Acinetobacter baumannii is a Gram-negative pathogen that causes a multitude of nosocomial infections. The Acinetobacter trimeric autotransporter adhesin (Ata) belongs to the superfamily of trimeric autotransporter adhesins which are important virulence factors in many Gram-negative species. Phylogenetic profiling revealed that ata is present in 78% of all sequenced A. baumannii isolates but only in 2% of the closely related species A. calcoaceticus and A. pittii. Employing a markerless ata deletion mutant of A. baumannii ATCC 19606 we show that adhesion to and invasion into human endothelial and epithelial cells depend on Ata. Infection of primary human umbilical cord vein endothelial cells (HUVECs) with A. baumannii led to the secretion of interleukin (IL)-6 and IL-8 in a time- and Ata-dependent manner. Furthermore, infection of HUVECs by WT A. baumannii was associated with higher rates of apoptosis via activation of caspases-3 and caspase-7, but not necrosis, in comparison to ∆ata. Ata deletion mutants were furthermore attenuated in their ability to kill larvae of Galleria mellonella and to survive in larvae when injected at sublethal doses. This indicates that Ata is an important multifunctional virulence factor in A. baumannii that mediates adhesion and invasion, induces apoptosis and contributes to pathogenicity in vivo.

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The Gram-negative bacterial pathogen Acinetobacter baumannii is a major cause of hospital-acquired opportunistic infections. The increasing spread of pan-drug resistant strains, paired with a surge in virulence, makes A. baumannii top-ranking among the ESKAPE pathogens for which novel routes of treatment are urgently needed. Comparative genomics approaches have successfully identified genetic changes that coincide with the emergence of pathogenicity in Acinetobacter. Genes that are prevalent both in pathogenic and a-pathogenic Acinetobacter species were not considered ignoring that virulence factors may emerge by the modification of evolutionarily old and widespread proteins. Here, we increased the resolution of comparative genomics analyses to also include lineage-specific changes in protein domain architectures. Using type IVa pili (T4aP) as an example, we show that two pilus components, FimU and the pilus tip adhesin ComC, exist with markedly differing domain architectures in pathogenic Acinetobacter species. ComC, has gained a von Willebrand Factor type A domain displaying a finger-like protrusion, and we provide experimental evidence that this finger conveys virulence-related functions in A. baumannii. Both genes together form an evolutionary cassette, which has been replaced at least twice during A. baumannii diversification. The resulting strain-specific differences in T4aP layout suggests differences in the way how individual strains interact with their host. Our study underpins the hypothesis that A. baumannii uses T4aP for host infection as it was shown previously for other pathogens. It also indicates that many more functional complexes may exist whose precise function can be rapidly adjusted by changing the domain architecture of individual proteins.

Covid-19 is the most devastating pandemic of the past 100 years. A zoonotic transfer presumably at a wildlife market introduced the causative virus, SARS-CoV-2 (sarbecovirus; beta-coronavirus), to humans in late 2019. Meanwhile, the mechanistic details of the infection process have been largely elucidated, and structural models explain binding of the virial spike to the human cell surface receptor ACE2. Yet, the evolutionary trajectory that gave rise to this pathogen is poorly understood. Here we scan SARS-CoV-2 protein sequences in-silico for innovations along the evolutionary lineage starting with the last common ancestor of coronaviruses. Substantial differences in the sets of proteins encoded by SARS-CoV-2 and viruses outside sarbecovirus, and in their domain architectures, indicate divergent functional demands. By contrast, sarbecoviruses themselves are almost fully conserved at these levels of resolution. However, profiling spike evolution on the sub-domain level using predicted linear epitopes reveals that this protein was gradually reshaped within sarbecovirus. The only epitope that is private to SARS-CoV-2 overlaps with the furin cleavage site. This lends phylogenetic support to the hypothesis that a change in strategy facilitated the zoonotic transfer of SARS-CoV-2 and its success as a human pathogen. Upon furin cleavage, spike switches from a “stealth mode” where immunodominant ACE2 binding epitopes are largely hidden to an “attack mode” where these epitopes are exposed. The resulting reinforcement of ACE2 binding extends the window of opportunity for cell entry. SARS-CoV-2 variants fine-tuning this mode switch will be particularly threatening as they optimize immune evasion.