HtrA Is Important for Stress Resistance and Virulence in Haemophilus parasuis

Haemophilus parasuisis an opportunistic pathogen that causes Glässer's disease in swine, with polyserositis, meningitis, and arthritis. The high-temperature requirement A (HtrA)-like protease, which is involved in protein quality control, has been reported to be a virulence factor in many pathogens. In this study, we showed that HtrA ofH. parasuis(HpHtrA) exhibited both chaperone and protease activities. Finally, nickel import ATP-binding protein (NikE), periplasmic dipeptide transport protein (DppA), and outer membrane protein A (OmpA) were identified as proteolytic substrates for HpHtrA. The protease activity reached its maximum at 40°C in a time-dependent manner. Disruption of thehtrAgene from strain SC1401 affected tolerance to temperature stress and resistance to complement-mediated killing. Furthermore, increased autoagglutination and biofilm formation were detected in thehtrAmutant. In addition, thehtrAmutant was significantly attenuated in virulence in the murine model of infection. Together, these data demonstrate that HpHtrA plays an important role in the virulence ofH. parasuis.

Ontogeny of dipeptide uptake and peptide transporter 1 (PepT1) expression along the gastrointestinal tract in the neonatal Yucatan miniature pig

The H+-coupled transporter, peptide transporter 1 (PepT1), is responsible for the uptake of dietary di- and tripeptides in the intestine. Using an in vivo continuously perfused gut loop model in Yucatan miniature pigs, we measured dipeptide disappearance from four 10 cm segments placed at equidistant sites along the length of the small intestine. Pigs were studied at 1, 2, 3 (suckling) and 6 weeks (post-weaning) postnatal age. Transport capability across the PepT1 transporter was assessed by measuring the disappearance of 3H-glycylsarcosine; real-time RT-PCR was also used to quantify PepT1 mRNA. Each of the regions of intestine studied demonstrated the capacity for dipeptide transport. There were no differences among age groups in transport rates measured in the most proximal intestine segment. Transport of 3H-glycylsarcosine was significantly higher in the ileal section in the youngest age group (1 week) compared with the other the suckling groups; however, all suckling piglet groups demonstrated lower ileal transport compared with the post-weaned pigs. Colonic PepT1 mRNA was maximal in the earliest weeks of development and decreased to its lowest point by week 6. These results suggest that peptide transport in the small intestine may be of importance during the first week of suckling and again with diet transition following weaning.

PepT2 transporter protein expression in human neoplastic glial cells and mediation of fluorescently tagged dipeptide derivative β-Ala-Lys-Nε-7-amino-4-methyl-coumarin-3-acetic acid accumulation

Object The present study was aimed at analyzing the accumulation of the fluorescently tagged dipeptide derivative, β-Ala-Lys-Nε-7-amino-4-methyl coumarin-3-acetic acid (AMCA), in primary cultures of human neoplastic glial cells. This molecule is a highly specific reporter used to investigate the dipeptide transport system hPepT2. Methods In this study the authors used immunocytochemical methods to determine the cell-specific accumulation of a small and fluorescently tagged reporter molecule named β-Ala-Lys-Nε-AMCA to detect dipeptide transport capacity of neoplastic glial cells. Furthermore, specific mRNA levels were quantified using Northern blot analysis and the tissue distribution of hPepT2 mRNA transcripts was demonstrated with in-situ hybridization histochemical analysis. Results Recent fluorescent immunocytochemical analyses have revealed that β-Ala-Lys-Nε-AMCA specifically accumulates within anaplastic cells of astrocytic lineage but not in anaplastic oligodendrocytes or neurons. Northern blot analysis demonstrated that human hPepT2 mRNA is specifically detected in primary cell cultures of human glioblastoma but not in oligodendroglioma. Moreover, in situ hybridization analyses revealed an astrocytic localization of hPepT2 transcripts in human glioblastoma and astrocytoma cells. The hPepT2 transcription levels were clearly dependent on the grade of glial cell differentiation: within low-grade gliomas (WHO Grade II), more hPepT2 mRNA was detected compared with tumors of a higher grade of dedifferentiation (WHO Grade IV). Analysis of expression levels of hPepT2 mRNA in human neoplastic glial cells xenografted into the brains of athymic rats (han rnu+/+) showed a markedly increased expression of hPepT2 after 2 weeks of growth in vivo compared with the primary counterparts grown in vitro. Conclusions The authors concluded that expression of the hPepT2 transporter protein is a characteristic of glial cells of astrocytic lineage, and is dependent on the grade of astroglial cell differentiation and the extracellular matrix (here brain neuropil). The authors found that β-Ala-Lys-Nε-AMCA is as an excellent reporter molecule for assessing neoplastic glial cell function and physiological characteristics.