Rapamycin Re-Directs Lysosome Network, Stimulates ER-Remodeling, Involving Membrane CD317 and Affecting Exocytosis, in Campylobacter Jejuni-Lysate-Infected U937 Cells.

The Gram-negative Campylobacter jejuni is a major cause of foodborne gastroenteritis in humans worldwide. The cytotoxic effects of Campylobacter have been mainly ascribed to the actions of the cytolethal distending toxin (CDT): it is mandatory to put in evidence risk factors for sequela development, such as reactive arthritis (ReA) and Guillain-Barré syndrome (GBS). Several researches are directed to managing symptom severity and the possible onset of sequelae. We found for the first time that rapamycin (RM) is able to largely inhibit the action of C. jejuni lysate CDT in U937 cells, and to partially avoid the activation of specific sub-lethal effects. In fact, we observed that the ability of this drug to redirect lysosomal compartment, stimulate ER-remodeling (highlighted by ER-lysosome and ER-mitochondria contacts), protect mitochondria network, and downregulate CD317/tetherin, is an important component of membrane microdomains. In particular, lysosomes are involved in the process of the reduction of intoxication, until the final step of lysosome exocytosis. Our results indicate that rapamycin confers protection against C. jejuni bacterial lysate insults to myeloid cells.

Pathogenesis of Streptococcus urinary tract infection depends on bacterial strain and ß-hemolysin/cytolysin that mediates cytotoxicity, cytokine synthesis, inflammation and virulence.

Streptococcus agalactiae can cause urinary tract infection (UTI) including cystitis and asymptomatic bacteriuria (ABU). The early host-pathogen interactions that occur during S. agalactiae UTI and subsequent mechanisms of disease pathogenesis are poorly defined. Here, we define the early interactions between human bladder urothelial cells, monocyte-derived macrophages, and mouse bladder using uropathogenic S. agalactiae (UPSA) 807 and ABU-causing S. agalactiae (ABSA) 834 strains. UPSA 807 adhered, invaded and killed bladder urothelial cells more efficiently compared to ABSA 834 via mechanisms including low-level caspase-3 activation, and cytolysis, according to lactate dehydrogenase release measures and cell viability. Severe UPSA 807-induced cytotoxicity was mediated entirely by the bacterial ß-hemolysin/cytolysin (ß-H/C) because an ß-H/C-deficient UPSA 807 isogenic mutant, UPSA 807ΔcylE, was not cytotoxic in vitro; the mutant was also significantly attenuated for colonization in the bladder in vivo. Analysis of infection-induced cytokines, including IL-8, IL-1ß, IL-6 and TNF-α in vitro and in vivo revealed that cytokine and chemokine responses were dependent on expression of ß-H/C that also elicited severe bladder neutrophilia. Thus, virulence of UPSA 807 encompasses adhesion to, invasion of and killing of bladder cells, pro-inflammatory cytokine/chemokine responses that elicit neutrophil infiltration, and ß-H/C-mediated subversion of innate immune-mediated bacterial clearance from the bladder.

The role of Mycobacterium tuberculosis Rv3166c protein-derived high-activity binding peptides in inhibiting invasion of human cell lines.

Given the urgent need for designing a new antituberculosis vaccine conferring total protection on patients of all ages, following the line of research adopted by our institute, this work has identified Mycobacterium tuberculosis (Mtb) Rv3166c protein high-activity binding peptides (HABPs) which are able to inhibit bacterial invasion of U937 (monocyte-derived macrophages) and A549 (type II alveolar epithelial cells) cell lines. The presence and transcription of the rv3166c gene in the Mtb species complex was confirmed by polymerase chain reaction (PCR) and reverse transcriptase-PCR; Rv3166c expression was evaluated by western blot and cellular localisation confirmed by immunoelectron microscopy. Its presence was mainly determined on cell surface. Sixteen peptides covering its entire length were chemically synthesised and tested for their ability to bind to U937 and A549 cells. Two U937 HABPs were identified and three for A549, one of them being shared by both cell lines. The four HABPs found inhibited Mtb entry by 15.07-94.06%. These results led us to including Rv3166c HABPs as candidates for further studies contributing towards the search for a multiepitope, chemically synthesised, subunit-based antituberculosis vaccine.

The Neisseria meningitidis capsule is important for intracellular survival in human cells.

While much data exist in the literature about how Neisseria meningitidis adheres to and invades human cells, its behavior inside the host cell is largely unknown. One of the essential meningococcal attributes for pathogenesis is the polysaccharide capsule, which has been shown to be important for bacterial survival in extracellular fluids. To investigate the role of the meningococcal capsule in intracellular survival, we used B1940, a serogroup B strain, and its isogenic derivatives, which lack either the capsule or both the capsule and the lipooligosaccharide outer core, to infect human phagocytic and nonphagocytic cells and monitor invasion and intracellular growth. Our data indicate that the capsule, which negatively affects bacterial adhesion and, consequently, entry, is, in contrast, fundamental for the intracellular survival of this microorganism. The results of in vitro assays suggest that an increased resistance to cationic antimicrobial peptides (CAMPs), important components of the host innate defense system against microbial infections, is a possible mechanism by which the capsule protects the meningococci in the intracellular environment. Indeed, unencapsulated bacteria were more susceptible than encapsulated bacteria to defensins, cathelicidins, protegrins, and polymyxin B, which has long been used as a model compound to define the mechanism of action of CAMPs. We also demonstrate that both the capsular genes (siaD and lipA) and those encoding an efflux pump involved in resistance to CAMPs (mtrCDE) were up-regulated during the intracellular phase of the infectious cycle.

Involvement of p38 mitogen-activated protein kinase in E. coli-induced U937 apoptosis.

OBJECTIVE: To investigate whether the effect of E. coli on U937 cell lines apoptosis is mediated via p38 mitogen-activated protein kinase (MAPK) activation. METHODS: The U937 cell lines were treated with E. coli at different time or together with SB203580, an inhibitor for p38. Cell apoptosis was analyzed by flow cytometry. p38 activities were detected by Western blotting. RESULTS: E. coli induced apoptosis in cultured U937 cell lines in a time-dependent manner. The phosphorylation of p38 was induced after 10 minutes infection, reached the peak after 20 minutes, and started to decline after 30 minutes. In contrast, the level of total p38 protein was not changed in whole experimental period. Inhibition of p38 with SB203580 significantly inhibited E. coli induced apoptosis in U937 cells. CONCLUSION: The activation of the p38 MAPK in U937 cell lines by E. coli is a major pathway to mediate the apoptosis.

fliP influences Citrobacter koseri macrophage uptake, cytokine expression and brain abscess formation in the neonatal rat.

Citrobacter koseri causes neonatal meningitis frequently complicated with multiple brain abscesses. During C. koseri central nervous system infection in the neonatal rat model, previous studies have documented many bacteria-filled macrophages within the neonatal rat brain and abscesses. Previous studies have also shown that C. koseri is taken up by, survives phagolysosomal fusion and replicates in macrophages in vitro and in vivo. In this study, in order to elucidate genetic and cellular factors contributing to C. koseri persistence, a combinatory technique of differential fluorescence induction and transposon mutagenesis was employed to isolate C. koseri genes induced while inside macrophages. Several banks of mutants were subjected to a series of enrichments to select for gfp : : transposon fusion into genes that are turned off in vitro but expressed when intracellular within macrophages. Further screening identified several mutants attenuated in their recovery from macrophages compared with the wild-type. A mutation within an Escherichia coli fliP homologue caused significant attenuation in uptake and hypervirulence in vivo, resulting in death within 24 h. Furthermore, analysis of the immunoregulatory interleukin (IL)-10/IL-12 cytokine response during infection suggested that C. koseri fliP expression may alter this response. A better understanding of the bacteria-macrophage interaction at the molecular level and its contribution to brain abscess formation will assist in developing preventative and therapeutic strategies.

Differences in protein synthesis between wild type and intracellular growth-deficient strains of Legionella pneumophila in U937 and Acanthamoeba polyphaga.

An important aspect of Legionnaires' disease is the growth of the causative agent, Legionella pneumophila, within infected host cells. Many proteins including stress proteins of L. pneumophila were strongly induced in a wild type strain that had been used to infect U937 human macrophage-like cells. In contrast, the expression of the proteins was much weaker within a protozoan host, Acanthamoeba polyphaga. The results suggested that active bacterial protein synthesis is required more within macrophages than within protozoa for adaptation of L. pneumophila to intracellular environments. The synthesis of these proteins was not observed in intracellular growth-deficient strains after infection in either type of host cells. The inability of protein synthesis in these strains is correlated with their inability of intracellular growth. Furthermore, on U937 infection, the synthesis of beta-galactosidase encoded in an inducible reporter construct immediately ceased in the in intracellular growth-deficient strains after infection, while the wild type strain was able to synthesize it during the course of infection. These results suggested that the intracellular growth of Legionella pneumophila within macrophages requires active protein synthesis from an earlier stage of bacterial infection.

lbtA and lbtB are required for production of the Legionella pneumophila siderophore legiobactin.

Under iron stress, Legionella pneumophila secretes legiobactin, a nonclassical siderophore that is reactive in the chrome azurol S (CAS) assay. Here, we have optimized conditions for legiobactin expression, shown its biological activity, and identified two genes, lbtA and lbtB, which are involved in legiobactin production. lbtA appears to be iron repressed and encodes a protein that has significant homology with siderophore synthetases, and FrgA, a previously described iron-regulated protein of L. pneumophila. lbtB encodes a protein homologous with members of the major facilitator superfamily of multidrug efflux pumps. Mutants lacking lbtA or lbtB were defective for legiobactin, producing 40 to 70% less CAS reactivity in deferrated chemically defined medium (CDM). In bioassays, mutant CDM culture supernatants, unlike those of the wild type, did not support growth of iron-limited wild-type bacteria in 2',2'-dipyridyl-containing buffered charcoal yeast extract (BCYE) agar and a ferrous iron transport mutant on BCYE agar without added iron. The lbtA mutant was modestly defective for growth in deferrated CDM containing the iron chelator citrate, indicating that legiobactin is required in conditions of severe iron limitation. Complementation of the lbt mutants restored both siderophore expression, as measured by the CAS assay and bioassays, and bacterial growth in deferrated, citrate-containing media. The lbtA mutant replicated as the wild type did in macrophages, amoebae, and the lungs of mice. However, L. pneumophila expresses lbtA in the macrophage, suggesting that legiobactin, though not required, may play a dispensable role in intracellular growth. The discovery of lbtAB represents the first identification of genes required for L. pneumophila siderophore expression.

Shigella and Salmonella: death as a means of survival.

Shigella and Salmonella kill host cells and trigger inflammatory responses by mechanisms that are not fully understood. The goal of this review is to reevaluate key observations reported over the past 15 years and, whenever possible, to provide a chronological perspective as to how our understanding of the pathways by which Shigella and Salmonella kill host cells has evolved.

Characterization of the major secreted zinc metalloprotease- dependent glycerophospholipid:cholesterol acyltransferase, PlaC, of Legionella pneumophila.

Legionella pneumophila, an intracellular pathogen causing a severe pneumonia, possesses distinct lipolytic activities which have not been completely assigned to specific enzymes so far. We cloned and characterized a gene, plaC, encoding a protein with high homology to PlaA, the major secreted lysophospholipase A of L. pneumophila and to other hydrolytic enzymes belonging to the GDSL family. Here we show that L. pneumophila plaC mutants possessed reduced phospholipase A and lysophospholipase A activities and lacked glycerophospholipid:cholesterol acyltransferase activity in their culture supernatants. The mutants' reduced phospholipase A and acyltransferase activities were complemented by reintroduction of an intact copy of plaC. Additionally, plaC conferred increased lysophospholipase A and glycerophospholipid:cholesterol acytransferase activities to recombinant Escherichia coli. Furthermore, PlaC was shown to be another candidate exported by the L. pneumophila type II secretion system and was activated by a factor present in the bacterial culture supernatant dependent on the zinc metalloprotease. Finally, the role of plaC in intracellular infection of Acanthamoeba castellanii and U937 macrophages with L. pneumophila was assessed, and plaC was found to be dispensable. Thus, L. pneumophila possesses another secreted lipolytic enzyme, a protein with acyltransferase, phospholipase A, and lysophospholipase A activities. This enzyme is distinguished from the previously characterized phospholipases A and lysophospholipases A by its capacity not only to cleave fatty acids from lipids but to transfer them to cholesterol. Cholesterol is an important compound of eukaryotic membranes, and an acyltransferase might be a tool for host cell modification to fit the needs of the bacterium.

OmpD but not OmpC is involved in adherence of Salmonella enterica serovar typhimurium to human cells.

Conflicting reports exist regarding the role of porins OmpC and OmpD in infections due to Salmonella enterica serovar Typhimurium. This study investigated the role of these porins in bacterial adherence to human macrophages and intestinal epithelial cells. ompC and ompD mutant strains were created by transposon mutagenesis using P22-mediated transduction of Tn10 and Tn5 insertions, respectively, into wild-type strain 14028. Fluorescein-labeled wild-type and mutant bacteria were incubated with host cells at various bacteria to cell ratios for 1 h at 37 degrees C and analyzed by flow cytometry. The mean fluorescence intensity of cells with associated wild-type and mutant bacteria was used to estimate the number of bacteria bound per host cell. Adherence was also measured by fluorescence microscopy. Neither assay showed a significant difference in binding of the ompC mutant and wild-type strains to the human cells. In contrast, the ompD mutant exhibited lowered binding to both cell types. Our findings suggest that OmpD but not OmpC is involved in the recognition of Salmonella serovar Typhimurium by human macrophages and intestinal epithelial cells.

Comparative assessment of virulence traits in Legionella spp.

Legionella pneumophila is a facultative intracellular pathogen that accounts for the majority of cases of Legionnaires' disease in the USA and Europe, but other Legionella spp. have been shown to cause disease. In contrast, Legionella longbeachae is the leading cause of Legionnaires' disease in Australia. The hallmark of Legionnaires' disease caused by L. pneumophila is the intracellular replication within phagocytes in the alveolar spaces, and the Dot/Icm type IV secretion system is essential for intracellular replication. Although it has been presumed that intracellular replication within phagocytes is the hallmark of other virulent legionellae, the virulence traits of Legionella spp. apart from L. pneumophila are not well defined. In this study, 27 strains of Legionella spp. belonging to 16 species that have been isolated from humans or from the environment were examined for five virulence traits exhibited by L. pneumophila: cytopathogenicity, intracellular replication within macrophages, induction of apoptosis/DNA fragmentation, pore-formation-mediated cytolysis of the host cell, and the presence of the dot/icm loci. The strains were divided into two broad groups (low and high cytopathogenic groups) based on cytopathogenicity assays using U937 human-derived macrophages. The other four virulence traits were evaluated in the low and high cytopathogenic groups of Legionella species. Most L. pneumophila serogroup 1 strains were highly cytopathogenic after 72 h, manifested high levels of intracellular growth, induced apoptosis/DNA fragmentation, and exhibited pore-forming activity. The majority of the other species were the low cytopathogenic group that did not induce apoptosis, neither did they exhibit pore-forming activity. All the species of legionellae tested have all the dot/icm loci, when examined by DNA hybridization. No correlation was found between cytopathogenicity and the other four pathogenic traits.

Interleukin-1beta responses to Mycoplasma pneumoniae infection are cell-type specific.

Interleukin-1beta (IL-1beta) is a major proinflammatory cytokine that is involved in many important cellular functions such as proliferation, differentiation, and activation of different cell types. Its mature form is released from the cells in response to various bacterial and viral infections, and it plays a significant role in host defense. Mycoplasma pneumoniae is a small bacterium without a cell wall that causes tracheobronchitis and atypical pneumonia in humans following attachment to respiratory epithelium, as well as extrapulmonary infections. Very little is known about the role of cytokines in pathogenesis or the response of target cells to M.pneumoniae attachment. The purpose of this study was to investigate the ability of M. pneumoniae to induce IL-1beta in human lung epithelial carcinoma A549 and in human monocytic U937 cell lines. Following M. pneumoniae infection, both IL-1beta mRNA and protein were induced in A549 cells vs. no induction in uninfected cells; however, the protein remained inside the A549 cells. Similarly, M. pneumoniae infection strongly increased mRNA and extracellular protein levels in U937 cells, which unlike A549 cells did exhibit baseline constitutive levels. De novo IL-1beta protein expression was verified by cycloheximide studies. M. pneumoniae infection did not affect constitutive caspase-1 mRNA or protein levels in either cell line. Reduced caspase-1 activity in A549 cell lysates suggests the presence of an endogenous caspase-1 inhibitory component in the A549 cells. These collective data confirm previous studies that show that M. pneumoniae is a potent inducer of cytokines following adherence to host target cells, and establish that IL-1beta release in response to M. pneumoniae infection is cell-type specific, thus emphasizing the importance of carefully considering multiple cell types in M. pneumoniae pathogenesis studies involving both immune cells and cytokine release patterns.

A mutation in an ompR-like gene on a Legionella longbeachae serogroup 1 plasmid attenuates virulence.

Examination of a panel of Legionella longbeachae serogroup 1 strains using a guinea pig model of virulence determined that this clonal species of Legionella shows a remarkable variation in symptoms and disease outcome (Doyle et al., Infect. Immun. 69, 5335-5344, 2001). The presence of plasmids was investigated, as plasmid encoded functions may contribute to the virulence of genetically similar strains. Partial sequence analysis of a large native plasmid (approximately 120 kb), designated pA5H5, from a highly virulent Australian isolate revealed a putative two-component regulatory system with inferred identity to the OmpR family of two-component transcriptional regulatory proteins and EnvZ sensor kinases. An isogenic mutant was constructed in the transcriptional regulatory gene, designated lrpR (L. longbeachae sg 1 regulatory protein) and this strain was tested in Acanthamoeba, U937 cells and in a guinea pig animal model. The mutant was reduced in intracellular multiplication within Acanthamoeba but not U937 macrophage-like cells. However, the lrpR mutant did appear reduced in invasion at the early stages of infection of U937 cells. The lrpR mutant was also attenuated for virulence in a guinea pig animal model of infection. The importance of plasmid-encoded functions for the pathogenicity of Legionella longbeachae serogroup 1 strains is discussed.

The C-terminus of IcmT is essential for pore formation and for intracellular trafficking of Legionella pneumophila within Acanthamoeba polyphaga.

We have shown previously that the five rib (release of intracellular bacteria) mutants of Legionella pneumophila are competent for intracellular replication but defective in pore formation-mediated cytolysis and egress from protozoan and mammalian cells. The rib phenotype results from a point mutation (deletion) DeltaG544 in icmT that is predicted to result in the expression of a protein truncated by 32 amino acids from the C-terminus. In contrast to the rib mutants that are capable of intracellular replication, an icmT null mutant was completely defective in intracellular replication within mammalian and protozoan cells, in addition to its defect in pore formation-mediated cytolysis. The icmT wild-type allele complemented the icmT null mutant for both defects of intracellular replication and pore formation-mediated cytolysis and egress from mammalian cells. In contrast, the icmTDeltaG544 allele complemented the icmT null mutant for intracellular growth, but not for the pore-forming activity. Consistent with their defect in pore formation-mediated cytotoxicity in vitro, both mutants failed to cause pulmonary inflammation in A/J mice. Interestingly, the rib mutant was severely defective in intracellular growth within Acanthamoeba polyphaga. Confocal laser scanning and electron microscopy confirmed that the rib mutant and the icmT null mutant were severely and completely defective, respectively, in intracellular growth in A. polyphaga, and the respective defects correlated with fusion of the bacterial phagosomes to lysosomes. Taken together, the data showed that the C-terminus domain of IcmT is essential for the pore-forming activity and is required for intracellular trafficking and replication within A. polyphaga, but not within mammalian cells.

Actinobacillus actinomycetemcomitans induces lethal effects on the macrophage-like human cell line U937.

We examined the cytotoxicity in culture medium of Actinobacillus actinomycetemcomitans against the human monocyte-macrophage-like cell line U937 using the trypan blue exclusion test and WST-1 test. We found that A. actinomycetemcomitans Y4 showed the highest cytotoxic activity among the three different serotype strains and the cytotoxic effects of both bacterial cells and culture supernatants in A. actinomycetemcomitans Y4 were stronger on phorbol-12-myristate 13-acetate (PMA)-induced U937 cells than uninduced U937 cells. Morphological changes in PMA-induced U937 cells treated with culture supernatants differed from those treated with leukotoxin, and a difference in the susceptibility to 56 degrees C heat treatment was found between culture supernatants and leukotoxin. The cytotoxic activity by WST-1 was determined more rapidly and strongly than that by trypan blue assay. These findings suggested that the cytotoxic effect of A. actinomycetemcomitans was influenced by the differentiation of U937 cells and may be more potent on the respiratory chain than the cell membrane.

How the parasitic bacterium Legionella pneumophila modifies its phagosome and transforms it into rough ER: implications for conversion of plasma membrane to the ER membrane.

Within five minutes of macrophage infection by Legionella pneumophila, the bacterium responsible for Legionnaires' disease, elements of the rough endoplasmic reticulum (RER) and mitochondria attach to the surface of the bacteria-enclosed phagosome. Connecting these abutting membranes are tiny hairs, which are frequently periodic like the rungs of a ladder. These connections are stable and of high affinity - phagosomes from infected macrophages remain connected to the ER and mitochondria (as they were in situ) even after infected macrophages are homogenized. Thin sections through the plasma and phagosomal membranes show that the phagosomal membrane is thicker (72+/-2 A) than the ER and mitochondrial membranes (60+/-2 A), presumably owing to the lack of cholesterol, sphingolipids and glycolipids in the ER. Interestingly, within 15 minutes of infection, the phagosomal membrane changes thickness to resemble that of the attached ER vesicles. Only later (e.g. after six hours) does the ER-phagosome association become less frequent. Instead ribosomes stud the former phagosomal membrane and L. pneumophila reside directly in the rough ER. Examination of phagosomes of various L. pneumophila mutants suggests that this membrane conversion is a four-stage process used by L. pneumophila to establish itself in the RER and to survive intracellularly. But what is particularly interesting is that L. pneumophila is exploiting a poorly characterized naturally occurring cellular process.

Mycobacterium avium grown in Acanthamoeba castellanii is protected from the effects of antimicrobials.

Mycobacterium avium is a common cause of systemic bacterial infection in patients with AIDS. Infection with M. avium has been linked to bacterial colonization of domestic water supplies and commonly occurs through the gastrointestinal tract. Acanthamoeba castellanii, a waterborne protozoan, may serve as an environmental host for M. avium. It has been shown that growth of M. avium in amoebae enhances invasion and intracellular replication of the bacteria in human macrophages and intestinal epithelial cell line HT-29 as well as in mice. We determined that growth of M. avium within A. castellanii influenced susceptibility to rifabutin, azithromycin, and clarithromycin. No significant activity against M. avium was seen with rifabutin, azithromycin, and clarithromycin when used to treat monolayers on both day 1 and day 4 after infection. When tested in a macrophage-like cell line (U937), all compounds showed significant anti-M. avium activity. Growth of M. avium in amoebae appears to reduce the effectiveness of the antimicrobials. These findings may have significant implications for prophylaxis of M. avium infection in AIDS.

Phagocytosis of mycobacteria by U937 cells: a rapid method for monitoring uptake and separating phagocytosed and free bacteria by magnetic beads.

A human-derived monocytic cell line (U937) was induced to phagocytose Mycobacterium phlei by the addition of phorbol myristate acetate (PMA) to the culture medium for 50-60 h. Cells not treated with PMA were unable to phagocytose M. phlei. Magnetic beads enabled a rapid and highly efficient separation of phagocytosed and free bacteria to be achieved, an approach which is particularly useful if colony plating is used to enumerate bacterial survival within phagocytic cells. Fluorescence-activated cell sorting (FACS) analysis showed that 98% of U937 cells contained viable bacteria after 3 h.

Escherichia coli promotes macrophage apoptosis.

BACKGROUND: Escherichia coli is the bacterium most commonly isolated from the urine of patients with urinary tract infection (UTI). Recurrent episodes of UTI lead to renal interstitial scarring. In interstitial fibrosis and scarring, infiltration of mononuclear cells has been reported to play a key role. MATERIALS AND METHODS: We evaluated the effect of two strains of E. coli--the pathogenic BH-5 and the plasmidless, nonfimbriated HB-101-on human monocyte and murine macrophage apoptosis. RESULTS: E. coli BH-5 enhanced apoptosis in a time- and dose-dependent manner. It also promoted necrosis in a time- and dose-dependent manner. Strain HB-101 promoted monocyte apoptosis in a dose-dependent manner. However, the magnitude of HB-101-induced monocyte apoptosis was lower than BH-5-induced macrophage apoptosis. CONCLUSION: The ability of E. coli to induce apoptosis may contribute to its virulence and play a role in renal interstitial scarring.