Bioinformatics analysis of the locus for enterocyte effacement provides novel insights into type-III secretion

Abstract

Background: Like many other pathogens, enterohaemorrhagic and enteropathogenic strains of Escherichia coli employ a type-III secretion system to translocate bacterial effector proteins into host cells, where they then disrupt a range of cellular functions. This system is encoded by the locus for enterocyte effacement. Many of the genes within this locus have been assigned names and functions through homology with the better characterised Ysc-Yop system from Yersinia spp. However, the functions and homologies of many LEE genes remain obscure.

Results: We have performed a fresh bioinformatics analysis of the LEE. Using PSI-BLAST we have been able to identify several novel homologies between LEE-encoded and Ysc-Yop-associated proteins: Orf2/YscE, Orf5/YscL, rORF8/EscI, SepQ/YscQ, SepL/YopN-TyeA, CesD2/LcrR. In addition, we highlight homology between EspA and flagellin, and report many new homologues of the chaperone CesT.

Conclusion: We conclude that the vast majority of LEE-encoded proteins do indeed possess homologues and that homology data can be used in combination with experimental data to make fresh functional predictions.

Figure 1

1a: A comparison between the E. coli O157:H7 LEE and the Yersinia enterocolitica Yop-Ysc clusters. 1b: Graphical representation of the LEE-encoded type-III secretion system. The LEE region between EspF and Ler is shown (4589000–4620000 of the E. coli O157:H7 RIMD 0509952 genome. Accession number: dbj|BA000007.2). The Yop-Ysc cluster between YopM and YscL is shown (15000–40000 of the Yersinia enterocolitica plasmid pYVe227 Accession number: dbj|BA000007.2gb|AF102990). Genes shaded in the same solid colour exhibit previously known homologies between the LEE and Ysc/Yop cluster. Genes shaded in the same colour but with horizontal hatching are homologues where we have added information in this paper. Genes which show no apparent homology to any the Ysc/Yop system, but which encode proteins that appear in our graphical representation are shaded with grey/white diagonal hatching (espB, espA, sepZ). Uncharacterized genes where there is no homology, and where their gene products do not appear in our cartoon are shaded solid grey. In the cartoon of the LEE-encoded type-III secretion system proteins are shaded in the same colour as their corresponding genes in the map of the gene cluster. Model based on the type III secretion model from KEGG , and additional published data and images [70, 73, 145, 156, 157]. IM, inner membrane; PG, peptidoglycan layer; OM, outer membrane; EM, eukaryotic membrane.

Figure 2

Multiple alignment of the FHA domain from EscD with other FHA domains. Alignment is presented using default CHROMA [155] settings: Consensus abbreviations (amino acids): a, aromatic (FHWY, blue lettering on a dark yellow background); b, big (EFHIKLMQRWY, blue on light yellow); h, hydrophobic (ACFGHILMTVWY, black on dark yellow); l, aliphatic (ILV, grey on dark yellow); p, polar (CDEHKNQRST, blue on white); s, small (ACDGNPSTV, dark green on white); t, tiny (AGS, light green on white); -, negatively-charged (DE, red on white); and, +, positively-charged (KR, blue on white), c, charged (DEKRH, pink on white. Organism and gene name abbreviations used: EHEC (Escherichia coli O157:H7) EscD (ECs4558/dbj|BAB37981), Vp (Vibrio parahaemolyticus) PscD(ref|NP_798074), Pa(Pseudomonas aeruginosa) PscD (ref|NP_250408), Ah (Aeromonas hydrophila) AscD(gb|AAS91829), Pl (Photorhabdus luminescens) LscD(gb|AAO18032), Yp (Yersinia enterocolitica) YscD(gb|AAC37021), Dv (Desulfovibrio vulgaris) YscD(ref|YP_009153), Ct (Chlamydia trachomatis) CT664 (NP_220183), Cp (Chlamydophila pneumoniae) CpB0739(gb|AAP98668), Xa (Xanthomonas axonopodis) HrpD5 (gb|BAD29996), Ea (Erwinia amylovora) HrpQ(gb|AAB06000). The remaining 12 sequences are representative members of the SMART FHA domain (SM00240). Gene names, numbering and alignment are as presented in the SMART FHA family alignment .

Figure 3

A multiple alignment of the phospholipid-binding (BON) domain from EscD with other BON domains. The most obvious BON domain from EscD is shown. However, from patterns of residue conservation centred on conserved glycines in the EscD family of proteins (data not shown), we suspect that there may be one or two more BON-like domains within the cytoplasmic portion of EscD. For sequences 1–11 organism and gene name abbreviations are as for Figure 2 legend. The remaining 16 sequences are representative members of the Pfam BON domain (PF04972). Gene names, numbering and alignment are as presented in the Pfam:BON family alignment . Alignment is presented using default CHROMA settings (see Figure 2 legend).

Figure 4

Multiple alignment of SepQ/EscQ with related type-III secretion proteins. The sequence and secondary structure of the crystal structure of the C-terminal domain of the HrcQb protein from Pseudomonas Syringae Pv. Phaseolicola (pdb|1OY9) are shown 56. Sequences 2–21 (including SepQ) are from COG1886. 1OY9 and the remaining 10 sequences are representative members of Pfam:SpoA. Organism and gene name abbreviations are abbreviated as follows: EHEC (Escherichia coli O157:H7) SepQ (ECs4565/gb|AAG58829), Yp (Yersinia pestis CO92) YscQ (ref|NP_403921), Ct YscQ (ref|NP_220191), At (Agrobacterium tumefaciens str. C58) AGRc988p(ref|NP_353589), Ba (Buchnera aphidicola) FliN (sp|P57183), Ci (Caulobacter vibrioides) FliN(sp|Q03593), Cc (Caulobacter crescentus) FliN(ref|NP_420978), Cj (Campylobacter jejuni) FliY (ref|NP_281274) and FliN (ref|NP_281542), EHEC EpaO (ECs3726/sp|Q8X6F0), Pa (Pseudomonas aeruginosa) PscQ (ref|NP_250385), Rs (Ralstonia solanacearum) HrcQ (ref|NP_522422), St (Salmonella typhimurium) SsaQ(sp|P74860) and SpaO (sp|P40699), Yp FliN(ref|NP_404342.1), Aa (Aquifex aeolicus) FliN (sp|O67495), Li (Listeria innocua) Lin0701 (ref|NP_470044), Lin0706 (ref|NP_470049), and Lin0708 (ref|NP_470051), Ml (Mesorhizobium loti) hrcQ (ref|NP_106868), At FliM (sp|Q44457), Hp (Helicobacter pylori) FliM (sp|O25675), Bs (Bacillus subtilis) FliM (sp|P23453), Bb (Borrelia burgdorferi) FliM(sp|57511), Tp (Treponema pallidum) FliM (sp|P74927), Vp FliM (sp|Q9Z6GI), Rp (Rhodobacter sphaeroides) FliM (O85118), Sf (Shigella flexneri) SpaO (sp|P35534), Bp (Burkholderia pseudomallei) SctQ (Q9ZGR1), Ea (Erwinia amylovora) HrcQ (Q46645). Alignment is presented using default CHROMA settings (see Figure 2 legend).

Figure 5

Multiple alignment of Orf5/EscL with related type-III secretion proteins. Organism and gene name abbreviations: EHEC (Escherichia coli O157:H7) EscL (ECs4584/sp|O85643), Yp(Yersinia pestis CO92) YscL(ref|NP_395195), Bb (Borrelia burgdorferi) FliH(ref|NP_212423), Tm(Thermotoga maritim) FliH(ref|NP_228034), Ph(Pyrococcus horikoshii OT3) Ph1978(ref|NP_143803), E. coli K-12 FliH (ref|NP_416450), Tm TM1614(ref|NP_229414). Note that Ph1978 and TM1614 are annotated as a hypothetical protein and an ATPase synthase F0, subunit b, respectively. Alignment is presented using default CHROMA settings (see Figure 2 legend).

Figure 6

Multiple alignment of rOrf8/EscI with related type-III secretion proteins. Organism and gene name abbreviations as follows: EHEC (Escherichia coli O157:H7) EscI (ECs4572/sp|O85634) and EprJ (ECs3717/sp|Q8X6G5), Sf (Shigella flexineri) MxiI (sp|Q06080) and MxiH (sp|Q06079), St (Salmonella typhimurium) PrgJ (sp|P41785) and PrgI (sp|P41784), and Yp (Yersinia pestis CO92) YscI (Q00933). Alignment is presented using default CHROMA settings (see Figure 2 legend).

Figure 7

Multiple alignment of EspA with other EspA-like proteins and flagellins. The sequence and secondary structure of S. typhimurium Sjw1665 R-type straight flagellar filament is shown (pdb|1UCU) [76]. Organism and gene names are abbreviated as follows: EHEC (Escherichia coli O157:H7) EspA (dbj|BAB37979), EPEC (Escherichia coli E2348/69) EspA (gb|AAC38394), Et (Edwardsiella tarda) EspA (gb|AAN52733), Cr (Citrobacter rodentium) EspA(gb|AAL06381), Cv (Chromobacterium violaceum) CseB1 (gb|AAQ60253), Cv CseB2 (gb|AAQ60252), Cv CseB3 (gb|AAQ60250), Cv CseB4 (gb|AAQ60249), Se (Salmonella enterica Typhi) SseB (ref|NP_456130), Bb (Bartonella bacilliformis) Fla1 (gb|AAO33576), Rm (Rhizobium meliloti) Fla2(gb|AAA26277), Ap (Aquifex pyrophilus) FlaA(gb|AAA88923), Bo (Bordetella bronchiseptica) FlaA (gb|AAA22977), Lm, (Listeria moncytogenes) FlaA (gb|EAL07249), Hm (Helicobacter mustelae) FlaB(gb|AAA25017), Vp (Vibrio parahaemolyticus) LafA (dbj|BAC62891), Bh (Bacillus halodurans) Hag (dbj|BAB07335), Bs (Bacillus subtilis) Hag (gb|AAA22437), Le (Legionella micdadei) Fla (emb|CAA59172), Th (Treponema hydodysenteriae) FlaB2 (emb|CAA45081), Pm (Proteus mirabilis) FliC1(gb|AAA62396), E. coli K-12 FliC(gb|AAC74990). Alignment is presented using default CHROMA settings (see Figure 2 legend).

Figure 8

Multiple alignment of SepL with related type-III secretion proteins, including YopN-like and TyeA-like proteins. The N-terminal (YopN-like) and C-terminal (TyeA-like) domains are shown separately for clarity. Organism and gene name abbreviations as follows: EHEC(Escherichia coli O157:H7) SepL (ECs4557/dbj|BAB37980), Se (Salmonella enterica Typhi) SsaL (gb|AAL20336), Cv (Chromobacterium violaceum) InvE (gb|AAQ60301), St (Salmonella typhimurium) InvE (pir||A46138), Sf (Shigella flexineri) MxiC (gb|AAL72332), Er (Erwinia chrysanthemi) HrpJ (gb|AAO34609), Pt (Pectobacterium atrosepticum) HrpJ (emb|CAD43175), Bp (Bordetella pertussis) BopN (emb|CAE42532), Cp (Chlamydophila pneumoniae) CopN (gb|AAD18473), Cc (Chlamydophila caviae) CopN (gb|AAP05204), Ah (Aeromonas hydrophila) AopN(gb|AAR26331) and Acr1(gb|AAR26332), Pl (Photorhabdus luminescens) LopN(gb|AAO18045) and LssA(gb|AAO18046), Pa (Pseudomonas aeruginosa) PopN(gb|AAC45939) and Pcr1(gb|AAC45940), Ye (Yersinia enterocolitica) YopN(gb|AAD16823) and TyeA(gb|AAN37519), Vp (Vibrio parahaemolyticus) PopN(dbj|BAC59930) and VP1666(dbj|BAC59929). Alignment is presented using default CHROMA settings (see Figure 2 legend).

Figure 9

Multiple alignment of CesT with related type-III secretion chaperones. Organism and gene name abbreviations as follows: As(Aeromonas salmonicida) orfX(gb|AAK83051), Pa(Pseudomonas aeruginosa) PA3842(ref|NP_252531), Cv(Chromobacterium violaceum) CV0975(ref|NP_900645), Pl(Photorhabdus luminescens) unkown(gb|AAO18077), Ye(Yersinia enterocolitica) SycT(ref|NP_783658), Bt(Bordetella pertussis) BP0499(ref|NP_879351), Pl plu4749(ref|NP_931911), Ps(Pseudomonas syringae) SchF(ref|NP_790352), Sf(Shigella flexineri) IpgE(gb|AAP78997), Ye YsaK(gb|AAB69191), Ea(Erwinia amylovora) DspF(gb|AAC04851), Ps AvrF(ref|NP_791203), EPEC(Escherichia coli E2348/69) CesF(gb|AAC38388), Ps Pspto1369(ref|NP_791196) and Pspto4589(ref|NP_794340), Pl plu0829 (ref|NP_928172), Ps Pspto4721(ref|NP_794464), Bt BP2236(ref|NP_880880), Pa ExsC(gb|AAC46214), Pl plu3753(ref|NP_930959), Ye Orf155(ref|NP_783720), Vp VP1687(ref|NP_798066), Ps SchA(ref|NP_795083), Dv Dvu2312(ref|YP_011525), Lm(Leishmania major) L4830.13(emb|CAC22615), Lm P883.10(emb|CAC37204), Ct CT043(ref|NP_219546), Xa(Xanthomonas axonopodis) HpaB(ref|NP_640751), Ps Pspto1374(ref|NP_791201), Vp(Vibrio parahaemolyticus) VP1684(ref|NP_798063), Bt BP2265(ref|NP_880908), Ye YscB(ref|NP_052412), Pa PscB(gb|AAC44773), Pl SctB(ref|NP_930983), Vp NosA(ref|NP_798076), Dv(Desulfovibrio vulgaris) CesT(ref|YP_011605), Ea ORFA(gb|AAF63396), Ct(Chlamydia trachomatis) SycE(ref|NP_219591), Ct CT663(ref|NP_220182), Ye SycH(ref|NP_863547), Vp VP1682(ref|NP_798060), Ye SycN(ref|NP_052398), As(Aeromonas salmonicida) Acr2(emb|CAD30215), Pl plu3764(ref|NP_930970), Pa Pcr2(gb|AAC45941), Vp VP1665(ref|NP_798044), and Bt BP2258(ref|NP_880901). Alignment is presented using default CHROMA settings (see Figure 2 legend).