A pathoadaptive deletion in an enteroaggregative Escherichia coli outbreak strain enhances virulence in a Caenorhabditis elegans model.

Enteroaggregative Escherichia coli (EAEC) strains are important diarrheal pathogens. EAEC strains are defined by their characteristic stacked-brick pattern of adherence to epithelial cells but show heterogeneous virulence and have different combinations of adhesin and toxin genes. Pathoadaptive deletions in the lysine decarboxylase (cad) genes have been noted among hypervirulent E. coli subtypes of Shigella and enterohemorrhagic E. coli. To test the hypothesis that cad deletions might account for heterogeneity in EAEC virulence, we developed a Caenorhabditis elegans pathogenesis model. Well-characterized EAEC strains were shown to colonize and kill C. elegans, and differences in virulence could be measured quantitatively. Of 49 EAEC strains screened for lysine decarboxylase activity, 3 tested negative. Most notable is isolate 101-1, which was recovered in Japan, from the largest documented EAEC outbreak. EAEC strain 101-1 was unable to decarboxylate lysine in vitro due to deletions in cadA and cadC, which, respectively, encode lysine decarboxylase and a transcriptional activator of the cadAB genes. Strain 101-1 was significantly more lethal to C. elegans than control strain OP50. Lethality was attenuated when the lysine decarboxylase defect was complemented from a multicopy plasmid and in single copy. In addition, restoring lysine decarboxylase function produced derivatives of 101-1 deficient in aggregative adherence to cultured human epithelial cells. Lysine decarboxylase inactivation is pathoadapative in an important EAEC outbreak strain, and deletion of cad genes could produce hypervirulent EAEC lineages in the future. These results suggest that loss, as well as gain, of genetic material can account for heterogeneous virulence among EAEC strains.

Aggregative Adherence and Intestinal Colonization by Enteroaggregative Escherichia coli Are Produced by Interactions among Multiple Surface Factors.

Enteroaggregative Escherichia coli (EAEC) bacteria are exceptional colonizers that are associated with diarrhea. The genome of EAEC strain 042, a diarrheal pathogen validated in a human challenge study, encodes multiple colonization factors. Notable among them are aggregative adherence fimbriae (AAF/II) and a secreted antiaggregation protein (Aap). Deletion of aap is known to increase adherence, autoaggregation, and biofilm formation, so it was proposed that Aap counteracts AAF/II-mediated interactions. We hypothesized that Aap sterically masks heat-resistant agglutinin 1 (Hra1), an integral outer membrane protein recently identified as an accessory colonization factor. We propose that this masking accounts for reduced in vivo colonization upon hra1 deletion and yet no colonization-associated phenotypes when hra1 is deleted in vitro. Using single and double mutants of hra1, aap, and the AAF/II structural protein gene aafA, we demonstrated that increased adherence in aap mutants occurs even when AAF/II proteins are genetically or chemically removed. Deletion of hra1 together with aap abolishes the hyperadherence phenotype, demonstrating that Aap indeed masks Hra1. The presence of all three colonization factors, however, is necessary for optimal colonization and for rapidly building stacked-brick patterns on slides and cultured monolayers, the signature EAEC phenotype. Altogether, our data demonstrate that Aap serves to mask nonstructural adhesins such as Hra1 and that optimal colonization by EAEC is mediated through interactions among multiple surface factors. IMPORTANCE Enteroaggregative Escherichia coli (EAEC) bacteria are exceptional colonizers of the human intestine and can cause diarrhea. Compared to other E. coli pathogens, little is known about the genes and pathogenic mechanisms that differentiate EAEC from harmless commensal E. coli. EAEC bacteria attach via multiple proteins and structures, including long appendages produced by assembling molecules of AafA and a short surface protein called Hra1. EAEC also secretes an antiadherence protein (Aap; also known as dispersin) which remains loosely attached to the cell surface. This report shows that dispersin covers Hra1 such that the adhesive properties of EAEC seen in the laboratory are largely produced by AafA structures. When the bacteria colonize worms, dispersin is sloughed off, or otherwise removed, such that Hra1-mediated adherence occurs. All three factors are required for optimal colonization, as well as to produce the signature EAEC stacked-brick adherence pattern. Interplay among multiple colonization factors may be an essential feature of exceptional colonizers.

Pick Your Poisson: An Educational Primer for Luria and Delbrück's Classic Paper.

The origin of beneficial mutations is fundamentally important in understanding the processes by which natural selection works. Using phage-resistant mutants in Escherichia coli as their model for identifying the origin of beneficial mutations, Luria and Delbrück distinguished between two different hypotheses. Under the first hypothesis, which they termed "acquired immunity," the phages induced bacteria to mutate to immunity; this predicts that none of the resistant mutants were present before infection by the phages. Under the second hypothesis, termed "mutation to immunity," resistant bacteria arose from random mutations independent of the presence of the phages; this predicts that resistant bacteria were present in the population before infection by the phages. These two hypotheses could be distinguished by calculating the frequencies at which resistant mutants arose in separate cultures infected at the same time and comparing these frequencies to the theoretical results under each model. The data clearly show that mutations arise at a frequency that is independent of the presence of the phages. By inference, natural selection reveals the genetic variation that is present in a population rather than inducing or causing this variation.

Crossover distribution and high interference for both the X chromosome and an autosome during oogenesis and spermatogenesis in Caenorhabditis elegans.

Regulation of both the number and the location of crossovers during meiosis is important for normal chromosome segregation. We used sequence-tagged site polymorphisms to examine the distribution of all crossovers on the X chromosome during oogenesis and on one autosome during both oogenesis and spermatogenesis in Caenorhabditis elegans. The X chromosome has essentially one crossover during oogenesis, with only three possible double crossover exceptions among 220 recombinant X chromosomes. All three had one of the two crossovers in the same chromosomal interval, suggesting that crossovers in that interval do not cause interference. No other interval was associated with double crossovers. Very high interference was also found on an autosome during oogenesis, implying that each chromosome has only one crossover during oogenesis. During spermatogenesis, recombination on this autosome was reduced by approximately 30% compared to oogenesis, but the relative distribution of the residual crossovers was only slightly different. In contrast to previous results with other autosomes, no double crossover chromosomes were observed. Despite an increased frequency of nonrecombinant chromosomes, segregation of a nonrecombinant autosome during spermatogenesis appears to occur normally. This indicates that an achiasmate segregation system helps to ensure faithful disjunction of autosomes during spermatogenesis.

SMG-ly knocking out gene expression in specific cells: an educational primer for use with "a novel strategy for cell-autonomous gene knockdown in caenorhabditis elegans defines a cell-specific function for the G-protein subunit GOA-1".

A recent article by Maher et al. in GENETICS introduces an alternative approach to cell-type-specific gene knockdown in Caenorhabditis elegans, using nonsense-mediated decay. This strategy has the potential to be applicable to other organisms (this strategy requires that animals can survive without nonsense-mediated decay-not all can). This Primer article provides a guide and resource for educators and students by describing different gene knockdown methodologies, by assisting with the technically difficult portions of the Maher et al. article, and by providing conceptual questions relating to the article.

Crossover distribution and frequency are regulated by him-5 in Caenorhabditis elegans.

Mutations in the him-5 gene in Caenorhabditis elegans strongly reduce the frequency of crossovers on the X chromosome, with lesser effects on the autosomes. him-5 mutants also show a change in crossover distribution on both the X and autosomes. These phenotypes are accompanied by a delayed entry into pachytene and premature desynapsis of the X chromosome. The nondisjunction, progression defects and desynapsis can be rescued by an exogenous source of double strand breaks (DSBs), indicating that the role of HIM-5 is to promote the formation of meiotic DSBs. Molecular cloning of the gene shows that the inferred HIM-5 product is a highly basic protein of 252 amino acids with no clear orthologs in other species, including other Caenorhabditis species. Although him-5 mutants are defective in segregation of the X chromosome, HIM-5 protein localizes preferentially to the autosomes. The mutant phenotypes and localization of him-5 are similar but not identical to the results seen with xnd-1, although unlike xnd-1, him-5 has no apparent effect on the acetylation of histone H2A on lysine 5 (H2AacK5). The localization of HIM-5 to the autosomes depends on the activities of both xnd-1 and him-17 allowing us to begin to establish pathways for the control of crossover distribution and frequency.

HIM-8 binds to the X chromosome pairing center and mediates chromosome-specific meiotic synapsis.

The him-8 gene is essential for proper meiotic segregation of the X chromosomes in C. elegans. Here we show that loss of him-8 function causes profound X chromosome-specific defects in homolog pairing and synapsis. him-8 encodes a C2H2 zinc-finger protein that is expressed during meiosis and concentrates at a site on the X chromosome known as the meiotic pairing center (PC). A role for HIM-8 in PC function is supported by genetic interactions between PC lesions and him-8 mutations. HIM-8 bound chromosome sites associate with the nuclear envelope (NE) throughout meiotic prophase. Surprisingly, a point mutation in him-8 that retains both chromosome binding and NE localization fails to stabilize pairing or promote synapsis. These observations indicate that stabilization of homolog pairing is an active process in which the tethering of chromosome sites to the NE may be necessary but is not sufficient.