Mouse Models of Escherichia coli O157:H7 Infection and Shiga Toxin Injection

Escherichia coli O157:H7 has been responsible for multiple food- and waterborne outbreaks of diarrhea and/or hemorrhagic colitis (HC) worldwide. More importantly, a portion of E. coli O157:H7-infected individuals, particularly young children, develop a life-threatening sequela of infection called hemolytic uremic syndrome (HUS). Shiga toxin (Stx), a potent cytotoxin, is the major virulence factor linked to the presentation of both HC and HUS. Currently, treatment of E. coli O157:H7 and other Stx-producing E. coli (STEC) infections is limited to supportive care. To facilitate development of therapeutic strategies and vaccines for humans against these agents, animal models that mimic one or more aspect of STEC infection and disease are needed. In this paper, we focus on the characteristics of various mouse models that have been developed and that can be used to monitor STEC colonization, disease, pathology, or combinations of these features as well as the impact of Stx alone

High Frequency, Spontaneous <i>motA</i> Mutations in <i>Campylobacter jejuni</i> Strain 81-176

<div><p><i>Campylobacter jejuni</i> is an important cause of bacterial diarrhea worldwide. The pathogenesis of <i>C. jejuni</i> is poorly understood and complicated by phase variation of multiple surface structures including lipooligosaccharide, capsule, and flagellum. When <i>C. jejuni</i> strain 81-176 was plated on blood agar for single colonies, the presence of translucent, non-motile colonial variants was noted among the majority of opaque, motile colonies. High-throughput genomic sequencing of two flagellated translucent and two opaque variants as well as the parent strain revealed multiple genetic changes compared to the published genome. However, the only mutated open reading frame common between the two translucent variants and absent from the opaque variants and the parent was <i>motA</i>, encoding a flagellar motor protein. A total of 18 spontaneous <i>motA</i> mutations were found that mapped to four distinct sites in the gene, with only one class of mutation present in a phase variable region. This study exemplifies the mutative/adaptive properties of <i>C. jejuni</i> and demonstrates additional variability in <i>C. jejuni</i> beyond phase variation.</p></div

Sequenced T (translucent or distinct) variants with a <i>motA</i> mutation listed by type (see Figure 3).

1<p>Percentage of variants out of 18 total.</p>2<p>Reversion of the mutation confirmed by sequence analysis. N/D indicates that analysis was not done for this single variant.</p

Amino acid alignment of MotA.

<p>The <i>motA</i> mutations are predicted to result in non-functional proteins. A missense mutation at base 262 (T1, Type B) resulted in an A to P mutation at amino acid 88; a nonsense mutation at base 612 (T3, Type D) resulted in a premature truncation; a duplication of 49 bp created a direct repeat within <i>motA</i> and led to the replacement of the last 73 residues in the C-terminus with a sequence of 39 new residues that are highlighted (T108, Type C); and a deletion at base 64 resulting in a truncated protein (T158, Type A).</p

Nucleotide alignment of <i>motA</i> and mutated alleles.

<p>The four mutations found within <i>motA</i> have been assigned a type for ease in distinguishing. Type A mutation is a deletion at base 64 (T158). Type B mutation is a missense mutation at base 262 (T1). Type C mutation is a duplication of 49 bp (boxed) creating a direct repeat (T108). Type D mutation is a nonsense mutation at base 612 (T3).</p

Derivation of 81-176/55 and the appearance of colonial variants linked with motility.

<p>(A) Cartoon depicting the origin of the sequenced strains. Abbreviations include O for opaque and T for translucent. (B) Appearance of colonial variants of <i>C. jejuni</i> 81-176 grown on CBA plates in microaerobic conditions following dilution and plating. Colony morphology was examined on CBA (C) as well as MH agar (D) and motility analysis made use of 0.6% BB agar 24-well plates with single colonies stabbed into the wells. (E) Transmission electron microscopy images of negatively stained WT, opaque, and translucent variants of <i>C. jejuni</i> strain 81-176.</p

Schematic illustration of the 69-176/55 and its 4 additional sequenced offspring.

<p>The assembly of the whole genome sequences revealed varying SNPs throughout the chromosome. One confirmed difference between the previously sequenced 81-176 and 81-176/55 (as well as the opaque and translucent progeny) was the presence of a 69 bp deletion in the intergenic region between <i>hup</i> and <i>cysK</i>. An incomplete direct repeat (IDR) bracketing the deletion is indicated.</p

The Polysaccharide Capsule of \u3ci\u3eCampylobacter jejuni\u3c/i\u3e Modulates the Host Immune Response

Campylobacter jejuni is a major cause of bacterial diarrheal disease worldwide. The organism is characterized by a diversity of polysaccharide structures, including a polysaccharide capsule. Most C. jejuni capsules are known to be decorated nonstoichiometrically with methyl phosphoramidate (MeOPN). The capsule of C. jejuni 81-176 has been shown to be required for serum resistance, but here we show that an encapsulated mutant lacking the MeOPN modification, an mpnC mutant, was equally as sensitive to serum killing as the nonencapsulated mutant. A nonencapsulated mutant, a kpsM mutant, exhibited significantly reduced colonization compared to that of wild-type 81-176 in a mouse intestinal colonization model, and the mpnC mutant showed an intermediate level of colonization. Both mutants were associated with higher levels of interleukin 17 (IL-17) expression from lamina propria CD4+ cells than from cells from animals infected with 81-176. In addition, reduced levels of Toll-like receptor 4 (TLR4) and TLR2 activation were observed following in vitro stimulation of human reporter cell lines with the kpsM and mpnC mutants compared to those with wild-type 81-176. The data suggest that the capsule polysaccharide of C. jejuni and the MeOPN modification modulate the host immune response