H-NOX and NosP Regulate Flagellar Protein and Virulence Factor Production in Vibrio cholerae

The ability of Vibrio cholerae to transition between motile and sessile forms in the environment and in the host is critical to its survival and virulence. The molecular cues, sensor proteins, and signaling pathways mediating these transitions are highly complex and often overlapping. Nevertheless, a detailed understanding of them is critical for understanding the persistence and pathogenesis of this deadly pathogen. Nitric oxide (NO) functions as an important signaling molecule in many bacteria, affecting biofilm formation, motility, and virulence, often through interaction with heme protein sensors. The genome of V. cholerae encodes two such sensors called H-NOX and NosP. Here we constructed a Δhnox/nosP mutant and employed a multi-omics methodology that combines tandem-mass-tag (TMT)-based quantitative proteomics, phosphoproteomics, and targeted metabolomics to investigate the function of these sensors. A set of 258 proteins was differentially expressed in the mutant that included many proteins involved in flagellar biosynthesis and motility as well as critical virulence factors, iron acquisition systems, and metabolic enzymes. Many of the identified genes are also part of the ferric uptake regulator (Fur) regulon and iron-dependent transcriptional repression of several Fur targets was disrupted. Phosphoproteomics analysis also revealed proteins involved in motility and virulence as differentially phosphorylated in the mutant strain. In most cases, these phosphoproteins have not been previously observed and provide a wealth of new targets for investigating mechanisms of V. cholerae signaling. Taken together, this work illustrates a role for H-NOX and NosP in promoting factors important for infection while suppressing those important for environmental survival, suggesting a function in priming the organism for infection and/or maintaining the infectious phenotype.