Enterohemorrhagic Escherichia coli O157:H7, a world-wide human food-borne pathogen, causes mild to severe diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. The ability of this pathogen to persist in the environment contributes to its dissemination to a wide range of foods and food processing surfaces. Biofilms are thought to be involved in persistence, but the process of biofilm formation is complex and poorly understood in E. coli O157:H7. To better understand the genetics of this process, a mini-Tn5 transposon insertion library was constructed in strain EDL933 and screened for biofilm-negative mutants using a microtiter plate assay. Ninety-five of 11,000 independent insertions (0.86%) were biofilm negative, and transposon insertions were located in 51 distinct genes/intergenic regions that must be involved either directly or indirectly in biofilm formation. All of the 51 biofilm-negative mutants showed reduced biofilm formation on both hydrophilic and hydrophobic surfaces. Thirty-six genes were unique to this study, including genes on the virulence plasmid pO157. The type V secreted autotransporter serine protease EspP and the enterohemolysin translocator EhxD were found to be directly involved in biofilm formation. In addition, EhxD and EspP were also important for adherence to T84 intestinal epithelial cells, suggesting a role for these genes in tissue interactions in vivo.

Mycoplasma hyopneumoniae causes swine pneumonia and contributes significantly to the porcine respiratory disease complex. The mechanisms of pathogenesis are difficult to address, since there is a lack of genetic tools, but microarrays are available and can be used to study transcriptional changes that occur during disease as a way to identify important virulence-related genes. Mycoplasmas were collected from bronchial alveolar lavage samples and compared to broth-grown cells using microarrays. Bronchial alveolar lavage was performed on pigs 28 days postinfection, and mycoplasmas were isolated by differential centrifugation. Mycoplasma RNAenriched preparations were then obtained from total RNA by subtracting eucaryotic ribosomal and messenger RNAs. Labeled cDNAs were generated with mycoplasma open reading frame-specific primers. Nine biological replicates were analyzed. During lung infection, our analysis indicated that 79 M. hyopneumoniae genes were differentially expressed (P < 0.01), at a false-discovery rate of <2. The reactions that invading pathogens have toward host responses determine their ability to colonize and cause disease. Most bacteria have inducible regulons whose products enhance colonization and assist in the disease process. With mycoplasmas, the simplest of bacterial pathogens, the story of virulence factors and their regulation has been poorly studied and is less well known. Some mycoplasma species can generate high rates of diversity in selected genes or gene families through small sequence changes and recombination events resulting in changes at the genetic level (32). This variation is usually expressed by the generation of new chimeric surface molecules with high rates of antigenic diversity that are thought to help evade the host immune system. Phase switching may result when variation occurs within poly(A) tracts in promoter regions or in structural gene sequences or by DNA inversion. The generation of chimeric genes by intragenic recombination also occurs. This variation is carried through subsequent generations. There is no evidence that any of these mechanisms are operative in Mycoplasma hyopneumoniae, however. Some surface molecules in M. hyopneumoniae undergo posttranslational modification through proteolytic processing as a form of variation identifiable by immunoblotting (4, 7).Previous studies have shown that gene regulation does occur in M. hyopneumoniae in regard to heat shock (13), iron deprivation (14), and exposure to hydrogen peroxide (23). Since these growth conditions only partially mimic those found in vivo, we wanted to determine how exposure to the host during disease alters the transcriptional profiles of M. hyopneumoniae. Thus, we obtained organisms from infected pig lungs and compared their transcriptome with that from organisms grown in the laboratory using microarray technology. This provided a "global" snapshot of the steady-state concentrations of mRNAs, a good indicator of gene expression. Surprisingly, our results indicate that more genes are down-regulated d...

Transcriptome analysis using microarrays has become a powerful tool to better understand the process of disease and other complex biological processes such as food spoilage and biofilm formation. This review is divided into two basic sections: 1) a short history and description of microarrays and 2) a discussion of studies involving bacterial food safety pathogens that focused on whole genome transcript analysis. Not included are the many studies using microarrays to identify, diagnose, or genetically characterize these organisms. This review focuses on studies involving Escherichia coli O157:H7, Salmonella spp., Campylobacter jejuni, Listeria monocytogenes, and Yersinia enterocolitica. Many of the studies involve altering the growth environment to simulate stress conditions and the use of host-pathogen model systems to explore virulence mechanisms. Few studies use conditions that might be considered unique to the food industry. Exceptions are studies of biofilm-specific transcriptome changes and analysis following pressure treatment. This review should not be considered as a comprehensive review, and where appropriate, species-specific reviews are cited that are more complete.

During the establishment of Escherichia coli O157:H7 infection, its capacity to adhere to host intestinal epithelial cells is the critical first step in pathogenesis. It also has the capability to form biofilms, and because both are surface activities, we sought to gain insight into a potential linkage between biofilm formation and adherence to epithelial cells. We conducted an adherence assay with 51 biofilm-negative mutants and two human epithelial cell lines, T84 and HEp2. Our results show that unlike wild-type cells, biofilm-negative mutants adhere poorly to epithelial cells. Some adhesin-negative mutants were fully competent in biofilm formation, however. Thus, biofilm-forming activity in E. coli O157:H7 EDL933 is required for adherence to T84 and HEp2 cells, but it is not sufficient.

In the last 15 years, crustacean fisheries have experienced billions of dollars in economic losses, primarily due to viral diseases caused by such pathogens as white spot syndrome virus (WSSV) in the Pacific white shrimp Litopenaeus vannamei and Asian tiger shrimp Penaeus monodon. To date, no effective measures are available to prevent or control disease outbreaks in these animals, despite their economic importance. Recently, double-stranded RNA-based vaccines have been shown to provide specific and robust protection against WSSV infection in cultured shrimp. However, the limited stability of double-stranded RNA is the most significant hurdle for the field application of these vaccines with respect to delivery within an aquatic system. Polyanhydride nanoparticles have been successfully used for the encapsulation and release of vaccine antigens. We have developed a double-stranded RNA-based nanovaccine for use in shrimp disease control with emphasis on the Pacific white shrimp L. vannamei. Nanoparticles based on copolymers of sebacic acid, 1,6-bis(p-carboxyphenoxy)hexane, and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane exhibited excellent safety profiles, as measured by shrimp survival and histological evaluation. Furthermore, the nanoparticles localized to tissue target replication sites for WSSV and persisted through 28 days postadministration. Finally, the nanovaccine provided ~80% protection in a lethal WSSV challenge model. This study demonstrates the exciting potential of a safe, effective, and field-applicable RNA nanovaccine that can be rationally designed against infectious diseases affecting aquaculture.

To better understand the genetics and function(s) of biofilm formation in Escherichia coli O157:H7, a series of studies were performed beginning with a genome wide mutational analysis. A mini-Tn5 transposon library was constructed and screened for biofilm phenotype using a microtiter plate assay. Ninety-five of 11,000 independent insertions (0.86%) were biofilm-negative, and transposon insertions were located in 51 distinct genes/intergenic regions. Thirty-six genes were unique to this study. All of the fifty-one biofilm-negative phenotype (bnp) mutants showed reduced biofilm formation on both hydrophilic and hydrophobic surfaces. Notably, the involvement of virulence plasmid pO157 genes ehxD, an enterohemolysin auto transporter, and espP were identified. In addition, EhxD and EspP were also shown to be important for adherence to T84 intestinal epithelial cells suggesting a role for these genes in tissue interactions in vivo.The involvement of Z2086, a phage-encoded putative regulator, in biofilm formation was confirmed, the first such demomstration of a function for the cryptic phage-encoded genes in biofilm formation. Transcriptional analysis of a Z2086 deletion (∆Z2086) compared to wild type showed that at p < 0.01, 363 genes were differentially expressed. Six (1.6%) were upregulated and 357 (98.3%) were down-regulated. The function of the differentially-regulated genes varied widely and included 11 biofilm-related genes, 20 virulence genes, genes of metabolic pathways, ABC transporters, ribosomal proteins and DNA binding proteins. Of the 357 down-regulated genes, 213 (59.6%) were phage-encoded, 16 (4.5%) were pO157encoded.During the establishment of E. coli O157:H7 infection, its capacity to adhere to host intestinal epithelial cells is the critical first step in pathogenesis. To gain insight into a potential linkage between biofilm formation and adherence to epithelial cells, the ability of the fifty-one bnp mutants to adhere to two human epithelial-like cell lines was examined. The analysis showed that all bnp mutants were incapable of adhering to T84 and HEp2 cells. In addition, adherence mutants ∆eae and ∆espAB were fully competent in biofilm formation. Thus, these results show that while the biofilm forming activity is required for adherence to these cells, it is not sufficient. Additional adhesins or other supplemental structures are also required; the loss of either activity results in an inability to adhere.

No abstract

No abstract