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Exposure of the vertebrate embryo to maternal hormones can have long-lasting effects on its phenotype, which has been studied extensively by experimentally manipulating maternal steroids, mostly androgens, in bird eggs. Yet, there is a severe lack of understanding of how and when these effects are actually mediated, hampering both underlying proximate and ultimate explanations. Here we report a novel finding that the embryo expresses androgen receptor (AR) and estrogen receptor (ERα) mRNA in its extraembryonic membranes (EMs) as early as before its own hormone production starts, suggesting a novel substrate for action of maternal hormones on the offspring. We also report the first experimental evidence for steroid receptor regulation in the avian embryo in response to yolk steroid levels: the level of AR is dependent on yolk androgen levels only in the EMs but not in body tissues, suggesting embryonic adaptation to maternal hormones. The results also solve the problem of uptake of lipophilic steroids from the yolk, why they affect multiple traits, and how they could mediate maternal effects without affecting embryonic sexual differentiation.

Dengue virus infects immune cells, including monocytes, macrophages and dendritic cells (DC). We compared virus infectivity in macrophages and DC, and found that the virus origin determined the cell tropism of progeny virus. The highest efficiency of re-infection was seen for macrophage-derived dengue virus. Furthermore, in the presence of enhancing antibodies, macrophage-derived virus gave greater enhancement of infection compared with immature DC derived virus. Taken together, our results highlight the importance of macrophages in dengue infection.

Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of "toxin-coated bacteria" proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or "free" in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and toxin-induced Ca2+-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system. Technical and human support provided by SGIker (Analytical and High-Resolution Microscopy in Biomedicine Service of UPV/EHU) and Rocio Alonso for excellent technical assistance are gratefully acknowledged. We also thank Dr. N. Guiso from the Pasteur Institute (Paris, France) for the Bordetella pertussis strains BP18323 and non-virulent bgv negative BP18323H-. This study was supported by grants from the Spanish Ministerio de Ciencia y Tecnologia (Project BFU 2012-36241), and the Basque Government (Grupos Consolidados IT849-13 and ETORTEK Program). K.B.U and D.G.B. were recipients of a fellowship from the Bizkaia Biophysics Foundation. A.E.G. was a recipient of a research contract (INNPACTO 2010) and A.E.L. was a recipient of a fellowship from the University of the Basque Country (UPV/EH). A patent with reference N/Ref.: P1683USPC (2004) was approved.

The Type VI secretion system (T6SS) mediates toxin delivery into both eukaryotic and prokaryotic cells. It is composed of a cytoplasmic structure resembling the tail of contractile bacteriophages anchored to the cell envelope through a membrane complex composed of the TssL and TssM inner membrane proteins and of the TssJ outer membrane lipoprotein. The C-terminal domain of TssM is required for its interaction with TssJ, and for the function of the T6SS. In Citrobacter rodentium, the tssM1 gene does not encode the C-terminal domain. However, the stop codon is preceded by a run of 11 consecutive adenosines. In this study, we demonstrate that this poly-A tract is a transcriptional slippery site that induces the incorporation of additional adenosines, leading to frameshifting, and hence the production of two TssM1 variants, including a full-length canonical protein. We show that both forms of TssM1, and the ratio between these two forms, are required for the function of the T6SS in C. rodentium. Finally, we demonstrate that the tssM gene associated with the Yersinia pseudotuberculosis T6SS-3 gene cluster is also subjected to transcriptional frameshifting., Author Summary Nonstandard decoding mechanisms lead to the synthesis of different protein variants from a single DNA sequence. These mechanisms are particularly important when the genome length has to be limited such as viral genomes, limited by the available space in the capsid, or to synthesize two different polypeptides that have distinct functional properties. Here, we report that tssM, a gene encoded within the Citrobacter rodentium Type VI secretion (T6S) gene cluster, is interrupted by a premature stop codon; however, the stop codon is preceded by a slippery site constituted by 11 consecutive adenosines. Reiterative transcription leads to the incorporation of additional nucleotides in the mRNA and therefore restores the original framing. As a consequence, two different TssM variants are created by transcriptional frameshifting, including a full-length 130-kDa protein and an 88-kDa truncated variant. We further show that both forms, and the ratio between these two forms, are required for the function of the transport apparatus. Interestingly, a similar mechanism regulates the synthesis of two TssM variants in Yersinia pseudotuberculosis.