Functional Characterization and Conditional Regulation of the Type VI Secretion System in Vibrio fluvialis

Abstract

Vibrio fluvialis is an emerging foodborne pathogen of increasing public health concern. The mechanism(s) that contribute to the bacterial survival and disease are still poorly understood. In other bacterial species, type VI secretion systems (T6SSs) are known to contribute to bacterial pathogenicity by exerting toxic effects on host cells or competing bacterial species. In this study, we characterized the genetic organization and prevalence of two T6SS gene clusters (VflT6SS1 and VflT6SS2) in V. fluvialis. VflT6SS2 harbors three “orphan” hcp-vgrG modules and was more prevalent than VflT6SS1 in our isolates. We showed that VflT6SS2 is functionally active under low (25°C) and warm (30°C) temperatures by detecting the secretion of a T6SS substrate, Hcp. This finding suggests that VflT6SS2 may play an important role in the survival of the bacterium in the aquatic environment. The secretion of Hcp is growth phase-dependent and occurs in a narrow range of the growth phase (OD600 from 1.0 to 2.0). Osmolarity also regulates the function of VflT6SS2, as evidenced by our finding that increasing salinity (from 170 to 855 mM of NaCl) and exposure to high osmolarity KCl, sucrose, trehalose, or mannitol (equivalent to 340 mM of NaCl) induced significant secretion of Hcp under growth at 30°C. Furthermore, we found that although VflT6SS2 was inactive at a higher temperature (37°C), it became activated at this temperature if higher salinity conditions were present (from 513 to 855 mM of NaCl), indicating that it may be able to function under certain conditions in the infected host. Finally, we showed that the functional expression of VflT6SS2 is associated with anti-bacterial activity. This activity is Hcp-dependent and requires vasH, a transcriptional regulator of T6SS. In sum, our study demonstrates that VflT6SS2 provides V. fluvialis with an enhanced competitive fitness in the marine environment, and its activity is regulated by environmental signals, such as temperature and osmolarity.

Transcriptional Regulation of the Type VI Secretion System 1 Genes by Quorum Sensing and ToxR in Vibrio parahaemolyticus

“…The highest transcript levels of hcp1 and VP1388 occurred at an OD 600 value of 0.6 and 0.8, respectively, due to the QS regulation ( Figure 1 ), suggesting V. parahaemolyticus T6SS1 functions only during the mid-logarithmic growth phase. Similar observations have been reported in other species, including Yersinia pseudotuberculosis ( Zhang et al, 2011 ), V. cholerae ( Ishikawa et al, 2009 ), V. alginolyticus ( Sheng et al, 2012 ), V. anguillarum ( Tang et al, 2016 ), and V. fluvialis ( Huang et al, 2017 ). Notably, the highest expression levels of AphA and ToxR occurred at an OD 600 value of 0.05 to 0.2, whereas that of OpaR occurred at 0.4–0.6.…”

Bacterial symbionts use a type VI secretion system to eliminate competitors in their natural host

“…4 A ), T6SS1 may be active under different conditions compared with T6SS2. Indeed, other Vibrio species show T6SS activity that is conditionally regulated ( 13 , 33 , 84 ), and future work will need to explore how free-living versus host-like conditions modulate V. fischeri T6SS gene expression and function. Alternatively, T6SS1 may be used to interact with eukaryotic cells ( 15 , 28 , 72 ).…”

Integration Host Factor Modulates the Expression and Function of T6SS2 in Vibrio fluvialis

“…The T6SS operating is energetically costly to bacterial cells, so its gene cluster is tightly controlled to adapt its expression and assembly to changing environmental conditions ( Journet and Cascales, 2016 ). Environmental cues, such as temperature ( Ishikawa et al, 2012 ; Salomon et al, 2013 ; Huang et al, 2017 ), salinity/osolarity ( Ishikawa et al, 2012 ; Huang et al, 2017 ), iron limitation ( Brunet et al, 2011 ), stresses ( Gueguen et al, 2013 ), cell lysates ( LeRoux et al, 2015 ) etc., affect the expression of T6SS in various species. At present, VasH is the first identified T6SS regulator encoded within the Vibrio cholerae T6SS major cluster, which works as a transactivator of T6SS in V. cholerae together with σ 54 ( Bernard et al, 2011 ; Kitaoka et al, 2011 ).…”

“…We previously sequenced the whole genome of a clinical isolate of V. fluvialis , an emerging foodborne pathogen of increasing public health concern, whose sequence analysis revealed the existence of T6SS homologous gene clusters ( Lu et al, 2014 ). Subsequently, we characterized the organization, function, and expression regulation of T6SS in V. fluvialis ( Huang et al, 2017 ). We showed that one of the two T6SSs, termed VflT6SS2, is functionally active under low (25°C) and warm (30°C) temperatures.…”

“…We showed that one of the two T6SSs, termed VflT6SS2, is functionally active under low (25°C) and warm (30°C) temperatures. The functional expression of VflT6SS2 is associated with antibacterial activity which is Hcp-dependent and requires the transcriptional regulator VasH as in V. cholerae ( Kitaoka et al, 2011 ; Huang et al, 2017 ). The genetic composition and organization of VflT6SS2 in V. fluvialis are highly homologous to the T6SS in V. cholerae , except possessing three hcp-vgrG orphan clusters, named tssD 2_ a - tssI 2_ a , tssD 2_ b - tssI 2_ b and tssD 2_ c - tssI 2_ c in V. fluvialis .…”