Potential Mechanisms in the Pathogenesis of Virulent Aeromonas hydrophila in Channel Catfish, Ictalurus punctatus

Abstract

Aeromonas hydrophila are Gram-negative bacteria ubiquitous in freshwater ecosystems. A. hydrophila are opportunistic pathogens with a broad host range that includes mammals, fish, reptiles, amphibians, and invertebrates. In Southeastern catfish production systems, A. hydrophila are common opportunistic pathogens causing muscle necrosis and, less often, septicemia, with outbreaks occurring following primary immune insult. In 2009, outbreaks of motile aeromonad septicemia (MAS) of epidemic proportions occurred in west Alabama catfish production systems resulting in significant mortalities, with some ponds exceeding 60% mortality of market-size catfish. The etiologic agent, a hypervirulent A. hydrophila, referred to herein as vAh, is capable of causing disease as a primary pathogen. Since 2009, vAh-induced MAS (vMAS) outbreaks in west Alabama and east Mississippi catfish productions systems have resulted in $60 - $70 million dollar in losses. Because no prerequisite environmental conditions have been identified, management practices have failed to limit or prevent vMAS outbreaks. In an attempt to elucidate mechanisms of vAh pathogenesis, a comparison of vAh proteins secreted under two primary ecological niches – biofilm and planktonic growth - was undertaken. To further understand how growth niche could influence pathogenicity, gene expression of putative virulence factors was compared. Lastly, to determine the importance of these secreted proteins in vAh virulence, a secretion-deficient mutant was created by recombineering, and functional screening of mutant and wild-type secretomes were performed in vitro, and in vivo challenges were performed in channel catfish. The results of this work show that vAh niche occupation significantly influences secretion of degradative and toxigenic proteins. Furthermore, gene expression is intimately tied to niche occupation, with 35% of all genes differentially expressed based on growth condition. Biofilm transcriptomes revealed upregulation of multiple putative virulence factor classes that, while required for biofilm production and maintenance, could act secondarily to increase host colonization and invasiveness. Toxin genes were upregulated in planktonic transcriptomes, suggesting that planktonic growth may more closely mimic bacterial growth in vivo and may prime vAh to cause disease within a host. Finally, functional screening of a T2SS mutant revealed complete loss of degradative and hemolytic ability in a secretion-deficient mutant in vitro as well as complete attenuation of virulence in vivo. Whole-pathway T2SS complementation completely restored proteolytic and hemolytic functionality in vitro and restored in vivo virulence, demonstrating that T2SS is the primary secretory pathway for degradative and hemolytic proteins in vAh, and that a functional T2SS is required for vAh virulence. These results established that T2SS and the T2S effectors play a vital role in the pathogenicity of vAh, and provide convincing evidence for the role of toxins in the development of vMAS. The research presented herein underscores the importance of considering pond ecology and natural bacterial niche ecology in the study of bacterial pathogenicity and virulence.