Felipe Del Canto

Distribution of Classical and Nonclassical Virulence Genes in Enterotoxigenic Escherichia coli Isolates from Chilean Children and tRNA Gene Screening for Putative Insertion Sites for Genomic Islands

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea. Three adhesins (Tia, TibA, EtpA), an iron acquisition system (Irp1, Irp2, and FyuA), a GTPase (LeoA), and an autotransporter (EatA) are ETEC virulence-related proteins that, in contrast to the classical virulence factors (enterotoxins and fimbrial colonization factors) have not heretofore been targets in characterizing isolates from epidemiological studies. Here, we determined the occurrence of these nonclassical virulence genes in 103 ETEC isolates from Chilean children with diarrhea and described their association with O serogroups and classical virulence determinants. Because tia, leoA, irp2, and fyuA are harbored by pathogenicity islands inserted into the selC and asnT tRNA genes (tDNAs), we analyzed the regions flanking these loci. Ten additional tDNAs were also screened to identify hot spots for genetic insertions. Associations between the most frequent serogroups and classical colonization factor (CF)-toxin profiles included O6/LT-STh/CS1-CS3-CS21 (i.e., O6 serogroup, heat-labile [LT] and human heat-stable [STh] enterotoxins, and CFs CS1, -3 and -21), O6/LT-STh/CS2-CS3-CS21, and O104-O127/STh/CFAI-CS21. The eatA and etpA genes were detected in more than 70% of the collection, including diverse serogroups and virulence profiles. Sixteen percent of the ETEC strains were negative for classical and nonclassical adhesins, suggesting the presence of unknown determinants of adhesion. The leuX, thrW, and asnT tDNAs were disrupted in more than 65% of strains, suggesting they are hot spots for the insertion of mobile elements. Sequences similar to integrase genes were identified next to the thrW, asnT, pheV, and selC tDNAs. We propose that the eatA and etpA genes should be included in characterizations of ETEC isolates in future epidemiological studies to determine their prevalence in other geographical regions. Sequencing of tDNA-associated genetic insertions might identify new ETEC virulence determinants.

Identification of Coli Surface Antigen 23, a Novel Adhesin of Enterotoxigenic Escherichia coli

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea, mainly in developing countries. Although there are 25 different ETEC adhesins described in strains affecting humans, between 15% and 50% of the clinical isolates from different geographical regions are negative for these adhesins, suggesting that additional unidentified adhesion determinants might be present. Here, we report the discovery of Coli Surface Antigen 23 (CS23), a novel adhesin expressed by an ETEC serogroup O4 strain (ETEC 1766a), which was negative for the previously known ETEC adhesins, albeit it has the ability to adhere to Caco-2 cells. CS23 is encoded by an 8.8-kb locus which contains 9 open reading frames (ORFs), 7 of them sharing significant identity with genes required for assembly of K88-related fimbriae. This gene locus, named aal (adhesion-associated locus), is required for the adhesion ability of ETEC 1766a and was able to confer this adhesive phenotype to a nonadherent E. coli HB101 strain. The CS23 major structural subunit, AalE, shares limited identity with known pilin proteins, and it is more closely related to the CS13 pilin protein CshE, carried by human ETEC strains. Our data indicate that CS23 is a new member of the diverse adhesin repertoire used by ETEC strains.

Vaccines for viral and bacterial pathogens causing acute gastroenteritis: Part I: Overview, vaccines for enteric viruses and Vibrio cholerae

Efforts to develop vaccines for prevention of acute diarrhea have been going on for more than 40 y with partial success. The myriad of pathogens, more than 20, that have been identified as a cause of acute diarrhea throughout the years pose a significant challenge for selecting and further developing the most relevant vaccine candidates. Based on pathogen distribution as identified in epidemiological studies performed mostly in low-resource countries, rotavirus, Cryptosporidium, Shigella, diarrheogenic E. coli and V. cholerae are predominant, and thus the main targets for vaccine development and implementation. Vaccination against norovirus is most relevant in middle/high-income countries and possibly in resource-deprived countries, pending a more precise characterization of disease impact. Only a few licensed vaccines are currently available, of which rotavirus vaccines have been the most outstanding in demonstrating a significant impact in a short time period. This is a comprehensive review, divided into 2 articles, of nearly 50 vaccine candidates against the most relevant viral and bacterial pathogens that cause acute gastroenteritis. In order to facilitate reading, sections for each pathogen are organized as follows: i) a discussion of the main epidemiological and pathogenic features; and ii) a discussion of vaccines based on their stage of development, moving from current licensed vaccines to vaccines in advanced stage of development (in phase IIb or III trials) to vaccines in early stages of clinical development (in phase I/II) or preclinical development in animal models. In this first article we discuss rotavirus, norovirus and Vibrio cholerae. In the following article we will discuss Shigella, Salmonella (non-typhoidal), diarrheogenic E. coli (enterotoxigenic and enterohemorragic), and Campylobacter jejuni.

Vaccines for viral and bacterial pathogens causing acute gastroenteritis: Part II: Vaccines for Shigella, Salmonella, enterotoxigenic E. coli (ETEC) enterohemorragic E. coli (EHEC) and Campylobacter jejuni

In Part II we discuss the following bacterial pathogens: Shigella, Salmonella (non-typhoidal), diarrheogenic E. coli (enterotoxigenic and enterohemorragic) and Campylobacter jejuni. In contrast to the enteric viruses and Vibrio cholerae discussed in Part I of this series, for the bacterial pathogens described here there is only one licensed vaccine, developed primarily for Vibrio cholerae and which provides moderate protection against enterotoxigenic E. coli (ETEC) (Dukoral®), as well as a few additional candidates in advanced stages of development for ETEC and one candidate for Shigella spp. Numerous vaccine candidates in earlier stages of development are discussed.

Locus of Adhesion and Autoaggregation (LAA), a pathogenicity island present in emerging Shiga Toxin–producing Escherichia coli strains

Shiga Toxin-producing Escherichia coli (STEC) are a group of foodborne pathogens associated with diarrhea, dysentery, hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Shiga toxins are the major virulence factor of these pathogens, however adhesion and colonization to the human intestine is required for STEC pathogenesis. A subset of STEC strains carry the Locus of Enterocyte Effacement (LEE) pathogenicity island (PAI), which encodes genes that mediate the colonization of the human intestine. While LEE-positive STEC strains have traditionally been associated with human disease, the burden of disease caused by STEC strains that lacks LEE (LEE-negative) has increased recently in several countries; however, in the absence of LEE, the molecular pathogenic mechanisms by STEC strains are unknown. Here we report a 86-kb mosaic PAI composed of four modules that encode 80 genes, including novel and known virulence factors associated with adherence and autoaggregation. Therefore, we named this PAI as Locus of Adhesion and Autoaggregation (LAA). Phylogenomic analysis using whole-genome sequences of STEC strains available in the NCBI database indicates that LAA PAI is exclusively present in a subset of emerging LEE-negative STEC strains, including strains isolated from HC and HUS cases. We suggest that the acquisition of this PAI is a recent evolutionary event, which may contribute to the emergence of these STEC.

Immunoproteomic Analysis To Identify Shiga Toxin-Producing Escherichia coli Outer Membrane Proteins Expressed during Human Infection

ABSTRACT Shiga-toxin producing Escherichia coli (STEC) is the etiologic agent of acute diarrhea, dysentery, and hemolytic-uremic syndrome (HUS). There is no approved vaccine for STEC infection in humans, and antibiotic use is contraindicated, as it promotes Shiga toxin production. In order to identify STEC-associated antigens and immunogenic proteins, outer membrane proteins (OMPs) were extracted from STEC O26:H11, O103, O113:H21, and O157:H7 strains, and commensal E. coli strain HS was used as a control. SDS-PAGE, two-dimensional-PAGE analysis, Western blot assays using sera from pediatric HUS patients and controls, and matrix-assisted laser desorption ionization–tandem time of flight analyses were used to identify 12 immunogenic OMPs, some of which were not reactive with control sera. Importantly, seven of these proteins have not been previously reported to be immunogenic in STEC strains. Among these seven proteins, OmpT and Cah displayed IgG and IgA reactivity with sera from HUS patients. Genes encoding these two proteins were present in a majority of STEC strains. Knowledge of the antigens produced during infection of the host and the immune response to those antigens will be important for future vaccine development.

Chaperone-Usher Pili Loci of Colonization Factor-Negative Human Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of diarrhea worldwide. Among the 25 different ETEC adhesins, 22 are known as “colonization factors” (CFs), of which 17 are assembled by the chaperone-usher (CU) mechanism. Currently, there is no preventive therapy against ETEC, and CFs have been proposed as components for vaccine development. However, studies of diarrhea-causing ETEC strains worldwide indicate that between 15 and 50% of these are negative for known CFs, hindering the selection of the most widespread structures and suggesting that unknown adhesins remain to be identified. Here, we report the result of a comprehensive analysis of 35 draft genomes of ETEC strains which do not carry known adhesin genes; our goal was to find new CU pili loci. The phylogenetic profiles and serogroups of these strains were highly diverse, a majority of which produced only the heat-labile toxin. We identified 10 pili loci belonging to CU families β (1 locus), γ2 (7 loci), κ (1 locus), and π (1 locus), all of which contained the required number of open reading frames (ORFs) to encode functional structures. Three loci were variants of previously-known clusters, three had been only-partially described, and four are novel loci. Intra-loci genetic variability identified would allow the synthesis of up to 14 different structures. Clusters of putative γ2-CU pili were most common (23 strains), followed by putative β-CU pili (12 strains), which have not yet been fully characterized. Overall, our findings significantly increase the number of ETEC adhesion genes associated with human infections.

TleA, a Tsh-Like Autotransporter Identified in a Human Enterotoxigenic Escherichia coli Strain

ABSTRACT Enterotoxigenic Escherichia coli (ETEC), a leading cause of acute diarrhea, colonizes the intestine by means of adhesins. However, 15 to 50% of clinical isolates are negative for known adhesins, making it difficult to identify antigens for broad-coverage vaccines. The ETEC strain 1766a, obtained from a child with watery diarrhea in Chile, harbors the colonization factor CS23 but is negative for other known adhesins. One clone, derived from an ETEC 1766a genomic library (clone G10), did not produce CS23 yet was capable of adhering to Caco-2 cells. The goal of this study was to identify the gene responsible for this capacity. Random transposon-based mutagenesis allowed the identification of a 4,110-bp gene that codes for a homologue of the temperature-sensitive hemagglutinin (Tsh) autotransporter described in avian E. coli strains (97% identity, 90% coverage) and that is called TleA (Tsh-like ETEC autotransporter) herein. An isogenic ETEC 1766a strain with a tleA mutation showed an adhesion level similar to that of the wild-type strain, suggesting that the gene does not direct attachment to Caco-2 cells. However, expression of tleA conferred the capacity for adherence to nonadherent E. coli HB101. This effect coincided with the detection of TleA on the surface of nonpermeabilized bacteria, while, conversely, ETEC 1766a seems to secrete most of the produced autotransporter to the medium. On the other hand, TleA was capable of degrading bovine submaxillary mucin and leukocyte surface glycoproteins CD45 and P-selectin glycoprotein ligand 1 (PSGL-1). These results suggest that TleA promotes colonization of the intestinal epithelium and that it may modulate the host immune response.

Genetic diversity and virulence determinants of Escherichia coli strains isolated from patients with Crohn's disease in Spain and Chile

Adherent-invasive Escherichia coli (AIEC) strains are genetically variable and virulence factors for AIEC are non-specific. FimH is the most studied pathogenicity-related protein, and there have been few studies on other proteins, such as Serine Protease Autotransporters of Enterobacteriacea (SPATEs). The goal of this study is to characterize E. coli strains isolated from patients with Crohns disease (CD) in Chile and Spain, and identify genetic differences between strains associated with virulence markers and clonality. We characterized virulence factors and genetic variability by pulse field electrophoresis (PFGE) in 50 E. coli strains isolated from Chilean and Spanish patients with CD, and also determined which of these strains presented an AIEC phenotype. Twenty-six E. coli strains from control patients were also included. PFGE patterns were heterogeneous and we also observed a highly diverse profile of virulence genes among all E. coli strains obtained from patients with CD, including those strains defined as AIEC. Two iron transporter genes chuA, and irp2, were detected in various combinations in 68–84% of CD strains. We found that the most significant individual E. coli genetic marker associated with CD E. coli strains was chuA. In addition, patho-adaptative fimH mutations were absent in some of the highly adherent and invasive strains. The fimH adhesin, the iron transporter irp2, and Class-2 SPATEs did not show a significant association with CD strains. The V27A fimH mutation was detected in the most CD strains. This study highlights the genetic variability of E. coli CD strains from two distinct geographic origins, most of them affiliated with the B2 or D E. coli phylogroups and also reveals that nearly 40% of Chilean and Spanish CD patients are colonized with E.coli with a characteristic AIEC phenotype.

Vaccination with DNA Encoding Truncated Enterohemorrhagic Escherichia coli (EHEC) Factor for Adherence-1 Gene (efa-1') Confers Protective Immunity to Mice Infected with E. coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is the predominant causative agent of hemorrhagic colitis in humans and is the cause of haemolytic uraemic syndrome and other illnesses. Cattle have been implicated as the main reservoir of this organism. Here, we evaluated the immunogenicity and protective efficacy of a DNA vaccine encoding conserved sequences of truncated EHEC factor for adherence-1 (efa-1′) in a mouse model. Intranasal administration of plasmid DNA carrying the efa-1′ gene (pVAXefa-1′) into C57BL/6 mice elicited both humoral and cellular immune responses. In animals immunized with pVAXefa-1′, EHEC-secreted protein-specific IgM and IgG antibodies were detected in sera at day 45. Anti-EHEC-secreted protein sIgA was also detected in nasal and bronchoalveolar lavages. In addition, antigen-specific T-cell-proliferation, IL-10, and IFN-γ were observed upon re-stimulation with either heat-killed bacteria or EHEC-secreted proteins. Vaccinated animals were also protected against challenge with E. coli O157:H7 strain EDL933. These results suggest that DNA vaccine encoding efa-1′ have therapeutic potential in interventions against EHEC infections. This approach could lead to a new strategy in the production of vaccines that prevent infections in cattle.