Article Review of “Bistable Expression of Virulence Genes in Salmonella Leads to the Formation of an Antibiotic-Tolerant Subpopulation”

Abstract

The August 2014 research article, “Bistable Expression of Virulence Genes in Salmonella Leads to the Formation of an Antibiotic-Tolerant Subpopulation,” (“Bistable expression…”) answers some interesting questions about gene expression variables among a species. However, it also brings to light the ever-evolving definition of the term species, and how a species might diverge over time. The article details several experiments and examines how differential expression in members of a genetically identical population results in different functions or consequences. In this case, the translation dictates the bacteria’s reproductive fitness growth rate and ability to withstand exposure to antibiotics (1). In this paper, I examine how these findings affect the progression of the species concept and how they might affect Salmonella typhimurium’s (S. typhimurium) place on the tree or web of life.

Diversity

Over the past decade, scientists have started to question if a bacterium’s heritage can truly be determined, and if so – how to do it. An organism’s ability to incorporate genes from other bacteria – even from those of a completely different species – and their ability to pick up helpful genetic information from the surrounding environment shows how complex the genome of any prokaryotic organism can become. Add to those complications the incredibly small percentage of bacteria that can be isolated and grown in a laboratory culture, and it becomes clear just how big of a challenge it is to acquire, organize and understand prokaryotic genetic data (3).

In addition, prokaryotes can excrete bacteriocins which inhibit the ability of competing bacteria to grow and prosper, thereby shielding a clone from habitat and resource loss to other similar species. These chemicals prevent progression of one species, while they allow the bacteriocin producer unrestricted access to nutrients (7). Other indirect variables may also affect bacterial diversity, including the presence of unrelated viral and parasite diseases like HIV and malaria. The addition of these contagions to the puzzle means researchers must consider even more variables, such as the medications patients with viral agents take that can alter the environment and affect bacterial diversity. In addition, these already immunocompromised people are more likely to succumb to a S. typhimurium infection (6).

Bacteria grow rapidly compared to eukaryotic organisms. This means that, from a genomic standpoint, researchers must consider mutations, horizontal gene transfer, bacteriocins not normally considered in an evolutionary context, selective pressures, and other possible methods of evolution, in a fast-paced, unpredictable model. (7). Metagenomics can offer insight into these intricate microbiological relationships used in communities of mixed populations, not with clonal populations. It helps compensate for the inability to culture all organisms of an environment in a laboratory setting. The process uses the genetic information in that environment to assess its inhabitants (5).

Research on bacterial diversity must begin with a decision on what data to compare. In “Intracontinental spread of human invasive Salmonella typhimurium pathovariants in sub-Saharan Africa,” they chose to sequence entire genomes and compare single nucleotide polymorphism (SNP), or differences in DNA nucleotides between populations. The authors of “Bistable Expression…” compared type three secretion system expression genes and used time lapse photography to monitor the activity of single experimental cells in relation to administered antibiotics and the changing environment.

Salmonella spp.

S. typhimurium, an invasive but self-limiting serovar of Salmonella enterica, is distributed worldwide and causes disease in both humans and other animals. In the United States, it caused a 2013 outbreak that involved 356 people in 39 states who purchased mail-order poultry to maintain backyard flocks. Sufferers presented with up to a week of fever and gastrointestinal discomfort including diarrhea and cramping (4).

One of the main virulence factors that makes S. typhimurium such a pathogenic concern is its use of the type three secretion system. The bacteria genetically codes for this two-component type three secretion system that helps it invadethe host cell through the membrane. Both parts of this virulence factor combination must be present for S. typhimurium to act as a pathogen. Through this important process, this facultative, intracellular organism inserts effector proteins into the target host cell – much like a syringe injects medication into a patient. Once inside, these effector proteins prepare the cell for pathogenic invasion and give S. typhimurium the edge it needs to take over the host cell for its own reproduction (2).

The Study

Sub-populations of bacteria with the same genome can differentially express genes leading to completely different traits. In this study, researchers evaluated the benefits of identical organisms that express different genes, leading to a more than one phenotype. They hypothesized two main reasons a clone might express genetic information differently. The first, population bet-hedging, allows two phenotypes to be expressed in different bacteria that share the same genetic information and exist in the same colony. This allows one of the two population subsets to survive environmental conditions that may decimate the other subset. They called the process bet-hedging because it allows the bacteria an extra opportunity to survive an adverse situation. The second reason, division of labor, allows two population subsets with the same genome to express different traits that both benefit the entire clone (1).

To test the efficacy of each phenotypic trait, they grew T1^– cells, best suited to a normal environment, and T1⁺ cells, capable of recovery after antibiotic exposure, in the same basic growth medium, lysogeny broth. The T1⁺ bacteria grew much slower under normal conditions while the T1^– cells grew rapidly. The researchers hypothesized that these obvious growth rate differences occurred when T1⁺ cells spent valuable resources creating the T1 virulence factor protein, while T1^– cells spent the same resources on population maintenance and subsequent growth.

Once the colonies were established and the research team knew which subset of the colony expressed which trait, they subjected the clones to a couple of different levels of two antibiotics – one a kanamycin and the other a ciprofloxacin. T1^– cells were unable to thrive when in contact with antibiotics, while T1⁺ cells had little to no issue with the environmental alteration.

Therefore, the expression of a gene helps the bacteria in one way, while costing it in another. Because it cannot create T1 without slowing growth of the clone, it has evolved to allow only part of the colony to produce this virulence factor. This explains why antibiotic resistance develops. The T1⁺ cells, or bacteria with similar virulence bet-hedging mechanisms, may be the ones remaining when a patient only completes a portion of their antibiotic regimen, allowing them to reproduce, yielding additional bacteria that contain the T1⁺ virulence factor that confers resistance to antibiotics (1).

In conclusion, the researchers determined that antibiotic resistance may not actually be the issue. Based on their data and results, it appears that diverse genetic expression of the exact same genome may be the culprit, leading to more and more bacteria not susceptible or less susceptible to mankind’s arsenal of antibiotics.

 

References

1. Arnoldini, M., Vizcarra, I. A., Peña-Miller, R., Stocker, N., Diard, M., Vogel, V., & … Ackermann, M. (2014). Bistable Expression of Virulence Genes in Salmonella Leads to the Formation of an Antibiotic-Tolerant Subpopulation. Plos Biology, 12(8), 1-8. doi:10.1371/journal.pbio.1001928
2. Baison-Olmo, F., Cardenal-Munoz, E., & Ramos-Morales, F. (n.d). PipB2 is a substrate of the Salmonella pathogenicity island 1-encoded type III secretion system. Biochemical And Biophysical Research Communications, 423(2), 240-246.
3. Bruijn, F. d. (2011). Handbook of molecular microbial ecology I [electronic resource] : metagenomics and complementary approaches / Frans J. de Bruijn. Hoboken, N.J. : Wiley-Blackwell, 2011.
4. Centers for Disease Control. (2013) <www.cdc.gov/salmonella/typhimurium-live-poultry-04-13/index.html>, link changed to <Outbreak of Salmonella Infections Linked to Backyard Poultry | CDC> Retrieved 31 Oct. 2014.
5. Nature Reviews (2014) <nrmicro/focus/metagenomics> Content has been removed. Retrieved 4 Nov. 2014.
6. Okoro, C. K., Kingsley, R. A., Connor, T. R., Harris, S. R., Parry, C. M., Al-Mashhadani, M. N., & … Dougan, G. (2012). Intracontinental spread of human invasive Salmonella Typhimurium pathovariants in sub-Saharan Africa. Nature Genetics, 44(11), 1215-1221. doi:10.1038/ng.2423
7. Pašić, L., Ambrožič Avguštin, J., Starčič Erjavec, M., Herzog-Velikonja, B., Podlesek, Z., & Žgur-Bertok, D. (2014). Two Tales of Prokaryotic Genomic Diversity: Escherichia coli and Halophiles. Food Technology & Biotechnology, 52(2), 158-169.