Nucleoid-associated Proteins Shape the Regulation of Virulence Factors and Defense Elements in Vibrio cholerae

Abstract

Human populations have battled with pandemics caused by bacterial infections throughout history. The severe diarrheal disease cholera is one of the ancient bacterial infections that is still relevant in parts of the world to this day. Vibrio cholerae is a Gram-negative comma-shaped bacterium, which is the causative agent of cholera, a disease that can be acquired via contaminated food and water. Cholera clinically presents itself in affected patients as rice watery diarrhea and has been estimated to cause between 21,000 to 143,000 deaths annually. The epidemic-causing V. cholerae strains have been successful in evolving to cause several cholera pandemics throughout recorded history. The success of its pathogenicity is due to its acquisition of various exogenous DNA via horizontal gene transfer (HGT) that harbor multiple pathogenicity factors, such as the main cholera toxin, responsible for the clinical manifestation of the disease. Other V. cholerae horizontally acquired elements (HAEs) include virulence factors to successfully colonize the host, resistance determinants allowing the bacterium to defend itself against plasmids, vibriophages (phages), and antibiotics. Because HAEs integrate into the bacterial genome, the new DNA must be domesticated and properly regulated with the existing regulatory networks. Many of the HAEs possess low adenine and thymine (AT) base composition compared to the progenitor genome to distinguish between the self- and non-self-DNA. H-NS is a widely studied xenogeneic silencer which binds such AT-rich elements, including HAEs. We investigated the regulation of some of the important HAEs in a clinical isolate of V. cholerae by deleting select genes encoding nucleoid-associated proteins (NAPs), including H-NS, that have been shown previously to regulate HAEs and other virulence factors in the bacterium. TsrA is another regulatory protein but without a predicted DNA-binding domain and has been shown to have an overlapping regulon with H-NS. In our work, I observed that H-NS plays a major role in the regulation of many HAEs in our strain background, and that the deletion of tsrA did not produce any additional effects at such loci in the ΔhnsΔtsrA double mutant. Some exceptions exist, such as the nitrate reductase operon, which is highly transcribed in the absence of both H-NS and TsrA compared to either single deletion strain, suggesting the important role of both proteins in regulation of diverse targets. We hypothesize the role of TsrA based on our computational modeling, that it modulates H-NS conformation by forming a potential heterotetramer with an H-NS dimer. Another key finding discovered in my work is that H-NS is responsible for partial repression of the phage-inducible chromosomal island-like element, PLE. PLEs are phage satellites that confer antiphage defense against the ICP1 phage, the inducer of the PLE transcriptional program. Following up with our initial findings, we assessed the role of PLE derepression from the lack of H-NS on phage infection. We observe that the PLE and ICP1 transcription are altered in the early infection when H-NS is absent, however, transcription of the phage is not inhibited at the end of infection but rather increased as opposed to our initial hypothesis. Our research work reports the regulatory implications of NAPs on V. cholerae, ICP1 phage and its phage satellite, PLE. Our findings provide directions to investigate for understanding the evolution and domestication of genes acquired via HGT in V. cholerae and other important pathogens.