Genes Driving Virulence in Streptococcus pneumoniae Identified with CRISPRi-seq

A team of infectious disease researchers at UC Santa Cruz has developed a new method to identify virulence genes in Streptococcus pneumoniae. Using CRISPRi-seq in a mouse model of pneumonia, they were able to gain new insights into the progression of the disease and its interaction with the flu virus.

"Bacterial pneumonia is a lot more common, and more deadly, after a viral infection. Historically, a lot of the deaths during flu outbreaks such as the 1918 pandemic have been attributed to pneumococcal pneumonia," said Jacqueline Kimme, co-first author of a paper published today in Cell Host Microbe.

Using their new approach, Kimmey and her colleagues were able to identify the genes that drive virulence in S. pneumoniae. The researchers created a pooled library of S. pneumoniae strains in which each of the bacteria's genes was targeted by CRISPR interference in one of the bacterial strains. The CRISPR interference system was inducible by the antibiotic doxycycline, so the genes were not silenced until the bacteria (which were resistant to the antibiotic) were introduced into mice given doxycycline-containing feed. In addition, a genetic "barcode" on the guide RNAs used to target the silenced genes enabled the researchers to easily track each strain after infection. With a single sequencing step, they could identify which strains had survived and caused infections in the mice.

The system also enabled the researchers to assess a crucial phase of the infection when most of the bacteria die off. Only a small number of bacteria survive this "bottleneck" and go on to cause invasive disease. The researchers estimated that as few as 25 bacterial cells could survive the bottleneck and cause disease. They also found a surprising amount of variation in the outcome of the bottleneck, even though the mice were genetically identical and were infected through a carefully controlled protocol. The effects of the bottleneck overshadowed the gene silencing effects, resulting in little difference between the control mice and those in which bacterial genes were silenced.

"There was no consistency in terms of which strains survived, and there was huge variability in the size of the bottleneck," Kimmey said. "We know there is a lot of variability in the clinical progression of the disease in humans, so it is very exciting to see so much variation in this highly controlled system."

The researchers then added flu to the system, infecting the mice with type A influenza prior to introducing S. pneumoniae. In mice pre-infected with influenza, there was no bottleneck, and a relatively small dose of bacteria caused rampant infection in the lungs. This enabled the researchers to assess the effects of gene silencing on the virulence of the bacteria.

The results pointed to several genes as having important roles in pneumococcal infections, including genes identified as virulence factors in previous studies, such as the bacterial capsule genes. Surprisingly, the gene for the bacteria's main toxin, pneumolysin, did not appear to be necessary for the development of infections. Together with other recent findings, this suggests that pneumolysin may be more important for transmission than for survival in the host, the researchers said.