Association of putative enteroaggregative Escherichia coli virulence genes and biofilm production in isolates from travelers to developing countries.

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen that causes acute and chronic diarrhea among children, human immunodeficiency virus-infected patients, and travelers to developing regions of the world. The pathogenesis of EAEC strains involves the production of biofilm. In this study, we determined the association between presence of putative EAEC virulence genes and biofilm formation in 57 EAEC isolates (as defined by HEp-2 adherence) from travelers with diarrhea and in 18 EAEC isolates from travelers without diarrhea. Twelve nondiarrheagenic E. coli isolates from healthy travelers were used as controls. Biofilm formation was measured by using a microtiter plate assay with the crystal violet staining method, and the presence of the putative EAEC virulence genes aap, aatA, aggR, astA, irp2, pet, set1A, and shf was determined by PCR. EAEC isolates were more likely to produce biofilm than nondiarrheagenic E. coli isolates (P = 0.027), and the production of biofilm was associated with the virulence genes aggR, set1A, aatA, and irp2, which were found in 16 (40%), 17 (43%), 10 (25%), and 27 (68%) of the biofilm producers versus only 4 (11%), 6 (6%), 2 (6%), and 15 (43%) in non-biofilm producers (P = 0.008 for aggR, P = 0.0004 for set1A, P = 0.029 for aatA, and P = 0.04 for irp2). Although the proportion of EAEC isolates producing biofilm in patients with diarrhea (51%) was similar to that in patients without diarrhea (61%), biofilm production was related to the carriage of aggR (P = 0.015), set1A (P = 0.001), and aatA (P = 0.025). Since aggR is a master regulator of EAEC, the presence of aap (P = 0.004), astA (P = 0.001), irp2 (P = 0.0006), pet (P = 0.002), and set1A (P = 0.014) in an aggR versus an aggR-lacking background was investigated and was also found to be associated with biofilm production. This study suggests that biofilm formation is a common phenomenon among EAEC isolates derived from travelers with or without diarrhea and that multiple genes associated with biofilm formation are regulated by aggR.

Proteomic identification of in vivo substrates for matrix metalloproteinases 2 and 9 reveals a mechanism for resolution of inflammation.

Clearance of allergic inflammatory cells from the lung through matrix metalloproteinases (MMPs) is necessary to prevent lethal asphyxiation, but mechanistic insight into this essential homeostatic process is lacking. In this study, we have used a proteomics approach to determine how MMPs promote egression of lung inflammatory cells through the airway. MMP2- and MMP9-dependent cleavage of individual Th2 chemokines modulated their chemotactic activity; however, the net effect of complementing bronchoalveolar lavage fluid of allergen-challenged MMP2(-/-)/MMP9(-/-) mice with active MMP2 and MMP9 was to markedly enhance its overall chemotactic activity. In the bronchoalveolar fluid of MMP2(-/-)/MMP9(-/-) allergic mice, we identified several chemotactic molecules that possessed putative MMP2 and MMP9 cleavage sites and were present as higher molecular mass species. In vitro cleavage assays and mass spectroscopy confirmed that three of the identified proteins, Ym1, S100A8, and S100A9, were substrates of MMP2, MMP9, or both. Function-blocking Abs to S100 proteins significantly altered allergic inflammatory cell migration into the alveolar space. Thus, an important effect of MMPs is to differentially modify chemotactic bioactivity through proteolytic processing of proteins present in the airway. These findings provide a molecular mechanism to explain the enhanced clearance of lung inflammatory cells through the airway and reveal a novel approach to target new therapies for asthma.

Enteroaggregative Escherichia coli as a cause of acute diarrhea

Enteroaggregative Escherichia coli (EAEC) are considered an important emerging enteric and food-borne pathogen. The groups importantly affected by EAEC include international travelers, children in the developing world, and patients with HIV infection. EAEC does not commonly cause diarrheal illness in all hosts. ^ The reasons for the observed clinical variation in EAEC infection are multifactorial and are dependant on the pathogen, the inoculum ingested and the host susceptibility. A major obstacle in identifying the mechanism of pathogenesis for EAEC is the heterogeneity in virulence of strains. No EAEC virulence gene is consistently present in all diarrheagenic strains. However, a recent report suggests that a package of plasmid borne and chromosomal virulence factors are under the control of the described transcriptional activator aggR. Although the exact inoculum required for EAEC diarrheal illness is not known, a volunteer study has shown that oral ingestion of 10 10 cfu of virulent EAEC elicited diarrhea. Ongoing studies are being conducted to better define the exact infectious dose. There are also host factors associated with increased susceptibility of persons to diarrheal illness with EAEC. ^ The following three manuscripts: (1) review EAEC as an emerging enteric pathogen; (2) identify EAEC as a cause of acute diarrhea among different subpopulations worldwide; (3) identify virulence characteristics and the molecular epidemiology of EAEC isolates among travelers with diarrheal illness and describe the pathogenesis of EAEC infection. ^

Origins and evolution of VNTR loci: The apolipoprotein B 3$\sp\prime$ VNTR

I studied the apolipoprotein (apo) B 3$\sp\prime$ variable number tandem repeat (VNTR) and did computer simulations of the stepwise mutation model to address four questions: (1) How did the apo B VNTR originate? (2) What is the mutational mechanism of repeat number change at the apo B VNTR? (3) To what extent are population and molecular level events responsible for the determination of the contemporary apo B allele frequency distribution? (4) Can VNTR allele frequency distributions be explained by a simple and conservative mutation-drift model? I used three general approaches to address these questions: (1) I characterized the apo B VNTR region in non-human primate species; (2) I constructed haplotypes of polymorphic markers flanking the apo B VNTR in a sample of individuals from Lorrain, France and studied the associations between the flanking-marker haplotypes and apo B VNTR size; (3) I did computer simulations of the one-step stepwise mutation model and compared the results to real data in terms of four allele frequency distribution characteristics.^ The results of this work have allowed me to conclude that the apo B VNTR originated after an initial duplication of a sequence which is still present as a single copy sequence in New World monkey species. I conclude that this locus did not originate by the transposition of an array of repeats from somewhere else in the genome. It is unlikely that recombination is the primary mutational mechanism. Furthermore, the clustered nature of these associations implicates a stepwise mutational mechanism. From the high frequencies of certain haplotype-allele size combinations, it is evident that population level events have also been important in the determination of the apo B VNTR allele frequency distribution. Results from computer simulations of the one-step stepwise mutation model have allowed me to conclude that bimodal and multimodal allele frequency distributions are not unexpected at loci evolving via stepwise mutation mechanisms. Short tandem repeat loci fit the stepwise mutation model best, followed by microsatellite loci. I therefore conclude that there are differences in the mutational mechanisms of VNTR loci as classed by repeat unit size. (Abstract shortened by UMI.) ^

A cladistic analysis of apolipoprotein B gene variation and plasma lipid and apolipoprotein B levels, using familial data

Any functionally important mutation is embedded in an evolutionary matrix of other mutations. Cladistic analysis, based on this, is a method of investigating gene effects using a haplotype phylogeny to define a set of tests which localize causal mutations to branches of the phylogeny. Previous implementations of cladistic analysis have not addressed the issue of analyzing data from related individuals, though in human studies, family data are usually needed to obtain unambiguous haplotypes. In this study, a method of cladistic analysis is described in which haplotype effects are parameterized in a linear model which accounts for familial correlations. The method was used to study the effect of apolipoprotein (Apo) B gene variation on total-, LDL-, and HDL-cholesterol, triglyceride, and Apo B levels in 121 French families. Five polymorphisms defined Apo B haplotypes: the signal peptide Insertion/deletion, Bsp 1286I, XbaI, MspI, and EcoRI. Eleven haplotypes were found, and a haplotype phylogeny was constructed and used to define a set of tests of haplotype effects on lipid and apo B levels.^ This new method of cladistic analysis, the parametric method, found significant effects for single haplotypes for all variables. For HDL-cholesterol, 3 clusters of evolutionarily-related haplotypes affecting levels were found. Haplotype effects accounted for about 10% of the genetic variance of triglyceride and HDL-cholesterol levels. The results of the parametric method were compared to those of a method of cladistic analysis based on permutational testing. The permutational method detected fewer haplotype effects, even when modified to account for correlations within families. Simulation studies exploring these differences found evidence of systematic errors in the permutational method due to the process by which haplotype groups were selected for testing.^ The applicability of cladistic analysis to human data was shown. The parametric method is suggested as an improvement over the permutational method. This study has identified candidate haplotypes for sequence comparisons in order to locate the functional mutations in the Apo B gene which may influence plasma lipid levels. ^