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

Abstract

Under conditions of activated type III secretion Shigella fiexneri up-regulates the expression of numerous genes, including the virulence plasmid (pINV)-encoded ospB and phoN2 genes. ospB and plwN2 are virulence-associated genes which are part of a bicistronic transcriptional unit encoding OspB, a protein (effector) of unknown function secreted by the type III secretion (TTS) apparatus, and PhoN2 (apyrase or ATP-diphosphohydrolase), a periplasmic protein involved in polar IcsA localization on the surface of S. flexneri. In this work we used real-time peR to measure transcription of ospB and phoN2 of wild-type S. ftexneri strain M90T as well as of derivative mutants impaired in definite virulence traits. The results obtained confirmed and extended previous reports indicating that the expression of ospB and phoN2 genes is modulated in a virB-dependent, mxiE-independent manner under conditions of non-activated secretion, while their expression is considerably induced in a 1I1xiE-dependent manner under conditions of activated secretion. That the expression of the ospB-phoN2 operon is up-regulated in condition of activated secretion, indicates that probably the expression of these two genes might be important, especially during the later stages of infection of S. fiexneri. Bacteria belonging to Shigella species are human intestinal pathogens which cause the most communicable of bacterial dysenteries, shigellosis. It has been calculated that shigellosis causes approximately 1.1 million deaths and over 164 million cases each year, with the majority of cases occurring in children of developing nations (I). Shigella strains contain a virulence plasmid (pINV) of approximately 220 kb which carries genes enabling these microrganisms to invade and disseminate into intestinal epithelial cells, leading to the destruction of the mucosa. Sequence and genetic analysis of pINVs from different Shigella flexneri strains indicated that the genes required for entry and diffusion into intestinal epithelial cells, and for the induction of apoptosis of macrophages are clustered in a piNY region of approximately 30 kb designated the entry region (2). The entry region encodes i) a type III secretion (TTS) apparatus (the mxi-spa operon), which is used by S. fiexneri

The Shigella flexneri virulence factor apyrase is released inside eukaryotic cells to hijack host cell fate

“…Immediately after its translation, apyrase localizes in the periplasm beneath IcsA and promotes actin-based motility, then it translocates on the bacterial surface where it hydrolyzes eATP and then, during infection, it is released to target iATP. This long-lasting activity is supported by the high stability of phoN2 mRNA, leading to its continuous expression during bacterial growth as well as during T3SS secretion ( 7 , 8 ). Furthermore, it is well established that Shigella temporally controls the release of T3SS effectors.…”

“…In particular, the transcriptional factor VirF activates the expression of icsA and the secondary transcriptional activator, VirB; VirB, in turn, activates the expression of genes coding for the Shigella T3SS, for the early effectors, and for MxiE. MxiE, under T3SS active secretion, is responsible for the expression of late effectors and the upregulation of the ospB-phoN2 operon ( 7 – 9 ). OspB is a T3SS effector, which modulates the host inflammatory response during the early stages of infection to promote bacterial dissemination ( 10 ).…”

The Shigella flexneri OspB effector: an early immunomodulator

“…cDNA samples were diluted 10-fold with RNAse free water and 2 l aliquots were used as templates in each Real-Time PCR reaction. Real-Time PCR were performed with a LightCycler thermocycler (Roche) by use of a QuantiTect SYBR Green PCR kit (Qiagen), as previously described (Nicoletti et al, 2008). At least three wells were run for each sample.…”

“…To determine the best timing for detecting OspB within host cell compartments, transcription of the ospB gene in the wild-type S. flexneri strain M90T was measured during a time course infection of HeLa cells by Real-Time PCR, as previously described (Nicoletti et al, 2008). As shown in Fig.…”