Hereditary hemochromatosis restores the virulence of plague vaccine strains.

Nonpigmented Yersinia pestis (pgm) strains are defective in scavenging host iron and have been used in live-attenuated vaccines to combat plague epidemics. Recently, a Y. pestis pgm strain was isolated from a researcher with hereditary hemochromatosis who died from laboratory-acquired plague. We used hemojuvelin-knockout (Hjv(-/-)) mice to examine whether iron-storage disease restores the virulence defects of nonpigmented Y. pestis. Unlike wild-type mice, Hjv(-/-) mice developed lethal plague when challenged with Y. pestis pgm strains. Immunization of Hjv(-/-) mice with a subunit vaccine that blocks Y. pestis type III secretion generated protection against plague. Thus, individuals with hereditary hemochromatosis may be protected with subunit vaccines but should not be exposed to live-attenuated plague vaccines.

YopR impacts type III needle polymerization in Yersinia species.

A hallmark of Yersinia type III machines is the presence of needles extending from the bacterial surface. Needles perform two functions, serving as the conduits for the transport of effectors into immune cells but also acting as a sensor. The polymerized needle protein, YscF, is thought to perceive threshold levels of environmental calcium ions to trigger secretion. yopR (yscH) is a gene downstream of yscEFG, encoding the chaperones and principal building blocks of the needle. Here we investigated the contribution of YopR towards type III secretion and pathogenesis. Yersinia pestis KIM D27 mutants lacking yopR were defective for virulence in a mouse model of septicemic plague. yopR variants of Yersinia enterocolitica W22703 displayed a reduced ability to inject effectors into macrophages and required lower calcium concentrations to activate type III secretion than wild-type yersiniae. Furthermore, yopR mutants failed to assemble YscF into needle complexes and instead secreted YscF into the medium. These results imply that YopR may be involved in controlling the secretion of YscF, thereby impacting the assembly of type III machines. An alternative possibility, which YopR participates directly in the polymerization of YscF, seems less likely as YopR is not associated with purified needles.

Yersinia enterocolitica type III secretion of YopR requires a structure in its mRNA.

Yersinia type III secretion machines transport substrate proteins into the extracellular medium or into the cytoplasm of host cells. Translational hybrids, involving genes that encode substrates as well as reporter proteins that otherwise cannot travel the type III pathway, identified signals that promote transport of effector Yops into host cells. Signals for the secretion of substrates into high calcium media were hitherto unknown. By exploiting attributes of translational hybrids between yopR, whose product is secreted, and genes that encode impassable proteins that jam the secretion machine, we isolated yopR mutations that abolish substrate recognition. Similar to effector Yops, an N-terminal or 5' signal in codons 1-11 is required to initiate YopR into the type III pathway. YopR secretion cannot be completed and translational hybrids cannot impose a block without a second signal, positioned at codons 131-149. Silent mutations in the second signal abrogate function and the phenotype of other mutations can be suppressed by secondary mutations predicted to restore base complementary in a 3' stem-loop structure of the yopR mRNA.

Secretion signal recognition by YscN, the Yersinia type III secretion ATPase.

Yersinia type III machines secrete protein substrates across the bacterial envelope. Secretion signals of some substrates have been identified; however, the mechanisms whereby these signals interact with type III machines are not known. Here we show that fusion of YopR, an early secretion substrate, to the N terminus of dihydrofolate reductase (DHFR) or other tightly folded proteins generates impassable hybrids that cannot travel the type III pathway. YopR hybrids capture YscN, the ATPase that provides energy for type III transport reactions, in the bacterial cytoplasm. Eleven N-terminal residues function as the YopR secretion signal, which is required for both binding to YscN and blocking the type III pathway. When expressed during type III machine assembly, YopR-DHFR blocks all secretion. Delayed expression of YopR-DHFR, when yersiniae have already engaged the type III pathway, blocks secretion of early (YscP) but not of late (effector Yops) substrates. These observations support a model whereby type III machines are programmed to secrete a sequence of proteins that can be disrupted when an impassable early substrate interacts with the YscN ATPase and blocks the transport of late substrates.

Characterization of the Yersinia enterocolitica type III secretion ATPase YscN and its regulator, YscL.

Type III secretion is a mechanism used by a broad range of gram-negative bacteria to neutralize eukaryotic defenses by enabling translocation of bacterial proteins directly into the cytoplasm of host cells. The bacterial energy source for secretion is ATP, which is consumed by an ATPase that couples ATP hydrolysis to the unfolding of secreted proteins and the dissociation of their chaperones just prior to secretion. By studying the biochemical properties of YscN and YscL of Yersinia enterocolitica, we have characterized them as the ATPase and ATPase regulator, respectively, of the type III secretion system of this organism. In vivo, YscL and YscN interact with each other, and the overexpression of glutathione S-transferase-YscL abolishes secretion and down-regulates the expression of secretion apparatus components.

Zipper-like interaction between proteins in adjacent daughter cells mediates protein localization.

Protein localization is crucial for cellular morphogenesis and intracellular signal transduction cascades. Here we describe an interaction between two membrane proteins expressed in different cells of the Bacillus subtilis sporangium, the mother cell protein SpoIIIAH and the forespore protein SpoIIQ. We used affinity chromatography, coimmunoprecipitation, and the yeast two-hybrid system to demonstrate that the extracellular domains of these proteins interact, tethering SpoIIIAH to the sporulation septum, and directing its assembly with SpoIIQ into helical arcs and foci around the forespore. We also demonstrate that this interaction can direct proteins made in the same cell to active division sites, as when SpoIIQ is made in the mother cell, it localizes to nascent septa in a SpoIIIAH-dependent manner. Both SpoIIIAH and SpoIIQ are necessary for activation of the second forespore-specific transcription factor (sigma(G)) after engulfment, and we propose that the SpoIIIAH-SpoIIQ complex contributes to a morphological checkpoint coupling sigma(G) activation to engulfment. In keeping with this hypothesis, SpoIIIAH localization depends on the first step of engulfment, septal thinning. The SpoIIQ-SpoIIIAH complex reaches from the mother cell cytoplasm to the forespore cytoplasm and is ideally positioned to govern the activity of engulfment-dependent transcription factors.