strain adhered similarly to HEp-2 cells, but the M protein-deficient strain exhibited greater penetration. Preincubation of HEp-2 cells with an N-terminal synthetic M3 peptide did not alter the adherence or penetration by an M3 strain. In summary, this study demonstrates that GAS from invasive and non-invasive disease adhere to and penetrate HEp-2 cells equally well and that multiple strains of GAS with various M protein types have the ability to adhere to and penetrate HEp-2 cells.     

The role of Tir, EspA, and NleB in the colonization of cattle by Shiga toxin producing Escherichia coli O26:H11

Shiga toxin producing Escherichia coli (STEC) O26:H11 is an enteric pathogen capable of causing severe hemorrhagic colitis that can lead to hemolytic uremic syndrome. This organism is able to colonize cattle and human intestinal epithelial cells by secreting effectors via a type III secretion system (T3SS). In this investigation, we examined the role of 2 effectors, Tir and NleB, and the structural translocator component EspA in the adherence of STEC to epithelial cells and in the colonization of cattle. Isogenic deletion mutants were constructed and using microscopy and flow cytometry compared to the wild-type strain in their ability to adhere to HEp-2 cells. A competitive assay was also used to measure the capacity of the mutants to colonize the intestinal tract of cattle, where both the mutant and the parental strains were introduced orally at the same time. Genomic DNA was extracted from enriched fecal samples collected at various time points, and quantitative real-time PCR was used to quantify bacteria. A significant reduction in fecal shedding was observed, and adherence to HEp-2 cells was decreased for the tir and espA mutants. Deletion of the nleB gene did not have a significant effect on the adherence of HEp-2 cells; however, in an in vivo model, it strongly reduced the ability of STEC O26:H11 to colonize the bovine intestinal tract.     

Influence of culture conditions on cell surface hydrophobicity of rods of genus Serratia

The cell surface hydrophobicity (CSH) is a non-specific adhesion factor that is important in the proliferation of microorganisms on solid surfaces. Serratia spp. is a bacterium that has been increasingly implicated as a primary pathogen in numerous human infections, particularly in urinary tract infections. CSH of 60 Serratia spp. strains isolated from clinical materials was evaluated using the ammonium sulfate salt aggregation test. Bacteria were grown for 24 h and 48 h at room temperature (22 degrees C) and 37 degrees C on enrichment broth and agar (Biomed), enrichment agar with 5% human blood and medium composed of agar granulated (Becton Dickinson), neopeptone (Difco) and 1% (v/v) glycerol. CSH was estimated most frequently when the analyzed strains in enrichment broth were cultured. When grown in enrichment broth cells of Serratia spp. at room temperature were more hydrophobic (43% after 24 h and 47% after 48 h) than those at 37 degrees C (30% after 24 h and 33% after 48 h). CSH of the examined Serratia spp. strains were depended on the temperature, time of the culture of bacteria and the kind of media. The influence of the culture conditions on the changes in CSH of the analyzed bacteria may suggest significance of these properties in the pathogenesis of Serratia spp.