The Longus type IV pilus of enterotoxigenic Escherichia coli (ETEC) mediates bacterial self-aggregation and protection from antimicrobial agents

Abstract

Enterotoxigenic Escherichia coli (ETEC) strains are leading causes of childhood diarrhea in developing countries. ETEC pili and non-pili adherence factors designated colonization surface antigens (CSA) are believed to be important in the pathogenesis of diarrhea. Longus, a type IV pilus identified as the CSA(21), is expressed in up to one-third of ETEC strains, and share similarities to the toxin-coregulated pilus of Vibrio cholerae, and the bundle-forming pilus of enteropathogenic E. coli. To identify longus phenotype and possible function, a site-directed mutation of the lngA major subunit gene in the E9034A wild type ETEC strain was constructed. Lack of longus expression from the lngA mutant was demonstrated by immunoblot analysis and electron microscopy using specific anti-LngA antibody. Formation of self-aggregates by ETEC was shown to be dependent on longus expression as the lngA mutant or wild type grown under poor longus expression conditions was unable to express this phenotype. Longus-expressing ETEC were also associated with improved survival when exposed to antibacterial factors including lysozyme and antibiotics. This suggests that longus-mediated bacterial self-aggregates protect bacteria against antimicrobial environmental agents and may promote gut colonization.

Figure 1. Predicted protein LngA model based on automated comparison with TcpA

Panel A. Protein sequence and structural alignment of LngA and TcpA. The alpha helices or beta strands are shown above or below each LngA or TcpA sequences, respectively. Panel B. Frontal view of the tertiary structure of LngA based on automated comparison with TcpA showing alpha spins and beta strands. Alpha1 and 2 helices N-terminus LngA regions are predicted to face the inner core of the alpha helix while the C-terminus alpha 3 and 4 are predicted to face the outer surface of the helix. Panel C. Diagram of the LngA subunit arrangement into an alpha helix, showing six subunits per helix turn. Three alpha helix is predicted to form the longus filaments as described for TCP of V. cholerae. Longus filaments may also group in bundles as shown by TEM.

Figure 2. Deletion mutation of the lngA gene

Panel A. Diagram of the cat cassette insertion into the lngA gene by homologous recombination. Diagram of the wild-type lngA gene and a recombinant lngA:cat DNA fragment that by homologous recombination event result in the lngA:cat insertion mutant. Primers pKD3-lngA-F/pKD3-lngA-R to construct lngA::FRT-cat exchange marker and primers lngA-F/lngA-708R to test for lngA sequences are shown. Panel B. Detection of lngA:cat DNA recombination by PCR amplification. i) DNA PCR amplification for detection of lngA DNA using primer set lngA-F/lngA708-R. Lane 1: E. coli DH5α; Lane 2: DH5α pKD46; Lane 3: DH5α pKD3; Lane 4: E9034A; Lane 5: E9034A(pKD46); Lane 6: DH5α pAClngA:FRT-cat; Lane 7: E9034AlngA:FRT-:cat. Arrows indicate DNA fragments of 1.1 and 0.7 kb. ii) DNA PCR amplification for detection of cat insertion using primer set C1-lngAR PCR. Lanes 1 to 7: same as panel B-I above. Arrow indicates a 0.84-Kb DNA fragment. Panel C. Diagram the isogenic lngA deletion mutation construction, showing the Flp-mediated cat cassette excision by homologous recombination at the FRT sites. The resulting lngA deletion mutant contains an FRT site in the middle of lngA. Primer c1/lngA-708R for recognition of cat sequences and primers lngA-F/lngA708R for detection of lngA sequences are shown. Panel D. Detection of lngA deletion mutation by DNA PCR amplification. i) PCR amplification for detection of lngA DNA using primer set lngA-F-lngA708R. Lane 1: DH5α; Lane 2: DH5α pCP20; Lane 3: E9034A; Lane 4: E9034AlngA::FRT-cat; Lane 5: E9034AlngA::FRT-cat(pCP20); Lane 6: E9034AΔlngA. Arrows indicate 1.1, 0.7, and 0.19-Kb DNA fragments. ii) PCR amplification for detection of cat DNA using primers C1-lngA708-R. Lanes 1 to 6: same as panel D-i above. Arrow indicates a 0.84-Kb DNA fragment. M represents molecular weight markers (Bioline, Tauton, MA). Panel E. Expression of LngA. The expression of LngA was determined by immunoblot in whole cells lysates using a anti-Longus monoclonal antibody. Lane 1, E coli DH5α(pAClngA); Lane2, E coli DH5α; Lane 3, E coli HB101; Lane 4, E9034A; Lane 5, E9034AΔlngA; Lane 6, E9034AΔlngA(pAC-lngA); Lane 7, E9034AΔlngA::FRT-cat; Lane 8, E9034AΔlngA::FRT-cat (pAC-lngA). Arrows indicate the 28 kDa LngA prepilin and the 22 kDa mature LngA pilin.

Figure 3. Transmission electron microscopy (TEM) of ETEC strains expressing longus

Panel A. Wild-type E9034A ETEC strain grown under optimal conditions for longus expression showing typical longus filaments. Panel B. Isogenic E9034AΔlngA mutant strain lacking pili. Panel C. E9034AΔlngA (pAClngA) complemented mutant strain showing Longus-like structures. Panel D. Immunogold labeling TEM of E9034A ETEC wild-type strain. Panel E. Immunogold labeling of E9034AΔlngA mutant strain. Panel F. Immunogold labeling of E9034AΔlngA (pAClngA) complemented mutant strain. Arrows indicate gold particles specifically attached to type IV pili. Scale bar size indicated in μM.

Figure 4. Longus-mediated microcolony formation phenotype

Panels A, D and G: E9034A wild-type ETEC strain; Panels B, E and H: E9034AΔlngA; Panel C, F and I: E9034AΔlngA(pAC-lngA) complemented mutant. Arrow indicates bacterial self-aggregates. Panels A, B, and C are pictures obtained from bacterial suspensions. Scale bar sizes indicated in mm. Panels D, E, and F are pictures obtained by inverted light microscope. Panels G, H, and I are scanning electron microscopy pictures. Arrow indicates pilin-bacterial interactions. Scale bars sizes for Panels D to I indicated in μM. All strains were grown in TB.

Figure 5. Effect of longus expression on ETEC survival after exposure to antimicrobials

Panel A. Effect of antimicrobials on bacterial suspensions preincubated in LB. After overnight incubations all suspensions were processed to determine CFU/ml. Panel B. Effect of antimicrobials on bacterial suspensions preincubated in TB. Antimicrobial concentrations used: Chloramphenicol (Cm) at 24 μg/ml, Gentamicin 96 μg/ml, Tetracycline 24 μg/ml, Kanamycin 192 μg/ml, and lysozyme 768 μg/ml. Black bars: E9034A: wild type ETEC strain; white bars: E9034AΔlngA mutant; and striped bars: E9034A:lngA(pBADlngA) complemented mutant. Error bars correspond to standard deviations from experiments done in triplicate.

Figure 6. Kinetics of survival after longus-expressing ETEC exposure to antimicrobials

Panel A. Kinetics of survival after exposure of preincubated wild type and mutant to lysozyme (350 μg/ml). Panel B. Kinetics of survival after exposure of preincubated wild type and longus mutant strains to Gentamicin (48 μg/ml). Black diamons: E9034A: wild type ETEC strain; black square: E9034A:lngA ETEC mutant strain. Error bars correspond to standard deviations from experiments done in triplicate.