Cell-contact-stimulated formation of filamentous appendages by Salmonella typhimurium does not depend on the type III secretion system encoded by Salmonella pathogenicity island 1

Abstract

The formation of filamentous appendages on Salmonella typhimurium has been implicated in the triggering of bacterial entry into host cells (C. C. Ginocchio, S. B. Olmsted, C. L. Wells, and J. E. Galán, Cell 76:717-724, 1994). We have examined the roles of cell contact and Salmonella pathogenicity island 1 (SPI1) in appendage formation by comparing the surface morphologies of a panel of S. typhimurium strains adherent to tissue culture inserts, to cultured epithelial cell lines, and to murine intestine. Scanning electron microscopy revealed short filamentous appendages 30 to 50 nm in diameter and up to 300 nm in length on many wild-type S. typhimurium bacteria adhering to both cultured epithelial cells and to murine Peyer's patch follicle-associated epithelia. Wild-type S. typhimurium adhering to cell-free culture inserts lacked these filamentous appendages but sometimes exhibited very short appendages which might represent a rudimentary form of the cell contact-stimulated filamentous appendages. Invasion-deficient S. typhimurium strains carrying mutations in components of SPI1 (invA, invG, sspC, and prgH) exhibited filamentous appendages similar to those on wild-type S. typhimurium when adhering to epithelial cells, demonstrating that formation of these appendages is not itself sufficient to trigger bacterial invasion. When adhering to cell-free culture inserts, an S. typhimurium invG mutant differed from its parent strain in that it lacked even the shorter surface appendages, suggesting that SPI1 may be involved in appendage formation in the absence of epithelia. Our data on S. typhimurium strains in the presence of cells provide compelling evidence that SPI1 is not an absolute requirement for the formation of the described filamentous appendages. However, appendage formation is controlled by PhoP/PhoQ since a PhoP-constitutive mutant very rarely possessed such appendages when adhering to any of the cell types examined.

FIG. 1

Scanning electron micrographs of S. typhimurium SL1344 adhering to Caco-2 cells (A and B), MDCK cells (C), murine Peyer’s patches (D and E) and cell-free culture supports (F). A proportion of wild-type S. typhimurium cells adhering to target cells formed numerous short filamentous appendages when they adhered to either Caco-2 cells (B) or MDCK cells (C), while others lacked such appendages (A). Similar appendages were present on some S. typhimurium SL1344 cells that adhered to M cells (D and E) after infection of murine Peyer’s patches in ligated intestinal loops. The asterisk in panel E indicates an area of membrane remodelling associated with bacterial invasion. A small proportion of S. typhimurium SL1344 cells attached to cell-free culture supports possessed appendages which exhibited a similar distribution but which were shorter than many of those on cell-adhered bacteria (F). Bars, 2 μm.

FIG. 2

Scanning electron micrographs of invA mutant S. typhimurium SB111 adhering to MDCK cells (A and B), Caco-2 cells (C), and cell-free culture supports (D). A heterogeneous adherence pattern is observed after infection of MDCK cell monolayers with S. typhimurium SB111 (A). The majority of bacteria formed aggregates linked by long filaments and lacked the shorter filamentous appendages observed on wild-type bacteria. Isolated bacteria (arrowheads in panel A) were largely proficient in the formation of these short filamentous appendages. The marked difference in surface morphology between the two bacterial subtypes is clear at higher magnification (B). Similar patterns of adherence and appendage expression were observed when S. typhimurium SB111 adhered to Caco-2 cells, with individual bacteria expressing short filamentous appendages (C). The appendages elaborated by S. typhimurium SB111 were generally thicker than those observed on wild-type strains (compare panels B and C with Fig. 1B and C). S. typhimurium SB111 adhering to cell-free culture supports produced much smaller and sparser appendages (D). Bars, 2 μm.

FIG. 3

Scanning electron micrographs of the S. typhimurium strains carrying mutations in invG (strain 83; A to E) or in sspC (strain EE638; F). When adhering to cell-free culture supports (A), S. typhimurium 83 lacked any of the short filamentous appendages observed on a proportion of cells of wild-type and invA mutant S. typhimurium strains (see, e.g., Fig. 1F and 2D), although longer filamentous appendages were occasionally observed. When adhering to MDCK cells (B) or Caco-2 cells (C), a variable proportion of S. typhimurium 83 cells formed numerous short filamentous appendages indistinguishable from those seen on wild-type bacteria. Similar surface features were also present on some S. typhimurium 83 cells adhering to M cells (D) and enterocytes (E) after infection of murine Peyer’s patches in ligated intestinal loops. S. typhimurium EE638 also produced short filamentous appendages when adhering to MDCK or Caco-2 cells (F); these appendages generally more closely resembled those observed on S. typhimurium invA mutant SB111. Bars, 2 μm.

FIG. 4

Scanning electron micrographs of invG mutant S. typhimurium strain SB161 adhering to MDCK cells. SB161 exhibited a heterogeneous adherence pattern after infection of MDCK cell monolayers (A). Adherent bacteria mainly formed aggregates linked by long filaments and lacked short filamentous appendages (A). Most bacteria which appeared to adhere independently of the aggregates also lacked short filamentous appendages (B). Bars, 2 μm.

FIG. 5

Scanning electron micrographs of PhoP^c S. typhimurium strain CS022 (A to D) and prgH mutant strain IB040 (E and F). Almost all of the S. typhimurium CS022 cells observed on Caco-2 cells lacked the short filamentous appendages observed on other strains (A), although two which exhibited such features were found (B). S. typhimurium CS022 cells observed on MDCK cells (data not shown) and on murine Peyer’s patch epithelia (C) invariably lacked these surface features, as did those adhering to cell-free culture supports (D), although longer filamentous appendages were occasionally observed. S. typhimurium IB040 cells frequently formed numerous short filamentous appendages when they adhered to Caco-2 cells (E), while such features were rarely observed on strain IB040 bacteria adhering to MDCK cells (F). Most of the cell-adhered bacteria of strain IB040 were present in dense aggregates linked by long filaments (F), which resembled the adherence pattern of invA mutant strain SB111 (compare Fig. 4F with Fig. 2A). Bars, 2 μm.