ABSTRACT
Vibrio parahaemolyticus cells transit from free-swimming to surface adapted lifestyles, such as swarming colonies and three-dimensional biofilms. These transitions are regulated by sensory modules and regulatory networks that involve the second messenger cyclic diguanylate monophosphate (c-di-GMP). In this work, we show that a previously uncharacterized c-di-GMP phosphodiesterase (VP1881) from V. parahaemolyticus plays an important role in modulating the c-di-GMP pool. We found that the product of VP1881 promotes its own expression when the levels of c-di-GMP are low or when the phosphodiesterase (PDE) is catalytically inactive. This behavior has been observed in a class of c-di-GMP receptors called trigger phosphodiesterases, and hence we named the product of VP1881 TpdA, for trigger phosphodiesterase A. The absence of tpdA showed a negative effect on swimming motility while, its overexpression from an isopropyl-ß-d-thiogalactopyranoside (IPTG)-inducible promoter showed a positive effect on both swimming and swarming motility and a negative effect on biofilm formation. Changes in TpdA abundance altered the expression of representative polar and lateral flagellar genes, as well as that of the biofilm-related gene cpsA. Our results also revealed that autoactivation of the native PtpdA promoter is sufficient to alter c-di-GMP signaling responses such as swarming and biofilm formation in V. parahaemolyticus, an observation that could have important implications in the dynamics of these social behaviors. IMPORTANCE c-di-GMP trigger phosphodiesterases (PDEs) could play a key role in controlling the heterogeneity of biofilm matrix composition, a property that endows characteristics that are potentially relevant for sustaining integrity and functionality of biofilms in a variety of natural environments. Trigger PDEs are not always easy to identify based on their sequence, and hence not many examples of these type of signaling proteins have been reported in the literature. Here, we report on the identification of a novel trigger PDE in V. parahaemolyticus and provide evidence suggesting that its autoactivation could play an important role in the progression of swarming motility and biofilm formation, multicellular behaviors that are important for the survival and dissemination of this environmental pathogen.
Subject(s)
Biofilms/growth & development , Cyclic GMP/analogs & derivatives , Phosphoric Diester Hydrolases/metabolism , Vibrio parahaemolyticus/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cyclic GMP/chemistry , Cyclic GMP/genetics , Cyclic GMP/metabolism , Gene Expression Regulation, Bacterial , Genes, Bacterial/genetics , Phosphoric Diester Hydrolases/chemistry , Phosphoric Diester Hydrolases/genetics , Second Messenger Systems , Vibrio parahaemolyticus/geneticsABSTRACT
Trypanosoma brucei is a protozoan flagellate that causes African sleeping sickness. Flagellar function in this organism is critical for life cycle progression and pathogenesis, however the regulation of flagellar motility is not well understood. The flagellar axoneme produces a complex beat through the precisely coordinated firing of many proteins, including multiple dynein motors. These motors are found in the inner arm and outer arm complexes. We are studying one of the inner arm dynein motors in the T. brucei flagellum: dynein-f. RNAi knockdown of genes for two components of dynein-f: DNAH10, the a heavy chain, and IC138, an intermediate chain, cause severe motility defects including immotility. To determine if motility defects result from structural disruption of the axoneme, we used two different flagellar preparations to carefully examine axoneme structure in these strains using transmission electron microscopy (TEM). Our analysis showed that inner arm dynein size, axoneme structural integrity and fixed central pair orientation are not significantly different in either knockdown culture when compared to control cultures. These results support the idea that immotility in knockdowns affecting DNAH10 or IC138 results from loss of dynein-f function rather than from obvious structural defects in the axoneme.
Subject(s)
Animals , Axoneme/metabolism , Dyneins/chemistry , Trypanosoma brucei brucei/metabolism , Cell Cycle , Cell Movement , Dyneins/metabolism , Flagella/metabolism , Models, Biological , Microscopy, Electron, Transmission/methods , RNA InterferenceABSTRACT
Trypanosoma brucei is a protozoan flagellate that causes African sleeping sickness. Flagellar function in this organism is critical for life cycle progression and pathogenesis, however the regulation of flagellar motility is not well understood. The flagellar axoneme produces a complex beat through the precisely coordinated firing of many proteins, including multiple dynein motors. These motors are found in the inner arm and outer arm complexes. We are studying one of the inner arm dynein motors in the T. brucei flagellum: dynein-f. RNAi knockdown of genes for two components of dynein-f: DNAH10, the a heavy chain, and IC138, an intermediate chain, cause severe motility defects including immotility. To determine if motility defects result from structural disruption of the axoneme, we used two different flagellar preparations to carefully examine axoneme structure in these strains using transmission electron microscopy (TEM). Our analysis showed that inner arm dynein size, axoneme structural integrity and fixed central pair orientation are not significantly different in either knockdown culture when compared to control cultures. These results support the idea that immotility in knockdowns affecting DNAH10 or IC138 results from loss of dynein-f function rather than from obvious structural defects in the axoneme.(AU)