Negative regulation of Salmonella pathogenicity island 2 is required for contextual control of virulence during typhoid

Salmonella enterica relies on a type III secretion system encoded in Salmonella pathogenicity island-2 (SPI-2) to survive and replicate within macrophages at systemic sites during typhoid. SPI-2 virulence is induced upon entry into macrophages, but the mechanisms of SPI-2 gene control in vivo remain unclear, particularly with regard to negative regulators that control the contextual activation of SPI-2. Here, we identified and characterized YdgT as a negative modulator of the SPI-2 pathogenicity island and established that this negative regulation is central to systemic pathogenesis because ydgT mutants overexpressing typhoid virulence genes were ultimately attenuated during infection. ydgT mutants displayed a biphasic virulence phenotype during in vivo competitive infections that consisted of an early “gain-of-virulence” dependent on SPI-2 activation, followed by attenuation later in infection indicating that proper contextual regulation of SPI-2 by YdgT is necessary for full virulence during systemic colonization. These data suggest that overexpression of virulence-associated type III secretion genes can have an adverse effect on bacterial pathogenesis in vivo

The Salmonella effector protein PipB2 is a linker for kinesin-1

Understanding the mechanisms of Salmonella virulence is an important challenge. The capacity of this intracellular bacterial pathogen to cause diseases depends on the expression of virulence factors including the second type III secretion system (TTSS-2), which is used to translocate into the eukaryotic cytosol a set of effector proteins that divert the biology of the host cell and shape the bacterial replicative niche. Yet little is known about the eukaryotic functions affected by individual Salmonella effectors. Here we report that the TTSS-2 effector PipB2 interacts with the kinesin light chain, a subunit of the kinesin-1 motor complex that drives anterograde transport along microtubules. Translocation of PipB2 is both necessary and sufficient for the recruitment of kinesin-1 to the membrane of the Salmonella-containing vacuole. In vivo, PipB2 contributes to the attenuation of Salmonella mutant strains in mice. Taken together, our data indicate that the TTSS-2-mediated fine-tuning of kinesin-1 activity associated with the bacterial vacuole is crucial for the virulence of Salmonella

Translocation of surface-localized effectors in type III secretion

Pathogenic Yersinia species suppress the host immune response by using a plasmid-encoded type III secretion system (T3SS) to translocate virulence proteins into the cytosol of the target cells. T3SS-dependent protein translocation is believed to occur in one step from the bacterial cytosol to the target-cell cytoplasm through a conduit created by the T3SS upon target cell contact. Here, we report that T3SS substrates on the surface of Yersinia pseudotuberculosis are translocated into target cells. Upon host cell contact, purified YopH coated on Y. pseudotuberculosis was specifically and rapidly translocated across the target-cell membrane, which led to a physiological response in the infected cell. In addition, translocation of externally added YopH required a functional T3SS and a specific translocation domain in the effector protein. Efficient, T3SS-dependent translocation of purified YopH added in vitro was also observed when using coated Salmonella typhimurium strains, which implies that T3SS-mediated translocation of extracellular effector proteins is conserved among T3SS-dependent pathogens. Our results demonstrate that polarized T3SS-dependent translocation of proteins can be achieved through an intermediate extracellular step that can be reconstituted in vitro. These results indicate that translocation can occur by a different mechanism from the assumed single-step conduit model

Immune complex-induced enhancement of bacterial antigen presentation requires Fcγ Receptor III expression on dendritic cells

Dendritic cells (DCs) are capable of initiating adaptive immune responses against infectious agents by presenting pathogen-derived antigens on MHC molecules to naïve T cells. Because of their key role in priming adaptive immunity, it is expected that interfering with DC function would be advantageous to the pathogen. We have previously shown that Salmonella enterica serovar Typhimurium (ST), is able to survive inside DCs and interfere with their function by avoiding activation of bacteria-specific T cells. In contrast, when ST is targeted to Fcγ receptors on the DC surface, bacteria are degraded and their antigens presented to T cells. However, the specific Fcγ receptor responsible of restoring presentation of antigens remains unknown. Here, we show that IgG-coated ST was targeted to lysosomes and degraded and its antigens presented on MHC molecules only when the low-affinity activating FcγRIII was expressed on DCs. FcγRIII-mediated enhancement of Ag presentation led to a robust activation of T cells specific for bacteria-expressed antigens. Laser confocal and electron microscopy analyses revealed that IgG-coated ST was rerouted to the lysosomal pathway through an FcγRIII-dependent mechanism. PI-3K activity was required for this process, because specific inhibitors promoted the survival of IgG-coated ST inside DCs and prevented DCs from activating bacteria-specific T cells. Our data suggest that the DC capacity to efficiently activate T cells upon capturing IgG-coated virulent bacteria is mediated by FcγRIII and requires PI-3K activity