MxiK and MxiN interact with the Spa47 ATPase and are required for transit of the needle components MxiH and MxiI, but not of Ipa proteins, through the type III secretion apparatus of Shigella flexneri.
The type III secretion (TTS) pathway is used by numerous Gram-negative pathogens to inject virulence factors into eukaryotic cells. The Shigella flexneri TTS apparatus (TTSA) spans the bacterial envelope and its assembly requires the products of approximately 20 mxi and spa genes. We present a functional analysis of the mxiK, mxiN and mxiL genes. Inactivation of mxiK and mxiN, but not mxiL, resulted in the assembly of a non-functional TTSA that lacked the outer needle. The amounts of needle components MxiH and MxiI were drastically reduced in mxiK and mxiN mutants and in the secretion defective spa47 mutant, indicating that MxiH and MxiI are degraded if they do not transit through the TTSA. Remarkably, expression of MxiH-His in the mxiN mutant and MxiI-His in the mxiK mutant restored assembly of a functional TTSA, as shown by the ability of these strains to enter into epithelial cells and to secrete Ipa proteins in response to activation by Congo red. Using a two-hybrid screen in yeast and immunoprecipitation assays from S. flexneri extracts, we identified interactions between MxiK and Spa33 and Spa47 and between MxiN and Spa33 and Spa47. These results suggest that transit of the needle components MxiH and MxiI through the TTSA involves the concerted action of the cytoplasmic proteins Spa47, Spa33, MxiK and MxiN. They also show that neither MxiK nor MxiN are absolutely required for secretion of Ipa proteins, provided that the TTSA is correctly assembled.
Jouihri
Noureddine
N
Laboratoire de Bactériologie Moléculaire, Faculté de Médecine, Université Libre de Bruxelles, 808 Route de Lennik, CP 614 b, B-1070 Bruxelles, Belgium.
Sory
Marie-Paule
MP
Page
Anne-Laure
AL
Gounon
Pierre
P
Parsot
Claude
C
Allaoui
Abdelmounaaïm
A
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000502:physiology*; D018829:Adhesins, Bacterial / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003224:Congo Red / Q000494:pharmacology; D004847:Epithelial Cells / Q000382:microbiology; D015964:Gene Expression Regulation, Bacterial; D046911:Macromolecular Substances; D010957:Plasmids / Q000235:genetics; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic / Q000235:genetics; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012763:Shigella flexneri / Q000187:drug effects / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D020798:Two-Hybrid System Techniques; D014774:Virulence / Q000235:genetics
D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D046911:Macromolecular Substances; C413572:MxiH protein, Shigella; C459782:MxiI protein, Shigella flexneri; D011993:Recombinant Fusion Proteins; C053476:invasin, Yersinia; D003224:Congo Red; D000251:Adenosine Triphosphatases; C478167:Spa47 protein, Shigella flexneri