Micromanaging virulence: A new role for Leucine-Responsive Regulatory Protein (Lrp)

New investigations conducted by Chang-Ho Baek, a researcher at the Biodesign Institute of Arizona State University, demonstrate for the first time that a particular protein—Lrp—regulates the ability of bacteria to invade host cells, colonize tissues and sustain illness.

Lrp, or leucine regulatory protein, accomplishes this through a global orchestration of key virulence genes. Salmonella typhimurium, causative agent of food poisoning and other maladies, was the focus of this study, though Lrp regulation is believed to play a critical role in a range of pathogenic organisms. Lrp—a DNA binding protein, has been recognized for some 30 years as a vital genetic component in archea and in bacterial pathogens like E. coli, where it has been shown to act as a global transcription regulator.

Baek’s current work on Lrp and virulence began in April of 2007, though considerable effort was required to convince a skeptical scientific community of research results indicating Lrp’s expanded responsibility for virulence regulation. An elegant series of experiments solidifying the hypothesis formed the basis of a paper by Baek and colleagues, published in the February, 2009 issue of the Journal of Bacteriology. Subsequently, Baek’s contribution was recognized by the prestigious Faculty of 1000 Biologists, an international consortium of 2300 of the world’s foremost life scientists, who select and evaluate outstanding papers.

Genes conferring virulence in salmonella are arranged in two critical clusters, known as Salmonella Pathogenicity Islands 1& 2 (SPI-1, SPI-2). Expression of Lrp was observed to repress hilA and invF— a pair of gene regulators situated in the SPI-1 cluster, which encodes components of the type, III secretion system—essential forsalmonella’s invasion of host cells. Further, Lrp expression repressed ssrA, part of the SPI-2 regulatory system controlling the bacteria’s ability to cause systemic infection and intracellular replication. Lrp accomplishes this multi-gene orchestration by directly binding to the respective promoter regions of these virulence genes, PhilA, PinvF, and PssrA.

Pathogenicity is critical for salmonella’s successful colonization of host cells, but delicate regulation is critical. As Baek explains, “at some point, a salmonella bacterium must turn on a specific set of SPI-1 virulence genes, allowing it to effectively invade the host cell. However, once salmonella gets into the tissue, switched-on SPI-1 genes are a disadvantage, because the host immune system can easily detect bacterial presence and attempt to eliminate it. At this stage, the Lrp level goes up, turning off the unnecessary genes. ”

To explore his suspicions concerning Lrp’s role in virulence, Baek produced two mutant strains of salmonella, one in which the Lrp gene had been deleted (deltaLrp) and another where Lrp was overexpressed, Lrp(Con). The deltaLrp mutant displayed heightened virulence in mouse studies while overexpression of Lrp in the Lrp(Con) mutant yielded severely attenuated virulence. In the case of Lrp(Con), defects were observed in the bacteria’s invasion, cytotoxicity and colonization of host cells, producing a 5-fold decrease in colonization of host tissue. DeltaLrp, on the other hand, improves invasion, cytotoxicity and colonization properties of salmonella.

Like most pathogenic prokaryotic cells, salmonella are able to keenly sense and respond to such environmental cues as iron, calcium, magnesium, temperature, osmolarity, anaerobiosis, pH, and host products. They are also acutely sensitive to nutritional conditions in their environment, displaying heightened virulence when they inhabit a nutritionally rich medium. Alternately, a minimal nutrient medium forces the bacteria to compete for sustenance with eukaryotic host cells. In this situation, virulence—regulated by Lrp expression, becomes attenuated.

Baek believes Lrp may be a global regulator of prokaryotic virulence in a wide range of pathogenic microorganisms, pointing out that the Lrp homologue in E. coli is 99% identical to the form present in Salmonella typhimurium, in terms of amino acid sequence. “I’m not sure if Lrp suppresses virulence in all pathogenic bacteria,” Baek says, “but I know that in salmonella, elevated Lrp produces markedly reduced virulence.”

The mechanism of global gene regulation allows for very rapid modification of bacterial virulence, and this was a tip-off for Baek, who stresses that timing is critical if bacteria are to survive host defense strategies. “When environmental conditions change and salmonella go from an invasive stage to systemic infection, they need to respond as quickly as possible,” he notes.

Lrp’s central role in bacterial pathogenicity makes it a particularly attractive therapeutic candidate. Specifically, Baek suggests that the deltaLrp mutant could be used as a potent live vaccine carrier, owing to its hypervirulent ability to infect host cells, thereby stimulating a robust immune response. In future research, Baek hopes to further explore the underlying mechanisms permitting Lrp’s cooperative binding to specific DNA sites, affecting the structure of the whole genome and rapidly modulating target genes in response to environmental signals.