LitR of Vibrio salmonicida is a salinity-sensitive quorum-sensing regulator of phenotypes involved in host interactions and virulence

Abstract

Vibrio (Aliivibrio) salmonicida is the causal agent of cold-water vibriosis, a fatal bacterial septicemia primarily of farmed salmonid fish. The molecular mechanisms of invasion, colonization, and growth of V. salmonicida in the host are still largely unknown, and few virulence factors have been identified. Quorum sensing (QS) is a cell-to-cell communication system known to regulate virulence and other activities in several bacterial species. The genome of V. salmonicida LFI1238 encodes products presumably involved in several QS systems. In this study, the gene encoding LitR, a homolog of the master regulator of QS in V. fischeri, was deleted. Compared to the parental strain, the litR mutant showed increased motility, adhesion, cell-to-cell aggregation, and biofilm formation. Furthermore, the litR mutant produced less cryptic bioluminescence, whereas production of acylhomoserine lactones was unaffected. Our results also indicate a salinity-sensitive regulation of LitR. Finally, reduced mortality was observed in Atlantic salmon infected with the litR mutant, implying that the fish were more susceptible to infection with the wild type than with the mutant strain. We hypothesize that LitR inhibits biofilm formation and favors planktonic growth, with the latter being more adapted for pathogenesis in the fish host.

Fig 1

Phylogenetic relationships of LitR homologs in vibrios. Accession numbers (NCBI) for the sequences are as follows: V. salmonicida LitR, gi:209696031; V. harveyi LuxR, gi:107933356; V. parahaemolyticus OpaR, gi:28899290; V. anguillarum VanT, gi:18104604; V. Vibrio cholerae HapR, gi:87133250; V. fischeri LitR, gi:59712784; and Vibrio vulnificus SmcR, gi:8101587. The bar indicates the number of substitutions/site. Bootstrap values are shown to show statistical support of branching.

Fig 2

Static biofilms of V. salmonicida LFI1238 and the ΔlitR strain formed in L15 medium at 4°C. The biofilms were visualized by phase-contrast microscopy after 3 days (A and B) and macroscopically after 6 days (C and D). No biofilm was formed by LFI1238 (A and C), whereas the ΔlitR strain (B and D) formed a thick and viscous biofilm under the chosen conditions.

Fig 3

Expression of litR mRNA in V. salmonicida LFI1238 as determined by qRT-PCR. The wild type was cultivated in LB medium supplemented with 1% NaCl and 3% NaCl and harvested at different optical densities (at 600 nm). The relative expression (RQ) was calculated by the 2^−ΔΔCT method to determined the level of expression relative to the transcription level in cultures harvested at an OD₆₀₀ of 0.1 in LB1 and LB3, respectively (38). The C_T values were normalized using the 16S rRNA level of V. salmonicida as a reference.

Fig 4

Survival plots after i.p. challenge of Atlantic salmon (Salmo salar) with V. salmonicida LFI1238 (blue line), the ΔlitRc strain (green line), and the ΔlitR strain (red line).