Characterization of lsa(D), a Novel Gene Responsible for Resistance to Lincosamides, Streptogramins A, and Pleuromutilins in Fish Pathogenic Lactococcus garvieae Serotype II.

Aims: Fish pathogenic Lactococcus garvieae serotype II has been isolated from cultured fish species in Japan. This study aimed to investigate the molecular mechanisms of lincomycin (LCM)-resistant L. garvieae serotype II and assess the molecular basis for lincosamides-streptogramins A-pleuromutilins (LSAP)-resistant phenotype. Results: We identified a novel lsa(D)-encoded 497-aa ATP-binding cassette F (ABC-F) protein in the LSAP-resistant strains. Amino acid identities of 41.25-54.73% were obtained between the deduced amino acids from Lsa(D) and other Lsa-type ABC-F proteins. Furthermore, comparative analysis revealed that the allele of lsa(D) with single point mutation at 233 aa position (TGG → TAG; tryptophan→premature termination codon [PTC]) in LSAP-sensitive strains. The minimum inhibitory concentrations of antimicrobials against the lsa(D) complementary strain and lsa(D)-disrupted mutant confirmed that lsa(D) conferred the LSAP-resistant phenotype. The reverse transcription-polymerase chain reaction could not detect the noncoding region of lsa(D) allelic variant in the LSAP-sensitive strains. Additionally, the PTC (TAG) in LCM-sensitive strains was replaced by TGG, CAG, or TAT in the laboratory-induced revertant mutants. Conclusions: The novel lsa(D) conferred the LSAP-resistant phenotype in clinically LCM-resistant L. garvieae serotype II strains. However, the allele of lsa(D) gene containing the PTC was found in L. garvieae serotype II, resulting in the LSAP-susceptible phenotype.

Endogenous molecules released by haemocytes receiving Sargassum oligocystum extract lead to downstream activation and synergize innate immunity in white shrimp Litopenaeus vannamei.

White shrimp Litopenaeus vannamei haemocytes receiving immunostimulating Sargassum oligocystum extract (SE) caused necrosis in haemocyte cells, which released endogenous EM-SE molecules. This study examined the immune response of white shrimp L. vannamei receiving SE and EM-SE in vitro and in vivo. Shrimp haemocytes receiving SE exhibited degranulation, changes in cell size and cell viability, necrosis and a release of EM-SE. Shrimp haemocytes receiving SE, EM-SE, and the SE + EM-SE mixture (SE + EM-SE) increased their phenoloxidase (PO) activity which was significantly higher in shrimp haemocytes receiving the SE + EM-SE mixture. Furthermore, shrimp haemocytes receiving EM-SE showed degranulation and changes in cell size and cell viability. Shrimp receiving SE, EM-SE, and SE + EM-SE all increased their immune parameters, phagocytic activity, clearance efficiency and resistance to Vibrio alginolyticus, being significantly higher in shrimp receiving SE + EM-SE. Meanwhile, the recombinant lipopolysaccharide- and ß-1,3-glucan binding protein of L. vannamei (rLvLGBP) was bound to SE, EM-SE, and SE + EM-SE. We conclude that in shrimp haemocytes receiving a non-self molecule, SE in dying cells released EM-SE which led to downstream activation and synergization of the immune response. This study demonstrated that the innate immunity of shrimp was elicited and enhanced by a mixture of endogenous molecules and exogenous substances (or immunostimulants).

Endogenous molecules induced by a pathogen-associated molecular pattern (PAMP) elicit innate immunity in shrimp.

Invertebrates rely on an innate immune system to combat invading pathogens. The system is initiated in the presence of cell wall components from microbes like lipopolysaccharide (LPS), ß-1,3-glucan (ßG) and peptidoglycan (PG), altogether known as pathogen-associated molecular patterns (PAMPs), via a recognition of pattern recognition protein (PRP) or receptor (PRR) through complicated reactions. We show herein that shrimp hemocytes incubated with LPS, ßG, and PG caused necrosis and released endogenous molecules (EMs), namely EM-L, EM-ß, and EM-P, and found that shrimp hemocytes incubated with EM-L, EM-ß, and EM-P caused changes in cell viability, degranulation and necrosis of hemocytes, and increased phenoloxidase (PO) activity and respiratory burst (RB) indicating activation of immunity in vitro. We found that shrimp receiving EM-L, EM-ß, and EM-P had increases in hemocyte count and other immune parameters as well as higher phagocytic activity toward a Vibrio pathogen, and found that shrimp receiving EM-L had increases in proliferation cell ratio and mitotic index of hematopoietic tissues (HPTs). We identified proteins of EMs deduced from SDS-PAGE and LC-ESI-MS/MS analyses. EM-L and EM-P contained damage-associated molecular patterns (DAMPs) including HMGBa, HMGBb, histone 2A (H2A), H2B, and H4, and other proteins including proPO, Rab 7 GPTase, and Rab 11 GPTase, which were not observed in controls (EM-C, hemocytes incubated in shrimp salt solution). We concluded that EMs induced by PAMPs contain DAMPs and other immune molecules, and they could elicit innate immunity in shrimp. Further research is needed to identify which individual molecule or combined molecules of EMs cause the results, and determine the mechanism of action in innate immunity.