Targeted therapy via oral administration of attenuated Salmonella expression plasmid-vectored Stat3-shRNA cures orthotopically transplanted mouse HCC.

The development of RNA interference-based cancer gene therapies has been delayed due to the lack of effective tumor-targeting delivery systems. Attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) has a natural tropism for solid tumors. We report here the use of attenuated S. Typhimurium as a vector to deliver shRNA directly into tumor cells. Constitutively activated signal transducer and activator of transcription 3 (Stat3) is a key transcription factor involved in both hepatocellular carcinoma (HCC) growth and metastasis. In this study, attenuated S. Typhimurium was capable of delivering shRNA-expressing vectors to the targeted cancer cells and inducing RNA interference in vivo. More importantly, a single oral dose of attenuated S. Typhimurium carrying shRNA-expressing vectors targeting Stat3 induced remarkably delayed and reduced HCC (in 70% of mice). Cancer in these cured mice did not recur over 2 years following treatment. These data demonstrated that RNA interference combined with Salmonella as a delivery system may offer a novel clinical approach for cancer gene therapy.

Enhanced tumor suppression in vitro and in vivo by co-expression of survivin-specific siRNA and wild-type p53 protein.

The development of malignant prostate cancer involves multiple genetic alterations. For example, alterations in both survivin and p53 are reported to have crucial roles in prostate cancer progression. However, little is known regarding the interrelationships between p53 and survivin in prostate cancer. Our data demonstrate that the expression of survivin is inversely correlated with that of wtp53 protein (r(s)=0.548) in prostate cancer and in normal prostate tissues. We have developed a therapeutic strategy, in which two antitumor factors, small interfering RNA-survivin and p53 protein, are co-expressed from the same plasmid, and have examined their effects on the growth of PC3, an androgen-independent prostate cancer cell line. When p53 was expressed along with a survivin-specific short hairpin RNA (shRNA), tumor cell proliferation was significantly suppressed and apoptosis occurred. In addition, this combination also abrogated the expression of downstream target molecules such as cyclin-dependent kinase 4 and c-Myc, while enhancing the expression of GRIM19. These changes in gene expression occurred distinctly in the presence of survivin-shRNA/wtp53 compared with control or single treatment groups. Intratumoral injection of the co-expressed construct inhibited the growth and survival of tumor xenografts in a nude mouse model. These studies revealed evidence of an interaction between p53 and survivin proteins plus a complex signaling network operating downstream of the wtp53-survivin pathway that actively controls tumor cell proliferation, survival and apoptosis.

Campylobacter jejuni--microtubule-dependent invasion.

Campylobacter jejuni is the leading bacterial cause of food-borne illness worldwide and a major cause of Guillain-Barré paralysis. Recent molecular and cellular studies of one well-characterized C. jejuni strain have begun to unravel the details of an unusual microtubule-dependent (actin-filament-independent) gut-invasion mechanism, through which at least some C. jejuni initiate disease. Although responsible for causing a human dysenteric syndrome remarkably similar to that triggered by Shigella spp., current evidence suggests that C. jejuni use some markedly different molecular mechanisms of pathogenesis compared with shigellae.

An alternative interpretation of nanobacteria-induced biomineralization.

The reported isolation of nanobacteria from human kidney stones raises the intriguing possibility that these microorganisms are etiological agents of pathological extraskeletal calcification [Kajander, E. O. & Ciftçioglu, N. (1998) Proc. Natl. Acad. Sci. USA 95, 8274-8279]. Nanobacteria were previously isolated from FBS after prolonged incubation in DMEM. These bacteria initiated biomineralization of the culture medium and were identified in calcified particles and biofilms by nucleic acid stains, 16S rDNA sequencing, electron microscopy, and the demonstration of a transferable biomineralization activity. We have now identified putative nanobacteria, not only from FBS, but also from human saliva and dental plaque after the incubation of 0.45-microm membrane-filtered samples in DMEM. Although biomineralization in our "cultures" was transferable to fresh DMEM, molecular examination of decalcified biofilms failed to detect nucleic acid or protein that would be expected from growth of a living entity. In addition, biomineralization was not inhibited by sodium azide. Furthermore, the 16S rDNA sequences previously ascribed to Nanobacterium sanguineum and Nanobacterium sp. were found to be indistinguishable from those of an environmental microorganism, Phyllobacterium mysinacearum, that has been previously detected as a contaminant in PCR. Thus, these data do not provide plausible support for the existence of a previously undiscovered bacterial genus. Instead, we provide evidence that biomineralization previously attributed to nanobacteria may be initiated by nonliving macromolecules and transferred on "subculture" by self-propagating microcrystalline apatite.

Involvement of a plasmid in virulence of Campylobacter jejuni 81-176.

Campylobacter jejuni strain 81-176 contains two, previously undescribed plasmids, each of which is approximately 35 kb in size. Although one of the plasmids, termed pTet, carries a tetO gene, conjugative transfer of tetracycline resistance to another strain of C. jejuni could not be demonstrated. Partial sequence analysis of the second plasmid, pVir, revealed the presence of four open reading frames which encode proteins with significant sequence similarity to Helicobacter pylori proteins, including one encoded by the cag pathogenicity island. All four of these plasmid-encoded proteins show some level of homology to components of type IV secretion systems. Mutation of one of these plasmid genes, comB3, reduced both adherence to and invasion of INT407 cells to approximately one-third that seen with wild-type strain 81-176. Mutation of comB3 also reduced the natural transformation frequency. A mutation in a second plasmid gene, a virB11 homolog, resulted in a 6-fold reduction in adherence and an 11-fold reduction in invasion compared to the wild type. The isogenic virB11 mutant of strain 81-176 also demonstrated significantly reduced virulence in the ferret diarrheal disease model. The virB11 homolog was detected on plasmids in 6 out of 58 fresh clinical isolates of C. jejuni, suggesting that plasmids are involved in the virulence of a subset of C. jejuni pathogens.

Differential bacterial survival, replication, and apoptosis-inducing ability of Salmonella serovars within human and murine macrophages.

Salmonella serovars are associated with human diseases that range from mild gastroenteritis to host-disseminated enteric fever. Human infections by Salmonella enterica serovar Typhi can lead to typhoid fever, but this serovar does not typically cause disease in mice or other animals. In contrast, S. enterica serovar Typhimurium and S. enterica serovar Enteritidis, which are usually linked to localized gastroenteritis in humans and some animal species, elicit a systemic infection in mice. To better understand these observations, multiple strains of each of several chosen serovars of Salmonella were tested for the ability in the nonopsonized state to enter, survive, and replicate within human macrophage cells (U937 and elutriated primary cells) compared with murine macrophage cells (J774A.1 and primary peritoneal cells); in addition, death of the infected macrophages was monitored. The serovar Typhimurium strains all demonstrated enhanced survival within J774A.1 cells and murine peritoneal macrophages, compared with the significant, almost 100-fold declines in viable counts noted for serovar Typhi strains. Viable counts for serovar Enteritidis either matched the level of serovar Typhi (J774A. 1 macrophages) or were comparable to counts for serovar Typhimurium (murine peritoneal macrophages). Apoptosis was significantly higher in J774A.1 cells infected with serovar Typhimurium strain LT2 compared to serovar Typhi strain Ty2. On the other hand, serovar Typhi survived at a level up to 100-fold higher in elutriated human macrophages and 2- to 3-fold higher in U937 cells compared to the serovar Typhimurium and Enteritidis strains tested. Despite the differential multiplication of serovar Typhi during infection of U937 cells, serovar Typhi caused significantly less apoptosis than infections with serovar Typhimurium. These observations indicate variability in intramacrophage survival and host cytotoxicity among the various serovars and are the first to show differences in the apoptotic response of distinct Salmonella serovars residing in human macrophage cells. These studies suggest that nonopsonized serovar Typhimurium enters, multiplies within, and causes considerable, acute death of macrophages, leading to a highly virulent infection in mice (resulting in death within 14 days). In striking contrast, nonopsonized serovar Typhi survives silently and chronically within human macrophages, causing little cell death, which allows for intrahost dissemination and typhoid fever (low host mortality). The type of disease associated with any particular serovar of Salmonella is linked to the ability of that serovar both to persist within and to elicit damage in a specific host's macrophage cells.

Campylobacter jejuni 81-176 associates with microtubules and dynein during invasion of human intestinal cells.

Campylobacter jejuni uptake into cultured INT407 cells was analyzed kinetically over a wide range of starting multiplicities of infection (MOI; from 0.02 to 20,000 bacteria/epithelial cell). The efficiency of internalization was the highest at MOI of 0.02 and decreased steadily at higher MOIs, presumably due to reported C. jejuni autoagglutination at higher densities. Total internalized Campylobacter CFU increased gradually from an MOI of 0.02 to a peak at an MOI of 200 (reaching an average of two bacteria internalized per epithelial cell) and decreased at higher MOIs. The invasion process was apparently saturated within 2 h at an MOI of 200, indicating stringent host cell limitations on this entry process. Furthermore, whereas control Salmonella typhi invaded all monolayer cells within 1 h, only two-thirds of monolayer cells were infected after 2 h with C. jejuni at MOIs of 200 to 2,000. The percentage of Campylobacter-infected host cells gradually increased to 85% after 7 h of infection, suggesting that C. jejuni entry may be host cell cycle dependent. Direct evidence of the involvement of microtubules in C. jejuni internalization, suggested previously by biochemical inhibitor studies, was obtained by time course immunofluorescence microscopic analyses. Bacteria initially bound to the tips of host cell membrane extensions containing microtubules, then aligned in parallel with microtubules during entry, colocalized specifically with microtubules and dynein but not with microfilaments, and moved over 4 h, presumably via microtubules to the perinuclear region of host cells. Orthovanadate, which inhibits dynein activity, specifically reduced C. jejuni 81-176 entry, suggesting that this molecular motor is involved in entry and endosome trafficking during this novel bacterial internalization process. Collectively, these data suggest that C. jejuni enters host cells in a targeted and tightly controlled process leading to uptake into an endosomal vacuole which apparently moves intracellularly along microtubules via the molecular motor, dynein, to the perinuclear region.

Physical limitations on Salmonella typhi entry into cultured human intestinal epithelial cells.

Kinetic studies of Salmonella typhi invasion of INT407 cells at different multiplicities of infection (MOIs) have revealed a strict physical limitation on S. typhi entry at MOIs of >/=40. Staining of infected monolayers to distinguish intracellular from extracellular bacteria revealed that all monolayer cells are susceptible to infection and that internalized bacteria are typically contained in one to three separate clusters per cell during the first 60 min. Scanning and transmission electron microscopic analyses of time course-infected monolayers showed that at early times postinfection, bacteria bind to shortened, coalesced microvilli in one to three focal aggregate structures per host cell surface. As reported previously for S. typhimurium, focal aggregates progress to conical membrane ruffles that appear to engulf one or a few centrally contained S. typhi cells by a macropinocytic process, which enhanced the entry of simultaneously added Escherichia coli HB101 about 30-fold. Additionally, kinetic studies showed that at an MOI of approximately 400, maximal S. typhi entry is virtually completed within 30 to 35 min. Monolayers pretreated with S. typhi for 30 min to saturate the entry process were severely reduced in the ability to internalize subsequently added kanamycin-resistant strains of S. typhi or S. typhimurium, but E. coli HB101(pRI203) expressing the cloned Yersinia inv gene was not reduced in entry. In invasion inhibition assays, anti-beta1 integrin antibodies markedly reduced E. coli HB101(pRI203) invasion efficiency but did not reduce S. typhi entry. Collectively, these data provide direct physical and visual evidence which indicates that S. typhi organisms are internalized at a limited number (i.e., two to four) of sites on host cells. S. typhi and S. typhimurium likely share INT407 cell entry receptors which do not appear to be members of the beta1 integrin superfamily.

Mutation in the peb1A locus of Campylobacter jejuni reduces interactions with epithelial cells and intestinal colonization of mice.

Campylobacter jejuni is one of the leading causes of bacterial diarrhea throughout the world. We previously found that PEB1 is a homolog of cluster 3 binding proteins of bacterial ABC transporters and that a C. jejuni adhesin, cell-binding factor 1 (CBF1), if not identical to, contains PEB1. A single protein migrating at approximately 27 to 28 kDa was recognized by anti-CBF1 and anti-PEB1. To determine the role that the operon encoding PEB1 plays in C. jejuni adherence, peb1A, the gene encoding PEB1, was disrupted in strain 81-176 by insertion of a kanamycin resistance gene through homologous recombination. Inactivation of this operon completely abolished expression of CBF1, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. In comparison to the wild-type strain, the mutant strain showed 50- to 100-fold less adherence to and 15-fold less invasion of epithelial cells in culture. Mouse challenge studies showed that the rate and duration of intestinal colonization by the mutant were significantly lower and shorter than with the wild-type strain. In summary, PEB1 is identical to a previously identified cell-binding factor, CBF1, in C. jejuni, and the peb1A locus plays an important role in epithelial cell interactions and in intestinal colonization in a mouse model.

Regulatory considerations for Campylobacter vaccine development.

The high, worldwide incidence of Campylobacter jejuni-associated diarrheal disease has recently prompted the development of anti-Campylobacter vaccines. However, the association of C. jejuni infections with subsequent development of Guillain-Barré syndrome has increased concerns from a pathogenesis standpoint and from a vaccine development and regulation standpoint. This brief overview describes the purpose and process of Food and Drug Administration review of vaccine products and highlights some important considerations pertinent to Campylobacter vaccine development.

Uptake of pathogenic intracellular bacteria into human and murine macrophages downregulates the eukaryotic 26S protease complex ATPase gene.

A differential PCR technique detected the transcriptional downregulation of the mss1 (mammalian suppressor of svg1) gene in murine J774A.1 macrophages following uptake of Salmonella typhimurium. This downregulation was also noted after entry of virulent strains of Listeria monocytogenes and Shigella flexneri, two other facultative intracellular bacterial species. In contrast, uptake of nonpathogenic Escherichia coli HB101, an aroA mutant of S. typhimurium, an invasion plasmid antigen B (ipaB) mutant of S. flexneri, hemolysin (hly) and positive-regulatory factor (prfA) mutants of L. monocytogenes, or latex beads produced mss1 expression levels similar to that of uninfected macrophages. Transcriptional downregulation of mss1 was also shown to occur in S. typhimurium-infected human U937 cells, albeit to an extent less than that in murine J774A.1 cells. In addition to a lower abundance of mss1 transcripts, we also demonstrate for the first time that less MSS1 protein was detected in intracellular-bacterium-infected cells (beginning about 1 h after entry of the pathogenic intracellular bacteria) than in noninfected cells. Some strains with specific mutations in characterized genes, such as an ipaB mutant strain of S. flexneri and an hly mutant strain of L. monocytogenes, did not elicit this lower level of expression of MSS1 protein. The decrease in MSS1 within infected macrophages resulted in an accumulation of ubiquitinated proteins, substrates for MSS1. Since MSS1 comprises the ATPase part of the 26S protease that degrades ubiquitinated proteins, we hypothesize that downregulation of the mss1 gene by intracellular bacterial entry may help subvert the host cell's normal defensive response to internalized bacteria, allowing the intracellular bacteria to survive.

Transcriptional downregulation of a mouse Golgi mannosidase gene following Salmonella typhimurium uptake into murine macrophages.

Uptake of Salmonella typhimurium into murine macrophage cells caused transcriptional downregulation of a mRNA population in eukaryotic cells after 2 h of infection. The cloned cDNA that was identified showed extensive homology with a murine alpha 1,2-mannosidase gene involved in N-oligosaccharide processing. These findings suggest a disruption of eukaryotic protein processing after phagocytosis of S. typhimurium by the murine macrophage cells.

Molecular characterization of the tia invasion locus from enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) shares with other diarrheal pathogens the capacity to invade epithelial cell lines originating from the human ileum or colon, although the role of invasion in ETEC pathogenesis remains undefined. Two distinct loci (tia and tib) that direct noninvasive E. coli to adhere to and invade intestinal epithelial cell lines have previously been isolated from cosmid libraries of the classical ETEC strain H10407. Here, we report the molecular characterization of the tia locus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cellular fractions of E. coli DH5alpha carrying the tia-positive cosmids and recombinant plasmid subclones revealed that this locus directs the production of a 25-kDa protein (the Tia protein) that is localized to the outer membrane. The tia locus was subcloned to a maximum of 2 kb and mutagenized with bacteriophage Mud. Synthesis of this protein was directly correlated with the ability of subclones and Mud transposon mutants to adhere to and invade epithelial cells. Sequencing of the tia locus identified a 756-bp open reading frame. All transposon insertions resulting in an invasion-negative phenotype mapped to this open reading frame. The open reading frame was amplified and directionally cloned behind the lac promoter of pHG165. This construct directed DHalpha to express a 25-kDa protein and to adhere to and invade epithelial cells. The role of the tia gene in directing epithelial adherence and invasion was further assessed by the construction of chromosomal tia deletion derivatives of the parent ETEC strain, H10407. These tia deletion strains were noninvasive and lacked the ability to adhere to human ileocecal cells. The tia gene shares limited homology with the Yersinia ail locus and significant homology with the hra1 agglutinin gene cloned from a porcine ETEC strain. Additionally, tia probes hybridized to geographically diverse ETEC strains, as well as some enteropathogenic E. coli, enteroaggregative E. coli, and Shigella sonnei strains.

Detection and characterization by differential PCR of host eukaryotic cell genes differentially transcribed following uptake of intracellular bacteria.

Host eukaryotic cell genes that are differentially transcribed after phagocytosis of various pathogenic and nonpathogenic bacterial cells were identified by a differential PCR (DPCR) system. This DPCR procedure favors detection and isolation of host genes affected at the transcriptional level by selecting for poly(A) tails but differs substantially from reverse transcription-PCR. Several unidentified macrophage gene fragments from genes that were either transcriptionally activated or downregulated following uptake of Listeria monocytogenes into J774 mouse macrophage cells were initially defined by this DPCR procedure. Because of the sensitivity of the DPCR technique, all of the genes exhibited less than a 10-fold difference in transcription compared with noninfected cells as measured by limiting-dilution PCR. One of the gene fragments has a very high level of homology with a mitogen-activated protein kinase phosphatase (MKP-1), whereas the other affected fragments showed no homologies to known gene sequences. In addition, one of the gene fragments (WS30-B2/1) was specifically downregulated after L. monocytogenes uptake and another gene was repressed by uptake of either Shigella flexneri or L. monocytogenes, while transcription of the genes represented by fragment WS13-B9/9, and to some extent MKP-1, was activated following general phagocytosis (i.e., following uptake of any species of bacterium tested). Further characterization of the affected genes was conducted by using mutants of L. monocytogenes. A hemolysin-negative mutant of L. monocytogenes failed to elicit transcriptional regulation of gene fragment WS10-B4/14 or WS30-B2/1, and it elicited only minimal regulation of MKP-1, suggesting that escape from the phagosome may be required to initiate these responses. Furthermore, mutants with mutations in mpl and actA, two genes whose gene products are involved in actin polymerization and intrahost spread, also did not induce regulation of WS10-B4/14. These results demonstrate that (i) DPCR can identify specific host cell genes which are differentially transcribed after infection with certain microorganisms and (ii) some of these genes may be new or may never before have been linked to interactions between hosts and pathogens.

Some structures and processes of human epithelial cells involved in uptake of enterohemorrhagic Escherichia coli O157:H7 strains.

Several enterohemorrhagic Escherichia coli (EHEC) strains of serotype O157:H7 isolated from patients with hemorrhagic colitis, ischemic colitis, or hemolytic uremic syndrome were all found to be able to invade certain human epithelial cell lines in vitro. Their ability to gain entry into epithelial cells was compared with those of known invasive Shigella flexneri and Salmonella typhi strains and the noninvasive E. coli strain HB101 in invasion assays utilizing gentamicin to kill extracellular bacteria. All EHEC strains under investigation were efficiently internalized into T24 bladder and HCT-8 ileocecal cells. In striking contrast to shigellae, the same EHEC strains were not taken up into human embryonic intestinal INT407 cells or HEp-2 cells any more than the noninvasive E. coli strain HB101. The mechanism(s) of EHEC internalization was characterized by comparing the invasion efficiencies in the absence and presence of a variety of inhibitors acting on structures and processes of prokaryotic or eukaryotic cells. Also, wild-type, plasmid-containing EHEC strains were compared with their plasmid-cured isogenic derivative strains to determine if plasmid genes affect invasion ability. Plasmid-cured EHEC invaded as well as wild-type EHEC, indicating that invasion ability is chromosomally encoded. Inhibition of bacterial protein synthesis by simultaneous addition of bacteria and chloramphenicol to the monolayer blocked EHEC uptake dramatically, suggesting the presence of an invasion protein(s) with a short half-life. Studies utilizing inhibitors which act on eukaryotic cells demonstrated a strong dependence on microfilaments in the process of uptake of all EHEC strains into both T24 and HCT-8 cells. In general, depolymerization of microtubules as well as inhibition of receptor-mediated endocytosis reduced the efficiency of EHEC invasion of T24 cells, whereas interference with endosome acidification reduced EHEC entry into only HCT-8 cells. Taxol-induced stabilization of microtubules did not inhibit internalization into T24 cells or into the HCT-8 cell line. In marked contrast, the ability of S. typhi Ty2 to invade either cell line was inhibited only by depolymerization of microfilaments. In addition to the cell line specificity of EHEC invasion, not all EHEC strains displayed uniform behavior in the presence of inhibitors, suggesting the existence of variant uptake pathways in different strains. Most importantly, previous reports of the inability of EHEC to invade INT407 or HEp-2 cell lines support the currently held belief that EHEC strains are noninvasive.(ABSTRACT TRUNCATED AT 400 WORDS)