From the Spring 2021 issue of "Microcosm."

COVID-19 Variants

The evolution of SARS-CoV-2 has posed the threat of new variants that may exhibit increased transmissibility or potential for antibody escape. This threat has amplified the importance of next-generation sequencing methods that can decipher and track how and when the virus is mutating. A study published in Nature demonstrated that chronic SARS-CoV-2 infection leads to the evolution of viruses with reduced sensitivity to neutralizing antibodies that are passively acquired through convalescent plasma treatment. Over the course of 101 days, researchers generated whole genome sequences from 23 time points, demonstrating the emergence of a dominant viral strain that contained a D796H substitution in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. Together, these mutations conferred a 2-fold decrease in sensitivity to convalescent plasma in an immunocompromised host. However, as serum antibodies from the treatment diminished, so did the concentration of viruses with these escape mutations, suggesting that selective pressure in the host was driving viral adaptation.

Growing evidence indicates that many SARS-CoV-2 variants share similar combinations of mutations and might not be as highly variable as we once feared. In an article published in Scientific American, Dr. Vaughn Cooper, evolutionary biologist and ASM COMS-elected Board Director, describes how increased genome surveillance of the coronavirus has allowed several recent studies to identify signatures of convergent evolution, indicating that certain mutations that repeatedly arise in independent lineages with increased frequency over time may be linked to increased viral fitness.

Dr. Cooper’s lab has identified at least 7 independent lineages that acquired a mutation at position 677 in the virus’ spike gene, and the E484K mutation that has been correlated with antibody escape appears in multiple SARS-CoV-2 variants, including B.1.352, P.1 and B.1.526. Other mutations, including the ΔH69/ΔV70 deletion, as well as substitutions in the receptor-binding domain (at positions 417, 452 and 501) and near the furin cleavage site (at 681 and 701), have also been identified in multiple SARS-CoV-2 lineages.

Although new major adaptations to the SARS-CoV-2 genome are relatively few and far between, Dr. Cooper warns that increased selective pressure, along with increased numbers of SARS-CoV-2 infections, facilitate continued virus evolution. In fact, a study in Nature Medicine describes the identification of 16 new lineages of SARS-CoV-2 isolated in South Africa between March 6 and August 26, 2020, many of which possess unique mutations that have not been identified elsewhere. Getting infection numbers under control and continuing to track SARS-CoV-2 variants are therefore key to managing the spread of the disease.

Role of Virus and Host in Human Disease

Ever since it became feasible, genomic sequencing has been used to understand host susceptibility to a variety of infectious diseases. Likewise, it was expected that genetic risk factors play a role in the progression of COVID-19. A genetic association study aiming to better explain the wide variation in clinical manifestations, ranging from asymptomatic to mild to severe disease and death, was published in Nature in September 2020. In this study, scientists identified a haplotype, located on chromosome 3, that is associated with increased respiratory failure and a 60% higher risk of being hospitalized with COVID-19. Phylogenetic analysis revealed that the gene cluster is inherited from Neanderthals and found in approximately 50% of people in south Asia and 16% of people in Europe.

The same team of scientists recently published a study in PNAS that identified a different Neanderthal haplotype, located on chromosome 12, with the opposite effect. According to the report, each copy of this OAS1 variant reduces the risk of developing severe COVID-19 by approximately 22%. This region encodes oligoadenylate synthetases, enzymes that are involved in host immune responses that degrade RNA viruses and activate additional antiviral mechanisms. Genes on the OAS locus have proved to be protective against at least 3 additional viruses, including West Nile virus, hepatitis C and SARS-CoV. There is still much to be learned about how genetic factors impact disease severity and host immune responses to COVID-19, but these studies have begun to unravel the mystery.

Microbiome Association

The gut microbiome is an important regulator of adaptive immunity. It has been shown to influence tumor development and modulate host responses to chemotherapy and immunotherapy. An article published in Nature Microbiology describes the identification of specific microbial signatures in patients with non-small-cell lung cancer (NSCLC ) and how the bacteria identified impact the therapeutic efficacy of cancer drugs.

Linear discriminant analysis Effect Size (LEfSe analysis) of 16S ribosomal (rRNA) sequencing data from 96 NSCLC patient stool samples and 139 healthy controls was used to determine how cancer therapeutics affect microbiome composition. Bifidobacterium bifidum was significantly enriched in those who responded to treatment, and qPCR confirmed these results. Interestingly, previous studies have demonstrated that Bifidobacterium spp. enhances therapeutic efficacy of PD-1 blockades through dendritic cell maturation.

Next, in order to evaluate the therapeutic potential of the bacterium, syngeneic mouse tumors were treated with commercial strains of B. bifidum. Scientists found that only specific B. bifidum strains worked synergistically with PD-1 blockade or oxaliplatin treatment to reduce tumor burdens in mice. It was therefore proposed that B. bifidum modulates antitumor immune responses through the biosynthesis of immune-stimulating molecules and metabolites that potentiate interferon-γ production.

One Health

In what some are calling an impressive case of scientific detective work, researchers finally have identified the cause of vacuolar myelinopathy (VM), the neurological disease linked to mass eagle deaths across the southeastern U.S. for nearly 2 decades. VM is known to cause lesions on the brains of waterfowl and birds of prey, and has recently been found to also affect fish, worms, amphibians and reptiles.

In 2014/2015, the occurrence of VM was tied to the presence of a cyanobacterium, Aetokthonos hydrillicola, which was discovered growing on an invasive plant called Hydrilla verticillate in manmade bodies of water. Since the discovery of A. hydrillicola, researchers have suspected a cyanobacteria-produced neurotoxin to be at the root of VM. They were able to confirm the toxicity of crude extracts of A. hydrillicola taken from VM sites; however, laboratory cultures were not found to be neurotoxic.

A study published in Science describes how mass spectrometry was used to expose the colocalization of VM-positive cyanobacteria samples with a brominated metabolite, subsequently named aetokthonotoxin (AETX). When researchers supplemented laboratory cultures with potassium bromide, A. hydrillicola readily produced AETX in the lab. Leghorn chickens that were gavaged with AETX developed VM brain lesions, while control chickens did not, confirming that AETX is the causative agent of VM. Furthermore, the metabolite is fat-soluble, suggesting that it has the potential to accumulate in tissues and pose a significant threat to the food web.

"One Health" emphasizes the relationship between environmental, animal and human health. This example demonstrates the role of environmental disruption in the transmission of neurological toxins and transfer through the food web. Understanding the ecological pressures and dynamics that drive this transfer can inform targeted mitigation strategies to protect both wildlife and human health.

Because AETX biosynthesis is dependent on the availability of bromide, an important step to controlling the spread of VM is identifying unnatural bromide sources and eliminating them from the environment. For example, the herbicide diquat bromide has been shown to accumulate in the leaves of the hydrilla plant upon which the cyanobacteriam feed. Rather than using herbicides to control this invasive species, stocking lakes with sterile fish that eat hydrilla could offer an alternative, chemical-free approach for containing this aggressive plant. Researchers suggest that coal-fired power plants, road salt, fracking fluids and brominated flame-retardants may also be sources of bromide in manmade bodies of water.

Molecular Microbiology

A fundamental question in molecular biology has to do with the coordination of bacterial replication: How do bacteria ensure that DNA replication and cell division proceed efficiently during metabolic conditions that are required for growth? A Journal of Bacteriology study characterized an essential role of the TusA protein that links translation efficiency to cell division in E. coli. TusA is a highly conserved and versatile protein. After transcription, TusA is responsible for inserting sulfur modifications into nucleosides of tRNAs that encode Lys, Gln and Glu at position 34. Posttranscriptional sulfur modifications, like the formation of 2-thiouridine, help ensure that translation proceeds accurately and efficiently. TusA-deficient mutants have been shown to exhibit poor viability, a defect in cell division and a filamentous morphology.

In this study, researchers discovered that lack of thiolation of wobble uridine (U34) nucleotides on Lys, Gln or Glu tRNAs deregulates the production of RpoS (Sigma S) and Fis (Factor for Inversion Stimulation) proteins. Together, these proteins are responsible for promoting, enhancing and regulating transcription. The results indicate that limiting production of RpoS and Fis results in delayed filament formation by altering FtsZ regulation. Thus, the absence of TusA changes translation efficiency, disrupts the cellular regulatory network and ultimately causes major defects in cell division.

A study in mBio discovered that another E. coli protein, HolC, is involved in coordinating bacterial replication and transcription by overcoming conflicts between the replication fork and transcription elongation complexes. DNA polymerase III holoenzyme is the primary enzyme complex that catalyzes DNA replication in bacteria. It consists of multiple subunits, including a clamp-loader complex, which loads and unloads the processivity clamp, a ring-like structure that encircles the DNA and associates DNA polymerase with its template. The clamp-loader complex has 2 accessory proteins, HolC and HolD. Together, these proteins help assemble and stabilize the clamp-loader complex, but HolC is the only protein of the complex that binds with single-stranded binding proteins, an interaction that directs DNA polymerase to RNA primers and stabilizes the interaction.

Although HolC is not essential for viability, deletion mutants exhibit poor growth and acquire suppressor mutations. One of those suppressors reduces the stability of RNAP. Another duplicates the ssb gene. On the other hand, transcription factors, DksA and Rho termination factor NusA remain viable, even when HolC is absent, but loss of DksA and NusA leads to synthetic growth defects with HolC.

Transcription elongation complexes can impede the progress of the replication fork. In the absence of HolC, it appears that DNA replication is incomplete and Rho-dependent termination is critical to maintaining chromosome integrity. This suggests a new role for HolC in preventing collisions between transcription elongation complexes and the replication fork. However, the mechanism by which it accomplishes this task has yet to be fully defined.

Vaccine and Drug Development

Tuberculosis (TB) is responsible for millions of deaths annually, presents a serious antimicrobial resistance (AMR) threat, and remains one of the most frequent causes of disease worldwide. Comparative genomics and protein analysis have become powerful tools for uncovering potential targets for effective vaccine and anti-mycobacterial drugs, both of which are desperately needed to manage this disease more effectively.

A study published in mSphere used publicly available sequence data across seven Mycobacterium tuberculosis (MTB) lineages to map 8,535 genome sequences against the H37Rv reference genome. From this data, single nucleotide polymorphisms (SNPs) and distribution frequency of nucleotide variants were identified, facilitating one of the largest-scale, most comprehensive analyses of MTB sequence variation to date. The researchers found that highly conserved genes were often associated with stress responses and the maintenance of redox balance, while highly variable genes were often associated with AMR. They also identified a number of highly conserved genes that could potentially be used as targets for novel vaccine candidates and antituberculous medications.

Efficacy of the Mycobacterium bovis BCG vaccine is geographically variable and is especially low in high-risk individuals. In an effort to identify new antigen targets for improvement or replacement of the BCG vaccine, a study published in Infection and Immunity describes a high-throughput proteome-wide protein-purification study that screened 1,781 proteins for antigenic activity. The study identified 49 antigens that induce antigen-specific gamma interferon (IFN-γ) release from the blood cells of TB patients and healthy donors. Of the 3 antigens that caused significant reduction in colony-forming unit (CFU) counts in the lungs of mice, 2 also induced protective T-cell immune response. Additional preclinical assessment of these 2 antigens (Rv1485 and Rv1705c) is needed to determine whether they have therapeutic potential.

Bacterial Evolution: Niche Restriction and the Path to Speciation

Bacteria from the same species can exhibit wide genetic variations. In fact, only about half of the genes in any given strain of E. coli are shared by all other strains. As a species, E. coli has adapted the ability to survive in multiple niches, including human and animal hosts, as well as environmental habitats, and as it turns out, not all core genes are always necessary for reproduction and survival. A study in Nature Microbiology sought to determine how variations in environmental conditions and genetic backgrounds impact gene essentiality in E. coli. This study offers direct comparisons of the essential character of core E. coli genes from 18 strains that are representative of the genetic diversity of the species.

Using a CRISPR interference platform (CRISPRi) to bind and silence expression of target genes, a single-guide RNA (sgRNA) library was generated that allowed the analysis of gene essentiality in different genetic backgrounds. The results indicate that certain genes can become more or less essential in the presence or absence of environmental stressors, including toxins and prophages. In addition to providing evolutionary insights, identifying core essential genes could aid in the design of new antimicrobials and genome-reduction efforts.

Microbial Survival

Can microbes survive in outer space? Thanks to a lot of patience, some ingenuity and the incredible utility of whole genome sequencing, researchers may be one step closer to finding out. According to a Frontiers in Microbiology article, 4 strains of bacteria in the Methylobacteriaceae family have been isolated from the International Space Station (ISS). Samples were collected during 2 consecutive flights (March 2015 and May 2015) from different locations on the ISS, including a research station, the cupola and the surface of a dining table. Whole genome sequencing, followed by phylogenetic analysis, revealed that 3 of the recovered strains, designated IF7SW-B2T, IIF1SW-B5 and IIF4SW-B5T, belonged to the same previously unidentified species of bacteria and were most closely related to Methylobacterium indicum. Researchers proposed the name Methylobacterium ajmalii sp. nov for the novel species. The fourth recovered strain was identified as Methylorubrum rhodesianum and was discovered in an old HEPA air filter that returned to Earth in May 2011.

All 4 microbes belong to a family of bacteria that are commonly found in air, soil, freshwater and sediments. Members of the Methylobacteriaceae family are involved in nitrogen fixation and abiotic stress tolerance, as well as plant growth and protection from pathogens. Astronauts have been growing vegetables, such as red romaine lettuce, on the ISS for years. Now researchers are searching for genes in the newly identified species that may promote plant growth in low-gravity conditions. For example, IF7SW-B2T exhibited a higher number of stress-tolerance genes and contained a gene that is essential for cytokinin production, as well as multiple genes that are involved in regulating the cobalamin synthesis pathway. Characterization of this new species of bacteria is just beginning, and there is much to learn about the incredible diversity and adaptability of microbes by studying microbial life on spacecraft.

Another example of microbial survival involves the long-term asymptomatic survival of a virus in humans. Genomic analyses conducted by 3 independent research groups have revealed that the Ebola virus responsible for the current outbreak in Guinea is nearly identical to the strain that caused the 2013-2016 epidemic. Now scientists are questioning whether the virus has remained dormant in a survivor for more than 5 years. A Science article recently addressed the plausibility and implications of such lengthy viral persistence. The genetic similarities between the 2 viruses make it highly unlikely that the source of the current outbreak was animal-to-human transmission. However, it is possible that the virus has remained in circulation through undocumented human-to-human transmission events over the years. In 2016, an Ebola resurgence, which also took place in Guinea, originated from a survivor who sexually transmitted the virus approximately 500 days after infection, but the evidence that Ebola may remain latent for at least half a decade is surprising. In addition to the public health implications, the potential for long-term persistence of this RNA virus may impact survivors who already face significant stigmatization because of the disease. Gaining a better understanding of how the virus behaves will help inform communities and guide public health practices, including vaccine distribution and the development of treatment options.