Pseudopilin residue E5 is essential for recruitment by the type 2 secretion system assembly platform.
Type II secretion systems (T2SSs) promote secretion of folded proteins playing important roles in nutrient acquisition, adaptation and virulence of Gram-negative bacteria. Protein secretion is associated with the assembly of type 4 pilus (T4P)-like fibres called pseudopili. Initially membrane embedded, pseudopilin and T4 pilin subunits share conserved transmembrane segments containing an invariant Glu residue at the fifth position, E5. Mutations of E5 in major T4 pilins and in PulG, the major pseudopilin of the Klebsiella T2SS abolish fibre assembly and function. Among the four minor pseudopilins, only PulH required E5 for secretion of pullulanase, the substrate of the Pul T2SS. Mass-spectrometry analysis of pili resulting from the co-assembly of PulG(E5A) variant and PulG(WT) ruled out an E5 role in pilin processing and N-methylation. A bacterial two-hybrid analysis revealed interactions of the full-length pseudopilins PulG and PulH with the PulJ-PulI-PulK priming complex and with the assembly factors PulM and PulF. Remarkably, PulG(E5A) and PulH(E5A) variants were defective in interaction with PulM but not with PulF, and co-purification experiments confirmed the E5-dependent interaction between native PulM and PulG. These results reveal the role of E5 in a recruitment step critical for assembly of the functional T2SS, likely relevant to T4P assembly systems.
Department of Microbiology, Laboratory of Macromolecular Systems and Signalling, Institut Pasteur, CNRS ERL3526, 25 rue du Dr Roux, 75724, Paris, Cedex 15, France.|Université Paris Diderot (Paris 7) Sorbonne Paris Cité
Mangayarkarasi
M
Nivaskumar
Université Paris Diderot (Paris 7) Sorbonne Paris Cité|Laboratory of Intercellular Communication and Microbial Infections, CIRB, Collège de France, Paris, France.|Institut National de la Santé et de la Recherche Médicale (Inserm) U1050, France.|Centre National de la Recherche Scientifique (CNRS), UMR7241, France.|MEMOLIFE Laboratory of Excellence and Paris Science Lettre, France.
Javier
J
Santos-Moreno
Structural Mass spectrometry and Proteomics unit, CNRS UMR3528, Institut Pasteur, Paris, France.
Christian
C
Malosse
Department of Microbiology, Laboratory of Macromolecular Systems and Signalling, Institut Pasteur, CNRS ERL3526, 25 rue du Dr Roux, 75724, Paris, Cedex 15, France.
Nathalie
N
Nadeau
Structural Mass spectrometry and Proteomics unit, CNRS UMR3528, Institut Pasteur, Paris, France.
Julia
J
Chamot-Rooke
Laboratory of Intercellular Communication and Microbial Infections, CIRB, Collège de France, Paris, France.|Institut National de la Santé et de la Recherche Médicale (Inserm) U1050, France.|Centre National de la Recherche Scientifique (CNRS), UMR7241, France.|MEMOLIFE Laboratory of Excellence and Paris Science Lettre, France.
Guy
G
Tran Van Nhieu
Department of Microbiology, Laboratory of Macromolecular Systems and Signalling, Institut Pasteur, CNRS ERL3526, 25 rue du Dr Roux, 75724, Paris, Cedex 15, France. ofrancet@pasteur.fr.
Olivera
O
Francetic
D000595:Amino Acid Sequence; D036781:Fimbriae Proteins / Q000235:genetics / Q000378:metabolism*; D010861:Fimbriae, Bacterial / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D007709:Klebsiella / Q000235:genetics / Q000378:metabolism*; D041121:Klebsiella oxytoca / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D000069178:Type II Secretion Systems / Q000378:metabolism*
D000069178:Type II Secretion Systems; D005973:Glutamine; D036781:Fimbriae Proteins; D006026:Glycoside Hydrolases; C100181:pullulanase