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Streptococcus agalactiae secrete virulence factors believed to be able of killing host tissues, especially under elevated water temperature. A direct effect of S. agalactiae secretory products on tilapia cells was tested on the tilapia kidney (TK‐1) cell culture. The bacteria were cultured under four different temperature levels: 22, 29, 32 and 37°C; the cell‐free portion was processed through SDS‐PAGE; and distinct bands were identified by LC‐MS/MS. At least, three virulence factors were identified, Bsp, PcsB and CAMP factor, with increasing levels as the cultured temperature rose. Expressions of bsp, pcsB and cfb were also up‐regulated with the rising of the temperature in S. agalactiae culture. The supernatant from the bacteria cultured under specified temperatures was added into TK‐1 cell‐cultured wells. Morphological damage and mortality of the cultured cells, as determined by MTT method, were increased progressively from the supernatant treatment according to the rise of temperature in S. agalactiae culture. This study suggests that the production of the three virulence factors of S. agalactiae reported herein is temperature‐dependent, and it is likely that CAMP factor directly kills the TK‐1 cells since the other two types of protein are involved in S. agalactiae cell division and the bacterial adherence to host tissues. [ABSTRACT FROM AUTHOR]

One of the clinical manifestations of streptococcosis is swimming errors of the infected fish, which is likely caused by lesions in the brain. As most studies described brain histopathology in streptococcosis as meningitis, with a limited description of lesions in the whole brain, the aim of this study was therefore to explore histopathology of the whole brain of red tilapia experimentally infected with Streptococcus agalactiae serotype III. Transcripts relating to motoneuron functions and inflammatory responses were also investigated. In the S. agalactiae‐infected fish, the parenchyma of the whole brain and its associated meninx primitiva were found to be markedly infiltrated by mononuclear cells and Gram‐positive cocci. Hemorrhage, neuronal necrosis, and localized spongiform histopathology were observed, especially within the midbrain and the cerebellum. The lesion was observed in the medial longitudinal fasciculus and its nucleus. Expressions of the transcripts CD166, GAP43, SMN, and SV2B of the infected fish did not change, while those of IL‐1β and TNF‐α were significantly upregulated. It is likely that S. agalactiae cause extensive damage to the fish brain, especially in areas that control swimming activities, through both direct invasion of the bacteria and acute inflammatory responses of the brain resident macrophages, or microglia. [ABSTRACT FROM AUTHOR]

Proprotein convertase subtilisin/kexin 4 (PCSK4) is implicated for sperm fertilizing ability, based on studies using Pcsk4-null mice. Herein we demonstrated proprotein convertase (PC) activity in intact sperm and acrosomal vesicles. To determine whether this activity was important for sperm fertilizing ability, a peptide inhibitor was designed based on PCSK4 prodomain sequence (proPC475-90), which contains its primary autocatalytic cleavage site. ProPC475-90 inhibited recombinant PCSK4's activity with a Ki value of 5.4 µM, and at 500 µM, it inhibited sperm PC activity almost completely. Treatment of sperm with proPC475-90 inhibited their egg fertilizing ability in a dose dependent manner. Correlation between sperm PC activity and fertilizing ability showed a high co-efficient value (>0.9), indicating the importance of sperm PC activity in fertilization. In particular, sperm PC activity was important for capacitation and zona pellucida (ZP)-induced acrosome reaction, since proPC475-90-treated sperm showed markedly decreased rates in these two events. These results were opposite to those observed in Pcsk4-null sperm, which contained higher PC activity than wild type sperm, possibly due to overcompensation by PCSK7, the other PCSK enzyme found in sperm. ADAM2 (45 kDa), a sperm plasma membrane protein, involved in sperm-egg plasma membrane interaction, was also processed into a smaller form (27 kDa) during capacitation at a much reduced level in proPC475-90-treated sperm. This result suggested that ADAM2 may be a natural substrate of sperm PCSK4 and its cleavage by the enzyme during acrosome reaction may be relevant to the fertilization process. J. Cell. Physiol. 226: 2817-2826, 2011. © 2011 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

One of the important peptide hormones that control reproduction in crustaceans is gonad-inhibiting hormone (GIH). GIH is known to modulate gonad maturation by inhibiting synthesis of vitellogenin (Vg), the precursor of yolk proteins. In this study, a cDNA encoding a GIH (Pem-GIH) from the eyestalk of Penaeus monodon was cloned using RT-PCR and RACE techniques. Pem-GIH cDNA is 861 bp in size with a single ORF of 288 bp. The deduced Pem-GIH consists of a 17-residue signal peptide and a mature peptide region of 79 amino acids with features typical of type II peptide hormones from the CHH family. Pem-GIH transcript was detected in eyestalk, brain, thoracic and abdominal nerve cords of adult P. monodon. The gonad-inhibiting activity of Pem-GIH was investigated using the RNA interference technique. Double-stranded RNA, corresponding to the mature Pem-GIH sequence, can trigger a decrease in Pem-GIH transcript levels both in eyestalk ganglia and abdominal nerve cord explant culture and in female P. monodon broodstock. The conspicuous increase in Vg transcript level in the ovary of GIH-knockdown shrimp suggests a negative influence for Pem-GIH on Vg gene expression, and thus implies its role as a gonad-inhibiting hormone. This is the first report to demonstrate the use of double-stranded RNA to elucidate the function of GIH in P. monodon. [ABSTRACT FROM AUTHOR]