Abstract
Hypervirulent Aeromonas hydrophila (vAh) is an emerging pathogen in freshwater aquaculture systems. In the U.S.A., outbreaks of motile aeromonad septicemia associated with vAh result in the loss of over 3 million pounds of channel catfish from Southeastern production systems each year. A. hydrophila is a well-known opportunistic pathogen that secretes degradative and potentially toxigenic proteins, and the rapid mortality that occurs when catfish are challenged with vAh by intraperitoneal injection suggests that vAh-induced motile aeromonad septicemia may be, in part, a toxin-mediated disease. While vAh isolates from carp isolated in China possess complete Type I, Type II, and Type VI secretion systems, many of the US catfish isolates only possess complete Type I and Type II secretions systems. In order to determine the role of secreted proteins in vAh-induced disease, and to determine the extent of protein secretion by the Type II secretion pathway, an exeD secretin mutant was constructed using a recombineering method in the well-characterized US vAh strain, ML09-119. Wild-type and mutant secretomes were analyzed for protein content by SDS-PAGE and by assays for specific enzymes and toxins. Type II secretion-deficient mutants had a near complete loss of secreted proteins and enzyme/toxin activity, including hemolytic and proteolytic activity. The intact Type II secretion system was cloned and used to complement the deletion mutant, ML09-119 exeD, which restored protein secretion and the degradative and toxigenic potential. In vivo challenges in channel catfish resulted in complete attenuation of virulence in ML09-119 exeD, while the complemented mutant was observed to have restored virulence. These results indicate that secreted proteins are critical to vAh virulence, and that the Type II secretion system is the primary secretory pathway utilized for multiple effectors of vAh pathogenesis.
Highlights
A. hydrophila is a ubiquitous Gram-negative bacterium capable of inhabiting a wide range of environments and acting as an opportunistic pathogen in fish, reptiles, amphibians, and mammals [1,2,3]
One study reported that the presence of elastase was required for A. hydrophila virulence in a rainbow trout (Oncorhynchus mykiss) model, with LD50 values two orders of magnitude higher in the elastase-deficient mutant [39]
Elastase may be capable of degradation of other structural proteins, such as collagen types III and IV, which may lead to the destruction of the basement membrane, granting bacterial ingress to the dermal tissue [39, 40]
Summary
A. hydrophila is a ubiquitous Gram-negative bacterium capable of inhabiting a wide range of environments and acting as an opportunistic pathogen in fish, reptiles, amphibians, and mammals [1,2,3]. In 2009 a novel, highly-virulent strain of A. hydrophila was isolated from channel catfish during epidemic outbreaks of peracute motile aeromonad septicemia (MAS) in Alabama catfish production ponds [6]. The bacterium continues to cause significant losses in the Southeastern catfish industry. This epidemic strain, referred to as hypervirulent Aeromonas hydrophila (vAh). VAh isolates within the sequence type 251 (ST251) pathotype are clonal in nature but show significant genetic variation from the typical, primarily opportunistic A. hydrophila [6, 7] and are capable of producing disease as a primary pathogen, resulting in rapid and devastating mortality in catfish production ponds [7, 8] This epidemic strain, referred to as hypervirulent Aeromonas hydrophila (vAh). vAh isolates within the sequence type 251 (ST251) pathotype are clonal in nature but show significant genetic variation from the typical, primarily opportunistic A. hydrophila [6, 7] and are capable of producing disease as a primary pathogen, resulting in rapid and devastating mortality in catfish production ponds [7, 8]
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