APOBEC3 enzymes are innate immune effectors that introduce mutations into viral genomes. These enzymes are cytidine deaminases which transform cytosine into uracil. They preferentially mutate cytidine preceded by thymidine making the 5'TC motif their favored target. Viruses have evolved different strategies to evade APOBEC3 restriction. Certain viruses actively encode viral proteins antagonizing the APOBEC3s, others passively face the APOBEC3 selection pressure thanks to a depleted genome for APOBEC3-targeted motifs. Hence, the APOBEC3s left on the genome of certain viruses an evolutionary footprint. The aim of our study is the identification of these viruses having a genome shaped by the APOBEC3s. We analyzed the genome of 33,400 human viruses for the depletion of APO-BEC3-favored motifs. We demonstrate that the APOBEC3 selection pressure impacts at least 22% of all currently annotated human viral species. The papillomaviridae and polyomaviridae are the most intensively footprinted families; evidencing a selection pressure acting genome-wide and on both strands. Members of the parvoviridae family are differentially targeted in term of both magnitude and localization of the footprint. Interestingly, a massive APOBEC3 footprint is present on both strands of the B19 erythroparvovirus; making this viral genome one of the most cleaned sequences for APOBEC3-favored motifs. We also identified the endemic coronaviridae as significantly footprinted. Interestingly, no such footprint has been detected on the zoonotic MERS-CoV, SARS-CoV-1 and SARS-CoV-2 coronaviruses. In addition to viruses that are footprinted genome-wide, certain viruses are footprinted only on very short sections of their genome. That is the case for the gamma-herpesviridae and adenoviridae where the footprint is localized on the lytic origins of replication. A mild footprint can also be detected on the negative strand of the reverse transcribing HIV-1, HIV-2, HTLV-1 and HBV viruses. Together, our data illustrate the extent of the APOBEC3 selection pressure on the human viruses and identify new putatively APOBEC3-targeted viruses.

is a class III zoonotic bacterial pathogen able to survive and replicate inside host cells, including macrophages. Here we report a multidimensional transposon sequencing analysis to identify genes essential for growth in rich medium and replication in RAW 264.7 macrophages. The construction of a dense transposon mutant library and mapping of 929,769 unique mini-Tn insertion sites in the genome allowed identification of 491 essential coding sequences and essential segments in the genome. Chromosome II carries a lower proportion (5%) of essential genes than chromosome I (19%), supporting the hypothesis of a recent acquisition of a megaplasmid as the origin of chromosome II. Temporally resolved transposon sequencing analysis as a function of macrophage infection stages identified 79 genes with a specific attenuation phenotype in macrophages, at either 2, 5, or 24 h postinfection, and 86 genes for which the attenuated mutant phenotype correlated with a growth defect on plates. We identified 48 genes required for intracellular growth, including the operon, encoding the type IV secretion system, which supports the validity of the screen. The remaining genes encode amino acid and pyrimidine biosynthesis, electron transfer systems, transcriptional regulators, and transporters. In particular, we report the need of an intact pyrimidine nucleotide biosynthesis pathway in order for to proliferate inside RAW 264.7 macrophages.

Brucella abortus is a pathogen infecting cattle, able to survive, traffic, and proliferate inside host cells. It belongs to the Alphaproteobacteria, a phylogenetic group comprising bacteria with free living, symbiotic, and pathogenic lifestyles. An essential regulator of cell cycle progression named CtrA was described in the model bacterium Caulobacter crescentus. This regulator is conserved in many alphaproteobacteria, but the evolution of its regulon remains elusive. Here we identified promoters that are CtrA targets using ChIP-seq and we found that CtrA binds to promoters of genes involved in cell cycle progression, in addition to numerous genes encoding outer membrane components involved in export of membrane proteins and synthesis of lipopolysaccharide. Analysis of a conditional B. abortus ctrA loss of function mutant confirmed that CtrA controls cell division. Impairment of cell division generates elongated and branched morphologies, that are also detectable inside HeLa cells. Surprisingly, abnormal bacteria are able to traffic to the endoplasmic reticulum, the usual replication niche of B. abortus in host cells. We also found that CtrA depletion affected outer membrane composition, in particular the abundance and spatial distribution of Omp25. Control of the B. abortus envelope composition by CtrA indicates the plasticity of the CtrA regulon along evolution.

Entry of the facultative intracellular pathogen into host cells results in the formation of endosomal-containing vacuoles (eBCVs) that initially traffic along the endocytic pathway. eBCV acidification triggers the expression of a type IV secretion system that translocates bacterial effector proteins into host cells. This interferes with lysosomal fusion of eBCVs and supports their maturation to replicative -containing vacuoles (rBCVs). Bacteria replicate in rBCVs to large numbers, eventually occupying most of the cytoplasmic volume. As rBCV membranes tightly wrap each individual bacterium, they are constantly being expanded and remodeled during exponential bacterial growth. rBCVs are known to carry endoplasmic reticulum (ER) markers; however, the relationship of the vacuole to the genuine ER has remained elusive. Here, we have reconstructed the 3-dimensional ultrastructure of rBCVs and associated ER by correlative structured illumination microscopy (SIM) and focused ion beam/scanning electron microscopic tomography (FIB/SEM). Studying-infected HeLa cells and trophoblasts derived from -infected mice, we demonstrate that rBCVs are complex and interconnected compartments that are continuous with neighboring ER cisternae, thus supporting a model that rBCVs are extensions of genuine ER.

Erythritol is the preferential carbon source for most brucellae, a group of facultative intracellular bacteria that cause a worldwide zoonosis. Since this polyol is abundant in genital organs of ruminants and swine, it is widely accepted that erythritol accounts at least in part for the characteristic genital tropism of brucellae. Nevertheless, proof of erythritol availability and essentiality during Brucella intracellular multiplication has remained elusive. To investigate this relationship, we compared ΔeryH (erythritol-sensitive and thus predicted to be attenuated if erythritol is present), ΔeryA (erythritol-tolerant but showing reduced growth if erythritol is a crucial nutrient) and wild type B. abortus in various infection models. This reporting system indicated that erythritol was available but not required for B. abortus multiplication in bovine trophoblasts. However, mice and humans have been considered to lack erythritol, and we found that it was available but not required for B. abortus multiplication in human and murine trophoblastic and macrophage-like cells, and in mouse spleen and conceptus (fetus, placenta and envelopes). Using this animal model, we found that B. abortus infected cells and tissues contained aldose reductase, an enzyme that can account for the production of erythritol from pentose cycle precursors.