Weimer Micro Lab

Contributed by Carol Huang

We have recently introduced a newer version of the Nanopore sequencing device, MinION MK 1B into the laboratory. It’s a portable, real-time sequencing device.

The Nanopore MinION sequencing platform has great advanced features. Upon reach objectives, it can generate 10 to 20 GB DNA sequencing data from each flow cell. With different fragmentation options ultra-long reads, hundreds of kb in length, were made possible. Since I have been engaged in NGS library construction for many years, the more impressive feature of this is the time saved on sequencing library preparation in comparison with other platforms. Sequencing libraries can be made in less than an hour using the library kit we picked, while to make long-read library could take 2 days by using other sequencing platforms. We were able to pick the sequencing data set from the first five minutes of sequencing run and identified the serotype of the microbe tested. These features will be very practical in the field of pathogen infection control.

One of the key factors to ensure high-quality data outcome is the quality of input materials, RNA, DNA, their integrity, and purity. Purity, 260/280 and 260/230 ratios, affects enzyme efficiency during library preparation, organic residuals would interfere with chemical reaction and might damage nanopore membranes. There are 512 nanopore channels available to capture library fragments. Small fragments always get captured, occupy nanopore channels first, which would prevent those bigger fragments from being sequenced and furthermore reduces sequencing efficiency. So for the best results, we need to have high purity input DNA /RNA and keep narrow final library size distribution, eliminate outsized final library fragments, small size in particular.

Contributed by Poyin Chen

The first step in bacterial interaction with host cells is host recognition, followed by host adhesion. All of these initial interactions take place at the host cell surface; however, we have only scratched the surface of what is known about bacterial-host membrane interactions. This host recognition is usually initiated by a bacterial protein transiently binding to a transmembrane host protein. The association begins with receptors on the host cell binding ligands on the microbial cell to form a partnership that initiates responses in both cells. Methods to determine the specific cognate partnerships are lacking. The transient nature of this initial interaction is a main contributor to our lack of understanding in the initial interactions between a pathogen and its host. One method to elucidate these interactions is by covalently linking the bacterial protein to the host protein during interaction so that we can purify these receptor-ligand complexes for further study without disrupting their interaction. This method was developed by the Weimer lab to discover cognate host-microbe receptor/ligand pairs using a covalent cross-linking strategy with whole cells. We have been successfully implemented this method to identify novel bacterial-host receptor/ligand partnerships, of which many partnerships have been validated by independent research groups. The identification of these protein pairings allows us to define biologically important events that are triggered during association of the microbiome. Furthermore, knowing what these pairings are is critical in defining the initiating signal from the host membrane that results in pathology or commensal association.

Stay tuned for the publication!

Contributed by Nguyet Kong

A study from Vanderbilt University found that mothers milk doesn’t just give babies nutrients, but the sugars help protects them from bacterial infections, making this a new class of antimicrobial. Mother’s milk is consisting of different protein, fats, and sugars. This study has discovered that some carbohydrates possess antibacterial properties that are non-toxic.

The results were presented at the American Chemical Society in Washington DC on August 20^th. The group started to look at different ways to defeat bacterial infections, and not look at the protein but look at the role and functions of the sugars, which is less known. The researchers collected human milk carbohydrates, also known as oligosaccharides, and profiled them using mass spectrometry. Then added the compounds to strep cultures to observe the outcome. They have found the sugars from the five samples produce a different outcome from killing the entire strep colony to breaking down the biofilm layer the bacteria produced to protect themselves. Also, the study shows that the milk sugar antimicrobial activity extends to other bacterial infections.

References

https://www.sciencedaily.com/releases/2017/08/170820075008.htm

Dorothy L. Ackerman, Ryan S. Doster, Jörn-Hendrik Weitkamp, David M. Aronoff, Jennifer A. Gaddy, Steven D. Townsend. Human Milk Oligosaccharides Exhibit Antimicrobial and Antibiofilm Properties against Group B Streptococcus. ACS Infectious Diseases, 2017; 3 (8): 595 DOI: 10.1021/acsinfecdis.7b00064

Contributed by Nguyet Kong

In a recent PLOS paper, researchers from Texas A&M found a bacterium, Streptococcus gallolyticus might lead to colon cancer by assisting with tumor growth that might cause more cancer-related deaths. Streptococcus has been known that there is an association with cancer, but little is known about Streptococcus role in that association. Dr. Yi Xu research group studied Streptococcus in cell culture, animal model and tissues from human tumors. The research shows that the methods complement each other. The cell culture model shows that in presence of bacteria, it promotes proliferation in the cancer cells, but the cell proliferation depends on the growth phage of Streptococcus when in contact with the cancer cells. Also, the cell culture work shows a production of Beta-catenin following the present of Streptococcus, so this might be showing that the bacteria pathway is causing cancer. In the animal models, Streptococcus develop more tumors have more beta-catenin and other sign of cancer. In the tissue study from human patients, they found most were infected with Streptococcus, which was unknown before. These results show that a tumor role of Streptococcus is involved specific bacteria and host factors and have important clinical implications.

http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1006440

https://www.sciencenews.org/article/these-bacteria-may-egg-colon-cancer?tgt=nr

Contributed by Azarene Foutouhi

Herpesviridae is a large family of Herpesviruses which are named for their tendency to result in recurrent infections. After clearance of lytic infections, these viruses enter latency, a mechanism which allows them to resist elimination and establish recurrent lifelong infections. More than 90% of people have become infected with one of these viruses, and latent forms result in most people.

While studying host response to latent Beta and Gamma-Herpesvirus infections, Barton et al. found that Herpesvirus latency confers protection from bacterial infection. In fact, mice latently infected with the Gammaherpesvirus HV68 had a nearly complete survival rate post L. monocytogenes challenge, while all control or mice with acute HV68 infections died by day 5.

MacDuff et al. found similar results while using HOIL-1 KO mice, and found that these genetically immunodeficient mice were rescued from Listeria lethality by latent HV68 infection. The HV68 latency protected the HOIL-1 KO mice from a dose of Listeria 1000-fold higher than the LD₅₀ for HV68 negative mice, suggesting the metagenome (including virome) can help link phenotype and genotype.

Barton et al. repeated their study on the effect of HV68 latency on bacterial infection by depleting the CD4+ and CD8+ cells of latently infected and Listeria inoculated mice prior to Listeria challenge. The Listeria immune mice showed levels of Listeria equal to that of control, as the lack of CD4+ and CD8+ cells rendered them unprepared to combat the infection. However, even when depleted of CD4+ and CD8+ cells the HV68 latently infected mice showed levels of listeria similar to that of the undepleted Listeria immune mice, suggesting HV68 latency confers protection in an unexpected way. These data suggest latent herpesvirus infections could be offering the host long-term protection (up to three months) from bacterial infection independently of the adaptive immune system.

1. Barton, Erik S., Douglas W. White, and Herbert W. Virgin. “Herpesvirus Latency and Symbiotic Protection from Bacterial Infection.” Viral Immunology22.1 (2009): 3–4. PMC. Web. 11 July 2017.
2. MacDuff, Donna A et al. “Phenotypic Complementation of Genetic Immunodeficiency by Chronic Herpesvirus Infection.” Ed. Stephen P Goff. eLife4 (2015): e04494. PMC. Web. 11 July 2017.