Uncovering strain- and age-dependent innate immune responses to SARS-CoV-2 infection in air-liquid-interface cultured nasal epithelia.

Continuous assessment of the impact of SARS-CoV-2 on the host at the cell-type level is crucial for understanding key mechanisms involved in host defense responses to viral infection. We investigated host response to ancestral-strain and Alpha-variant SARS-CoV-2 infections within air-liquid-interface human nasal epithelial cells from younger adults (26-32 Y) and older children (12-14 Y) using single-cell RNA-sequencing. Ciliated and secretory-ciliated cells formed the majority of highly infected cell-types, with the latter derived from ciliated lineages. Strong innate immune responses were observed across lowly infected and uninfected bystander cells and heightened in Alpha-infection. Alpha highly infected cells showed increased expression of protein-refolding genes compared with ancestral-strain-infected cells in children. Furthermore, oxidative phosphorylation-related genes were down-regulated in bystander cells versus infected and mock-control cells, underscoring the importance of these biological functions for viral replication. Overall, this study highlights the complexity of cell-type-, age- and viral strain-dependent host epithelial responses to SARS-CoV-2.

Horizontal gene transfer facilitates the molecular reverse-evolution of antibiotic sensitivity in experimental populations of H. pylori.

Reversing the evolution of traits harmful to humans, such as antimicrobial resistance, is a key ambition of applied evolutionary biology. A major impediment to reverse evolution is the relatively low spontaneous mutation rates that revert evolved genotypes back to their ancestral state. However, the repeated re-introduction of ancestral alleles by horizontal gene transfer (HGT) could make reverse evolution likely. Here we evolve populations of an antibiotic-resistant strain of Helicobacter pylori in growth conditions without antibiotics while introducing an ancestral antibiotic-sensitive allele by HGT. We evaluate reverse evolution using DNA sequencing and find that HGT facilitates the molecular reverse evolution of the antibiotic resistance allele, and that selection for high rates of HGT drives the evolution of increased HGT rates in low-HGT treatment populations. Finally, we use a theoretical model and carry out simulations to infer how the fitness costs of antibiotic resistance, rates of HGT and effects of genetic drift interact to determine the probability and predictability of reverse evolution.

Recombination resolves the cost of horizontal gene transfer in experimental populations of Helicobacter pylori.

Horizontal gene transfer (HGT) is important for microbial evolution, yet we know little about the fitness effects and dynamics of horizontally transferred genetic variants. In this study, we evolve laboratory populations of Helicobacter pylori, which take up DNA from their environment by natural transformation, and measure the fitness effects of thousands of transferred genetic variants. We find that natural transformation increases the rate of adaptation but comes at the cost of significant genetic load. We show that this cost is circumvented by recombination, which increases the efficiency of selection by decoupling deleterious and beneficial genetic variants. Our results show that adaptation with HGT, pervasive in natural microbial populations, is shaped by a combination of selection, recombination, and genetic drift not accounted for in existing models of evolution.

Revisiting Antibiotic Resistance: Mechanistic Foundations to Evolutionary Outlook.

Antibiotics are the pivotal pillar of contemporary healthcare and have contributed towards its advancement over the decades. Antibiotic resistance emerged as a critical warning to public wellbeing because of unsuccessful management efforts. Resistance is a natural adaptive tool that offers selection pressure to bacteria, and hence cannot be stopped entirely but rather be slowed down. Antibiotic resistance mutations mostly diminish bacterial reproductive fitness in an environment without antibiotics; however, a fraction of resistant populations 'accidentally' emerge as the fittest and thrive in a specific environmental condition, thus favouring the origin of a successful resistant clone. Therefore, despite the time-to-time amendment of treatment regimens, antibiotic resistance has evolved relentlessly. According to the World Health Organization (WHO), we are rapidly approaching a 'post-antibiotic' era. The knowledge gap about antibiotic resistance and room for progress is evident and unified combating strategies to mitigate the inadvertent trends of resistance seem to be lacking. Hence, a comprehensive understanding of the genetic and evolutionary foundations of antibiotic resistance will be efficacious to implement policies to force-stop the emergence of resistant bacteria and treat already emerged ones. Prediction of possible evolutionary lineages of resistant bacteria could offer an unswerving impact in precision medicine. In this review, we will discuss the key molecular mechanisms of resistance development in clinical settings and their spontaneous evolution.

Copper homeostasis at the host vibrio interface: lessons from intracellular vibrio transcriptomics.

Recent studies revealed that several vibrio species have evolved the capacity to survive inside host cells. However, it is still often ignored if intracellular stages are required for pathogenicity. Virulence of Vibrio tasmaniensis LGP32, a strain pathogenic for Crassostrea gigas oysters, depends on entry into hemocytes, the oyster immune cells. We investigated here the mechanisms of LGP32 intracellular survival and their consequences on the host-pathogen interaction. Entry and survival inside hemocytes were required for LGP32-driven cytolysis of hemocytes, both in vivo and in vitro. LGP32 intracellular stages showed a profound boost in metabolic activity and a major transcription of antioxidant and copper detoxification genes, as revealed by RNA sequencing. LGP32 isogenic mutants showed that resistance to oxidative stress and copper efflux are two main functions required for vibrio intracellular stages and cytotoxicity to hemocytes. Copper efflux was also essential for host colonization and virulence in vivo. Altogether, our results identify copper resistance as a major mechanism to resist killing by phagocytes, induce cytolysis of immune cells and colonize oysters. Selection of such resistance traits could arise from vibrio interactions with copper-rich environmental niches including marine invertebrates, which favour the emergence of pathogenic vibrios resistant to intraphagosomal killing across animal species.

RNA at 92 °C: the non-coding transcriptome of the hyperthermophilic archaeon Pyrococcus abyssi.

The non-coding transcriptome of the hyperthermophilic archaeon Pyrococcus abyssi is investigated using the RNA-seq technology. A dedicated computational pipeline analyzes RNA-seq reads and prior genome annotation to identify small RNAs, untranslated regions of mRNAs, and cis-encoded antisense transcripts. Unlike other archaea, such as Sulfolobus and Halobacteriales, P. abyssi produces few leaderless mRNA transcripts. Antisense transcription is widespread (215 transcripts) and targets protein-coding genes that are less conserved than average genes. We identify at least three novel H/ACA-like guide RNAs among the newly characterized non-coding RNAs. Long 5' UTRs in mRNAs of ribosomal proteins and amino-acid biosynthesis genes strongly suggest the presence of cis-regulatory leaders in these mRNAs. We selected a high-interest subset of non-coding RNAs based on their strong promoters, high GC-content, phylogenetic conservation, or abundance. Some of the novel small RNAs and long 5' UTRs display high GC contents, suggesting unknown structural RNA functions. However, we were surprised to observe that most of the high-interest RNAs are AU-rich, which suggests an absence of stable secondary structure in the high-temperature environment of P. abyssi. Yet, these transcripts display other hallmarks of functionality, such as high expression or high conservation, which leads us to consider possible RNA functions that do not require extensive secondary structure.

Transcriptomic profiling of the oyster pathogen Vibrio splendidus opens a window on the evolutionary dynamics of the small RNA repertoire in the Vibrio genus.

Work in recent years has led to the recognition of the importance of small regulatory RNAs (sRNAs) in bacterial regulation networks. New high-throughput sequencing technologies are paving the way to the exploration of an expanding sRNA world in nonmodel bacteria. In the Vibrio genus, compared to the enterobacteriaceae, still a limited number of sRNAs have been characterized, mostly in Vibrio cholerae, where they have been shown to be important for virulence, as well as in Vibrio harveyi. In addition, genome-wide approaches in V. cholerae have led to the discovery of hundreds of potential new sRNAs. Vibrio splendidus is an oyster pathogen that has been recently associated with massive mortality episodes in the French oyster growing industry. Here, we report the first RNA-seq study in a Vibrio outside of the V. cholerae species. We have uncovered hundreds of candidate regulatory RNAs, be it cis-regulatory elements, antisense RNAs, and trans-encoded sRNAs. Conservation studies showed the majority of them to be specific to V. splendidus. However, several novel sRNAs, previously unidentified, are also present in V. cholerae. Finally, we identified 28 trans sRNAs that are conserved in all the Vibrio genus species for which a complete genome sequence is available, possibly forming a Vibrio "sRNA core."

Different expression and activity of the alpha1 and alpha4 isoforms of the Na,K-ATPase during rat male germ cell ontogeny.

Two catalytic isoforms of the Na,K-ATPase, alpha1 and alpha4, are present in testis. While alpha1 is ubiquitously expressed in tissues, alpha4 predominates in male germ cells. Each isoform has distinct enzymatic properties and appears to play specific roles. To gain insight into the relevance of the Na,K-ATPase alpha isoforms in male germ cell biology, we have studied the expression and activity of alpha1 and alpha4 during spermatogenesis and epididymal maturation. This was explored in rat testes at different ages, in isolated spermatogenic cells and in spermatozoa from the caput and caudal regions of the epididymis. Our results show that alpha1 and alpha4 undergo differential regulation during development. Whereas alpha1 exhibits only modest changes, alpha4 increases with gamete differentiation. The most drastic changes for alpha4 take place in spermatocytes at the mRNA level, and with the transition of round spermatids into spermatozoa for expression and activity of the protein. No further changes are detected during transit of spermatozoa through the epididymis. In addition, the cellular distribution of alpha4 is modified with development, being diffusely expressed at the plasma membrane and intracellular compartments of immature cells, finally to localize to the midregion of the spermatozoon flagellum. In contrast, the alpha1 isoform is evenly present along the plasma membrane of the developing and mature gametes. In conclusion, the Na,K-ATPase alpha1 and alpha4 isoforms are functional in diploid, meiotic and haploid male germ cells, alpha4 being significantly upregulated during spermatogenesis. These results support the importance of alpha4 in male gamete differentiation and function.