Streptococcus suis (S. suis) causes meningitis, arthritis and endocarditis in piglets. The aim of this study was to characterize the IgM degrading enzyme of S. suis (IdeSsuis) and to investigate the role of IgM cleavage in evasion of the classical complement pathway and pathogenesis. Targeted mutagenesis of a cysteine in the putative active center of IdeSsuis abrogated IgM cleavage completely. In contrast to wt rIdeSsuis, point mutated rIdeSsuis_C195S did not reduce complement-mediated hemolysis indicating that complement inhibition by rIdeSsuis depends on the IgM proteolytic activity. A S. suis mutant expressing IdeSsuis_C195S did not reduce IgM labeling, whereas the wt and complemented mutant showed less IgM F(ab’)2 and IgM Fc antigen on the surface. IgM cleavage increased survival of S. suis in porcine blood ex vivo and mediated complement evasion as demonstrated by blood survival and C3 deposition assays including the comparative addition of rIdeSsuis and rIdeSsuis_C195S. However, experimental infection of piglets disclosed no significant differences in virulence between S. suis wt and isogenic mutants without IgM cleavage activity. This work revealed for the first time in vivo labeling of S. suis with IgM in the cerebrospinal fluid of piglets with meningitis. In conclusion, this study classifies IdeSsuis as a cysteine protease and emphasizes the role of IgM cleavage for bacterial survival in porcine blood and complement evasion though IgM cleavage is not crucial for the pathogenesis of serotype 2 meningitis. S. suis disease is one of the main reasons for high economic losses in pig husbandry worldwide [S. suis colonizes the mucosal surfaces of healthy pigs but can lead to invasive disease, mainly in growing piglets of approximately five to ten weeks of age [S. suis infection [S. suis is also an emerging zoonotic agent especially in Southeast Asia [orldwide . S. suiss of age ,3. Thesenfection . S. suisast Asia .S. suis is very heterogeneous. It has been divided into 35 serotypes and more than 700 sequence types (ST) [S suis [Ssuis [Ssuis, expressed universally among S. suis serotypes, is a highly specific protease, with porcine IgM as its sole substrate [Ssuis shows homology to other streptococcal immunoglobulin degrading enzymes such as IdeS of S. pyogenes, IdeZ of S. equi subsp. zooepidemicus, IdeE of S. equi subsp. equi and IdeP of S. phocae subsp. phocae [Ssuis, however, remains the only specific IgM protease and, in contrast to the above mentioned IgG proteases, possesses a long C-terminal domain not involved in protease activity [The species pes (ST) . Howeverpes (ST) . However [S suis ,7. Virul [S suis , factor  [S suis  and lasts [Ssuis . IdeSsuiubstrate . IdeSsui. phocae . IdeSsuiactivity .Ssuis reduced IgM-mediated C3 deposition on the bacterial surface and has a positive effect on survival of S. suis in porcine blood in the presence of specific IgM titers [Ssuis is a cysteine protease and to specifically test if the IgM cleavage activity of IdeSsuis is involved in complement evasion and virulence.IgM has important effector functions in immunity. First of all, IgM represents not only the main class of natural antibodies , but is M titers . The aimSsuis is a cysteine protease. In order to proof this hypothesis and further elucidate the role of IgM cleavage by IdeSsuis in complement evasion and virulence, a point mutation was introduced into the putative active center of rIdeSsuis. The cysteine at position 195 was replaced by a serine, the rest of the gene remaining intact. The point mutation was also introduced into the truncated construct rIdeSsuis_homologue lacking the large C-terminal domain. Expression and purification of full length protein was verified for the different recombinant IdeSsuis constructs by Coomassie staining . Analyzing the IgM cleaving activity of cysteine to serine point mutated recombinant rIdeSsuis_C195S and rIdeSsuis_homologue_C195S by Western Blot analysis revealed that both recombinant constructs are unable to cleave porcine IgM . In concordance with the results obtained by the hemolysis assay, IgM labeling of sheep erythrocytes was significantly reduced by rIdeSsuis and rIdeSsuis_homologue, but not by the point mutated constructs rIdeSsuis_C195S and rIdeSsuis_homologue_C195S as shown by flow cytometry  as elicC_domain ). Thus, C-domain ). Hemolyytometry ).10.1080Ssuis is attenuated in survival in porcine blood with high specific anti-S. suis IgM titers ex vivo [Ssuis and non-functional rIdeSsuis_C195S were performed. In these assays, S. suis 10∆ideSsuis could survive significantly better in porcine blood of growing piglets if rIdeSsuis was externally added . Surviv20 µg/ml ). In theanalysis ). Cleavaactivity ). In conS. suis mutants were generated to investigate the role of IgM cleavage by IdeSsuis in the pathogenesis of S. suis diseases further. Firstly, 10∆ideSsuis was chromosomally complemented to render 10∆ideSsuis∇ideSsuis_ EcoRI expressing wt IdeSsuis. A silent mutation (EcoRI site) was introduced in this strain for further discrimination of wt and complemented mutant. Secondly, the cysteine to serine point mutation of IdeSsuis was implemented in the chromosome of S. suis itself to render ∆ideSsuis∇ideSsuis_C195S. Site-directed mutagenesis was confirmed using sequencing and Southern blot analysis including confirmation of the silent mutation leading to the new EcoRI site in 10∆ideSsuis∇ideSsuis_ EcoRI . Anti-IgM Western blot analysis showed that the complemented strain 10∆ideSsuis∇ideSsuis_EcoRI possesses full IgM cleavage activity whereas 10∆ideSsuis∇ideSsuis_C195S is unable to cleave porcine IgM .Two complemented cine IgM ) even thSsuis leads to reduction of surface bound IgM [Ssuis to reduce the amount of surface-bound IgM. S. suis wt, 10∆ideSsuis and the complemented mutants 10∆ideSsuis∇ideSsuis_EcoRI and 10∆ideSsuis∇ideSsuis_C195S were therefore analyzed regarding their ability to reduce labeling of bacteria with IgM. Flow cytometry 2 fragments might remain attached to it as IdeSsuis cleaves the heavy chain of IgM at the N-terminus of the C3 domain [S. suis population were reduced by IdeSsuis activity 2 fragments as well.Opsonization induced by antibodies binding to the bacterial surface is an important immune defense mechanism. Previous work showed that the expression of Ideound IgM . We hypoytometry ) and immytometry ) showed activity . Analogoactivity . The redS. suis strains led us to hypothesize that the loss of IgM cleavage activity of expressed IdeSsuis leads to increased activation of the classical complement pathway and thus increased labeling of bacteria with C3b antigen. Using a serum depleted of IgG antibodies, it was shown that 10∆ideSsuis and 10∆ideSsuis∇ideSsuis_C195S were indeed significantly more labeled with C3 than S. suis wt . When rIdeSsuis was added to 10∆ideSsuis, C3 labeling was reduced by 91% . Furthermore, addition of rIdeSsuis to 10∆ideSsuis led to a significant reduction in C3 labeling even when compared to the level of C3 labeling of the wt . Thence it can be concluded that activation of the classical complement pathway and subsequent C3 labeling of S. suis is reduced by the IgM cleaving activity of rIdeSsuis.Differences in surface-bound IgM in the investigated  suis wt . Surprisd by 91% . A signiS. suis in porcine blood of growing piglets with high titers of S. suis specific IgM. Blood survival assays revealed significantly higher survival factors (SF) for S. suis wt than for 10∆ideSsuis in the blood of eight-week-old piglets with high anti-S. suis IgM (71.4 ELISA units) and high anti-S. suis IgG (77.6 ELISA units) antibody titers  had lower survival factors than S. suis wt and 10∆ideSsuis∇ideSsuis_EcoRI .The ability to survive in blood is an important feature for any pathogen causing bacteremia and systemic diseases after dissemination in the blood. We hypothesized that loss of IgM cleavage would lead to increased killing of y titers ). S. suinificant ). NevertS. suis and mutants in porcine blood, the C3 convertase inhibitor vaccinia virus complement control protein (VCP) was added to the blood prior to the addition of bacteria. VCP inhibits both the classical and the alternative C3 convertase and has been shown to inhibit the complement cascade in porcine blood [S. suis increased significantly above levels without complement inhibitor. Addition of 4 µg/ml VCP led to a doubling of the survival factor of the wt, a triplication of the survival factor of 10∆ideSsuis and 10∆ideSsuis∇ideSsuis_EcoRI and a 2.4-fold increase of the survival factor of 10∆ideSsuis∇ideSsuis_C195S, but differences to the samples without VCP were not significant. Furthermore, at 4 µg/ml VCP the survival factors of S. suis wt and 10∆ideSsuis∇ideSsuis_EcoRI were higher than those of 10∆ideSsuis and 10∆ideSsuis∇ideSsuis_C195S although differences were not significant . Interestingly, addition of 4 µg/ml VCP to 10∆ideSsuis led to an increased survival of the mutant approximating S. suis wt levels . As a read-out parameter for complement activation in this assay, soluble C5b-9 (sC5b-9) was measured after the described blood survival assay. The sC5b-9 ELISA revealed a concentration dependent decrease in sC5b-9 levels in samples with VCP . SignifS. suis is mainly infiltrated by neutrophils, opsonophagocytosis assays were conducted to specifically investigate the role of IgM cleavage in evasion of killing by these immune cells. Opsonophagocytosis assays did indeed reveal that the survival factor of S. suis strain 10  was significantly higher than that of 10∆ideSsuis  and 10∆ideSsuis ∇ideSsuis C195S  . However, the survival factor of S. suis wt was also significantly higher than that of 10∆ideSsuis ∇ideSsuis_EcoRI .As tissue infected with Ssuis∇ideSsuis_EcoRI and the wt. Despite full IgM cleavage activity, the complemented mutant showed lower survival factors in blood survival and opsonophagocytosis assays and was significantly more C3 labeled than the wt. We conducted comparative transmission electron microscopy of S. suis strain 10, 10∆ideSsuis, 10∆ideSsuis∇ideSsuis_EcoRI and 10∆ideSsuis∇ideSsuis_C195S to verify intact capsule expression in these strains. However, electron microscopy indicated that the morphology of the capsule of 10∆ideSsuis∇ideSsuis_EcoRI differed from that of the three other investigated strains . Differences in capsule expression between S. suis strain 10, 10∆ideSsuis and 10∆ideSsuis∇ideSsuis_C195S were not recorded. PCR analysis of 16 kb of the cps2 locus of S. suis strain 10 and 10∆ideSsuis ∇ideSsuis_EcoRI was conducted to investigate if larger deletions or insertions in cps genes were detectable in the complemented mutant. However, the cps amplification products did not differ in size between S. suis strain 10 and 10∆ideSsuis ∇ideSsuis_EcoRI . In conclusion, the attenuated phenotype of 10∆ideSsuis∇ideSsuis_EcoRI might be due to an aberrant capsule morphology, but the reason for this phenotypic alteration is not known.Blood survival, opsonophagocytosis and C3 deposition assays revealed phenotypic differences between the complemented mutant 10∆ide strains . The capSsuis in prime-vaccinated growing piglets [S. suis mutants without IgM cleavage activity. An animal infection experiment was consequently conducted to investigate the role of IgM cleavage by IdeSsuis in the pathogenesis of S. suis serotype 2 infection by comparing the mutant 10∆ideSsuis∇ideSsuis_C195S to the wt. Immunological screening of the piglets prior to infection revealed moderate to high anti-S. suis IgM  and IgG  titers and very low anti-IdeSsuis antibody titers  . Antibody titers did not differ significantly between the infection groups. Sixty-seven percent of piglets infected with either the wt, 10∆ideSsuis∇ideSsuis_EcoRI or 10∆ideSsuis∇ideSsuis_C195S survived the experiment versus 44.4% of piglets infected with 10∆ideSsuis . In theectively ). Patholningitis . Thus, mω = 1.89 . The resω = 1.89 . Additioω = 1.89 . EcoRI rS. suis burden (3–5 x 107 CFU/ml) but varying intracerebrospinal anti-S. suis IgM titers (< 0.1–7.7 ELISA units/ml). IgM cleavage products were not detected in the CSF of these experimentally infected piglets with acute meningitis 2 and anti IgM Fc specific antibodies. S. suis in the CSF of experimentally infected piglets was labeled with porcine IgM regardless of the infection strain 2 positive and IgM Fc positive cells than those with lower IgM titers. In conclusion, flow cytometric analysis indicated that IgM might still be bound to the bacterial surface of S. suis wt in the CSF of piglets with acute meningitis, at least in the case of intracerebrospinal IgM concentrations above five ELISA units.The CSF of one piglet with meningitis per infection group was investigated for detection of IgM cleavage products by anti-IgM Western Blot analysis. The chosen piglets all had a high intracerebrospinal ningitis , left. Aproducts , right. n strain . HoweverSsuis of S. suis is involved in complement evasion [Ssuis only, it remained to be shown that the identified phenotypes depend on the lack of the IgM cleavage activity. Noteworthy, IdeSsuis is a very large protein compared to other immunoglobulin proteases and the C-terminal 674 amino acids of the protein are not necessary for cleavage of IgM [Ssuis might carry out an additional function, which might also be related to complement evasion. Thus, we designed experiments to specifically investigate the role of IgM cleavage, which is a unique phenotype of this bacterial pathogen. To this end, a single point mutation (cysteine to serine) was introduced in the putative active center of the IgM cleaving domain of rIdeSsuis and the isogenic mutant 10∆ideSsuis was chromosomally complemented to express wt IdeSsuis (10∆ideSsuis∇ideSsuisEcoRI), as well as point mutated IdeSsuis (10∆ideSsuis∇ideSsuis_C195S). As strain 10∆ideSsuis∇ideSsuis_C195S expresses stable, point mutated IdeSsuis lacking the IgM cleavage activity, this strain is a state-of-art tool to reveal phenotypes determined by this specific protease activity. Comparative investigations with this strain demonstrated that IdeSsuis-mediated modulation of IgM labeling is due to the IgM cleaving activity alone. Loss of function experiments and comparative phenotypic analysis adding rIdeSsuis and rIdeSsuis_C195S indicated further that IdeSsuis-mediated modulation of C3 deposition is also mainly due to the IgM cleavage activity. However, as addition of rIdeSsuis_C195S also reduced C3 deposition on the bacterial surface significantly, it is not unlikely that the large C-terminus carries out a further function related to complement evasion.Previous research indicated, that the IgM protease Ide evasion . Howevere of IgM . This suSsuis is a cysteine protease. The inability of cysteine to serine point mutated rIdeSsuis to cleave porcine IgM, as shown in this study, confirms IdeSsuis as a cysteine protease. Thus, IdeSsuis is a true member of the IdeS-family of cysteine proteases annotated under the name of C66 in the MEROPS peptidase database (https://www.ebi.ac.uk/merops/cgi-bin/famsum?family=C66).Homology to known cysteine proteases and an inhibitor profile  suggesteStreptococcus pyogenes for example has been shown to use the C5a and C3 peptidase ScpA [Borrelia burgdorferi inhibits complement via multiple FH binding proteins [Escherichia coli can cleave C1-INH and thus enhance its complement inhibitory function [S. suis complement evasion has not been intensively studied and so far only the sialic acid containing capsule of certain serotypes and factor H binding molecules have been described as complement evasion factors [S. suis disease. By using the complement inhibitor VCP we show here that survival of S. suis in porcine blood is very much restricted by active complement. Accordingly, C3 knockout mice are known to be highly susceptible to disseminated S. suis infection [Since IgM is the most important activator of the classical complement cascade, we investigated the impact of IgM cleavage on the complement system. Mechanisms of complement evasion are multiple in pathogenic bacteria, viruses and yeast. ase ScpA , M protease ScpA  and the ase ScpA  to circuproteins  and entefunction . For S.  factors ,27. HoweSsuis, suggesting that IdeSsuis is involved in a novel complement evasion mechanism [Ssuis. Since C195S point mutated rIdeSsuis did not influence complement-mediated hemolysis, the IgM cleaving activity of IdeSsuis alone must be responsible for reduction of complement mediated hemolysis. In accordance, IgM labeling of sheep erythrocytes was only significantly reduced by functional rIdeSsuis but not by rIdeSsuis_C195S.Hemolysis caused by a porcine anti-erythrocyte immune serum with high IgM titers is inhibited by addition of rIdeechanism . In thisS. suis can be a prerequisite for subsequent surface-associated complement activation. We therefore analyzed IgM labeling of S. suis wt and mutants deficient in IgM cleavage and could show that labeling of S. suis with IgM is significantly reduced by IdeSsuis activity and that this reduction depends on the presence of a cysteine in the active center of IdeSsuis. Interestingly, not only the Fc fragments of porcine IgM detach from the bacterial surface upon IdeSsuis-mediated IgM cleavage, but the F(ab’)2 fragments disengage also. This phenomenon contrasts with labeling of bacteria with IgG antibodies upon cleavage by IdeS. Björk et al. showed that IgG Fab fragments remain attached to the bacterial surface upon IgG cleavage [IgM labeling of cleavage . We propcleavage . Disruptcleavage . Since n2 and unencapsulated S. suis showed that C3 deposition on S. suis depends on the classical complement pathway [Streptococcus pneumoniae in mice [Ssuis expression of S. suis wt depends mainly on its IgM cleavage activity. The increased C3 deposition of the complemented mutant 10∆ideSsuis∇ideSsuisEcoRI is, to our minds, related to a defective capsule architecture of the mutant which was detected by transmission electron microscopy. Noteworthy, C3 deposition is also significantly enhanced on the surface of an isogenic unencapsulated mutant of S. suis serotype 2 strain 10 [Ssuis and 10∆ideSsuis∇ideSsuis_C195S showed a significant increase in C3 labeling in comparison to the wt and 10∆ideSsuis showed significantly less C3 deposition after addition of rIdeSsuis than after addition of rIdeSsuis_C195S. It was recently pointed out by Kerr et al. that C3 is not only important during systemic disease but also at the site of infection during the initial steps of infection with pneumococci [S. suis asymptomatically colonizes the nasopharynx of pigs, reduced C3b deposition mediated by preceding IgM cleavage could also be important for colonization of the porcine nasopharynx by S. suis and eventual spreading. Reduced opsonization with complement nonetheless remains very important for survival in blood which is a prerequisite for S. suis to reach sites of infection, especially the cerebrospinal fluid and the meninges. Multiple virulence mechanisms must play in concert to enable S. suis to circumvent killing during bacteremia. Both mutants deficient in IgM proteolysis, 10∆ideSsuis and 10∆ideSsuis∇ideSsuis_C195S, are attenuated in whole blood survival assays. Importantly, attenuation of 10∆ideSsuis in porcine blood is abolished by addition of rIdeSsuis but not rIdeSsuis_C195S. We thus conclude that survival of S. suis in porcine blood with specific IgM titers is influenced by the ability to cleave IgM and that survival is very much restricted by IgM. In this study, we could also demonstrate that the two S. suis mutants 10∆ideSsuis and 10∆ideSsuis∇ideSsuis_C195S survive less well in opsonophagocytosis assays with purified neutrophils than the wt. This distinguishes IdeSsuis from IdeS of S. pyogenes because the IgG cleaving activity of IdeS does not impact killing by neutrophils as shown by Okumura et al [Previous experiments with the specific classical complement pathway inhibitor EGTA MgCl pathway . The imp in mice . We coultrain 10 . We are umococci . Additioumococci –33 and tumococci . Since Sra et al .Ssuis is not an essential virulence factor, despite ex vivo data suggesting a role of IgM cleavage by IdeSsuis in pathogenicity. Animal experimental infection as conducted in this study confirmed that IdeSsuis activity is not crucial for induction of severe pathologies, in particular meningitis, and associated mortality in growing piglets. Analogously, IgM-labeled S. suis bacteria were detected in the CSF of piglets from all infection groups. Differences in IgM labeling between the individual animals could possibly be due to varying stages of disease progression and inflammation and thus different amounts of IgM in the CSF at the time of CSF acquisition. Different peak patterns of the fluorescence intensity for IgM-labeled S. suis wt and 10∆ideSsuis∇ideSsuis_EcoRI in the CSF of experimentally infected piglets could not be explained by differing IgM titers or bacterial load within the CSF. Disparate peak patterns might be due to as jet unknown host factors or a divergent behavior of the complemented ideSsuis mutant in the CSF under in vivo conditions, particularly considering its aberrant capsule morphology. However, none of our conclusions on the impact of IgM cleavage are depended on finding an explanation for the two peaks of the complemented S. suis mutant since it expresses stable IdeSsuis and is able to cleave IgM. We conclude that IdeSsuis activity does not suffice to reduce labeling of S. suis with IgM in the CSF, especially in a highly inflammatory setting such as acute meningitis. This would also explain why we could not detect significant differences in the number of animals with meningitis among infection groups. However, to our knowledge, this is the first detection of IgM-labeled streptococci in the CSF of experimentally infected animals. IdeSsuis activity might not be enough to reduce labeling of S. suis with IgM completely in an inflammatory setting but IgM cleavage could have an important role for S. suis’ ability to survive in the nasopharynx since IgM concentrations are lower in nasal secretion and saliva than in serum [Ssuis-mediated IgM cleavage does not influence virulence significantly, resemble results published for IdeS of S. pyogenes which was also shown unessential for virulence of a highly invasive Group A streptococcus strain [Ssuis since all investigations were carried out with a highly virulent serotype 2 strain and derived mutants.Animal experiments conducted by Xiao et al.  and our in serum  or inflas strain . Okumuras strain . The samSsuis related virulence were not specifically designed to reveal the role of IgM proteolysis in pathogenesis and a complemented mutant was not included. The experimental design of this study, including a mutant differing from the wt in one amino acid only, allowed to investigate the role of IdeSsuis-mediated IgM cleavage in virulence more thoroughly. Accordingly, we can exclude that the phenotypic comparison is influenced by functions of the large C-terminus of IdeSsuis, which are not yet known.Former studies on IdeSsuis is not an essential virulence factor, the fact that this protease is conserved among S. suis serotypes [Ssuis might not be essential for induction of disease in growing piglets but it might play a role in colonization of mucous membranes. It has recently been shown that human secretory IgM, actively secreted to mucous membranes, can activate complement and offer protection at mucosal surfaces [Ssuis might be crucial for S. suis to circumvent activation of complement on mucous membranes and thus facilitate colonization. Furthermore, since monomeric IgM is the only virgin B-cell receptor in swine, pigs lacking IgD [Ssuis-mediated IgM cleavage. Complement inhibition through IgM cleavage could for example reduce C3d mediated B-cell co-stimulation via CD21. Further studies are needed to investigate this question, in particular the importance of IdeSsuis in colonization as well as in disease progression and immune modulation.Despite the finding that Ideerotypes  points tsurfaces . Therefoking IgD , IgM cleking IgD . It is tSsuis of S. suis was investigated regarding complement modulation, survival in porcine blood and virulence. Whereas the IgM cleaving activity of IdeSsuis was shown to be important for reduction of complement-mediated hemolysis, labeling of sheep erythrocytes with IgM and opsonization of S. suis with IgM and complement, an impact of IgM cleavage in induction of disease, especially meningitis, could not be demonstrated.In summary, in this study, the IgM cleaving activity of IdeSsuis of S. suis is a cysteine protease and its IgM cleaving activity is important for bacterial survival in porcine blood and evasion of the classical complement pathway, but not for virulence in growing piglets. Including numerous in vitro assays as well as an in vivo model of infection, this study provides extensive insight into the role of IgM cleavage in S. suis pathogenesis.We conclude, that IdeS. suis strain 10 is a virulent serotype 2 strain that has been used by different groups in studies on pathogenesis [Ssuis (∆ideSsuis) carries an in-frame deletion of the complete ideSsuis gene except the signal sequence [Ssuis∇ideSsuis_EcoRI (∇ideSsuis_EcoRI) and 10∆ideSsuis∇ideSsuis_C195S (∇ideSsuis_C195S) were constructed in this study. S. suis was grown in Todd-Hewitt broth (THB)  and on Columbia agar plates with 6% sheep blood  at 37°C. Escherichia coli was grown in Luria-Bertani (LB) medium  at 37°C under constant shaking. The following antibiotics were added to the medium if needed: 100 µg/ml ampicillin for E. coli carrying a pET45b-derived plasmid, 8 µg/ml and 3.5 µ/ml chloramphenicol for E. coli and S. suis carrying a pSET5s-derived plasmid, respectively.ogenesis ,41,42. Asequence . The othS. suis strain 10 served as template for PCR. Primers were designed based on the ideSsuis sequence of S. suis P1/7 (Gene ID: 8153996) which is identical to the ideSsuis sequence of strain 10 [Standard DNA manipulations were performed as described . Chromostrain 10 . Primer Ssuis [Ssuis at the position 195 resulting in the replacement of a cysteine by a serine in the final protein. Following thermal cycling, a DpnI digest was conducted and the product electro transformed into competent E. coli BL21. A similar procedure was carried out to generate rIdeSsuis_homologue_C195S using QuikChange Lightning Site Directed Mutagenesis Kit . More precisely, pETideSsuis_homologue, a plasmid encoding the His-tagged N-terminal domain of IdeSsuis (aa 36 to 432), sufficient for IgM proteolysis [Ssuis_C195S and pETideSsuis_homologue_C195S were sequenced to verify the original sequences and the codon encoding the C195S point mutation.The plasmid pETideSsuis  was pointeolysis , was useE. coli bacteria harboring the respective plasmids were incubated in LB-broth with ampicillin until an OD600 of 0.5. Once the OD was reached, isopropyl-β-D-thiogalactopyranoside (IPTG)  at a final concentration of 1mM was added and the cultures were then incubated at room temperature (RT) for 2.5h under constant shaking. Following centrifugation, E. coli were subjected to a lysozyme digest (1 mg/ml lysozyme 4°C 30 min) followed by ultrasound treatment . Recombinant proteins were then purified under native conditions via Ni-TED columns as recommended by the manufacturer  and dialyzed against phosphate buffered saline using dialysis membranes with a molecular weight cut off of 6000–8000Da  and frozen in liquid nitrogen. Recombinant muramidase-released protein (rMrp) was purified as previously described [escribed . His-tagSDS-PAGE was performed with 10 or 8% separating gels under reducing conditions or 6% separating gels under non-reducing conditions. Stacking gels were uniformly 4%. Following gel electrophoresis proteins were blotted onto nitrocellulose membranes  and blocked with 5% skim milk powder in Tris-buffered saline with 0.05% Tween 20 (TBST). Incubation with primary and secondary antibodies was performed in TBST with 1% skim milk powder. Antibody dilutions are specified in Table S2. Protein detection was performed using chemiluminescent substrates SuperSignal West Pico  or Quantum .Determination of IgM cleavage was performed as described previously . BrieflySsuis, rIdeSsuis_C195S, rIdeSsuis_homologue, rIdeSsuis_homologue_C195S, rIdeSsuis_C-domain, rMrp) were incubated in a concentration of 18 µg/ml with a 1:100 dilution of porcine anti-sheep erythrocyte (αEry) serum drawn either 7 or 14 days post immunization with sheep erythrocytes [Ssuis and rIdeSsuis_C195S were added to the sera prior to the hemolysis assay to reach final concentrations of 5, 20, 100, 200 µg/ml. Sheep erythrocytes were purified as described [405 of the supernatant was measured.Recombinant proteins . Temperature shifts, performed as described previously [S. suis.r pSET5s  to rendeeviously , ensuredSsuis was constructed using the primers Sonde450bpfor and Sonde450bprev. The second probe, 412bp in size, binding within the pSET5s backbone was generated with the primer pair pSET5sSondefor and pSET5sSonderev. After hybridization, the membrane was incubated with peroxidase-labeled streptavidin  and signals detected by chemiluminescence . PCR analysis with the primers pSET5spreMCSfor and postMCSpSET5s was conducted to verify complete excision of the pSET5s plasmid from the S. suis chromosome.To verify the correct chromosomal complementation, mutants were sequenced, tested in restriction enzyme digest with EcoRI and analyzed by non-radioactive Southern blot. For Southern blotting, chromosomal DNA was digested with HincII, blotted onto a positively charged nylon membrane  and subsequently hybridized with two different biotinylated probes. Between hybridizations with the different probes, the membrane was stripped with 0.2 N NaOH, 1% sodium dodecyl sulfate (SDS). The first probe, 450 bp in size, binding a 685bp fragment within ideSsuis was confirmed in bacterial supernatants by anti-IdeSsuis Western blot analysis as described [S. suis strain 10, 10∆ideSsuis, 10∆ideSsuis ∇ideSsuis_EcoRI, ∆ideSsuis∇ideSsuis_C195S were grown to an OD600 of 0.8, centrifuged and the supernatants concentrated 24-fold using 30 kDa cut off centrifugal filters . One hundred microliter of the 24-fold concentrated supernatants was incubated with a 1:100 dilution of porcine serum, followed by anti-IgM Western blotting.Expression of Ideescribed . Furtherescribed . BrieflyS. suis strain 10, 10∆ideSsuis, 10∆ideSsuis∇ideSsuis_C195S and 10∆ideSsuis∇ideSsuis_EcoRI was investigated by transmission electron microscopy using lysine-ruthenium red (LRR) staining as described previously [S. suis strain 10 and 10∆ideSsuis∇ideSsuis_EcoRI was measured 25 times. Measurements were only conducted of bacteria which capsule had been cut in a 90° angle.The expression and thickness of the capsule of eviously . The capSsuis and F(ab’)2 fragments, purified as described below, were used as antigen for immunization of a rabbit. IgM-Fc specific antibodies were purified from a commercial polyclonal anti-pig IgM antibody . All chromatographic purification procedures were performed with the ÄCTA prime plus chromatography system . In detail, the gamma globulin fraction of porcine serum was twice precipitated with 40% ammonium sulfate, followed by dialysis against phosphate buffered saline with 300 kDa cut off membranes  for five days. The dialysate was then affinity chromatographed over protein G columns  to exclude porcine IgG. The flow through from protein G columns was purified by thiophilic adsorption chromatography . The eluate from thiophilic adsorption chromatography was purified further via size exclusion chromatography . The eluates corresponding in size to pentameric IgM were pooled and concentrated via 10 kDa cut off centrifugal filters  to render a total volume of 2 ml. Purified IgM was next subjected to a digest with rIdeSsuis (20 µg rIdeSsuis/mg IgM) for 2.5h at 37°C. F(ab’)2 fragments were purified from the digest via size exclusion chromatography. The eluates corresponding to 130kDa F(ab’)2 fragments were pooled and subsequently purified via affinity chromatography with a Ni-sepharose column  to exclude remaining rIdeSsuis that had been used to digest IgM. The flow through of this chromatography was then used as antigen for immunization of a rabbit (BioGenes GmbH). The rabbit was immunized three times with 250–500 µg antigen being used for priming and 100 µg antigen for the two booster immunizations. Final bleeding serum was precipitated with 40% ammonium sulfate, the precipitate was then purified by affinity chromatography with Sepharose columns covalently coupled with porcine IgG to exclude those antibodies cross reacting with the light chain of porcine IgG. The flow through was purified further by affinity chromatography with Sepharose columns covalently coupled with porcine IgM-F(ab’)2 fragments generated also by rIdeSsuis digest. The eluate from this chromatography was analyzed for its specificity for porcine IgM-F(ab’)2 portion in Western blot analysis as described above . Western blot analysis confirmed that the antibody does not cross-react with IdeSsuis and rMrp (results not shown). For purification of anti-IgM Fc specific antibodies, a commercial polyclonal goat anti-pig IgM antibody was purified by affinity chromatography with Sepharose columns coupled with IgM-F(ab’)2. The flow through from this procedure was then applied to Sepharose columns coupled with intact pentameric porcine IgM. The eluate from this chromatography was also verified in Western blot analysis to recognize only IgM Fc fragments .To generate an antibody specifically against porcine IgM F(ab’)2 fragments, porcine IgM was purified from serum, digested with rIdeS. suis bacterin based on strain 10, essentially as described previously for a bacterin vaccination trial [A pig was prime-booster-booster vaccinated with a on trial  except tS. suis strain 10, 10∆ideSsuis, 10∆ideSsuis ∇ideSsuis_EcoRI and ∆ideSsuis∇ideSsuis_C195S were grown to an OD600 of 0.8, followed by incubation at 4°C with the anti-S. suis hyperimmune serum against S. suis serotype 2 for 0.5 h. Hereafter, bacteria were incubated in THB at 37°C for 4h. In the first staining protocol, bacteria were labeled with a mouse anti-pig IgM primary antibody  and a PE-labeled goat anti-mouse IgG secondary antibody  as described by Seele et al. [S. suis using flow cytometry  and immunofluorescent microscopy. The percent reduction of IgM labeling was calculated as follows: The amount of IgM labeled bacteria after opsonization with serum and before incubation at 37°C was set at 100%. The reduction in IgM labeling was then calculated by subtracting the % of IgM positive bacteria after incubation at 37°C from the initial 100%. Staining was also performed with antibodies directed specifically against the F(ab’)2- and Fc-regions of porcine IgM. Prior to this staining, opsonized bacteria were blocked with 1:100 diluted donkey serum  at 4°C for 0.5h. Staining with primary and secondary antibodies was performed for 0.5 h at 4°C each. Rabbit anti-pig IgM-F(ab’)2 and goat anti-pig IgM-Fc primary antibodies were both used at a dilution of 1:100. As secondary antibodies, 1:250 dilutedPE-labeled F(ab’)2 donkey anti-goat IgG  and 1:200 diluted fluorescein isothiocyanate (FITC)- labeled donkey anti-rabbit IgG  antibodies were used. Detection of IgM labeled S. suis was again performed by flow cytometry . The % reduction in the IgM F(ab’)2 and IgM Fc signal was calculated as described above. To detect IgM on the surface of S. suis in the CSF of experimentally infected piglets, CSF of piglets with high intracerebrospinal S. suis burden was centrifuged and stained with anti-IgM F(ab’)2 and anti IgM Fc specific antibodies. Cerebrospinal fluid was frozen within five minutes after acquisition and stored at −80°C until analysis. Flow cytometry  was used to detect IgM F(ab’)2 and IgM Fc labeling on S. suis. To gate S. suis in the CSF correctly, preliminary tests were conducted with S. suis wt incubated in the CSF of healthy but S. suis infected piglets or in the CSF of the same piglets supplemented with serum with moderate IgM levels (50 ELISA units) or IgG levels (29.1 ELISA units), respectively. The frequencies of parent of IgM F(ab’)2 and IgM Fc-positive S. suis were determined via FlowJoTM_V10 software.For detection of IgM on the bacterial surface e et al. . PresencS. suis strain 10 serum as source of IgM and complement. Briefly, S. suis strain 10, 10∆ideSsuis, 10∆ideSsuis ∇ideSsuis_EcoRI, ∆ideSsuis∇ideSsuis_C195S were grown to an OD600 of 0.8. Then, 75µl of the respective cultures were centrifuged and incubated for 1h at 37°C on a rotator with 150 µl of a 1:2 dilution of an anti-S. suis strain 10 hyperimmune serum depleted of all IgG. Staining of C3 labeled bacteria was conducted with 250µl of a 1:150 diluted FITC-labeled cross-reactive rabbit anti-human C3c antibody  for 1h at 4°C under constant rotation. As positive control, undiluted serum was used, as negative control, serum that had been heat inactivated at 56°C for 30 min was used. For samples with the addition of recombinant protein, 5µg of rIdeSsuis or rIdeSsuis_C195S were added to 10∆ideSsuis prior to incubation with serum. Samples were measured using BD, FACSFortessa and analyzed using FlowJoTM_V10 software.Complement deposition on the streptococcal surface was assessed basically as described , with th-S. suis strain 10 IgM, anti-S. suis strain 10 IgG and anti-IdeSsuis antibody titers were determined by ELISA as described by Seele et al. [Antie et al. . AntibodS. suis wt and mutants in porcine blood was analyzed using whole heparinized blood from eight-week old piglets from a commercial pig farm (infected with various S. suis serotypes). The withdrawal of blood was approved under the permit number N19/14 by the responsible authorities of the state of Saxony, Germany (Landesdirektion Sachsen). Assays with the addition of rIdeSsuis_homologue and rIdeSsuis_homologue_C195S were conducted with blood from eleven-week old piglets known to be free of S. suis sly+mrp+epf+cps2, cps7 and cps9 that served as placebo animals in an infection experiment approved by the responsible authorities of the state of Saxony, Germany under the permit number TVV 37–17. Survival factors were determined by dividing the number of CFUs after a 2 h incubation period at 37°C by the number of CFUs at t = 0 . As indicated, 0.5 ml porcine whole blood was mixed with 0.1, 1, 10 µg rIdeSsuis or 10 µg rIdeSsuis_C195S, rIdeSsuis_homologue, rIdeSsuis_homologue_C195S. Then 1.5 x 106 CFU S. suis wt or 10∆ideSsuis were added. Bacteria were added from glycerol stocks prepared at the late exponential growth phase. The mixture was incubated for 2 h at 37°C on a rotator. For the detection of IgM cleavage products after the 2 h incubation period, samples were centrifuged and the supernatant analyzed by anti-pig IgM Western blot analysis under reducing conditions as described above.Survival of vaccinia virus complement control protein , 10, 2, 0.2 µg of VCP were added to 0.5 ml whole porcine blood and incubated at 37°C for 5 min on a rotator. S. suis wt, 10∆ideSsuis, 10∆ideSsuis∇ideSsuis_EcoRI, 10∆ideSsuis∇ideSsuis_C195S were then added at 4 × 106 CFU/ml and the whole incubated at 37°C for 2h. VCP was used at concentrations where it is known to inhibit complement significantly but not completely [For survival assays with the complement inhibitor 5 CFU S. suis strain 10, 10∆ideSsuis, 10∆ideSsuis ∇ideSsuis_EcoRI, ∆ideSsuis∇ideSsuis_C195S were incubated with 100 µl of porcine serum for 0.5h at 37°C on a rotator. Bacteria were added from glycerol stocks prepared at the late exponential growth phase. The chosen experimental serum was drawn from a piglet with meningitis five days after experimental infection with S. suis strain 10 within a different animal experiment approved under the permit number TVV 11/16 by the ethics committee of the Landesdirektion Sachsen. This serum has moderate levels of specific anti-S. suis IgM (20.4 ELISA units/ml) and relatively low anti-S. suis IgG (35.8 ELISA units/ml) as confirmed by anti-S. suis IgM and IgG ELISA. Serum of colostrum-deprived piglets (CDS) served as positive control, anti-S. suis hyperimmune serum as negative control. Porcine neutrophils were separated as described by Seele et al. 2015 using ficoll density centrifugation [6 purified porcine neutrophils were added to the pre-incubated serum containing the above mentioned 1.5 x 105 S. suis corresponding to a multiplicity of infection (MOI) of 0.03. Samples were then incubated for 2h at 37°C on a rotator and the survival factor determined as described for the whole blood survival assays above.For opsonophagocytosis assays with purified porcine neutrophils, 1.5 x 10Porcine sC5b-9 was measured by ELISA as described by Messias-Reason et al. . with misly+mrp+epf+cps2, cps7 and cps9 S. suis were divided into four infection groups with nine animals per group. The animals in the respective groups were intranasally challenged with 1.5 x 109 CFU S. suis strain 10, 10∆ideSsuis, 10∆ideSsuis ∇ideSsuis_EcoRI, 10∆ideSsuis∇ideSsuis_C195S at an age of 8 weeks after predisposition with 1% acetic acid as described [S. suis strains were screened in a MP-PCR for detection of the cps1, cps2, cps7, cps9, mrp, epf, sly, arcA and gdh [Ssuis infection group were tested in an ideSsuis PCR with the primer pair 800vorEcoRI, 800nachEcoRI to confirm the absence of the ideSsuis gene. All isolates from the CSF, the tonsils and chosen inner organs of the 10∆ideSsuis∇ideSsuis _C195S infection group were subjected to an ideSsuis PCR with the primer pair IdeSsuis_con_fo, IdeSsuis_con_rev, followed by sequencing to confirm the presence of the C195S point mutation. All isolates from the 10∆ideSsuis∇ideSsuis EcoRI infection group as well as all CSF isolates from the wt and 10∆ideSsuis∇ideSsuis _C195S infection groups were tested in a PCR with the primer pair 800vorEcoRI, 800nachEcoRI. The resulting 1619 bp PCR products weres cut with EcoRI and separated by 1% agarose gel electrophoresis to discriminate between S. suis wt and the complemented strain 10∆ideSsuis∇ideSsuis _ EcoRI.Thirty-six specific-pathogen free German Landrace piglets known to be free of escribed . Bacteriescribed . Isolate and gdh . All isohttps://www.graphpad.com/quickcalcs/grubbs1/) and excluded from analysis. Probabilities were considered as follows p < 0.05 *, p < 0.01 **, p < 0.001 ***. Flow cytometric data was analyzed using FlowJoTM _V10 or Flowing Software version 2.5.1.All experiments were repeated at least three times. Data was analyzed for normal distribution by the Kolmogorov-Smirnov normality test. In case of normal distribution, student’s unpaired, two-tailed t-test was conducted. If data was not normally distributed, the nonparametric two tailed Mann–Whitney test was applied. Kaplan–Meier diagrams representing mortality and morbidity were analyzed with the log-rank test. Concentration dependency was analyzed by calculating the Person correlation coefficient. A confidence interval of 95% was chosen for all analyzes. Figures of all experiments represent the mean and standard deviation. Outliers were calculated by Graphpad QuickCalcs (


Diabetic nephropathy (DN) is one of the most devastating complications of diabetes mellitus. Carbohydrate response element binding protein (ChREBP) is a basic helix–loop–helix leucine zipper transcription factor that primarily mediates glucose homeostasis in the body. The present study investigated the role of ChREBP in the pathogenesis of DN. The expression of ChREBP was detected in patients with type 2 diabetes mellitus (T2DM), diabetic mice, and mesangial cells. ELISA was used to measure cytokine production in mesangial cells. Flow cytometry analysis was performed to detect the apoptosis of mesangial cells in the presence of high glucose. The expression levels of ChREBP and several cytokines  were up-regulated in T2DM patients. The mRNA and protein levels of ChREBP were also significantly elevated in the kidneys of diabetic mice. Moreover, glucose treatment promoted mRNA levels of TNF-α, IL-1β, and IL-6 in mesangial cells. Glucose stimulation induced significant apoptosis of SV40 MES 13 cells. In addition, transfection with ChREBP siRNA significantly inhibited ChREBP expression. Consequently, the inflammatory responses and apoptosis were inhibited in SV40 MES 13 cells. These results demonstrated that ChREBP could mediate the inflammatory response and apoptosis of mesangial cells, suggesting that ChREBP may be involved in the pathogenesis of DN. Diabetic nephropathy (DN) is one of the most devastating complications of diabetes mellitus and is characterized by macroalbuminuria, hypertension, and decreased glomerular filtration rate (GFR) . RecentlCarbohydrate response element binding protein (ChREBP) is a basic helix–loop–helix leucine zipper transcription factor that primarily mediates glucose homeostasis in the body. After forming a heterodimeric complex in the nucleus, this protein activates carbohydrate response element motifs for transcriptional regulation of its target genes in a glucose-dependent manner ,5. AmounIn the present study, we detected the expression levels of ChREBP and its target genes in patients with type II diabetes and further investigated the roles of ChREBP in glomerular mesangial cells.Patients (62 cases) with type 2 diabetes mellitus (T2DM) were recruited in the Second Hospital of Jilin University from March 2015 to November 2017. Diagnostic criteria: meeting the 1999 WHO diagnosis of diabetes  and the classification criteria, with kidney dysfunction to various degree. According to the 24 h-urinary albumin excretion rate (24 h-UAER), patients were divided to type 2 diabetes mellitus group  and diabetic nephropathy group . Thirty cases healthy subjects (NC group) without family history of diabetes were enrolled in the hospital within the same period. The blood samples were collected from the cubital vein, and the serum was isolated from the whole blood sample post coagulation. The present study was approved by the Internal Review Board (IRB) of Second Hospital of Jilin University, and each participant signed the informed consent.2.Renal mesangial SV40-MES 13 cells were obtained from American Type Culture Collection (ATCC)  and were cultured in low glucose Dulbecco’s Modified Eagle’s Medium , supplemented with 10% fetal bovine serum (FBS) . The cells were incubated at 37°C, with 5% COMale C57BL/6 mice were purchased from the Shanghai Laboratory Animal Company . Mice were fasted for 4 h and injected intraperitoneally with 60 mg/kg STZ  or vehicle control for five consecutive days. All animal experiments were approved by the Animal Ethics Committee of Jilin University.CT method.RNA was isolated from cultured cells using the RNeasy mini-kit . The quantity and quality of total RNA samples were checked by Bioanalyzer RNA 6000 Nano assay . Total RNA (1 µg) was reverse transcribed into cDNA with the PrimeScript RT Master Mix Perfect Real Time (TaKaRa). Real-time PCR conditions were 25–30 cycles at 95°C for 30s, 56°C for 30s, and 72°C for 1 min. All reactions were performed in triplicate. The relative amounts of mRNA were calculated by using the comparative The production of ChREBP and inflammatory cytokines including TNF-α, IL-1β, and IL-6 was determined using a commercially available ELISA kit . These concentrations were interpolated from the standard curves and all the samples were assayed in triplicate.Whole cells were lysed in 1× SDS sample buffer and resolved by electrophoresis using SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with primary antibodies overnight, and then incubated with appropriate horseradish peroxide-conjugated secondary antibodies for 3 h followed by detection with a Super Signal Enhanced Chemiluminescence kit . For sequential blotting, the membranes were stripped with Stripping Buffer (Pierce) and re-probed with proper antibodies.t-test and one-way ANOVA. Values with P<0.05 were considered statistically significant.The results were calculated as the mean ± the standard derivation (SD). Significances between groups were evaluated using Student’s P=0.390). The detailed values of BMI, FPG, HbA1c, TC, TG, HDL-C, LDL-C, FINS, Scr, or HOMA-IR in the DM, DN, or control group were shown in Main metabolic and biochemical characteristics of all subjects are summarized in P<0.05) (P<0.05)  A–C. ChRE(P<0.05) D. Additi(P<0.05) A–C.C57BL/6 mice with diabetic models were induced by administration with streptozotocin (STZ). Consequently, we found that the mRNA and protein levels of ChREBP were significantly elevated in the kidneys of diabetic mice A,B. MoreTo confirm the inflammatory reaction to the high glucose in mesangial cells, we detected the expression of several pro-inflammatory cytokines  in SV40 MES 13 cells. Results showed that glucose treatment promoted mRNA levels of TNF-α, IL-1β, and IL-6 A. MoreovTo explore the role of ChREBP in the inflammatory response, we manipulated the ChREBP level in mesangial cells using RNAi method. We found that transfection with ChREBP siRNA significantly inhibited the mRNA and protein expression of ChREBP in SV40 MES 13 cells A,B. As aChREBP is a critical transcription factor that primarily mediates glucose homeostasis . RecentlIt is widely considered that DN is a chronic low-grade inflammatory disease . Many inin vivo data of depletion of ChREBP were absent. In our future study, we will knockdown ChREBP to validate its role in the rat DN model. At last, the specific underlying mechanism by which ChREBP exerts its biological role in diabetes is not fully elucidated.There are several limitations of this study. First, we detected the expression of ChREBP in a relatively small sample size, which should be further validated in a larger cohort population. Second, the knockdown of ChREBP was only performed in renal mesangial cells, and thus the in vivo or in vitro, in an association with an elevated inflammatory response. Moreover, the strategy of ChREBP inhibition reduced the inflammatory cytokines in response to high glucose. The present study demonstrates that ChREBP may be developed as a novel therapeutic target for controlling the progression of DN.In summary, the present study reveals that ChREBP is elevated by the glucose


Chronic diseases account for over 70% of health gaps between Aboriginal people and the rest of the Australian population. The 1 Deadly Step program involves community-based events that use a sporting platform and cultural ambassadors to improve chronic disease prevention and management in New South Wales (NSW).This study aimed to evaluate the feasibility and acceptability of a community-based chronic disease screening program for Aboriginal people.In 2015, the program was enhanced to include an iPad app for screening assessments, a results portal for nominated care providers, and a reporting portal for program administrators and implemented in 9 NSW community events. A mixed methods evaluation comprising survey data, analytics obtained from iPad and Web portal usage, and key informant interviews was conducted.Overall, 1046 people were screened between April 2015 and April 2016  female, 957 (91.49%) Aboriginal or Torres Strait Islander). High chronic disease rates were observed (231 [22.08%] participants at high cardiovascular disease (CVD) risk, 173 [16.54%] with diabetes, and 181 [17.30%] with albuminuria). A minority at high risk of CVD (99/231 [42.9%]) and with diabetes (73/173 [42.2%]) were meeting guideline-recommended management goals. Overall, 297 participants completed surveys (response rate 37.4%) with 85.1% reporting satisfaction with event organization and information gained and 6.1% experiencing problems with certain screening activities. Furthermore, 21 interviews were conducted. A strong local working group and processes that harnessed community social networks were key to implementation success. Although software enhancements facilitated screening and data management, some technical difficulties  impeded smooth processing of information. Only 51.43% of participants had a medical review recorded postevent with wide intersite variability (10.5%-85.6%). Factors associated with successful follow-up included clinic managers with overall program responsibility and availability of medical staff for immediate discussion of results on event day. The program was considered highly resource intensive to implement and support from a central coordinating body and integration with existing operational processes was essential.1 Deadly Step offers an effective and acceptable strategy to engage Aboriginal communities in chronic disease screening. High rates of risk factors and management gaps were encountered, including people with no previous knowledge of these issues. Strategies to improve linkage to primary care could enhance the program’s impact on reducing chronic disease burden. Chronic diseases, including cardiovascular disease (CVD), diabetes, chronic kidney disease (CKD), chronic respiratory disease and cancer, account for over 70% of health gaps between Aboriginal people and the rest of the Australian population . Aborigideadly is used by many Aboriginal people to mean awesome, great, excellent and the term 1 Deadly Step makes a link between rugby league and making a step toward good health. First implemented in 2012, it uses a culturally safe, innovative, community-based model in which annual events are held to increase awareness of chronic diseases and to promote prevention, early detection, and evidence-based management of chronic diseases through timely referral and follow-up. At each community event, consenting participants are taken through specific stations to assess chronic disease risk factors. Drawing on the popularity of rugby league in Aboriginal communities, the program uses this sporting platform to encourage local communities to participate. High-profile Aboriginal rugby league players from the local community are engaged as cultural ambassadors and are available on the event day to promote the importance of looking after one’s health.Community-based strategies that improve the uptake of best practice recommendations could both substantially reduce the disease burden from chronic diseases and help improve health system efficiencies. The 1 Deadly Step program was developed in partnership with New South Wales (NSW) Health and the Australian Rugby League to address the high prevalence of chronic diseases in NSW Aboriginal communities. The term In this paper, we describe the development of an electronic platform to support implementation of the 1 Deadly Step program and outline the findings from a mixed methods evaluation. It draws on the key findings from the full evaluation report prepared for the commissioning agency . The objAn earlier evaluation of the program in 2012 concluded that 1 Deadly Step events represented a successful community screening approach with high acceptability by the participating communities . HoweverA screening algorithm was developed as an iPad app. ACCHS staff were engaged to inform the design via a series of workshops and iterative testing of software prototypes. A local Aboriginal artist also provided the software design to improve the visual appeal of the app. The core elements of the system built are shown in A Web-based portal allows program staff to manage events . This inFor consenting participants, a clinical summary report is accessible from a password protected, secure data repository. The ACCHS or general practice can view data for the participants who have nominated them as their care provider . Up to 3Program administrators can access aggregated data reports for each event, which includes the demographic and health profile of participants, numbers of assessments completed, and the nominated care providers. A site evaluation report was also provided by the research team postevent for dissemination to participating health care providers.The enhanced program was implemented by the NSW Agency for Clinical Innovation in 2015-2016. A program logic model  and evalA mixed methods approach was taken to data collection, and 4 main data sources were used to inform the evaluation . Deident1c), and the capillary blood glucose level taken on the event day. CKD risk was defined as any of the following: body mass index (BMI) greater than 30 kg/m2, current smoker, the presence of CVD, family history of CKD, and the presence of diabetes ... and so we winged it on the day...it took probably about a minute with each person and everyone had the hang of it. So that just proves how simple the app is to use I guess.LHD coordinatorOne manager commented that the training could be more structured and that less experienced staff might benefit from small group learning. Several participants also suggested dedicating more training in the use of the point-of-care machinery.Interviewees were generally positive about event day, viewing it as an opportunity for family and friends to come together. Holding events outdoors rather than inside a clinic facility was seen as particularly important:it’s a good get together...you can’t put money on the value of community getting together... having a yarn and catching up with each other.ACCHS registered nurseIt was also perceived to be a fun way to learn more about risk factors for chronic disease. One hospital district nurse felt that it went beyond the usual “deficit” model of Aboriginal health and served to empower individuals:Aboriginal people must get... bored of the statistics thrown at them about chronic disease...and their lifestyle management is causing all of these problems. Whereas I don’t think that’s what 1 Deadly Step did. I think it was a really positive way of getting the message across that you can do something about this and we’re here to help you.LHD Clinical Nurse Consultantdemedicalize the program:It also gave staff an opportunity to strengthen relationships with staff from other organizations. These events also have potential to boost ACCHS staff morale and make their work more visible to board members. Some events were held concurrently with a longstanding national cultural event  and this was an effective strategy to NAIDOC draws everybody…They don’t really see it as coming here to get screened. They see it as—if I do all these steps I get that cool jersey and I get to have a feed and I get to have a day out with my family.ACCHS practice managerHowever, running an event alongside another community event also increased operational complexity. Aboriginal staff, in particular, have community and family responsibilities at these events in addition to their work responsibilities. This appeared to make management and oversight of the day more difficult. Despite many participants highlighting the importance of holding outdoor events, inclement weather also poses additional challenges such as exposed electrical cords, marquees becoming unstable in the wind, and an increased potential for biological samples to be incorrectly processed, misplaced, or tipped over.Most people considered the clinical information collected to be important; however, this needed to be balanced against managing the workflow associated with large-scale screening. Consequently, there were mixed views concerning the optimal amount of information that should be collected. Some interviewees considered that all of the data were important as they could support other service activities such as completion of government-rebated Aboriginal health assessments. One ACCHS manager also considered this information of particular importance for improving the quality of their key performance indicator data that is provided to funding bodies. Other interviewees questioned the relative merits of conducting point-of-care testing for cholesterol, diabetes, and kidney disease for all participants (as discussed further).The use of Country Rugby League ambassadors was considered as a useful community engagement strategy, making the event more fun for children and freeing up parent or carer time for screening. Some sites used the ambassadors to leverage additional marketing opportunities through free local media advertising or via a Facebook page. Although viewed favorably, some interviewees commented that the ambassadors need to be more committed to supporting the program:there’s a role for ambassadors, but...if...the ambassadors can’t speak passionately about it, people can see straight through that...Often it’s sold as the rugby league player’s going to be there and then they’re not...it’s like a con job.ACCHS CEO1 Deadly Step shirts were also critically important incentives to enhance event attendance:in all the years I’ve been working in Aboriginal health, shirts are a really big incentive, people...love to wear them and they love to promote them.ACCHS clinic managerEncouraging the staff to wear the event shirts in the weeks leading to the event was a successful marketing strategy, as patients were asking “How do I get one of the shirts?” Some participants were disappointed that the shirt design remained unchanged from the previous year, further highlighting the importance of refreshing the designs regularly.. The main issue raised by some interviewees was related to the challenges of entering results at the blood test station. Additional problems included an inability to enter an error code when a patient’s result was outside the range of the machine. Some interviewees suggested taking a modular approach to screening events where event organizers could adapt the program to their specific requirements. This included provision of a light version of the app that did not include all of the mandatory screening stations.There was general consensus that the iPad app was easy to use and required minimal trainingSeveral interviewees raised the issue of bottlenecks associated with the blood and urine testing stations. The point-of-care machines take several minutes to process a result, and problems particularly arose when samples had to be rerun because of errors:...it’s like the brake lights on the highway...Once you start having to...rerun samples and potentially getting the same error…it slows down the flow of people going through.ACCHS CEOSome interviewees reported problems in printing patient reports, which was related to the use of older model iPads. Other interviewees commented on problems related to insufficient network capacity and problems with printers. These delays resulted in some participants leaving before receiving their report or having a discussion with GPs and nurses.Most interviewees considered follow-up activities to be resource intensive. At 1 high-performing site with follow-up rates over 80%, the ACCHS clinic manager reflected that it was around a two-month process to implement adequately:It was six to eight weeks, and there are still people with low level risk...and we’re still capturing them...But all the ones that we listed as priority, have been followed up.ACCHS clinic managerOne ACCHS CEO felt that these requirements should be made clearer in the working group when preparing for an event:...there’s a lot of work in the event, but potentially there’s a heap of work after the event and you need to think through what your follow up strategies are going to be...ACCHS CEOMuch of the follow-up processes were implemented by managers, administrators, and Aboriginal Health Workers. At sites with staff shortages, these processes were particularly difficult to operationalize. The major challenge was related to participants who nominated a care provider other than the local ACCHS for follow-up. For non-ACCHS participants, it was originally envisaged that the nominated GPs would be registered into the system and notified of the patients who had requested follow-up through them. However, in practice, this process was difficult to implement, and consequently, the coordinating agency modified the process such that a hospital district staff member was given responsibility for follow-up of non-ACCHS participants.Coordinating follow-up processes between different sectors was seen as a valuable outcome from LHD participation in the program. However, some staff suggested this needed to translate into more tangible benefits to justify their participation. For example, 1 LHD staff member would have liked the follow-up process to go one step further and allow for uploads of patient reports into the hospital record system:...I suppose the question is what was in it for us?...quite a lot of staff were involved–paid for by the LHD...if we didn't get any access to the results and be able to have some input into those patient's care then why would we be involved in the first place?LHD care coordinatorOne solution to improve follow-up care at some sites was availability of GPs and/or senior nurses on the event day itself for immediate discussion with participants. These sites ensured sufficient privacy for the participant, and it reduced the managerial and administrative staff workload postevent.The principal process for follow-up was through the use of the provider-reporting portal. Although not all providers consistently used the portal, those that had used it were generally positive and appreciated its simplicity. In addition to the patient-specific reports, the overall event report summary that was provided to key stakeholders was also generally viewed positively:I thought it [the event report Summary] was really good...it gives you exactly how many people were screened, how many people nominated the stakeholders as their provider...It highlighted for us as a health provider that you can target programs around some of that data[ACCHS project officerAlthough this report was also reviewed by the working groups, it was unclear to what extent the information was used to inform population health activities. One ACCHS CEO felt that they could benefit from strategic advice on implementation of chronic disease management programs across their community in light of the findings:it would be useful for somebody to work with the services around what they might do with that information...these are your risk factors in the community...and do some projections around...where it could head.ACCHS CEOSeveral interviewees also commented on the need for greater integration of the data into routine service provision. This included having the ability to upload participant reports directly into the electronic health records and to generate referrals for specific services such as smoking cessation. The current systems allowed only for uploading of static documents into the patient file. There was a strong interest in being able to upload results into the coded fields of the patient record that could then be used to autopopulate items required for key performance indicator reports and for Medicare-rebated health assessments.Although stakeholders were generally enthusiastic about involvement in 1 Deadly Step, the trade-off between investing in this program and other activities was raised, particularly by non-ACCHS interviewees:...they’re really pushing hard for activity-based funding...we certainly had discussions early on about whether it would be possible (to justify)...the time away from people’s usual...work activities.LHD Clinical Nurse ConsultantThe funding provided to sites was generally considered insufficient to cover actual costs, and it appears that a considerable amount of in-kind support  was harnessed to support implementation. Some interviewees suggested that multiple stakeholders should pool resources from existing budgets to rationalize costs:...we weren’t trying to delegate to each other which can cause controversy with who’s bossing who...We knew exactly what we had to do and, with that, we found we were more inclined to give more back into it.LHD coordinatorAn additional factor that may influence sustainability was the need to establish a system whereby shared learnings from sites could be made available to other sites. One interviewee, for instance, raised the idea that an experienced person from a site that had held a 1 Deadly Step event earlier might mentor those responsible for conducting an event elsewhere.In this paper, we examined multiple data sources to evaluate a community-based chronic disease screening and management program for Aboriginal communities in 1 Australian state. There are three main findings from the evaluation: (1) the clinical profile of participants suggested a high burden of chronic diseases and their risk factors, (2) the program had high satisfaction and acceptability rates with several implementation barriers and enablers identified, and (3) factors that might influence the maintenance and sustainability of the program were observed. These findings have important implications for future iterations of the program.It is difficult to determine representativeness of the communities in which events were held as people often travel large distances to attend. Therefore, we used a large geographic boundary to determine the draining population and estimated that 6% of the population were screened at these events. When compared with the 2012-2013 Australian Aboriginal and Torres Strait Islander Health Survey (AATSIHS) several observations can be made of the 1 Deadly Step participant profile ,15. In tThere are two important implications from the risk factor and care management information. First, 1 Deadly Step is a useful strategy for identifying people at high risk of chronic disease. To enhance the reach of the program, repeated events are likely to be needed to increase the proportion of eligible community members participating. The risk factor prevalence rates of 1 Deadly Step participants is considerably higher and occurs at younger ages than for non-Indigenous people, and in several areas, these rates are higher than reported in representative surveys of Aboriginal and Torres Strait Islander people. There are clearly substantial opportunities to use the program to address access barriers for this group. Second, substantial gaps in optimal care for those with or at high risk of chronic diseases were observed, and consequently, there are also major opportunities for providing higher quality care for these groups through better linkages with their primary care providers.community capital is perhaps the strongest asset of the program. Implementation of traditional clinical processes into such a setting requires considerable planning and harnessing of resources, and the ability to effectively engage communities in this process is noteworthy. The use of existing community events such as NAIDOC, judicious marketing through local and social media, involvement of country rugby league ambassadors, and coordination of activities via a local working group were highly effective strategies to support program participation. Staff from all health service sectors and levels within their organizations demonstrated immense enthusiasm for conducting activities that could address the high–chronic disease burden experienced by Aboriginal communities. Staff welcomed the opportunity to work collaboratively and showcase their efforts to the community. The coordinating agency played a critical role in supporting implementation of the program. Given the resource constraints under which these staff were working, this level of support for the program was an essential enabler to its successful implementation. The ability to be locally responsive while at the same time providing a macrolevel view of the program is an important success factor.Several factors influenced program implementation. Participants were generally highly satisfied with the program, and staff involved in its implementation were positive about their involvement. The ability of 1 Deadly Step to draw on existing A number of areas were also identified where the program was hindered by specific challenges. The most substantial issue was related to the follow-up of participants, particularly those who nominated another service provider other than the local ACCHS. Follow-up rates were highly variable across sites and suggest that there are particular local service issues at play. This has been documented in previous research conducted in the Northern Territory and Queensland . ServiceThere were also a range of technical hurdles that needed to be overcome. These appeared related to the use of point-of-care machines, network connectivity, and entering data on the iPad software itself. Aside from fixing technical bugs, a major implication is determining the most feasible amount of clinical information that can be collected. Some software modifications could be considered to reduce the amount of information collected at an event. A modular approach could be taken to screening where only certain high-risk subgroups need to have blood and urine testing. A “low-information” algorithm for risk prediction could also be used that was less reliant on complete risk factor information, and this could considerably shorten screening time and reduce bottlenecks. Such algorithms have been developed and validated in overseas populations but would likely need some adaptation and validation work before being used in this setting . Balancelearning collaborative in which nominated representatives could share resources, determine optimal operational approaches, benchmark their performance, and develop data-driven strategies to address follow-up challenges [There are several opportunities that could be derived from 1 Deadly Step. The most important is to integrate 1 Deadly Step data into service processes, thus making it a part of routine business planning and operations . Integraallenges . As consallenges .This evaluation comes with some important limitations. The sample attending 1 Deadly Step events was a relatively small convenience sample, and therefore, it may not be representative of the local communities participating. The 9 events were from a variety of locations across rural and urban settings in 1 Australian state; therefore, they may not be generalizable to other parts of Australia. The satisfaction survey response rate was low and could be prone to respondent bias. The assessment of the use of guideline-recommended medications was based on self-reported data that is subject to recall bias . The infTaking into consideration some of the existing strengths of the current program, the major challenges faced by 1 Deadly Step relate to its ability to sustain current levels of activity and scale-up activity across NSW. Considerable effort was expended to run these 9 events, and there is a risk that the goodwill associated with this may diminish, particularly as competing demands may displace 1 Deadly Step for other higher-priority areas. Therefore, it is important that the value proposition to staff and stakeholder organizations be high . Real pr1 Deadly Step was implemented in 9 communities in 2015-2016 and assessed the chronic disease risks for over 1000 Aboriginal people residing in these communities. The clinical data strongly support the justification for such a program given the high levels of risk factors encountered, often including people who would otherwise have had no knowledge of these issues before the events. Overall, the event implementation was highly successful and demonstrated high satisfaction by participants and staff alike. However, several challenges were highlighted, particularly in relation to resource constraints and follow-up processes. Several opportunities were identified to address these issues, and these are likely to play a critical role in influencing program sustainability. It is important to note that despite the successful implementation of the program, its effects in improving health outcomes remain unknown and a more detailed impact evaluation with long-term follow-up is needed to assess downstream system and health benefits.


Background: There is limited data on personal use fall prevention programs, and the relationship of race and ethnicity on fall risk awareness, personal beliefs, behavior change, and response to intervention. Objective: The aim of this study was to develop an educational program to prevent falls for ethnically diverse older adults. This program will be a culturally attuned program that values diversity and seeks to eliminate words and behaviors that might be discriminatory based on racial/ethnic or cultural identity. Methods: Three steps were used to develop the program: 1) constructing content domains; 2) generating the program draft; and 3) judging the program domain and content. The content domains were constructed based on data from a conventional content analysis of four focus groups from older participants (n=28) and their family caregivers (n=4), and individual in-depth interviews from health care providers (n=8). We generated the program outline with three response choices. Eight older participants and two health care providers rated it. Results: The program consisted of risk assessment, outreaching and raising awareness and knowledge. Risk assessment: all participants suggested that risk assessment should consists of objective and subjective measures. Outreaching: participants agreed that group-teaching and individual learning by peer coaching based on their culture, new blasts, brochures, and family-based approaches were the best outreaching methods that they preferred. They identified that raising awareness and knowledge should include the following topics: performing physical activity with fall risk awareness, medication management, visual care, behavioral adaptation with appropriate accessories /equipment, and environmental safety.