Exploring phage–host interactions in <i>Burkholderia cepacia</i> complex bacterium to reveal host factors and phage resistance genes using CRISPRi functional genomics and transcriptomics

Complex interactions of bacteriophages with their bacterial hosts determine phage host range and infectivity. While phage defense systems and host factors have been identified in model bacteria, they remain challenging to predict in non-model bacteria. In this paper, we integrate functional genomics and transcriptomics to investigate phage-host interactions, revealing active phage resistance and host factor genes in <i>Burkholderia cenocepacia</i> K56-2. <i>Burkholderia cepacia</i> complex species are commonly found in soil and are opportunistic pathogens in immunocompromised patients. We studied infection of <i>B. cenocepacia</i> K56-2 with Bcep176, a temperate phage isolated from <i>Burkholderia multivorans</i>. A genome-wide dCas9 knockdown library targeting <i>B. cenocepacia</i> K56-2 was constructed, and a pooled infection experiment identified 63 novel genes or operons coding for candidate host factors or phage resistance genes. The activities of a subset of candidate host factor and resistance genes were validated via single-gene knockdowns. Transcriptomics of <i>B. cenocepacia K56-2</i> during Bcep176 infection revealed that expression of genes coding for host factor and resistance candidates identified in this screen was significantly altered during infection by 4 h post-infection. Identifying which bacterial genes are involved in phage infection is important to understand the ecological niches of <i>B. cenocepacia</i> and its phages<i>,</i> and for designing phage therapies.IMPORTANCE<i>Burkholderia cepacia</i> complex bacteria are opportunistic pathogens inherently resistant to antibiotics, and phage therapy is a promising alternative treatment for chronically infected patients. <i>Burkholderia</i> bacteria are also ubiquitous in soil microbiomes. To develop improved phage therapies for pathogenic <i>Burkholderia</i> bacteria, or engineer phages for applications, such as microbiome editing, it's essential to know the bacterial host factors required by the phage to kill bacteria, as well as how the bacteria prevent phage infection. This work identified 65 genes involved in phage-host interactions in <i>Burkholderia cenocepacia</i> K56-2 and tracked their expression during infection. These findings establish a knowledge base to select and engineer phages infecting or transducing <i>Burkholderia</i> bacteria.