The Streptococcos suis sortases SrtB and SrtF are essential for disease in pigs.

The porcine pathogen Streptococcus suis colonizes the upper respiratory tracts of pigs, potentially causing septicaemia, meningitis and death, thus placing a severe burden on the agricultural industry worldwide. It is also a zoonotic pathogen that is known to cause systemic infections and meningitis in humans. Understanding how S. suis colonizes and interacts with its hosts is relevant for future strategies of drug and vaccine development. As with other Gram-positive bacteria, S. suis utilizes enzymes known as sortases to attach specific proteins bearing cell wall sorting signals to its surface, where they can play a role in host-pathogen interactions. The surface proteins of bacteria are often important in adhesion to and invasion of host cells. In this study, markerless in-frame deletion mutants of the housekeeping sortase srtA and the two pilus-associated sortases, srtB and srtF, were generated and their importance in S. suis infections was investigated. We found that all three of these sortases are essential to disease in pigs, concluding that their cognate-sorted proteins may also be useful in protecting pigs against infection

Serovar-dependent differences in Hfq-regulated phenotypes in actinobacillus pleuropneumoniae

The RNA chaperone Hfq regulates diverse processes in numerous bacteria. In this study, we compared phenotypes (growth rate, adherence, response to different stress conditions, and virulence in Galleria mellonella) of wild-type (WT) and isogenic hfq mutants of three serovars (1, 8 and 15) of the porcine pathogen A. pleuropneumoniae. Similar growth in rich broth was seen for all strains except Ap1∆hfq, which showed slightly reduced growth throughout the 24 hour time course, and the complemented Ap8∆hfqC mutant had a prolonged lag phase. Differences were seen between the three serovar WT strains regarding adherence, stress response and virulence in G. mellonella, and deletion of hfq affected some, but not all of these phenotypes, depending on serovar. Complementation by expression of cloned hfq from an endogenous promoter only restored some WT phenotypes, indicating that complex regulatory networks may be involved, and that levels of Hfq may be as important as presence/absence of the protein regarding its contribution to gene regulation. Our results support that Hfq is a pleiotropic global regulator in A. pleuropneumoniae, but serovar-related differences exist. These results highlight the importance of testing multiple strains/serovars within a given species when determining contributions of global regulators, such as Hfq, to expression of complex phenotypes

Pneumococcal interactions with mucin

The nasopharynx is covered by mucin. Mucin is a glycoprotein and the carbohydrate moieties are potential fermentable substrates for Streptococcus pneumoniae. Previous in vitro studies showed that S. pneumoniae can grow on mucin. This study was undertaken to investigate how S. pneumoniae degrades the mucin carbohydrates to mono and disaccharides for subsequent fermentation. In silico search of pneumococcal genome identified fourteen putative glycosidases. Pneumococcal mutants of each were made and tested for growth in defined medium with mucin as the only source of carbon. Of these, two genes SPD0065 and SPD0247 were chosen for further study. Consequently two novel glycosidases were described, β-galactosidase (BgaC), encoded by gene SPD0065 and a 6-phospho-β-glucosidase (BglA) encoded by gene SPD0247. The knocked-out mutants SPD0065M and SPD0247M could not grow in Sicard’s defined medium supplemented with mucin and exhibited decreased enzymatic activity when compared to the wild type D39. Since gene SPD0562 had been previously identified as encoding a β-galactosidase (BgaA), the relative contribution of BgaA and BgaC to total β-galactosidase activity was investigated by introducing mutations in these genes individually and together. Mutation in the individual genes resulted in significant decrease in the enzymatic activity but the double mutation did not totally abolished activity. BgaC had specificity for galactose (β1,3)-N-acetylgalactosamine. Furthermore, BgaC released galactose from desialylated fetuin. The expression of SPD0065 and SPD0247 in S. pneumoniae grown in mucin containing medium or harvested from tissues from infected animals was significantly up-regulated compared to growth in glucose containing medium. When the mutants were tested in vivo, it was noted that SPD0065M had attenuated growth in the nasopharynx and SPD0247M in the lungs. In this study was demonstrated that BgaC has a role in the sequential deglycosylation of host glycoproteins and that BglA is involved in the further degradation of mucin-derived sugars

Pneumococcal interactions with mucin

The nasopharynx is covered by mucin. Mucin is a glycoprotein and the carbohydrate moieties are potential fermentable substrates for Streptococcus pneumoniae. Previous in vitro studies showed that S. pneumoniae can grow on mucin. This study was undertaken to investigate how S. pneumoniae degrades the mucin carbohydrates to mono and disaccharides for subsequent fermentation. In silico search of pneumococcal genome identified fourteen putative glycosidases. Pneumococcal mutants of each were made and tested for growth in defined medium with mucin as the only source of carbon. Of these, two genes SPD0065 and SPD0247 were chosen for further study. Consequently two novel glycosidases were described, β-galactosidase (BgaC), encoded by gene SPD0065 and a 6-phospho-β-glucosidase (BglA) encoded by gene SPD0247. The knocked-out mutants SPD0065M and SPD0247M could not grow in Sicard’s defined medium supplemented with mucin and exhibited decreased enzymatic activity when compared to the wild type D39. Since gene SPD0562 had been previously identified as encoding a β-galactosidase (BgaA), the relative contribution of BgaA and BgaC to total β-galactosidase activity was investigated by introducing mutations in these genes individually and together. Mutation in the individual genes resulted in significant decrease in the enzymatic activity but the double mutation did not totally abolished activity. BgaC had specificity for galactose (β1,3)-N-acetylgalactosamine. Furthermore, BgaC released galactose from desialylated fetuin. The expression of SPD0065 and SPD0247 in S. pneumoniae grown in mucin containing medium or harvested from tissues from infected animals was significantly up-regulated compared to growth in glucose containing medium. When the mutants were tested in vivo, it was noted that SPD0065M had attenuated growth in the nasopharynx and SPD0247M in the lungs. In this study was demonstrated that BgaC has a role in the sequential deglycosylation of host glycoproteins and that BglA is involved in the further degradation of mucin-derived sugars.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Pneumococcal 6-Phospho-β-Glucosidase (BglA3) Is Involved in Virulence and Nutrient Metabolism.

For the generation of energy, the important human pathogen Streptococcus pneumoniae relies on host-derived sugars, including β-glucoside analogs. The catabolism of these nutrients involves the action of 6-phospho-β-glucosidase to convert them into usable monosaccharaides. In this study, we characterized a 6-phospho-β-glucosidase (BglA3) encoded by SPD_0247. We found that this enzyme has a cell membrane localization and is active only against a phosphorylated substrate. A mutated pneumococcal ΔSPD0247 strain had reduced 6-phospho-glucosidase activity and was attenuated in growth on cellobiose and hyaluronic acid compared to the growth of wild-type D39. ΔSPD0247-infected mice survived significantly longer than the wild-type-infected cohort, and the colony counts of the mutant were lower than those of the wild type in the lungs. The expression of SPD_0247 in S. pneumoniae harvested from infected tissues was significantly increased relative to its expression in vitro on glucose. Additionally, ΔSPD0247 is severely impaired in its attachment to an abiotic surface. These results indicate the importance of β-glucoside metabolism in pneumococcal survival and virulence

Galleria mellonella is an effective model to study Actinobacillus pleuropneumoniae infection.

Actinobacillus pleuropneumoniae is responsible for swine pleuropneumonia, a respiratory disease that causes significant global economic loss. Its virulence depends on many factors, such as capsular polysaccharides, RTX toxins and iron-acquisition systems. Analysis of virulence may require easy-to-use models that approximate mammalian infection and avoid ethical issues. Here, we investigate the potential use of the wax moth Galleria mellonella as an informative model for A. pleuropneumoniae infection. Genotypically distinct A. pleuropneumoniae clinical isolates were able to kill larvae at 37 °C but had different LD50 values, ranging from 10(4) to 10(7) c.f.u. per larva. The most virulent isolate (1022) was able to persist and replicate within the insect, while the least virulent (780) was rapidly cleared. We observed a decrease in haemocyte concentration, aggregation and DNA damage post-infection with isolate 1022. Melanization points around bacterial cells were observed in the fat body and pericardial tissues of infected G. mellonella, indicating vigorous cell and humoral immune responses close to the larval dorsal vessel. As found in pigs, an A. pleuropneumoniae hfq mutant was significantly attenuated for infection in the G. mellonella model. Additionally, the model could be used to assess the effectiveness of several antimicrobial agents against A. pleuropneumoniae in vivo. G. mellonella is a suitable inexpensive alternative infection model that can be used to study the virulence of A. pleuropneumoniae, as well as assess the effectiveness of antimicrobial agents against this pathogen