Expression of the VapD protein by Helicobacter pylori during intracellular infection

<i>Helicobacter pylori</i> can persist intracellularly, offering protection against the immune system and antimicrobial treatments, which promote chronic infection. Previous studies revealed that the virulence-associated protein D (<i>vapD</i>) gene is transcriptionally induced during <i>H. pylori</i> intracellular infection and was associated with bacterial persistence, as deletion of <i>vapD</i> impairs bacterial intracellular survival. However, whether VapD protein is expressed and localized during intracellular infection had not been demonstrated. The aim of this study was to detect the VapD protein when <i>H. pylori</i> is inside eukaryotic cells. Polyclonal antibodies against VapD and immunofluorescence microscopy were used to detect the VapD expression in co-cultures of <i>H. pylori</i> strain 26695 and AGS cells. The <i>H. pylori</i> strain Tx30a, which lacks the <i>vapD</i> gene, was used as a negative control. VapD expression was detected exclusively in AGS cells infected with strain 26695. Confocal microscopy confirmed the intracellular localization of the VapD signal, which coincided with the localization of <i>H. pylori</i> within AGS cells. In addition, a high proportion of <i>H. pylori</i>-infected AGS cells were positive for VapD signal. Together, these results provide first direct evidence of VapD expression during <i>H. pylori</i> intracellular infection and extend previous transcriptional and genetic studies by confirming VapD production at the protein level, supporting its association with the intracellular state of <i>H. pylori</i>.