Differential production and secretion of potentially toxigenic extracellular proteins from hypervirulent Aeromonas hydrophila under biofilm and planktonic culture

Abstract

Background Hypervirulent Aeromonas hydrophila (vAh) is an emerging pathogen in freshwater aquaculture that results in the loss of over 3 million pounds of marketable channel catfish, Ictalurus punctatus, and channel catfish hybrids (I. punctatus, ♀ x blue catfish, I. furcatus, ♂) each year from freshwater catfish production systems in Alabama, U.S.A. vAh isolates are clonal in nature and are genetically unique from, and significantly more virulent than, traditional A. hydrophila isolates from fish. Even with the increased virulence, natural infections cannot be reproduced in aquaria challenges making it difficult to determine modes of infection and the pathophysiology behind the devastating mortalities that are commonly observed. Despite the intimate connection between environmental adaptation and plastic response, the role of environmental adaption on vAh pathogenicity and virulence has not been previously explored. In this study, secreted proteins of vAh cultured as free-living planktonic cells and within a biofilm were compared to elucidate the role of biofilm growth on virulence. Results Functional proteolytic assays found significantly increased degradative activity in biofilm secretomes; in contrast, planktonic secretomes had significantly increased hemolytic activity, suggesting higher toxigenic potential. Intramuscular injection challenges in a channel catfish model showed that in vitro degradative activity translated into in vivo tissue destruction. Identification of secreted proteins by HPLC-MS/MS revealed the presence of many putative virulence proteins under both growth conditions. Biofilm grown vAh produced higher levels of proteolytic enzymes and adhesins, whereas planktonically grown cells secreted higher levels of toxins, porins, and fimbrial proteins. Conclusions This study is the first comparison of the secreted proteomes of vAh when grown in two distinct ecological niches. These data on the adaptive physiological response of vAh based on growth condition increase our understanding of how environmental niche partitioning could affect vAh pathogenicity and virulence. Increased secretion of colonization factors and degradative enzymes during biofilm growth and residency may increase bacterial attachment and host invasiveness, while increased secretion of hemolysins, porins, and other potential toxins under planktonic growth (or after host invasion) could result in increased host mortality. The results of this research underscore the need to use culture methods that more closely mimic natural ecological habitat growth to improve our understanding of vAh pathogenesis.

Persistence of a Wild-Type Virulent Aeromonas hydrophila Isolate in Pond Sediments from Commercial Catfish Ponds: A Laboratory Study

“…Through experimental trials, Zhang et al [ 66 ] reported that vAh isolate ML-10-51K could rapidly proliferate using colloidal chitin and chitin flakes as a sole carbon source at the same rate as if it were supplied glucose. Additionally, virulence factors expressed by establishing biofilm colonies of vAh [ 18 ] may allow for more specific niche partitioning [ 67 ] between vAh and closely related bacteria such as Pseudomonas and non-virulent Aeromonas spp.…”

“…For example, A. hydrophila is efficient in using siderophores to thrive in iron-limited conditions, and this aspect allows for enhanced virulence, as observed in laboratory settings [ 16 ]. In addition, the virulence of attenuated vAh isolates can be reduced by removing certain O-antigens [ 17 ], and vAh colonies can produce varying concentrations of proteolytic enzymes, adhesins, and toxins depending on their culture status [ 18 ]. Thus, components of biofilm formation and secretion systems are also integral to the virulence of vAh and its ability to evade fish defenses [ 10 ].…”

Development of Bioluminescent Virulent Aeromonas hydrophila for Understanding Pathogenicity

“…The virulence factors of A. hydrophila include biofilm, capsule, O-antigen, S-layer protein, secretion systems, hemolysins, degradative enzymes, enterotoxin, extracellular products, outer membrane proteins, flagella, pili, and fimbria [ 14 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ].…”

The Antivirulence Activity of Umbelliferone and Its Protective Effect against A. hydrophila-Infected Grass Carp

“…When environmental conditions deteriorate, the biofilm produced by A. hydrophila can provide a protective effect on autochthonous bacteria. Bacteria residing in the biofilm can continuously secrete proteolytic enzymes and adhesins to enhance bacterial virulence and antibiotic resistance [ 6 ].…”