Summary LcrQ is a regulatory protein unique to Yersinia. Previous study in Yersinia pseudotuberculosis and Yersinia enterocolitica prompted the model in which LcrQ negatively regulates the expression of a set of virulence proteins called Yops, and its secretion upon activation of the Yop secretion (Ysc) type III secretion system permits full induction of Yops expression. In this study, we tested the hypothesis that LcrQ’s effects on Yops expression might be indirect. Excess LcrQ was found to exert an inhibitory effect specifically at the level of Yops secretion, independent of production, and a normal inner Ysc gate protein LcrG was required for this activity. However, overexpression of LcrQ did not prevent YopH secretion, suggesting that LcrQ’s effects at the Ysc discriminate among the Yops. We tested this idea by determining the effects of deletion or overexpression of LcrQ, YopH and their common chaperone SycH on early Yop secretion through the Ysc. Together, our findings indicated that LcrQ is not a negative regulator directly, but it acts in partnership with SycH at the Ysc gate to control the entry of a set of Ysc secretion substrates. A hierarchy of YopH secretion before YopE appears to be imposed by SycH in conjunction with both LcrQ and YopH. LcrQ and SycH in addition influenced the deployment of LcrV, a component of the Yops delivery mechanism. Accordingly, LcrQ appears to be a central player in determining the substrate specificity of the Ysc.
The V antigen (LcrV) of the plague bacterium Yersinia pestis is a potent protective antigen that is under development as a vaccine component for humans. LcrV is multifunctional. On the bacterial surface it mediates delivery of a set of toxins called Yops into host cells, and as a released protein it can cause production of the immunosuppressive cytokine interleukin-10 (IL-10) and can inhibit chemotaxis of polymorphonuclear neutrophils. It is not known how these mechanisms of LcrV operate, what their relative importance is, when they function during plague, and which are critical to protection by antibody. This study investigated several of these issues. C57BL/6 mice, mice unable to express IL-10, or mice with the macrophage lineage eliminated were treated with a protective anti-LcrV antibody or a nonprotective antibody against YopM and infected intravenously by Y. pestis KIM5 or a strain that lacked the genes encoding all six effector Yops. Viable bacterial numbers were determined at various times. The data indicated that Yops were necessary for Yersinia growth after the bacteria had seeded liver and spleen. Anti-LcrV antibody prevented this growth, even in IL-10 ؊/؊ mice, demonstrating that one protective mechanism for anti-LcrV antibody is independent of IL-10. Anti-LcrV antibody had no effect on persistence in organs of Y. pestis lacking effector Yops, even though the yersiniae could strongly express LcrV, suggesting that Yops are necessary for building sufficient bacterial numbers to produce enough LcrV for its immunosuppressive effects. In vitro assays showed that anti-LcrV antibody could partially block delivery of Yops and downstream effects of Yops in infected macrophage-like J774A.1 cells. However, cells of the macrophage lineage were found to be dispensable for protection by anti-LcrV antibody in spleen, although they contributed to protection in liver. Taken together, the data support the hypothesis that one protective effect of the antibody is to block delivery of Yops to host cells and prevent early bacterial growth. The findings also identified the macrophage lineage as one host cell type that mediates protection.
Prevotella ruminicola B 1 4 is a gram-negative, anaerobic gastrointestinal bacterium. A 2.4-kbp chromosomal fragment from P. ruminicola encoding an 87-kDa aryl-glucosidase (CdxA) with cellodextrinase activity was cloned into Escherichia coli DH5␣ and sequenced. CdxA activity was found predominantly in the membrane fraction of both P. ruminicola and E. coli, but P. ruminicola localized the protein extracellularly while E. coli did not. The hydrolase had the highest activity on cellodextrins (3.43 to 4.13 mol of glucose released min ؊1 mg of protein ؊1) and p-nitrophenyl--D-glucoside (3.54 mol min ؊1 mg of protein ؊1). Significant activity (70% of p-nitrophenyl--D-glucoside activity) was also detected on arbutin and prunasin. Less activity was obtained with cellobiose, amygdalin, or gentiobiose. CdxA attacks cellodextrins from the nonreducing end, releasing glucose units, and appears to be an exo-1,4--glucosidase (EC 3.2.1.74) which also is able to attack -1,6 linkages. Comparison of the deduced amino acid sequence with other glycosyl-hydrolases suggests that this enzyme belongs to family 3 (B. Henrissat, Biochem. J. 280:309-316, 1991). On the basis of this sequence alignment, the catalytic residues are believed to be Asp-275 and Glu-265. This is the first report of a cloned ruminal bacterial enzyme which can cleave cyanogenic plant compounds and which may therefore contribute to cyanide toxicity in ruminants.Ruminant animals such as cattle and sheep have evolved to utilize the extensive fermentative abilities of their reticuloruminal microflora. This symbiotic relationship gives the ruminant animal access to a variety of herbaceous foodstuffs such as cellulose and hemicellulose which are nutritionally unavailable to monogastric animals. In addition to cellobiose, cellodextrins are a major product of cellulose hydrolysis in the rumen. Ruminal bacteria that can utilize cellodextrins are common. Prevotella ruminicola B 1 4 possesses extracellular cellodextrinase activity (27) and can utilize water-soluble cellodextrins (up to seven glucose units in length) as carbon and energy sources. In this paper, we report the cloning, sequencing, and partial characterization of an 87-kDa membrane-associated exo--glucosidase.Although normally beneficial, the expanded digestive repertoire of the ruminant animal can also be a detriment. A significant number of range forages such as serviceberry (Amelanchier alnifolia), bird's foot trefoil (Lotus corniculatus), chokeberry (Prunus virginiana), various sorghum species, white clover (Trifolium repens), and arrowgrass (Triglochin spp.) contain cyanogenic glycosides which are hydrolyzed during ruminal fermentation. These glycosides constitute a prominent class of toxic plant compounds (7, 9, 31) which presumably are synthesized to discourage grazing. For a review of the plant biosynthetic routes that produce these glycosides, see the work of Vennesland et al. (31).The P. ruminicola B 1 4 -glucosidase catalyzes the first step in cyanide release from cyanogenic plant glycosides. Sin...
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