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Group A Streptococcus (GAS) causes the life‐threatening infection in humans known as necrotizing fasciitis (NF). Infected subcutaneous tissues from an NF patient and mice challenged with the same GAS strain possessed high bacterial loads but a striking paucity of infiltrating polymorphonuclear leukocytes (PMNs). Impaired PMN recruitment was attributed to degradation of the chemokine IL‐8 by a GAS serine peptidase. Here, we use bioinformatics approach coupled with target mutagenesis to identify this peptidase as ScpC. We show that SilCR pheromone downregulates scpC transcription via the two‐component system—SilA/B. In addition, we demonstrate that in vitro , ScpC degrades the CXC chemokines: IL‐8 (human), KC, and MIP‐2 (both murine). Furthermore, using a murine model of human NF, we demonstrate that ScpC, but not the C5a peptidase ScpA, is an essential virulence factor. An ScpC‐deficient mutant is innocuous for untreated mice but lethal for PMN‐depleted mice. ScpC degrades KC and MIP‐2 locally in the infected skin tissues, inhibiting PMN recruitment. In conclusion, ScpC represents a novel GAS virulence factor functioning to directly inactivate a key element of the host innate immune response.

Casein kinase 1 (CK1) plays an important role in eukaryotic signaling pathways, and their substrates include key regulatory proteins involved in cell differentiation, proliferation and chromosome segregation. The Leishmania genome encodes six potential CK1 isoforms, of which five have orthologs in other trypanosomatidae. Leishmania donovani CK1 isoform 4 (Ldck1.4, orthologous to LmjF27.1780) is unique to Leishmania and contains a putative secretion signal peptide. The full-length gene and three shorter constructs were cloned and expressed in E. coli as His-tag proteins. Only the full-length 62.3 kDa protein showed protein kinase activity indicating that the N-terminal and C-terminal domains are essential for protein activity. LdCK1.4-FLAG was stably over expressed in L. donovani, and shown by immunofluorescence to be localized primarily in the cytosol. Western blotting using anti-FLAG and anti-CK1.4 antibodies showed that this CK1 isoform is expressed and secreted by promastigotes. Over expression of LdCK1.4 had a significant effect on promastigote growth in culture with these parasites growing to higher cell densities than the control parasites (wild-type or Ld:luciferase, P<0.001). Analysis by flow cytometry showed a higher percentage, ∼4-5-fold, of virulent metacyclic promastigotes on day 3 among the LdCK1.4 parasites. Finally, parasites over expressing LdCK1.4 gave significantly higher infections of mouse peritoneal macrophages compared to wild-type parasites, 28.6% versus 6.3%, respectively (p = 0.0005). These results suggest that LdCK1.4 plays an important role in parasite survival and virulence. Further studies are needed to validate CK1.4 as a therapeutic target in Leishmania.

Necrotising soft-tissue infections due to group A streptococcus (GAS) are rare (about 0·2 cases per 100000 people). The disease progresses rapidly, causing severe necrosis and hydrolysis of soft tissues. Histopathological analysis of necrotic tissue debrided from two patients (one with necrotising fasciitis and one with myonecrosis) showed large quantities of bacteria but no infiltrating neutrophils. We aimed to investigate whether the poor neutrophil chemotaxis was linked with the ability of group A streptococcus (GAS) to degrade host chemokines. We did RT-PCR, ELISA, and dot-blot assays to establish whether GAS induces synthesis of interleukin 8 mRNA, but subsequently degrades the released chemokine protein. Class-specific protease inhibitors were used to characterise the protease that degraded the chemokine. We used a mouse model of human soft-tissue infections to investigate the pathogenic relevance of GAS chemokine degradation, and to test the therapeutic effect of a GAS pheromone peptide (SilCR) that downregulates activity of chemokine protease. The only isolates from the necrotic tissue were two β-haemolytic GAS strains of an M14 serotype. A trypsin-like protease released by these strains degraded human interleukin 8 and its mouse homologue MIP2. When innoculated subcutaneously in mice, these strains produced a fatal necrotic soft-tissue infection that had reduced neutrophil recruitment to the site of injection. The M14 GAS strains have a missense mutation in the start codon of silCR, which encodes a predicted 17 aminoacid pheromone peptide, SilCR. Growth of the M14 strain in the presence of SilCR abrogated chemokine proteolysis. When SilCR was injected together with the bacteria, abundant neutrophils were recruited to the site of infection, bacteria were cleared without systemic spread, and the mice survived. The therapeutic effect of SilCR was also obtained in mice challenged with M1 and M3 GAS strains, a leading cause of invasive infections. The unusual reduction in neutrophils in necrotic tissue of people with GAS soft-tissue infections is partly caused by a GAS protease that degrades interleukin 8. In mice, degradation can be controlled by administration of SilCR, which downregulates GAS chemokine protease activity. This downregulation increases neutrophil migration to the site of infection, preventing bacterial spread and development of a fulminant lethal systemic infection.

Casein kinase 1 (CK1) plays an important role in eukaryotic signaling pathways, and their substrates include key regulatory proteins involved in cell differentiation, proliferation and chromosome segregation. The Leishmania genome encodes six potential CK1 isoforms, of which five have orthologs in other trypanosomatidae. Leishmania donovani CK1 isoform 4 (Ldck1.4, orthologous to LmjF27.1780) is unique to Leishmania and contains a putative secretion signal peptide. The full-length gene and three shorter constructs were cloned and expressed in E. coli as His-tag proteins. Only the full-length 62.3 kDa protein showed protein kinase activity indicating that the N-terminal and C-terminal domains are essential for protein activity. LdCK1.4-FLAG was stably over expressed in L. donovani, and shown by immunofluorescence to be localized primarily in the cytosol. Western blotting using anti-FLAG and anti-CK1.4 antibodies showed that this CK1 isoform is expressed and secreted by promastigotes. Over expression of LdCK1.4 had a significant effect on promastigote growth in culture with these parasites growing to higher cell densities than the control parasites (wild-type or Ld:luciferase, P<0.001). Analysis by flow cytometry showed a higher percentage, ~4-5-fold, of virulent metacyclic promastigotes on day 3 among the LdCK1.4 parasites. Finally, parasites over expressing LdCK1.4 gave significantly higher infections of mouse peritoneal macrophages compared to wild-type parasites, 28.6% versus 6.3%, respectively (p = 0.0005). These results suggest that LdCK1.4 plays an important role in parasite survival and virulence. Further studies are needed to validate CK1.4 as a therapeutic target in Leishmania.