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Heterologously expressed genes require adaptation to the host organism to ensure adequate levels of protein synthesis, which is typically approached by replacing codons by the target organism’s preferred codons. In view of frequently encountered suboptimal outcomes we introduce the codon-specific elongation model (COSEM) as an alternative concept. COSEM simulates ribosome dynamics during mRNA translation and informs about protein synthesis rates per mRNA in an organism- and context-dependent way. Protein synthesis rates from COSEM are integrated with further relevant covariates such as translation accuracy into a protein expression score that we use for codon optimization. The scoring algorithm further enables fine-tuning of protein expression including deoptimization and is implemented in the software OCTOPOS. The protein expression score produces competitive predictions on proteomic data from prokaryotic, eukaryotic, and human expression systems. In addition, we optimized and tested heterologous expression of manA and ova genes in Salmonella enterica serovar Typhimurium. Superiority over standard methodology was demonstrated by a threefold increase in protein yield compared to wildtype and commercially optimized sequences.
Correction to: Scientifc Reports https://doi.org/10.1038/s41598-019-43857-5, published online 17 May 2019. In the original version of this Article, Jan-Hendrik Trösemeier was incorrectly affiliated with ‘Division of Allergology, Paul Ehrlich Institut, Langen, Germany’. Te correct afliations are listed below...
Viruses rely completely on the hosts' machinery for translation of viral transcripts. However, for most viruses infecting humans, codon usage preferences (CUPrefs) do not match those of the host. Human papillomaviruses (HPVs) are a showcase to tackle this paradox: they present a large genotypic diversity and a broad range of phenotypic presentations, from asymptomatic infections to productive lesions and cancer. By applying phylogenetic inference and dimensionality reduction methods, we demonstrate first that genes in HPVs are poorly adapted to the average human CUPrefs, the only exception being capsid genes in viruses causing productive lesions. Phylogenetic relationships between HPVs explained only a small proportion of CUPrefs variation. Instead, the most important explanatory factor for viral CUPrefs was infection phenotype, as orthologous genes in viruses with similar clinical presentation displayed similar CUPrefs. Moreover, viral genes with similar spatiotemporal expression patterns also showed similar CUPrefs. Our results suggest that CUPrefs in HPVs reflect either variations in the mutation bias or differential selection pressures depending on the clinical presentation and expression timing. We propose that poor viral CUPrefs may be central to a trade-off between strong viral gene expression and the potential for eliciting protective immune response.
The human immunodeficiency virus type 1 (HIV-1) coreceptor use and viral evolution were analyzed in blood samples from an HIV-1 infected patient undergoing allogeneic stem cell transplantation (SCT). Coreceptor use was predicted in silico from sequence data obtained from the third variable loop region of the viral envelope gene with two software tools. Viral diversity and evolution was evaluated on the same samples by Bayesian inference and maximum likelihood methods. In addition, phenotypic analysis was done by comparison of viral growth in peripheral blood mononuclear cells and in a CCR5 (R5)-deficient T-cell line which was controlled by a reporter assay confirming viral tropism. In silico coreceptor predictions did not match experimental determinations that showed a consistent R5 tropism. Anti-HIV directed antibodies could be detected before and after the SCT. These preexisting antibodies did not prevent viral rebound after the interruption of antiretroviral therapy during the SCT. Eventually, transplantation and readministration of anti-retroviral drugs lead to sustained increase in CD4 counts and decreased viral load to undetectable levels. Unexpectedly, viral diversity decreased after successful SCT. Our data evidence that only R5-tropic virus was found in the patient before and after transplantation. Therefore, blocking CCR5 receptor during stem cell transplantation might have had beneficial effects and this might apply to more patients undergoing allogeneic stem cell transplantation. Furthermore, we revealed a scenario of HIV-1 dynamic different from the commonly described ones. Analysis of viral evolution shows the decrease of viral diversity even during episodes with bursts in viral load.
The specific temporal evolution of bacterial and phage population sizes, in particular bacterial depletion and the emergence of a resistant bacterial population, can be seen as a kinetic fingerprint that depends on the manifold interactions of the specific phage–host pair during the course of infection. We have elaborated such a kinetic fingerprint for a human urinary tract Klebsiella pneumoniae isolate and its phage vB_KpnP_Lessing by a modeling approach based on data from in vitro co-culture. We found a faster depletion of the initially sensitive bacterial population than expected from simple mass action kinetics. A possible explanation for the rapid decline of the bacterial population is a synergistic interaction of phages which can be a favorable feature for phage therapies. In addition to this interaction characteristic, analysis of the kinetic fingerprint of this bacteria and phage combination revealed several relevant aspects of their population dynamics: A reduction of the bacterial concentration can be achieved only at high multiplicity of infection whereas bacterial extinction is hardly accomplished. Furthermore the binding affinity of the phage to bacteria is identified as one of the most crucial parameters for the reduction of the bacterial population size. Thus, kinetic fingerprinting can be used to infer phage–host interactions and to explore emergent dynamics which facilitates a rational design of phage therapies.
Thrombopoietin (Thpo) signals via its receptor Mpl and regulates megakaryopoiesis, hematopoietic stem cell (HSC) maintenance and post-transplant expansion. Mpl expression is tightly controlled and deregulation of Thpo/Mpl-signaling is linked to hematological disorders. Here, we constructed an intracellular-truncated, signaling-deficient Mpl protein which is presented on the cell surface (dnMpl). The transplantation of bone marrow cells retrovirally transduced to express dnMpl into wildtype mice induced thrombocytopenia, and a progressive loss of HSC. The aplastic BM allowed the engraftment of a second BM transplant without further conditioning. Functional analysis of the truncated Mpl in vitro and in vivo demonstrated no internalization after Thpo binding and the inhibition of Thpo/Mpl-signaling in wildtype cells due to dominant-negative (dn) effects by receptor competition with wildtype Mpl for Thpo binding. Intracellular inhibition of Mpl could be excluded as the major mechanism by the use of a constitutive-dimerized dnMpl. To further elucidate the molecular changes induced by Thpo/Mpl-inhibition on the HSC-enriched cell population in the BM, we performed gene expression analysis of Lin-Sca1+cKit+ (LSK) cells isolated from mice transplanted with dnMpl transduced BM cells. The gene expression profile supported the exhaustion of HSC due to increased cell cycle progression and identified new and known downstream effectors of Thpo/Mpl-signaling in HSC (namely TIE2, ESAM1 and EPCR detected on the HSC-enriched LSK cell population). We further compared gene expression profiles in LSK cells of dnMpl mice with human CD34+ cells of aplastic anemia patients and identified similar deregulations of important stemness genes in both cell populations. In summary, we established a novel way of Thpo/Mpl inhibition in the adult mouse and performed in depth analysis of the phenotype including gene expression profiling.