Growth Phase Regulation of Vibrio cholerae RTX Toxin Export

Abstract

Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, secretes several "accessory" toxins, including RTX toxin, which causes the cross-linking of the actin cytoskeleton. RTX toxin is exported to the extracellular milieu by an atypical type I secretion system (T1SS), and we previously noted that RTXassociated activity is detectable only in supernatant fluids from log phase cultures. Here, we investigate the mechanisms for regulating RTX toxin activity in supernatant fluids. We find that exported proteases are capable of destroying RTX activity and may therefore play a role in the growth phase regulation of toxin activity. We determined that the absence of RTX toxin in stationary-phase culture supernatant fluids is also due to a lack of toxin secretion and not attributable to solely proteolytic degradation. We ascertained that the T1SS apparatus is regulated at the transcriptional level by growth phase control that is independent of quorum sensing, unlike other virulence factors of V. cholerae. Additionally, in stationary-phase cultures, all RTX toxin activity is associated with bacterial membranes or outer membrane vesicles.Oral ingestion of Vibrio cholerae by human hosts via contaminated food and water can result in cholera, a severe diarrheal disease that can quickly cause death if left untreated (34). The secreted ADP-ribosylating cholera toxin (CT) causes the profuse diarrhea that is a hallmark of cholera disease and is one of the main virulence factors produced by the bacterium. V. cholerae also expresses the toxin-coregulated pilus (TCP), which is essential for sustained colonization of the human small intestine. In addition, hemagglutinin/protease (HAP), hemolysin, and RTX toxin have been identified as secreted "accessory" toxins that also contribute to disease pathogenesis (11).RTX toxin is a very large toxin with a deduced molecular mass of 484 kDa and causes cell rounding and the depolymerization of the actin cytoskeleton in a broad range of cell types. Concurrent with actin stress fiber disassembly, actin monomers are covalently cross-linked into dimers, trimers, and higher multimers which can be visualized by Western blotting (13,25). rtxA, the 13,635-bp gene encoding the RTX toxin, is the largest open reading frame of the V. cholerae genome (17,25). rtxA is found in both clinical and environmental isolates of V. cholerae, but not in O1 classical biotypes (7,10,25).V. cholerae RTX toxin is a member of the RTX (repeat-intoxin) family of toxins (25) that includes Escherichia coli hemolysin (HlyA) and Bordetella pertussis adenylate cyclase-hemolysin toxin. All RTX exoproteins are exported out of the cell by type I secretion systems (T1SS) (41). These secretion systems commonly consist of three components: an inner membrane transport ATPase, a transmembrane linker protein, and an outer membrane porin, exemplified by the E. coli hemolysin T1SS consisting of HlyB, HlyD, and TolC, respectively (1, 19). V. cholerae RTX toxin is secreted by an atypical T1SS that is composed of four comp...

Cytotoxic and Inflammatory Responses Induced by Outer Membrane Vesicle-Associated Biologically Active Proteases from Vibrio cholerae

“…Several studies have shown that toxins such as CT, RTX, VCC, and metalloprotease PrtV in V. cholerae are transported to host cells through OMVs in a biologically active form (19,20,41). Our present study showed that both HAP-and VesC-associated OMVs are internalized into human intestinal epithelial cells (Int407) in a protease-independent manner.…”

ppGpp Conjures Bacterial Virulence

“…For example, the RTX toxin is not secreted during the stationary phase, and the promoters of the genes encoding both the toxin and cognate secretion system contain a GC-rich discriminator region, which is a hallmark of negatively controlled stringent promoters ( Fig. 3) (23). Furthermore, relA transcripts are upregulated ϳ3-fold when cells are in the resilient VBNC state (74).…”

SPI-9 of Salmonella enterica serovar Typhi is constituted by an operon positively regulated by RpoS and contributes to adherence to epithelial cells in culture

“…In addition, STY2875 is found in the membrane fraction. It has been reported that an ORF adjacent to genes encoding a T1SS and belonging to the same operon usually encodes a protein that must be exported into the extracellular milieu to exert its function, such as Vibrio cholerae rtxA and E. coli hlyA (Bakkes et al, 2010;Boardman et al, 2007). Nevertheless, other proteins secreted by the T1SS remain attached to the bacterial surface.…”