Clinical and in vitro models identify distinct adaptations enhancing Staphylococcus aureus pathogenesis in human macrophages.

Staphylococcus aureus is a facultative intracellular pathogen of human macrophages, which facilitates chronic infection. The genotypes, pathways, and mutations influencing that phenotype remain incompletely explored. Here, we used two distinct strategies to ascertain S. aureus gene mutations affecting pathogenesis in macrophages. First, we analyzed isolates collected serially from chronic cystic fibrosis (CF) respiratory infections. We found that S. aureus strains evolved greater macrophage invasion capacity during chronic human infection. Bacterial genome-wide association studies (GWAS) identified 127 candidate genes for which mutation was significantly associated with macrophage pathogenesis in vivo. In parallel, we passaged laboratory S. aureus strains in vitro to select for increased infection of human THP-1 derived macrophages, which identified 15 candidate genes by whole-genome sequencing. Functional validation of candidate genes using isogenic transposon mutant knockouts and CRISPR interference (CRISPRi) knockdowns confirmed virulence contributions from 37 of 39 tested genes (95%) implicated by in vivo studies and 7 of 10 genes (70%) ascertained from in vitro selection, with one gene in common to the two strategies. Validated genes included 17 known virulence factors (39%) and 27 newly identified by our study (61%), some encoding functions not previously associated with macrophage pathogenesis. Most genes (80%) positively impacted macrophage invasion when disrupted, consistent with the phenotype readily arising from loss-of-function mutations in vivo. This work reveals genes and mechanisms that contribute to S. aureus infection of macrophages, highlights differences in mutations underlying convergent phenotypes arising from in vivo and in vitro systems, and supports the relevance of S. aureus macrophage pathogenesis during chronic respiratory infection in CF. Additional studies will be needed to illuminate the exact mechanisms by which implicated mutations affect their phenotypes.

Transposon sequencing identifies genes impacting Staphylococcus aureus invasion in a human macrophage model.

Staphylococcus aureus is a facultative intracellular pathogen in many host cell types, facilitating its persistence in chronic infections. The genes contributing to intracellular pathogenesis have not yet been fully enumerated. Here, we cataloged genes influencing S. aureus invasion and survival within human THP-1 derived macrophages using two laboratory strains (ATCC2913 and JE2). We developed an in vitro transposition method to produce highly saturated transposon mutant libraries in S. aureus and performed transposon insertion sequencing (Tn-Seq) to identify candidate genes with significantly altered abundance following macrophage invasion. While some significant genes were strain-specific, 108 were identified as common across both S. aureus strains, with most (n = 106) being required for optimal macrophage infection. We used CRISPR interference (CRISPRi) to functionally validate phenotypic contributions for a subset of genes. Of the 20 genes passing validation, seven had previously identified roles in S. aureus virulence, and 13 were newly implicated. Validated genes frequently evidenced strain-specific effects, yielding opposing phenotypes when knocked down in the alternative strain. Genomic analysis of de novo mutations occurring in groups (n = 237) of clonally related S. aureus isolates from the airways of chronically infected individuals with cystic fibrosis (CF) revealed significantly greater in vivo purifying selection in conditionally essential candidate genes than those not associated with macrophage invasion. This study implicates a core set of genes necessary to support macrophage invasion by S. aureus, highlights strain-specific differences in phenotypic effects of effector genes, and provides evidence for selection of candidate genes identified by Tn-Seq analyses during chronic airway infection in CF patients in vivo.

In Vitro Selection Identifies Staphylococcus aureus Genes Influencing Biofilm Formation.

Staphylococcus aureus generates biofilms during many chronic human infections, which contributes to its growth and persistence in the host. Multiple genes and pathways necessary for S. aureus biofilm production have been identified, but knowledge is incomplete, and little is known about spontaneous mutations that increase biofilm formation as infection progresses. Here, we performed in vitro selection of four S. aureus laboratory strains (ATCC 29213, JE2, N315, and Newman) to identify mutations associated with enhanced biofilm production. Biofilm formation increased in passaged isolates from all strains, exhibiting from 1.2- to 5-fold the capacity of parental lines. Whole-genome sequencing identified nonsynonymous mutations affecting 23 candidate genes and a genomic duplication encompassing sigB. Six candidate genes significantly impacted biofilm formation as isogenic transposon knockouts: three were previously reported to impact S. aureus biofilm formation (icaR, spdC, and codY), while the remaining three (manA, narH, and fruB) were newly implicated by this study. Plasmid-mediated genetic complementation of manA, narH, and fruB transposon mutants corrected biofilm deficiencies, with high-level expression of manA and fruB further enhancing biofilm formation over basal levels. This work recognizes genes not previously identified as contributing to biofilm formation in S. aureus and reveals genetic changes able to augment biofilm production by that organism.

Biomaterial-induced conversion of quiescent cardiomyocytes into pacemaker cells in rats.

Pacemaker cells can be differentiated from stem cells or transdifferentiated from quiescent mature cardiac cells via genetic manipulation. Here we show that the exposure of rat quiescent ventricular cardiomyocytes to a silk-fibroin hydrogel activates the direct conversion of the quiescent cardiomyocytes to pacemaker cardiomyocytes by inducing the ectopic expression of the vascular endothelial cell-adhesion glycoprotein cadherin. The silk-fibroin-induced pacemaker cells exhibited functional and morphological features of genuine sinoatrial-node cardiomyocytes in vitro, and pacemaker cells generated via the injection of silk fibroin in the left ventricles of rats functioned as a surrogate in situ sinoatrial node. Biomaterials with suitable surface structure, mechanics and biochemistry could facilitate the scalable production of biological pacemakers for human use.

Roles of Silk Fibroin on Characteristics of Hyaluronic Acid/Silk Fibroin Hydrogels for Tissue Engineering of Nucleus Pulposus.

Silk fibroin (SF) and hyaluronic acid (HA) were crosslinked by horseradish peroxidase (HRP)/H2O2, and 1,4-Butanediol di-glycidyl ether (BDDE), respectively, to produce HA/SF-IPN (interpenetration network) (HS-IPN) hydrogels. HS-IPN hydrogels consisted of a SF strain with a high content of tyrosine (e.g., strain A) increased viscoelastic modules compared with those with low contents (e.g., strain B and C). Increasing the quantities of SF in HS-IPN hydrogels (e.g., HS7-IPN hydrogels with weight ratio of HA/SF, 5:7) increased viscoelastic modules of the hydrogels. In addition, the mean pores size of scaffolds of the model hydrogels were around 38.96 ± 5.05 µm which was between those of scaffolds H and S hydrogels. Since the viscoelastic modulus of the HS7-IPN hydrogel were similar to those of human nucleus pulposus (NP), it was chosen as the model hydrogel for examining the differentiation of human bone marrow-derived mesenchymal stem cell (hBMSC) to NP. The differentiation of hBMSC induced by transforming growth factor ß3 (TGF-ß3) in the model hydrogels to NP cells for 7 d significantly enhanced the expressions of glycosaminoglycan (GAG) and collagen type II, and gene expressions of aggrecan and collagen type II while decreased collagen type I compared with those in cultural wells. In summary, the model hydrogels consisted of SF of strain A, and high concentrations of SF showed the highest viscoelastic modulus than those of others produced in this study, and the model hydrogels promoted the differentiation of hBMSC to NP cells.

Morphological transformation of hBMSC from 2D monolayer to 3D microtissue on low-crystallinity SF-PCL patch with promotion of cardiomyogenesis.

The effects of the stiffness of substrates on the cell behaviours of human bone marrow-derived mesenchymal stem cells (hBMSC) have been investigated, but the effects of the secondary structures of proteins in the substrates on the morphological transformation and differentiation of hBMSC have yet been elucidated. To investigate these issues, silk fibroin-poly(ε-caprolactone) SP cardiac patches of poly(ε-caprolactone; P), on which is grafted by silk fibroin (SF) with various ß-sheet contents (or crystallinity) to provide various degrees of stiffness, were produced to examine the in vitro behaviours of hBMSC during proliferation, and cardiomyogenesis on the SP patches. ß-sheet contents of SF from 20% to 44% (SP20 to SP44, respectively) were induced on patches, which were examined by attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, and analysed using the Fourier self-deconvolution method. The stiffness of the SP patches, quantified by their Young's moduli and elasticities, increased with the crystallinity of the SF. During 3 days of proliferation, hBMSC migrated and morphologically transformed into 3D microtissues with diameters of approximately 150-200 µm on low-stiffness SP20 and SP30 patches, whereas 2D monolayers were observed on the SP37 and SP44 patches. The 3D microtissues/patch yielded more extensive in vitro cardiomyogenesis of hBMSC than the 2D cell monolayer with significantly higher expressions of all examined cardiac-specific proteins after induction by 5-aza. Notably, in vivo subcutaneously growing 3D microtissues on SP20 patches and a 2D monolayer on SP44 patches were preliminarily demonstrated in a rat model. Morphological transformations of hBMSC from a 2D monolayer to a 3D microtissue by low-stiffness SP cardiac patches, promoting cardiomyogenesis, provide a new opportunity for cardiac tissue engineering.

Promoting Cardiomyogenesis of hBMSC with a Forming Self-Assembly hBMSC Microtissues/HA-GRGD/SF-PCL Cardiac Patch Is Mediated by the Synergistic Functions of HA-GRGD.

Bone marrow-derived mesenchymal stem cell microtissues (BMSCMT) enhanced cardiomyogenesis in vitro and cardiac repairs of myocardial infarcted hearts in vivo are documented. Producing human BMSCMT onto patches in vitro for cardiac tissue engineering has not been reported. For possibly producing human bone marrow-derived mesenchymal stem cell microtissues (hBMSCMT) on an elastic silk fibroin (SF)-poly(ε-caprolactone) (PCL) based patches is hereby designed. After an elastic SF-PCL (SP) patch is fabricated, hyaluronic acid (HA)/SF-PCL(HSP) and HA-GRGD/SF-PCL(HGSP) patches are fabricated by photochemically grafting HA and HA-GRGD onto SP surfaces. The results show that the proliferations of hBMSC on HGSP patches significantly exceed those on the other patches, as determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Notably, the formation of 5-aza inducing cardiomyogenic differentiations of hBMSCMT/HGSP patches is observed with typical sizes of ≈317 µm wide and 26 µm high. The cardiomyogenesis of hBMSCMT/HGSP patches including the expressions of cardiac-specific genes (e.g., Gata4) and proteins (e.g., connexin43 (CX43)) significantly exceeds those of hBMSC monolayer on the HSP and SP patches. Promoting in vitro cardiomyogenesis of hBMSC with forming cardiomyogenic differentiation of hBMSCMT/HGSP hybrid patch is possibly mediated by the synergistic functions of HA-GRGD on enhancing the activity of F-actin. The hBMSCMT/HGSP cardiac patch may be further employed to cardiac tissue engineering.

Fetal alcohol spectrum disorder: report of one case.

One seven-year-old aboriginal boy visited our outpatient department for survey of study difficulty. The physical examination revealed microcephaly, broad depressed nasal bridge, thin upper lip, smooth philtrum, epicanthal folds and clinodactyly. He also had mild mental retardation and abnormal findings on brain MRI. His mother had confirmed daily alcoholic consumption (72 to 144 gm) during pregnancy. Besides the short stature and microcephaly, the patient had developmental delay in language. According to the history, clinical presentation, and the finding of brain imaging, this case matches the diagnosis of fetal alcohol spectrum disorder. It seems reasonable to consider that some cases with the idiopathic developmental delay may fit in this disorder, thus suggesting the importance of total abstinence from alcohol during pregnancy.