Summary: The plant pathogen Candidatus Phytoplasma mali (P. mali) is the causative agent of apple proliferation, a disease of increasing importance in apple‐growing areas within Europe. Despite its economic importance, little is known about the molecular mechanisms of disease manifestation within apple trees. In this study, we identified two TCP (TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR) transcription factors of Malus x domestica as binding partners of the P. mali SAP11‐like effector ATP_00189. Phytohormone analyses revealed an effect of P. mali infection on jasmonates, salicylic acid and abscisic acid levels, showing that P. mali affects phytohormonal levels in apple trees, which is in line with the functions of the effector assumed from its binding to TCP transcription factors. To our knowledge, this is the first characterization of the molecular targets of a P. mali effector and thus provides the basis to better understand symptom development and disease progress during apple proliferation. As SAP11 homologues are found in several Phytoplasma species infecting a broad range of different plants, SAP11‐like proteins seem to be key players in phytoplasmal infection.

The intracellular bacterium Chlamydia trachomatis causes infections of urogenital tract, eyes or lungs. Alignment reveals homology of CT166, a putative effector protein of urogenital C. trachomatis serovars, with the N-terminal glucosyltransferase domain of clostridial glucosylating toxins (CGTs). CGTs contain an essential DXD-motif and mono-glucosylate GTP-binding proteins of the Rho/Ras families, the master regulators of the actin cytoskeleton. CT166 is preformed in elementary bodies of C. trachomatis D and is detected in the host-cell shortly after infection. Infection with high MOI of C. trachomatis serovar D containing the CT166 ORF induces actin re-organization resulting in cell rounding and a decreased cell diameter. A comparable phenotype was observed in HeLa cells treated with the Rho-GTPase-glucosylating Toxin B from Clostridium difficile (TcdB) or HeLa cells ectopically expressing CT166. CT166 with a mutated DXD-motif (CT166-mut) exhibited almost unchanged actin dynamics, suggesting that CT166-induced actin re-organization depends on the glucosyltransferase motif of CT166. The cytotoxic necrotizing factor 1 (CNF1) from E. coli deamidates and thereby activates Rho-GTPases and transiently protects them against TcdB-induced glucosylation. CNF1-treated cells were found to be protected from TcdB- and CT166-induced actin re-organization. CNF1 treatment as well as ectopic expression of non-glucosylable Rac1-G12V, but not RhoA-G14A, reverted CT166-induced actin re-organization, suggesting that CT166-induced actin re-organization depends on the glucosylation of Rac1. In accordance, over-expression of CT166-mut diminished TcdB induced cell rounding, suggesting shared substrates. Cell rounding induced by high MOI infection with C. trachomatis D was reduced in cells expressing CT166-mut or Rac1-G12V, and in CNF1 treated cells. These observations indicate that the cytopathic effect of C. trachomatis D is mediated by CT166 induced Rac1 glucosylation. Finally, chlamydial uptake was impaired in CT166 over-expressing cells. Our data strongly suggest CT166's participation as an effector protein during host-cell entry, ensuring a balanced uptake into host-cells by interfering with Rac-dependent cytoskeletal changes.

The complement system modulates the intensity of innate and specific immunity. While it protects against infections by extracellular bacteria its role in infection with obligate intracellular bacteria, such as the avian and human pathogen Chlamydia (C.) psittaci, is still unknown. In the present study, knockout mice lacking C3 and thus all main complement effector functions were intranasally infected with C. psittaci strain DC15. Clinical parameters, lung histology, and cytokine levels were determined. A subset of infections was additionally performed with mice lacking C5 or C5a receptors. Complement activation occurred before symptoms of pneumonia appeared. Mice lacking C3 were ∼100 times more susceptible to the intracellular bacteria compared to wild-type mice, with all C3^−/− mice succumbing to infection after day 9. At a low infective dose, C3^−/− mice became severely ill after an even longer delay, the kinetics suggesting a so far unknown link of complement to the adaptive, protective immune response against chlamydiae. The lethal phenotype of C3^−/− mice is not based on differences in the anti-chlamydial IgG response (which is slightly delayed) as demonstrated by serum transfer experiments. In addition, during the first week of infection, the absence of C3 was associated with partial protection characterized by reduced weight loss, better clinical score and lower bacterial burden, which might be explained by a different mechanism. Lack of complement functions downstream of C5 had little effect. This study demonstrates for the first time a strong and complex influence of complement effector functions, downstream of C3 and upstream of C5, on the outcome of an infection with intracellular bacteria, such as C. psittaci.

We analyzed the defensive role of the cytosolic innate recognition receptor nucleotide oligomerization domain 1 (NOD1) during infection withListeria monocytogenes. Mice lacking NOD1 showed increased susceptibility to systemic intraperitoneal and intravenous infection with high or low doses ofL. monocytogenes, as measured by the bacterial load and survival. NOD1 also controlled dissemination ofL. monocytogenesinto the brain. The increased susceptibility to reinfection of NOD1^−/−mice was not associated with impaired triggering of listeria-specific T cells, and similar levels of costimulatory molecules or activation of dendritic cells was observed. Higher numbers of F480⁺Gr1⁺inflammatory monocytes and lower numbers of F480⁻Gr1⁺neutrophils were recruited into the peritoneum of infected WT mice than into the peritoneum of infected NOD1^−/−mice. We determined that nonhematopoietic cells accounted for NOD1-mediated resistance toL. monocytogenesin bone marrow radiation chimeras. The levels of NOD1 mRNA in fibroblasts and bone marrow-derived macrophages (BMM) were upregulated after infection withL. monocytogenesor stimulation with different Toll-like receptor ligands. NOD1^−/−BMM, astrocytes, and fibroblasts all showed enhanced intracellular growth ofL monocytogenescompared to WT controls. Gamma interferon-mediated nitric oxide production and inhibition ofL. monocytogenesgrowth were hampered in NOD1^−/−BMM. Thus, NOD1 confers nonhematopoietic cell-mediated resistance to infection withL. monocytogenesand controls intracellular bacterial growth in different cell populations in vitro.