In vitro/vivo Mechanism of Action of MP1102 With Low/Nonresistance Against Streptococcus suis Type 2 Strain CVCC 3928.

Streptococcosis is recognized as a leading infectious disease in the swine industry. Streptococcus suis serotype 2 is regarded as the most virulent species, which threatens human and pig health and causes serious economic losses. In this study, multiple in vitro and in vivo effects of MP1102 on multidrug resistant S. suis was studied for the first time. MP1102 exhibited significant antibacterial activity against S. suis (minimum inhibitory concentration, MIC = 0.028-0.228 µM), rapid bacteriocidal action, a longer postantibiotic effect than ceftriaxone, and a synergistic or additive effect with lincomycin, penicillin, and ceftriaxone (FICI = 0.29-0.96). No resistant mutants appeared after 30 serial passages of S. suis in the presence of MP1102. Flow cytometric analysis and electron microscopy observations showed that MP1102 destroyed S. suis cell membrane integrity and affected S. suis cell ultrastructure and membrane morphology. Specifically, a significantly wrinkled surface, intracellular content leakage, and cell lysis were noted, establishing a cyto-basis of nonresistance to this pathogen. DNA gel retardation and circular dichroism analysis indicated that MP1102 interacted with DNA by binding to DNA and changing the DNA conformation, even leading to the disappearance of the helical structure. This result further supported the mechanistic basis of nonresistance via interaction with an intracellular target, which could serve as a means of secondary injury after MP1102 is transported across the membrane. Upon treatment with 2.5-5.0 mg/kg MP1102, the survival of mice challenged with S. suis was 83.3-100%. MP1102 decreased bacterial translocation in liver, lung, spleen, and blood; inhibited the release of interleukin-1ß and tumor necrosis factor-α; and relieved the lung, liver, and spleen from acute injury induced by S. suis. These results suggest that MP1102 is a potent novel antibacterial agent for the treatment of porcine streptococcal disease.

A novel endolysin disrupts Streptococcus suis with high efficiency.

Streptococcus suis serotype 2 (S. suis 2) is a zoonotic pathogen that exhibits high-level resistance and multi-drug resistance to classic antibiotics and causes serious human casualties and heavy economic losses in the swine industry worldwide. Therefore, alternative therapies or novel antibacterial agents need to be developed to combat this pathogen. A novel endolysin derived from the S. suis temperate phage phi7917, termed Ly7917, was identified, which had broad lytic activity against S. suis type 1, 2, 7 and 9. Ly7917 consisted of an N-terminal cysteine, histidine-dependent amidohydrolases/peptidase catalytic domain and C-terminal SH3b cell wall binding domain. The endolysin maintained activity at high pH and its catalytic activity could be improved by addition of 10 µM 1.5 mM Ca(2+). In animal studies, 90% of BALB/c mice challenged with typical virulent strain HA9801 of S. suis 2 were protected by Ly7917 treatment. The bacterial load in the blood of HA9801-challenged mice was efficiently reduced almost 50% by Ly7917 while that of penicillin-G-treated mice kept almost unchanged. Our data suggest that Ly7917 may be an alternative therapeutic agent for infections caused by virulent S. suis strains.

Subcytolytic effects of suilysin on interaction of Streptococcus suis with epithelial cells.

Suilysin is a pore-forming cholesterol-dependent cytolysin secreted by Streptococcus suis (S. suis), an important swine and zoonotic pathogen. The role of suilysin in S. suis host-cell interaction is still unclear. We found a higher adherence and invasion rate of an unencapsulated sly-positive strain in comparison to its isogenic sly-negative mutant. Electron microscopy revealed that formation of membrane ruffles accompanying invasion of the sly-positive strain was abolished in the sly-negative mutant. Inhibition experiments showed that the actin cytoskeleton was involved in suilysin-mediated effects. Point-mutation of the domain putatively responsible for macropore-formation resulted in abolished hemolytic and cytolysin activity, but had no effect on S. suis host cell association. Concluding, our results indicate that subcytolytic suilysin promotes S. suis association with epithelial cells.

Pleiotropic effects of polysaccharide capsule loss on selected biological properties of Streptococcus suis.

In this study, an unencapsulated Streptococcus suis mutant was used to investigate the pleiotropic effects resulting from capsule loss. The capsule deficient mutant of S. suis acquired a biofilm-positive phenotype, which was associated with significantly increased cell surface hydrophobicity. Cell-associated fibrinogen-binding and chymotrypsin-like activities were decreased in the unencapsulated mutant. The mutant did not differ significantly from the encapsulated parent strain for minimal inhibitory concentrations to penicillin G, ampicillin, and tetracycline. However, while the encapsulated strain was highly resistant to the bactericidal action of penicillin G and ampicillin, the unencapsulated mutant was approximately 60-fold more sensitive. Compared with the parent strain, the unencapsulated mutant induced a much higher inflammatory response in monocyte-derived macrophages resulting in an increased secretion of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, and IL-8. The capsule appears to hinder important adhesins or hydrophobic molecules that mediate biofilm formation, as well as cell wall components capable of stimulating immune cells.

Non-encapsulated strains reveal novel insights in invasion and survival of Streptococcus suis in epithelial cells.

Streptococcus suis is a porcine and human pathogen causing invasive diseases, such as meningitis or septicaemia. Host cell interactions of S. suis have been studied mainly with serotype 2 strains, but multiple capsular serotypes as well as non-typeable strains exist with diverse virulence features. At present, S. suis is considered an extracellular pathogen. However, whether or not it can also invade host cells is a matter of controversial discussions. We have assessed adherence and invasion of S. suis for HEp-2 epithelial cells by comparing 10 serotype 2 strains and four non-typeable (NT) strains. Only the NT strains and a non-encapsulated serotype 2 mutant strain, but none of the serotype 2 strains, adhered strongly and were invasive. Invasion seemed to be affected by environmental signals, as suggested from comparison of strains grown in different media. Further phenotypic and genotypic characterization revealed a high diversity among the different strains. Electron microscopic analysis of invasion of selected invasive NT strains indicated different uptake mechanisms. One strain induced large invaginations comparable to those seen in 'caveolae' mediated uptake, whereas invasion of the other strains was accompanied by formation of filipodia-like membrane protrusions. Invasion of all strains, however, was similarly susceptible to hypertonic sucrose, which inhibits receptor-mediated endocytosis. Irrespective of the uptake pathway, streptococci resided in acidified phago-lysosome like vacuoles. All strains, except one, survived intracellularly as well as extracellular acidic conditions. Survival seemed to be associated with the AdiS protein, an environmentally regulated arginine deiminase of S. suis. Concluding, invasion and survival of NT strains of S. suis in epithelial cells revealed novel evidence that S. suis exhibits a broad variety of virulence-associated features depending on genetic variation and regulation.

Cloning and characterization of the gene encoding the glutamate dehydrogenase of Streptococcus suis serotype 2.

Given the lack of effective vaccines to control Streptococcus suis infection and the lack of a rapid and reliable molecular diagnostic assay to detect its infection, a polyclonal antibody was raised against the whole-cell protein of S. suis type 2 and used to screen an S. suis gene library in an effort to identify protective antigen(s) and antigens of diagnostic importance. A clone that produced a 45-kDa S. suis-specific protein was identified by Western blotting. Restriction analysis showed that the gene encoding the 45-kDa protein was present on a 1.6-kb pair DraI region on the cloned chromosomal fragment. The nucleotide sequence contained an open reading frame that encoded a polypeptide of 448 amino acid residues with a calculated molecular mass of 48.8 kDa, in close agreement with the size observed on Western blots. A GenBank database search revealed that the derived amino acid sequence is homologous to the sequence of glutamate dehydrogenase (GDH) protein isolated from various sources, including conserved motifs and functional domains typical of the family 1-type hexameric GDH proteins, thus placing it in that family. Because of these similarities, the protein was designated the GDH of S. suis. Hybridization studies showed that the gene is conserved among the S. suis type 2 strains tested. Antiserum raised against the purified recombinant protein was reactive with a protein of the same molecular size as the recombinant protein in S. suis strains, suggesting expression of the gene in all of the isolates and antigenic conservation of the protein. The recombinant protein was reactive with serum from pigs experimentally infected with a virulent strain of S. suis type 2, suggesting that the protein might serve as an antigen of diagnostic importance to detect S. suis infection. Activity staining showed that the S. suis GDH activity is NAD(P)H dependent but, unlike the NAD(P)H-dependent GDH from various other sources, that of S. suis utilizes L-glutamate rather than alpha-ketoglutarate as the substrate. Highly virulent strains of S. suis type 2 could be distinguished from moderately virulent and avirulent strains on the basis of their GDH protein profile following activity staining on a nondenaturing gel. We examined the cellular location of the protein using a whole-cell enzyme-linked immunosorbent assay and an immunogold-labeling technique. Results showed that the S. suis GDH protein is exposed at the surface of intact cells.

Epithelial invasion and cell lysis by virulent strains of Streptococcus suis is enhanced by the presence of suilysin.

Streptococcus suis is an important pathogen of pigs causing arthritis, pneumonia and meningitis and is an occupational disease of farmers and those in the meat industry. As with other streptococci, both virulent and avirulent strains of S. suis are frequently carried asymptomatically in the tonsillar crypts and nasal cavities. Little is known about the process by which virulent strains cross the mucosal epithelia to generate systemic disease and whether this process requires expression of specific bacterial virulence factors. Although putative virulence factors have been postulated, no specific role in the disease process has yet been demonstrated for these factors. This study is the first demonstration that virulent strains of S. suis both invade and lyse HEp-2 cells, a continuous laryngeal epithelial cell line, and that at least one bacterial virulence factor, suilysin, is involved in this process.