Identification of in vivo Essential Genes of Vibrio vulnificus for Establishment of Wound Infection by Signature-Tagged Mutagenesis

Abstract

Vibrio vulnificus can cause severe necrotic lesions within a short time. Recently, it has been reported that the numbers of wound infection cases in healthy hosts are increasing, for which surgical procedures are essential in many instances to eliminate the pathogen owing to its rapid proliferation. However, the mechanisms by which V. vulnificus can achieve wound infection in healthy hosts have not been elucidated. Here, we advance a systematic understanding of V. vulnificus wound infection through genome-wide identification of the relevant genes. Signature-tagged mutagenesis (STM) has been developed to identify functions required for the establishment of infection including colonization, rapid proliferation, and pathogenicity. Previously, STM had been regarded to be unsuitable for negative selection to detect the virulence genes of V. vulnificus owing to the low colonization and proliferation ability of this pathogen in the intestinal tract and systemic circulation. Alternatively, we successfully identified the virulence genes by applying STM to a murine model of wound infection. We examined a total of 5418 independent transposon insertion mutants by signature-tagged transposon mutagenesis and detected 71 clones as attenuated mutants consequent to disruption of genes by the insertion of a transposon. This is the first report demonstrating that the pathogenicity of V. vulnificus during wound infection is highly dependent on its characteristics: flagellar-based motility, siderophore-mediated iron acquisition system, capsular polysaccharide, lipopolysaccharide, and rapid chromosome partitioning. In particular, these functions during the wound infection process and are indispensable for proliferation in healthy hosts. Our results may thus allow the potential development of new strategies and reagents to control the proliferation of V. vulnificus and prevent human infections.

Structure, Function, and Regulation of the Essential Virulence Factor Capsular Polysaccharide of Vibrio vulnificus

“…Bacteria recovered from mice infected with MO6-24/T all had an opaque morphology, indicating that phase switching from translucent back to opaque had likely occurred in vivo by an undetermined mechanism [11]. Attenuation of virulence was a common property seen for acapsular transposon mutants of V. vulnificus in other studies as well [12][13][14].…”

Chemotactic invasion in deep soft tissue by Vibrio vulnificus is essential for the progression of necrotic lesions

“…Phenotypes in the motility of ΔpomA, ΔcheY, and Q93R, the number of detections from spleen at the two-time points, and the number of dead mice after infection. We previously showed that ΔpomA and ΔcheY were attenuated mutants that could not proliferate enough to be detected from spleen [17]. The detailed study of these mutants here revealed that their ability to invade the muscle and the systemic circulation was reduced (Figures 3(c) and 5(a)).…”

“…The Δ pomA and Δ cheY deletion mutants were constructed as described previously [ 17 ]. Briefly, the pomA gene and the cheY gene were amplified and cloned into the suicide vector pYAK1, retaining sacB .…”

“…Flagellar-based motility and chemotaxis were identified as the essential factors to establish wound infection of V. vulnificus in our previous study [ 17 ]. The flagellum is one of the main structures for motile bacteria and a major antigen for recognition by the host cells through the toll-like receptor [ 18 , 19 ], and its mutant shows a striking phenotype both in vitro and in vivo .…”

“…Chemotactic receptors of H. pylori sense acids, and the bacterium migrates to the gastric gland by chemotaxis and can proliferate in the stomach [ 27 ]. In these study on bacterial chemotaxis and pathogenicity, nonchemotactic mutant Δ cheY was used to find a preferred colonization site where the mutant cannot invade due to lacking chemotaxis [ 17 , 27 , 28 ]. Interestingly, Butler and Camilli found a niche, where Vibrio cholerae invades and proliferates, by using two phenotypes; mutants of Δ cheY and a point mutant of CheY.…”