Abstract
BackgroundVibrio vulnificus is a marine bacterial species that causes opportunistic infections manifested by serious skin lesions and fulminant septicemia in humans. We have previously shown that the multifunctional autoprocessing repeats in toxin (MARTXVv1) of a biotype 1 V. vulnificus strain promotes survival of this organism in the host by preventing it from engulfment by the phagocytes. The purpose of this study was to further explore how MARTXVv1 inhibits phagocytosis of this microorganism by the macrophage.MethodsWe compared between a wild-type V. vulnificus strain and its MARTXVv1-deficient mutant for a variety of phagocytosis-related responses, including morphological change and activation of signaling molecules, they induced in the macrophage. We also characterized a set of MARTXVv1 domain-deletion mutants to define the regions associated with antiphagocytosis activity.ResultsThe RAW 264.7 cells and mouse peritoneal exudate macrophages underwent cell rounding accompanied by F-actin disorganization in the presence of MARTXVv1. In addition, phosphorylation of some F-actin rearrangement-associated signaling molecules, including Lyn, Fgr and Hck of the Src family kinases (SFKs), focal adhesion kinase (FAK), proline-rich tyrosine kinase 2 (Pyk2), phosphoinositide 3-kinase (PI3K) and Akt, but not p38, was decreased. By using specific inhibitors, we found that these kinases were all involved in the phagocytosis of MARTXVv1-deficient mutant in an order of SFKs-FAK/Pyk2-PI3K-Akt. Deletion of the effector domains in the central region of MARTXVv1 could lead to reduced cytotoxicity, depending on the region and size of deletion, but did not affect the antiphagocytosis activity and ability to cause rounding of macrophage. Reduced phosphorylation of Akt was closely associated with inhibition of phagocytosis by the wild-type strain and MARTXVv1 domain-deletion mutants, and expression of the constitutively active Akt, myr-Akt, enhanced the engulfment of these strains by macrophage.ConclusionsMARTXVv1 could inactivate the SFKs-FAK/Pyk2-PI3K-Akt signaling pathway in the macrophages. This might lead to impaired phagocytosis of the V. vulnificus-infected macrophage. The majority of the central region of MARTXVv1 is not associated with the antiphagocytosis activity.
Highlights
Vibrio vulnificus is a marine bacterial species that causes opportunistic infections manifested by serious skin lesions and fulminant septicemia in humans
It has been demonstrated that MARTXVv1 exerts comparable cytotoxicity and antiphagocytosis effect in the RAW 264.7 cells and mouse peritoneal exudate macrophages [10]
Involvement of MARTXVv1 effector domains in cytotoxicity and antiphagocytosis To determine the roles of various domains of MARTXVv1 (Fig. 5a) in antiphagocytosis and lysis of Effect of myrAkt on phagocytosis of macrophages We showed that MARTXVv1 could cause dephosphorylation of Src family kinases (SFKs) Y418, focal adhesion kinase (FAK) Y861, proline-rich tyrosine kinase 2 (Pyk2) Y402, phosphoinositide 3-kinase (PI3K) p85 Y458 and Akt S473, and inactivation of Akt alone resulted in reduced phagocytosis in RAW 264.7 cells (Fig. 4)
Summary
Vibrio vulnificus is a marine bacterial species that causes opportunistic infections manifested by serious skin lesions and fulminant septicemia in humans. We have previously shown that the multifunctional autoprocessing repeats in toxin (MARTXVv1) of a biotype 1 V. vulnificus strain promotes survival of this organism in the host by preventing it from engulfment by the phagocytes. Vibrio vulnificus is a gram-negative bacillus distributed worldwide in estuaries. Strains of this species are currently divided into biotypes 1, 2 and 3 based on their biochemical traits and host range [1, 2]. Biotype 1 V. vulnificus comprises most of the clinical and environmental isolates, and may cause serious skin lesions and/or fulminant septicemia in humans contracting this organism via wounds or ingestion of contaminated seafood [3]. Deletion of the effector domains of the MARTX in V. vulnificus, which causes necrotic death of a variety of eukaryotic cells [10, 12, 17, 18], abolishes the ability to cause rounding, but not lysis, of HeLa cells [16]
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