Monday, April 18, 2016

Semen microbiome


The classical microbiology had established that there are several sites in the body that are not physiologically accessible to the microbial community. Much of them are shown to harbour some microbial content and the list of sterile sites gets smaller. With access to better and deeper sequencing technologies and increasing computational ability we are finding microbiome in everything. For example placenta, amniotic fluid etc was considered as the "No No" for microbial community in physiological context, but a lot of current literature is suggestive of "It mayn't be the case".

One of the places in human body that was unexpectedly shown to harbour microbiome was seminal fluid. I like the phrase by Roger Meissen, "It’s a strange place to call home, but seminal fluid offers the perfect environment for particular types of bacteria". As the story goes, Seminal microbiome is a highly under studied system in terms of microbiome. In the last coupe of years, research has shed light on the subject and it has been speculated that the microbiome composition has a correlation with reproductive health. There are a variety of organisms proposed to be part of seminal microbiome (Lactobacillus, Veillonella, Streptococcus, Porphyromonas and Atopobium), which draws nutrition from seminal fluid rich in Fructose.

Fig 1: Overlap between seminal fluid and fecal samples.
In the current study, the authors examined the microbiome of seminal fluid in mice model. The most common question in human studies is the source of microbiome. Since the seminal fluid passes down the urinary tract, the organisms in the tract are also bound to show up in the study. In the current study the researchers withdrew seminal fluid directly from seminal vesicles and sequenced the 16S rRNA. For a comparison they also sequenced the fecal microbiome. researchers identified several groups of bacteria such as Proteobacteria, Actinobacteria, Fusobacteria, Flavobacteria, and Acidobacteria. More interesting finding was that the seminal microbiome was consistent throughout the mice age while the fecal microbiome kept changing. I haven't fully understood the logic of comparing with fecal microbiome. I had rather do it with urinary microbiome, since this is anatomically closer.

This study is a proof of concept that seminal fluid harbours a unique microbiome. The study also identified that the seminal fluid microbiome is influential of metabolism. For example, P. acnes, Streptophyta spp, Corynebacterium spp, Pseudomonas veronii, and Acinetobacter spp are positively associated with significant amounts of metabolic pathway changes in the seminal fluid. Bioinformatic analysis of the sequence revealed a huge lot of interesting possibilities.

Rosenfeld, one of the investigators involved in the study comments, "The data showed that the bacterial composition found in the male reproductive tract contained potentially detrimental bacteria that can be transmitted to female reproductive partners and offspring. The bacteria also could be the causative agent of chronic prostatitis, a possible precursor to prostate cancer in males. Additionally, further testing showed this bacterial community contains the bacteria that may cause obesity in rats. Understanding how these genetic and environmental factors influence this particular microbiome could help in understanding how possible developmental disorders and diseases are passed down by fathers to their offspring."
Liu CM, Osborne BJ, Hungate BA, Shahabi K, Huibner S, Lester R, Dwan MG, Kovacs C, Contente-Cuomo TL, Benko E, Aziz M, Price LB, & Kaul R (2014). The semen microbiome and its relationship with local immunology and viral load in HIV infection. PLoS pathogens, 10 (7) PMID: 25058515

Javurek AB, Spollen WG, Ali AM, Johnson SA, Lubahn DB, Bivens NJ, Bromert KH, Ellersieck MR, Givan SA, & Rosenfeld CS (2016). Discovery of a Novel Seminal Fluid Microbiome and Influence of Estrogen Receptor Alpha Genetic Status. Scientific reports, 6 PMID: 26971397

Saturday, April 09, 2016

CRISPR for HIV- We are not yet there


HIV is not something that needs an introduction. And I have written about it in this blog in great details ranging from basics to vaccine research. I have also written posts on CRISPR Cas technology and how it is useful for gene editing. In fact CRISPR based system has made gene editing a much simpler work. I had also covered the story of editing human embryo using CRISPR that has lead to a lot debate. The news now is a group led by Yong Fan from Guangzhou, China has published a paper describing attempt to attack HIV genome in embryo cells and another group led by Wang etal from McGill University AIDS Centre Canada showing that HIV can resist CRISPR attack.

Fig 1: CCR5Δ32 renders cell
resistant to HIV. Source
Using CRISPR to snip out the HIV gene from the cell is not a new idea. In fact over the past 3 years or so, there are a lot of studies. The basic issue with Anti Retroviral treatments is the rebound problem. HIV genes tend to persist in Quiescent immune cells and there was no way to remove the HIV genome out. One of the first, was to use Zinc finger nuclease. The idea was to knock-out the co receptor for HIV such as CCR5 and CXCR4 which effectively renders the cell impermeable to HIV. This method has not been completely effective as theoretically predicted due to a variety of reasons. Interestingly, a mutation in CCR5 called as CCR5Δ32 renders the cells resistant to HIV. In a paper published in 2014, showed that using CRISPR system based on a guided RNA, attacking HIV is practically possible at least in lab conditions.

Let's put it this way. The best way to completely cure a person from HIV is to render the HIV genome non functional. So you could either edit out the HIV genome directly or create a non functional receptor thus effectively rendering new cells resistant to infection. With this in mind, lets look into two recently published papers.

In the first study by Yong Fan and group 213 fertilized human eggs where collected from 87 patients, which were not fit for implantation due to chromosomal abnormalities. The team shot a CRISPR system to attack CCR5 gene to introduce Δ32 mutation. Subsequent analysis showed that 4 of 26 embryo's were successfully modified. Not all the cells had been modified and some had acquired other mutations. This is not surprising and consistent with previous only paper on genetic editing in similar 3PN system, where they also found many off target changes. This paper doesn't offer much other than the spark of ethical debate of using embryonic cell for genetic research and just re-enforces what we already have known- It doesn't work much. "It just emphasizes that there are still a lot of technical difficulties to doing precision editing in human embryo cells,” says Xiao-Jiang Li.

Fig 2: Attacking HIV genome.
The second paper by Wang etal is interesting. They used a CRISPR Cas system, that can attack the HIV target gene. The team used single guide RNAs (sgRNAs) and the Cas9 enzyme to target and HIV-1 DNA from the genome of human T cells in vitro. The cut DNA naturally undergoes a process called as NHEJ (non-homologous end joining) which leads to insertions and deletions (commonly called together as indels) that often impair DNA function. In other words there is deliberate mutation introduction. By two weeks hey however saw that the T cells were producing copies of HIV particles that had escaped the CRISPR attack. Sequencing revealed that the CRISPR had induced minor changes and indels which didn't effect viral replication but was sufficient enough to be resistant to CRISPR attack. As predicted mutations where heavily clustered in the CRISPR target region. The very same mutating mode of CRISPR activity that beats HIV down has also induced resistance. This study highlights a potential problem of interest.

The moral of the story is we are not yet ready for gene therapy against HIV. We still need major developments to reduce the off target activity, increase efficiency.
Hu W, Kaminski R, Yang F, Zhang Y, Cosentino L, Li F, Luo B, Alvarez-Carbonell D, Garcia-Mesa Y, Karn J, Mo X, & Khalili K (2014). RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection. PNAS, 111 (31), 11461-6 PMID: 25049410

Kang X, He W, Huang Y, Yu Q, Chen Y, Gao X, Sun X, & Fan Y (2016). Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing. Journal of assisted reproduction and genetics PMID: 27052831

Zhen Wang, Qinghua Pan, Patrick Gendron, Weijun Zhu, Fei Guo, Shan Cen, Mark A. Wainberg, Chen Liang (2016). CRISPR/Cas9-Derived Mutations Both Inhibit HIV-1 Replication and Accelerate Viral Escape Cell Reports : doi:10.1016/j.celrep.2016.03.042