Intraspecies variation in the emergence of hyperinfectious bacterial strains in nature.

Salmonella is a principal health concern because of its endemic prevalence in food and water supplies, the rise in incidence of multi-drug resistant strains, and the emergence of new strains associated with increased disease severity. Insights into pathogen emergence have come from animal-passage studies wherein virulence is often increased during infection. However, these studies did not address the prospect that a select subset of strains undergo a pronounced increase in virulence during the infective process- a prospect that has significant implications for human and animal health.

In vivo-selected mutations in methyl-directed mismatch repair suppress the virulence attenuation of Salmonella dam mutant strains following intraperitoneal, but not oral, infection of naïve mice.

Salmonella enterica serovar Typhimurium that lacks the DNA adenine methylase (Dam) ectopically expresses multiple genes that are preferentially expressed during infection, is attenuated for virulence, and confers heightened immunity in vaccinated hosts. The safety of dam mutant Salmonella vaccines was evaluated by screening within infected mice for isolates that have an increased capacity to cause disease relative to the attenuated parental strain. Since dam mutant strains are sensitive to the DNA base analog 2-aminopurine (2-AP), we screened for 2-AP-resistant (2-AP(r)) isolates in systemic tissues of mice infected with dam mutant Salmonella.

Altered levels of Salmonella DNA adenine methylase are associated with defects in gene expression, motility, flagellar synthesis, and bile resistance in the pathogenic strain 14028 but not in the laboratory strain LT2.

Comparative genomic analysis has revealed limited strain diversity between Salmonella pathogenic and nonpathogenic isolates. Thus, some of the relative virulence and host-immune response disparities may be credited to differential gene regulation rather than gross differences in genomic content. Here we show that altered levels of Salmonella DNA adenine methylase (Dam) resulted in acute defects in virulence-associated gene expression, motility, flagellin synthesis, and bile resistance in the Salmonella pathogenic strain 14028 but not in avirulent laboratory strain LT2.

Beyond therapy..

In response to the Blackburn and Rowley essay on the President's Council on Bioethics, several thought-provoking opinions on ethical challenges in biomedical research are expressed by prominent stakeholders

DNA adenine methylase overproduction in Yersinia pseudotuberculosis alters YopE expression and secretion and host immune responses to infection.

Yersinia pseudotuberculosis mutants that overproduce the DNA adenine methylase (Dam) are highly attenuated, confer fully protective immune responses, and secrete several Yersinia virulence proteins (Yersinia outer proteins [Yops]) under conditions that are nonpermissive for secretion in wild-type strains. We examined here the effects of Dam overproduction on Yersinia virulence determinant expression and secretion, as well as the host immune response to Yersinia antigens. Western blot analysis with convalescent antisera identified several low-calcium-responsive antigens whose synthesis was affected by Dam overproduction.

DNA adenine methylase is essential for viability and plays a role in the pathogenesis of Yersinia pseudotuberculosis and Vibrio cholerae.

Salmonella strains that lack or overproduce DNA adenine methylase (Dam) elicit a protective immune response to different Salmonella species. To generate vaccines against other bacterial pathogens, the dam genes of Yersinia pseudotuberculosis and Vibrio cholerae were disrupted but found to be essential for viability. Overproduction of Dam significantly attenuated the virulence of these two pathogens, leading to, in Yersinia, the ectopic secretion of virulence proteins (Yersinia outer proteins) and a fully protective immune response in vaccinated hosts.

Salmonella DNA adenine methylase mutants confer cross-protective immunity.

Salmonella isolates that lack or overproduce DNA adenine methylase (Dam) elicited a cross-protective immune response to different Salmonella serovars. The protection afforded by the Salmonella enterica serovar Typhimurium Dam vaccine was greater than that elicited in mice that survived a virulent infection. S.

Assessment of bacterial pathogenesis by analysis of gene expression in the host.

A number of techniques have been developed to assess the expression of microbial virulence genes within the host (in vivo). These studies have shown that bacteria employ a wide variety of mechanisms to coordinately regulate the expression of these genes during infection. Two tenets have emerged from these studies: bacterial adaptation responses are critical to growth within the host, and interactions between microorganisms and the microenvironments of their hosts cannot be revealed from in vitro studies alone.

In vivo gene expression and the adaptive response: from pathogenesis to vaccines and antimicrobials.

Microbial pathogens possess a repertoire of virulence determinants that each make unique contributions to fitness during infection. Analysis of these in vivo-expressed functions reveals the biology of the infection process, encompassing the bacterial infection strategies and the host ecological and environmental retaliatory strategies designed to combat them (e.g.

An essential role for DNA adenine methylation in bacterial virulence.

Salmonella typhimurium lacking DNA adenine methylase (Dam) were fully proficient in colonization of mucosal sites but showed severe defects in colonization of deeper tissue sites. These Dam- mutants were totally avirulent and were effective as live vaccines against murine typhoid fever. Dam regulated the expression of at least 20 genes known to be induced during infection; a subset of these genes are among those activated by the PhoP global virulence regulator.

Differential patterns of acquired virulence genes distinguish Salmonella strains.

Analysis of several Salmonella typhimurium in vivo-induced genes located in regions of atypical base composition has uncovered acquired genetic elements that cumulatively engender pathogenicity. Many of these regions are associated with mobile elements, encode predicted adhesin and invasin-like functions, and are required for full virulence. Some of these regions distinguish broad host range from host-adapted Salmonella serovars and may contribute to inherent differences in host specificity, tissue tropism, and disease manifestation.

Applications for atomic force microscopy of DNA.

Tapping mode atomic force microscopy (AFM) of DNA in propanol, dry helium, and aqueous buffer each have specific applications. Resolution is best in propanol, which precipitates and immobilizes the DNA and provides a fluid imaging environment where adhesive forces are minimized. Resolution on exceptional images of DNA appears to be approximately 2 nm, sufficient to see helix turns in detail, but the smallest substructures typically seen on DNA in propanol are approximately 6-10 nm in size.

Recent advances in atomic force microscopy of DNA.

Three advances involving DNA in atomic force microscopy (AFM) are reported here. First a HEPES-Mg buffer has been used that improves the spreading of DNA and provides good DNA coverage with as little as 200-500 picograms per sample. Second, the new "tapping" mode has been used to improve the ease and resolution of AFM-imaging of DNA in air.

Atomic force microscopy of DNA in aqueous solutions.

DNA on mica can be imaged in the atomic force microscope (AFM) in water or in some buffers if the sample has first been dehydrated thoroughly with propanol or by baking in vacuum and if the sample is imaged with a tip that has been deposited in the scanning electron microscope (SEM). Without adequate dehydration or with an unmodified tip, the DNA is scraped off the substrate by AFM-imaging in aqueous solutions. The measured heights and widths of DNA are larger in aqueous solutions than in propanol.

Atomic force microscopy of single- and double-stranded DNA.

A method has been developed for imaging single-stranded DNA with the atomic force microscope (AFM). phi X174 single-stranded DNA in formaldehyde on mica can be imaged in the AFM under propanol or butanol or in air. Measured lengths of most molecules are on the order of 1 mu, although occasionally more extended molecules with lengths of 1.

Reproducible imaging and dissection of plasmid DNA under liquid with the atomic force microscope.

Reproducible images of uncoated DNA in the atomic force microscope (AFM) have been obtained by imaging plasmid DNA on mica in n-propanol. Specially sharpened AFM tips give images with reproducible features several nanometers in size along the DNA. Plasmids can be dissected in propanol by increasing the force applied by the AFM tip at selected locations.

Imaging single-stranded DNA, antigen-antibody reaction and polymerized Langmuir-Blodgett films with an atomic force microscope.

The combination of an (AFM) atomic force microscope together with microfabricated cantilevers that have integrated tips opens many possibilities for imaging systems of great importance in biology. We have imaged single-stranded 25mer DNA that was adsorbed on treated mica or that was covalently bound with a crosslinker to a polymerized Langmuir-Blodgett (LB) film, the top monolayer of a bilayer system. At low magnification the AFM shows cracks between solid domains, like in an image taken with a fluorescence microscope.