Dennis J. Kopecko

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

ABSTRACT 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 atetO 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 pyloriproteins, 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 ofC. jejuni pathogens.

Differential Bacterial Survival, Replication, and Apoptosis-Inducing Ability of Salmonella Serovars within Human and Murine Macrophages

ABSTRACT 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. entericaserovar 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 Salmonellawere 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 distinctSalmonella 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 Salmonellais linked to the ability of that serovar both to persist within and to elicit damage in a specific hosts macrophage cells.

Down-regulation of signal transducer and activator of transcription 3 expression using vector-based small interfering RNAs suppresses growth of human prostate tumor in vivo.

Purpose: Signal transducer and activator of transcription 3 (Stat3) is constitutively activated in a variety of cancers and it is a common feature of prostate cancer. Thus, Stat3 represents a promising molecular target for tumor therapy. We applied a DNA vector–based Stat3-specific RNA interference approach to block Stat3 signaling and to evaluate the biological consequences of Stat3 down-modulation on tumor growth using a mouse model. Experimental Design: To investigate the therapeutic potential of blocking Stat3 in cancer cells, three small interfering RNAs (siRNA; Stat3-1, Stat3-2, and Stat3-3) specific for different target sites on Stat3 mRNA were designed and used with a DNA vector–based RNA interference approach expressing short hairpin RNAs to knockdown Stat3 expression in human prostate cancer cells in vitro as well as in vivo. Results: Of the three equivalently expressed siRNAs, only Stat3-3 and Stat3-2, which target the region coding for the SH2 domain and the coiled-coil domain, respectively, strongly suppressed the expression of Stat3 in PC3 and LNCaP cells. The Stat3-1 siRNA, which targeted the DNA-binding domain, exerted no effect on Stat3 expression, indicating that the gene silencing efficiency of siRNA may be dependent on the local structure of Stat3 mRNA. The Stat3 siRNAs down-regulated the expression of Bcl-2 (an antiapoptotic protein), and cyclin D1 and c-Myc (cell growth activators) in prostate cancer cells. Inhibition of Stat3 and its related genes was accompanied by growth suppression and induction of apoptosis in cancer cells in vitro and in tumors implanted in nude mice. Conclusions: These data indicate that Stat3 signaling is a promising molecular target for prostate cancer therapy and that vector-based Stat3 siRNA may be useful as a therapeutic agent for treatment of prostate cancer.

Intratumoral Delivery and Suppression of Prostate Tumor Growth by Attenuated Salmonella enterica serovar typhimurium Carrying Plasmid-Based Small Interfering RNAs

The facultative anaerobic, invasive Salmonella enterica serovar typhimurium (S. typhimurium) has been shown to retard the growth of established tumors. We wondered if a more effective antitumor response could be achieved in vivo if these bacteria were used as tools for delivering specific molecular antitumor therapeutics. Constitutively activated transcription factor signal transducer and activator of transcription 3 (STAT3) promotes the survival of a number of human tumors. In this study, we investigated the relative efficacies of attenuated S. typhimurium alone or combined with Stat3-specific small interfering RNA (siRNA) in terms of tumor growth and metastasis. The bacteria preferentially homed into tumors over normal liver and spleen tissues in vivo. S. typhimurium expressing plasmid-based Stat3-specific siRNAs significantly inhibited tumor growth, reduced the number of metastastic organs, and extended the life time for C57BL6 mice bearing an implanted prostate tumor, versus bacterial treatment alone. These results suggest that attenuated S. typhimurium combined with an RNA interference approach might be more effective for the treatment of primary as well as metastatic cancer.

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.

Effects of Plasmid-Based Stat3-Specific Short Hairpin RNA and GRIM-19 on PC-3M Tumor Cell Growth

Purpose: Persistent activation of signal transducers and activators of transcription 3 (Stat3) and its overexpression contribute to the progression and metastasis of several different tumor types. For this reason, Stat3 is a reasonable target for RNA interference–mediated growth inhibition. Blockade of Stat3 using specific short hairpin RNAs (shRNA) can significantly reduce prostate tumor growth in mice. However, RNA interference does not fully ablate target gene expression in vivo, owing to the idiosyncrasies associated with shRNAs and their targets. To enhance the therapeutic efficacy of Stat3-specific shRNA, we applied a combination treatment involving gene associated with retinoid-IFN–induced mortality 19 (GRIM-19), another inhibitor of STAT3, along with shRNA. Experimental Design: The coding sequences for GRIM-19, a cellular STAT3-specific inhibitor, and Stat3-specific shRNAs were used to create a dual expression plasmid vector and used for prostate cancer therapy in vitro and in mouse xenograft models in vivo. Results: The coexpressed Stat3-specific shRNA and GRIM-19 synergistically and more effectively suppressed prostate tumor growth and metastases when compared with treatment with either single agent alone. Conclusion: The simultaneous use of two specific, but mechanistically different, inhibitors of STAT3 activity exerts enhanced antitumor effects.

Breaching the mucosal barrier by stealth: an emerging pathogenic mechanism for enteroadherent bacterial pathogens

The clinical and molecular understanding of bacterial diseases of the gastrointestinal tract, developed largely over the past 50 years, has led to our current assignment of these causative agents into an evolving set of mechanistic categories (see Table ​Table1).1). Early studies revealed that some organisms (Staphylococcus aureus among them) produce toxins in contaminated foods and that, upon ingestion, these preformed toxins trigger a rapid-onset, net jejunal secretion of electrolytes and water, typically lasting less than 24 hours. In these illnesses, the causative organism need not be present in the intestine. In contrast, the “enterotoxigenic” pathogens, typified by Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC), are noninflammatory and must colonize the gastrointestinal tract in order to deliver toxins to the mucosa. These toxins typically effect cAMP- or cGMP-mediated net Cl– secretion, often resulting in a large-volume diarrhea. Recent studies of certain strains of Bacteroides fragilis indicate that these agents can serve as large bowel mediators of enterotoxigenic disease via a toxin that disrupts the mucosal barrier (1). Table 1 Pathogenic mechanisms used by diarrheogenic bacteria Another long-recognized grouping of enteric bacterial diseases is the “invasive” category, which can be subdivided based upon the extent of dissemination within the host. The most superficial invaders, Shigella spp. and enteroinvasive E. coli, have similar, if not identical, virulence properties. These organisms invade the epithelium of the colon and terminal ileum, spread intercellularly via actin-filament projections (2), and cause focal microulcerations of the ileocecal mucosa. The submucosal group is exemplified by Yersinia enterocolitica and Yersinia pseudotuberculosis, which transcytose across the mucosal epithelium and proceed to the regional lymph nodes, causing severe abdominal pain and, occasionally, mesenteric lymphadenitis. Recent clinical and experimental data suggest that Campylobacter jejuni is an invasive pathogen that can translocate across the mucosa, and that may survive submucosally, but does not typically reach the bloodstream (3). The third invasive subgroup is typified by certain Salmonella serovars (e.g., Typhi and Paratyphi) that invade and translocate across the epithelium. These organisms enter and apparently survive for many hours in submucosal monocytes, which may serve to transport these pathogens to distal sites in the host (e.g., spleen, liver, and bone marrow). The characterization of a previously unrecognized group of pathogenic E. coli (i.e., enteropathogenic E. coli [EPEC], enteroaggregative E. coli [EAEC], diffusely adherent E. coli [DAEC], and enterohemmorhagic E. coli [EHEC]) since the 1950s has led to the establishment of the enteroadherent category of disease. These agents colonize the small and/or large intestine, do not appear to be invasive to any appreciable extent in vivo, and, with the exception of shiga-like toxins of EHEC, are not known to elaborate any of the classical enterotoxins, yet they cause diarrheal illness. Classical EPEC serotypes are known to induce “pedestal” or “cup-like” rearrangements, referred to as attaching and effacing lesions, of epithelial cell membranes. The attaching bacteria erode the microvilli via a rearrangement of cytoskeletal actin into host membrane pedestal structures that support the organism. EAEC and DAEC display characteristic, but different, adherence patterns on cultured intestinal cells and have not been shown to cause any striking membrane effacement. Although certain aspects of EPEC pathogenesis, such as adherence and the attaching and effacing phenotype, have been studied elegantly and in detail (4), the mechanisms by which these and other enteroadherent organisms induce inflammation and diarrhea have only recently started to emerge (5). Salmonella enterica serovar Typhimurium (now abbreviated S. Typhimurium) have previously been considered members of the invasive category, because of their invasiveness in cultured cells and their ability to cause a typhoid-like illness in mice. Indeed, S. Typhimurium may occasionally be isolated from extraintestinal sites in humans including the bloodstream, particularly in immunocompromised hosts. However, these pathogens are more typically associated in humans with diarrheal illness of mild to moderate severity accompanied by fever and the appearance of fecal leukocytes. Although S. Typhimurium may trigger limited invasion of the intestine, as may occur with other enteroadherent pathogens, mucosal invasion does not appear to be a prominent aspect of normal disease. As now shown in this issue of the JCI (6), S. Typhimurium appear to be prime examples of organisms that cause diarrheal disease through a general mechanism that may characterize many bacteria commonly placed in the enteroadherent category of intestinal diseases.

Cronobacter spp. (previously Enterobacter sakazakii) invade and translocate across both cultured human intestinal epithelial cells and human brain microvascular endothelial cells

The mechanism of Cronobacter pathogenesis in neonatal meningitis and potential virulence factors (aside from host cell invasion ability) remain largely unknown. To ascertain whether Cronobacter can invade and transcytose across intestinal epithelial cells, enter into the blood stream and then transcytose across the blood-brain-barrier, we have utilized human intestinal INT407 and Caco-2 cells and brain microvascular endothelial cell (HBMEC) monolayers on Transwell filters as experimental model systems. Our data indicate a wide range of heterogeneity with respect to invasion efficiency among twenty-three Cronobacter isolates screened. For selected isolates, we observed significant levels of transcytosis for Cronobacter sakazakii across tight monolayers of both Caco-2 and HBMEC, mimicking in vivo ability to cross the intestine as well as the blood brain barrier, and at a frequency equivalent to that of a control meningitis-causing Escherichia coli K1 strain. Finally, EM analysis demonstrated intracellular Cronobacter bacteria within host vacuoles in HBMEC, as well as transcytosed bacteria at the basolateral surface. These data reveal that certain Cronobacter isolates can invade and translocate across both cultured human intestinal epithelial cells and HBMEC, thus demonstrating a potential path for neonatal infections of the central nervous system (CNS) following oral ingestion.

Genetic stability of vaccine strain Salmonella Typhi Ty21a over 25 years

The attenuated live bacterial vaccine strain Salmonella enterica Serovar Typhi Ty21a is the main constituent of Vivotif, the only licensed oral vaccine against typhoid fever. The strain was developed in the 1970s by chemical mutagenesis. In the course of this mutagenesis, a number of mutations were introduced into the vaccine strain. Characterisation of the vaccine strain during development as well as release of master- and working seed lots (MSL and WSL) and commercial batches is based on phenotypic assays assessing microbiological and biochemical characteristics of Ty21a. In the current study, we have analysed by DNA sequencing the specific mutations originally correlated with the attenuation of strain Ty21a. These data demonstrate the stability of these mutations for MSLs and WSLs of Ty21a produced between 1980 and 2005. Finally, we have confirmed the correlation of these genetic mutations with the expected phenotypic attenuations for the seed lots used in vaccine manufacture over 25 years.

Bacterial delivery of siRNAs: a new approach to solid tumor therapy.

RNAi is a powerful research tool for specific gene silencing and may also lead to promising novel therapeutic strategies. However, the development of RNAi-based therapies has been slow due to the lack of targeted delivery methods. The biggest challenge in the use of siRNA-based therapies is the delivery to target cells. There are many additional obstacles to in vivo delivery of siRNAs, such as degradation by endogenous enzymes and interaction with blood components leading to nonspecific uptake into cells, which govern biodistribution and availability of siRNA in the body. Naked unmodified synthetic siRNA including plasmid-carried-shRNA-expression constructs cannot penetrate cellular membranes, and therefore, systemic application is unlikely to be successful. The success of gene therapy by siRNAs relies on the development of safe, economical, and efficacious in vivo delivery systems into the target cells. Attenuated Salmonella have been employed recently as vectors to deliver silencing hairpin RNA (shRNA) expression plasmids into mammalian cells. This approach has achieved gene silencing in vitro and in vivo. The facultative anaerobic, invasive Salmonella have a natural tropism for solid tumors including metastatic tumors. Genetically modified, attenuated Salmonella have been used recently both as potential antitumor agents by themselves, and to deliver specific tumoricidal therapies. This chapter describes the use of attenuated bacteria as tumor-targeting delivery systems for cancer therapy.