Type III secretion enables bacteria to intoxicate eukaryotic cells with anti-host effectors. A class of secreted cargo are the two hydrophobic translocators that form a translocon pore in the host cell plasma membrane through which the translocated effectors may gain cellular entry. In pathogenic Yersinia, YopB and YopD shape this translocon pore. Here, four in cis yopD mutations were constructed to disrupt a predicted α-helix motif at the C-terminus. Mutants YopD(I262P) and YopD(K267P) poorly localized Yop effectors into target eukaryotic cells and failed to resist uptake and killing by immune cells. These defects were due to deficiencies in host-membrane insertion of the YopD-YopB translocon. Mutants YopDA(263P) and YopD(A270P) had no measurable in vitro translocation defect, even though they formed smaller translocon pores in erythrocyte membranes. Despite this, all four mutants were attenuated in a mouse infection model. Hence, YopD variants have been generated that can spawn translocons capable of targeting effectors in vitro, yet were bereft of any lethal effect in vivo. Therefore, Yop translocators may possess other in vivo functions that extend beyond being a portal for effector delivery into host cells.

Type III secretion is a tightly controlled virulence mechanism utilized by many gram negative bacteria to colonize their eukaryotic hosts. To infect their host, human pathogenic Yersinia spp. translocate protein toxins into the host cell cytosol through a preassembled Ysc-Yop type III secretion device. Several of the Ysc-Yop components are known for their roles in controlling substrate secretion and translocation. Particularly important in this role is the YopN and TyeA heterodimer. In this study, we confirm that Y. pseudotuberculosis naturally produce a 42 kDa YopN-TyeA hybrid protein as a result of a +1 frame shift near the 3 prime of yopN mRNA, as has been previously reported for the closely related Y. pestis. To assess the biological role of this YopN-TyeA hybrid in T3SS by Y. pseudotuberculosis, we used in cis site-directed mutagenesis to engineer bacteria to either produce predominately the YopN-TyeA hybrid by introducing +1 frame shifts to yopN after codon 278 or 287, or to produce only singular YopN and TyeA polypeptides by introducing yopN sequence from Y. enterocolitica, which is known not to produce the hybrid. Significantly, the engineered 42 kDa YopN-TyeA fusions were abundantly produced, stable, and were efficiently secreted by bacteria in vitro. Moreover, these bacteria could all maintain functionally competent needle structures and controlled Yops secretion in vitro. In the presence of host cells however, bacteria producing the most genetically altered hybrids (+1 frameshift after 278 codon) had diminished control of polarized Yop translocation. This corresponded to significant attenuation in competitive survival assays in orally infected mice, although not at all to the same extent as Yersinia lacking both YopN and TyeA proteins. Based on these studies with engineered polypeptides, most likely a naturally occurring YopN-TyeA hybrid protein has the potential to influence T3S control and activity when produced during Yersinia-host cell contact.

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The teeth play importa nt roles in food mastication, prehens ion and defe ns e against predato rs . Althoug h severa l studie s demons tra te d the developm e nt of teeth in differe nt mammalia n species , no data are, to our knowle d g e , available in Egyptia n buffalos . Therefore, the present investig a tion was conducte d to study the develop me n t of incisor teeth in buffaloe . Design: Descriptive study. Animals : sevente e n buffalo embryos and fetuses of both sexes were used. Their crown verte bra l rump lengths (CVRL) ranged from 2 to 46 cm (equiva le nt to 38 -177 days old). Proce dure: Embryos and fetuses were fixed in 10% neutra l buffere d formalin and decalcifie d by 14% EDTA solution for several weeks . The samples were dehydra te d , cleare d and embedde d in paraffin wax using standa rd technique s . Sections were cut on Leitz microtom e and mounte d on uncoa te d slides. For general histolog ica l structure , a selection of slides was routine ly stained with haematoxylin -e osin and examine d by the light micros cope . Result: The present study reporte d for the first time that the prenata l developme nt of incisors in buffalo passes throug h three sequentia l stages: the bud, cap and bell stag e s . Althoug h each stage was easily differe ntia te d based on its form and its distinc t developm e ntal features , the three stages were overla ppe d with each other's . Genera ll y , the bud stage was observe d in 11 cm CVRL buffalo fetuses, meanwhile the cap and be ll stages were detecte d in 23 cm and 30 cm CVRL fetuses respective ly. Conclus ion and clinical relevance:These results can be used as inductive index for determina tion of the ages of buffalo embryos and fetuses.