Uncovering new function for small RNAs in Salmonella infections

Millions of people get sick from Salmonella each year because they are food-borne pathogens. These bacteria rely on a sophisticated network of genes and gene products that give them the ability to sense environmental conditions to do this. In a recent study, researchers looked into how small RNAs aid Salmonella in expressing its virulence genes.

The bacteria first invade intestinal cells using a needle-like structure known as a type 3 secretion system before infecting humans. This structure directly injects proteins into the cells, triggering a series of events that lead to inflammation and, ultimately, diarrhea.

The Salmonella pathogenicity island 1 is a region of DNA that contains the genes for this system and additional genes required for invasion.

SPI-1 needs to be well controlled. If the type 3 secretion system needle apparatus is not made, Salmonella cannot cause an infection, and if too much of the needle apparatus is made, it makes Salmonella sick.”

Sabrina Abdulla, Study First Author and Graduate Student, Carl R. Woese Institute for Genomic Biology

An extensive regulatory network manages SPI-1. First, the expression of DNA is regulated by three transcription factors: HilD, HilC, and RtsA. Additionally, they activate HilA, another transcription factor, which in turn activates the remaining SPI-1 genes.

Not only that, but in order for SPI-1 to successfully infect its host, it also needs to sense a variety of environmental cues and adjust the expression of its genes.

Abdulla added, “We have known for a long time that there are a lot of environmental factors that feed into the gene regulation in Salmonella. However, we didn’t know how. That is when researchers started looking at small RNAs.”

In bacterial cells, the way genes function is largely governed by small RNAs. The mRNA, which contains the instructions for making proteins, or proteins, are typically the objects of these molecules’ interactions. Consequently, sRNAs have an impact on a number of bacterial processes, such as virulence and reactions to the environment.

In this study, the researchers examined the sRNAs that control the hilD mRNA in particular, the region of the mRNA known as the 3′ untranslated region, which is not used to produce the HilD protein. The 3′ UTRs in bacteria are typically 50–100 nucleotides long. The hilD mRNA’s 3′ UTR, however, was 300 nucleotides long.

“The starting point for my work was the observation that when we deleted the 3’ UTR, the expression of the hilD gene went up 60-fold. We then decided to look for sRNAs that might be interacting with this region,” Abdulla stated.

The scientists found that while the sRNAs Spot 42 and SdsR can both target the 3′ UTR, they do so in various parts of the splice donor.

Abdulla further stated, “This result suggests that the entire 3′ UTR is important for regulation. We showed that the sRNAs stabilize the hilD mRNA and protect it from being degraded. Such long 3′ UTRs have not been well studied. With more genomic research, people are realizing more and more that these longer regions exist and that they are important for regulation.”

The researchers also examined whether Spot 42 and SdsR could influence how Salmonella causes infections using mice. To determine which mutant bacteria survive and spread infection, they used mouse competition assays in which they introduced bacteria with and without sRNAs.

Abdulla pointed out, “We found that when the sRNAs are deleted, the bacteria cannot survive in the host. We also showed that the sRNAs play a role in helping SPI-1 invade the host cells.”

Cari Vanderpool (MME/IGOH), a professor of microbiology, added, “Now that we know that sRNAs play an important role in controlling SPI-1 through their regulatory effects on the hilD 3′ UTR, we want to extend our studies in two directions.”

She further stated, “We would like to understand more about how, at a molecular level, the sRNAs influence hilD mRNA levels. We would also like to better understand how sRNAs participate in regulating expression of other important SPI-1 genes.”