AUTHOR=Monfardini Mariane V. , Souza Renata T. , Rojas Thais C. G. , Guerrieri Caroline G. , Orikaza Cristina , Scaletsky Isabel C. A. 

TITLE=Transposon mutagenesis of atypical enteroaggregative Escherichia coli reveals a hemagglutinin-associated protein that mediates cell adhesion and contributes to the Galleria mellonella virulence

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=13

YEAR=2023

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2023.1166158

DOI=10.3389/fcimb.2023.1166158

ISSN=2235-2988

ABSTRACT=<p>Twenty-two atypical enteroaggregative <italic>Escherichia coli</italic> isolates from a previous epidemiological study harboring EAEC virulence genes were examined for their adhesion properties. Nine strains showed a typical aggregative adherence (AA) pattern, while 13 strains showed variant AA, such as AA with lined up cells characteristic of the chain-like adhesion (CLA) and AA mainly to HeLa cells characteristic of the diffuse adherence (DA). The aggregative forming pilus (AFP) genes <italic>afpA2</italic> and <italic>afpR</italic> were detected only in strain Q015B, which exhibited an AA/DA pattern. Using Tn5-based transposon mutagenesis on Q015B strain, we identified a 5517-bp open reading frame (ORF) encoding a predicted 1838-amino-acid polypeptide that is genetically related to a putative filamentous hemagglutinin identified in <italic>E. coli</italic> strain 7-233-03_S3_C2. Therefore, the ORF was named <italic>orfHA</italic>. The regions flanking <italic>orfHA</italic> were sequenced and two ORFs were found; upstream, an ORF that encodes a 603-amino-acid polypeptide with 99% identity to hemolysin secretion/activation proteins of the ShlB/FhaC/HecB family, and downstream, another ORF, which encodes a 632-amino-acid polypeptide with 72% identity to the glycosyltransferase EtpC. An <italic>orfHA</italic> mutant (Q015BΔ<italic>orfHA</italic>) was constructed from strain Q015B. Q015BΔ<italic>orfHA</italic> strain did not adhere to HeLa cells, whereas Q015BΔ <italic>orfHA</italic> transformed with a pACYC184 plasmid carrying <italic>orfHA</italic> restored the AA/DA phenotype of strain Q015B. Furthermore, the Q015Δ<italic>orfHA</italic> mutant had a marked effect on the ability of strain Q015B to kill the larvae of <italic>Galleria mellonella</italic>. Our results suggest that the AA/DA pattern of strain Q015B is mediated by a hemagglutinin-associated protein which also contributes to its virulence in the <italic>G. mellonella</italic> model.</p>