Table 1.
Summation of phenotypes exhibited by strains with in cis mutations in yopN and tyeA.
| Variant | Stabilitya | Growthb | Synthesis and secretion | Viabilitye | Virulence attenuationf | TyeA bindingg | |||
|---|---|---|---|---|---|---|---|---|---|
| Surface YscFc | YopN (Hybrid)d | Other Yopsd | YTH | BACTH | |||||
| YopN288(scramble)293 | WT | WT | WT | WT (WT) | WT | WT | WT | WT | WT |
| YopN288STOP | WT | WT | WT | WT (↓) | WT | Null-like | WT | WT | WT |
| YopN279(F+1), 287(F−1) | ↓ | Null | WT | Null (↓↓) | Null | Null | ND | Null | Null |
| YopN279(F+1), 287STOP | WT | Null | WT | Null-like (↓) | Null-like | Null-like | ND | Null | Null* |
| YopN279STOP | (↓) | Null | WT | Null (↓) | Null | Null | ND | Null | Null* |
| YopNW279G | ↓ | Null | WT | Null (↑) | Null | Null-like | ND | Null | Null* |
| TyeAY3A | WT | WT | WT | WT (WT) | WT | WT | ND | WT | WT |
| TyeAL5A | (↓) | WT | WT | WT (WT) | WT | WT | ND | WT | WT |
| TyeAF8A | ↓ | Null | WT | Null (↑) | Null | Null-like | ND | Null | Null* |
| TyeAF33A | ↓ | Null-like | WT | Null-like (WT) | Null-like | Null-like | ND | WT-like | Null* |
A summary of the intrabacterial stability of each YopN and TyeA variant shown in Figure 4 and as determined by the method of Feldman et al. (2002). WT: normal stability; (↓): slight instability; ↓: moderate instability.
Analysis of growth Y. pseudotuberculosis phenotypes was performed as previously described (Amer et al., 2011, 2013). Results shown in electronic Supplementary Material, Figure S1 are summarized as wild type (WT) that represents the phenotype of parental bacteria (YPIII/pIB102) or conversely as “Null” that represents the single ΔyopN or ΔtyeA null mutants or the double ΔyopN, tyeA null mutant. “WT” growth refers to calcium dependency (CD) at 37°C and reflects wild type regulatory control of Yop synthesis by virtue of a functional YopN-TyeA regulatory complex, whereas “Null” growth refers to temperature sensitivity (TS) at 37°C and echoes defective regulatory control whereby Yop synthesis is constitutive due to a defective YopN–TyeA regulatory complex (Iriarte et al., 1998; Cheng et al., 2001; Schubot et al., 2005). Null-like reflects a growth phenotype the lies between CD and TS, where bacteria grow only modestly at 37°C in the presence of calcium.
Analysis of cross-linked YscF higher-order structures derived from the bacterial surface was used to gage if Ysc T3SS's are correctly assembled and competent for Yops substrate secretion (Amer et al., 2013). Results shown in electronic Supplementary Material, Figure S2 are summarized as like wild type (WT) or the ΔyscU, lcrQ null mutant (Null).
A summary of the degree of controlled Yop synthesis and secretion generated from bacterial strains harboring the yopN and tyeA mutations as determined for production of YopN (Figures 2A, 7A) as well as the YopD injectisome component and the injected YopE cytotoxic effector (Figures 2B, 7B). WT: normal substrate synthesis and secretion in inducing conditions; Null: deregulated (constitutive) Yops synthesis and secretion; Null-like: partial deregulation. In parenthesis is an assessment of YopN-TyeA hybrid formation (Figures 2A, 7A). WT: normal formation; ↓: low level formation; ↓↓: not readily detectable by standard immunoblot; ↑: deregulated (constitutive) YopN-TyeA hybrid synthesis and secretion.
As a gage for measuring the effectiveness of Ysc-Yop T3SS activity, we analyzed the degree in which Yersinia could resist engulfment by professional phagocytic cells and subsequent intracellular killing by host antimicrobial activities (Bartra et al., 2001; Amer et al., 2011, 2013; Costa et al., 2012, 2013). The results are a summary of data presented in Figure 3. WT: bacteria maintain a high degree of viability being indistinguishable from wild type; Null-like: the ability to maintain viability is impaired; Null: bacteria are as susceptible to immune cell killing as is the ΔyopN null mutant.
Groups of five mice were co-infected with a the parental strain and strains containing yopN mutated alleles. The degree of attenuation was determined by competitive index measurements as detailed in electronic Supplementary Material, Table S1 and previously (Amer et al., 2013). WT: virulence of mutant bacteria was not statistically different from the parent; ND, not determined.
Determined from conventional yeast two-hybrid assay (YTH; Figure 5; Francis et al., 2000) and bacterial adenylate cyclase two hybrid (BACTH; electronic Supplementary Material, Figure S3; Thanikkal et al., 2012). WT: robust interaction between YopN and TyeA; Null: no detectable binding between YopN and TyeA; WT-like: a modest interaction between YopN and TyeA. The asterisk (*) indicates that one or both fusion proteins were unstable or not detected by immunoblot analysis.