Abstract
Enterotoxigenic Escherichia coli (ETEC) is a major cause of morbidity in children under 5 years of age in low- and middle-income countries and a leading cause of traveler's diarrhea worldwide. The ability of ETEC to colonize the intestinal epithelium is mediated by fimbrial adhesins, such as CS21 (Longus). This adhesin is a type IVb pilus involved in adherence to intestinal cells in vitro and bacterial self-aggregation. Fourteen open reading frames have been proposed to be involved in CS21 assembly, hitherto only the lngA and lngB genes, coding for the major (LngA) and minor (LngB) structural subunit, have been characterized. In this study, we investigated the role of the LngA, LngB, LngC, LngD, LngH, and LngP proteins in the assembly of CS21 in ETEC strain E9034A. The deletion of the lngA, lngB, lngC, lngD, lngH, or lngP genes, abolished CS21 assembly in ETEC strain E9034A and the adherence to HT-29 cells was reduced 90%, compared to wild-type strain. Subcellular localization prediction of CS21 proteins was similar to other well-known type IV pili homologs. We showed that LngP is the prepilin peptidase of LngA, and that ETEC strain E9034A has another peptidase capable of processing LngA, although with less efficiency. Additionally, we present immuno-electron microscopy images to show that the LngB protein could be localized at the tip of CS21. In conclusion, our results demonstrate that the LngA, LngB, LngC, LngD, LngH, and LngP proteins are essential for CS21 assembly, as well as for bacterial aggregation and adherence to HT-29 cells.
Highlights
Enterotoxigenic Escherichia coli (ETEC) remains as a major bacterial pathogen associated to high morbidity, mainly in children less than 5 years of age in low- and middle-income countries
Several proteins involved in TCP, CFA/III, and BFP assembly are homologous with proteins encoded in the lng cluster (Gomez-Duarte et al, 2007; Roux et al, 2012)
Quantitative analysis of confocal images allowed us to measure CS21 length; we found that the average length of CS21 from the E9034A strain was 9.01 ± 2.05 μm and the E9034A lngB was 22.62 ± 6.77 μm (Figure 7D, Supplemental Figure 3)
Summary
Enterotoxigenic Escherichia coli (ETEC) remains as a major bacterial pathogen associated to high morbidity, mainly in children less than 5 years of age in low- and middle-income countries. CS21, called longus, is a class b type IV pilus which is encoded in a 14 kb lng gene cluster located in a large virulence plasmid (50–90 kb) (Girón et al, 1994; Gomez-Duarte et al, 2007). Girón et al (1994) reported for the first time that CS21 structural gene (lngA) is encoded in a large plasmid, and that a 5-kb Bam HI restriction fragment from this plasmid was sufficient for pilus assembly in an Escherichia coli (E. coli) strain K-12 (DH5αF′IQ). Gomez-Duarte et al (2007) reported a cluster of 14 genes (lngR, lngS, lngT, lngA, lngB, lngC, lngD, lngE, lngF, lngG, lngH, lngI, lngJ, and lngP) of 13.93 kb located in tandem that might be required for expression of CS21
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