Apyrase, the product of the virulence plasmid-encoded phoN2 (apy) gene of Shigella flexneri, is necessary for proper unipolar IcsA localization and for efficient intercellular spread

Abstract

The role in virulence of the Shigella flexneri ospB-phoN2 operon has been evaluated. Here we confirm that OspB is an effector and show that apyrase, the product of phoN2, may be a virulence factor, since it is required for efficient intercellular spreading. Apyrase may be important in a deoxynucleoside triphosphate-hydrolyzing activity-independent manner, suggesting that it may act as an interaction partner in the process of IcsA localization.

FIG. 1.

Secretion of OspB by the TTS machinery. Western blot analysis with anti-FLAG, anti-IpaB, anti-IpaC, and anti-apyrase antibodies of whole-cell extracts and culture supernatants of HND549 (ospB-3×FLAG), of HND5311 (mxiA ospB::3×FLAG), and of HND5311 [(mxiA ospB::3×FLAG)/pHN111(P_BADmxiA)] in the absence (MxiA⁻) or in the presence (MxiA⁺) of 0.2% of the inducer l-arabinose (L-ara). A plus sign denotes culture supernatant extracts of bacteria resuspended in phosphate-buffered saline and incubated at 37°C for 30 min in the presence of 7 μg/ml of Congo red in order to induce activation of TTS-mediated secretion of effector proteins. The locations of 3× FLAG-tagged OspB recombinant protein as well as of IpaB, IpaC, and apyrase proteins are indicated. na, not added.

FIG. 2.

phoN2 mutant strain HND115 forms small plaques on Caco-2 cell monolayers. Plaque formation by wild-type strain M90T, HND115 (phoN2), and HND115/pHN28(P_BADphoN2) supplemented with 0.2% of the inducer l-arabinose (L-ara) (apyrase⁺) within the agarose layer is shown. Dishes were photographed after 48 h of infection.

FIG. 3.

IcsA expression and secretion in phoN2 mutant strain HND115. Western analysis with anti-IcsA antibodies of whole-cell extracts and culture supernatants of wild-type M90T and of its derivatives SC560 (icsA) and HND115 (phoN2). IcsA and IcsA* are indicated.

FIG. 4.

Indirect immunofluorescence labeling of IcsA. Panels show IcsA localization using an anti-IcsA antiserum in wild-type strain M90T, in phoN2 deletion strain HND115, and in HND115/pHN28(P_BADphoN2) and their corresponding phase-contrast images. In the panels of M90T and HND115/pHN28(P_BADphoN2) supplemented with the inducer l-arabinose (L-ara) (PhoN2⁺), arrows point to representative bacteria exhibiting polar IcsA localization, while in HND115 (apyrase⁻) arrows point to bacteria presenting IcsA apparently localized over the entire bacterial surface.

FIG. 5.

Fluorescence images of actin tail formations inside HeLa cells infected by phoN2 deletion strain HND115. HeLa cells were infected with wild-type strain M90T, HND115, or HND115/pHN28(P_BADphoN2) in the presence of 0.2% of the inducer l-arabinose (L-ara) (apyrase⁺). Actin was stained with rhodamine-conjugated phalloidin. Nuclei and bacterial DNA were counterstained in blue (4′,6′-diamidino-2-phenylindole). Images were recorded with a Leica camera and processed using Qwin software (Leica). Arrows in M90T and in HND115/pHN28 (apyrase⁺) panels point to representative bacteria exhibiting normal actin tail formation at a pole of a bacterium moving within the cytoplasm. Arrows in the HND115 (apyrase⁻) panel point to representative bacteria impaired in their ability to form normal actin tails (altered phenotype).

FIG. 6.

phoN2 mutant strain HND115 forms plaques of approximately wild-type size on Caco-2 cell monolayers when complemented with plasmid pFB1-4. pFB1-4 (Table 1) contains a randomly generated mutant of the apy gene, the orthologue of phoN2 in the EIEC strain HN280, encoding a recombinant apyrase presenting the R192P substitution which inactivates its dNTP-hydrolyzing activity. (A) Western blot analysis with anti-apyrase antibodies of whole-cell extracts of M90T, HND115 (phoN2), and HND115/pFB1-4 (pBluescript SKapy_R192P). (B) Plaques formed by M90T, HND115 (phoN2), and HND115/pFB1-4 (pBluescript SKapy_R192P). Dishes were photographed after 48 h of infection.