We have shown that bacteria injected intravenously into live animals entered and replicated in solid tumors and metastases. The tumor-specific amplification process was visualized in real time using luciferase-catalyzed luminescence and green fluorescent protein fluorescence, which revealed the locations of the tumors and metastases. Escherichia coli and three attenuated pathogens (Vibrio cholerae, Salmonella typhimurium, and Listeria monocytogenes) all entered tumors and replicated. Similarly, the cytosolic vaccinia virus also showed tumor-specific replication, as visualized by real-time imaging. These findings indicate that neither auxotrophic mutations, nor vaccinia virus deficient for the thymidine kinase gene, nor anaerobic growth conditions were required for tumor specificity and intratumoral replication. We observed localization of tumors by light-emitting microorganisms in immunocompetent and in immunocompromised rodents with syngeneic and allogeneic tumors. Based on their 'tumor-finding' nature, bacteria and viruses may be designed to carry multiple genes for detection and treatment of cancer.
Eukaryotic expression vectors can be delivered to macrophages using attenuated self-destructing Listeria monocytogenes. L. monocytogenes cells are preferentially lysed in the host cell macrophage cytosol by the production of a PactA-dependent Listeria-specific phage lysin. Efficient expression of the cloned reporter genes by the macrophages and subsequent antigen presentation were achieved after the delivery of eukaryotic expression vectors by the attenuated suicide L. monocytogenes strain. After delivery by L. monocytogenes plasmid DNAs were found to integrate into the macrophage cell's genome at a frequency of about 10(-7).
Vaccination provides the most potent measure against infectious disease, and recombinant (r) viable vaccines expressing defined pathogen-derived antigens represent powerful candidates for future vaccination strategies.In a new approach we constructed r-aroA-Salnonella typhimurium displaying p60 or listeriolysin (Hly) antigen ofListeria monocytogenes in secreted or somatic form in the host cell. Vaccination of mice with r-aroA-S. typhimurium induced protection against the intracellular pathogen L. monocytogenes only with secreted and not with somatic antigen. Secreted Hly was slightly more potent in inducing protective immunity than secreted p60. Both r-aroA-S. typhimurium secreting p60 in the endosome and r-aroA-S. typhimurium secreting Hly in the cytosol induced protective CD4+ and CD8+ T-cells suggesting CD8+ T-cell stimulation independent from intracellular residence of r-aroA-S. typhimurium carriers. Hence, not only the type of antigen but also its display by the r-carrier within the host cell critically influences vaccine efficacy.
Protein p60 encoded by the iap gene is regarded as an essential gene product of Listeria monocytogenes. Here we report, however, the successful construction of a viable iap deletion mutant of L. monocytogenes EGD. The mutant, which produces no p60, shows abnormal septum formation and tends to form short filaments and hooked forms during logarithmic growth. These abnormal bacterial cells break into almost normal sized single bacteria in the late-stationary-growth phase. The iap mutant is strongly attenuated in a mouse model after intravenous injection, demonstrating the importance of p60 during infection, and the invasiveness of the ⌬iap mutant for 3T6 fibroblasts and Caco-2 epithelial cells is slightly reduced. Upon uptake by epithelial cells and macrophages, the iap mutant escapes from the phagosome into the cytosol with the same efficiency as the wild-type strain, and the mutant bacteria also grow intracellularly at a rate similar to that of the wild-type strain. Intracellular movement and cell-to-cell spread are drastically reduced in various cell lines, since the iap-negative bacteria fail to induce the formation of actin tails. However, the bacteria are covered with actin filaments. Most intracellular bacteria show a nonpolar and uneven distribution of ActA around the cell, in contrast to that for the wild-type strain, where ActA is concentrated at the old pole. In an iap ؉ revertant strain that produces wild-type levels of p60, intracellular movement, cell-to-cell spread, and polar distribution of ActA are fully restored. In vitro analysis of ActA distribution on the filaments of the ⌬iap strain shows that the loss of bacterial septum formation leads to ActA accumulation at the presumed division sites. In the light of data presented here and elswhere, we propose to rename iap (invasion-associated protein) cwhA (cell wall hydrolase A).
Mutants of Listeria monocytogenes with deletions in genes of the common branch of the biosynthesis pathway leading to aromatic compounds were constructed as possible virulence-attenuated carrier strains for protein antigens or vaccine DNA. aroA, aroB, and in particular aroE mutants showed strongly reduced growth rates in epithelial cells and even in rich culture media. The metabolism of the aro mutants under these conditions was predominantly anaerobic. Aerobic metabolism and a wild-type growth rate were, however, regained upon the addition of vitamin K 2 , suggesting that the aro mutants are deficient in oxidative respiration due to the lack of menaquinone. Replication of the aro mutants in the host cell's cytosol and cell-to-cell spread were drastically slowed down, and all aro mutants showed high virulence attenuation in mice, i.e., the 50% lethal dose in BALB/c mice was increased at least 10 4 -fold for the aroA, aroB, and aroA/B mutants and >10 5 -fold for the aroE mutant compared to the parent strain. Nevertheless, mice preimmunized with aro mutant bacteria elicited good T-cell response and full protection against a subsequent challenge with the virulent wild-type strain. A total of 5 ؋ 10 6 aroA, aroB, and aroA/B mutant bacteria were sufficient to obtain a protective T-cell response, while 5 ؋ 10 8 aroE or aroA/E mutants were necessary to achieve comparable numbers of antigen-specific T cells. These numbers were well tolerated without causing any signs of disease, indicating that Listeria strains with deletions in genes of the basic branch of the aromatic amino acid pathway could be useful vaccine carriers for inducing T-cell immunity.
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