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.