The Role of Long Polar Fimbriae in Escherichia coli O104:H4 Adhesion and Colonization.

A renewed interest in Shiga toxin-producing Escherichia coli (STEC) strains was sparked due to the appearance of an outbreak in 2011, causing 3,816 diarrheal cases and some deaths in Europe. The causative strain was classified as enteroaggregative E. coli of serotype O104:H4 that had acquired Shiga toxin genes. The ability of STEC O104:H4 to cause disease relies greatly on the bacteria's capacity to colonize, persist, and produce Shiga toxin. However, not much is known about the colonization factors of this strain. Because long polar fimbriae (lpf) lpf1 and lpf2 operons encode important colonization factors in other STEC isolates and E. coli O104:H4 possesses both loci, we hypothesized that Lpf is required for adhesion and colonization. In this study, isogenic lpfA1 and lpfA2 major fimbrial subunit mutants were constructed. To determine their role in O104:H4's virulence, we assessed their ability to adhere to non-polarized and polarized intestinal epithelial cells. The ΔlpfA1 showed decreased adherence in both cell systems, while the ΔlpfA2 only showed a decrease in adherence to polarized Caco-2 cells. We also tested the O104:H4 mutants' ability to form biofilm and found that the ΔlpfA1 was unable to form a stable biofilm. In an in vivo murine model of intestinal colonization, the ΔlpfA1 had a reduced ability to colonize the cecum and large intestine, consistent with the in vitro data. Further, we tested the lpfA1 mutants' ability to compete against the wild type. We found that in the in vitro and in vivo models, the presence of the wild type O104:H4 facilitates increased adherence of the ΔlpfA1 to levels exceeding that of the wild type. Overall, our data demonstrated that Lpf1 is one of the factors responsible for O104:H4 intestinal adhesion and colonization.

EHEC Adhesins.

Adhesins are a group of proteins in enterohemorrhagic Escherichia coli (EHEC) that are involved in the attachment or colonization of this pathogen to abiotic (plastic or steel) and biological surfaces, such as those found in bovine and human intestines. This review provides the most up-to-date information on these essential adhesion factors, summarizing important historical discoveries and analyzing the current and future state of this research. In doing so, the proteins intimin and Tir are discussed in depth, especially regarding their role in the development of attaching and effacing lesions and in EHEC virulence. Further, a series of fimbrial proteins (Lpf1, Lpf2, curli, ECP, F9, ELF, Sfp, HCP, and type 1 fimbriae) are also described, emphasizing their various contributions to adherence and colonization of different surfaces and their potential use as genetic markers in detection and classification of different EHEC serotypes. This review also discusses the role of several autotransporter proteins (EhaA-D, EspP, Saa and Sab, and Cah), as well as other proteins associated with adherence, such as flagella, EibG, Iha, and OmpA. While these proteins have all been studied to varying degrees, all of the adhesins summarized in this chapter have been linked to different stages of the EHEC life cycle, making them good targets for the development of more effective diagnostics and therapeutics.

Enterohemorrhagic Escherichia coli Adhesins.

Adhesins are a group of proteins in enterohemorrhagic Escherichia coli (EHEC) that are involved in the attachment or colonization of this pathogen to abiotic (plastic or steel) and biological surfaces, such as those found in bovine and human intestines. This review provides the most up-to-date information on these essential adhesion factors, summarizing important historical discoveries and analyzing the current and future state of this research. In doing so, the proteins intimin and Tir are discussed in depth, especially regarding their role in the development of attaching and effacing lesions and in EHEC virulence. Further, a series of fimbrial proteins (Lpf1, Lpf2, curli, ECP, F9, ELF, Sfp, HCP, and type 1 fimbria) are also described, emphasizing their various contributions to adherence and colonization of different surfaces and their potential use as genetic markers in detection and classification of different EHEC serotypes. This review also discusses the role of several autotransporter proteins (EhaA-D, EspP, Saa and Sab, and Cah), as well as other proteins associated with adherence, such as flagella, EibG, Iha, and OmpA. While these proteins have all been studied to varying degrees, all of the adhesins summarized in this article have been linked to different stages of the EHEC life cycle, making them good targets for the development of more effective diagnostics and therapeutics.

Identification of novel and cross-species seroreactive proteins from Bacillus anthracis using a ligation-independent cloning-based, SOS-inducible expression system.

The current standard for Bacillus anthracis vaccination is the Anthrax Vaccine Adsorbed (AVA, BioThrax). While effective, the licensed vaccine schedule requires five intramuscular injections in the priming series and yearly boosters to sustain protection. One potential approach to maintain or improve the protection afforded by an anthrax vaccine, but requiring fewer doses, is through the use of purified proteins to enhance an antibody response, which could be used on their own or in combination with the current vaccine. This study describes a novel, high-throughput system to amplify and clone every gene in the B. anthracis pXO1 and pXO2 virulence plasmids. We attempted to express each cloned gene in Escherichia coli, and obtained full-length expression of 57% of the proteins. Expressed proteins were then used to identify immunogens using serum from three different mammalian infection models: Dutch-belted rabbits, BALB/c mice, and rhesus macaque monkeys. Ten proteins were detected by antibodies in all of these models, eight of which have not been identified as immunoreactive in other studies to date. Serum was also collected from humans who had received the AVA vaccine, and similar screens showed that antigens that were detected in the infection models were not present in the serum of vaccinated humans, suggesting that antibodies elicited by the current AVA vaccine do not react with the immunoreactive proteins identified in this study. These results will contribute to the future selection of targets in antigenicity and protection studies as one or more of these proteins may prove to be worthy of inclusion in future vaccine preparations.

Paradoxical DNA repair and peroxide resistance gene conservation in Bacillus pumilus SAFR-032.

BACKGROUND: Bacillus spores are notoriously resistant to unfavorable conditions such as UV radiation, gamma-radiation, H2O2, desiccation, chemical disinfection, or starvation. Bacillus pumilus SAFR-032 survives standard decontamination procedures of the Jet Propulsion Lab spacecraft assembly facility, and both spores and vegetative cells of this strain exhibit elevated resistance to UV radiation and H2O2 compared to other Bacillus species. PRINCIPAL FINDINGS: The genome of B. pumilus SAFR-032 was sequenced and annotated. Lists of genes relevant to DNA repair and the oxidative stress response were generated and compared to B. subtilis and B. licheniformis. Differences in conservation of genes, gene order, and protein sequences are highlighted because they potentially explain the extreme resistance phenotype of B. pumilus. The B. pumilus genome includes genes not found in B. subtilis or B. licheniformis and conserved genes with sequence divergence, but paradoxically lacks several genes that function in UV or H2O2 resistance in other Bacillus species. SIGNIFICANCE: This study identifies several candidate genes for further research into UV and H2O2 resistance. These findings will help explain the resistance of B. pumilus and are applicable to understanding sterilization survival strategies of microbes.