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.ospB and phoN2 (apy) are genes located on the virulence plasmid (pINV) of Shigella species and of related enteroinvasive Escherichia coli (EIEC) strains (3,6,35). Indirect evidence indicates that ospB and phoN2 may contribute to pathogenicity (1,3,5,8,17,23,25,35). ospB encodes a protein of unknown function (OspB) surmised to be an effector secreted by the type III secretion (TTS) apparatus of Shigella flexneri (6,20). The two genes are organized in a single highly conserved bicistronic operon. Although phoN2 encodes a periplasmic protein (5), their expression is regulated by the VirF-VirB cascade (35) and by MxiE in concert with IpgC so that they are expressed in a VirB-dependent, MxiE-independent manner under conditions of nonactivated secretion and are up-regulated, in a MxiEdependent manner, under conditions of activated secretion (8,17,20,26,30), indicating that these genes may be important in postinvasion events related to virulence.phoN2 encodes apyrase, a periplasmic enzyme which belongs to the family of ATP-hydrolyzing enzymes (3, 5, 18) able to sequentially hydrolyze nucleoside triphosphates to diphosphates and then to monophosphates. However, it is not active against monophosphates, a distinguishing feature from acid phosphatases (1,3,41). Due to its catalytic activity and primary structure (apyrase presents an exposed N-terminal poly-proline sequence), it has been implicated in the decrease of host cell intracellular ATP, a characteristic metabolic event following S. flexneri infection, as well as in the S. flexneri-induced actin-polymerization process (1,3,5,25,35). Thus, these findings strongly suggest that OspB and apyrase may be virulence factors. The purpose of this study was to investigate the role of both genes in the mechanism of pathogenicity of S. flexneri. OspB is an effector secreted by the TTS apparatus of S. flexneri. To confirm and extend previous reports indicatingOspB as an effector secreted by the TTS apparatus (6, 20), we transduced a nonpolar deletion encompassing mxiA into the S. flexneri M90T derivative strain HND549 (ospB::3ϫFLAG), thus generating HND5311 (mxiA ospB::3ϫFLAG) ( Table 1). C-terminal 3ϫ FLAG tagging was achieved essentially as described by Uzzau et al. (42) using the specific primer pair shown in Table S1 in the supplemental material. Whole-cell extracts and supernatants of exponentially growing bacteria were analyzed by immunoblotting using monoclonal antibodies against the 3ϫ FLAG epitope (Sigma) and mouse polyclonal antibodies preparation directed against apyrase (Fig. 1). Apyrase antiserum was obtained by immunizing BALB/...

Bacteriophage 2851 Is a Prototype Phage for Dissemination of the Shiga Toxin Variant Gene 2c in Escherichia coli O157:H7

“…ORF73 (position 54,419) may encode an effector for a type III secretion system, as its gene product shows similarity to OspB of Shigella. In Shigella species, the gene for OspB is located on a virulence plasmid and secreted by a plasmid-encoded type III secretion system; however, a function for the OspB protein has yet to be determined (41,55). The last putative gene of the phage genome (ORF75) encodes a hypothetical protein which shows similarity to hypothetical protein ECs2227 of E. coli EHEC strain O157 Sakai.…”

Molecular Pathogenesis ofShigellaspp.: Controlling Host Cell Signaling, Invasion, and Death by Type III Secretion

“…The essential mediators of efficient movement by actin nucleation at one pole of the bacterium are IcsA/VirG, SopA/IscP, VirA, and PhoN2 (apyrase). The corresponding genes are also located outside of the entry region on the virulence plasmid (22,63,150,156,258,338,339). Among these proteins, only the secretion of VirA depends on the T3SS.…”

Evaluation by Real-Time PCR of the Expression of S. Flexneri Virulence-Associated Genes ospB and phoN2 under Different Genetical Backgrounds

“…ospB encodes for an effector of unknown function (OspB) secreted by the TTS apparatus of S. fiexneri (2,7,14). phoN2 encodes for apyrase, a periplasmic enzyme which belongs to the family of ATPhydrolyzing enzymes (5,9,23), able to sequentially hydrolyze nucleoside triphosphates to diphosphates and then to monophosphates.…”

“…Oligonucleotide primers were designed based on the S. flexneri pINY sequence (2). M90T non-polar deletion mutants of virB, mxiA and mxiE were constructed by the PCR-based gene-disruption method reported by Datsenko and Wanner, as previously described (7,20). In this way, M90T derivative strains HND54 (AvirB), HND43 (AmxiA) and HND63 (AmxiE) were generated.…”